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LA1512

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val_pubmed_ORC_4096_1592

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the anatomy of craniovertebral junction bones of the tiger , horse , and deer was analyzed and compared with human bones . the evolutionary changes and structural alterations that have occurred due to the functional variations are clearly seen in the comparison . understanding the anatomy of these animals clarifies the function of the various components of the bones of the craniovertebral junction . the evolutionary changes in the shapes and architectural design of the craniovertebral junction bones in each of these animals have been perfected to suit the job at hand . two dried bones each of the adult tiger , horse , and deer were procured . one of the coauthors ( s.a.g . ) has a special permission from the government of india to procure and handle cadavers of tigers for scientific purposes . the craniovertebral region bones were collected , and their major anatomical features was studied and compared with those of the adult human bones [ figures 14 ] . the shape , orientation , size , texture , foramina , and borders of each bone were studied and compared . relationship of the vertebral artery and c1 and c2 spinal nerves to the craniovertebral junction were studied on the basis of literature survey.[13 ] view of the posterior surfaces of the c1 and c2 vertebrae of tiger ( a ) , deer ( b ) , and horse ( c ) . note the differences in the sizes and shapes of the bones of each animal in relationship with the human bones ( d ) images of the craniovertebral junction bones of a horse . ( a ) inferior view of the posterior aspect of the skull showing the large occipital condyles ( 1 ) . ( b ) superior ( anterior ) surface of the atlas , as seen from the superior and anterior perspective , showing the deep cup - shaped articular surface ( 1 ) for the articulation with occipital condyles . ( e ) ventral view of the c1 vertebra showing the ventral arch ( 1 ) , ventral tubercle ( 2 ) , transverse process ( 3 ) , transverse foramen ( 4 ) , atlantal fossa ( 5 ) , and superior articular facets ( 6 ) . ( f ) dorsal view of the c1 vertebra showing the dorsal arch ( 1 ) , the inferior articular facets ( 2 ) , transverse foramen ( 3 ) , alar foramen ( 4 ) , and lateral vertebral foramen ( 5 ) . ( g ) superior view of the axis vertebra of the horse showing the c - shaped configuration of the odontoid process ( 1 ) and the deep impressions for the longitudinal ligament ( 2 ) . the superior articular facets of the axis are seen in relationship with the odontoid process . the atlantoaxial joints are relatively flat when compared with the deep concavity of the superior facets of the atlas in relationship with the occipital bone . the notch for the c2 spinal nerve is converted into the lateral vertebral foramen ( 1 ) following ossification of the ligament . ( a ) ventral view of the c1 vertebra showing the ventral arch ( 1 ) , ventral tubercle ( 2 ) , transverse process ( 3 ) , and superior articular facets ( 4 ) . ( b ) dorsal view of the c1 vertebra showing the dorsal arch ( 1 ) and the alar foramen ( 2 ) . the articular surface of the c - shaped odontoid process ( 1 ) can be seen in continuity with the superior articular facets forming a saddle - shaped joint . ( d ) superior view of the axis vertebra of the deer showing the c - shaped odontoid process ( 1 ) and the confluent superior articular surfaces ( 2 ) . the saddle - shaped articular surface can be vividly seen . ( h ) lateral view of the head of the deer . note the location of the occipital condyles and the prominence of the occipital crest images of the bones of a tiger . ( a ) ventral view of the c1 vertebra showing the ventral arch ( 1 ) , transverse process ( 2 ) , superior articular facets ( 3 ) , and alar foramen ( 4 ) . ( b ) dorsal view of the c1 vertebra showing the dorsal arch ( 1 ) and the transverse process ( 2 ) . ( c ) lateral view of the axis vertebra showing the denslike odontoid process ( 1 ) and the characteristic spinous process ( 2 ) . note the shape of the odontoid process and the articular surface of the facet that resemble the human c2 vertebra . ( f ) lateral view of the skull of the tiger showing the large occipital crest in quadrupeds , the cervical spine is a vertical part of the entire vertebral column and the thoracic spine is more or less horizontally oriented . the head of the tiger , deer , and horse protrudes anteriorly in such a fashion that it is in the maximally flexed position at the occipitoatlantal joint articulation [ figure 2 ] . on the other hand , the cervicothoracic junction is aligned in the maximally extended position . this asymmetric placement of the vertebrae in quadrupeds ensures an energy - saving balance of the head when the animal is in the resting position . when in the resting position , the movements permitted at the occipitoatlantal articulation are primarily of extension ( flexion being gravity - assisted passive movement ) ; accordingly , the posterior cervical neck musculature is markedly strong in these animals . the occipital crest is most remarkably thick in the tiger , as seen in figure 4 . in humans , the entire spine assumes a general vertical orientation and its curvatures are much less pronounced when compared with the quadrupeds studied . the vertical stance of the human being places the head directly over the neck in line of the weight bearing of the rest of the spine . the muscles of the nape of the neck and the occipital crest in humans are significantly small in dimension . the atlantoaxial bone and joint complex of the tiger have much more remarkable resemblance to human beings than the bones of herbivorous animals such as horse and deer . the brain size is relatively small and the olfactory nerves well developed and long , reaching to a length of about a foot in the horse . the cerebellum is proportionately large in animals as compared with the cerebral hemispheres . in humans , the angulation of the anterior skull base in relationship with the clivus is probably related to the relatively large size of the cerebral hemispheres . the superior articular surface of the atlas ( referred to as anterior articular facet in quadrupreds ) and the occipital condyles are much larger , thicker , and stronger in all the three animals studied as compared with the corresponding human bones . the large occipital condyles of these animals sit deep into the cup - shaped anterior ( superior ) articular facets of the atlas [ figures 24 ] and form a joint that appears like a ginglymus or a hinge joint , providing an opportunity for extra stability and enhanced mobility as compared with the human occipitoatlantal joint . the superior facet of the atlas is much deeper in all the three animals as compared with the human superior facet , which is almost flat . the range of movements at the occipitoatlantal articulation is chiefly of extension and flexion , with a small amount of lateral oblique movements . the cervical part of the vertebral column is most mobile in horses ; the mouth may be brought around to reach the flank on full lateral flexion of the neck and ventrally to reach the pasture on ventral flexion . the atlas bone has ring - shaped anterior and posterior arches and has wide lateral platelike projections or wings of the transverse processes in all the three animals studied [ figures 24 ] . the transverse process in the human atlas is reduced to only the vertebral artery canal . the ventral ( anterior ) and dorsal ( posterior ) arches of the atlas are much thicker in the tiger , horse , and deer as compared with the humans . it is perforated on either side near its cranial margin by the lateral vertebral foramen [ figure 2 ] . in these animals , the term lateral vertebral foramen is used instead of the term intervertebral foramen in the case of the atlas and axis as this foramen does not lie between the two vertebrae as the term the ventral arch is thicker , narrower , and less curved than the dorsal arch . on the ventral surface is the ventral tubercle , which is more prominent in the horse and deer as compared with humans and tiger . although the transverse processes are large in the horse and deer as compared with humans , they are proportionately smaller in size as compared with those of the tiger . the ventral surfaces of the transverse process of the atlas in the horse and deer have a greater depth than those of a tiger , in which they are shallower but wider . between the ventral aspect of the transverse process and each wing is perforated by two foramina : the cranial one is the alar foramen , which connects with the lateral vertebral foramen by a short groove ; and the caudal one is the transverse foramen [ figure 2 ] . in tigers , there is a lateral vertebral foramen for the first cervical nerve close to the cranial border of the dorsal arch . the alar foramen is replaced by a notch in the cranial border of the wing that transmits the ventral branch of the first cervical nerve . while the human transverse process is horizontally oriented ( transverse ) , it is vertically aligned in the animals studied . the articular surface of the superior facet of the atlas accounts for roughly 75% of the entire ring of the atlas as compared with less than 40% in the atlas of humans . the joint surfaces are separated by a wide notch dorsally and a narrow one ventrally . the atlantodental joint is remarkably prominent in the horse and deer , providing articulation to the large and c - shaped odontoid process in these herbivorous animals [ figures 2 and 3 ] . the inferior articular surfaces ( referred to as posterior articular surface in quadrupeds ) of the lateral mass of the atlas are confluent anteriorly with the joint surface on the posterior arch of the atlas to form a saddle - shaped articular surface . in the horse and deer , the axis is the longest of all vertebrae . it measures 16 cm in the horse and 4.9 cm in the deer in its vertical length . the odontoid process is denslike in human and tiger bones , whereas it is a c - shaped , relatively thin and flat ring that has a wide area of joint formation with the posterior surface of the anterior arch of the atlas [ figures 24 ] . this wide area of the atlantodental joint is seen uniformly in herbivorous animals as against the denslike odontoid process in carnivorous animals . the anterior surface of the odontoid process forms a well - defined joint with the posterior surface of the anterior arch of the atlas . the joints of the horse and deer are much larger as compared with those of humans and tiger . the rotatory movements of the neck at the craniovertebral junction are superior in the horse and deer as compared with those of the tiger and humans . the limitations of the rotatory movements at the craniovertebral junction and the placement of eyeballs in a more anterior perspective of the head in the tiger and humans as compared with those in the horse and deer are adaptations that suit their lifestyle and preying , hunting , and survival needs . the dorsal surface of the odontoid process has two deep impressions on either side of the midline in a horse and deer for the attachment of the thick and fan - shaped longitudinal ligament [ figure 2 ] . this ligament extends from the rough concave dorsal surface of the dens , widens cranially , and is attached to the transverse rough area on the inner surface of the ventral arch of the atlas . the atlantoaxial joints are relatively similar in their inclination and depth in human beings and in all the three animals studied . in the horse , the lamina or the arch of the axis has a notch on each side of its cranial border that is converted into a lateral vertebral foramen ( intervertebral foramen ) by a ligament that ossifies later [ figure 2 ] . a groove extends ventrally and caudally from this foramen and houses the ventral branch of the second cervical spinal nerve . in the deer and tiger , the cranial border has a deep notch that is not converted into a foramen . in humans the inferior articular processes of the three animals are vertically oriented as compared with their more horizontal orientation in humans . the transverse process in the deer and horse is small and single and projects caudally . the transverse process and the vertebral artery foramen in the axis of the tiger are similar to those in humans . the spinous processes of the axis of all the three animals studied are large , strong , and bifid [ figures 14 ] . the axis in the tiger is characterized by its length and its enormous spinous process , which overhangs both the dorsal arch of the atlas and the laminae of the c3 vertebra . the cranial extent of the spinous process matches that of the dens [ figure 4 ] . the c2 spinous process of humans is short , stubby , and bifid and is smaller than that of the other three animals . the vertebral artery has a peculiar relationship with the transverse process of the horse . after exiting from the transverse foramen of the axis , it crosses the capsule of the atlantoaxial joint and enters the transverse foramen of the atlas . after coursing through the atlantal fossa it then runs dorsally through the alar foramen and enters the vertebral canal through the lateral vertebral foramen ( intervertebral foramen ) . in the tiger , the vertebral artery , after exiting from the transverse process of the axis , courses over the dorsal arch of the atlas and enters the transverse foramen , which is present in the base of the wing of the transverse process it then exits on the ventral aspect , loops posteriorly , and enters the lateral vertebral foramen to pursue its intracranial course . the first cervical nerve emerges through the lateral vertebral foramen of the atlas and supplies several large muscles of the nape of the neck in all the three animals studied . rudimentary in nature and function . in the horse and deer , the dorsal branch of the nerve passes dorsolaterally and supplies the dorsal neck musculature . in the horse , the ventral branch descends through the alar foramen of the atlas and passes anteriorly into the neck . in the tiger , the c1 nerve exits from the lateral vertebral foramen and its ventral branch exits through the notch in the cranial border of the atlas along the course of the vertebral artery . the second cervical nerve is larger than the first one in all the three animals , as in humans . it emerges from the spinal canal through the lateral vertebral foramen on the cranial border of the lamina of the axis . the brain size is relatively small and the olfactory nerves well developed and long , reaching to a length of about a foot in the horse . the cerebellum is proportionately large in animals as compared with the cerebral hemispheres . in humans , the angulation of the anterior skull base in relationship with the clivus is probably related to the relatively large size of the cerebral hemispheres . the superior articular surface of the atlas ( referred to as anterior articular facet in quadrupreds ) and the occipital condyles are much larger , thicker , and stronger in all the three animals studied as compared with the corresponding human bones . the large occipital condyles of these animals sit deep into the cup - shaped anterior ( superior ) articular facets of the atlas [ figures 24 ] and form a joint that appears like a ginglymus or a hinge joint , providing an opportunity for extra stability and enhanced mobility as compared with the human occipitoatlantal joint . the superior facet of the atlas is much deeper in all the three animals as compared with the human superior facet , which is almost flat . the range of movements at the occipitoatlantal articulation is chiefly of extension and flexion , with a small amount of lateral oblique movements . the cervical part of the vertebral column is most mobile in horses ; the mouth may be brought around to reach the flank on full lateral flexion of the neck and ventrally to reach the pasture on ventral flexion . the atlas bone has ring - shaped anterior and posterior arches and has wide lateral platelike projections or wings of the transverse processes in all the three animals studied [ figures 24 ] . the transverse process in the human atlas is reduced to only the vertebral artery canal . the ventral ( anterior ) and dorsal ( posterior ) arches of the atlas are much thicker in the tiger , horse , and deer as compared with the humans . it is perforated on either side near its cranial margin by the lateral vertebral foramen [ figure 2 ] . in these animals , the term lateral vertebral foramen is used instead of the term intervertebral foramen in the case of the atlas and axis as this foramen does not lie between the two vertebrae as the term the ventral arch is thicker , narrower , and less curved than the dorsal arch . on the ventral surface is the ventral tubercle , which is more prominent in the horse and deer as compared with humans and tiger . although the transverse processes are large in the horse and deer as compared with humans , they are proportionately smaller in size as compared with those of the tiger . the ventral surfaces of the transverse process of the atlas in the horse and deer have a greater depth than those of a tiger , in which they are shallower but wider . between the ventral aspect of the transverse process and the lateral mass is a depression called the atlantal fossa . in horses , each wing is perforated by two foramina : the cranial one is the alar foramen , which connects with the lateral vertebral foramen by a short groove ; and the caudal one is the transverse foramen [ figure 2 ] . in tigers , there is a lateral vertebral foramen for the first cervical nerve close to the cranial border of the dorsal arch . the alar foramen is replaced by a notch in the cranial border of the wing that transmits the ventral branch of the first cervical nerve . while the human transverse process is horizontally oriented ( transverse ) , it is vertically aligned in the animals studied . the articular surface of the superior facet of the atlas accounts for roughly 75% of the entire ring of the atlas as compared with less than 40% in the atlas of humans . the joint surfaces are separated by a wide notch dorsally and a narrow one ventrally . the atlantodental joint is remarkably prominent in the horse and deer , providing articulation to the large and c - shaped odontoid process in these herbivorous animals [ figures 2 and 3 ] . the inferior articular surfaces ( referred to as posterior articular surface in quadrupeds ) of the lateral mass of the atlas are confluent anteriorly with the joint surface on the posterior arch of the atlas to form a saddle - shaped articular surface . it measures 16 cm in the horse and 4.9 cm in the deer in its vertical length . the odontoid process is denslike in human and tiger bones , whereas it is a c - shaped , relatively thin and flat ring that has a wide area of joint formation with the posterior surface of the anterior arch of the atlas [ figures 24 ] . this wide area of the atlantodental joint is seen uniformly in herbivorous animals as against the denslike odontoid process in carnivorous animals . the anterior surface of the odontoid process forms a well - defined joint with the posterior surface of the anterior arch of the atlas . the joints of the horse and deer are much larger as compared with those of humans and tiger . the rotatory movements of the neck at the craniovertebral junction are superior in the horse and deer as compared with those of the tiger and humans . the limitations of the rotatory movements at the craniovertebral junction and the placement of eyeballs in a more anterior perspective of the head in the tiger and humans as compared with those in the horse and deer are adaptations that suit their lifestyle and preying , hunting , and survival needs . the dorsal surface of the odontoid process has two deep impressions on either side of the midline in a horse and deer for the attachment of the thick and fan - shaped longitudinal ligament [ figure 2 ] . this ligament extends from the rough concave dorsal surface of the dens , widens cranially , and is attached to the transverse rough area on the inner surface of the ventral arch of the atlas . the atlantoaxial joints are relatively similar in their inclination and depth in human beings and in all the three animals studied . in the horse , the lamina or the arch of the axis has a notch on each side of its cranial border that is converted into a lateral vertebral foramen ( intervertebral foramen ) by a ligament that ossifies later [ figure 2 ] . a groove extends ventrally and caudally from this foramen and houses the ventral branch of the second cervical spinal nerve . in the deer and tiger , the cranial border has a deep notch that is not converted into a foramen . in humans the inferior articular processes of the three animals are vertically oriented as compared with their more horizontal orientation in humans . the transverse process in the deer and horse is small and single and projects caudally . the transverse process and the vertebral artery foramen in the axis of the tiger are similar to those in humans . the spinous processes of the axis of all the three animals studied are large , strong , and bifid [ figures 14 ] . the axis in the tiger is characterized by its length and its enormous spinous process , which overhangs both the dorsal arch of the atlas and the laminae of the c3 vertebra . the cranial extent of the spinous process matches that of the dens [ figure 4 ] . the c2 spinous process of humans is short , stubby , and bifid and is smaller than that of the other three animals . the vertebral artery has a peculiar relationship with the transverse process of the horse . after exiting from the transverse foramen of the axis , it crosses the capsule of the atlantoaxial joint and enters the transverse foramen of the atlas . after coursing through the atlantal fossa , it anastomoses with the occipital artery . it then runs dorsally through the alar foramen and enters the vertebral canal through the lateral vertebral foramen ( intervertebral foramen ) . in the tiger , the vertebral artery , after exiting from the transverse process of the axis , courses over the dorsal arch of the atlas and enters the transverse foramen , which is present in the base of the wing of the transverse process it then exits on the ventral aspect , loops posteriorly , and enters the lateral vertebral foramen to pursue its intracranial course . the first cervical nerve emerges through the lateral vertebral foramen of the atlas and supplies several large muscles of the nape of the neck in all the three animals studied . the dorsal branch of the nerve passes dorsolaterally and supplies the dorsal neck musculature . in the horse , the ventral branch descends through the alar foramen of the atlas and passes anteriorly into the neck . in the tiger , the c1 nerve exits from the lateral vertebral foramen and its ventral branch exits through the notch in the cranial border of the atlas along the course of the vertebral artery the second cervical nerve is larger than the first one in all the three animals , as in humans . it emerges from the spinal canal through the lateral vertebral foramen on the cranial border of the lamina of the axis . the variations in morphometry have a relationship with the quadruped stance , acute flexion position of the neck in the neutral position , and hunting and survival needs of the individual animal .

aim : to compare the osseous anatomy of the craniovertebral junction of a horse , deer , and tiger with that of a human being . the variation in the structure of bones in these animals is analyzed.materials and methods : various dimensions of the bones of the craniovertebral junction of the horse , deer , and tiger were quantitatively measured , and their differences with those of human bones were compared and analyzed.results:apart from the sizes and weights , there are a number of structural variations in the bones of these animals that depend on their functional needs . the more remarkable difference in joint morphology is noticed in the occipitoatlantal joint . the occipitoatlantal articulation is remarkably large and deep , resembling a hinge joint in all the three animals studied . the odontoid process is c shaped in the deer and horse and is denslike in the tiger and humans . the transverse processes of the atlas are in the form of large wings in all the three animals . the arches of the atlas are large and flat , but the traverse of the vertebral artery resembles , to an extent , to that of human vertebral artery . the rotatory movements of the head at the craniovertebral junction are wider ranged in the horse and deer as compared with those of the tiger and humans . the bones of the craniovertebral junction of all the three animals are adapted to the remarkable thickness and strength of the extensor muscles of the nape of the neck.conclusions:despite the wide variations in the size of the bones , the basic patterns of structure , vascular and neural relationship , and joint alignments have remarkable similarities and a definite pattern of differences .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Platybasia Occipitoatlantal articulation Atlas Axis Vertebral artery C1 spinal nerve C2 spinal nerve CONCLUSIONS

the anatomy of craniovertebral junction bones of the tiger , horse , and deer was analyzed and compared with human bones . understanding the anatomy of these animals clarifies the function of the various components of the bones of the craniovertebral junction . the evolutionary changes in the shapes and architectural design of the craniovertebral junction bones in each of these animals have been perfected to suit the job at hand . two dried bones each of the adult tiger , horse , and deer were procured . the craniovertebral region bones were collected , and their major anatomical features was studied and compared with those of the adult human bones [ figures 14 ] . relationship of the vertebral artery and c1 and c2 spinal nerves to the craniovertebral junction were studied on the basis of literature survey. [13 ] view of the posterior surfaces of the c1 and c2 vertebrae of tiger ( a ) , deer ( b ) , and horse ( c ) . note the differences in the sizes and shapes of the bones of each animal in relationship with the human bones ( d ) images of the craniovertebral junction bones of a horse . ( b ) superior ( anterior ) surface of the atlas , as seen from the superior and anterior perspective , showing the deep cup - shaped articular surface ( 1 ) for the articulation with occipital condyles . ( e ) ventral view of the c1 vertebra showing the ventral arch ( 1 ) , ventral tubercle ( 2 ) , transverse process ( 3 ) , transverse foramen ( 4 ) , atlantal fossa ( 5 ) , and superior articular facets ( 6 ) . ( f ) dorsal view of the c1 vertebra showing the dorsal arch ( 1 ) , the inferior articular facets ( 2 ) , transverse foramen ( 3 ) , alar foramen ( 4 ) , and lateral vertebral foramen ( 5 ) . ( g ) superior view of the axis vertebra of the horse showing the c - shaped configuration of the odontoid process ( 1 ) and the deep impressions for the longitudinal ligament ( 2 ) . the superior articular facets of the axis are seen in relationship with the odontoid process . the atlantoaxial joints are relatively flat when compared with the deep concavity of the superior facets of the atlas in relationship with the occipital bone . ( d ) superior view of the axis vertebra of the deer showing the c - shaped odontoid process ( 1 ) and the confluent superior articular surfaces ( 2 ) . ( h ) lateral view of the head of the deer . note the location of the occipital condyles and the prominence of the occipital crest images of the bones of a tiger . note the shape of the odontoid process and the articular surface of the facet that resemble the human c2 vertebra . ( f ) lateral view of the skull of the tiger showing the large occipital crest in quadrupeds , the cervical spine is a vertical part of the entire vertebral column and the thoracic spine is more or less horizontally oriented . the head of the tiger , deer , and horse protrudes anteriorly in such a fashion that it is in the maximally flexed position at the occipitoatlantal joint articulation [ figure 2 ] . on the other hand , the cervicothoracic junction is aligned in the maximally extended position . this asymmetric placement of the vertebrae in quadrupeds ensures an energy - saving balance of the head when the animal is in the resting position . when in the resting position , the movements permitted at the occipitoatlantal articulation are primarily of extension ( flexion being gravity - assisted passive movement ) ; accordingly , the posterior cervical neck musculature is markedly strong in these animals . in humans , the entire spine assumes a general vertical orientation and its curvatures are much less pronounced when compared with the quadrupeds studied . the vertical stance of the human being places the head directly over the neck in line of the weight bearing of the rest of the spine . the muscles of the nape of the neck and the occipital crest in humans are significantly small in dimension . the atlantoaxial bone and joint complex of the tiger have much more remarkable resemblance to human beings than the bones of herbivorous animals such as horse and deer . the brain size is relatively small and the olfactory nerves well developed and long , reaching to a length of about a foot in the horse . in humans , the angulation of the anterior skull base in relationship with the clivus is probably related to the relatively large size of the cerebral hemispheres . the superior articular surface of the atlas ( referred to as anterior articular facet in quadrupreds ) and the occipital condyles are much larger , thicker , and stronger in all the three animals studied as compared with the corresponding human bones . the large occipital condyles of these animals sit deep into the cup - shaped anterior ( superior ) articular facets of the atlas [ figures 24 ] and form a joint that appears like a ginglymus or a hinge joint , providing an opportunity for extra stability and enhanced mobility as compared with the human occipitoatlantal joint . the superior facet of the atlas is much deeper in all the three animals as compared with the human superior facet , which is almost flat . the range of movements at the occipitoatlantal articulation is chiefly of extension and flexion , with a small amount of lateral oblique movements . the cervical part of the vertebral column is most mobile in horses ; the mouth may be brought around to reach the flank on full lateral flexion of the neck and ventrally to reach the pasture on ventral flexion . the atlas bone has ring - shaped anterior and posterior arches and has wide lateral platelike projections or wings of the transverse processes in all the three animals studied [ figures 24 ] . the transverse process in the human atlas is reduced to only the vertebral artery canal . the ventral ( anterior ) and dorsal ( posterior ) arches of the atlas are much thicker in the tiger , horse , and deer as compared with the humans . in these animals , the term lateral vertebral foramen is used instead of the term intervertebral foramen in the case of the atlas and axis as this foramen does not lie between the two vertebrae as the term the ventral arch is thicker , narrower , and less curved than the dorsal arch . on the ventral surface is the ventral tubercle , which is more prominent in the horse and deer as compared with humans and tiger . although the transverse processes are large in the horse and deer as compared with humans , they are proportionately smaller in size as compared with those of the tiger . the ventral surfaces of the transverse process of the atlas in the horse and deer have a greater depth than those of a tiger , in which they are shallower but wider . in tigers , there is a lateral vertebral foramen for the first cervical nerve close to the cranial border of the dorsal arch . while the human transverse process is horizontally oriented ( transverse ) , it is vertically aligned in the animals studied . the articular surface of the superior facet of the atlas accounts for roughly 75% of the entire ring of the atlas as compared with less than 40% in the atlas of humans . the atlantodental joint is remarkably prominent in the horse and deer , providing articulation to the large and c - shaped odontoid process in these herbivorous animals [ figures 2 and 3 ] . the inferior articular surfaces ( referred to as posterior articular surface in quadrupeds ) of the lateral mass of the atlas are confluent anteriorly with the joint surface on the posterior arch of the atlas to form a saddle - shaped articular surface . in the horse and deer , the axis is the longest of all vertebrae . it measures 16 cm in the horse and 4.9 cm in the deer in its vertical length . the odontoid process is denslike in human and tiger bones , whereas it is a c - shaped , relatively thin and flat ring that has a wide area of joint formation with the posterior surface of the anterior arch of the atlas [ figures 24 ] . this wide area of the atlantodental joint is seen uniformly in herbivorous animals as against the denslike odontoid process in carnivorous animals . the anterior surface of the odontoid process forms a well - defined joint with the posterior surface of the anterior arch of the atlas . the joints of the horse and deer are much larger as compared with those of humans and tiger . the rotatory movements of the neck at the craniovertebral junction are superior in the horse and deer as compared with those of the tiger and humans . the limitations of the rotatory movements at the craniovertebral junction and the placement of eyeballs in a more anterior perspective of the head in the tiger and humans as compared with those in the horse and deer are adaptations that suit their lifestyle and preying , hunting , and survival needs . the dorsal surface of the odontoid process has two deep impressions on either side of the midline in a horse and deer for the attachment of the thick and fan - shaped longitudinal ligament [ figure 2 ] . this ligament extends from the rough concave dorsal surface of the dens , widens cranially , and is attached to the transverse rough area on the inner surface of the ventral arch of the atlas . the atlantoaxial joints are relatively similar in their inclination and depth in human beings and in all the three animals studied . in the horse , the lamina or the arch of the axis has a notch on each side of its cranial border that is converted into a lateral vertebral foramen ( intervertebral foramen ) by a ligament that ossifies later [ figure 2 ] . in the deer and tiger , the cranial border has a deep notch that is not converted into a foramen . in humans the inferior articular processes of the three animals are vertically oriented as compared with their more horizontal orientation in humans . the transverse process in the deer and horse is small and single and projects caudally . the transverse process and the vertebral artery foramen in the axis of the tiger are similar to those in humans . the spinous processes of the axis of all the three animals studied are large , strong , and bifid [ figures 14 ] . the axis in the tiger is characterized by its length and its enormous spinous process , which overhangs both the dorsal arch of the atlas and the laminae of the c3 vertebra . the c2 spinous process of humans is short , stubby , and bifid and is smaller than that of the other three animals . the vertebral artery has a peculiar relationship with the transverse process of the horse . after exiting from the transverse foramen of the axis , it crosses the capsule of the atlantoaxial joint and enters the transverse foramen of the atlas . in the tiger , the vertebral artery , after exiting from the transverse process of the axis , courses over the dorsal arch of the atlas and enters the transverse foramen , which is present in the base of the wing of the transverse process it then exits on the ventral aspect , loops posteriorly , and enters the lateral vertebral foramen to pursue its intracranial course . the first cervical nerve emerges through the lateral vertebral foramen of the atlas and supplies several large muscles of the nape of the neck in all the three animals studied . in the horse and deer , the dorsal branch of the nerve passes dorsolaterally and supplies the dorsal neck musculature . in the horse , the ventral branch descends through the alar foramen of the atlas and passes anteriorly into the neck . in the tiger , the c1 nerve exits from the lateral vertebral foramen and its ventral branch exits through the notch in the cranial border of the atlas along the course of the vertebral artery . the second cervical nerve is larger than the first one in all the three animals , as in humans . in humans , the angulation of the anterior skull base in relationship with the clivus is probably related to the relatively large size of the cerebral hemispheres . the superior articular surface of the atlas ( referred to as anterior articular facet in quadrupreds ) and the occipital condyles are much larger , thicker , and stronger in all the three animals studied as compared with the corresponding human bones . the large occipital condyles of these animals sit deep into the cup - shaped anterior ( superior ) articular facets of the atlas [ figures 24 ] and form a joint that appears like a ginglymus or a hinge joint , providing an opportunity for extra stability and enhanced mobility as compared with the human occipitoatlantal joint . the superior facet of the atlas is much deeper in all the three animals as compared with the human superior facet , which is almost flat . the range of movements at the occipitoatlantal articulation is chiefly of extension and flexion , with a small amount of lateral oblique movements . the cervical part of the vertebral column is most mobile in horses ; the mouth may be brought around to reach the flank on full lateral flexion of the neck and ventrally to reach the pasture on ventral flexion . the atlas bone has ring - shaped anterior and posterior arches and has wide lateral platelike projections or wings of the transverse processes in all the three animals studied [ figures 24 ] . the transverse process in the human atlas is reduced to only the vertebral artery canal . the ventral ( anterior ) and dorsal ( posterior ) arches of the atlas are much thicker in the tiger , horse , and deer as compared with the humans . in these animals , the term lateral vertebral foramen is used instead of the term intervertebral foramen in the case of the atlas and axis as this foramen does not lie between the two vertebrae as the term the ventral arch is thicker , narrower , and less curved than the dorsal arch . on the ventral surface is the ventral tubercle , which is more prominent in the horse and deer as compared with humans and tiger . although the transverse processes are large in the horse and deer as compared with humans , they are proportionately smaller in size as compared with those of the tiger . the ventral surfaces of the transverse process of the atlas in the horse and deer have a greater depth than those of a tiger , in which they are shallower but wider . in tigers , there is a lateral vertebral foramen for the first cervical nerve close to the cranial border of the dorsal arch . while the human transverse process is horizontally oriented ( transverse ) , it is vertically aligned in the animals studied . the articular surface of the superior facet of the atlas accounts for roughly 75% of the entire ring of the atlas as compared with less than 40% in the atlas of humans . the atlantodental joint is remarkably prominent in the horse and deer , providing articulation to the large and c - shaped odontoid process in these herbivorous animals [ figures 2 and 3 ] . the inferior articular surfaces ( referred to as posterior articular surface in quadrupeds ) of the lateral mass of the atlas are confluent anteriorly with the joint surface on the posterior arch of the atlas to form a saddle - shaped articular surface . it measures 16 cm in the horse and 4.9 cm in the deer in its vertical length . the odontoid process is denslike in human and tiger bones , whereas it is a c - shaped , relatively thin and flat ring that has a wide area of joint formation with the posterior surface of the anterior arch of the atlas [ figures 24 ] . the anterior surface of the odontoid process forms a well - defined joint with the posterior surface of the anterior arch of the atlas . the joints of the horse and deer are much larger as compared with those of humans and tiger . the rotatory movements of the neck at the craniovertebral junction are superior in the horse and deer as compared with those of the tiger and humans . the limitations of the rotatory movements at the craniovertebral junction and the placement of eyeballs in a more anterior perspective of the head in the tiger and humans as compared with those in the horse and deer are adaptations that suit their lifestyle and preying , hunting , and survival needs . the dorsal surface of the odontoid process has two deep impressions on either side of the midline in a horse and deer for the attachment of the thick and fan - shaped longitudinal ligament [ figure 2 ] . this ligament extends from the rough concave dorsal surface of the dens , widens cranially , and is attached to the transverse rough area on the inner surface of the ventral arch of the atlas . the atlantoaxial joints are relatively similar in their inclination and depth in human beings and in all the three animals studied . in the horse , the lamina or the arch of the axis has a notch on each side of its cranial border that is converted into a lateral vertebral foramen ( intervertebral foramen ) by a ligament that ossifies later [ figure 2 ] . in the deer and tiger , the cranial border has a deep notch that is not converted into a foramen . in humans the inferior articular processes of the three animals are vertically oriented as compared with their more horizontal orientation in humans . the transverse process in the deer and horse is small and single and projects caudally . the transverse process and the vertebral artery foramen in the axis of the tiger are similar to those in humans . the spinous processes of the axis of all the three animals studied are large , strong , and bifid [ figures 14 ] . the axis in the tiger is characterized by its length and its enormous spinous process , which overhangs both the dorsal arch of the atlas and the laminae of the c3 vertebra . the c2 spinous process of humans is short , stubby , and bifid and is smaller than that of the other three animals . the vertebral artery has a peculiar relationship with the transverse process of the horse . after exiting from the transverse foramen of the axis , it crosses the capsule of the atlantoaxial joint and enters the transverse foramen of the atlas . in the tiger , the vertebral artery , after exiting from the transverse process of the axis , courses over the dorsal arch of the atlas and enters the transverse foramen , which is present in the base of the wing of the transverse process it then exits on the ventral aspect , loops posteriorly , and enters the lateral vertebral foramen to pursue its intracranial course . the first cervical nerve emerges through the lateral vertebral foramen of the atlas and supplies several large muscles of the nape of the neck in all the three animals studied . in the horse , the ventral branch descends through the alar foramen of the atlas and passes anteriorly into the neck . in the tiger , the c1 nerve exits from the lateral vertebral foramen and its ventral branch exits through the notch in the cranial border of the atlas along the course of the vertebral artery the second cervical nerve is larger than the first one in all the three animals , as in humans . it emerges from the spinal canal through the lateral vertebral foramen on the cranial border of the lamina of the axis . the variations in morphometry have a relationship with the quadruped stance , acute flexion position of the neck in the neutral position , and hunting and survival needs of the individual animal .

molecular dynamics ( md ) and monte carlo ( mc ) simulations have grown to become a central tool in physics , chemistry , and biology over the past three decades . however , in spite of the huge advancement of both algorithms and hardware , there are still some unresolved methodological issues . arguably , the most persistent of these is the question of how to handle long - range electrostatic ( coulomb and dipole the basic problem is that the integral1diverges for all finite values of the cutoff radius rcut as long as the intermolecular potential v(r ) does not decay faster than r. thus , applying a simple ( spherical or cubic ) cutoff to the electrostatic potentials may often lead to serious artifacts in the structure and thermodynamics of the system under study . although several solutions to the infinite - range interaction problem have been proposed , the most common way to circumvent this problem is the use of lattice - based summation techniques , or periodic boundary conditions ( pbcs ) . these methods compose a plethora of different algorithms that all rest on the same basic assumption , namely that the ( finitely sized ) simulation cell is duplicated in all directions to create an infinite lattice . the original implementation of this idea was developed by ewald and is built upon a separation of the interaction into short - range and long - range parts , where the former is summed up in real space and the latter in reciprocal space . the original ewald method has since been developed in many ways , and today different mesh - based methods are numerically faster alternatives to the classical ewald summation . when simulating a fluid phase , the assumption of periodicity is clearly not a correct description of the real system . this criticism has been put forward several times in the literature but was originally noted by valleau and whittington , who gave a qualitative argument about the inability of lattice summation methods to correctly reproduce long - range fluctuations in fluid systems . furthermore , several studies have addressed the issue of periodicity effects on the properties of lennard - jones fluids , ionic solutions , and biomolecules . in the context of dipolar systems , boresch and steinhauser conducted a careful study of dipole fluctuations and correlations in spc water simulated using the ewald summation technique . in particular , they addressed the importance of the so - called surface term , which describes the solvation from the dielectric surroundings of the infinite lattice on structural properties such as the dielectric permittivity , dipole time correlation functions , and the kirkwood g factor . however , the total dipole moment of the simulation box is a special property , in the sense that its total interaction with all its periodic images is identically zero , as long as the contributions are summed in spherical shells . therefore , the periodicity effects on the fluctuating dipole moment of the whole simulation box ( and related properties ) are expected to be small . in a recent contribution , we showed , however , that the fluctuations of higher order electric multipole moments of the whole simulation box are greatly influenced by the interaction between each instantaneous multipole and all of its periodic images . this effect is manifested through a difference of as much as 50% between the dielectric permittivities calculated from different multipole components , depending on whether the multipole component has an attractive or a repulsive ( or , in some cases , zero ) interaction with its neighbors . a schematic picture of the coupling of different multipole components in a system under pbcs is given in figure 1 . schematic picture of the coupling of the total dipole ( left ) and higher multipoles ( right ) of a simulation cell subjected to pbcs . see its neighbors since its self - interaction energy is zero but is solvated by the dielectric response from the surrounding medium through the surface term . 1 ) couple to its neighbors through their nonzero self - interaction but are not affected by the surface term . in addition , the dipole as well as the higher multipoles interact with the set of unconstrained multipoles qlm , l l and/or m m , ( not depicted ) in the surrounding cells , giving an ( approximately ) isotropic contribution to the solvation . in addition to the cubic simulation cell used in the majority of computer simulations , some alternative simulation cell geometries have been suggested and implemented , most notably the rhombic dodecahedron ( rd ) and the truncated octahedron ( to ) . these two bodies have the appealing property of more closely resembling the geometry of solvated spherical solutes , in the sense that they have larger inscribed spheres than a cubic simulation cell of the same volume . even though these alternative geometries are implemented in major simulation packages , there are only a few studies probing the effect of changing the cell geometry on the thermodynamic properties of the system under study . because these cells pack in lattice structures different from that of the cube , it seems reasonable to expect their periodicity effects to differ qualitatively from those of a cubic cell . in the present contribution , we will extend our previous analysis of the periodicity effects on a dipolar model system from the qualitative to the quantitative level , as well as from cubic to noncubic simulation cells . we will develop a heuristic model describing the solvation of and electrostatic fluctuations in a spherical subvolume of a dielectric medium exhibited to pbcs . this model will be compared to values of the dielectric constant calculated from simulations of a simple dipolar model system . in the following , the electrostatic fluctuations in a spherical subvolume of a dipolar model system treated using the ewald summation technique will be described by dividing the long - range solvation energy of this subvolume into two contributions:an approximately isotropic part , coming from the interaction between the instantaneous multipole qlm of a spherical volume in the central simulation cell and its noncorrelated neighbors , i.e. , qlm with l l and/or m m , in the other cells . this interaction is , at least partly , boltzmann - weighted in a simulation , and we will thus attempt to describe it using formulas valid for an isotropic dielectric medium.a strongly anisotropic part , coming from the self - interaction between qlm in the central cell and its fully correlated replicas ( qlm with l = l and m = m ) in the rest of the lattice . this part of the interaction is not boltzmann - weighted , because of the perfect periodicity imposed by the pbcs . we will thus describe this interaction using the reduced lattice - interaction tensors introduced previously . an approximately isotropic part , coming from the interaction between the instantaneous multipole qlm of a spherical volume in the central simulation cell and its noncorrelated neighbors , i.e. , qlm with l l and/or m m , in the other cells . this interaction is , at least partly , boltzmann - weighted in a simulation , and we will thus attempt to describe it using formulas valid for an isotropic dielectric medium . a strongly anisotropic part , coming from the self - interaction between qlm in the central cell and its fully correlated replicas ( qlm with l = l and m = m ) in the rest of the lattice . this part of the interaction is not boltzmann - weighted , because of the perfect periodicity imposed by the pbcs . we will thus describe this interaction using the reduced lattice - interaction tensors introduced previously . on the basis of this description , we will present a heuristic derivation of the long - range solvation free energy of the spherical subvolume . this will be compared to the behavior expected from a spherical subvolume inside an infinite isotropic dielectric medium and the analysis will thus enable us to directly probe the magnitude of the periodicity effect introduced by the pbcs . in the present study , the term periodic boundary conditions refers to a system with a potential energy upot of the form2where n = ( nx , ny , nz ) is a vector that runs over all lattice points in the particular ( unit length ) lattice and a denotes the side length of the unit cell . furthermore , the primed sum indicates that the term with i = j for n = 0 should be excluded , and v(rij , i , j ) denotes the intermolecular potential between particles i and j , depending in general on their separation rij and orientations i and j . in practice , v(rij , i , j ) is usually long - range in the sense that it decays no faster than r , the two most important examples being the coulomb and dipole since the sum in eq 2 is slowly ( and conditionally ) convergent , more elaborate methods to evaluate the potential energy in a pbc system need to be used in practice . the by far most popular technique to achieve a fast convergence of the potential energy is the technique originally due to ewald and different mesh - based variants thereof . within the ewald - based methods , the short - range ( n = 0 ) part of upot is screened through the addition of a gaussian charge ( dipole ) cloud and is thereafter summed within a , usually spherical , cutoff after considering the nearest image convention . the long - range ( n 0 ) part of the potential energy is summed up in fourier space , leading to a quickly ( and absolutely ) convergent sum . although cubic simulation cells are used for the majority of simulation studies , the use of alternative simulation cell geometries has started to become increasingly popular . in total , five classes of geometrical bodies are translationally space - filling and can thus be used for simulating a periodic system ; however , due to their relatively sphere - like symmetry , the two most useful alternatives to the cube , at least for the simulation of bulk systems , are the rhombic dodecahedron ( rd ) and truncated octahedron ( to ) . while the cube , of course , packs in a simple cubic ( sc ) lattice structure , the natural choice for the lattice structures of the rd and to are face - centered cubic ( fcc ) and body - centered cubic ( bcc ) , respectively . however , smith and fincham showed that the use of a body - centered tetragonal ( bct ) lattice structure for the rd , with one side of the unit cell elongated by a factor 2 compared to the other two , greatly facilitates the implementation of ewald summation for this geometry , by simply excluding k - space terms of certain parity . thus , we will use the bcc and bct lattice structures as the basis of our analysis . in figure 2 , the rhombic dodecahedron ( left ) and truncated octahedron ( right ) inscribed in their bct and bcc unit cells , respectively . in the following subsections , we will treat relevant parts of the theory of solvation and fluctuations in dielectric media . first , we will review the theory for the solvation of a polarizable dipole in a dielectric medium ( section ) . subsequently , in section , we will treat electrostatic fluctuations and solvation in isotropic dielectric media . finally , in section , we will use tools from the two preceding parts to develop a heuristic model for the solvation of a dielectric subvolume in a pbc system . generally , the collective electrostatic fluctuations will be quantified through the spherical multipole moments qlm , defined through3where (r ) denotes the charge density in a point r = ( r , ) = ( r , , ) v and clm( ) represents racah s unnormalized spherical harmonics . the index l denotes the order of the multipole , whereas m describes its orientation in an external coordinate frame . just as for the spherical harmonics , m takes on all integer values between l and + l. however , the m and + m components are related according to4where * denotes complex conjugation ; thus , qlm and ql m are not independent degrees of freedom . instead , we will adopt the approach taken previously and treat separately the real and imaginary parts of qlm , denoted respectively by superscripts r and i , for m 0 . since ql0 is real , these l + 1 real and l imaginary multipole components form 2l + 1 linearly independent fluctuation modes . furthermore , we will use the bracketed superscripts ( r ) and ( i ) to denote quantities that are somehow related to the real and imaginary parts of qlm , although not themselves complex quantities . the solvation energy usolv of a polarizable point dipole of magnitude , radius r , and polarizability embedded in a dielectric medium of dielectric permittivity is given by5where6quantifies the reaction field , parallel to the dipole , coming from the surrounding dielectric medium . a physical interpretation of the expression for usolv is facilitated by expanding eq 5 in a geometric series , i.e.7from this expression , we can identify the prefactor g/2 as the solvation energy of a permanent dipole immersed in a dielectric medium , whereas the factor n(g ) takes into account the increase of the solvation energy due to the additional polarization of the particle by the reaction field . the infinite sum is due to the incremental nature of this process ; the reaction field increases the total dipole moment of the particle , which in turn polarizes the dielectric to yield a larger reaction field , etc . in section , we will show how this partitioning of the solvation energy can be mapped onto the solvation of a dielectric subvolume exhibited to pbcs . the ( unnormalized ) probability distribution pvac(qlm ) of the 2-pole moment of a spherical dielectric volume with radius r and dielectric permittivity in a vacuum is given by the gaussian function8where uvac , given by9denotes the ( free ) energy cost for creating an instantaneous multipole moment qlmx in the dielectric volume , = ( kbt ) is the inverse thermal energy , and x { r , i}. if the dielectric volume is immersed in an infinite dielectric medium with the same value of as the sphere itself , u is decreased due to the depolarizing reaction field from the surrounding medium , changing the probability distribution to10where11and the second equality is accurate for not too small values of . the energy expression in eq 9 is roughly independent of for high - dielectric media and thus not numerically useful for determining from a computer simulation . in contrast , the right - hand - side of eq 11 shows that udiel for large and intermediate values of , and thus determining the width of pdiel in a computer simulation can be used to determine the dielectric permittivity of the system under study . more specifically , eq 10 can be transformed into a formula for the mean - square quantity (qlmx) by noting that the gaussian form implies that (qlmx) = ( 2clm ) . after some rearrangements , still using the simplified form for high and intermediate , we get12 the l = 1 case of eq 12 applied to the total dipole moment has been widely used to determine from computer simulations of fluids , although care needs to be taken to use a form of the formula proper for the particular boundary conditions being used . in an infinite , isotropic dielectric medium , is by definition independent of l , m , and r , but for a finite and/or molecular system , this does not necessarily hold . in particular , as we will show below , is not independent of l and m for a system exposed to pbcs . in addition to the dielectric permittivity , the above formulas can be used to obtain the free energy change avacdiel of bringing the dielectric sphere from vacuum into its own medium . to this end , we will employ the standard relationship13where zdiel and zvac denote the configuration integrals in the solvated and nonsolvated states , respectively . inserting eqs 811 and carrying out the integrations gives14thus , avacdiel is ( i ) always negative , ( ii ) independent of m and r , and ( iii ) only weakly dependent on l , a dependence that disappears quickly in the limit l . finally , we note that avacdiel diverges logarithmically as for all l. we will now propose a mapping of the energy expression in eq 7 for a polarizable dipole in a dielectric medium onto the solvation of a dielectric subvolume in a system exposed to pbcs . as a first assumption , we will describe the energy of creating an instantaneous multipole moment qlmx in the spherical volume , excluding the anisotropic part of the solvation , by the same expression as in an infinite dielectric medium . using eq 11 and eq 7 , we thus make the assignment15obviously , udiel is qualitatively different from the prefactor g/2 of eq 7 ; most importantly , it has a positive rather than a negative sign , since it also includes the energetic cost of creating the multipole moment in the dielectric medium , whereas the energy in eq 7 is valid for a permanent dipole , i.e. , excluding the self - energy of the charge distribution . in addition to the isotropic solvation , the instantaneous multipole moment induces a generalized reaction field coming from its own replicas in all of the surrounding boxes , which in turn polarizes the dielectric volume . this behavior is fully analogous to the polarization of a polarizable dipole by its own reaction field ; however , in the case of pbcs the reaction field is not proportional to the factor g of eq 6 but rather to the lattice interaction tensor slm(x ) quantifying the interaction between the multipole component qlmx and all its replicas in the lattice . the use of eqs 44 and 47 of ref ( 38 ) leads us to the following definition of slm(x):16where a is the side length of the unit cell , is the kronecker delta , and the function f is defined by17with ( ) representing the wigner 3j symbol . the two terms for m > 0 come from the interaction between qlmx in the central unit cell and ( i ) qlmx and ( ii ) ql mx in the surrounding cells . we furthermore note that ( see appendix a)18where the sum runs over all multipole components with a given l. thus , the ( unweighted ) mean value of the lattice interaction for any l 1 is zero for all multipoles and lattices . it should finally be clarified that , whereas g always yields an attractive coupling between the polarizable dipole and the reaction field , slm(x ) can represent attractive as well as repulsive couplings , depending on the symmetry properties of each multipole . finally , we make the assumption that the polarizability in eq 7 can be mapped according to kselfr , where kself is a positive constant related to the magnitude of the anisotropic solvation . on the basis of the above discussion , we suggest that the ( free ) energy for creating an instantaneous multipole moment in a spherical subvolume of a dielectric exhibited to pbcs is given by19 in accordance with eq 10 , we also form the corresponding probability distribution ppbc(qlmx):20 we note that , just like in the case of isotropic dielectric solvation , ppbc is gaussian ( as long as kselfrslm(x ) < 1 ) , but with the important difference that its exponent is now m - dependent through the dependence on slm(x ) . the gaussian form with respect to qlmx implies that the mean - square multipole moment (qlmx)pbc can be expressed as21 in analogy with eq 12 , we now define the apparent dielectric permittivity pbc , lm(x ) as22which from the above reasoning now becomes dependent on l and m due to the anisotropic polarization induced by the pbcs . finally , inserting eq 21 into eq 22 gives the relation23where we have added the subscript diel to , to stress that it represents the true ( m - independent ) dielectric permittivity that the fluid would have if it behaved as an isotropic dielectric medium . for a molecular system , pbc , lm(x ) can be obtained by sampling (qlmx)pbc in a computer simulation . by plotting pbc , lm(x ) as a function of slm(x ) , the two constants diel and kself appearing in eq 23 can be determined from the intercept and slope of a linear fit to the data points . just as in the case of dielectric solvation , we may use the analogy of eq 13 to define the free energy change avacpbc of bringing a dielectric sphere from a vacuum into a system under pbcs . using eqs 8 , 9 , and 1920 gives , after performing the integrations,24 since avacpbc describes the long - range part of the electrostatic free energy of the simulated system , it should ideally not differ too much from avacdiel , and therefore a comparison between these two quantities may be a good way of assessing the accuracy of the particular boundary conditions being used . in particular , for slm(x ) = 0 or kself = 0 , avacpbc reduces to eq 14 for an isotropic dielectric medium . the molecular model system is composed by particles possessing a pairwise additive interparticle potential v(rij , i , j ) , composed of a dipolar and a lennard - jones ( lj ) part according to25where26and27 in the above equations , i represents the dipole of particle i , rij is the vector pointing from particle i to particle j , rij = |rij| , and lj and lj are the lj parameters . two different values of the molecular dipole moment = || were employed : = 0.45 atomic units ( 0.23813e , * /(40ljlj3 ) = 1.290 ) and = 0.65 atomic units ( 0.34397e , * = 1.863 ) . the lj parameters were set to lj = 2.8863 and lj = 1.97023 kj mol . the thermodynamic properties of the model system were determined by performing md simulations in the canonical ( constant n , v , t ) ensemble , using n = 1000 particles in a cell of volume v = 2.601 10 for all three simulation cell geometries . the temperature was kept constant at t = 315.78 k ( t * kbt/lj = 1.333 ) . toroidal boundaries for the noncubic simulation cells were applied according to the procedures devised by smith , whereas ewald summation with tinfoil boundaries were implemented using the formulas due to smith and fincham . a spherical cutoff in real space of rcut = 14 was used in conjunction with the ewald screening parameter = 3.2/rcut . the cutoff ncut in reciprocal space was set to 7 , 10 , and 9 for the cube , rd , and to geometries , respectively , to yield a constant relative error in the k - space energy of 10 . for all simulations , the integrated mc / md / brownian dynamics simulation package molsim was used . for further details about the simulation parameters , the multipole moments qlm , 1 l 4 , of a sphere with radius r were evaluated after every 100th time step . the contribution qlm , i from a molecular dipole q1m , i located at r = ( r , ) to the total multipole moment qlm = iqlm , i was calculated according to28where all terms containing clm with |m| > l should be excluded . for each sampled configuration , qlm was calculated with each particle used as the origin , giving in total n sampled values of qlm per configuration . in addition , reference values of diel for various r values were calculated using eq 12 from a simulation in a cubic simulation cell and n = 10 particles , i.e. , 100 times as large as the primary systems . this large ( compared to r ) system size was used in order to ensure that the values of diel thus obtained are unaffected by the boundary . in figure 3 , numerically calculated values of the reduced interaction tensor rslm(x ) for l = 2 and 3 are given for the sc , bct , and bcc lattices . we note that rslm(x ) is highly dependent on m , taking on both positive , corresponding to repulsive net interaction energies , and negative , corresponding to attractive net interactions , values . we also note that there is no obvious correlation between either the sign or the magnitude of rslm(x ) obtained from the three different lattices . for l = 2 , the sc lattice yields significantly ( 100% ) higher absolute values of rslm(x ) than the other two lattices ; however , for l = 3 , the situation is the opposite . this means that the magnitude ( and sign ) of the coupling of a certain multipole depends strongly on its symmetry in relation to the symmetry of the particular lattice where it resides . we furthermore note ( results not shown ) that slm(x ) = 0 for all m and all lattices , meaning that the total dipole dipole interaction is zero . this is a well - known fact for all cubic lattices , as long as the lattice sum is carried out in spherical shells . values of the reduced interaction tensor rslm(x ) for ( a ) l = 2 and ( b ) l = 3 relevant for the three different simulation cell geometries . the values were obtained from eq 16 using a spherical cutoff of nmax = 50 . in figure 4 , the probability distribution ppbc(qlmx ) for two different octupole components , obtained from a simulation of the = 0.45 system in rd geometry , is shown . clearly , there is a significant difference between the widths of the two probability distributions , although both distributions follow the predicted gaussian form very well . the width of ppbc(qlmx ) should be compared to the corresponding values of slm(x ) given in figure 3 . obviously , the narrower one of the two probability distributions corresponds to a repulsive value of slm(x ) ( rs32(r ) = 0.88 ) , whereas the wider distribution corresponds to an attractive net interaction ( rs32(i ) = 0.66 ) . thus , there is at least qualitative reason in our assumption that the width of ppbc(qlmx ) , and thus the magnitude of pbc , can be described by the lattice interaction tensor slm(x ) . probability distribution ppbc(qlmx ) of two octupole components obtained from a simulation with = 0.45 in rd geometry . the values of the corresponding reduced interaction tensors are rs32(r ) = 0.88 and rs32(i ) = 0.66 . in order to quantitatively assess the dependence of ppbc(qlmx ) on slm(x ) , we evaluated the former quantity in terms of the apparent ( m - dependent ) dielectric permittivity pbc , lm(x ) , defined through eq 22 . in figure 5 , plots of pbc , lm(x ) versus rslm(x ) obtained for both dipole strengths and all three simulation cell geometries are presented . clearly , the proposed linear relationship between pbc , lm(x ) and rslm(x ) is very well reproduced by the simulation data in all cases . furthermore , the results obtained from the = 0.45 systems exhibit slopes that are essentially independent of geometry and l. the slopes of the = 0.65 data show a larger variation , although no systematic dependence on geometry and l is apparent . we also note the perhaps somewhat nonintuitive fact that the magnitude of the self - interaction ( quantified through rslm(x ) ) does not decay with increasing l , at least not for l 4 . furthermore , the self - interaction magnitudes do not show any clear trend between the different cell geometries . as an example , we note that the cubic geometry exhibits the largest quadrupole ( l = 2 ) self - interactions , whereas the octupole ( l = 3 ) self - interaction has its largest magnitude in the rd and to geometries . apparent dielectric permittivity pbc , lm(x ) obtained from simulations of dipoles with ( a ) = 0.45 and ( b ) = 0.65 versus rslm(x ) for the corresponding lattice types . the results for l = 3 ( l = 4 ) have been shifted vertically by 5 ( 10 ) units for = 0.45 and 20 ( 40 ) units for = 0.65 to enhance readability . because of the good linearity of the data , eq 23 can be used to obtain values of diel and kself from the intercept and slope , respectively , of the data in figure 5 . in table 1 , fitted values of diel and kself are given for both dipole strengths and all three cell geometries , together with values of diel independently calculated from a simulation with n = 10 using eq 12 and the same values of the sampling radius r. from this data , we note that1.the fitted values of diel are close ( within 5% ) to the ones calculated from eq 12 for all fittings except those with = 0.65 and l = 2 , where the fittings generally yield too low values of diel.2.the fitted values of kself are slightly larger and exhibit larger variations for = 0.65 than for = 0.45 . observation 1 shows that our assumption that the interaction between noncorrelated multipoles ( i.e. excluding the self - interaction ) can be described using formulas for an isotropic dielectric medium ( eq 15 ) is reasonable , perhaps with the exception for the l = 2 and = 0.65 case . observation 2 indicates that there may be a slight variation of kself with diel ( or ) , although the source of this variation is not clear . the larger variation in kself for = 0.65 we attribute to the larger statistical noise present in the more strongly coupled system . the fitted values of diel are close ( within 5% ) to the ones calculated from eq 12 for all fittings except those with = 0.65 and l = 2 , where the fittings generally yield too low values of diel . the fitted values of kself are slightly larger and exhibit larger variations for = 0.65 than for = 0.45 . we furthermore note that diel decreases with increasing l , in line with what we have observed before . the apparent geometry dependence of diel is not due directly to the geometry but rather to the slightly different radii of the inscribed spheres in the three simulation cells ; this behavior is also consistent with our previous observation that diel increases with increasing sampling sphere radius r for a given l. in figure 6 , the data corresponding to figure 5a , but obtained using sampling radii half as large , are presented . in this case , it is obvious that the magnitude of the self - interaction quickly becomes less significant for increasing l , due to its r dependence . the linear fits are however still satisfactory , even though the very small variation in pbc , lm(x ) over the range of rslm(x ) values leads to larger statistical errors in the fittings , especially for l = 3 and 4 . the apparent shift in the y direction between curves obtained using different geometries is merely due to the values used for the sampling radius r being geometry dependent , leading to different values of the intrinsic dielectric permittivity diel . in fact , the same shift is present in figure 5 , although it is not visible due to the much wider range of the ordinate axis . according to our theoretical assumptions , the value of kself should be independent of r , meaning that the slope of the lines in figures 5a and 6 should be identical . in table 2 , although the variation in kself is larger than in table 1 due to the larger statistical noise , our assumption for kself is not obviously contradicted . using the smaller sampling radii for the = 0.65 system ( data not shown ) , however , seems to yield somewhat larger values of kself than in table 1 , although the statistical significance of these values can be questioned . note the different scale on both axes and that the data have not been shifted in the y direction . figure 7 gives the free energy avacpbc for l = 3 , = 0.65 , and rd geometry calculated using eq 24 . clearly , the anisotropy in pbc discussed in the previous paragraphs also corresponds to a large anisotropy in the solvation free energy of the subvolume . 1.30 ( eq 14 , red solid line in figure 7 ) . however , the average of avacpbc over all seven octupole components is avacpbc(avg ) 1.26 ( black dashed line in figure 7 ) , i.e. , very close to the dielectric value . thus , the total solvation free energy ( at least on the octupolar level ) of the simulation box is very close to that of an isotropic system , even though it is distributed in a highly anisotropic way . a possible key to understanding this behavior is to be found in eq 18 , namely , that the ( unweighted ) interaction tensors for any given l 1 cancel out when summed over all multipole components . thus , the suppression of some fluctuation modes is exactly compensated by the enhancement of others , leading to a reasonable mean value of the energy . this behavior is reproduced for all multipole components and geometries ( results not shown ) , in the sense that avacpbc(avg ) and avacdiel are always within 10% of each other . solvation free energy avacpbc for l = 3 ( black solid line ) obtained from a = 0.65 system in rd geometry using eq 24 and values of kself and diel(fit ) from table 1 . the red solid line gives avacdiel obtained from eq 14 using diel(sim ) from table 1 , and the black dashed line gives the mean value of avacpbc , averaged over all seven octupole components ( black symbols , two doubly degenerate values ) . in the present study , we have presented a quantitative analysis of the periodicity effects induced in a dipolar system by the use of pbcs . using classical electrostatics and statistical thermodynamics , we developed a heuristic model relating the apparent , anisotropic dielectric permittivity pbc , lm(x ) to the reduced lattice interaction tensors slm(x ) . the theory exhibits excellent agreement with results from md simulations of stockmayer fluids with two different dipole strengths and three different simulation cell geometries . although the anisotropy in the electrostatic fluctuations is independent of l on the length scale of the simulation box , it is shown that the range of the boundary effects ( i.e. , the minimum value of r needed to induce significant boundary effects ) decreases strongly with increasing l. furthermore , it was shown that the large ( 200% ) anisotropy in the solvation free energy on the length scale of the simulation box disappears when averaged over all fluctuation modes , leading to the total solvation free energy being practically identical to the value predicted for an isotropic system . even though the simulation part of our study is based on a stockmayer model system , we argue that our use of a dielectric continuum model as the theoretical basis means that the effects are fully transferable to any polar system which may be described as a dielectric medium , in particular the many popular water models used in molecular simulations . we also expect that any structural property , i.e. , not only the dielectric permittivity , evaluated on the length - scale of the full simulation box is equally affected by the boundary effects . one of the most important observations from this study is that the total solvation free energy is , in spite of the large anisotropy of the individual contributions , very close to the correct , isotropic value . this observation is indeed closely analogous to the corresponding averaging in the anisotropy of the radial distribution function for a lennard - jones fluid under pbcs observed by pratt and haan . we argue that this property explains the success of ewald summation and related techniques , at least when it comes to evaluating energies and relatively short - range structural properties . nevertheless , as we have also shown previously , one should use caution when evaluating structural properties on length scales larger than half the length of the simulation box . another relevant question is whether there is any rationale behind using a noncubic ( rd or to ) simulation cell in order to reduce periodicity effects , as has been suggested previously . first of all , it is clear that the periodicity effects when taking the full simulation cell into account are as strong for all three cell geometries ( figure 5 ) , albeit not identical for a given l. we note , however , that the influence from the periodicity on the quadrupolar ( l = 2 ) fluctuations is significantly lower in the rd and to geometries than for the cube . since the magnitude of the boundary effect for a given l decays as r , this means that the leading - order contribution to the boundary effects is about 50% smaller ( figure 6 ) in the rd and to geometries compared to when using a cubic simulation cell . on the other hand , which simulation cell to be used also depends on the specific system under study . for example , if one wants to simulate a macromolecule ( e.g. , a protein ) with a particularly large molecular octupole moment , a cubic box may be the most appropriate one , due to its lower lattice coupling for the system octupole moment . furthermore , it may also be advantageous , when using rotational constraints , to orient the axis of the largest electrostatic moment along the axis with the lowest value of slm(x ) ; for example , orienting the octupole moment in the s33(r ) or s33(i ) direction gives a lattice interaction of less than 3% of the value obtained when the octupole is oriented along the s32(i ) axis . the present study provides a quantitative understanding of the isotropic and anisotropic parts of the solvation in a polar system under pbcs and puts them in relation to the behavior of an isotropic system . this understanding is essential for the possibility to remedy the periodicity effects , for example by imposing suitable bias functions in an mc simulation in order to remove the anisotropic self - interaction .

a heuristic model based on dielectric continuum theory for the long - range solvation free energy of a dipolar system possessing periodic boundary conditions ( pbcs ) is presented . the predictions of the model are compared to simulation results for stockmayer fluids simulated using three different cell geometries . the boundary effects induced by the pbcs are shown to lead to anisotropies in the apparent dielectric constant and the long - range solvation free energy of as much as 50% . however , the sum of all of the anisotropic energy contributions yields a value that is very close to the isotropic one derived from dielectric continuum theory , leading to a total system energy close to the dielectric value . it is finally shown that the leading - order contribution to the energetic and structural anisotropy is significantly smaller in the noncubic simulation cell geometries compared to when using a cubic simulation cell .

Introduction Theory Computational Details Results and Discussion Conclusions

arguably , the most persistent of these is the question of how to handle long - range electrostatic ( coulomb and dipole the basic problem is that the integral1diverges for all finite values of the cutoff radius rcut as long as the intermolecular potential v(r ) does not decay faster than r. thus , applying a simple ( spherical or cubic ) cutoff to the electrostatic potentials may often lead to serious artifacts in the structure and thermodynamics of the system under study . although several solutions to the infinite - range interaction problem have been proposed , the most common way to circumvent this problem is the use of lattice - based summation techniques , or periodic boundary conditions ( pbcs ) . the original implementation of this idea was developed by ewald and is built upon a separation of the interaction into short - range and long - range parts , where the former is summed up in real space and the latter in reciprocal space . this criticism has been put forward several times in the literature but was originally noted by valleau and whittington , who gave a qualitative argument about the inability of lattice summation methods to correctly reproduce long - range fluctuations in fluid systems . however , the total dipole moment of the simulation box is a special property , in the sense that its total interaction with all its periodic images is identically zero , as long as the contributions are summed in spherical shells . in a recent contribution , we showed , however , that the fluctuations of higher order electric multipole moments of the whole simulation box are greatly influenced by the interaction between each instantaneous multipole and all of its periodic images . this effect is manifested through a difference of as much as 50% between the dielectric permittivities calculated from different multipole components , depending on whether the multipole component has an attractive or a repulsive ( or , in some cases , zero ) interaction with its neighbors . in addition , the dipole as well as the higher multipoles interact with the set of unconstrained multipoles qlm , l l and/or m m , ( not depicted ) in the surrounding cells , giving an ( approximately ) isotropic contribution to the solvation . in addition to the cubic simulation cell used in the majority of computer simulations , some alternative simulation cell geometries have been suggested and implemented , most notably the rhombic dodecahedron ( rd ) and the truncated octahedron ( to ) . these two bodies have the appealing property of more closely resembling the geometry of solvated spherical solutes , in the sense that they have larger inscribed spheres than a cubic simulation cell of the same volume . because these cells pack in lattice structures different from that of the cube , it seems reasonable to expect their periodicity effects to differ qualitatively from those of a cubic cell . in the present contribution , we will extend our previous analysis of the periodicity effects on a dipolar model system from the qualitative to the quantitative level , as well as from cubic to noncubic simulation cells . this model will be compared to values of the dielectric constant calculated from simulations of a simple dipolar model system . in the following , the electrostatic fluctuations in a spherical subvolume of a dipolar model system treated using the ewald summation technique will be described by dividing the long - range solvation energy of this subvolume into two contributions:an approximately isotropic part , coming from the interaction between the instantaneous multipole qlm of a spherical volume in the central simulation cell and its noncorrelated neighbors , i.e. an approximately isotropic part , coming from the interaction between the instantaneous multipole qlm of a spherical volume in the central simulation cell and its noncorrelated neighbors , i.e. on the basis of this description , we will present a heuristic derivation of the long - range solvation free energy of the spherical subvolume . this will be compared to the behavior expected from a spherical subvolume inside an infinite isotropic dielectric medium and the analysis will thus enable us to directly probe the magnitude of the periodicity effect introduced by the pbcs . in the present study , the term periodic boundary conditions refers to a system with a potential energy upot of the form2where n = ( nx , ny , nz ) is a vector that runs over all lattice points in the particular ( unit length ) lattice and a denotes the side length of the unit cell . in practice , v(rij , i , j ) is usually long - range in the sense that it decays no faster than r , the two most important examples being the coulomb and dipole since the sum in eq 2 is slowly ( and conditionally ) convergent , more elaborate methods to evaluate the potential energy in a pbc system need to be used in practice . within the ewald - based methods , the short - range ( n = 0 ) part of upot is screened through the addition of a gaussian charge ( dipole ) cloud and is thereafter summed within a , usually spherical , cutoff after considering the nearest image convention . the long - range ( n 0 ) part of the potential energy is summed up in fourier space , leading to a quickly ( and absolutely ) convergent sum . although cubic simulation cells are used for the majority of simulation studies , the use of alternative simulation cell geometries has started to become increasingly popular . in total , five classes of geometrical bodies are translationally space - filling and can thus be used for simulating a periodic system ; however , due to their relatively sphere - like symmetry , the two most useful alternatives to the cube , at least for the simulation of bulk systems , are the rhombic dodecahedron ( rd ) and truncated octahedron ( to ) . however , smith and fincham showed that the use of a body - centered tetragonal ( bct ) lattice structure for the rd , with one side of the unit cell elongated by a factor 2 compared to the other two , greatly facilitates the implementation of ewald summation for this geometry , by simply excluding k - space terms of certain parity . first , we will review the theory for the solvation of a polarizable dipole in a dielectric medium ( section ) . finally , in section , we will use tools from the two preceding parts to develop a heuristic model for the solvation of a dielectric subvolume in a pbc system . a physical interpretation of the expression for usolv is facilitated by expanding eq 5 in a geometric series , i.e.7from this expression , we can identify the prefactor g/2 as the solvation energy of a permanent dipole immersed in a dielectric medium , whereas the factor n(g ) takes into account the increase of the solvation energy due to the additional polarization of the particle by the reaction field . the infinite sum is due to the incremental nature of this process ; the reaction field increases the total dipole moment of the particle , which in turn polarizes the dielectric to yield a larger reaction field , etc . the ( unnormalized ) probability distribution pvac(qlm ) of the 2-pole moment of a spherical dielectric volume with radius r and dielectric permittivity in a vacuum is given by the gaussian function8where uvac , given by9denotes the ( free ) energy cost for creating an instantaneous multipole moment qlmx in the dielectric volume , = ( kbt ) is the inverse thermal energy , and x { r , i}. if the dielectric volume is immersed in an infinite dielectric medium with the same value of as the sphere itself , u is decreased due to the depolarizing reaction field from the surrounding medium , changing the probability distribution to10where11and the second equality is accurate for not too small values of . after some rearrangements , still using the simplified form for high and intermediate , we get12 the l = 1 case of eq 12 applied to the total dipole moment has been widely used to determine from computer simulations of fluids , although care needs to be taken to use a form of the formula proper for the particular boundary conditions being used . in addition to the dielectric permittivity , the above formulas can be used to obtain the free energy change avacdiel of bringing the dielectric sphere from vacuum into its own medium . as a first assumption , we will describe the energy of creating an instantaneous multipole moment qlmx in the spherical volume , excluding the anisotropic part of the solvation , by the same expression as in an infinite dielectric medium . using eq 11 and eq 7 , we thus make the assignment15obviously , udiel is qualitatively different from the prefactor g/2 of eq 7 ; most importantly , it has a positive rather than a negative sign , since it also includes the energetic cost of creating the multipole moment in the dielectric medium , whereas the energy in eq 7 is valid for a permanent dipole , i.e. in addition to the isotropic solvation , the instantaneous multipole moment induces a generalized reaction field coming from its own replicas in all of the surrounding boxes , which in turn polarizes the dielectric volume . this behavior is fully analogous to the polarization of a polarizable dipole by its own reaction field ; however , in the case of pbcs the reaction field is not proportional to the factor g of eq 6 but rather to the lattice interaction tensor slm(x ) quantifying the interaction between the multipole component qlmx and all its replicas in the lattice . the use of eqs 44 and 47 of ref ( 38 ) leads us to the following definition of slm(x):16where a is the side length of the unit cell , is the kronecker delta , and the function f is defined by17with ( ) representing the wigner 3j symbol . we furthermore note that ( see appendix a)18where the sum runs over all multipole components with a given l. thus , the ( unweighted ) mean value of the lattice interaction for any l 1 is zero for all multipoles and lattices . finally , we make the assumption that the polarizability in eq 7 can be mapped according to kselfr , where kself is a positive constant related to the magnitude of the anisotropic solvation . on the basis of the above discussion , we suggest that the ( free ) energy for creating an instantaneous multipole moment in a spherical subvolume of a dielectric exhibited to pbcs is given by19 in accordance with eq 10 , we also form the corresponding probability distribution ppbc(qlmx):20 we note that , just like in the case of isotropic dielectric solvation , ppbc is gaussian ( as long as kselfrslm(x ) < 1 ) , but with the important difference that its exponent is now m - dependent through the dependence on slm(x ) . the gaussian form with respect to qlmx implies that the mean - square multipole moment (qlmx)pbc can be expressed as21 in analogy with eq 12 , we now define the apparent dielectric permittivity pbc , lm(x ) as22which from the above reasoning now becomes dependent on l and m due to the anisotropic polarization induced by the pbcs . using eqs 8 , 9 , and 1920 gives , after performing the integrations,24 since avacpbc describes the long - range part of the electrostatic free energy of the simulated system , it should ideally not differ too much from avacdiel , and therefore a comparison between these two quantities may be a good way of assessing the accuracy of the particular boundary conditions being used . the molecular model system is composed by particles possessing a pairwise additive interparticle potential v(rij , i , j ) , composed of a dipolar and a lennard - jones ( lj ) part according to25where26and27 in the above equations , i represents the dipole of particle i , rij is the vector pointing from particle i to particle j , rij = |rij| , and lj and lj are the lj parameters . the thermodynamic properties of the model system were determined by performing md simulations in the canonical ( constant n , v , t ) ensemble , using n = 1000 particles in a cell of volume v = 2.601 10 for all three simulation cell geometries . toroidal boundaries for the noncubic simulation cells were applied according to the procedures devised by smith , whereas ewald summation with tinfoil boundaries were implemented using the formulas due to smith and fincham . the cutoff ncut in reciprocal space was set to 7 , 10 , and 9 for the cube , rd , and to geometries , respectively , to yield a constant relative error in the k - space energy of 10 . in addition , reference values of diel for various r values were calculated using eq 12 from a simulation in a cubic simulation cell and n = 10 particles , i.e. this large ( compared to r ) system size was used in order to ensure that the values of diel thus obtained are unaffected by the boundary . this means that the magnitude ( and sign ) of the coupling of a certain multipole depends strongly on its symmetry in relation to the symmetry of the particular lattice where it resides . values of the reduced interaction tensor rslm(x ) for ( a ) l = 2 and ( b ) l = 3 relevant for the three different simulation cell geometries . obviously , the narrower one of the two probability distributions corresponds to a repulsive value of slm(x ) ( rs32(r ) = 0.88 ) , whereas the wider distribution corresponds to an attractive net interaction ( rs32(i ) = 0.66 ) . clearly , the proposed linear relationship between pbc , lm(x ) and rslm(x ) is very well reproduced by the simulation data in all cases . furthermore , the self - interaction magnitudes do not show any clear trend between the different cell geometries . in table 1 , fitted values of diel and kself are given for both dipole strengths and all three cell geometries , together with values of diel independently calculated from a simulation with n = 10 using eq 12 and the same values of the sampling radius r. from this data , we note that1.the fitted values of diel are close ( within 5% ) to the ones calculated from eq 12 for all fittings except those with = 0.65 and l = 2 , where the fittings generally yield too low values of diel.2.the fitted values of kself are slightly larger and exhibit larger variations for = 0.65 than for = 0.45 . the apparent geometry dependence of diel is not due directly to the geometry but rather to the slightly different radii of the inscribed spheres in the three simulation cells ; this behavior is also consistent with our previous observation that diel increases with increasing sampling sphere radius r for a given l. in figure 6 , the data corresponding to figure 5a , but obtained using sampling radii half as large , are presented . in this case , it is obvious that the magnitude of the self - interaction quickly becomes less significant for increasing l , due to its r dependence . the apparent shift in the y direction between curves obtained using different geometries is merely due to the values used for the sampling radius r being geometry dependent , leading to different values of the intrinsic dielectric permittivity diel . in fact , the same shift is present in figure 5 , although it is not visible due to the much wider range of the ordinate axis . according to our theoretical assumptions , the value of kself should be independent of r , meaning that the slope of the lines in figures 5a and 6 should be identical . clearly , the anisotropy in pbc discussed in the previous paragraphs also corresponds to a large anisotropy in the solvation free energy of the subvolume . , very close to the dielectric value . thus , the total solvation free energy ( at least on the octupolar level ) of the simulation box is very close to that of an isotropic system , even though it is distributed in a highly anisotropic way . thus , the suppression of some fluctuation modes is exactly compensated by the enhancement of others , leading to a reasonable mean value of the energy . in the present study , we have presented a quantitative analysis of the periodicity effects induced in a dipolar system by the use of pbcs . using classical electrostatics and statistical thermodynamics , we developed a heuristic model relating the apparent , anisotropic dielectric permittivity pbc , lm(x ) to the reduced lattice interaction tensors slm(x ) . the theory exhibits excellent agreement with results from md simulations of stockmayer fluids with two different dipole strengths and three different simulation cell geometries . although the anisotropy in the electrostatic fluctuations is independent of l on the length scale of the simulation box , it is shown that the range of the boundary effects ( i.e. , the minimum value of r needed to induce significant boundary effects ) decreases strongly with increasing l. furthermore , it was shown that the large ( 200% ) anisotropy in the solvation free energy on the length scale of the simulation box disappears when averaged over all fluctuation modes , leading to the total solvation free energy being practically identical to the value predicted for an isotropic system . even though the simulation part of our study is based on a stockmayer model system , we argue that our use of a dielectric continuum model as the theoretical basis means that the effects are fully transferable to any polar system which may be described as a dielectric medium , in particular the many popular water models used in molecular simulations . , not only the dielectric permittivity , evaluated on the length - scale of the full simulation box is equally affected by the boundary effects . one of the most important observations from this study is that the total solvation free energy is , in spite of the large anisotropy of the individual contributions , very close to the correct , isotropic value . this observation is indeed closely analogous to the corresponding averaging in the anisotropy of the radial distribution function for a lennard - jones fluid under pbcs observed by pratt and haan . first of all , it is clear that the periodicity effects when taking the full simulation cell into account are as strong for all three cell geometries ( figure 5 ) , albeit not identical for a given l. we note , however , that the influence from the periodicity on the quadrupolar ( l = 2 ) fluctuations is significantly lower in the rd and to geometries than for the cube . since the magnitude of the boundary effect for a given l decays as r , this means that the leading - order contribution to the boundary effects is about 50% smaller ( figure 6 ) in the rd and to geometries compared to when using a cubic simulation cell . furthermore , it may also be advantageous , when using rotational constraints , to orient the axis of the largest electrostatic moment along the axis with the lowest value of slm(x ) ; for example , orienting the octupole moment in the s33(r ) or s33(i ) direction gives a lattice interaction of less than 3% of the value obtained when the octupole is oriented along the s32(i ) axis . the present study provides a quantitative understanding of the isotropic and anisotropic parts of the solvation in a polar system under pbcs and puts them in relation to the behavior of an isotropic system .

systemic sclerosis ( ssc ) is an often fatal disease characterized by autoimmunity and inflammation , leading to widespread vasculopathy and fibrosis of multiple organs 1 . there are two distinct subsets of the disease , limited and diffuse , which are based on the extent of skin involvement however , one of the hallmarks of the vasculopathy associated with both subsets of ssc is platelet activation , which has been implicated as a key mediator of the fibrosis that underlies ssc 3 . phospholipid growth factors ( plgfs ) are a family of lipids with growth factor - like properties . lpa targets cells through at least six well - characterized extracellular receptors and one nuclear receptor ( the ppar receptor ) 4 , 5 . lpa is found at physiologically and pathophysiologically significant concentrations in plasma and serum , respectively . lpa is typically produced by the action of plasma lysophospholipase d ( lysopld ) , also known as autotaxin , on lysophosphatidylcholine ( lpc ) 6 . in serum , activated platelets are one of the primary sources of lpa , which is produced via the action of lysopld on lpc and other lysophospholipids 7 . lpa has been found to stimulate cell division and migration and to inhibit apoptosis 8 . a similar differentiation step is critical to the pathogenesis of ssc , in that myofibroblasts are one of the primary sources of ssc fibrotic tissue 10 , 11 . rho - associated kinases ( rock ) , which are activated by lpa , have recently been found to stimulate myofibroblast differentiation from cultured human skin ssc fibroblasts . sphingosine 1-phosphate ( s1p ) , another major member of the plgf family , acts as an agonist to membrane receptors that have close evolutionary links to several of the lpa receptors . both b- and t - cells require s1p activity to allow their movement out of lymphoid organs , and thymocytes require s1p receptor activation for release from the thymus 13 . although there is currently no known association between s1p and ssc , ssc has an autoimmune component with reported lymphocyte involvement that suggests there may be a role for s1p in the development and/or progression of ssc . in view of the high level of platelet activation seen in ssc 14 , along with the increased myofibroblast activity and impaired myofibroblast apoptosis , we hypothesized that elevated plgfs play a role in the pathogenesis of ssc , and specifically that lpa would be elevated in ssc subjects relative to healthy controls . therefore , we designed this study to determine whether differences exist in serum lpa concentrations and in lysopld activities of ssc and control subjects . we chose to look at a range of different lpa species because of the likelihood that we would find differences in either a particular species or class of lpa 15 . in addition to lpa measurement , we also measured and compared s1p concentrations in the blood of ssc versus control subjects because of the likely autoimmune pathogenesis of ssc and the link between s1p and immune cell function . because of significant differences that are present between limited and diffuse ssc , we also examined the differences in lipid products and lysopld activity between subjects with these two subgroups of the disease . finally , we measured in ssc and control subjects the concentrations of additional bioactive lysophospholipids , including lysophosphatidylethanolamine ( lpe ) , lysophosphatidylinositol ( lpi ) and lysophosphatidylserine ( lps ) , which are precursors of lpa . ethics statement . written consent for participation in the study was obtained from all participants in accordance with the helsinki ii declaration , and the protocol was approved by the uthsc institutional review board . subjects . ssc subjects with a history of limited or diffuse ssc were recruited from the rheumatology clinic of the university of tennessee health science center ( uthsc ) . all ssc subjects met the 1980 acr classification criteria for clinical diagnosis of limited or diffuse ssc 16 . those with a history of an organ or stem cell transplant , those who had used prednisone , cyclophosphamide , d - penicillamine , cyclosporine a , methotrexate , azathioprine , or other immune modulator therapies within one month of study start , and those who were less than 18 years old were excluded from the study . after an overnight fast , blood was collected from 10 patients with ssc ( 7 with diffuse disease and 3 with limited disease ) and from 13 healthy controls . serum was drawn off after the collected blood had been left 1 h at room temperature and then spun at 2500 rpm for 15 min . samples were stored at -80 c until overnight shipping ( on dry ice ) to japan for biochemical analysis . the laboratory performing the analyses was provided the sample key only after the analyses were complete and reported . 1-heptadecanoyl ( 17:0)-lpc and c17-s1p were purchased from avanti polar lipids ( alabaster , al , usa ) . 17:0-lpa was prepared from 17:0-lpc by the action of phospholipase d from streptomyces chromofuscus , as described previously 17 . 17:0-lpe was prepared from 17:0-lpc and ethanolamine hydrochloride by a transphosphatidylation reaction with phospholipase d from actinomadura sp . ( seikagaku kogyo ; tokyo , japan ) according to the method for preparation of phosphatidylglycerol from phosphatidylcholine 18 . in brief , a mixture of 0.1 ml of 5 m ethanolamine hydrochloride/0.2 m acetate buffer ( ph 5.5 ) containing 0.1 mmol 17:0-lpc , 0.05 ml of 0.22 m cacl2/0.02 m nacl , and 0.1 ml of phospholipase d solution ( 8 mg / ml ) in 0.2 m acetate buffer was incubated while being stirred for 2 h at 30c . after the reaction was stopped by addition of 2 ml chloroform / methanol mixture ( 1:1 , v / v ) , the mixture was centrifuged at 1300 x g for 10 min . lipids recovered into the organic layer were separated by thin - layer chromatography on a silica gel plate ( silica gel g60 ; merck ; darmstadt , germany ) developed with chloroform / methanol/20% ammonia ( 60:35:8 , v / v ) . 17:0-lpe was extracted from silica in the corresponding lipid band by the method of bligh and dyer 19 . purities of 17:0-lpa and 17:0-lpe recovered from the silica were checked by liquid chromatography - tandem mass spectrometry ( lc - ms - ms ) as described below . lipids were extracted from human serum samples by the modified method of bligh and dyer 19 after adjusting the aqueous phase ph to 9 - 9.5 with 20% ammonium hydroxide . to the lipid extract were added 5 nmol 17:0-lpc , 0.05 nmol 17:0-lpe , 0.1 nmol 17:0-lpa , and 0.2 nmol c17 s1p . most of the lipids , including lpc and lpe , were extracted into the organic layer . the lipid extract was dried under a stream of nitrogen gas , reconstituted with 0.5 ml of methanol / water mixture ( 1:1 , v / v ) containing 5 mm ammonium formate , and termed the neutral lipid fraction . the remaining aqueous layer was acidified to ph 2 - 2.5 with 1 n hydrochloric acid , and acidic lipids such as lpa , lpi and lps and s1p were extracted into the organic layer by the method of bligh and dyer 19 . the second lipid extract ( acidic lysophospholipid fraction ) was dried down under a stream of nitrogen gas and dissolved in 0.1 ml of acetonitrile / isopropanol / methanol / water ( 1:1:1:1 , v / v ) mixture containing 0.2% formic acid for lc - ms - ms . lc - ms - ms was performed on a quadrupole - linear iontrap hybrid ms , 4000 qtrap ( applied biosystems / mds sciex ; concord , on , canada ) , with an agilent 1100 lc system combined with an autosampler ( agilent technologies ; wilmington , de , usa ) . separation of neutral lipid fractions by lc was achieved using an agilent zorbax eclipse xdb - c18 column ( 50 mm x 1 mm ; 3.5-m particle size silica ) . the composition of the mobile phase was methanol / water ( 4:1 , v / v ) containing 5 mm ammonium formate , which was pumped at a flow rate of 0.1 ml / min for an isocratic elution . separation of acidic lysophospholipids by lc was performed with a tosoh tsk - ods-100z column ( 150 mm x 2 mm ; silica with 5-m particle size ) developed with methanol / water ( 19:1 , v / v ) containing 5 mm ammonium formate at a flow rate of 0.22 ml / min in an isocratic elution mode . routinely , 5 l aliquots of test solutions were applied to the mass spectrometer for analysis . lysophospholipids were analyzed by multiple reaction monitoring ( mrm ) in a positive ion mode for lpc and lpe or in a negative ion mode for lpa , lpi , lps , and s1p . in the positive ion mrm , q1 and q3 were set for the protonated molecular ion and [ phosphorylcholine ] at m / z 184 for lpc or [ m - 141 ] for lpe . in the negative ion mrm , q3 was set to [ cyclic glycerol phosphate ] at m / z 153 for lpa and lps , [ phosphorylinositol - h2o ] at m / z 241 for lpi or [ po3 ] at m / z 79 for s1p , in combination with the deprotonated molecular ion as q1 for all lysophospholipids tested . amounts of the different molecular species of lpc and lpe were calculated from the ratios of their areas of positive ions to those of 17:0-lpc or lpe internal standards . similarly , the amounts of molecular species of lpa were calculated from the ratios of their peak areas of negative ions to that of 17:0-lpa , an internal standard . the amounts of molecular species of lps and lpi were calculated from both their peak areas of negative ions to that of 17:0-lpa and the correction factors for lpi and lps . these correction factors were determined to be 10.5 and 6.4 , respectively , based on both the extraction efficiencies of 16:0- and 18:1-lps and bovine liver lpi , and the relative ion efficiency for lpi and lps against 17:0-lpa by mrm under our conditions . lysopld activity in serum was measured by the enzyme - linked fluorometric method for determination of choline produced together with lpa from exogenously added 0.15 mm 18:2-lpc . in our assay , performed on a 96-well microplate , 0.05 ml of 3.3-fold diluted serum was mixed with 0.025 ml of saline and 0.025 ml of lpc solution at 0.6 mm in saline containing 0.25% bovine serum albumin . the mixtures in the wells were incubated at 37 c for 9 or 24 h , and were then mixed with 0.2 ml of assay buffer . the assay buffer was composed of 0.1 ml of tris - hcl buffer ( ph 8.5 ) , 0.04 ml of 7.5 mm 3-(4-hydroxyphenyl)propionic acid , 0.02 ml of 25 u / ml choline oxidase and 0.04 ml of 2 u / ml horseradish peroxidase . with the aid of a standard line obtained with 0 , 0.1 , 0.3 , 1 , 3 , 10 , and 30 nmol / ml of water solution of choline chloride , the choline concentration was determined from the intensity of fluorescence measured at 320 nm ( excitation ) and 404 nm ( emission ) , and lysopld activity was calculated as nmol choline / ml of serum . alternatively , serum lysopld activity of subjects with diffuse ssc and healthy controls was measured by quantifying lpa production using lc - ms - ms as described above . statistical analysis . ten female ssc subjects and 14 female age- and race - matched healthy controls were included in this study . among the ssc subjects , seven had diffuse disease and three had limited disease . the mean duration of disease in the ssc subjects was 10.3 years , with a range of 3 - 28 years . measured serum concentrations of lpc , lpa , s1p , and dihydrosphingosine 1-phosphate ( dihydro s1p ) , which is identical to s1p except that it lacks one double bond and acts as an agonist for all s1p receptors but with a 20-fold lower affinity for s1p2 20 , 21 , from the control and ssc groups are summarized in fig . 1a ) , a precursor of lpa , had the highest concentrations ( 16:0>18:2>18:0>18:1 and 5 minor species ) followed by lpa ( fig . 1 b ) ( 18:2>20:4>16:0=18:1 and 5 minor species ) and then s1p ( fig . figure 2 shows serum concentrations of lpe , lpi , and lps from control and ssc subjects . of these lysophospholipids , lpe had the highest concentrations ( 18:2>18:1>16:0=20:4 and 3 minor species ) , which were less than the concentrations of lpa , followed by lpi ( 20:4>18:2>18:0>18:1 and 5 minor species ) and then lps ( 18:0>18:1>20:4 ) . thus , the most predominant precursor for lpa produced by serum lysopld would be lpc . it is noteworthy that the percentages of saturated molecular species of lpc were higher than those of lpa ( fig . 1b ) versus the control group as well as in the limited ssc group versus control ( 2.54 0.15 nmol / ml vs. 1.15 0.37 nmol / ml for control , p= 0.026 ) , with no differences measured between controls and the diffuse ssc group ( 1.99 0.49 nmol / ml , p = 0.088 ) . in addition , the s1p levels were significantly higher in ssc vs. control groups ( fig . 1c ) , and also in the diffuse ssc group versus controls ( data not shown ) . essentially no differences were observed for serum levels of lpc , lpe , lps , and lpi , as shown in figs . these ratios in part can show the conversion status of lpc to lpa for the individual species and for the combined total of species , with values closer to 1 indicating more conversion of lpc to lpa . here we found a significant difference in the total lpc / total lpa ratio between control and ssc ( p < 0.01 ) groups . there was also a significant difference between control and diffuse ssc groups ( p < 0.02 , data not shown ) . in addition , the values for 20:4 ratios were significantly different between control and ssc groups ( p < 0.01 ) , control and limited ssc ( p < 0.02 , data not shown ) groups , and control and diffuse ssc ( p < 0.05 , data not shown ) groups . in addition , there was a significant difference in the 20:3 ratio between control and ssc groups ( p < 0.05 ) and control and limited ssc groups ( p < 0.05 , data not shown ) . no significant differences were found between groups in the lysopld activity toward 150 m 18:2-lpc ( fig . there were also no significant differences between total and individual levels of lpa produced during 12 h incubation of sera from ssc patients and control subjects ( fig . for this study , we chose to measure not only total lpa levels in ssc versus control subjects , but also to measure several different specific saturated and unsaturated lpa species . it is now known that the different species of lpa have variable specificities to lpa receptors as well as differing potencies to cause cell specific cell responses 22 - 25 . in addition , lpa agonists and antagonists are being modeled after different lpa species 26 - 28 . the major findings of this study were significantly elevated concentrations of 20:4 lpa and s1p in the serum of ssc subjects versus controls . the total serum lpa : lpc ratio , and within this , the subgroups of 20:3 and 20:4 lpa : lpc ratios , were also elevated in the ssc subjects versus controls . no significant differences in other lysophospholipid mediators , including lpe , lpi , and lps , were found between control and ssc subjects . in addition , there were no differences observed in lysopld enzymatic activity between the ssc subjects and controls . the serum level of lpa appears to be controlled by a balance between lpa - producing and lpa - degrading enzymatic activities . we postulate that the elevated lpa and lpa : lpc ratios found in ssc subjects may be linked to reduced activity of a postulated lpa - degrading lysophospholipase in the serum of ssc subjects that would preferentially hydrolyze highly polyunsaturated lpas , such as the 20:4 species , over other lpa species . increased lpa synthesis is likely not responsible for these elevated values because we found no increase in lysopld activity in any of the ssc groups . our findings of significantly higher lpa : lpc ratios in the polyunsaturated species over saturated and monounsaturated species in serum samples from ssc patients and control subjects is consistent with a previous finding that sn-1-lyso - lpc , formed via hydrolysis of phosphatidylcholine by phospholipase a1 , is a superior substrate for lysopld in human serum and plasma as compared to sn-2-lyso - lpc , which is generated by hydrolysis of phosphatidylcholine by phospholipase a2 29 . it will be interesting to measure pla1 vs. pla2 activity in control and diffuse ssc subjects once the molecular identity of pla1 is determined . among the many different lpa subtypes measured in this study , 20:4 lpa was consistently elevated in the ssc subjects ( both limited and diffuse ) versus control subjects . 20:4 lpa has been used in a number of studies as an example of an sn-1 unsaturated lpa . overall , 20:4 has been shown to have broad specificity for the different lpa receptors 23 . in regard to ssc , 20:4 lpa has been found to have a high platelet - activating potency 24 , 25 . while activated platelets are a common feature of ssc , and activated platelets are known to release lpa , the presence of elevated 20:4 lpa could act in a positive feedback manner to perpetuate platelet activation . from an immune standpoint , unsaturated lpas , with 20:4 lpa being one of the species tested , this is potentially important , given recent evidence for altered antigen processing in ssc 31 . when stratifying by ssc disease types , the total serum lpa : lpc ratio and serum 20:4 lpa : lpc ratio were significantly elevated in the diffuse ssc subjects versus controls . although we had a small sample size of subjects with limited ssc , we found that serum 20:4 lpa and the 20:3 and 20:4 lpa : lpc ratios were significantly higher in the limited ssc group versus control subjects . in addition , elevated serum s1p concentrations approached significance in diffuse ssc and limited ssc subjects versus controls ( p = 0.05 and 0.09 , respectively ) . these lpa : lpc ratio and s1p patterns all follow the general pattern of these parameters being higher in ssc subjects versus controls . platelets are known to play a pathological role as a main source of circulating s1p by accumulating and releasing s1p after local blood coagulation , whereas erythrocytes are considered to be the main source of plasma s1p 32 . it is quite likely that the elevated level of s1p in sera from ssc patients is due to the activation of platelets during blood coagulation in vitro . the biological / clinical significance of our findings relates to both the immune etiology and fibrotic disposition of ssc . in regard to the immune system , the elevated s1p concentrations found in ssc subjects may relate to the lymphocyte involvement in the autoimmune genesis of ssc . s1p is required for lymphocyte egress from lymphoid organs ; in addition , thymocytes require s1p receptor activation to be released from the thymus 13 . therefore , elevated s1p may act to increase the pool of immune cells available for autoimmune responses . interestingly , an experimental s1p agonist , fty720 , is being tested as a possible pharmacological agent to treat immune rejection following transplants and autoimmune diseases 33 . although it acts as an s1p receptor agonist , fty720 actually leads to s1p receptor internalization , leaving the receptors unavailable for binding with s1p and thus antagonizing the effects of s1p . the result is the trapping of lymphocytes within lymph nodes and peyer 's patches and a marked reduction of lymphocytes in the circulation 34 . thus , it is theoretically possible that treatment with fty720 or other agents directed at s1p might have effects in ssc . elevated lpa and lpa / lpc ratios may be involved in the fibrotic component of ssc . a recent study found that elevated plasma lpa levels and higher serum lysopld activity were associated with hepatitis c and its associated liver fibrosis 35 . the authors of this study postulate that elevated lpa contributed to the hepatic fibrosis through its proliferative and anti - apoptotic effect on hepatic stellate cells . in a second recent study linking lpa to a fibrotic pathology , lpa was found to be elevated in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis , and inhibition of the lpa1 receptor reduced the chemotactic activity of this fluid towards fibroblasts 36 . the authors speculate that increased vascular leakage associated with lpa1 receptor activation may lead to increased fibrin deposition and a corresponding fibrotic cascade in injured airspaces . it is possible that elevations in lpa and/or s1p concentrations may have altered vascular permeability in our ssc subjects , although this awaits further study . our previous work indicates that lpa - induced myofibroblast differentiation may be involved in ssc fibrotic activity and possibly pathological fibrosis in general . myofibroblasts are cells with smooth muscle characteristics ( they contain a smooth muscle actin and are contractile ) and are typically derived from fibroblasts or epithelial cells . in ssc , they also appear to be derived from circulating blood - derived mononuclear cells 38 . myofibroblasts are widely accepted to be major contributors to tissue and organ fibrosis 39 - 41 . not only did we demonstrate that lpa could trigger fibroblast to myofibroblast differentiation 9 , but we have also described an lpa - activated cl current ( icllpa ) that is active in myofibroblasts but not myofibroblast precursors . we have observed icllpa in myofibroblasts from the lung 9 and cornea 42 , 43 and from the skin , heart , and liver ( unpublished data ) . we have demonstrated that blocking icllpa activity can prevent fibroblast to myofibroblast differentiation 9 . in conclusion , we measured 10 different lpc and lpa species ( 2 saturated and 8 unsaturated ) along with s1p and lysopld activity in the serum of control subjects and subjects with confirmed ssc . we demonstrated significantly increased serum 20:4 lpa and s1p concentrations in ssc subjects versus controls , along with elevated lpa : lpc ratios of two different unsaturated ( but not saturated ) phospholipid species , as well as the ratio of all species combined ( total ) . we also determined that there was no difference in the enzymatic activity of lysopld , which generates lpa from lpc , between control and ssc subjects . our results are potentially of real clinical significance in a disease for which there remains no cure and no directed therapies . although we can control some manifestations of the disease , no pharmacological therapies are currently available that alter the underlying systemic fibrosis that is the hallmark of ssc . our study suggests that directed pharmacological inhibition of lpa and s1p receptors ( novel lpa and s1p receptor agonists and antagonists are currently under development ) 34 , 44 may represent an innovative pathway for targeting the fibrosis and autoimmunity in ssc . the major limitation of this study is the relative rarity of the disease and subsequent difficulty in recruiting large numbers of ssc subjects , especially with both diffuse and limited disease , making it difficult to adequately power these comparisons . despite this limitation , we were able to see significant results between the groups tested , indicating what is likely clinically significant differences in the levels of these bioactive lipids and the lpa : lpc ratios between control and ssc subjects .

systemic sclerosis ( ssc ) is an often fatal disease characterized by autoimmunity and inflammation , leading to widespread vasculopathy and fibrosis . lysophosphatidic acid ( lpa ) , a bioactive phospholipid in serum , is generated from lysophospholipids secreted from activated platelets in part by the action of lysophospholipase d ( lysopld ) . sphingosine 1-phosphate ( s1p ) , a member of the bioactive lysophospholipid family , is also released from activated platelets . because activated platelets are a hallmark of ssc , we wanted to determine whether subjects with ssc have altered serum lysophospholipid levels or lysopld activity . lysophospholipid levels were measured using mass spectrometric analysis . lysopld activity was determined by quantifying choline released from exogenous lysophosphatidylcholine ( lpc ) . the major results were that serum levels of arachidonoyl ( 20:4)-lpa and s1p were significantly higher in ssc subjects versus controls . furthermore , serum lpa : lpc ratios of two different polyunsaturated phospholipid molecular species , and also the ratio of all species combined , were significantly higher in ssc subjects versus controls . no significant differences were found between other lysophospholipid levels or lysopld activities . elevated 20:4 lpa , s1p levels and polyunsaturated lpa : lpc ratios may be markers for and/or play a significant role in the etiology of ssc and may be future pharmacological targets for ssc treatment .

Introduction Subjects and Methods Results Discussion Limitations

systemic sclerosis ( ssc ) is an often fatal disease characterized by autoimmunity and inflammation , leading to widespread vasculopathy and fibrosis of multiple organs 1 . there are two distinct subsets of the disease , limited and diffuse , which are based on the extent of skin involvement however , one of the hallmarks of the vasculopathy associated with both subsets of ssc is platelet activation , which has been implicated as a key mediator of the fibrosis that underlies ssc 3 . lpa is found at physiologically and pathophysiologically significant concentrations in plasma and serum , respectively . lpa is typically produced by the action of plasma lysophospholipase d ( lysopld ) , also known as autotaxin , on lysophosphatidylcholine ( lpc ) 6 . in serum , activated platelets are one of the primary sources of lpa , which is produced via the action of lysopld on lpc and other lysophospholipids 7 . a similar differentiation step is critical to the pathogenesis of ssc , in that myofibroblasts are one of the primary sources of ssc fibrotic tissue 10 , 11 . rho - associated kinases ( rock ) , which are activated by lpa , have recently been found to stimulate myofibroblast differentiation from cultured human skin ssc fibroblasts . sphingosine 1-phosphate ( s1p ) , another major member of the plgf family , acts as an agonist to membrane receptors that have close evolutionary links to several of the lpa receptors . both b- and t - cells require s1p activity to allow their movement out of lymphoid organs , and thymocytes require s1p receptor activation for release from the thymus 13 . although there is currently no known association between s1p and ssc , ssc has an autoimmune component with reported lymphocyte involvement that suggests there may be a role for s1p in the development and/or progression of ssc . in view of the high level of platelet activation seen in ssc 14 , along with the increased myofibroblast activity and impaired myofibroblast apoptosis , we hypothesized that elevated plgfs play a role in the pathogenesis of ssc , and specifically that lpa would be elevated in ssc subjects relative to healthy controls . therefore , we designed this study to determine whether differences exist in serum lpa concentrations and in lysopld activities of ssc and control subjects . we chose to look at a range of different lpa species because of the likelihood that we would find differences in either a particular species or class of lpa 15 . in addition to lpa measurement , we also measured and compared s1p concentrations in the blood of ssc versus control subjects because of the likely autoimmune pathogenesis of ssc and the link between s1p and immune cell function . because of significant differences that are present between limited and diffuse ssc , we also examined the differences in lipid products and lysopld activity between subjects with these two subgroups of the disease . finally , we measured in ssc and control subjects the concentrations of additional bioactive lysophospholipids , including lysophosphatidylethanolamine ( lpe ) , lysophosphatidylinositol ( lpi ) and lysophosphatidylserine ( lps ) , which are precursors of lpa . written consent for participation in the study was obtained from all participants in accordance with the helsinki ii declaration , and the protocol was approved by the uthsc institutional review board . ssc subjects with a history of limited or diffuse ssc were recruited from the rheumatology clinic of the university of tennessee health science center ( uthsc ) . all ssc subjects met the 1980 acr classification criteria for clinical diagnosis of limited or diffuse ssc 16 . after an overnight fast , blood was collected from 10 patients with ssc ( 7 with diffuse disease and 3 with limited disease ) and from 13 healthy controls . 17:0-lpa was prepared from 17:0-lpc by the action of phospholipase d from streptomyces chromofuscus , as described previously 17 . in brief , a mixture of 0.1 ml of 5 m ethanolamine hydrochloride/0.2 m acetate buffer ( ph 5.5 ) containing 0.1 mmol 17:0-lpc , 0.05 ml of 0.22 m cacl2/0.02 m nacl , and 0.1 ml of phospholipase d solution ( 8 mg / ml ) in 0.2 m acetate buffer was incubated while being stirred for 2 h at 30c . after the reaction was stopped by addition of 2 ml chloroform / methanol mixture ( 1:1 , v / v ) , the mixture was centrifuged at 1300 x g for 10 min . 17:0-lpe was extracted from silica in the corresponding lipid band by the method of bligh and dyer 19 . lipids were extracted from human serum samples by the modified method of bligh and dyer 19 after adjusting the aqueous phase ph to 9 - 9.5 with 20% ammonium hydroxide . to the lipid extract were added 5 nmol 17:0-lpc , 0.05 nmol 17:0-lpe , 0.1 nmol 17:0-lpa , and 0.2 nmol c17 s1p . most of the lipids , including lpc and lpe , were extracted into the organic layer . the lipid extract was dried under a stream of nitrogen gas , reconstituted with 0.5 ml of methanol / water mixture ( 1:1 , v / v ) containing 5 mm ammonium formate , and termed the neutral lipid fraction . the remaining aqueous layer was acidified to ph 2 - 2.5 with 1 n hydrochloric acid , and acidic lipids such as lpa , lpi and lps and s1p were extracted into the organic layer by the method of bligh and dyer 19 . lc - ms - ms was performed on a quadrupole - linear iontrap hybrid ms , 4000 qtrap ( applied biosystems / mds sciex ; concord , on , canada ) , with an agilent 1100 lc system combined with an autosampler ( agilent technologies ; wilmington , de , usa ) . lysophospholipids were analyzed by multiple reaction monitoring ( mrm ) in a positive ion mode for lpc and lpe or in a negative ion mode for lpa , lpi , lps , and s1p . in the positive ion mrm , q1 and q3 were set for the protonated molecular ion and [ phosphorylcholine ] at m / z 184 for lpc or [ m - 141 ] for lpe . in the negative ion mrm , q3 was set to [ cyclic glycerol phosphate ] at m / z 153 for lpa and lps , [ phosphorylinositol - h2o ] at m / z 241 for lpi or [ po3 ] at m / z 79 for s1p , in combination with the deprotonated molecular ion as q1 for all lysophospholipids tested . amounts of the different molecular species of lpc and lpe were calculated from the ratios of their areas of positive ions to those of 17:0-lpc or lpe internal standards . similarly , the amounts of molecular species of lpa were calculated from the ratios of their peak areas of negative ions to that of 17:0-lpa , an internal standard . the amounts of molecular species of lps and lpi were calculated from both their peak areas of negative ions to that of 17:0-lpa and the correction factors for lpi and lps . lysopld activity in serum was measured by the enzyme - linked fluorometric method for determination of choline produced together with lpa from exogenously added 0.15 mm 18:2-lpc . the mixtures in the wells were incubated at 37 c for 9 or 24 h , and were then mixed with 0.2 ml of assay buffer . the assay buffer was composed of 0.1 ml of tris - hcl buffer ( ph 8.5 ) , 0.04 ml of 7.5 mm 3-(4-hydroxyphenyl)propionic acid , 0.02 ml of 25 u / ml choline oxidase and 0.04 ml of 2 u / ml horseradish peroxidase . with the aid of a standard line obtained with 0 , 0.1 , 0.3 , 1 , 3 , 10 , and 30 nmol / ml of water solution of choline chloride , the choline concentration was determined from the intensity of fluorescence measured at 320 nm ( excitation ) and 404 nm ( emission ) , and lysopld activity was calculated as nmol choline / ml of serum . alternatively , serum lysopld activity of subjects with diffuse ssc and healthy controls was measured by quantifying lpa production using lc - ms - ms as described above . among the ssc subjects , seven had diffuse disease and three had limited disease . the mean duration of disease in the ssc subjects was 10.3 years , with a range of 3 - 28 years . measured serum concentrations of lpc , lpa , s1p , and dihydrosphingosine 1-phosphate ( dihydro s1p ) , which is identical to s1p except that it lacks one double bond and acts as an agonist for all s1p receptors but with a 20-fold lower affinity for s1p2 20 , 21 , from the control and ssc groups are summarized in fig . 1a ) , a precursor of lpa , had the highest concentrations ( 16:0>18:2>18:0>18:1 and 5 minor species ) followed by lpa ( fig . figure 2 shows serum concentrations of lpe , lpi , and lps from control and ssc subjects . of these lysophospholipids , lpe had the highest concentrations ( 18:2>18:1>16:0=20:4 and 3 minor species ) , which were less than the concentrations of lpa , followed by lpi ( 20:4>18:2>18:0>18:1 and 5 minor species ) and then lps ( 18:0>18:1>20:4 ) . 1b ) versus the control group as well as in the limited ssc group versus control ( 2.54 0.15 nmol / ml vs. 1.15 0.37 nmol / ml for control , p= 0.026 ) , with no differences measured between controls and the diffuse ssc group ( 1.99 0.49 nmol / ml , p = 0.088 ) . in addition , the s1p levels were significantly higher in ssc vs. control groups ( fig . 1c ) , and also in the diffuse ssc group versus controls ( data not shown ) . essentially no differences were observed for serum levels of lpc , lpe , lps , and lpi , as shown in figs . these ratios in part can show the conversion status of lpc to lpa for the individual species and for the combined total of species , with values closer to 1 indicating more conversion of lpc to lpa . here we found a significant difference in the total lpc / total lpa ratio between control and ssc ( p < 0.01 ) groups . there was also a significant difference between control and diffuse ssc groups ( p < 0.02 , data not shown ) . in addition , the values for 20:4 ratios were significantly different between control and ssc groups ( p < 0.01 ) , control and limited ssc ( p < 0.02 , data not shown ) groups , and control and diffuse ssc ( p < 0.05 , data not shown ) groups . in addition , there was a significant difference in the 20:3 ratio between control and ssc groups ( p < 0.05 ) and control and limited ssc groups ( p < 0.05 , data not shown ) . no significant differences were found between groups in the lysopld activity toward 150 m 18:2-lpc ( fig . there were also no significant differences between total and individual levels of lpa produced during 12 h incubation of sera from ssc patients and control subjects ( fig . for this study , we chose to measure not only total lpa levels in ssc versus control subjects , but also to measure several different specific saturated and unsaturated lpa species . the major findings of this study were significantly elevated concentrations of 20:4 lpa and s1p in the serum of ssc subjects versus controls . the total serum lpa : lpc ratio , and within this , the subgroups of 20:3 and 20:4 lpa : lpc ratios , were also elevated in the ssc subjects versus controls . no significant differences in other lysophospholipid mediators , including lpe , lpi , and lps , were found between control and ssc subjects . in addition , there were no differences observed in lysopld enzymatic activity between the ssc subjects and controls . we postulate that the elevated lpa and lpa : lpc ratios found in ssc subjects may be linked to reduced activity of a postulated lpa - degrading lysophospholipase in the serum of ssc subjects that would preferentially hydrolyze highly polyunsaturated lpas , such as the 20:4 species , over other lpa species . increased lpa synthesis is likely not responsible for these elevated values because we found no increase in lysopld activity in any of the ssc groups . our findings of significantly higher lpa : lpc ratios in the polyunsaturated species over saturated and monounsaturated species in serum samples from ssc patients and control subjects is consistent with a previous finding that sn-1-lyso - lpc , formed via hydrolysis of phosphatidylcholine by phospholipase a1 , is a superior substrate for lysopld in human serum and plasma as compared to sn-2-lyso - lpc , which is generated by hydrolysis of phosphatidylcholine by phospholipase a2 29 . among the many different lpa subtypes measured in this study , 20:4 lpa was consistently elevated in the ssc subjects ( both limited and diffuse ) versus control subjects . in regard to ssc , 20:4 lpa has been found to have a high platelet - activating potency 24 , 25 . while activated platelets are a common feature of ssc , and activated platelets are known to release lpa , the presence of elevated 20:4 lpa could act in a positive feedback manner to perpetuate platelet activation . from an immune standpoint , unsaturated lpas , with 20:4 lpa being one of the species tested , this is potentially important , given recent evidence for altered antigen processing in ssc 31 . when stratifying by ssc disease types , the total serum lpa : lpc ratio and serum 20:4 lpa : lpc ratio were significantly elevated in the diffuse ssc subjects versus controls . although we had a small sample size of subjects with limited ssc , we found that serum 20:4 lpa and the 20:3 and 20:4 lpa : lpc ratios were significantly higher in the limited ssc group versus control subjects . in addition , elevated serum s1p concentrations approached significance in diffuse ssc and limited ssc subjects versus controls ( p = 0.05 and 0.09 , respectively ) . these lpa : lpc ratio and s1p patterns all follow the general pattern of these parameters being higher in ssc subjects versus controls . platelets are known to play a pathological role as a main source of circulating s1p by accumulating and releasing s1p after local blood coagulation , whereas erythrocytes are considered to be the main source of plasma s1p 32 . the biological / clinical significance of our findings relates to both the immune etiology and fibrotic disposition of ssc . in regard to the immune system , the elevated s1p concentrations found in ssc subjects may relate to the lymphocyte involvement in the autoimmune genesis of ssc . s1p is required for lymphocyte egress from lymphoid organs ; in addition , thymocytes require s1p receptor activation to be released from the thymus 13 . thus , it is theoretically possible that treatment with fty720 or other agents directed at s1p might have effects in ssc . elevated lpa and lpa / lpc ratios may be involved in the fibrotic component of ssc . a recent study found that elevated plasma lpa levels and higher serum lysopld activity were associated with hepatitis c and its associated liver fibrosis 35 . in a second recent study linking lpa to a fibrotic pathology , lpa was found to be elevated in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis , and inhibition of the lpa1 receptor reduced the chemotactic activity of this fluid towards fibroblasts 36 . it is possible that elevations in lpa and/or s1p concentrations may have altered vascular permeability in our ssc subjects , although this awaits further study . our previous work indicates that lpa - induced myofibroblast differentiation may be involved in ssc fibrotic activity and possibly pathological fibrosis in general . in ssc , they also appear to be derived from circulating blood - derived mononuclear cells 38 . we have observed icllpa in myofibroblasts from the lung 9 and cornea 42 , 43 and from the skin , heart , and liver ( unpublished data ) . in conclusion , we measured 10 different lpc and lpa species ( 2 saturated and 8 unsaturated ) along with s1p and lysopld activity in the serum of control subjects and subjects with confirmed ssc . we demonstrated significantly increased serum 20:4 lpa and s1p concentrations in ssc subjects versus controls , along with elevated lpa : lpc ratios of two different unsaturated ( but not saturated ) phospholipid species , as well as the ratio of all species combined ( total ) . we also determined that there was no difference in the enzymatic activity of lysopld , which generates lpa from lpc , between control and ssc subjects . although we can control some manifestations of the disease , no pharmacological therapies are currently available that alter the underlying systemic fibrosis that is the hallmark of ssc . our study suggests that directed pharmacological inhibition of lpa and s1p receptors ( novel lpa and s1p receptor agonists and antagonists are currently under development ) 34 , 44 may represent an innovative pathway for targeting the fibrosis and autoimmunity in ssc . the major limitation of this study is the relative rarity of the disease and subsequent difficulty in recruiting large numbers of ssc subjects , especially with both diffuse and limited disease , making it difficult to adequately power these comparisons . despite this limitation , we were able to see significant results between the groups tested , indicating what is likely clinically significant differences in the levels of these bioactive lipids and the lpa : lpc ratios between control and ssc subjects .

all mice were bred in the charles river facility ( charles river , l'arbresle , france ) and transported to the laboratory on request . male c57bl/6j mice and knockouts for the 2-subunit of nicotinic receptors ( 2 ) , aged 78 wk on the arrival were used in this study . both c57bl/6j and 2 mice arrived at the same time from the breeding facility in two distinct transportation boxes . both strains were treated similarly : they were initially group housed ( 4 mice / cage ) for 1 wk to acclimatize to the animal facility under a 12/12-h light - dark cycle ( lights on at 7:30 am ) . some c57bl/6j and all 2 mice were then separated and housed individually for 4 wk , while some c57bl/6j mice remained group housed ( 4 mice / cage ) . the 2 mice were originally generated from a 129/sv es cell line as described previously ( 36 ) and backcrossed onto the c57bl/6j strain for 20 generations , which is twice the number of backcrosses suggested by the bandburry conference guidelines ( 37 ) . they were shown to be at > 99.99% c57bl/6j by a genomic analysis using 400 markers . for these reasons , and because littermates are not available in the breeding facilities , we used c57bl/6j mice as controls . groups consisting of 812 mice were formed for the 3 experimental procedures : global ne depletion and behavior ( experiment 1 ) ; ex vivo assessment of brain monoamine levels ( experiment 2 ) ; and prl ne depletion and behavior ( experiment 3 ) . each experimental procedure consisted of 4 groups : sham - treated ( c57bl/6 and 2 ) and lesioned ( c57bl/6 and 2 ) mice . in total , 95 animals were used . however , a small number of animals were not included in the final analysis due to the presence of gliosis ( 10 mice ) or due to outliers ( 4 mice ) , as confirmed by the dixon test ( 38 ) . control groups used for behavioral experiments 1 ( 10 c57bl/6 and 7 2 mice ) and 3 ( 15 c57bl/6 and 14 2 mice ) were pooled since there were no statistical differences between them ( anova main group effect , p=0.66 , ns ) . animals were returned to their individual home cage for 1 wk after surgery and before behavioral testing . all experimental procedures were carried out in accordance with the ethical standard defined by the french centre national de la recherche scientifique and european community guidelines for care of laboratory animals ( no . all procedures were conducted to reduce the number of mice used when possible and to reduce their level of pain and discomfort as much as possible . louis , mo , usa ) was dissolved in 0.9% saline containing 0.1% ascorbic acid . gbr 12909 dihydrochloride ( sigma ) was dissolved in dmso ( 25 mg / ml ) . the selective noradrenergic neurotoxin n-(2-chloroethyl)-n - ethyl-2-bromobenzylamine hydrochloride ( dsp-4 ) ; tocris cookson , ellisville , mo , usa ) was dissolved in distilled water ( 50 mg / kg ) . global ne depletion was achieved by systemic administration of dsp-4 . at 8 d before the behavioral experiment , adult male c57bl/6 mice and 2 mice were injected intraperitoneallly with dsp-4 ( 50 mg / kg ) or vehicle ( sham - treatment group ) . at 30 min before the dsp-4 or vehicle injections , animals were pretreated with gbr 12909 dihydrochloride ( 25 mg / ml ) to protect dopaminergic neurons ( 39 ) . mice were anesthetized with a mix of ketamine / xylazine [ 1000:20 mg / ml , 300:50 l , respectively , in phosphate - buffered saline ( pbs ) , qs 20.3 ml / mouse ] and positioned in a stereotactic instrument ( stoelting , wood dale , il usa ) . bilateral infusions were made into the prl of the pfc under stereotactic guidance ( 40 ) ( from bregma : + 2.2 mm anteroposterior , 0.3 mm mediolateral , 2.2 mm dorsoventral ) . animals were infused with 6-ohda ( 10 g in 0.5 l / site of injection ) or vehicle ( 0.5 l of 0.9% saline containing 0.1% ascorbic acid / site of injection ) . infusions were made through a fine needle with a flat tip ( 36 gauge ; coopers needle works , birmingham , uk ) connected via peg tubing to a 5-l syringe ( hamilton ) held by a microliter infusion pump . the delivery rate of injection was 0.2 l / min . to allow sufficient time for diffusion , the needle was left in place for 10 min after each infusion . mice were given 7 d to recover from surgery . to minimize nonspecific damage , mice lesioned with 6-ohda were pretreated with gbr 12909 ( 15 mg / kg i.p . ) to protect dopaminergic neurons . the magnitude and neurochemical selectivity of the lesion were verified afterward by immunofluorescence for norepinephrine transporter ( net ; see materials and methods and supplemental data ) . at 8 d after surgery , male mice were tested in the social interaction paradigm to evaluate the effects of local or global ne depletion on social behavior . mice socially isolated were shown to display a higher motivation for social contact than mice reared in groups ( 24 , 41 ) . as described previously ( 23 , 33 , 42 ) , each mouse previously isolated was placed individually in a large transparent testing cage for a 30-min habituation period . this mouse was called the isolated - host ( ih ) mouse . at the end of the habituation period , a drug / behavior - naive , group - housed c57bl/6 mouse [ called the social - visitor ( sv ) mouse ] of same weight , age , and sex was introduced into the testing cage . each sv mouse was gently placed in a corner opposite to the ih mouse for an 8-min test session recorded via a camera on a computer for offline scoring . behavioral features of affiliative social behavior , i.e. , social contact duration and follow behavior ( 24 , 33 , 43 ) , were first manually measured and compared between c57bl/6 and 2 mice , as well as between lesioned and sham - treated mice . furthermore , a more detailed analysis was carried out using miceprofiler software ( 33 ) . briefly , with this semiautomatic analysis , we distinguished contact events , defined as any position in which mice were at whiskers distance or less apart . within contact events , we discriminated oral - oral , oral - genital , and side - by - side contacts . apart from contact events , we scored different postures in each mouse of the dyad . among them , we distinguished stop behaviors , back - to - back postures , and follow behaviors . several previous studies have defined stop behaviors , that is short bouts of stopping in between locomotor sequences , as being important for the organization of actions in different animals ( 4447 ) . stop behaviors may be very brief and last only one image frame ( 1/15 s ) . in addition , animals may not stop completely ( speed < 1.75 cm / s ) but can scan the environment , rear , sniff , and make head movements ( 47 ) . therefore , as stop behaviors have been shown to be important choice points in various animal models during novelty exploration and locomotion , we included them in the repertoire we defined in our social interaction task that allows large movements and novelty seeking ( 33 ) . back - to - back postures were scored when both mice were looking in opposite directions , were relatively static , not touching each other , and had their heads at an angle that did not allow them to see each other . follow behaviors were defined by mice moving at a speed > 1.75 cm / s with one mouse behind the other and a distance between them of < 1.5 cm ( 33 ) . these complex events that have been shown to be indicative of behavioral flexibility were virtually impossible to analyze manually in such a detail . dominance was characterized as a threat behavior that may be made to reinforce a dominant position or to territory protection . this behavior may progress to aggression , depending on the flight / submissive or defensive behavior of the congener ( 4850 ) . here , like in our previous studies ( 51 , 52 ) , we defined the index of social dominance as the combination of two types of behaviors : paw control and aggressive grooming . paw control events were scored each time the ih mouse put at least one of its forepaws on the back or head of the sv mouse . we also calculated an index of aggressiveness , which included the number of times the ih mouse circled around , bit , and rattled its tail in front of the sv mouse . at 24 h after the social interaction test , animals were assessed for 10 min in a light / dark box to evaluate anxiety . the apparatus consisted of a rectangular acrylic box , divided into two compartments : one dark that was covered by a dark opaque top , and one white that was open and brightly illuminated ( 400 lux ; ref . the mouse was gently placed in the corner of the light box and could move freely from one compartment to the other through a dark corridor . a video camera was fixed above the apparatus to record the animal 's behavior for offline analyses , so that the test was conducted in absence of an experimenter . the criteria used to assess an animal 's level of anxiety were the initial latency to escape the light box , the number of entries into the dark box , and the total time spent in the light compartment . an entry into a compartment was registered if the mouse placed both forepaws into the box . the standard epm test is commonly used to assess anxiety - like behavior in rodents . this task relies on conflict between the innate fear that rodents exhibit toward elevated and open areas vs. natural exploration of novel environments . the maze was a cross - shaped elevated maze with two open arms facing each other and two walled arms . in such a maze , the natural tendency of rodents is to prefer enclosed dark spaces to open brightly lit spaces . the day following the dark - light test , animals were tested for 10 min in epm . a video camera fixed on the ceiling of the experimental room right above the central platform allowed the experimenter to record and track offline the mouse position within the maze . total time spent in open and closed arms and numbers of entries into the open arms were measured . entrances were counted when subjects put both forepaws on an arm or on the platform . from these measures , we derived the percentage of time spent on the open arms , expressed as a percentage of time spent on the open and closed arms . at the completion of the last behavioral test , mice were deeply anesthetized with intraperitoneal pentobarbital and transcardially perfused with 20 ml of ice - cold 0.1 m pbs ( ph 7.4 ) , followed by 50 ml of ice - cold 4% paraformaldehyde in 0.1 m phosphate buffer ( pb ) . brains were rapidly removed and postfixed overnight in the same solution at 4c . then , 60-m coronal sections were cut on a vibratome . one section in 4 was used for cresyl violet histology , tyrosine hydroxylase ( th ) and serotonin [ 5-hydroxytryptamine ( 5-ht ) ] immunohistochemistry , and net immunofluorescence . cresyl violet staining was used to verify the most ventral position of injectors in the pfc as well as the integrity of the adjacent tissue . immunohistochemistry was used to demonstrate the preservation of both dopaminergic and serotoninergic systems ( i.e. , to ensure of the selectivity of our procedure with dsp4 or 6-ohda injection coupled to gbr 12909 injection ) . in experiment 2 , we determined and compared constitutive brain levels of monoamines and ach in c57bl/6 and 2 mice . twelve animals of each group ( c57bl/6 and 2 ) were killed by exposure to a rising concentration of carbon dioxide and cervical dislocation ( 53 ) . frozen sections were dissected using a punch from 6 regions of interest ( 40 ) : rostral prelimbic cortex ( rprl ) , caudal prelimbic cortex ( cprl ) , orbitofrontal cortex ( ofc ) , hippocampus ( h ) , nucleus accumbens ( acb ) , and amygdala ( a ) . tissue aliquots were derived from both hemispheres and homogenized in 200 ml of 0.2 m perchloric acid by an ultrasonic cell disruptor ( microson , barcelona , spain ) . levels of ne , da , and 5-ht and their metabolites ( dopac , 5hiaa , and mhpg ) were determined in the supernatant by reversed - phase , high - performance liquid chromatography ( hplc ) , as described previously ( 53 ) . the analysis of ach was also performed by hplc , with the mobile phase containing 75 mm na2hpo4 and 5 l / ml proclin reagent ( bas , congleton , uk ) , ph 8.0 ( adjusted with 46/48% naoh ) , and a flow rate of 120 l / min ( 582 solvent delivery module ; esa inc . , chelmsford , ma , usa ) . samples were injected manually ( unijet microbore valve ; bas ) onto a microbore analytical column ( 5301 mm , 10 m ; bas ) , as described previously ( 54 ) . to verify possible differences in the balance between these neurotransmitters after global noradrenergic lesion , 12 c57bl/6 and 12 2 mice were pretreated with the selective noradrenergic neurotoxin dsp-4 ( 50 mg / kg i.p . ) . these animals were assessed postmortem for brain tissue levels of monoamines and ach , as described above . the main effects of genotype ( 2 levels : c57bl/6 and 2 ) and of lesion ( 2 levels : sham treatment and global ne lesion , or ne prl depletion ) , as well as statistical interactions between these factors , were assessed with anova and statview 4.57 ( sas institute , cary , nc , usa ) . post hoc analyses were performed on significant main effects or significant interactions using bonferonni 's test . dixon 's q test ( 38 ) was used before any other statistical treatment to objectively detect a single outlier within a group of data for which a gaussian distribution applies . as flexible social behaviors are strongly modulated by ne and ach systems , we first investigated the effect of global ne depletion on social behavior in c57bl/6 and 2 mice . for that purpose , we used a social interaction task in which a previously isolated male mouse was confronted with a novel male c57bl/6 mouse in a novel , large environment . this behavioral setting triggers frequent switching between social and nonsocial actions in both animals , thereby generating frequent and varied behavioral choices . we quantified various social behavioral sequences reflecting flexible behavior ( 33 ) such as physical contact , dynamic approaches and escape behaviors , and relative position events . we also quantified dominance behavior and aggressiveness . in agreement with our previous results ( 23 , 24 , 33 , 43 ) , c57bl/6 mice spent less time in social contact than 2 mice ( p<0.0001 ) and made fewer follow behavioral sequences ( p<0.0001 ; fig . effects of global ne lesion by injection of dsp-4 on social interaction and on social aggressive - like behavior in c57bl/6 mice ( sham treated n=25 , lesioned n=10 ) and 2 mice ( sham treated n=21 ; lesioned n=8 ) . a ) contact duration , showing the total time spent in social contact , which increases for c57bl/6 mice and decreases for 2 mice , after the lesion . dsp-4 lesions produced a decrease of this behavior in 2 mice but had no effect in c57bl/6 mice . c ) 2 mice show a higher index of dominance ( paw control and aggressive grooming ) . d ) lack of effect of dsp-4 on aggressiveness ( circling , bite , and tail rattling ) in c57bl/6 and 2 mice . histograms show mean se values . * p 0.05 , * * p 0.005 , * * * p 0.0005 . in this social interaction paradigm , the time spent in social contact and the frequency of follow sequences are the first obvious features that are indicative of social behavior flexibility . a flexible behavior will thus be reflected by a reduction of frequency with time of some behavioral sequences , postures , or contact and by the emergence of novel behavioral features . thus , these results corroborate the rigid social phenotype of 2 mice described previously ( 23 , 24 , 33 , 43 ) . global depletion of brain ne led to opposite effects in c57bl/6 and 2 mice during social testing . specifically , the total time spent in social contact and the total number of follow sequences were significantly affected by the lesion ( p<0.0001 and p=0.001 , respectively ) but differently according to genotype : in c57bl/6 mice , global ne depletion led to a 40% increase in the duration of social contact ( p=0.003 ) . by contrast , in 2 mice , ne depletion led to a 33% decrease in the duration of social contact ( p=0.0007 ) and a 43% decrease in the number of follow sequences ( p=0.0002 ; fig . 2 mice exhibited higher dominance scores compared with c57bl/6 mice ( p=0.02 ) but a similar low level of aggressiveness ( p=0.3317 ) . global ne depletion increased dominance behavior only in c57bl/6 mice ( p<0.005 ) and had no significant effect on aggressive behavior in any group of mice ( fig . it is known that monoamine and ach contribute to flexible behaviors mainly via prefrontal modulation . however , no information as to the levels of any of the major nt was available in 2 mice . we thus measured basal levels of da , ne , 5-ht , and ach , as well as their metabolites , in both groups of mice and tested the hypothesis that the opposite behavioral effects after global ne depletion could be explained by constitutive biochemical differences . the biochemical quantification was performed by hplc on frozen punches of pfc , ofc , acb , h , and a regions . basal levels of ne , da , 5-ht , and ach were increased in 2 mice compared with c57bl/6 mice in the pfc ( p=0.0014 , p=0.0075 , p=0.0022 , and p<0.0001 , respectively ; fig . 2 ) . moreover , in the ofc and a , basal levels of ne and 5-ht were also increased . in the acb , only ne levels were significantly higher , while in the h , only the da levels were increased ( supplemental fig . overall , the 5-ht activity was significantly increased in 2 mice ( p=0.02 ) , while the activity of ne and da remained unchanged , as we could assess by measuring their respective metabolites ( p=0.9866 and p=0.0991 , respectively ) . levels of monoamines and ach in the prl of c57bl/6 ( n=12 ) and 2 mice ( n=12 ) . tissue content of all monoamines and ach increased in 2 mice compared with c57bl/6 mice . histograms show mean se values . * * p 0.005 , * * * p 0.0005 . neurochemical analysis revealed that basal levels of monoamines and ach were higher in 2 mice . to elucidate whether this effect was associated with an increase in monoaminergic fiber density , we measured the density of th , 5-ht , and net fibers in the pfc of c57bl/6 and 2 mice . the 2 mice showed a significantly lower density of th , 5-ht , and net fibers ( p=0.048 , p=0.0001 , and p=0.0047 , respectively ) compared with c57bl/6 mice ( supplemental fig . s2 ) . therefore , increased basal levels of monoamines in the prl was unlikely to be due to an increase in the density of monoaminergic fibers in this region . by contrast , 5-ht fiber density was reduced in c57bl/6 mice after ne depletion ( p=0.001 ) . neurochemical and neuroanatomical analyses therefore indicated that 2 mice exhibited increased basal levels of monoamines , although the density of monoaminergic innervations was decreased . moreover , the increase in basal neurotransmitter levels was not always followed by an increase in monoaminergic activity . collectively , these results suggest that vesicular storage of monoamines may be increased in 2 mice . ne depletion of the prl had no significant effect on anxiety - related behavior in the dark - light paradigm in c57bl/6 or 2 genotypes . however , in the epm , the percentage of time spent in open arms was altered differently in c57bl/6 and in 2 mice by pfc ne depletion ( genotype x treatment f(1,43)=6.89 , p=0.012 ) . post hoc t tests revealed that ne depletion of the pfc led to a significant anxiolytic effect in 2 mice ( sham treatment : 19 2% ; ne depletion : 28 2.76% ; t=2.7 , df=20 , p=0.013 ) but had no effect in c57bl/6 mice ( t=0.88 , df=23 , p=0.39 ) . in previous experiments , we showed that the profound alteration in social interaction exhibited by 2 mice was similar to that obtained after damage to the prl of the pfc . notably , we showed that expression of functional 2*nachrs in the prl of 2 mice was necessary and sufficient for restoring flexible social interaction ( 24 ) . here , we observed that global ne depletion produced opposite effects in social behavior of 2 and c57bl/6 mice . to further investigate the putative role of the ne transmission in the prl , we carried out specific prl ne depletion and tested its effect in the same social paradigm . dominant - like behaviors associated with 2 mice were not affected by prl ne depletion ( p=0.40 , ns ; fig . dominant behavior was also not altered in c57bl/6 mice ( p=0.1 , ns ; fig . by contrast , in c57bl/6 mice , the index of aggressiveness increased dramatically by 6-fold following the selective depletion of ne from the prl ( p=0.0002 ; fig . this was unlikely to be due to anxiogenic effects , as anxiety was not altered by ne prl depletion in c57bl/6 mice ( see above ) . effects of ne prl depletion on dominance and aggressive - like behavior in c57bl/6 ( sham treated n=25 ; lesioned n=10 ) and 2 mice ( sham treated n=21 ; lesioned n=8 ) . 2 mice show a higher index of dominance ( paw control and aggressive grooming , a ) but not of aggressiveness ( b ) as compared with c57bl/6 mice . after lesion , there was a significant increase in aggressiveness in c57bl/6 mice but not in 2 mice . histograms show mean se values . * p 0.05 , * * * p 0.0005 . the same results obtained after global ne lesion were found in 2 mice after specific ne prl depletion . to investigate whether these effects were due to fine changes in the social repertoire , we assessed the key behavioral sequences of the social repertoire that were specifically altered by ne depletion . for this purpose , we analyzed social data with miceprofiler ( 33 ) software that we designed for underpinning decision points during complex behavioral chains . ne prl depletion produced a similar effect than that of the global lesion in 2 mice , i.e. , a 44% decrease in social contact duration ( p<0.0001 ; fig . 4 ) and a 36% decrease in the number of follow behaviors ( p=0.007 ; supplemental fig . a more detailed temporal analysis of some behavioral parameters indicated that the duration of social contact decreased steadily from the beginning of the experiment and followed a normal pattern in 2 mice after the prl ne lesion ( fig . 4 ) . global effects of ne prl depletion on social interaction in c57bl/6 ( sham treated n=25 ; lesioned n=10 ) and 2 mice ( sham treated n=21 ; lesioned n=8 ) . contact duration ( top panel ) decreased for 2 mice following the lesion but not for c57bl/6 mice . detailed analysis ( bottom panel ) illustrates the restoration , in lesioned 2 mice , of the temporal evolution of the contact events throughout the 8-min experiment , as compared with 2 sham - treated mice . histograms show mean se values . * * * p 0.0005 . while the duration of social contact was not altered in c57bl/6 mice after ne prl depletion , the global organization of behavior and the richness of repertoire were drastically modified ( fig . these parameters were assessed by the construction of graphs representing the probability of transition from an event to another ( the thicker the arrow , the more probable the event ) . the symbols representing both mice and their respective postures are provided in supplemental table s2 . transitions are computed for the first 4 min and the 4 last min of the experiment . black arrows represent events that occur steadily ( with the same probability ) for the first and the last 4 min . red and green arrows represent events that occur only in the 4 first and the 4 last minutes respectively . transitional graphs showed an increased proportion of unchanged behavioral sequences ( black arrows ) in 2 mice as compared with c57bl/6 mice . after ne - prl depletion , behaviors conserved with time ( black arrows ) were increased in c57bl/6 mice and decreased in 2 mice . transitional behavioral graphs after prefrontal ne depletion in c57bl/6 ( sham treated n=25 ; lesioned n=10 ) and 2 mice ( sham treated n=21 ; lesioned n=8 ) . these graphs represent the probability of transition from an event to another ( the thicker the arrow , the more probable the event ) . the symbols representing both mice and their respective postures are provided in supplemental table s2 . the transition is computed for the first 4 min and the 4 last min of the experiment . black arrows represent events that occur steadily ( with the same probability with an overlap of 1 of their respective sd ) for the first and the last 4 min . red and green arrows represent events that occur only in the 4 first and the 4 last minutes , respectively . transitional graphs showed an impoverishment of the social repertoire in c57bl/6 mice after ne prefrontal depletion , with less various behavioral events than in sham - treated mice . temporal evolution of behavioral sequences , illustrated by the number of red and green arrows , by comparison with events linked by black arrows , was also drastically lower in c57bl/6 mice after ne prefrontal depletion . therefore , c57bl/6 mice exhibited more rigid social behavior after ne prefrontal depletion . by contrast , in 2 mice , ne prl depletion favored a recovery of the temporal evolution of contact and follow events , thus reducing significantly rigid behaviors . stop behaviors , which were significantly less frequent in 2 mice than in c57bl/6 mice , were restored after ne lesion . detailed social behavioral analyses allowed us to assess other key behavioral markers of flexibility beyond social contact duration and the frequency of follow sequences . within the behavioral repertoire , we pinpointed two important markers of social flexibility ( 33 ) : stop behaviors , which constitutes behavioral choice points . this parameter indicates a capability to stop moving and disengage from an ongoing action to make a novel movement based on environmental cues . the other marker is reflected by a relative position of both mice . in this posture , both mice are almost immobile , looking in opposite directions and are not touching or seeing each other . this back - to - back behavior is a risk - prone posture that provides the dyad with a full panoramic view of the environment , reflecting a form of cooperative social behavior . both types of behaviors were previously shown to occur much less frequently in 2 mice compared with control mice ( 33 ) . stop behaviors , which were significantly decreased in 2 mice compared with c57bl/6 mice ( p<0.0001 ) were restored after prl ne depletion ( lesion effect in 2 mice p<0.0001 ; fig . the number of back - to - back sequences that was decreased in 2 mice compared with c57bl/6 mice ( p=0.001 ) was also restored in 2 mice after ne prl depletion ( lesion effect in 2 mice , p=0.002 ; fig . these results show that specific ne prl depletion normalized flexible social behavior in 2 mice by restoring social contact duration , follow sequences , immobility periods and back - to - back postures . by contrast , stop behaviors and back - to - back sequences , like contact duration , were not affected by ne depletion in c57bl/6 mice ( depletion effect in c57bl/6 mice all p>0.4 , ns ) . detailed effects of ne prl depletion on key elements of social interaction in c57bl/6 ( sham treated n=25 , lesioned n=10 ) and 2 mice ( sham treated n=21 , lesioned n=8 ) . ne depletion of the pfc restored both immobility ( a ) and back - to - back ( b ) sequences in 2 mice , both behaviors drastically impaired before lesion . furthermore , dominance and aggressiveness were shown to be functionally dissociated , with the former being modulated by the cholinergic nicotinic system and the latter potentiated by prefrontal ne but only in presence of functional 2*nachrs . in a social context , mice make multiple decisions that take into account their own motivations and choices , as well as the unpredictable actions of a conspecific . such encounters maximize uncertainty , generating high attentional states ( 55 ) that bias behavior toward socially rewarding stimuli ( 56 , 57 ) . the social paradigm used in the present study requires flexible decision - making processes that depend on the functional integrity of the pfc and 2*nachrs in this region ( 24 ) . here we demonstrated that global depletion of ne in the brain produced strongly divergent effects on social behavior in c57bl/6 and 2 mice . while it caused rigid social behavior in c57bl/6 mice , it restored behavioral flexibility in 2 mice . it has been shown that prefrontal ne is necessary for attentional processing , especially when the task is novel and challenging , and stimuli are unpredictable ( 25 , 5860 ) . individuals , whether animals or humans , naturally orient to novel stimuli with high sensory salience , particularly when attentional demands are low ( 60 , 61 ) . ne signaling has been postulated to provide an interrupt ( 62 ) or reset signal ( 58 ) thereby allowing the flexible detection of an unexpected target in the environment . according to the hypothesis that ne modulates the salience of external stimuli ( 58 , 6062 ) and that an optimal level of ne activity is necessary to optimize attention and decision - making performance ( 31 ) , our results show that 2 mice were more susceptible than c57bl/6 control mice to external stimuli during a social encounter . this susceptibility may be driven by disrupted ne transmission in the prl and by the salience of social stimuli being augmented by prior social isolation ( 24 ) . the prosocial behavior observed in 2 mice is likely due to a hypersalience of external stimuli , which in the social context is mainly generated by the congener . the loci of this effect appears to be the pfc and functional nachrs , as specific ne pfc depletion normalized major social flexible behaviors ( e.g. , stop behaviors , contact duration and follow behaviors ) in 2 mice . in addition , prl - ne depletion restored back - to - back sequences that were virtually absent in these mice . finally , we built transitional graphs ( see fig . 5 ) that illustrate the probability of transition from 1 behavioral state to another , confirming the more rigid behavior in 2 mice ( 33 , 43 ) , with a majority of behavioral sequences that remain unchanged throughout the experiment . moreover , transitional graphs unraveled an impoverishment of the social repertoire in c57bl/6 mice after ne prefrontal depletion , with less various behavioral events than in sham - treated mice . therefore , c57bl/6 mice exhibited more rigid social behavior after ne prefrontal depletion . by contrast , in 2 mice , ne prl depletion favored a recovery of the temporal evolution of contact and follow events , thus reducing significantly rigid behaviors . we have previously shown that 2 mice are hyperactive ( 63 ) and exhibit increased c - fos expression in the pfc , specifically when exposed to a novel environment ( 64 ) . these findings indicate an increased arousal / vigilance state when mice are confronted with highly arousing stimuli , such as a novel social interaction ( 23 ) , shown to trigger pfc activation ( 24 ) . this increased arousal / vigilance state can be , in part , explained by the high basal levels of monoamines and ach , observed especially in the pfc and that is likely to be due to an increased storage of these neurotransmitters . in a previous work , we showed that whole - brain ne depletion increased global aggressiveness in c57bl/6 mice ( 52 ) . here , distinguishing aggressive and dominance behaviors , we found that global ne depletion affected neither aggressiveness nor dominance . by contrast , neurochemically selective ne depletion of the prl led to a 6-fold increase in c57bl/6 control mouse aggressiveness , while it had no effect on 2 mice . thus , our data show that a lack of prefrontal ne transmission promotes aggressive - like behaviors . 2 mice exhibited more dominant behavior than c57bl/6 mice , which was not perturbed by ne - prl depletion , suggesting therefore that the dominant behavior we observed was related to the absence of 2*nachrs . by contrast , the balance between basal levels of monoamines and of ach appears to modulate aggressiveness , and this modulation requires functional 2*nachrs . in 2 mice , which exhibited constitutively increased levels of monoamines and ach in the pfc , ne depletion did not favor aggressiveness . by contrast , in c57bl/6 mice , ne - depletion led to a profound increase in aggressive - like behavior . our biochemical analysis showed that , in c57bl/6 mice , an increase in da levels after global ne depletion is sufficient to cause the 2-behavioral phenotype ( see fig . after ne global depletion in c57bl/6 mice , da levels increased in the pfc ( data not shown ) , suggesting an involvement of da in prosocial behaviors . this may be relevant to an analogous behavioral profile in da transporter - knockout ( dat ) mice , which shows increased levels of da , hyperactivity , impaired decision - making , and behavioral rigidity that includes an inability to shift and adjust behaviors in a new context ( 65 , 66 ) . our current results may also be relevant to understanding how cholinergic and monoaminergic systems interact in the pfc to modulate social cognition in schizophrenia . one of the proposed contributions of the cholinergic system to the pathophysiology of schizophrenia is through an imbalance between the cholinergic and dopaminergic systems ( 68 ) . recent data suggest that cholinergic perturbations in schizophrenia are not simply due to changes in ach levels but rather to the balance between activation of both nicotinic and muscarinic receptors , which collectively determine the functional outcome of central cholinergic stimulation ( 67 , 69 ) . while the putative role of 7 * nachrs in schizophrenia is controversial and not well established , 2*nachrs have been shown to be up - regulated in this disorder ( 70 ) , control epigenetic modifications ( 71 ) , and also mediate the effects of cholinergic transmission on ne , 5-ht , and da function in patients with schizophrenia ( 72 ) . in particular , cholinergic modulation of da release by 2*nachr stimulation has been proposed to contribute specifically to schizophrenia symptoms ( 7375 ) . furthermore , ne has been suggested to underlie the pathophysiogical mechanisms of schizophrenia , depression , and parkinson 's disease - associated depression through its regulatory interactions with the da and serotonergic systems ( 7681 ) . hyperdopaminergia was also observed in c57bl/6 mice after ne depletion and is likely to be sufficient to cause the 2-prosocial phenotype in these mice . by contrast , in the absence of 2*nachrs , there was no change in the basal level of da or 5-ht after ne depletion , which corroborates the role of these receptors in modulating da or 5-ht release . therefore , these data are compatible with the notion that the hypervigilant states associated with the positive symptoms of schizophrenia are associated with an overactivity of brain ne function whereas the negative symptoms of this disorder are related to an underactivity of brain ne ( 76 ) . in addition , our results suggest that 2*nachrs may contribute to the pathophysiology of schizophrenia through complex and intricate underlying interactions between cholinergic , dopaminergic and noradrenergic systems in the pfc . in summary , the present findings point to a clear and crucial role of 2*nachrs in the modulation of monoamine and cholinergic ach activity and their effect on social and flexible behavior . in particular , our findings indicate the ne innervation of the pfc has a pivotal role in inhibitory response control and adaptive behavior , as well as in the modulation of aggression , which depend on functional interactions with the cholinergic system in this region .

social animals establish flexible behaviors and integrated decision - making processes to adapt to social environments . such behaviors are impaired in all major neuropsychiatric disorders and depend on the prefrontal cortex ( pfc ) . we previously showed that nicotinic acetylcholine receptors ( nachrs ) and norepinephrine ( ne ) in the pfc are necessary for mice to show adapted social cognition . here , we investigated how the cholinergic and ne systems converge within the pfc to modulate social behavior . we used a social interaction task ( sit ) in c57bl/6 mice and mice lacking 2*nachrs ( 2/ mice ) , making use of dedicated software to analyze > 20 social sequences and pinpoint social decisions . we performed specific pfc ne depletions before sit and measured monoamines and acetylcholine ( ach ) levels in limbic corticostriatal circuitry . after pfc - ne depletion , c57bl/6 mice exhibited impoverished and more rigid social behavior and were 6-fold more aggressive than sham - lesioned animals , whereas 2/ mice showed unimpaired social behavior . our biochemical measures suggest a critical involvement of da in sit . in addition , we show that the balance between basal levels of monoamines and of ach modulates aggressiveness and this modulation requires functional 2*nachrs . these findings demonstrate the critical interplay between prefrontal ne and nachrs for the development of adapted and nonaggressive social cognition.coura , r. s. , cressant , a. , xia , j. , de chaumont , f. , olivo - marin , j. c. , pelloux , y. , dalley , j. w. , granon , s. nonaggressive and adapted social cognition is controlled by the interplay between noradrenergic and nicotinic receptor mechanisms in the prefrontal cortex .

MATERIALS AND METHODS RESULTS DISCUSSION

male c57bl/6j mice and knockouts for the 2-subunit of nicotinic receptors ( 2 ) , aged 78 wk on the arrival were used in this study . the 2 mice were originally generated from a 129/sv es cell line as described previously ( 36 ) and backcrossed onto the c57bl/6j strain for 20 generations , which is twice the number of backcrosses suggested by the bandburry conference guidelines ( 37 ) . for these reasons , and because littermates are not available in the breeding facilities , we used c57bl/6j mice as controls . groups consisting of 812 mice were formed for the 3 experimental procedures : global ne depletion and behavior ( experiment 1 ) ; ex vivo assessment of brain monoamine levels ( experiment 2 ) ; and prl ne depletion and behavior ( experiment 3 ) . each experimental procedure consisted of 4 groups : sham - treated ( c57bl/6 and 2 ) and lesioned ( c57bl/6 and 2 ) mice . however , a small number of animals were not included in the final analysis due to the presence of gliosis ( 10 mice ) or due to outliers ( 4 mice ) , as confirmed by the dixon test ( 38 ) . control groups used for behavioral experiments 1 ( 10 c57bl/6 and 7 2 mice ) and 3 ( 15 c57bl/6 and 14 2 mice ) were pooled since there were no statistical differences between them ( anova main group effect , p=0.66 , ns ) . at 8 d before the behavioral experiment , adult male c57bl/6 mice and 2 mice were injected intraperitoneallly with dsp-4 ( 50 mg / kg ) or vehicle ( sham - treatment group ) . at 8 d after surgery , male mice were tested in the social interaction paradigm to evaluate the effects of local or global ne depletion on social behavior . , social contact duration and follow behavior ( 24 , 33 , 43 ) , were first manually measured and compared between c57bl/6 and 2 mice , as well as between lesioned and sham - treated mice . in addition , animals may not stop completely ( speed < 1.75 cm / s ) but can scan the environment , rear , sniff , and make head movements ( 47 ) . therefore , as stop behaviors have been shown to be important choice points in various animal models during novelty exploration and locomotion , we included them in the repertoire we defined in our social interaction task that allows large movements and novelty seeking ( 33 ) . here , like in our previous studies ( 51 , 52 ) , we defined the index of social dominance as the combination of two types of behaviors : paw control and aggressive grooming . a video camera fixed on the ceiling of the experimental room right above the central platform allowed the experimenter to record and track offline the mouse position within the maze . cresyl violet staining was used to verify the most ventral position of injectors in the pfc as well as the integrity of the adjacent tissue . in experiment 2 , we determined and compared constitutive brain levels of monoamines and ach in c57bl/6 and 2 mice . frozen sections were dissected using a punch from 6 regions of interest ( 40 ) : rostral prelimbic cortex ( rprl ) , caudal prelimbic cortex ( cprl ) , orbitofrontal cortex ( ofc ) , hippocampus ( h ) , nucleus accumbens ( acb ) , and amygdala ( a ) . levels of ne , da , and 5-ht and their metabolites ( dopac , 5hiaa , and mhpg ) were determined in the supernatant by reversed - phase , high - performance liquid chromatography ( hplc ) , as described previously ( 53 ) . the analysis of ach was also performed by hplc , with the mobile phase containing 75 mm na2hpo4 and 5 l / ml proclin reagent ( bas , congleton , uk ) , ph 8.0 ( adjusted with 46/48% naoh ) , and a flow rate of 120 l / min ( 582 solvent delivery module ; esa inc . to verify possible differences in the balance between these neurotransmitters after global noradrenergic lesion , 12 c57bl/6 and 12 2 mice were pretreated with the selective noradrenergic neurotoxin dsp-4 ( 50 mg / kg i.p . ) these animals were assessed postmortem for brain tissue levels of monoamines and ach , as described above . the main effects of genotype ( 2 levels : c57bl/6 and 2 ) and of lesion ( 2 levels : sham treatment and global ne lesion , or ne prl depletion ) , as well as statistical interactions between these factors , were assessed with anova and statview 4.57 ( sas institute , cary , nc , usa ) . as flexible social behaviors are strongly modulated by ne and ach systems , we first investigated the effect of global ne depletion on social behavior in c57bl/6 and 2 mice . for that purpose , we used a social interaction task in which a previously isolated male mouse was confronted with a novel male c57bl/6 mouse in a novel , large environment . in agreement with our previous results ( 23 , 24 , 33 , 43 ) , c57bl/6 mice spent less time in social contact than 2 mice ( p<0.0001 ) and made fewer follow behavioral sequences ( p<0.0001 ; fig . effects of global ne lesion by injection of dsp-4 on social interaction and on social aggressive - like behavior in c57bl/6 mice ( sham treated n=25 , lesioned n=10 ) and 2 mice ( sham treated n=21 ; lesioned n=8 ) . a ) contact duration , showing the total time spent in social contact , which increases for c57bl/6 mice and decreases for 2 mice , after the lesion . dsp-4 lesions produced a decrease of this behavior in 2 mice but had no effect in c57bl/6 mice . d ) lack of effect of dsp-4 on aggressiveness ( circling , bite , and tail rattling ) in c57bl/6 and 2 mice . specifically , the total time spent in social contact and the total number of follow sequences were significantly affected by the lesion ( p<0.0001 and p=0.001 , respectively ) but differently according to genotype : in c57bl/6 mice , global ne depletion led to a 40% increase in the duration of social contact ( p=0.003 ) . by contrast , in 2 mice , ne depletion led to a 33% decrease in the duration of social contact ( p=0.0007 ) and a 43% decrease in the number of follow sequences ( p=0.0002 ; fig . 2 mice exhibited higher dominance scores compared with c57bl/6 mice ( p=0.02 ) but a similar low level of aggressiveness ( p=0.3317 ) . global ne depletion increased dominance behavior only in c57bl/6 mice ( p<0.005 ) and had no significant effect on aggressive behavior in any group of mice ( fig . we thus measured basal levels of da , ne , 5-ht , and ach , as well as their metabolites , in both groups of mice and tested the hypothesis that the opposite behavioral effects after global ne depletion could be explained by constitutive biochemical differences . basal levels of ne , da , 5-ht , and ach were increased in 2 mice compared with c57bl/6 mice in the pfc ( p=0.0014 , p=0.0075 , p=0.0022 , and p<0.0001 , respectively ; fig . moreover , in the ofc and a , basal levels of ne and 5-ht were also increased . overall , the 5-ht activity was significantly increased in 2 mice ( p=0.02 ) , while the activity of ne and da remained unchanged , as we could assess by measuring their respective metabolites ( p=0.9866 and p=0.0991 , respectively ) . levels of monoamines and ach in the prl of c57bl/6 ( n=12 ) and 2 mice ( n=12 ) . tissue content of all monoamines and ach increased in 2 mice compared with c57bl/6 mice . neurochemical analysis revealed that basal levels of monoamines and ach were higher in 2 mice . to elucidate whether this effect was associated with an increase in monoaminergic fiber density , we measured the density of th , 5-ht , and net fibers in the pfc of c57bl/6 and 2 mice . therefore , increased basal levels of monoamines in the prl was unlikely to be due to an increase in the density of monoaminergic fibers in this region . by contrast , 5-ht fiber density was reduced in c57bl/6 mice after ne depletion ( p=0.001 ) . neurochemical and neuroanatomical analyses therefore indicated that 2 mice exhibited increased basal levels of monoamines , although the density of monoaminergic innervations was decreased . ne depletion of the prl had no significant effect on anxiety - related behavior in the dark - light paradigm in c57bl/6 or 2 genotypes . however , in the epm , the percentage of time spent in open arms was altered differently in c57bl/6 and in 2 mice by pfc ne depletion ( genotype x treatment f(1,43)=6.89 , p=0.012 ) . post hoc t tests revealed that ne depletion of the pfc led to a significant anxiolytic effect in 2 mice ( sham treatment : 19 2% ; ne depletion : 28 2.76% ; t=2.7 , df=20 , p=0.013 ) but had no effect in c57bl/6 mice ( t=0.88 , df=23 , p=0.39 ) . in previous experiments , we showed that the profound alteration in social interaction exhibited by 2 mice was similar to that obtained after damage to the prl of the pfc . notably , we showed that expression of functional 2*nachrs in the prl of 2 mice was necessary and sufficient for restoring flexible social interaction ( 24 ) . here , we observed that global ne depletion produced opposite effects in social behavior of 2 and c57bl/6 mice . to further investigate the putative role of the ne transmission in the prl , we carried out specific prl ne depletion and tested its effect in the same social paradigm . by contrast , in c57bl/6 mice , the index of aggressiveness increased dramatically by 6-fold following the selective depletion of ne from the prl ( p=0.0002 ; fig . this was unlikely to be due to anxiogenic effects , as anxiety was not altered by ne prl depletion in c57bl/6 mice ( see above ) . effects of ne prl depletion on dominance and aggressive - like behavior in c57bl/6 ( sham treated n=25 ; lesioned n=10 ) and 2 mice ( sham treated n=21 ; lesioned n=8 ) . after lesion , there was a significant increase in aggressiveness in c57bl/6 mice but not in 2 mice . to investigate whether these effects were due to fine changes in the social repertoire , we assessed the key behavioral sequences of the social repertoire that were specifically altered by ne depletion . 4 ) and a 36% decrease in the number of follow behaviors ( p=0.007 ; supplemental fig . global effects of ne prl depletion on social interaction in c57bl/6 ( sham treated n=25 ; lesioned n=10 ) and 2 mice ( sham treated n=21 ; lesioned n=8 ) . while the duration of social contact was not altered in c57bl/6 mice after ne prl depletion , the global organization of behavior and the richness of repertoire were drastically modified ( fig . transitional graphs showed an increased proportion of unchanged behavioral sequences ( black arrows ) in 2 mice as compared with c57bl/6 mice . after ne - prl depletion , behaviors conserved with time ( black arrows ) were increased in c57bl/6 mice and decreased in 2 mice . transitional behavioral graphs after prefrontal ne depletion in c57bl/6 ( sham treated n=25 ; lesioned n=10 ) and 2 mice ( sham treated n=21 ; lesioned n=8 ) . transitional graphs showed an impoverishment of the social repertoire in c57bl/6 mice after ne prefrontal depletion , with less various behavioral events than in sham - treated mice . temporal evolution of behavioral sequences , illustrated by the number of red and green arrows , by comparison with events linked by black arrows , was also drastically lower in c57bl/6 mice after ne prefrontal depletion . therefore , c57bl/6 mice exhibited more rigid social behavior after ne prefrontal depletion . stop behaviors , which were significantly less frequent in 2 mice than in c57bl/6 mice , were restored after ne lesion . within the behavioral repertoire , we pinpointed two important markers of social flexibility ( 33 ) : stop behaviors , which constitutes behavioral choice points . these results show that specific ne prl depletion normalized flexible social behavior in 2 mice by restoring social contact duration , follow sequences , immobility periods and back - to - back postures . by contrast , stop behaviors and back - to - back sequences , like contact duration , were not affected by ne depletion in c57bl/6 mice ( depletion effect in c57bl/6 mice all p>0.4 , ns ) . detailed effects of ne prl depletion on key elements of social interaction in c57bl/6 ( sham treated n=25 , lesioned n=10 ) and 2 mice ( sham treated n=21 , lesioned n=8 ) . ne depletion of the pfc restored both immobility ( a ) and back - to - back ( b ) sequences in 2 mice , both behaviors drastically impaired before lesion . furthermore , dominance and aggressiveness were shown to be functionally dissociated , with the former being modulated by the cholinergic nicotinic system and the latter potentiated by prefrontal ne but only in presence of functional 2*nachrs . the social paradigm used in the present study requires flexible decision - making processes that depend on the functional integrity of the pfc and 2*nachrs in this region ( 24 ) . here we demonstrated that global depletion of ne in the brain produced strongly divergent effects on social behavior in c57bl/6 and 2 mice . while it caused rigid social behavior in c57bl/6 mice , it restored behavioral flexibility in 2 mice . it has been shown that prefrontal ne is necessary for attentional processing , especially when the task is novel and challenging , and stimuli are unpredictable ( 25 , 5860 ) . according to the hypothesis that ne modulates the salience of external stimuli ( 58 , 6062 ) and that an optimal level of ne activity is necessary to optimize attention and decision - making performance ( 31 ) , our results show that 2 mice were more susceptible than c57bl/6 control mice to external stimuli during a social encounter . this susceptibility may be driven by disrupted ne transmission in the prl and by the salience of social stimuli being augmented by prior social isolation ( 24 ) . the prosocial behavior observed in 2 mice is likely due to a hypersalience of external stimuli , which in the social context is mainly generated by the congener . the loci of this effect appears to be the pfc and functional nachrs , as specific ne pfc depletion normalized major social flexible behaviors ( e.g. in addition , prl - ne depletion restored back - to - back sequences that were virtually absent in these mice . moreover , transitional graphs unraveled an impoverishment of the social repertoire in c57bl/6 mice after ne prefrontal depletion , with less various behavioral events than in sham - treated mice . therefore , c57bl/6 mice exhibited more rigid social behavior after ne prefrontal depletion . we have previously shown that 2 mice are hyperactive ( 63 ) and exhibit increased c - fos expression in the pfc , specifically when exposed to a novel environment ( 64 ) . these findings indicate an increased arousal / vigilance state when mice are confronted with highly arousing stimuli , such as a novel social interaction ( 23 ) , shown to trigger pfc activation ( 24 ) . this increased arousal / vigilance state can be , in part , explained by the high basal levels of monoamines and ach , observed especially in the pfc and that is likely to be due to an increased storage of these neurotransmitters . in a previous work , we showed that whole - brain ne depletion increased global aggressiveness in c57bl/6 mice ( 52 ) . here , distinguishing aggressive and dominance behaviors , we found that global ne depletion affected neither aggressiveness nor dominance . thus , our data show that a lack of prefrontal ne transmission promotes aggressive - like behaviors . 2 mice exhibited more dominant behavior than c57bl/6 mice , which was not perturbed by ne - prl depletion , suggesting therefore that the dominant behavior we observed was related to the absence of 2*nachrs . by contrast , the balance between basal levels of monoamines and of ach appears to modulate aggressiveness , and this modulation requires functional 2*nachrs . in 2 mice , which exhibited constitutively increased levels of monoamines and ach in the pfc , ne depletion did not favor aggressiveness . by contrast , in c57bl/6 mice , ne - depletion led to a profound increase in aggressive - like behavior . our biochemical analysis showed that , in c57bl/6 mice , an increase in da levels after global ne depletion is sufficient to cause the 2-behavioral phenotype ( see fig . after ne global depletion in c57bl/6 mice , da levels increased in the pfc ( data not shown ) , suggesting an involvement of da in prosocial behaviors . this may be relevant to an analogous behavioral profile in da transporter - knockout ( dat ) mice , which shows increased levels of da , hyperactivity , impaired decision - making , and behavioral rigidity that includes an inability to shift and adjust behaviors in a new context ( 65 , 66 ) . our current results may also be relevant to understanding how cholinergic and monoaminergic systems interact in the pfc to modulate social cognition in schizophrenia . one of the proposed contributions of the cholinergic system to the pathophysiology of schizophrenia is through an imbalance between the cholinergic and dopaminergic systems ( 68 ) . recent data suggest that cholinergic perturbations in schizophrenia are not simply due to changes in ach levels but rather to the balance between activation of both nicotinic and muscarinic receptors , which collectively determine the functional outcome of central cholinergic stimulation ( 67 , 69 ) . hyperdopaminergia was also observed in c57bl/6 mice after ne depletion and is likely to be sufficient to cause the 2-prosocial phenotype in these mice . by contrast , in the absence of 2*nachrs , there was no change in the basal level of da or 5-ht after ne depletion , which corroborates the role of these receptors in modulating da or 5-ht release . in addition , our results suggest that 2*nachrs may contribute to the pathophysiology of schizophrenia through complex and intricate underlying interactions between cholinergic , dopaminergic and noradrenergic systems in the pfc . in particular , our findings indicate the ne innervation of the pfc has a pivotal role in inhibitory response control and adaptive behavior , as well as in the modulation of aggression , which depend on functional interactions with the cholinergic system in this region .

diesel exhaust , the dominant pollutant in ambient air , has been classified as a potential or probable human carcinogen by the international agency for research in cancer studies have found that diesel exhaust particles ( deps ) are associated with various health effects such as inflammation of the respiratory tract , lung cancer and cardiovascular diseases , and their extracts , diesel particulate extract ( dpe ) , are thought to be mainly responsible for these malignant effects . dpe is a complex mixture composed of hundreds of organic chemical compounds including polycyclic aromatic hydrocarbons ( pahs ) , quinines , ketones , heterocyclic compounds , aldehydes , and other unidentified constituents , many of which are promutagens that require subsequent activation by biotic and abiotic factors to show their mutagenic or carcinogenic effects . for instance , the organic dep extract and oxidized phospholipids synergistically affected the expression profile of several genes involved in pathways relevant to vascular inflammatory processes . deps and bacterial lipopolysaccharides were reported to synergistically induce the generation of free radicals and neutrophilic inflammation in the lungs of rats . the methtylation of t helper genes and ige production were changed when mice were exposed to deps in combination with an allergen . hamster hybrid system , the cytotoxicity and genotoxicity of dpe at a low dose ( 20 g / ml ) could be activated by environmental physical factor ultraviolet a ( uva ) radiation ( 0.5 j / cm ) . ultraviolet ( uv ) radiation ( uv - a , 320400 nm ; uv - b , 280320 nm ; uv - c , < 290 nm ) is the carcinogenic component of sunlight , and 95% of uv reaching the surface of earth is uva . relative to the high carcinogenicity of uvb , uva is usually considered to be less carcinogenic due to the weak absorption of uva by dna molecules . however , recent evidence showed that uva also caused various forms of dna damage , such as cyclobutane pyrimidine dimers , single strand breaks , and dna furthermore , it was reported that uva - induced dna damage can be enhanced in the presence of either endogenous or exogenous photosensitizers , such as the diuretic agent hydrochlorothiazide and lomefloxacin . although the exposure to either diesel exhaust or uva radiation alone or in combination with other agents has been identified as an essential risk factor for various benign or malignant human diseases , the synergistic effects of diesel exhaust and uva remain to be clarified , especially in an in vivo system . caenorhabditis elegans ( c. elegans ) , a free - living nematode , is a simple multicellular eukaryote . because of its short life cycle , small size of body , transparent body , and easy of cultivation in a laboratory , c. elegans has been adopted as an excellent model in vivo for toxicological tests and environmental evaluation . importantly , c. elegans shares cellular and molecular structures and signaling pathways with higher organisms ; thus , biological information learned from c. elegans may be directly applicable to more complex organisms . moreover , genetically deficient strains of c. elegans are easily available , which facilitates further genetic dissection for the molecular mechanisms underlying the related biological events . within c. elegans , the germ line is an intrinsic part of oogenesis , which establishes an unbroken chain between generations . abnormal germ line development , such as the induction of germ line apoptosis , would not only harm the organism but also disturb the species balance from generation to generation . normally , germ cell apoptosis occurs physiologically under normal conditions . however , upon environmental stresses germ cell apoptosis was also induced sensitively through the signaling pathways that are distinct genetically from physiological apoptosis . it was reported that genotoxic insults ( such as ionizing radiation , uv radiation , mutagens , oxidative stresses , heat , and salt etc . ) induced germ line apoptosis likewise employed core apoptotic components but was dependent on the dna damage checkpoint hus-1 and regulator cep-1 . in the present study , with the level of germ cell apoptosis as a main checking end point , our results showed that the coexposure of l4-stage or young adult worms to dpe plus uva at low doses significantly enhanced the induction of germ cell apoptosis . the induction of germ cell apoptosis by dpe plus uva might be triggered by dna damage and involve erk , jnk , and p38/mapk signaling pathways . in addition , the mutant strains ced-3(n717 ) and ced-4(n1162 ) were used for determining the nature of germ cell death . strains with single - gene mutations of dna damage - induced germ cell death machinery , cep-1(w40 ) , cep-1(lg12501 ) , and hus-1(op241 ) , were employed for investigating the signaling pathways involved in the induction of germ cell death by dpe and/or uva . a worm line transgenic for hus-1::gfp , ws1433 : hus-1(op241 ) i ; unc-119(ed3)iii ; opis34 , was used for detecting the dna damage in germ cells . moreover , the strains deficient in the extracellular signaling - regulated protein kinases ( erk ) signaling cascade , lin-45(ku51 ) , mek-2 ( n1989 ) , and mpk-1 ( ku1 ) ; jun n - terminal kinases ( jnk ) signaling cascade , mek-1 ( ks54 ) , jnk-1 ( gk7 ) , and mkk-4 ( ju91 ) ; and p38 mapk signaling cascade , nsy-1 ( ag3 ) , sek-1 ( ag1 ) , and pmk-1 ( km25 ) , were also adopted . maintenance and genetic manipulation of c. elegans were carried out according to the standard procedures as described by brenner . all strains were grown at 20 c on nematode growth medium ( ngm ) and fed with the bacterium escherichia coli op50 . to obtain synchronized cultures , gravid hermaphrodites were lysed in an alkaline hypochlorite solution . in the present study , dpe ( standard reference material 1975 ) was provided by the national institute of standards and technology ( nist ; gaithersburg , md , usa ) . srm 1975 is a dichloromethane extract of the diesel particulate matter srm 2975 , which was generated by a forklift truck using an industrial diesel - powered engine and collected under specifically designed heavy - duty conditions ( nist 2000 ) . briefly , dpe was diluted to final concentrations in k - medium ( containing 52 mm nacl and 32 mm kcl ) . for the measurement of apoptosis , the mitotic germ cells , the brood size , the foci of hus-1::gfp , and the production of ros , age - synchronized young hermaphrodites were transferred into 30 mm - diameter petri dishes containing k - medium with op50 as a food source and treated with either dpe ( 20400 g / ml ) or uva ( 0.25.0 j / cm ) alone or in combination ( dpe + uva ) for determined times at 20 . for the measurement of body size , the life span , and the percentage of adult worms , the hatched l1-stage larvae were employed to investigate the possible developmental effects of dpe plus uva . in the dpe plus uva groups , worms were pretreated with 20 g / ml dpe for 1 h and then irradiated with a determined dose of uva . for uva radiation , three uv lamps ( ble - it151 , spectronics co. , westbury , new york , usa ) with an emission wavelength peak at 365 nm were used . the dishes were placed on a table that was 15 cm away from the uv lamps . during uv exposure , the dose rate was simultaneously measured by a radiometer ( photoelectric instrument factory of beijing normal university , beijing , china ) with a 365 nm detector located the same distance as the culture plates from the uv source . germ cell corpses were measured by acridine orange ( ao , sigma ) staining using a modified procedure developed by kelly et al . briefly , the treated worms were stained for 1 h in the dark at 20 c by transferring worms into a costar 24-well plate containing 500 l of 25 g / ml ao and op50 in m9 buffer ( 3 g of kh2po4 , 6 g of na2hpo4 , 5 g of nacl , 1 ml of 1 m mgso4 , and h2o to 1 l ) and then transferred to ngm and allowed to recover for 40 min on bacterial lawns also in the dark . ao staining positive cell corpses were assessed under an olympus ix71 fluorescence microscope ( olympus , tokyo , japan ) . the apoptotic cells appeared yellow or yellow - orange , representing increased dna fragmentation , while intact cells were uniformly green in color . the procedures used to assess mitotic germ cells were developed by craig et al . to clearly assess the mitotic germ cells , the dissected gonads were stained by 1 g / ml 4,6-diamidino-2-phenylindole ( dapi ) for 10 min in the dark , rinsed 3 times for 5 min in pbst ( pbs and 0.1% tween-20 ) , mounted in mounting solution ( 90% glycerol , 20 mm tris at ph 8.0 , and 1 mg / ml p - phenylenediamine ) , and then covered with a coverslip . the mitotic germ cells within 20-cell distance from the distal tip cell the procedures for brood size assay were conducted as described by craig et al . synchronized young adult hermaphrodites were treated with either dpe ( 20 g / ml ) or uva ( 0.2 , 0.5 , and 1.0 j / cm ) alone or in combination ( dpe + uva ) for 24 h. worms were then transferred individually onto a ngm plate containing a bacterial lawn 1 cm in diameter in the center of the dish . the adult worms were removed onto a fresh ngm plate daily or every other day , and the number of eggs and hatched f1 larvae were counted under a dissection microscope . the brood size was calculated by combining the number of embryos and hatched larvae . worms were photographed under a stereomicroscope equipped with a ccd camera at the time point of 72 h after l1-stage larvae were treated with either dpe ( 20 g / ml ) or uva ( 0.2 , 0.5 , and 1.0 j / cm ) alone or in combination ( dpe + uva ) . the body size was determined by measuring the flat surface area of the worms using imagej software . the life cycle was assayed by counting the percentage of adult worms in each treatment . l1-stage larvae were treated with either dpe ( 20 g / ml ) or uva ( 0.2 , 0.5 , and 1.0 j / cm ) alone or in combination ( dpe + uva ) throughout their life . in the experiment , worms were cultured individually in 96-well plates using op50 as food at 20 c . when the hermaphrodites developed to the gravid stage , they were transferred to fresh plates every other day to avoid confusing them with their progenies . worms were checked every day and would be scored as dead when they would not respond to tapping with a pick . dna damage in the c. elegans germ line was assessed with the strain hus-1::gfp as described previously . synchronized young adult hermaphrodites were treated with either dpe ( 20 g / ml ) or uva ( 0.5 j / cm ) alone or in combination ( dpe + uva ) for 24 h. worms were then mounted onto microscope slides in 0.2 mm of levamisole ( sigma ) , and foci were counted in a single z stack under a laser confocal microscope ( lsm710 zeiss , germany ) , where about 40 mitotic germ cells in c. elegans were observed . age - synchronized young hermaphrodites were treated with 0.5% and 1.0% dimethyl sulfoxide ( dmso ) or 10 m and 100 m sodium azide ( nan3 ) with or without concurrent treatment with dpe ( 20 g / ml ) for 1 h and then irradiated with uva ( 0.5 j / cm ) . then germ cell apoptosis was counted as described above . the dose of dmso and nan3 in the present study was nontoxic and nonmutagenic . the level of ros in c. elegans was measured with 2,7-dichlorodihydrofluorescein diacetate ( dcf - da ) , which is a general molecular probe that is used as an indicator of global ros flux in intact animals . after treatment , the worms were transferred into the wells of a costar 24-well microtiter plate ( black , clear , and flat - bottom wells ) containing dcf - da ( final concentration of 10 m in pbs ) and incubated for 30 min in the dark at 20 c . the relative fluorescence for worms was individually determined and analyzed using an olympus ix71 fluorescence microscope with a ccd camera and image - pro plus , version 6.0 . to detect o2 , we used the trap probe 2,2,6,6-tetramethyl-4-piperidone hydrochloride ( temp ; purity of 95% ) . the probe , which has been shown to be specific for o2 detection , reacts with o2 to yield a stable nitroxide radical 4-oxo-2,2,6,6-tetramethyl - piperidine - n - oxyl ( 4-o - tempo ) , having a known three - line esr spectrum . age - synchronized young adult hermaphrodites were treated with dpe ( 20 g / ml ) for 1 h at 20 c , and then temp ( sigma ; 0.05 m ) or the stable radical 2,2,6,6-tetramethylpiperidine - n - oxyl ( tempo ; 10 m ; sigma ) was added 30 min before uva radiation . the treated worms were collected immediately and transferred into 25 l capillaries after radiation . to eliminate the interference of o2 generation in the culture medium , the remaining medium in capillaries samples in 25 l capillaries inserted into 4 mm quartz tubes were used for esr analysis . esr spectra were recorded at room temperature on a emx-10/12 esr spectrometer ( bruker , german ) . we set the microwave source of the esr at 9.0 ghz and the power at 3.0 mw . the time constant was 0.3 s , and scan time was 120 s. the relative signal intensity of 4-o - tempo is represented by dividing the ratio of the 4-o - tempo signal intensity of the treated group by that of the control group . significant differences at the p < 0.05 level were tested using anova followed by tukey s multiple comparison test . for comparisons between different strains , dpe or uva has been reported to exhibit significant genotoxicity and cytotoxicity in several cell models . in this study , the genotoxicity of dpe or uva was assessed with germ cell death as an end point . as shown in figure 1 , following treatment with dpe ranging from 20 to 50 g / ml , germ cell death exhibited a basal level compared to that of the control populations ( in all cases , p > 0.05 ) , whereas the higher doses of dpe led to significant increases in the level of germ cell death in a dose - dependent manner ( in all cases , p < 0.05 ) . similarly , exposure to uva at low doses made no difference in germ cell death ( in all cases , p > 0.05 ) , and significant increases were observed when the exposure doses exceeded 2.5 j / cm ( in both cases , p < 0.05 ) . the results agreed with our previous reports that the treatments with dpe at 20 g / ml or uva less than 1.0 j / cm caused little toxic and mutagenic effects in the cell culture system . synchronized young adult hermaphrodites were exposed to the indicated doses of dpe ( a ) or uva ( b ) , and germ cell corpses were scored 24 h after exposure . all values are presented as the means se ; n 40 , and * represents p < 0.05 . to further clarify whether there are synergistic effects on the induction of germ cell death by low - doses of dpe plus uva , worms at young adulthood were exposed to low doses of dpe plus uva and then checked for the induction of germ cell death 24 h after cotreatment . as shown in figure 2a , the application of 20 g / ml dpe + 0.5 j / cm uva and 20 g / ml dpe + 1.0 j / cm uva both led to a significantly enhanced induction of germ cell death ( in both cases , p < 0.05 ) . synchronized young adult hermaphrodites were treated with dpe and/or uva , and germ cell death ( a ) and mitotic germ cells ( b ) were scored 24 h after exposure . all values are presented as the means se ; n 20 , and * represents p < 0.05 . moreover , to avoid the interference of germ cell proliferation by dpe plus uva on germ cell death , the numbers of mitotic germ cells were examined in the distal germ line . as shown in figure 2b , there was no significant changes in the number of mitotic germ cells in the groups of 20 g / ml dpe + 0.2 j / cm uva , 20 g / ml dpe + 0.5 j / cm uva , and 20 g / ml dpe + 1.0 j / cm uva compared with that in the untreated worms ( in all cases , p > 0.05 ) . the results suggested that the enhanced germ cell death induced by dpe plus uva was not due to the reduction of mitotic germ cell proliferation . in c. elegans , the spatial and temporal organization of the germ line allows one to investigate damage effects in various meiotic progressions through a reverse time course analysis . as shown in figure 3a , compared to the control or single - treated populations , the worms coexposed to 20 g / ml dpe + 0.2 j / cm uva exhibited slight increases in germ cell death at the time points of 6 and 12 h ( in both cases , p < 0.05 ) and recovered to the basal level at the time points of 24 and 36 h ( in both cases , p > 0.05 ) . however , for the groups of 20 g / ml dpe + 0.5 j / cm uva and 20 g / ml dpe + 1.0 j / cm uva , the worms both exhibited significant increases in germ cell death at all of the tested time points ( in all cases , p < 0.05 ) , and the largest induction of germ cell death occurred at the time point of 24 h. time course of germ cell death in c. elegans induced by 20 g / ml dpe + 0.2 j / cm uva ( a ) , 20 g / ml dpe + 0.5 j / cm uva ( b ) , and 20 g / ml dpe + 1.0 j / cm uva ( c ) . synchronized young adult hermaphrodites were exposed to dpe , uva , or dpe + uva , and germ cell corpses were scored at time points of 6 , 12 , 24 , and 36 h , respectively . all values are presented as the means se ; n 20 , and * represents p < 0.05 . to further clarify the nature of germ cell death induced after coexposure to dpe plus uva , c. elegans strains with single - gene mutations of the ced-3(n717 ) and ced-4(n1162 ) genes were employed . ced-3 and ced-4 are two critical components of the core apoptotic pathway within c. elegans . as shown in figure 4 , the synergistic induction of germ cell death was significantly inhibited in both ced-3(n717 ) and ced-4(n1162 ) mutant strains ( in both cases , p > 0.05 ) , suggesting that the germ cell death induced by coexposure to dpe plus uva might be apoptotic death in nature . the muations of the ced-3 and ced-4 genes significantly inhibited the induction of germ cell death by exposure to dpe plus uva . all values are presented as the means se ; n 40 , and * represents p < 0.05 . environmental stresses could modify the developmental processes when the larvae were exposed to toxicants either in embryonic development or early developmental stages . in c. elegans , germ cell apoptosis commences in early adulthood and increases over time . to exclude the changes of background value , we investigated the developmental effects by dpe plus uva at different stages . as shown in figure 2b and figure 5a , worms coexposed to dpe plus uva at the l4 stage had little effect on the index of mitotic germ cells and brood size . in addition , the body size and the life span of worms exposed to dpe plus uva at the l1 stage were not changed obviously as well ( figure 5b and c ) . however , there was a slight decrease in the percentage of adult worms compared to that in the single treatment of dpe or uva , or to the control ( in all cases , p > 0.05 ) when worms were coexposed to dpe plus uva at the l1 stage ( figure 5d ) . the results indicated that the enhanced levels of germ cell apoptosis after coexposure to dpe plus uva at the late stage did not result from the modification of the developmental procedure . ( a ) age - synchronized young hermaphrodites were treated with either dpe ( 20 g / ml ) or uva ( 0.21.0 j / cm ) alone or in combination ( dpe + uva ) for 24 h at 20 c , then the brood size was counted . ( b ) the body sizes were determined by measuring the flat surface area of the worms using imagej software , and there was no difference among all treatments after l1-stage larvae were treated with dpe and/or uva for 72 h. ( c ) life span curves of worms and ( d ) the percentage of adult worms were scored after l1-stage larvae were treated with dpe and/or uva for 72 h. data were pooled from three independent experiments . all values are presented as the means se ; n 20 , and * represents p < 0.05 . the classic dna damage - induced germ cell death machinery has been reported to be involved in the induction of apoptosis in addition to physiological germ cell apoptosis in c. elegans . to clarify whether c. elegans employed this death machinery for the induction of germ cell apoptosis after coexposure to dpe plus uva , worm strains with single - gene loss - of - function mutations of this death machinery , cep-1(w40 ) , cep-1(lg12501 ) , and hus-1(op241 ) , were used . as shown in figure 6a , in the worms with null mutations of the hus-1 and cep-1 genes , the induction of germ cell death was significantly inhibited after coexposure to dpe ( 20 g / ml ) plus uva ( 0.5 j / cm ) ( in all cases , p > 0.05 ) , while the wild type and the strain with partial loss - of - function of the cep-1 gene showed a significant induction of germ cell apoptosis . role of dna damage in the induction of germ cell apoptosis by exposure to dpe , uva , or dpe + uva . ( a ) there was significantly enhanced induction of germ cell apoptosis in the partial loss - of - function strain of cep-1(w40 ) , while the null mutation strains of hus-1(op241 ) and cep-1(lg12501 ) significantly inhibited the induction of germ cell apoptosis by dpe plus uva . ( b ) quantification of hus-1::gfp foci in the mitotic germ cells after worms were treated with dpe plus uva for 24 h. foci were scored in 40 proliferating germ cells . distinct foci of hus-1::gfp could be observed in a small number of the mitotic germ cells in c. elegans coexposed to dpe plus uva at the time point of 24 h. the scale bar represents 5 m . these results suggested that the classic dna damage - induced germ cell death machinery might be employed in germ cell apoptosis induced by dpe plus uva . all values are presented as the means se ; n 40 , and * represents p < 0.05 . to further determine the role of dna damage in the induction of germ cell apoptosis by dpe plus uva , the worms transgenic for hus-1::gfp were employed . in the c. elegans germ line , hus-1::gfp diffuses in proliferating germ nuclei , which relocalize and form distinct foci following dna damage . as shown in figure 6b , distinct foci of hus-1::gfp could be observed in a small number of mitotic germ cells at the time point of 24 h after worms were coexposed to dpe ( 20 g / ml ) plus uva ( 0.5 j / cm ) but nearly none in the single treatment of dpe or uva , or in the control worms . these results indicated that the dna - damage - induced germ cell death machinery played a pivotal role in the synergistic induction of germ cell apoptosis by dpe plus uva . it has been shown that the p53 protein can functionally interact with the mitogen - activated protein kinases ( mapks ) . once map kinases are activated , they function as effectors to phosphorylate and activate p53 , leading to a p53-mediated cellular response , including apoptosis . to explore the possible role of mapk signaling pathways in the induction of germ cell apoptosis of dpe plus uva , the strains with the loss - of - function of genes related to mapk pathways were used . the mapk signaling pathways mainly include erk , jnk , and p38 mapk cascades in c. elegans . in c. elegans , lin-45 ( mapkkk ) , mek-2 ( mapkk ) , and mpk-1 ( mapk ) are the components of the erk signaling pathway . as shown in figure 7a , the worm strains with loss - of - function of the lin-45(ku51 ) , mek-2 ( n1989 ) , and mpk-1 ( ku1 ) genes exhibited a basal level of germ cell apoptosis after coexposure to dpe plus uva compared to that of their respective controls ( in all cases , p > 0.05 ) . jkk-1 and mek-1 are members of mapk kinase ( mapkk ) , and jnk-1 is a member of the jnk homologue . in our experiments , the loss - of - function of these genes significantly inhibited the induction of germ cell apoptosis by coexposure to dpe plus uva ( in all cases , p > 0.05 ) , as shown in figure 7b . in the p38 mapk pathway of c. elegans , nsy-1 encodes a mapk kinase kinase ( mapkkk ) , sek-1 is a member of mapkk , and pmk-1 is the p38 mapk homologue . in the present study , the strains with single - gene loss - of - function mutations of the nsy-1 ( ag3 ) , sek-1 ( ag1 ) , and pmk-1 ( km25 ) genes were coexposed to dpe plus uva , respectively , and no significant induction of germ cell apoptosis was observed in all of the mutant strains ( in all cases , p > 0.05 ) , as shown in figure 7c . the results suggested that mapk signal pathways , including erk , jnk , and p38/mapk , might play a pivotal role in the induction of germ cell apoptosis by coexposure to dpe plus uva . induction of germ cell apoptosis by exposure to dpe , uva , or dpe + uva in worms deficient in erk ( a ) , jnk ( b ) , and p38/mapk ( c ) signaling pathways . germ cell apoptosis was significantly inhibited in all of the mutant strains after exposure to dpe plus uva , suggesting that the mapk signaling pathways play a pivotal role in germ cell apoptosis induced by dpe plus uva . all values are presented as the means se ; n 20 , and * represents p < 0.05 . ros was reported to activate the mitogen - activated protein kinases , and played an important role in the induction of dna damage . to find out the role of ros in the induction of germ cell apoptosis by dpe plus uva , the ros quenchers , nan3 and dmso , were employed . as shown in figure 8a , the induction of germ cell apoptosis by coexposure to dpe ( 20 g / ml ) + uva ( 0.5 j / cm ) was significantly inhibited in the presence of nan3 ( in both cases , p < 0.05 ) but only partially inhibited in the presence of dmso ( in both cases , p > 0.05 ) . in addition , the production of ros in individual worm coexposure to dpe plus uva increased in a time - dependent manner and reached the highest level at a time point of 24 h compared with that of the control or single - treated populations and decreased afterward ( figure 8b and c ) . ros , especially o2 , play a crucial role in germ cell apoptosis induced by dpe ( 20 g / ml ) plus uva ( 0.5 j / cm ) . ( a ) the induction of germ cell apoptosis by dpe plus uva was effectively rescued by nan3 , a specific o2 scavenger . ( b ) the in situ expression of fluorescence was measured using dcf - da ( a molecular probe ) in single whole worms . ( c ) the relative fluorescence was determined using image - pro plus , version 6.0 . ( d ) three - line esr spectra of the 4-o - tempo signal . all these results suggested that ros , especially o2 , play a pivotal role in the induction of germ cell apoptosis by dpe plus uva . all values are presented as the means se ; n 40 , and * represents p < 0.05 . since nan3 has been found to be an efficient quencher for singlet oxygen ( o2 ) , we further analyzed the o2 production by the o2 trapping probe , 2,2,6,6-tetramethyl-4-piperidone hydrochloride ( temp ) , coupled with electron spin resonance ( esr ) spectroscopy . as shown in figure 8d and e , 4-o - tempo triplet spectra and the relative signal intensity increased considerably in worms coexposed to dpe ( 20 g / ml ) plus uva ( 0.5 j / cm ) compared with those in the single treatment of dpe or uva , or with the control worms , and nan3 ( 100 m ) significantly reduced this signal ( p < 0.05 ) . taken together , the results indicated that the ros , especially o2 , played a pivotal role in the induction of germ cell apoptosis within c. elegans by coexposure to dpe plus uva . epidemiologic studies have shown that exposure to diesel exhaust is associated with various health effects , such as cancer induction . however , the cytotoxicity and genotoxicity of dpe in in vitro or in vivo studies were normally discovered at relatively higher doses . it was reported that cell death and apoptosis in macrophages were only significantly enhanced following an exposure dose of dpes higher than 100 g / ml . the organic extract of deps at the dose of 140 g / ml increased ros production in human neutrophil granulocytes and rat alveolar macrophages in vitro assayed with dcfh - da . consistent with these results , we found that a significant induction of germ cell death was only shown at a dose of dpe greater than 100 g / ml . in our previous study , we found that lower concentrations of dpe could manifest its cytotoxicity ( 10 g / ml ) and genotoxcity ( 20 g / ml ) in an al cell culture system with 0.5 j / cm of uva radiation . the question is whether the deleterious effects of diesel exhaust could be manifested by the environmental factor of uva at low doses in an in vivo system , as well as the underlying mechanisms . with a c. elegans system , we further demonstrated that the cyto- and genotoxicity of low - dose exposure of dpe could be activated synergistically by uva radiation ( 0.5 j / cm ) in the context of the whole organism . it is notable that this dose of uva radiation is much lower than those to show the genetic effects in single - exposure experiments ( > 24 j / cm ) . for the activation of dpe by uva radiation in synergistic effects , after absorbing sufficient uva light energy , xenobiotics in dpe can be elevated from ground state to an excited state . the excited molecules can not only react with biological molecules but also transfer their energy to molecular oxygen to create ros . it was reported that benzo[]pyrene , a component of dpe , became highly toxic or carcinogenic in in vitro and in vivo experiments in the manner of photoactivation . their metabolic products , such as diol epoxides and diones , are highly carcinogenic and can induce covalent dna adducts and oxidative dna lesions . metabolically activated xenobiotics in dpe also exerted stimulatory or toxic effects via the generation of ros . by employing a ros probe ( dcf - da ) and quenchers ( nan3 and dmso ) , the present study found that ros levels in worms coexposed to dpe ( 20 g / ml ) plus uva ( 0.5 j / cm ) significantly increased in a time - dependent manner , and the induction of germ cell apoptosis in worms treated with dpe plus uva was effectively restored to the basal level but not for 0.5% and 1.0% dmso treatment groups ( figure 8a ) . nan3 has been reported to be an efficient o2 quencher , and dmso mainly eliminates the effect of the hydroxyl radical ( ho ) . . showed that 1 mm nan3 efficiently quenched o2 formation in murine leukemia l1210 cells , while 1.0% dmso had no effect . to further elucidate the pivotal role of o2 , using a o2 trapping probe , temp , coupled with esr spectroscopy , we found increased o2 production in worms coexposed to dpe plus uva . these results indicated that the production of ros , especially o2 , played a pivotal role in the induction of germ cell apoptosis by dpe plus uva in c. elegans , which was consistent with the previous findings that o2 was mainly responsible for uva - activated toxicity of dpe in mammalian cells . in c. elegans , germ line apoptosis could be physiological and also stress - induced . unlike stress - induced apoptosis , physiological germ cell apoptosis is a highly controlled process , which commences in early adulthood and increases over time . as physiological germ cell apoptosis that is usually scored as background value in the measurement of germ cell death could be affected with worm development , it is quite important to assess the modification of developments by dpe plus uva under different worm stages . by exposing worms at the l1 or young adult stage , it was found that worms coexposed to dpe plus uva at young adult stage had little effect on the index of the mitotic germ cells and the brood size . in addition , there were no effects on the body size and the life span when worms were exposed at the l1 stage ( figure 5b and c ) . however , a slight decrease was found in the percentage of adult worms compared to the single treatment of dpe or uva , or to the control ( in all cases , p > 0.05 ) when worms were exposed at the l1 stage ( figure 5d ) . these findings were consistent with the results by xing et al . , showing that a significant decrease in locomotion was observed after l1-stage larvae were exposed to pb and hg at a concentration of 2.5 m , while no obvious difference was observed in young adult worms exposed to 100 m of the examined metals . therefore , germ cell apoptosis induced by uva plus dpe in young adult worms in the present study was not interfered , or was less , by the changes of physiological germ cell apoptosis . to find out the nature of apoptosis induced by dpe plus uva , we used mutant strains , such as dna damage response checkpoint protein hus-1 and the regulator cep-1/p53 . it has been reported that uv radiation - induced germ cell apoptosis in c. elegans was dependent on both the cep-1/p53 and the checkpoint hus-1 . although there is no evidence yet for the role of cep-1/p53 in the induction of germ cell apoptosis by dpe in c. elegans , p53-dependent cell apoptosis was reported in the j774a.1 macrophage cell line after exposure to dpe . in the present study , the lack of induction of germ cell apoptosis by coexposure in hus-1 and cep-1 mutants suggested that dna - damage - induced germ cell death machinery was involved in the synergistic induction of germ cell apoptosis by dpe plus uva . the enhanced induction of germ cell apoptosis in the w40 strain might be due to the partial loss - of - function of cep-1 and could not effectively and completely block damage signaling transduction . moreover , using the strain of hus-1::gfp , we found that distinct foci of hus-1::gfp could be observed in a small number of mitotic germ cells after worms were coexposed to dpe ( 20 g / ml ) plus uva ( 0.5 j / cm ) ( figure 6b ) . hus-1 is a part of the 9:1:1 complex , which encodes one of the checkpoint proteins that act as the dna damage sensors in c. elegans . it was reported that hus-1::gfp diffuses in proliferating germ nuclei and can be relocalized to distinct foci following dna damage . hence , the foci of hus-1::gfp in c. elegans germ cells indicated clearly that dna - damage - induced germ cell death machinery played a pivotal role in the synergistic induction of germ cell apoptosis by dpe plus uva . furthermore , the decreased survival rates in the f1 progenies of young adult worms with dpe ( 20 g / ml ) plus uva ( 0.2 , 0.5 , and 1.0 j / cm ) also proposed the occurrence of dna damage in the process ( figure s1 , supporting information ) . in addition to the oxidative damage to dna molecules , increased oxidative stress ( ros ) can also activate mapk signaling cascades . in this study , the mapk signaling pathways including erk , jnk , and p38 mapk were shown to take part in the synergistic induction of germ cell apoptosis by dpe plus uva . each of them is essential for germ cell apoptosis induced by coexposure to dpe plus uva , and blockage of any one can inhibit induction , suggesting an elaborate cooperation among three signal cascades in the synergistic induction of germ cell apoptosis . it has been shown that activation of mapks can phosphorylate and activate a number of signaling pathways , including p53 . therefore , in light of the above results , we hypothesize that synergistic germ cell apoptosis induced by dpe plus uva in c. elegans occur via dpe plus uva - induced ros generation that activates mapk signaling pathways ; subsequently , activation of p53 induces ced-4 and ced-3 , which finally leads to apoptosis . moreover , it is not excluded that these signaling pathways were separately used by the dpe plus uva - initiated events due to their distinct activation mechanisms . in addition , the blockage of dna - damage - induced signaling pathway ( hus-1 ) could also inhibit the synergistic induction of germ cell apoptosis in the presence of mapk signaling pathways , suggesting interplay between two types of signaling pathways . this might be another possible reason for the necessity of each signaling pathway for the induction of germ cell apoptosis by coexposure to dpe plus uva . in summary , our results suggested that uva radiation synergistically enhanced the toxicity of dpe at low - dose exposures in the context of the animal in vivo . the synergistic induction of germ cell apoptosis by dpe plus uva should mainly be triggered by dna damage , and the dpe plus uva generated ros , especially o2 , might be one of the factors that lead to dna damage . these data might have some significant implications for exactly assessing the health risk of diesel exhaust and for adopting protective measures for the population exposed to diesel exhaust .

diesel exhaust has been classified as a potential carcinogen and is associated with various health effects . a previous study showed that the doses for manifesting the mutagenetic effects of diesel exhaust could be reduced when coexposed with ultraviolet - a ( uva ) in a cellular system . however , the mechanisms underlying synergistic effects remain to be clarified , especially in an in vivo system . in the present study , using caenorhabditis elegans ( c. elegans ) as an in vivo system we studied the synergistic effects of diesel particulate extract ( dpe ) plus uva , and the underlying mechanisms were dissected genetically using related mutants . our results demonstrated that though coexposure of wild type worms at young adult stage to low doses of dpe ( 20 g / ml ) plus uva ( 0.2 , 0.5 , and 1.0 j / cm2 ) did not affect worm development ( mitotic germ cells and brood size ) , it resulted in a significant induction of germ cell death . using the strain of hus-1::gfp , distinct foci of hus-1::gfp was observed in proliferating germ cells , indicating the dna damage after worms were treated with dpe plus uva . moreover , the induction of germ cell death by dpe plus uva was alleviated in single - gene loss - of - function mutations of core apoptotic , checkpoint hus-1 , cep-1/p53 , and mapk dependent signaling pathways . using a reactive oxygen species ( ros ) probe , it was found that the production of ros in worms coexposed to dpe plus uva increased in a time - dependent manner . in addition , employing a singlet oxygen ( 1o2 ) trapping probe , 2,2,6,6-tetramethyl-4-piperidone , coupled with electron spin resonance analysis , we demonstrated the increased 1o2 production in worms coexposed to dpe plus uva . these results indicated that uva could enhance the apoptotic induction of dpe at low doses through a dna damage - triggered pathway and that the production of ros , especially 1o2 , played a pivotal role in initiating the synergistic process .

Introduction Materials and Methods Results Discussion

diesel exhaust , the dominant pollutant in ambient air , has been classified as a potential or probable human carcinogen by the international agency for research in cancer studies have found that diesel exhaust particles ( deps ) are associated with various health effects such as inflammation of the respiratory tract , lung cancer and cardiovascular diseases , and their extracts , diesel particulate extract ( dpe ) , are thought to be mainly responsible for these malignant effects . hamster hybrid system , the cytotoxicity and genotoxicity of dpe at a low dose ( 20 g / ml ) could be activated by environmental physical factor ultraviolet a ( uva ) radiation ( 0.5 j / cm ) . although the exposure to either diesel exhaust or uva radiation alone or in combination with other agents has been identified as an essential risk factor for various benign or malignant human diseases , the synergistic effects of diesel exhaust and uva remain to be clarified , especially in an in vivo system . in the present study , with the level of germ cell apoptosis as a main checking end point , our results showed that the coexposure of l4-stage or young adult worms to dpe plus uva at low doses significantly enhanced the induction of germ cell apoptosis . strains with single - gene mutations of dna damage - induced germ cell death machinery , cep-1(w40 ) , cep-1(lg12501 ) , and hus-1(op241 ) , were employed for investigating the signaling pathways involved in the induction of germ cell death by dpe and/or uva . for the measurement of apoptosis , the mitotic germ cells , the brood size , the foci of hus-1::gfp , and the production of ros , age - synchronized young hermaphrodites were transferred into 30 mm - diameter petri dishes containing k - medium with op50 as a food source and treated with either dpe ( 20400 g / ml ) or uva ( 0.25.0 j / cm ) alone or in combination ( dpe + uva ) for determined times at 20 . synchronized young adult hermaphrodites were treated with either dpe ( 20 g / ml ) or uva ( 0.2 , 0.5 , and 1.0 j / cm ) alone or in combination ( dpe + uva ) for 24 h. worms were then transferred individually onto a ngm plate containing a bacterial lawn 1 cm in diameter in the center of the dish . worms were photographed under a stereomicroscope equipped with a ccd camera at the time point of 72 h after l1-stage larvae were treated with either dpe ( 20 g / ml ) or uva ( 0.2 , 0.5 , and 1.0 j / cm ) alone or in combination ( dpe + uva ) . l1-stage larvae were treated with either dpe ( 20 g / ml ) or uva ( 0.2 , 0.5 , and 1.0 j / cm ) alone or in combination ( dpe + uva ) throughout their life . synchronized young adult hermaphrodites were treated with either dpe ( 20 g / ml ) or uva ( 0.5 j / cm ) alone or in combination ( dpe + uva ) for 24 h. worms were then mounted onto microscope slides in 0.2 mm of levamisole ( sigma ) , and foci were counted in a single z stack under a laser confocal microscope ( lsm710 zeiss , germany ) , where about 40 mitotic germ cells in c. elegans were observed . age - synchronized young hermaphrodites were treated with 0.5% and 1.0% dimethyl sulfoxide ( dmso ) or 10 m and 100 m sodium azide ( nan3 ) with or without concurrent treatment with dpe ( 20 g / ml ) for 1 h and then irradiated with uva ( 0.5 j / cm ) . age - synchronized young adult hermaphrodites were treated with dpe ( 20 g / ml ) for 1 h at 20 c , and then temp ( sigma ; 0.05 m ) or the stable radical 2,2,6,6-tetramethylpiperidine - n - oxyl ( tempo ; 10 m ; sigma ) was added 30 min before uva radiation . as shown in figure 1 , following treatment with dpe ranging from 20 to 50 g / ml , germ cell death exhibited a basal level compared to that of the control populations ( in all cases , p > 0.05 ) , whereas the higher doses of dpe led to significant increases in the level of germ cell death in a dose - dependent manner ( in all cases , p < 0.05 ) . to further clarify whether there are synergistic effects on the induction of germ cell death by low - doses of dpe plus uva , worms at young adulthood were exposed to low doses of dpe plus uva and then checked for the induction of germ cell death 24 h after cotreatment . as shown in figure 2a , the application of 20 g / ml dpe + 0.5 j / cm uva and 20 g / ml dpe + 1.0 j / cm uva both led to a significantly enhanced induction of germ cell death ( in both cases , p < 0.05 ) . moreover , to avoid the interference of germ cell proliferation by dpe plus uva on germ cell death , the numbers of mitotic germ cells were examined in the distal germ line . as shown in figure 2b , there was no significant changes in the number of mitotic germ cells in the groups of 20 g / ml dpe + 0.2 j / cm uva , 20 g / ml dpe + 0.5 j / cm uva , and 20 g / ml dpe + 1.0 j / cm uva compared with that in the untreated worms ( in all cases , p > 0.05 ) . as shown in figure 3a , compared to the control or single - treated populations , the worms coexposed to 20 g / ml dpe + 0.2 j / cm uva exhibited slight increases in germ cell death at the time points of 6 and 12 h ( in both cases , p < 0.05 ) and recovered to the basal level at the time points of 24 and 36 h ( in both cases , p > 0.05 ) . however , for the groups of 20 g / ml dpe + 0.5 j / cm uva and 20 g / ml dpe + 1.0 j / cm uva , the worms both exhibited significant increases in germ cell death at all of the tested time points ( in all cases , p < 0.05 ) , and the largest induction of germ cell death occurred at the time point of 24 h. time course of germ cell death in c. elegans induced by 20 g / ml dpe + 0.2 j / cm uva ( a ) , 20 g / ml dpe + 0.5 j / cm uva ( b ) , and 20 g / ml dpe + 1.0 j / cm uva ( c ) . to further clarify the nature of germ cell death induced after coexposure to dpe plus uva , c. elegans strains with single - gene mutations of the ced-3(n717 ) and ced-4(n1162 ) genes were employed . ( a ) age - synchronized young hermaphrodites were treated with either dpe ( 20 g / ml ) or uva ( 0.21.0 j / cm ) alone or in combination ( dpe + uva ) for 24 h at 20 c , then the brood size was counted . the classic dna damage - induced germ cell death machinery has been reported to be involved in the induction of apoptosis in addition to physiological germ cell apoptosis in c. elegans . to clarify whether c. elegans employed this death machinery for the induction of germ cell apoptosis after coexposure to dpe plus uva , worm strains with single - gene loss - of - function mutations of this death machinery , cep-1(w40 ) , cep-1(lg12501 ) , and hus-1(op241 ) , were used . as shown in figure 6a , in the worms with null mutations of the hus-1 and cep-1 genes , the induction of germ cell death was significantly inhibited after coexposure to dpe ( 20 g / ml ) plus uva ( 0.5 j / cm ) ( in all cases , p > 0.05 ) , while the wild type and the strain with partial loss - of - function of the cep-1 gene showed a significant induction of germ cell apoptosis . ( a ) there was significantly enhanced induction of germ cell apoptosis in the partial loss - of - function strain of cep-1(w40 ) , while the null mutation strains of hus-1(op241 ) and cep-1(lg12501 ) significantly inhibited the induction of germ cell apoptosis by dpe plus uva . ( b ) quantification of hus-1::gfp foci in the mitotic germ cells after worms were treated with dpe plus uva for 24 h. foci were scored in 40 proliferating germ cells . distinct foci of hus-1::gfp could be observed in a small number of the mitotic germ cells in c. elegans coexposed to dpe plus uva at the time point of 24 h. the scale bar represents 5 m . to further determine the role of dna damage in the induction of germ cell apoptosis by dpe plus uva , the worms transgenic for hus-1::gfp were employed . as shown in figure 6b , distinct foci of hus-1::gfp could be observed in a small number of mitotic germ cells at the time point of 24 h after worms were coexposed to dpe ( 20 g / ml ) plus uva ( 0.5 j / cm ) but nearly none in the single treatment of dpe or uva , or in the control worms . these results indicated that the dna - damage - induced germ cell death machinery played a pivotal role in the synergistic induction of germ cell apoptosis by dpe plus uva . to explore the possible role of mapk signaling pathways in the induction of germ cell apoptosis of dpe plus uva , the strains with the loss - of - function of genes related to mapk pathways were used . as shown in figure 7a , the worm strains with loss - of - function of the lin-45(ku51 ) , mek-2 ( n1989 ) , and mpk-1 ( ku1 ) genes exhibited a basal level of germ cell apoptosis after coexposure to dpe plus uva compared to that of their respective controls ( in all cases , p > 0.05 ) . in our experiments , the loss - of - function of these genes significantly inhibited the induction of germ cell apoptosis by coexposure to dpe plus uva ( in all cases , p > 0.05 ) , as shown in figure 7b . in the present study , the strains with single - gene loss - of - function mutations of the nsy-1 ( ag3 ) , sek-1 ( ag1 ) , and pmk-1 ( km25 ) genes were coexposed to dpe plus uva , respectively , and no significant induction of germ cell apoptosis was observed in all of the mutant strains ( in all cases , p > 0.05 ) , as shown in figure 7c . the results suggested that mapk signal pathways , including erk , jnk , and p38/mapk , might play a pivotal role in the induction of germ cell apoptosis by coexposure to dpe plus uva . germ cell apoptosis was significantly inhibited in all of the mutant strains after exposure to dpe plus uva , suggesting that the mapk signaling pathways play a pivotal role in germ cell apoptosis induced by dpe plus uva . to find out the role of ros in the induction of germ cell apoptosis by dpe plus uva , the ros quenchers , nan3 and dmso , were employed . as shown in figure 8a , the induction of germ cell apoptosis by coexposure to dpe ( 20 g / ml ) + uva ( 0.5 j / cm ) was significantly inhibited in the presence of nan3 ( in both cases , p < 0.05 ) but only partially inhibited in the presence of dmso ( in both cases , p > 0.05 ) . in addition , the production of ros in individual worm coexposure to dpe plus uva increased in a time - dependent manner and reached the highest level at a time point of 24 h compared with that of the control or single - treated populations and decreased afterward ( figure 8b and c ) . ros , especially o2 , play a crucial role in germ cell apoptosis induced by dpe ( 20 g / ml ) plus uva ( 0.5 j / cm ) . all these results suggested that ros , especially o2 , play a pivotal role in the induction of germ cell apoptosis by dpe plus uva . since nan3 has been found to be an efficient quencher for singlet oxygen ( o2 ) , we further analyzed the o2 production by the o2 trapping probe , 2,2,6,6-tetramethyl-4-piperidone hydrochloride ( temp ) , coupled with electron spin resonance ( esr ) spectroscopy . as shown in figure 8d and e , 4-o - tempo triplet spectra and the relative signal intensity increased considerably in worms coexposed to dpe ( 20 g / ml ) plus uva ( 0.5 j / cm ) compared with those in the single treatment of dpe or uva , or with the control worms , and nan3 ( 100 m ) significantly reduced this signal ( p < 0.05 ) . taken together , the results indicated that the ros , especially o2 , played a pivotal role in the induction of germ cell apoptosis within c. elegans by coexposure to dpe plus uva . consistent with these results , we found that a significant induction of germ cell death was only shown at a dose of dpe greater than 100 g / ml . in our previous study , we found that lower concentrations of dpe could manifest its cytotoxicity ( 10 g / ml ) and genotoxcity ( 20 g / ml ) in an al cell culture system with 0.5 j / cm of uva radiation . the question is whether the deleterious effects of diesel exhaust could be manifested by the environmental factor of uva at low doses in an in vivo system , as well as the underlying mechanisms . with a c. elegans system , we further demonstrated that the cyto- and genotoxicity of low - dose exposure of dpe could be activated synergistically by uva radiation ( 0.5 j / cm ) in the context of the whole organism . by employing a ros probe ( dcf - da ) and quenchers ( nan3 and dmso ) , the present study found that ros levels in worms coexposed to dpe ( 20 g / ml ) plus uva ( 0.5 j / cm ) significantly increased in a time - dependent manner , and the induction of germ cell apoptosis in worms treated with dpe plus uva was effectively restored to the basal level but not for 0.5% and 1.0% dmso treatment groups ( figure 8a ) . to further elucidate the pivotal role of o2 , using a o2 trapping probe , temp , coupled with esr spectroscopy , we found increased o2 production in worms coexposed to dpe plus uva . these results indicated that the production of ros , especially o2 , played a pivotal role in the induction of germ cell apoptosis by dpe plus uva in c. elegans , which was consistent with the previous findings that o2 was mainly responsible for uva - activated toxicity of dpe in mammalian cells . by exposing worms at the l1 or young adult stage , it was found that worms coexposed to dpe plus uva at young adult stage had little effect on the index of the mitotic germ cells and the brood size . in the present study , the lack of induction of germ cell apoptosis by coexposure in hus-1 and cep-1 mutants suggested that dna - damage - induced germ cell death machinery was involved in the synergistic induction of germ cell apoptosis by dpe plus uva . moreover , using the strain of hus-1::gfp , we found that distinct foci of hus-1::gfp could be observed in a small number of mitotic germ cells after worms were coexposed to dpe ( 20 g / ml ) plus uva ( 0.5 j / cm ) ( figure 6b ) . hence , the foci of hus-1::gfp in c. elegans germ cells indicated clearly that dna - damage - induced germ cell death machinery played a pivotal role in the synergistic induction of germ cell apoptosis by dpe plus uva . furthermore , the decreased survival rates in the f1 progenies of young adult worms with dpe ( 20 g / ml ) plus uva ( 0.2 , 0.5 , and 1.0 j / cm ) also proposed the occurrence of dna damage in the process ( figure s1 , supporting information ) . in this study , the mapk signaling pathways including erk , jnk , and p38 mapk were shown to take part in the synergistic induction of germ cell apoptosis by dpe plus uva . the synergistic induction of germ cell apoptosis by dpe plus uva should mainly be triggered by dna damage , and the dpe plus uva generated ros , especially o2 , might be one of the factors that lead to dna damage .

according to the 2014 report from the world health organization , 39% of the world 's adult population is overweight , and 13% is obese . in the near future an important feature of obesity and aging is dysregulation of fat in relation to morbidity and mortality . adipokines , proteins secreted by the adipose tissue ( at ) , can trigger metabolic syndromes such as obesity and type 2 diabetes . metabolic diseases are mostly caused by excessive energy storage in the lipid droplets of adipocytes , which results in at expansion . it is therefore of interest to determine the causes and the molecular mechanisms of at expansion in order to find opportunities to control it . over - nutrition leads to at expansion , which is regulated by two events : excessive energy storage into the lipid droplets of adipocytes , a process leading to hypertrophy ( increase in adipocyte size ) , and increased adipogenesis , also known as adipocyte hyperplasia . adipogenesis is a process of differentiation of multipotent mesenchymal stem cells ( msc ) into adipocytes . several transcription factors have been identified as master regulators for preadipocyte determination , such as zinc finger protein 423 ( zfp423 ) and early b cell factor 1 ( ebf1 ) . whereas zfp423 induces early commitment , terminal differentiation is tightly controlled by a transcriptional cascade , whereby peroxisome proliferator - activated receptor ( ppar ) is the essential transcription factor . further key transcriptional factors are the ccaat / enhancer - binding protein ( c / ebp ) family members ( i.e. , c / ebp , c / ebp , and c / ebp ) , kruppel - like factors ( klfs ) , camp responsive element binding protein ( creb ) and early growth response 20 ( krox20 ) . recently , it has been shown that the activator protein-1 ( ap-1 ) family is involved in the adipocyte differentiation process . the ap-1 family is formed by a dimeric protein complex , composed of fos , jun and/or activating transcription factor ( atf ) members . fos - related antigen 1 and 2 ( fra-1 and fra-2 ) are able to regulate adipocyte differentiation . fra-1 impairs adipocyte differentiation by inhibiting c / ebp , whereas fra-2 controls adipocyte turnover . fra-2 thereby not only decreases the adipocyte number by repressing ppar2 expression during adipocyte differentiation , but also decreases adipocyte apoptosis through direct repression of hypoxia - inducible factors ( hifs ) expression . the hif family is a heterodimeric transcription factor complex , composed of hif-1 , hif-2 and hif-1. the heterodimers consist of an oxygen - sensitive hif- protein ( hif-1 or hif-2 ) and the oxygen - insensitive hif-1 subunit . during normoxia , hif- proteins are poly - ubiquitinylated and are finally degraded by proteasomes . under hypoxic conditions , occurring in at during expansion they therefore become stabilized and form dimers with the constitutively expressed hif-1. transcriptional activation of genes controlled by the hif response elements is involved in the regulation of angiogenesis , metabolism , and inflammation . indeed , hif-1 promotes at dysfunction by inducing glucose tolerance , inhibiting energy expenditure and peripheral use of lipid , as well as by increasing leptin level and hfd - induced hepatic steatosis . the present protocol describes methods for studying at status to unravel the molecular characteristics of adipocyte homeostasis in adult mice . it shows how apoptosis , proliferation and differentiation of adipocytes in vivo and in vitro can be regulated by hypoxia . to do so , we use mice with adipocyte specific deletion of fra-2 generated by crossing mice carrying the fra-2 floxed alleles with fabp4-creert mice . by using fabp4-cre ert mice , the deletion is adipocyte specific and inducible by tamoxifen injection . for the adult model , intra peritoneal injections of tamoxifen are performed over 5 consecutive days starting at the age of 6 weeks . thus , the mice are subjected to a normal diet or high - fat diet for 6 weeks before the analysis is done . the mice used in this study were male based on a c57bl6 background to avoid female hormones , such as estrogens , shown to regulate the body fat distribution . using another genetic background might also alter the metabolic phenotype , due to strain - related differences in lipid management . this protocol demonstrates how to analyze at under hypoxia using histology and how to quantify adipocyte apoptosis , proliferation and differentiation in vivo using immunohistochemistry and gene profiling analyses . the study is completed by in vitro experiments , showing how to analyze primary adipocyte differentiation and apoptosis altered by exposure to hypoxia . ethics statement : animals are housed in standardized conditions following the guidelines of the german animal welfare act . animals are fed a standard diet and water ad libitum and kept with a 12 hr day / night cycle . all experiments with animals are authorized by the local ethics committee . to quantify hypoxia in vivo , first determine the body weight of the mice , then inject 60 mg / kg body weight of solid pimonidazole hydrochloride intra - peritoneally ( for example : pimonidazole is an effective hypoxic marker , which forms adducts with thiol groups in proteins , peptides and amino acids and is detected by a specific antibody . 45 min after injection , sacrifice mice by co2 asphyxiation and subsequent cervical dislocation.pin down the limbs of mice ( as illustrated in figure 1 ) and open the peritoneal cavity . remove the left and the right perigonadal ( epididymal ) fat pad inside the peritoneal cavity . note : fat pad are bound to the epididymis by the peritoneal leaflets as shown in figure 1 ( perigonadal fat pads are indicated by arrows).take care to remove the gonadal tissues from the fat pad . determine fat pad weights to calculate the ratio : fat pad weight ( g ) per body weight ( g ) . pin down the limbs of mice ( as illustrated in figure 1 ) and open the peritoneal cavity . remove the left and the right perigonadal ( epididymal ) fat pad inside the peritoneal cavity . note : fat pad are bound to the epididymis by the peritoneal leaflets as shown in figure 1 ( perigonadal fat pads are indicated by arrows ) . determine fat pad weights to calculate the ratio : fat pad weight ( g ) per body weight ( g ) . please click here to view a larger version of this figure . to compile a quantitative gene expression profile , use one perigonadal fat pad to isolate rna . note : until the tissue is processed , store tissue samples in rna stabilization solution at -80 c or in liquid nitrogen . to homogenize the fat pad , add the fat pad to 1 ml single - phase solution of guanidine isothiocyanate and phenol . use tubes containing ceramic beads ( 1.4 mm ) to crush the tissue in a homogenizer at 6,500 rpm ( 2 times 20 sec , with 30 sec pause).isolate the rna as follows ( single - step method by chomczynski and sacchi ) . to separate the phases , transfer homogenized fat pad to microcentrifuge tube , add 0.2 ml volume chloroform , shake for 15 sec , incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . transfer the upper aqueous phase ( around 400 l ) , which contains the rna , into a new microcentrifuge tube . do not include the dna - containing interphase or the protein - containing phenol phase.to precipitate the rna , add 1 volume of isopropanol , mix and incubate for 15 min at 4 c ( it is also possible to incubate at -20 c overnight ) . remove isopropanol supernatant carefully . wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min.after drying the precipitated rna for around 10 min at room temperature , dissolve it in 50 l h2o ( rnase - free ) . to facilitate this , incubate for 2 min at 65 c . note : keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage.quantify the rna preparations by a260/280 ( optimal quotient is between 1.8 and 2.2 ) and calculate the concentration by a260 : to avoid dna contamination , digest 1 g of the rna preparation with 1 u dnase i for 30 min at 37 c in a volume of 10 l . note : this step is optional.use 10 l of rna preparation containing 1 g rna for the reverse transcriptase reaction to generate single - stranded cdna , suitable for quantitative pcr application . the components and their amounts are listed in table 1.use a pcr master mix for a quantitative real - time pcr reaction . to determine metabolic changes in the whole fat pad , use specific primers for genes involved in at homeostasis ( table 2 ) and the pcr conditions listed in table 3.real-time pcr data analysis . define the baseline ( figure 2 ) , normally cycle 1 to 15 , where there is no change in fluorescence signals . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn).set the threshold within the exponential phase of the amplification curve . based on the ct value , calculate the relative expression ( ct ) and the fold change ( ct ) : to homogenize the fat pad , add the fat pad to 1 ml single - phase solution of guanidine isothiocyanate and phenol . use tubes containing ceramic beads ( 1.4 mm ) to crush the tissue in a homogenizer at 6,500 rpm ( 2 times 20 sec , with 30 sec pause ) . isolate the rna as follows ( single - step method by chomczynski and sacchi ) . to separate the phases , transfer homogenized fat pad to microcentrifuge tube , add 0.2 ml volume chloroform , shake for 15 sec , incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . transfer the upper aqueous phase ( around 400 l ) , which contains the rna , into a new microcentrifuge tube . do not include the dna - containing interphase or the protein - containing phenol phase.to precipitate the rna , add 1 volume of isopropanol , mix and incubate for 15 min at 4 c ( it is also possible to incubate at -20 c overnight ) . wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min.after drying the precipitated rna for around 10 min at room temperature , dissolve it in 50 l h2o ( rnase - free ) . to facilitate this , incubate for 2 min at 65 c . note : keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage.quantify the rna preparations by a260/280 ( optimal quotient is between 1.8 and 2.2 ) and calculate the concentration by a260 : to separate the phases , transfer homogenized fat pad to microcentrifuge tube , add 0.2 ml volume chloroform , shake for 15 sec , incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . transfer the upper aqueous phase ( around 400 l ) , which contains the rna , into a new microcentrifuge tube . do not include the dna - containing interphase or the protein - containing phenol phase . to precipitate the rna , add 1 volume of isopropanol , mix and incubate for 15 min at 4 c ( it is also possible to incubate at -20 c overnight ) . wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min . after drying the precipitated rna for around 10 min at room temperature , dissolve it in 50 l h2o ( rnase - free ) . to facilitate this , incubate for 2 min at 65 c . note : keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage . quantify the rna preparations by a260/280 ( optimal quotient is between 1.8 and 2.2 ) and calculate the concentration by a260 : to avoid dna contamination , digest 1 g of the rna preparation with 1 u dnase i for 30 min at 37 c in a volume of 10 l . use 10 l of rna preparation containing 1 g rna for the reverse transcriptase reaction to generate single - stranded cdna , suitable for quantitative pcr application . use a pcr master mix for a quantitative real - time pcr reaction . to determine metabolic changes in the whole fat pad , use specific primers for genes involved in at homeostasis ( table 2 ) and the pcr conditions listed in table 3 . real - time pcr data analysis . define the baseline ( figure 2 ) , normally cycle 1 to 15 , where there is no change in fluorescence signals . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn).set the threshold within the exponential phase of the amplification curve . based on the ct value , calculate the relative expression ( ct ) and the fold change ( ct ) : define the baseline ( figure 2 ) , normally cycle 1 to 15 , where there is no change in fluorescence signals . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn ) . based on the ct value , calculate the relative expression ( ct ) and the fold change ( ct ) : table 1 : components with respective volume for the reverse transcriptase reaction to generate single - stranded cdna . table 2 : list of genes with sequence of the respective primers used for analyzing adipocyte homeostasis . to perform histological analysis of the adipocyte homeostasis , use the second perigonadal fat pad . do not desiccate the tissue ! fix the fat pad in 3.7% pbs - buffered formaldehyde overnight , embed in paraffin ( follow the instructions as described elsewhere ) and cut the embedded tissue into 2 - 5 m thick sections ( maximum 5 m).determine the number of adipocytes per field and adipocyte size in a bright - field microscope after hematoxylin and eosin ( h&e ) staining : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o for 5 min.stain with hematoxylin , diluted 1:5 with distilled h2o , for 10 min at room temperature and wash in h2o for 5 min . stain with eosin solution ( 20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid ) for 30 sec and wash again with h2o for 5 min.dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . mount the sections with anhydrous mounting agent.evaluate the sections under a bright - field microscope . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3 : number of cells per area ( m ) , cell area ( x 10 m ) , cell size ( m ) . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . go to analyze - > set scale . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b.count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . for immunohistochemistry , prepare the section for antibody and tdt - mediated dutp - biotin nick end labeling ( tunel ) staining as follows : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o.for antigen retrieval , digest the tissue section for 30 min at 37 c with proteinase k working solution ( 20 g / ml in 10 mm tris / hcl , ph 7.4 - 8 ) and rinse with pbs . perform antibody staining in wet chambers : to block the endogenous peroxidase , use 3% hydrogen peroxide in pbs for 10 min , with subsequently washing 2 times for 5 min in pbs . to block the unspecific binding of antibodies , use 10% serum in pbs . use the serum of the host of the secondary antibody , in this case goat.for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . dilute antibodies in pbs/10% goat serum . wash the sections 3 times for 5 min in pbs.to enhance the signal use a biotinylated secondary antibody , with the dilution as listed in table 5 . for hypoxia detection , use hrp conjugated rabbit anti note : for the hypoxia staining with fitc - mab1 , skip step 1.4.4.4 ) and continue with step 1.4.4.5).for each slide , pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature ( this method is also referred to as avidin / biotin abc complex formulation ) and then add to the sections for another 45 min . wash 2 times for 5 min with pbs.incubate the sections in peroxidase substrate solution until the staining become more intense ( between 5 to 10 min ) . wash for 5 min with distilled h2o.for counterstaining , stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min.dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . determine apoptosis by tunel assay and follow manufacturer 's instructions : add 50 l enzyme solution into 450 l label solution . then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi ( 4',6-diamidino-2-phenylindole ) for counterstaining.evaluate the section under a fluorescence microscope with 100 fold magnification . for measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm ( green laser ) ; dapi excites at about 360 nm and emits at about 460 nm when bound to dna ( blue laser).quantify the overlays of dapi and fluorescein : fix the fat pad in 3.7% pbs - buffered formaldehyde overnight , embed in paraffin ( follow the instructions as described elsewhere ) and cut the embedded tissue into 2 - 5 m thick sections ( maximum 5 m ) . determine the number of adipocytes per field and adipocyte size in a bright - field microscope after hematoxylin and eosin ( h&e ) staining : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o for 5 min.stain with hematoxylin , diluted 1:5 with distilled h2o , for 10 min at room temperature and wash in h2o for 5 min . stain with eosin solution ( 20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid ) for 30 sec and wash again with h2o for 5 min.dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . mount the sections with anhydrous mounting agent.evaluate the sections under a bright - field microscope . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3 : number of cells per area ( m ) , cell area ( x 10 m ) , cell size ( m ) . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . go to analyze - > set scale . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b.count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o for 5 min . stain with hematoxylin , diluted 1:5 with distilled h2o , for 10 min at room temperature and wash in h2o for 5 min . stain with eosin solution ( 20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid ) for 30 sec and wash again with h2o for 5 min . dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3 : number of cells per area ( m ) , cell area ( x 10 m ) , cell size ( m ) . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b.count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale . select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . confirm with ok . the size of the picture and the analysis of the parameters are indicated in the unit of length given . to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b . count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting . to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m . to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . for immunohistochemistry , prepare the section for antibody and tdt - mediated dutp - biotin nick end labeling ( tunel ) staining as follows : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o.for antigen retrieval , digest the tissue section for 30 min at 37 c with proteinase k working solution ( 20 g / ml in 10 mm tris / hcl , ph 7.4 - 8 ) and rinse with pbs . deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o . for antigen retrieval , digest the tissue section for 30 min at 37 c with proteinase k working solution ( 20 g / ml in 10 mm tris / hcl , ph 7.4 - 8 ) and rinse with pbs . perform antibody staining in wet chambers : to block the endogenous peroxidase , use 3% hydrogen peroxide in pbs for 10 min , with subsequently washing 2 times for 5 min in pbs . to block the unspecific binding of antibodies , use 10% serum in pbs . use the serum of the host of the secondary antibody , in this case goat.for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . dilute antibodies in pbs/10% goat serum . wash the sections 3 times for 5 min in pbs.to enhance the signal use a biotinylated secondary antibody , with the dilution as listed in table 5 . for hypoxia detection , use hrp conjugated rabbit anti note : for the hypoxia staining with fitc - mab1 , skip step 1.4.4.4 ) and continue with step 1.4.4.5).for each slide , pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature ( this method is also referred to as avidin / biotin abc complex formulation ) and then add to the sections for another 45 min . wash 2 times for 5 min with pbs.incubate the sections in peroxidase substrate solution until the staining become more intense ( between 5 to 10 min ) . wash for 5 min with distilled h2o.for counterstaining , stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min.dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . evaluate the sections under a bright - field microscope . to block the endogenous peroxidase , use 3% hydrogen peroxide in pbs for 10 min , with subsequently washing 2 times for 5 min in pbs . to block the unspecific binding of antibodies , use 10% serum in pbs . use the serum of the host of the secondary antibody , in this case goat . for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . dilute antibodies in pbs/10% goat serum . wash the sections 3 times for 5 min in pbs . to enhance the signal use a biotinylated secondary antibody , with the dilution as listed in table 5 . for hypoxia detection , use hrp conjugated rabbit anti note : for the hypoxia staining with fitc - mab1 , skip step 1.4.4.4 ) and continue with step 1.4.4.5 ) . for each slide , pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature ( this method is also referred to as avidin / biotin abc complex formulation ) and then add to the sections for another 45 min . incubate the sections in peroxidase substrate solution until the staining become more intense ( between 5 to 10 min ) . for counterstaining , stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min . dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . determine apoptosis by tunel assay and follow manufacturer 's instructions : add 50 l enzyme solution into 450 l label solution . then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi ( 4',6-diamidino-2-phenylindole ) for counterstaining.evaluate the section under a fluorescence microscope with 100 fold magnification . for measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm ( green laser ) ; dapi excites at about 360 nm and emits at about 460 nm when bound to dna ( blue laser).quantify the overlays of dapi and fluorescein : add 50 l enzyme solution into 450 l label solution . then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi ( 4',6-diamidino-2-phenylindole ) for counterstaining . evaluate the section under a fluorescence microscope with 100 fold magnification . for measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm ( green laser ) ; dapi excites at about 360 nm and emits at about 460 nm when bound to dna ( blue laser ) . quantify the overlays of dapi and fluorescein : figure 3 : analyzing adipocyte characteristics in fat pad sections . section pictures of the perigonadal fat pad of male mice treated with a high - fat diet ( hfd ) or normal diet ( nd ) with a bright - field microscope ( a ) ; the threshold is adjusted into black and white ( b ) and the adipocyte size ( length ; c ) , area ( d ) and adipocyte cell number per mm ( e ) are quantified with imagej 1.48v . table 4 : antibodies with respective dilution used for the immunohistological staining of at sections . table 5 : secondary antibodies with dilution used for immunohistological staining . sacrifice mice and remove the subcutaneous adipose tissue . sacrifice the mice by co2 asphyxiation and subsequent cervical dislocation.pin down the limbs of mice as illustrated in figure 4 . detach the skin from the upper leg , loin and flank and pin it down with needles as in figure 4 . then remove the subcutaneous adipose tissue , which is located posterior at the base of the hind legs , surrounding the inguinal lymph nodes ( as shown in figure 4 , left : subcutaneous fat pads indicated by the arrows ; right : inguinal lymph node indicated by the arrow ) . detach the skin from the upper leg , loin and flank and pin it down with needles as in figure 4 . then remove the subcutaneous adipose tissue , which is located posterior at the base of the hind legs , surrounding the inguinal lymph nodes ( as shown in figure 4 , left : subcutaneous fat pads indicated by the arrows ; picture of subcutaneous fat pad ; the left arrows indicate the subcutaneous fat pad and the right arrow indicates the inguinal lymph node . seed adsc 4,000 cells / cm in dulbecco 's modified eagle 's medium - ham 's f-12 supplemented with 10% normal calf serum , 1% penicillin / streptomycin , 0.5% amphotericin b , 16 m biotin , 18 m pantothenic acid and 100 m ascorbic acid and grow culture to confluence around 70 to 80% , which is reached after 4 to 6 days of culture . induce adipogenic differentiation . remove the adherent adsc from the surface by trypsin treatment . remove medium , wash with pbs and add 0.025% trypsin solution ( preheated to 37 c ) for 2 min ( until cells detach from the surface ) . count the cells using a neubauer chamber . put the glass cover on the central area of the neubauer chamber . dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . count the cells in 4 squares located at the corners , each composed of 16 smaller squares . calculate the cell number per ml : seed adsc ( as described in point 2.2 ) in 12-well culture plates and grow culture to confluence around 70 to 80% ( reached after 4 to 6 days).induce adipogenic differentiation by adding 5 g / ml insulin , 1 m dexamethasone and 5 m 3-isobutyl-1-methylxanthine ( ibmx ) to the cultures . remove the adherent adsc from the surface by trypsin treatment . remove medium , wash with pbs and add 0.025% trypsin solution ( preheated to 37 c ) for 2 min ( until cells detach from the surface ) . immediately add medium and wash the cells . remove medium , wash with pbs and add 0.025% trypsin solution ( preheated to 37 c ) for 2 min ( until cells detach from the surface ) . immediately add medium and wash the cells . count the cells using a neubauer chamber . put the glass cover on the central area of the neubauer chamber . dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . count the cells in 4 squares located at the corners , each composed of 16 smaller squares . calculate the cell number per ml : put the glass cover on the central area of the neubauer chamber . dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . count the cells in 4 squares located at the corners , each composed of 16 smaller squares . calculate the cell number per ml : seed adsc ( as described in point 2.2 ) in 12-well culture plates and grow culture to confluence around 70 to 80% ( reached after 4 to 6 days ) . induce adipogenic differentiation by adding 5 g / ml insulin , 1 m dexamethasone and 5 m 3-isobutyl-1-methylxanthine ( ibmx ) to the cultures . cells will be fully differentiated after 7 days . to analyze the adipogenic differentiation , stain with oil red o , which stains triglycerides of mature adipocytes . note : work must be performed under a fume hood ! remove the medium , wash the adipocytes gently with pbs and fix the cells for 60 min with 2 ml 10% formalin.to prepare oil red o staining solution , mix 3 parts of the red oil o stock solution ( 300 mg red oil o powder dissolved in 100 ml 99% isopropanol ) with 2 parts distilled h2o and incubate for 10 min at room temperature . note : the working solution is stable for 2 hr.for the oil red o staining , remove the formalin , wash adipocytes with h2o , incubate with 2 ml 60% isopropanol for 5 min , remove the isopropanol and add 2 ml oil red o working solution for 5 min . counterstain with hematoxylin as in point 1.4.4.5).evaluate the plates under a phase contrast microscope with 100 fold magnification . the lipids of the adipocytes will appear red and the nuclei will appear blue . remove the medium , wash the adipocytes gently with pbs and fix the cells for 60 min with 2 ml 10% formalin . to prepare oil red o staining solution , mix 3 parts of the red oil o stock solution ( 300 mg red oil o powder dissolved in 100 ml 99% isopropanol ) with 2 parts distilled h2o and incubate for 10 min at room temperature . o staining , remove the formalin , wash adipocytes with h2o , incubate with 2 ml 60% isopropanol for 5 min , remove the isopropanol and add 2 ml oil red o working solution for 5 min . rinse the cells with tap water until the water is clear . optional step : silence the gene of interest by transfection with shrna . change the medium and add serum - free medium.for the transfection of adipocytes , use lipofection . follow the manufacturer 's instructions and use 1 g shrna per 12-well tissue plates . after addition of the lipid - dna - complex , incubate adipocytes for 48 hr at 37 c . change the medium and add serum - free medium . for the transfection of adipocytes , use lipofection . follow the manufacturer 's instructions and use 1 g shrna per 12-well tissue plates . after addition of the lipid - dna - complex , incubate adipocytes for 48 hr at 37 c . to analyze adipocytes subjected to hypoxia , use a hypoxic work station or hypoxic incubator to maintain the cells under hypoxic conditions . gather adipocytes with an extra soft cell scraper , wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer ( 10 mm hepes / naoh , ph 7.4 ; 140 mm nacl ; 2.5 mm cacl2 ) . add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min.for flow cytometry measurement , add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells ( figure 5 ) . add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [ using single - step method by chomczynski and sacchi ( step 1.3.2 ) ] and proceed as in steps 1.3.2.1 ) to 1.3.5.2 ) . analyze apoptosis by fitc - labeled annexin v and subsequent flow cytometry analyses . gather adipocytes with an extra soft cell scraper , wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer ( 10 mm hepes / naoh , ph 7.4 ; 140 mm nacl ; 2.5 mm cacl2 ) . add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min.for flow cytometry measurement , add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells ( figure 5 ) . gather adipocytes with an extra soft cell scraper , wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer ( 10 mm hepes / naoh , ph 7.4 ; 140 mm nacl ; 2.5 mm cacl2 ) . add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min . for flow cytometry measurement , add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells ( figure 5 ) . add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [ using single - step method by chomczynski and sacchi ( step 1.3.2 ) ] and proceed as in steps 1.3.2.1 ) to 1.3.5.2 ) . add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [ using single - step method by chomczynski and sacchi ( step 1.3.2 ) ] and proceed as in steps 1.3.2.1 ) to 1.3.5.2 ) . dot plot presentations of the facs for annexin v - fitc and to - pro-3 staining of adipocytes . to quantify hypoxia in vivo , first determine the body weight of the mice , then inject 60 mg / kg body weight of solid pimonidazole hydrochloride intra - peritoneally ( for example : inject 1.5 mg into a 25 g mouse ) . pimonidazole is an effective hypoxic marker , which forms adducts with thiol groups in proteins , peptides and amino acids and is detected by a specific antibody . 45 min after injection , sacrifice mice by co2 asphyxiation and subsequent cervical dislocation.pin down the limbs of mice ( as illustrated in figure 1 ) and open the peritoneal cavity . remove the left and the right perigonadal ( epididymal ) fat pad inside the peritoneal cavity . note : fat pad are bound to the epididymis by the peritoneal leaflets as shown in figure 1 ( perigonadal fat pads are indicated by arrows).take care to remove the gonadal tissues from the fat pad . determine fat pad weights to calculate the ratio : fat pad weight ( g ) per body weight ( g ) . pin down the limbs of mice ( as illustrated in figure 1 ) and open the peritoneal cavity . remove the left and the right perigonadal ( epididymal ) fat pad inside the peritoneal cavity . note : fat pad are bound to the epididymis by the peritoneal leaflets as shown in figure 1 ( perigonadal fat pads are indicated by arrows ) . take care to remove the gonadal tissues from the fat pad . determine fat pad weights to calculate the ratio : fat pad weight ( g ) per body weight ( g ) . please click here to view a larger version of this figure . to compile a quantitative gene expression profile , use one perigonadal fat pad to isolate rna . note : until the tissue is processed , store tissue samples in rna stabilization solution at -80 c or in liquid nitrogen . to homogenize the fat pad , add the fat pad to 1 ml single - phase solution of guanidine isothiocyanate and phenol . use tubes containing ceramic beads ( 1.4 mm ) to crush the tissue in a homogenizer at 6,500 rpm ( 2 times 20 sec , with 30 sec pause).isolate the rna as follows ( single - step method by chomczynski and sacchi ) . to separate the phases , transfer homogenized fat pad to microcentrifuge tube , add 0.2 ml volume chloroform , shake for 15 sec , incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . transfer the upper aqueous phase ( around 400 l ) , which contains the rna , into a new microcentrifuge tube . do not include the dna - containing interphase or the protein - containing phenol phase.to precipitate the rna , add 1 volume of isopropanol , mix and incubate for 15 min at 4 c ( it is also possible to incubate at -20 c overnight ) . wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min.after drying the precipitated rna for around 10 min at room temperature , dissolve it in 50 l h2o ( rnase - free ) . to facilitate this , incubate for 2 min at 65 c . note : keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage.quantify the rna preparations by a260/280 ( optimal quotient is between 1.8 and 2.2 ) and calculate the concentration by a260 : to avoid dna contamination , digest 1 g of the rna preparation with 1 u dnase i for 30 min at 37 c in a volume of 10 l . note : this step is optional.use 10 l of rna preparation containing 1 g rna for the reverse transcriptase reaction to generate single - stranded cdna , suitable for quantitative pcr application . the components and their amounts are listed in table 1.use a pcr master mix for a quantitative real - time pcr reaction . to determine metabolic changes in the whole fat pad , use specific primers for genes involved in at homeostasis ( table 2 ) and the pcr conditions listed in table 3.real-time pcr data analysis . define the baseline ( figure 2 ) , normally cycle 1 to 15 , where there is no change in fluorescence signals . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn).set the threshold within the exponential phase of the amplification curve . based on the ct value , calculate the relative expression ( ct ) and the fold change ( ct ) : to homogenize the fat pad , add the fat pad to 1 ml single - phase solution of guanidine isothiocyanate and phenol . use tubes containing ceramic beads ( 1.4 mm ) to crush the tissue in a homogenizer at 6,500 rpm ( 2 times 20 sec , with 30 sec pause ) . isolate the rna as follows ( single - step method by chomczynski and sacchi ) . to separate the phases , transfer homogenized fat pad to microcentrifuge tube , add 0.2 ml volume chloroform , shake for 15 sec , incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . transfer the upper aqueous phase ( around 400 l ) , which contains the rna , into a new microcentrifuge tube . do not include the dna - containing interphase or the protein - containing phenol phase.to precipitate the rna , add 1 volume of isopropanol , mix and incubate for 15 min at 4 c ( it is also possible to incubate at -20 c overnight ) . wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min.after drying the precipitated rna for around 10 min at room temperature , dissolve it in 50 l h2o ( rnase - free ) . to facilitate this , incubate for 2 min at 65 c . note : keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage.quantify the rna preparations by a260/280 ( optimal quotient is between 1.8 and 2.2 ) and calculate the concentration by a260 : to separate the phases , transfer homogenized fat pad to microcentrifuge tube , add 0.2 ml volume chloroform , shake for 15 sec , incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . transfer the upper aqueous phase ( around 400 l ) , which contains the rna , into a new microcentrifuge tube . do not include the dna - containing interphase or the protein - containing phenol phase . to precipitate the rna , add 1 volume of isopropanol , mix and incubate for 15 min at 4 c ( it is also possible to incubate at -20 c overnight ) . wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min . after drying the precipitated rna for around 10 min at room temperature , dissolve it in 50 l h2o ( rnase - free ) . to facilitate this , incubate for 2 min at 65 c . note : keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage . quantify the rna preparations by a260/280 ( optimal quotient is between 1.8 and 2.2 ) and calculate the concentration by a260 : to avoid dna contamination , digest 1 g of the rna preparation with 1 u dnase i for 30 min at 37 c in a volume of 10 l . use 10 l of rna preparation containing 1 g rna for the reverse transcriptase reaction to generate single - stranded cdna , suitable for quantitative pcr application . use a pcr master mix for a quantitative real - time pcr reaction . to determine metabolic changes in the whole fat pad , use specific primers for genes involved in at homeostasis ( table 2 ) and the pcr conditions listed in table 3 . define the baseline ( figure 2 ) , normally cycle 1 to 15 , where there is no change in fluorescence signals . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn).set the threshold within the exponential phase of the amplification curve . based on the ct value , calculate the relative expression ( ct ) and the fold change ( ct ) : define the baseline ( figure 2 ) , normally cycle 1 to 15 , where there is no change in fluorescence signals . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn ) . based on the ct value , calculate the relative expression ( ct ) and the fold change ( ct ) : table 1 : components with respective volume for the reverse transcriptase reaction to generate single - stranded cdna . table 2 : list of genes with sequence of the respective primers used for analyzing adipocyte homeostasis . please click here to view a larger version of this figure . to perform histological analysis of the adipocyte homeostasis , use the second perigonadal fat pad . fix the fat pad in 3.7% pbs - buffered formaldehyde overnight , embed in paraffin ( follow the instructions as described elsewhere ) and cut the embedded tissue into 2 - 5 m thick sections ( maximum 5 m).determine the number of adipocytes per field and adipocyte size in a bright - field microscope after hematoxylin and eosin ( h&e ) staining : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o for 5 min.stain with hematoxylin , diluted 1:5 with distilled h2o , for 10 min at room temperature and wash in h2o for 5 min . stain with eosin solution ( 20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid ) for 30 sec and wash again with h2o for 5 min.dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . mount the sections with anhydrous mounting agent.evaluate the sections under a bright - field microscope . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3 : number of cells per area ( m ) , cell area ( x 10 m ) , cell size ( m ) . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . go to analyze - > set scale . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . confirm with ok . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b.count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . for immunohistochemistry , prepare the section for antibody and tdt - mediated dutp - biotin nick end labeling ( tunel ) staining as follows : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o.for antigen retrieval , digest the tissue section for 30 min at 37 c with proteinase k working solution ( 20 g / ml in 10 mm tris / hcl , ph 7.4 - 8 ) and rinse with pbs . perform antibody staining in wet chambers : to block the endogenous peroxidase , use 3% hydrogen peroxide in pbs for 10 min , with subsequently washing 2 times for 5 min in pbs . to block the unspecific binding of antibodies , use 10% serum in pbs . use the serum of the host of the secondary antibody , in this case goat.for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . dilute antibodies in pbs/10% goat serum . wash the sections 3 times for 5 min in pbs.to enhance the signal use a biotinylated secondary antibody , with the dilution as listed in table 5 . for hypoxia detection , use hrp conjugated rabbit anti note : for the hypoxia staining with fitc - mab1 , skip step 1.4.4.4 ) and continue with step 1.4.4.5).for each slide , pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature ( this method is also referred to as avidin / biotin abc complex formulation ) and then add to the sections for another 45 min . wash 2 times for 5 min with pbs.incubate the sections in peroxidase substrate solution until the staining become more intense ( between 5 to 10 min ) . wash for 5 min with distilled h2o.for counterstaining , stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min.dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . determine apoptosis by tunel assay and follow manufacturer 's instructions : add 50 l enzyme solution into 450 l label solution . then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi ( 4',6-diamidino-2-phenylindole ) for counterstaining.evaluate the section under a fluorescence microscope with 100 fold magnification . for measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm ( green laser ) ; dapi excites at about 360 nm and emits at about 460 nm when bound to dna ( blue laser).quantify the overlays of dapi and fluorescein : fix the fat pad in 3.7% pbs - buffered formaldehyde overnight , embed in paraffin ( follow the instructions as described elsewhere ) and cut the embedded tissue into 2 - 5 m thick sections ( maximum 5 m ) . determine the number of adipocytes per field and adipocyte size in a bright - field microscope after hematoxylin and eosin ( h&e ) staining : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o for 5 min.stain with hematoxylin , diluted 1:5 with distilled h2o , for 10 min at room temperature and wash in h2o for 5 min . stain with eosin solution ( 20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid ) for 30 sec and wash again with h2o for 5 min.dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . mount the sections with anhydrous mounting agent.evaluate the sections under a bright - field microscope . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3 : number of cells per area ( m ) , cell area ( x 10 m ) , cell size ( m ) . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . go to analyze - > set scale . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . confirm with ok . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b.count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o for 5 min . stain with hematoxylin , diluted 1:5 with distilled h2o , for 10 min at room temperature and wash in h2o for 5 min . stain with eosin solution ( 20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid ) for 30 sec and wash again with h2o for 5 min . dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3 : number of cells per area ( m ) , cell area ( x 10 m ) , cell size ( m ) . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . go to analyze - > set scale . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . confirm with ok . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b.count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale . select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . go to analyze - > set scale . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . the size of the picture and the analysis of the parameters are indicated in the unit of length given . to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b . count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting . to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m . to determine the adipocyte area , go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . for immunohistochemistry , prepare the section for antibody and tdt - mediated dutp - biotin nick end labeling ( tunel ) staining as follows : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o.for antigen retrieval , digest the tissue section for 30 min at 37 c with proteinase k working solution ( 20 g / ml in 10 mm tris / hcl , ph 7.4 - 8 ) and rinse with pbs . deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o . for antigen retrieval , digest the tissue section for 30 min at 37 c with proteinase k working solution ( 20 g / ml in 10 mm tris / hcl , ph 7.4 - 8 ) and rinse with pbs . perform antibody staining in wet chambers : to block the endogenous peroxidase , use 3% hydrogen peroxide in pbs for 10 min , with subsequently washing 2 times for 5 min in pbs . to block the unspecific binding of antibodies , use 10% serum in pbs . use the serum of the host of the secondary antibody , in this case goat.for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . dilute antibodies in pbs/10% goat serum . wash the sections 3 times for 5 min in pbs.to enhance the signal use a biotinylated secondary antibody , with the dilution as listed in table 5 . for hypoxia detection , note : for the hypoxia staining with fitc - mab1 , skip step 1.4.4.4 ) and continue with step 1.4.4.5).for each slide , pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature ( this method is also referred to as avidin / biotin abc complex formulation ) and then add to the sections for another 45 min . wash 2 times for 5 min with pbs.incubate the sections in peroxidase substrate solution until the staining become more intense ( between 5 to 10 min ) . wash for 5 min with distilled h2o.for counterstaining , stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min.dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . evaluate the sections under a bright - field microscope . to block the endogenous peroxidase , use 3% hydrogen peroxide in pbs for 10 min , with subsequently washing 2 times for 5 min in pbs . to block the unspecific binding of antibodies , use 10% serum in pbs . use the serum of the host of the secondary antibody , in this case goat . for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . dilute antibodies in pbs/10% goat serum . wash the sections 3 times for 5 min in pbs . to enhance the signal use a biotinylated secondary antibody , with the dilution as listed in table 5 . for hypoxia detection , use hrp conjugated rabbit anti - fitc as the secondary antibody . note : for the hypoxia staining with fitc - mab1 , skip step 1.4.4.4 ) and continue with step 1.4.4.5 ) . for each slide , pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature ( this method is also referred to as avidin / biotin abc complex formulation ) and then add to the sections for another 45 min . incubate the sections in peroxidase substrate solution until the staining become more intense ( between 5 to 10 min ) . for counterstaining , stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min . dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . determine apoptosis by tunel assay and follow manufacturer 's instructions : add 50 l enzyme solution into 450 l label solution . then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi ( 4',6-diamidino-2-phenylindole ) for counterstaining.evaluate the section under a fluorescence microscope with 100 fold magnification . for measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm ( green laser ) ; dapi excites at about 360 nm and emits at about 460 nm when bound to dna ( blue laser).quantify the overlays of dapi and fluorescein : add 50 l enzyme solution into 450 l label solution . then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi ( 4',6-diamidino-2-phenylindole ) for counterstaining . evaluate the section under a fluorescence microscope with 100 fold magnification . for measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm ( green laser ) ; dapi excites at about 360 nm and emits at about 460 nm when bound to dna ( blue laser ) . quantify the overlays of dapi and fluorescein : figure 3 : analyzing adipocyte characteristics in fat pad sections . section pictures of the perigonadal fat pad of male mice treated with a high - fat diet ( hfd ) or normal diet ( nd ) with a bright - field microscope ( a ) ; the threshold is adjusted into black and white ( b ) and the adipocyte size ( length ; c ) , area ( d ) and adipocyte cell number per mm ( e ) are quantified with imagej 1.48v . table 4 : antibodies with respective dilution used for the immunohistological staining of at sections . sacrifice mice and remove the subcutaneous adipose tissue . sacrifice the mice by co2 asphyxiation and subsequent cervical dislocation.pin down the limbs of mice as illustrated in figure 4 . detach the skin from the upper leg , loin and flank and pin it down with needles as in figure 4 . then remove the subcutaneous adipose tissue , which is located posterior at the base of the hind legs , surrounding the inguinal lymph nodes ( as shown in figure 4 , left : subcutaneous fat pads indicated by the arrows ; right : inguinal lymph node indicated by the arrow ) . detach the skin from the upper leg , loin and flank and pin it down with needles as in figure 4 . then remove the subcutaneous adipose tissue , which is located posterior at the base of the hind legs , surrounding the inguinal lymph nodes ( as shown in figure 4 , left : subcutaneous fat pads indicated by the arrows ; picture of subcutaneous fat pad ; the left arrows indicate the subcutaneous fat pad and the right arrow indicates the inguinal lymph node . seed adsc 4,000 cells / cm in dulbecco 's modified eagle 's medium - ham 's f-12 supplemented with 10% normal calf serum , 1% penicillin / streptomycin , 0.5% amphotericin b , 16 m biotin , 18 m pantothenic acid and 100 m ascorbic acid and grow culture to confluence around 70 to 80% , which is reached after 4 to 6 days of culture . induce adipogenic differentiation . remove the adherent adsc from the surface by trypsin treatment . remove medium , wash with pbs and add 0.025% trypsin solution ( preheated to 37 c ) for 2 min ( until cells detach from the surface ) . count the cells using a neubauer chamber . put the glass cover on the central area of the neubauer chamber . dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . count the cells in 4 squares located at the corners , each composed of 16 smaller squares . calculate the cell number per ml : seed adsc ( as described in point 2.2 ) in 12-well culture plates and grow culture to confluence around 70 to 80% ( reached after 4 to 6 days).induce adipogenic differentiation by adding 5 g / ml insulin , 1 m dexamethasone and 5 m 3-isobutyl-1-methylxanthine ( ibmx ) to the cultures . remove the adherent adsc from the surface by trypsin treatment . remove medium , wash with pbs and add 0.025% trypsin solution ( preheated to 37 c ) for 2 min ( until cells detach from the surface ) . immediately add medium and wash the cells . remove medium , wash with pbs and add 0.025% trypsin solution ( preheated to 37 c ) for 2 min ( until cells detach from the surface ) . immediately add medium and wash the cells . count the cells using a neubauer chamber . put the glass cover on the central area of the neubauer chamber . dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . count the cells in 4 squares located at the corners , each composed of 16 smaller squares . calculate the cell number per ml : put the glass cover on the central area of the neubauer chamber . dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . count the cells in 4 squares located at the corners , each composed of 16 smaller squares . calculate the cell number per ml : seed adsc ( as described in point 2.2 ) in 12-well culture plates and grow culture to confluence around 70 to 80% ( reached after 4 to 6 days ) . induce adipogenic differentiation by adding 5 g / ml insulin , 1 m dexamethasone and 5 m 3-isobutyl-1-methylxanthine ( ibmx ) to the cultures . cells will be fully differentiated after 7 days . to analyze the adipogenic differentiation , stain with oil red o , which stains triglycerides of mature adipocytes . note : work must be performed under a fume hood ! remove the medium , wash the adipocytes gently with pbs and fix the cells for 60 min with 2 ml 10% formalin.to prepare oil red o staining solution , mix 3 parts of the red oil o stock solution ( 300 mg red oil o powder dissolved in 100 ml 99% isopropanol ) with 2 parts distilled h2o and incubate for 10 min at room temperature . note : the working solution is stable for 2 hr.for the oil red o staining , remove the formalin , wash adipocytes with h2o , incubate with 2 ml 60% isopropanol for 5 min , remove the isopropanol and add 2 ml oil red o working solution for 5 min . counterstain with hematoxylin as in point 1.4.4.5).evaluate the plates under a phase contrast microscope with 100 fold magnification . the lipids of the adipocytes will appear red and the nuclei will appear blue . remove the medium , wash the adipocytes gently with pbs and fix the cells for 60 min with 2 ml 10% formalin . to prepare oil red o staining solution , mix 3 parts of the red oil o stock solution ( 300 mg red oil o powder dissolved in 100 ml 99% isopropanol ) with 2 parts distilled h2o and incubate for 10 min at room temperature . note : the working solution is stable for 2 hr . for the oil red o staining , remove the formalin , wash adipocytes with h2o , incubate with 2 ml 60% isopropanol for 5 min , remove the isopropanol and add 2 ml oil red o working solution for 5 min . optional step : silence the gene of interest by transfection with shrna . change the medium and add serum - free medium.for the transfection of adipocytes , use lipofection . follow the manufacturer 's instructions and use 1 g shrna per 12-well tissue plates . after addition of the lipid - dna - complex , incubate adipocytes for 48 hr at 37 c . change the medium and add serum - free medium . for the transfection of adipocytes , use lipofection . after addition of the lipid - dna - complex , incubate adipocytes for 48 hr at 37 c . to analyze adipocytes subjected to hypoxia , use a hypoxic work station or hypoxic incubator to maintain the cells under hypoxic conditions . analyze apoptosis by fitc - labeled annexin v and subsequent flow cytometry analyses . gather adipocytes with an extra soft cell scraper , wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer ( 10 mm hepes / naoh , ph 7.4 ; 140 mm nacl ; 2.5 mm cacl2 ) . add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min.for flow cytometry measurement , add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells ( figure 5 ) . add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [ using single - step method by chomczynski and sacchi ( step 1.3.2 ) ] and proceed as in steps 1.3.2.1 ) to 1.3.5.2 ) . analyze apoptosis by fitc - labeled annexin v and subsequent flow cytometry analyses . gather adipocytes with an extra soft cell scraper , wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer ( 10 mm hepes / naoh , ph 7.4 add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min.for flow cytometry measurement , add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells ( figure 5 ) . gather adipocytes with an extra soft cell scraper , wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer ( 10 mm hepes / naoh , ph 7.4 ; 140 mm nacl ; 2.5 mm cacl2 ) . add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min . for flow cytometry measurement , add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells ( figure 5 ) . add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [ using single - step method by chomczynski and sacchi ( step 1.3.2 ) ] and proceed as in steps 1.3.2.1 ) to 1.3.5.2 ) . add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [ using single - step method by chomczynski and sacchi ( step 1.3.2 ) ] and proceed as in steps 1.3.2.1 ) to 1.3.5.2 ) . dot plot presentations of the facs for annexin v - fitc and to - pro-3 staining of adipocytes . we show how to determine adipocyte homeostasis in vivo and in vitro using the example of fra-2 fabp4-creert mice compared to wild - type littermates . our protocol defines how increased hif expression by hypoxia is correlated with adipocyte dysfunction as indicated by increased adipocyte apoptosis . increased adipocyte size and area in high - fat diet ( hfd ) treated mice over - nutrition , among other factors , results in adipocyte hypertrophy , caused by excessive energy storage in the lipid droplets . sections of the fat pad from normal ( nd ; figure 6a ) and high - fat diet ( hfd ; figure 6b ) mice as well as quantifications of the adipocyte size and area clearly show adipocyte hypertrophy after 6 weeks of hfd , which is indicated by increased adipocyte size in the hfd treated mice ( figure 6c and d ) . increased hypoxia in the adipose tissue ( at ) of adult fra-2 fabp4-creert mice leads to increased hif-1 level and adipocyte apoptosis to determine the in vivo status of hypoxia in at , fra-2 fabp4-creert mice are analyzed 6 weeks after fra-2 deletion at the age of 12 weeks and compared with wild - type littermates . pimonidazole is administered to the mice intraperitoneally as an effective hypoxia marker ; it is nontoxic and able to distribute into the at . hypoxic adipocytes in the at in vivo are defined by immunohistochemical antibody staining ( figure 7a ) . furthermore , the increased hypoxic status of the at in mice is accompanied by increased hif-1 positive adipocytes as indicated by the immunohistochemical staining figure 7b ) , which is confirmed by quantification of hif-1 expression levels and its targets genes . additionally , tunel staining of at sections from fra-2 fabp4-creert mice and control littermates ( figure 7c ) shows that increased adipocyte apoptosis is correlated with the presence of hypoxia and hif-1 expression . increased hif-1 expression in primary adipocytes through hypoxia induced adipocyte apoptosis to analyze adipocyte apoptosis in vitro , we use adipocytes generated from subcutaneous fat pads as described elsewhere . as expected , the hif-1 expression in adipocytes is increased after 24 hr of hypoxia ( figure 8a ) . to further analyze hif-1 activities , the rna levels of hif target genes such as inos ( inducible nitric oxide synthase ) are quantified by qpcr . figure 8b shows that the increased expression of hif-1 under hypoxic conditions leads to increased inos mrna level . since we have already shown in vivo ( figure 8) that increased hif-1 expression in adipocytes correlates with increased adipocyte apoptosis , apoptosis is also quantified in in vitro cultures by annexin v staining under hypoxic conditions . consistent with the in vivo data ( figure 7 ) , an increased hif-1 level is accompanied by increased adipocyte apoptosis induced by hypoxic conditions ( figure 8b ) . moreover , to prove that hypoxic - induced apoptosis is hif - dependent ; hif-1 or hif-2 is silenced by rna interference in adipocytes derived from wild - type or fra-2 deficient mice . the increased adipocyte apoptosis is restored by silencing hif-1 or hif-2 as shown by annexin v staining in figure 8b , proving that the hypoxia sensor hif- regulates the adipocyte apoptosis . figure 6 : increased adipocyte size and area in high - fat diet ( hfd ) mice . ( a , b ) h&e staining of sections from the perigonadal fat pad of male wild - type mice fed with normal diet ( nd ) ( a ) or high - fat diet ( hfd ) ( b ) for 6 weeks . ( c , d ) quantifications of adipocyte size ( c ) and area ( d ) from the perigonadal fat pad of wild - type mice fed with nd ( a ) or hfd ( b ) for 6 weeks . statistical analysis was performed using students t - test . * * * p < 0.0001.please click here to view a larger version of this figure . figure 7 : increased hif-1 level and apoptosis in adipocytes of adult fra-2 fabp4-creert mice . hypoxia ( a ) and hif-1 ( b ) staining in the at of male fra-2 fabp4-creert mice and male control littermates 6 weeks after tamoxifen injection . ( c ) tunel staining in fra-2 fabp4-creert mice and control littermates at 6 weeks after tamoxifen injection . this figure has been modified from luther et al .. please click here to view a larger version of this figure . ( a ) real - time pcr analysis of hif-1 and hifs target inos mrna levels in primary adipocytes placed in hypoxic chambers analyzed at the indicated time points . ( b ) quantification of apoptosis by annexin v facs staining in primary adipocytes isolated from fra-2 fabp4-creert mice or wild - type controls transfected with sh control or sh plasmid against hif-1 or hif-2 and placed under hypoxia ( 1% o2 ) for 24 hr . statistical analysis was performed using student 's t - test . * p < 0.05 and * * p < 0.01 were accepted as significant adipocytes are characterized phenotypically by their size , numbers and area , revealing adipocyte hyperplasia and hypertrophy , induced by excessive energy storage due to over - nutrition . these events leading to fatty acid dysregulation and subsequent metabolic syndromes are also states of increased fat mass with preserved metabolisms , which is also referred to as " healthy " fat expansion . for example , kusminski et al . showed that mice with massive fat expansion remain metabolically healthy , suggesting that fat expansion is not necessarily linked to metabolic syndromes and needs to be carefully determined to evaluate the characteristics of the adipocytes . the adipose tissue ( at ) plays a pivotal role in the regulation of body metabolism . at is the biggest endocrine organ that could influence dyslipidemia , atherosclerosis , hyperinsulinemia and hyperglycemia . evaluating at homeostasis and the molecular mechanisms regulating it could allow a better understanding of metabolic system disorders . therefore , unravelling the mechanisms regulating adipocyte differentiation , adipocyte size and fat pad mass would help to develop new therapeutic treatment for obesity disorders . using in vivo and in vitro methods , it is possible to determine the role of food and gene expression impacts on adipocyte differentiation and activity . to determine the at homeostasis , determining the balance between adipocyte differentiation , proliferation and apoptosis as suggested by our protocol is as important as analyzing the glucose and insulin metabolic response . expression profiling analyses of genes involved in adipogenesis , lipogenesis , lipolysis , fatty acid uptake , hypoxia , apoptosis and proliferation in primary adipocytes and visceral at is a high throughput method for obtaining an overview on adipocyte homeostasis and their possible dysfunction . interesting candidates should be further analyzed at the protein level by western blot or immunohistological staining . to obtain optimal results through real - time pcr system using unsymmetrical cyanine dyes , the concentrations of cdna ranging from 1 to 10 ng and the optimal primer concentrations ranging from 50 to 900 nm the critical components are the primers ; for each run , the melting curves need to be strictly controlled to ensure the specificity and to exclude the formation of primer dimers . furthermore , commercial available unsymmetrical cyanine dyes are provided as master mixes that contain a passive reference dye ( such as rox ) to provide an internal reference signal . the cdna signal is normalized during data analysis to the rox signals to correct well - to - well signal fluctuations . another point to be considered in order to establish a good qpcr system is the choice of the housekeeping gene . for each condition , several housekeeping genes are used , e.g. , hprt , -actin , gapdh , -2-mg or hsp90 . hif proteins stabilized by hypoxic conditions are master regulators determining not only adipocytes survival , but also metabolic changes , such as glucose , insulin tolerance and lipid metabolism . to ascertain hypoxic areas in the fat pad , hif-1 is determined in at sections by immunohistochemistry . since hif proteins are rapidly degraded within 5 to 10 min under normoxic conditions , the procedure and the fixation of the fat pad for the histological analysis should be tightly controlled to avoid latency time . therefore , to ensure hypoxia not only through hif staining , pimonidazole is used to determine hypoxic areas in the at . pimonidazole is able to distribute into tissues , as it was already shown in bones , and effectively mark hypoxic areas by binding to thiol - containing proteins specifically in hypoxic cells , which is further detected in histological sections by specific antibody binding . for example , the involvement of the prolyl hydroxylase ( phd ) enzyme , which induce hydroxylation of proline residues under normoxia , as well as the von hippel - lindau ( vhl ) protein , which recognize hydroxylated prolines and induce the poly - ubiquitination to mediate proteasomal hif degradation , need to be analyzed for a full overview of the pathway . moreover , ubiquitous detection of hifs would also determine the protein stability and degradation that can be alter . furthermore , proliferation by ki67 and apoptosis by tunel staining are determined in vivo through staining of at histological sections . quantification of proliferation by ki67 and apoptosis by tunel or annexin v staining through flow cytometry analysis is also carried out . proliferation could of course be measured by other techniques such as the analyses of the adipocyte cell cycle , which is not addressed by the measurement of ki67 positive cells . moreover , apoptosis study by tunel can be completed by facs analyses of annexin v and top - pr-3 which will determine the levels of necrosis versus the apoptosis cell death process . apoptosis is a fundamental process for the program of cell death , which is important for at homeostasis . indeed , dysregulation of adipocyte apoptosis has been implicated previously in processes contributing to obesity and lipodystrophy . moreover , in 2011 , keuper et al . linked adipose tissue inflammation to adipocyte apoptosis . they showed that macrophages induced apoptosis in preadipocytes and adipocytes , which in turn attract macrophages . the recruitment of macrophages accelerates inflammation , which contributes to metabolic syndromes such as glucose and insulin tolerance . however , adipocyte apoptosis is still a poorly studied phenomenon , despite the hypothesis that induced adipocyte apoptosis could lead to decreased weight . the present protocol uses immunohistochemical approaches to study different phenomenon such as proliferation , apoptosis and hypoxia in vivo . therefore , the tissue was fixed with 4% formaldehyde , which is a critical step . an extended tissue fixation time leads to change of the epitopes , which become non - accessible for the antibody . moreover , the thickness of the sections also influences the antibodies binding to their epitopes ; optimal thickness is between 2 and 5 m . sections thicker than 5 m will give false positive results due to increased binding sites . in contrast , sections thinner than 2 m contain less binding sites and positive areas are not well defined . further critical factors are the antibody itself , incubation time , concentration and even temperature , which influence the quality of the specific binding to the epitopes . therefore , validating antibody concentration and incubation time is necessary for each condition . to complete the study , we provide an in vitro adipocyte differentiation protocol , which could be extended by different treatments , stimulation or co - cultures . by using in vitro adipocyte cultures , it is feasible to determine defects in adipocyte differentiation and functions . to obtain reliable results , as for all primary cells , the healthy behavior and appearance of the adscs and adipocytes is quite important . the granularity , cytoplasmic vacuolations and/or detachment are signs of deterioration , indicating inadequate medium , microbial contamination or senescence of the primary cells . this protocol is using isolated adipocytes from the fat pad tissue , whereas it is as well possible to use mesenchymal stem cell isolated from bone marrow as described by other protocols . the latest includes stromal progenitor cells , which might reflect additional differentiation problems occurring at the very early step of adipocyte differentiation , this might be missed in our current protocol . moreover , adscs can be expanded rapidly ( more than 10 times within one week ) , and long - term cultured adscs after some passages still retain their mesenchymal pluripotency . another advantage using adscs is that one can easily switch to human , since adscs can be harvested from patients by liposuction which is a simple and minimally invasive method . as at influences several other organs in an endocrine manner , the protocol should be extended to adipokines . adipokines , such as leptin , adiponectin , tumor necrosis factor- ( tnf ) and resistin , secreted by adipocytes are known to affect metabolic diseases by controlling fat metabolism , energy homeostasis and insulin sensitivity . therefore , serum and adipocyte secretome analyses should be performed . in the case of at dysfunction , adipokines and pro - inflammatory cytokines , such as il-6 , can lead to dysregulation of organs such as the liver and pancreas , and of muscle function . in order to exclude systemic organ dysfunction , animal models or cell cultures we provide a protocol for analyzing the basic state of the at and adipocytes in vivo and in vitro to reveal molecular mechanisms of adipocyte homeostasis and functionality .

considering that adipose tissue ( at ) is an endocrine organ , it can influence whole body metabolism . excessive energy storage leads to the dysregulation of adipocytes , which in turn induces abnormal secretion of adipokines , triggering metabolic syndromes such as obesity , dyslipidemia , hyperglycemia , hyperinsulinemia , insulin resistance and type 2 diabetes . therefore , investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies . our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the at , which correlates with adipocyte apoptosis in adult mice . this protocol describes how to analyze at in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation , proliferation and apoptosis during hypoxia exposure , ascertained through staining of hypoxic cells or hif-1 protein . furthermore , in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes .

Introduction Protocol 1. 2. Representative Results Discussion Disclosures

according to the 2014 report from the world health organization , 39% of the world 's adult population is overweight , and 13% is obese . adipokines , proteins secreted by the adipose tissue ( at ) , can trigger metabolic syndromes such as obesity and type 2 diabetes . metabolic diseases are mostly caused by excessive energy storage in the lipid droplets of adipocytes , which results in at expansion . it is therefore of interest to determine the causes and the molecular mechanisms of at expansion in order to find opportunities to control it . over - nutrition leads to at expansion , which is regulated by two events : excessive energy storage into the lipid droplets of adipocytes , a process leading to hypertrophy ( increase in adipocyte size ) , and increased adipogenesis , also known as adipocyte hyperplasia . recently , it has been shown that the activator protein-1 ( ap-1 ) family is involved in the adipocyte differentiation process . fra-2 thereby not only decreases the adipocyte number by repressing ppar2 expression during adipocyte differentiation , but also decreases adipocyte apoptosis through direct repression of hypoxia - inducible factors ( hifs ) expression . the present protocol describes methods for studying at status to unravel the molecular characteristics of adipocyte homeostasis in adult mice . it shows how apoptosis , proliferation and differentiation of adipocytes in vivo and in vitro can be regulated by hypoxia . this protocol demonstrates how to analyze at under hypoxia using histology and how to quantify adipocyte apoptosis , proliferation and differentiation in vivo using immunohistochemistry and gene profiling analyses . the study is completed by in vitro experiments , showing how to analyze primary adipocyte differentiation and apoptosis altered by exposure to hypoxia . to quantify hypoxia in vivo , first determine the body weight of the mice , then inject 60 mg / kg body weight of solid pimonidazole hydrochloride intra - peritoneally ( for example : pimonidazole is an effective hypoxic marker , which forms adducts with thiol groups in proteins , peptides and amino acids and is detected by a specific antibody . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn).set the threshold within the exponential phase of the amplification curve . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn ) . to perform histological analysis of the adipocyte homeostasis , use the second perigonadal fat pad . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . use the serum of the host of the secondary antibody , in this case goat.for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . the size of the picture and the analysis of the parameters are indicated in the unit of length given . to determine the parameters of the adipocytes , adjust the threshold . go to analyze - > measure and a new window will appear , with the length of the diameter in m . use the serum of the host of the secondary antibody , in this case goat.for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . use the serum of the host of the secondary antibody , in this case goat . for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . then remove the subcutaneous adipose tissue , which is located posterior at the base of the hind legs , surrounding the inguinal lymph nodes ( as shown in figure 4 , left : subcutaneous fat pads indicated by the arrows ; right : inguinal lymph node indicated by the arrow ) . then remove the subcutaneous adipose tissue , which is located posterior at the base of the hind legs , surrounding the inguinal lymph nodes ( as shown in figure 4 , left : subcutaneous fat pads indicated by the arrows ; picture of subcutaneous fat pad ; the left arrows indicate the subcutaneous fat pad and the right arrow indicates the inguinal lymph node . to analyze the adipogenic differentiation , stain with oil red o , which stains triglycerides of mature adipocytes . for the transfection of adipocytes , use lipofection . to analyze adipocytes subjected to hypoxia , use a hypoxic work station or hypoxic incubator to maintain the cells under hypoxic conditions . dot plot presentations of the facs for annexin v - fitc and to - pro-3 staining of adipocytes . to quantify hypoxia in vivo , first determine the body weight of the mice , then inject 60 mg / kg body weight of solid pimonidazole hydrochloride intra - peritoneally ( for example : inject 1.5 mg into a 25 g mouse ) . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn).set the threshold within the exponential phase of the amplification curve . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn).set the threshold within the exponential phase of the amplification curve . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn ) . to perform histological analysis of the adipocyte homeostasis , use the second perigonadal fat pad . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . use the serum of the host of the secondary antibody , in this case goat.for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . to determine the parameters of the adipocytes , adjust the threshold . go to analyze - > measure and a new window will appear , with the length of the diameter in m . use the serum of the host of the secondary antibody , in this case goat.for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . use the serum of the host of the secondary antibody , in this case goat . for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . then remove the subcutaneous adipose tissue , which is located posterior at the base of the hind legs , surrounding the inguinal lymph nodes ( as shown in figure 4 , left : subcutaneous fat pads indicated by the arrows ; right : inguinal lymph node indicated by the arrow ) . then remove the subcutaneous adipose tissue , which is located posterior at the base of the hind legs , surrounding the inguinal lymph nodes ( as shown in figure 4 , left : subcutaneous fat pads indicated by the arrows ; picture of subcutaneous fat pad ; the left arrows indicate the subcutaneous fat pad and the right arrow indicates the inguinal lymph node . to analyze the adipogenic differentiation , stain with oil red o , which stains triglycerides of mature adipocytes . change the medium and add serum - free medium.for the transfection of adipocytes , use lipofection . for the transfection of adipocytes , use lipofection . to analyze adipocytes subjected to hypoxia , use a hypoxic work station or hypoxic incubator to maintain the cells under hypoxic conditions . dot plot presentations of the facs for annexin v - fitc and to - pro-3 staining of adipocytes . we show how to determine adipocyte homeostasis in vivo and in vitro using the example of fra-2 fabp4-creert mice compared to wild - type littermates . our protocol defines how increased hif expression by hypoxia is correlated with adipocyte dysfunction as indicated by increased adipocyte apoptosis . increased adipocyte size and area in high - fat diet ( hfd ) treated mice over - nutrition , among other factors , results in adipocyte hypertrophy , caused by excessive energy storage in the lipid droplets . sections of the fat pad from normal ( nd ; figure 6a ) and high - fat diet ( hfd ; figure 6b ) mice as well as quantifications of the adipocyte size and area clearly show adipocyte hypertrophy after 6 weeks of hfd , which is indicated by increased adipocyte size in the hfd treated mice ( figure 6c and d ) . increased hypoxia in the adipose tissue ( at ) of adult fra-2 fabp4-creert mice leads to increased hif-1 level and adipocyte apoptosis to determine the in vivo status of hypoxia in at , fra-2 fabp4-creert mice are analyzed 6 weeks after fra-2 deletion at the age of 12 weeks and compared with wild - type littermates . pimonidazole is administered to the mice intraperitoneally as an effective hypoxia marker ; it is nontoxic and able to distribute into the at . hypoxic adipocytes in the at in vivo are defined by immunohistochemical antibody staining ( figure 7a ) . furthermore , the increased hypoxic status of the at in mice is accompanied by increased hif-1 positive adipocytes as indicated by the immunohistochemical staining figure 7b ) , which is confirmed by quantification of hif-1 expression levels and its targets genes . additionally , tunel staining of at sections from fra-2 fabp4-creert mice and control littermates ( figure 7c ) shows that increased adipocyte apoptosis is correlated with the presence of hypoxia and hif-1 expression . increased hif-1 expression in primary adipocytes through hypoxia induced adipocyte apoptosis to analyze adipocyte apoptosis in vitro , we use adipocytes generated from subcutaneous fat pads as described elsewhere . figure 8b shows that the increased expression of hif-1 under hypoxic conditions leads to increased inos mrna level . since we have already shown in vivo ( figure 8) that increased hif-1 expression in adipocytes correlates with increased adipocyte apoptosis , apoptosis is also quantified in in vitro cultures by annexin v staining under hypoxic conditions . consistent with the in vivo data ( figure 7 ) , an increased hif-1 level is accompanied by increased adipocyte apoptosis induced by hypoxic conditions ( figure 8b ) . * p < 0.05 and * * p < 0.01 were accepted as significant adipocytes are characterized phenotypically by their size , numbers and area , revealing adipocyte hyperplasia and hypertrophy , induced by excessive energy storage due to over - nutrition . these events leading to fatty acid dysregulation and subsequent metabolic syndromes are also states of increased fat mass with preserved metabolisms , which is also referred to as " healthy " fat expansion . showed that mice with massive fat expansion remain metabolically healthy , suggesting that fat expansion is not necessarily linked to metabolic syndromes and needs to be carefully determined to evaluate the characteristics of the adipocytes . the adipose tissue ( at ) plays a pivotal role in the regulation of body metabolism . at is the biggest endocrine organ that could influence dyslipidemia , atherosclerosis , hyperinsulinemia and hyperglycemia . evaluating at homeostasis and the molecular mechanisms regulating it could allow a better understanding of metabolic system disorders . therefore , unravelling the mechanisms regulating adipocyte differentiation , adipocyte size and fat pad mass would help to develop new therapeutic treatment for obesity disorders . using in vivo and in vitro methods , it is possible to determine the role of food and gene expression impacts on adipocyte differentiation and activity . to determine the at homeostasis , determining the balance between adipocyte differentiation , proliferation and apoptosis as suggested by our protocol is as important as analyzing the glucose and insulin metabolic response . furthermore , commercial available unsymmetrical cyanine dyes are provided as master mixes that contain a passive reference dye ( such as rox ) to provide an internal reference signal . hif proteins stabilized by hypoxic conditions are master regulators determining not only adipocytes survival , but also metabolic changes , such as glucose , insulin tolerance and lipid metabolism . since hif proteins are rapidly degraded within 5 to 10 min under normoxic conditions , the procedure and the fixation of the fat pad for the histological analysis should be tightly controlled to avoid latency time . pimonidazole is able to distribute into tissues , as it was already shown in bones , and effectively mark hypoxic areas by binding to thiol - containing proteins specifically in hypoxic cells , which is further detected in histological sections by specific antibody binding . for example , the involvement of the prolyl hydroxylase ( phd ) enzyme , which induce hydroxylation of proline residues under normoxia , as well as the von hippel - lindau ( vhl ) protein , which recognize hydroxylated prolines and induce the poly - ubiquitination to mediate proteasomal hif degradation , need to be analyzed for a full overview of the pathway . furthermore , proliferation by ki67 and apoptosis by tunel staining are determined in vivo through staining of at histological sections . proliferation could of course be measured by other techniques such as the analyses of the adipocyte cell cycle , which is not addressed by the measurement of ki67 positive cells . indeed , dysregulation of adipocyte apoptosis has been implicated previously in processes contributing to obesity and lipodystrophy . linked adipose tissue inflammation to adipocyte apoptosis . they showed that macrophages induced apoptosis in preadipocytes and adipocytes , which in turn attract macrophages . the recruitment of macrophages accelerates inflammation , which contributes to metabolic syndromes such as glucose and insulin tolerance . therefore , the tissue was fixed with 4% formaldehyde , which is a critical step . an extended tissue fixation time leads to change of the epitopes , which become non - accessible for the antibody . further critical factors are the antibody itself , incubation time , concentration and even temperature , which influence the quality of the specific binding to the epitopes . to complete the study , we provide an in vitro adipocyte differentiation protocol , which could be extended by different treatments , stimulation or co - cultures . by using in vitro adipocyte cultures , it is feasible to determine defects in adipocyte differentiation and functions . this protocol is using isolated adipocytes from the fat pad tissue , whereas it is as well possible to use mesenchymal stem cell isolated from bone marrow as described by other protocols . the latest includes stromal progenitor cells , which might reflect additional differentiation problems occurring at the very early step of adipocyte differentiation , this might be missed in our current protocol . adipokines , such as leptin , adiponectin , tumor necrosis factor- ( tnf ) and resistin , secreted by adipocytes are known to affect metabolic diseases by controlling fat metabolism , energy homeostasis and insulin sensitivity . in order to exclude systemic organ dysfunction , animal models or cell cultures we provide a protocol for analyzing the basic state of the at and adipocytes in vivo and in vitro to reveal molecular mechanisms of adipocyte homeostasis and functionality .

in a previous report , global pertussis initiative ( gpi ) participants described the difficulties in defining pertussis from a clinical perspective . most case definitions are supplemented with laboratory and epidemiologic data so that reports may be categorized as confirmed , probable , or suspect . for example , in vaccine efficacy trials , specificity is expected to be close to 100% . yet , in outbreak situations in states or countries , specificity is sacrificed to achieve high sensitivity , which is important for disease prevention and control . in the prevaccine era , pertussis was considered a disease of children , and all the present clinical case definitions reflect this bias . with the current awareness that pertussis is common in adolescents and adults and that disease manifestations may be different in older persons , it is apparent that the one - size - fits - all clinical pertussis case definition is no longer optimal . in addition , there is an increasing awareness that pertussis in early infancy has many unique characteristics that should be recognized in a separate case definition in order to improve recognition of disease in this population . in this communication , we provide background data relating to current case definitions and then propose age - stratified case definitions that we believe will increase diagnostic specificity without decreasing sensitivity . selected , currently used , clinical case definitions and additional laboratory and epidemiologic requirements are presented in table 1 . most primary clinical case definitions , such as those by the world health organization ( who ) , centers for disease control and prevention ( cdc ) , massachusetts department of health , european union ( eu ) , pan american health organization ( paho ) , and australian department of health and ageing , have in common a requirement for 2 weeks of cough . to increase specificity , most definitions require at least 1 additional symptom , such as paroxysms , inspiratory whoop , or posttussive vomiting . table 1.selected presently used pertussis case definitionsorganization / country , yearclinical criterialaboratory and epidemiologic criteriacommentwho , 2000a case diagnosed as pertussis by a physician , or a person with a cough lasting 2 weeks with 1 of the following symptoms : paroxysms ( ie , fits ) of coughinginspiratory whoopingposttussive vomiting ( ie , vomiting immediately after coughing ) without other apparent causeisolation of b. pertussis , or detection of genomic sequences by pcr , or positive paired serologycase classification : clinical case : a case that meets the clinical case definition , but is not laboratory confirmed . laboratory - confirmed case : a case that meets the clinical case definition and is laboratory confirmed.cste/cdc , 2010a cough illness lasting 2 weeks with 1 of the following : paroxysms of coughing , inspiratory whoop , or posttussive vomiting , without other apparent cause ( as reported by a health professional)isolation of b. pertussis from clinical specimen pcr positive for pertussiscase classification : probable : in the absence of a more likely diagnosis , a cough illness lasting 2 weeks , with 1 of the following symptoms : paroxysms of coughing orinspiratory whooporposttussive vomiting and absence of laboratory confirmation , andno epidemiologic linkage to a laboratory- confirmed case of pertussisconfirmed : acute cough illness of any duration , with isolation of b. pertussis from a clinical specimen , or cough illness lasting 2 weeks , with 1 of the following symptoms : paroxysms of coughing orinspiratory whoop orposttussive vomitingand 1 of the following : pcr positive for pertussis orcontact with a laboratory - confirmed case of pertussisfrance , 2009patient coughing 14 days with 1 or more of the following : whoopvomitingcyanosisapneapatient coughing 14 days with : positive pcr / culture>100 iu / ml of anti - pt antibodies > 3 year from vaccination or 100% change in the antibody titer between 2 serologies at 1-month intervalepidemiologically confirmed : patient coughing 7 days and in contact in the past 20 days with a biologically confirmed casecanada , 2009suspect case : one or more of the following , with no other known cause : paroxysmal cough of any durationcough with inspiratory whoopcough ending in vomiting or gagging , or associated with apnea probable case : cough lasting 2 weeks or longer in the absence of appropriate laboratory tests and not epidemiologically linked to a laboratory confirmed case and 1 of the following , with no other known cause : paroxysmal cough of any durationcough with inspiratory whoopcough ending in vomiting or gagging , or associated with apneaconfirmed case : laboratory confirmation of infection : isolation of b. pertussis from an appropriate clinical specimen ordetection of b. pertussis dna from an appropriate clinical specimen and 1 of the following : - cough lasting 2 weeks - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending with vomiting or gagging , or associated with apnea orepidemiologic link to a laboratory- confirmed case and 1 of the following for which there is no other known cause : - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending in vomiting or gagging , or associated with apneamassachusetts , 200919891992 : 1 week with paroxysms or posttussive vomiting from 1993 : cough 2 weeks with 1 of the following : paroxysms , whoop , or posttussive vomiting ( cdc definition)bacteriologic cases : positive culture ( or + dfa until 1992 ) , pcr added 2004 serologic case : positive single - serum anti - pertussis toxin antibody ( persons 11 years only ) + clinical case definition epidemiologically linked case : contact with a laboratory confirmed case + clinical case definitioneu , 2008cough 2 weeks with 1 of the following : paroxysmsinspiratory whoopingposttussive vomitingorany person diagnosed as pertussis by a physician or apnea episodes in infantsisolation of b. pertussis nucleic acids of b. pertussis b. pertussis specific antibody response epidemiologic link by human - to - human transmissionpossible case : any person with clinical criteria probable case : person with clinical criteria and epidemiologic link confirmed case : person meeting the clinical and laboratory criteriaaustralia , 2004coughing 2 weeks or paroxysms of coughing or inspiratory whoop or posttussive vomitingculture of b. pertussis pcr for b. pertussis seroconversion or significant increase of antibodies ( without recent vaccination ) single iga titer to whole cells detection of b. pertussis by dfaprobable case : any person with clinical criteria confirmed case : person meeting the clinical and laboratory criteria or epidemiologic linkabbreviations : cdc , centers for disease control and prevention ; cste , council of state and territorial epidemiologists ; dfa , direct fluorescent antibody ; eu , european union ; iga , immunoglobin a ; iu , international units ; pcr , polymerase chain reaction ; pt , pertussis toxin ; who , world health organization . selected presently used pertussis case definitions paroxysms ( ie , fits ) of coughing inspiratory whooping posttussive vomiting ( ie , vomiting immediately after coughing ) without other apparent cause paroxysms of coughing or inspiratory whoopor absence of laboratory confirmation , and no epidemiologic linkage to a laboratory- confirmed case of pertussis paroxysms of coughing or inspiratory whoop or pcr positive for pertussis or contact with a laboratory - confirmed case of pertussis > 100 iu / ml of anti - pt antibodies > 3 year from vaccination or 100% change in the antibody titer between 2 serologies at 1-month interval paroxysmal cough of any duration cough with inspiratory whoop cough ending in vomiting or gagging , or associated with apnea paroxysmal cough of any duration cough with inspiratory whoop cough ending in vomiting or gagging , or associated with apnea isolation of b. pertussis from an appropriate clinical specimen or detection of b. pertussis dna from an appropriate clinical specimen 1 of the following : - cough lasting 2 weeks - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending with vomiting or gagging , or associated with apnea or - cough lasting 2 weeks - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending with vomiting or gagging , or associated with apnea or epidemiologic link to a laboratory- confirmed case and 1 of the following for which there is no other known cause : - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending in vomiting or gagging , or associated with apnea - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending in vomiting or gagging , or associated with apnea inspiratory whooping abbreviations : cdc , centers for disease control and prevention ; cste , council of state and territorial epidemiologists ; dfa , direct fluorescent antibody ; eu , european union ; iga , immunoglobin a ; iu , international units ; pcr , polymerase chain reaction ; pt , pertussis toxin ; who , world health organization . france requires that cough be present for more than 7 days , whereas australia accepts cough of any duration if it is accompanied by paroxysms , whooping , or vomiting . the eu also accepts any physician 's diagnosis of pertussis and apnea as a clinically defining symptom in infants . almost all case definitions require laboratory or epidemiologic linkage data , and such data may affect whether the case is categorized as confirmed , probable , or possible . laboratory confirmation tests include culture of bordetella pertussis and polymerase chain reaction ( pcr ) assays that are specific for b. pertussis . some countries , such as australia , also accept direct fluorescent antibody ( dfa ) testing , whereas the paho definition specifically discourages dfa . differences are also found concerning confirmation by serology : the cdc definition does not include serology , the who definition requires paired serology , and the eu definition elegantly compromises by requiring a b. pertussis specific antibody response . france and massachusetts also accept single serum serology with an elevated anti pertussis toxin ( anti - pt ) titer , and australia accepts an immunoglobin a ( iga ) response to whole b. pertussis . recently , ghanaie and associates studied the sensitivity and specificity of the who pertussis clinical case definition in 328 children aged 614 years with a persistent cough for 2 weeks . pertussis was diagnosed by culture and an is481 pcr for b. pertussis or is1001 pcr for b. parapertussis in nasopharyngeal swabs . all but 1 of these children had received 3 or more doses of whole - cell dtp vaccine . the sensitivity was 95.2% and the specificity was 15.0% with cough 2 weeks plus 1 of the who clinical criteria . posttussive emesis was the symptom that had the most pronounced effect in increasing specificity . as the entry criterion for this study was cough of > 2 weeks duration , the mean duration of cough in the study population was 20 days , and because diagnosis was made by pcr without serologic study , it is likely that cases were missed . this would lead to an artificially low specificity . between 2001 and 2005 , harnden et al . performed a prospective cohort study involving 172 children aged 516 years who had a cough lasting 14 days . bordetella pertussis infection was diagnosed by the demonstration of a 4-fold change in immunoglobin g ( igg ) antibody to pertussis toxin ( pt ) in paired samples or a single igg titer to pt that was greater than 100 enzyme - linked immunosorbent assay units / ml . in a subsequent analysis , wang and harnden used the clinical data from the 20012005 study to examine the sensitivity and specificity of defined clinical features . in children with a persistent cough that was characterized as paroxysmal , the sensitivity was 86% and the specificity was 23% ; persistent cough with posttussive vomiting had a sensitivity of 70% and a specificity of 61% ; persistent cough with whooping gave a sensitivity of 50% and a specificity of 74% . to obtain clinical case definition data in adolescents and adults , wang and harnden also used the data in the prospective pertussis surveillance study of strebel et al . performed in minnesota during 19951996 . in this study , persons 1049 years old who presented with an acute paroxysmal cough or a persistent cough illness of 734 days duration were enrolled . b. pertussis infection was diagnosed by culture , pcr , or serologic evidence of a titer rise or high single - serum specimen titer to pt . for paroxysmal cough , with posttussive vomiting , the sensitivity was 56% and the specificity was 68% ; for whooping , the sensitivity was 28% and the specificity was 85% . in 1998 , patriarca et al . evaluated 15 clinical case definitions for pertussis during community outbreaks and concluded that a definition of 14 days of cough was both sensitive ( 77%91% ) and specific ( 54%71% ) for monitoring culture - positive cases . however , in nonoutbreak situations , the use of their case definitions had low sensitivity . the present clinical case definitions of pertussis are inconsistent and are not used everywhere . in addition , they are not universally applicable . furthermore , resource - rich and resource - limited countries have unique problems related to the control of pertussis and its diagnosis . however , in all situations , the true burden of pertussis is unknown and is significantly underestimated . in resource - rich countries , a major priority relates to the education , awareness , and recognition of pertussis in adolescents and adults and its transmission to infants [ 1517 ] . in order to improve the awareness and recognition of the disease in these populations , awareness of proper sampling techniques for obtaining nasopharyngeal specimens ( nasopharyngeal swabs , nasopharyngeal aspiration ) for culture and pcr , as well as the usefulness of single - serum serology in diagnosis should also be fostered . finally , awareness of appropriate treatment and chemoprophylactic regimens for pertussis should be promulgated . ? how can it be distinguished from staccato coughing? is a pertussis - related cough dry ? when is it most likely to occur? how can a pertussis - related cough be differentiated from the cough seen with sinusitis ? bronchitis ? and that due to other infectious agents? is the cough worse at night ? are we able to quantify worse? does the cough significantly disturb ability to sleep ? considerations related to adolescent and adult pertussis in resource - limited countries , pertussis burden is especially underestimated because of a number of factors , including misdiagnosis , lack of recognition , and absent requirements for notification . as a result , adding to the problem is that surveillance systems are often not established or data are collected only sparsely . nevertheless , pertussis continues to be a serious health problem , especially among infants , in terms of both morbidity and mortality . in addition , because adolescent and adult pertussis is largely unrecognized , these age groups are not targeted for prevention and infected individuals are not treated , thereby facilitating spread of the disease in the community , including to vulnerable young infants . finally , laboratory access for confirmatory diagnosis is very limited . until healthcare professionals in both resource - rich and research - poor countries diagnose their adolescent and adult pertussis patients correctly , the burden of disease will continue to be significantly underestimated . without knowing that the disease predominantly occurs in this population , attempts to increase vaccine use in adolescents and adults are unlikely to be made . in persons with pertussis , the median time from cough onset to seeking medical care differs by age group . for example , in 1 study setting , children aged 712 years were seen after 7.8 days of coughing , whereas adolescents aged 1318 years were seen after 12.5 days and adults 17.3 days after symptoms began [ 18 and riffelmann m , et al . unpublished data ] . the interval from the onset of cough to when the patient seeks medical care has a major effect on the laboratory diagnosis of b. pertussis infection [ 1922 ] . culture obtained during the first 3 weeks of cough has 100% specificity , but low sensitivity , ranging from 20% to 80% , when compared with pcr and/or serology . in general , other factors that may influence the sensitivity of culture are the type and quality of specimen , the type of transport media , and the duration of transport ( optimally within 48 hours ) . real - time ( rt)pcr is more sensitive than culture and is the diagnostic method of choice in patients with cough illness of 3 weeks duration . selected issues with rt - pcr are presented in table 3 . in general , by the time most adults seek medical care , the time windows for both culture and rt - pcr have passed ; therefore , serologic diagnosis should be the method of choice [ 18 , 19 ] . table 3.issues relating to real - time polymerase chain reaction and pertussis serologypcr: more expensive than culture may be difficult to perform ( requires trained staff ) and to implement outside the hospital setting ( requires dedicated laboratory space) sensitivity decreases with increasing cough duration commercial kits are not widely available subject to contamination , especially during outbreak situationsserology: testing is mostly done in immunologically nonnaive populations testing is done with an antigen ( pertussis toxin ) that is contained in all acellular vaccines immune response to vaccine antigens can not be distinguished from response to infection interpretation of serology depends on vaccination history population - based cutoffs may need verification after change of vaccination calendar problems in serodiagnosis of b. parapertussis infectionsabbreviation : pcr , polymerase chain reaction . issues relating to real - time polymerase chain reaction and pertussis serology abbreviation : pcr , polymerase chain reaction . since all adults and most adolescents will have had a previous b. pertussis infection and/or pertussis immunization , they will have a rapid anamnestic antibody response to new b. pertussis infection ; consequently , by the time they seek care for a persistent cough illness , they are likely to have developed high antibody levels to b. pertussis antigens . pt is unique to b. pertussis and is highly immunogenic ; therefore , it is the antigen that should be used for single serum diagnosis of b. pertussis cough illness . single - serum igg anti - pt testing has been used successfully in massachusetts and in various countries in europe for approximately 2 decades [ 2426 ] . high levels of iga and/or igg antibodies to pt were described in many studies of prolonged cough illness as an accurate indicator of recent pertussis disease [ 21 , 22 , 2429 ] . in europe , single - serum serology for the diagnosis of pertussis has been intensively studied in the netherlands , and commercial test kits with a variety of pertussis antigens and varying degrees of sensitivity and specificity are available . eu reference laboratories have recently suggested recommendations for the serologic diagnosis of pertussis ; these include mainly quantifying igg antibodies to pt and reporting results in international units / ml [ 32 , 33 ] . in the united states , tests done in commercial laboratories have varying degrees of sensitivity and specificity . however , the tests with the greatest sensitivity and specificity are those that quantifiably measure igg and iga antibodies to pt ( personal clinical observation of one of the authors [ j. d. c. ] ) in recognition of the fact that the signs and symptoms of pertussis differ by age , we have tailored criteria for pertussis diagnosis in 3 different age cohorts ( 03 months , 4 months9 years , and 10 years ) . these criteria are presented in figure 1 . in figure 2 , clinical case definitions of pertussis for surveillance purposes are presented . abbreviations : igg , immunoglobin g ; pcr , polymerase chain reaction ; pt , pertussis toxin ; rsv , respiratory syncytial virus ; wbc , white blood cell . in resource - limited areas where pcr is not available serology not useful in this age cohort . figure 2.clinical case definition of pertussis for surveillance purposes . abbreviations : igg , immunoglobin g ; pcr , polymerase chain reaction ; pt , pertussis toxin ; rsv , respiratory syncytial virus ; wbc , white blood cell . in resource - limited areas where pcr is not available , these case definitions are intended to : ( 1 ) be more specific and/or more sensitive than existing case definitions of pertussis ( which were developed more than 40 years ago and were primarily designed either for surveillance purposes or for vaccine efficacy studies ) , ( 2 ) be applicable to both resource - rich and resource - poor settings , ( 3 ) encourage the increased use of laboratory confirmation , and ( 4 ) increase the sensitivity and specificity of pertussis reporting . if a patient meets 1 or more of the criteria for pertussis diagnosis , the physician should treat the patient and report the case to the appropriate health agencies . general comments on the clinical presentation of pertussis and its laboratory diagnosis are presented in tables 4 and 5 , respectively . table 4.general comments on clinical presentation of pertussis pertussis should be increasingly suspected in patients who are afebrile with increasing cough duration and severity coryza is associated with illness onset and , in contrast with most viral respiratory infections , does not become purulent the key to identifying a paroxysmal cough is that the patient does not inhale until he has run out of breath ( possibly resulting in an inspiratory whoop) paroxysmal cough episodes are more disturbing to the patient at night among young infants , apnea and seizures may not be noted to occur with recognized paroxysms most infants with pertussis will have had a close exposure to an adolescent or adult ( usually a family member ) with a prolonged afebrile cough illness the cough in pertussis is not truly productive sweating episodes occur in adolescents and adults in time periods when coughing is not occurring table 5.general comments on laboratory diagnostics of pertussis pcr and culture are most useful in the first 3 weeks after illness onset serology should not be used to diagnose pertussis in patients < 1 year after inoculation with an acellular or whole - cell vaccine formulation igg anti - pt elisa is preferred to iga anti - pt testing because the iga response following infection is less common , and thus a negative iga anti - pt test should not be relied upon as diagnostic evidence of a pertussis infection the attendees strongly discouraged the use of dfa to detect b. pertussis . they also strongly discouraged the use of elisa tests that employed whole b. pertussis as the antigenabbreviations : dfa , direct fluorescent antibody ; elisa , enzyme - linked immunosorbent assay ; ig , immunoglobin ; pcr , polymerase chain reaction ; pt , pertussis toxin . general comments on clinical presentation of pertussis general comments on laboratory diagnostics of pertussis abbreviations : dfa , direct fluorescent antibody ; elisa , enzyme - linked immunosorbent assay ; ig , immunoglobin ; pcr , polymerase chain reaction ; pt , pertussis toxin . there are a number of strong indicators of pertussis that differ by age group . in young infants , when these young infants are seen by physicians , they are thought to have a viral respiratory infection , and the parents are reassured . however , over the next day or two , the parents recognize the worsening of symptoms , but more often than not , the physicians do not ( based on author experience in california in 2010 [ j. d. c. ] ) . the key indicators of pertussis in these young infant cases are the afebrile nature of the illness combined with a cough that is increasing in frequency and severity and a coryza that remains watery . therefore , the presence of this triad would be expected to have high sensitivity and good specificity . the addition of apnea , seizures , cyanosis , emesis , or pneumonia would result in both high sensitivity and specificity . in these young infant cases , an elevated white blood cell count ( 20 000 cells/l ) with absolute lymphocytosis is virtually diagnostic . in older children ( 4 months to 9 years ) , the presence of a worsening paroxysmal , nonproductive cough of 7 days duration in an afebrile child with coryza that has not become purulent as noted with current case definitions , the addition of whoop , apnea , and posttussive emesis will each increase specificity . in those persons 10 years of age , the same triad listed above for those 4 months to 9 years would also result in high sensitivity with good specificity . in addition , the notation of sweating episodes between paroxysms will significantly increase specificity . in dealing with adult patients adults will often say that the cough is productive , but on further questioning , it is apparent that they actually do not produce purulent sputum . the case definitions of pertussis delineated here should first be tested in clinical trials to determine their utility to the average clinician and then be compared with existing case definitions to determine whether they confer increased sensitivity and/or specificity . although retrospective analyses are subject to bias , such analyses could be performed first in a proof - of - principle approach . if the new case definitions appear promising , a prospective study should be conducted to evaluate the proposed diagnostic criteria in the 3 different age categories ( 03 months , 4 months to 9 years , 10 years ) . the protocol we propose would involve the prospective evaluation of all persons in a defined population with cough illnesses of 7 days duration stratified into the 3 different age categories . the study populations should include geographic regions with different vaccine usage patterns ( acellular , whole - cell , or both ) . protocols could be adopted from the adult pertussis trial ( apert ) and the vaccine efficacy trial in erlangen , germany [ 12 , 35 ] . in both of these studies , investigators contacted participants every 2 weeks , and all subjects with cough illness of 7 days that was not improving were evaluated . studies should be of such duration that they cover the cyclical epidemiologic patterns of pertussis and include populations of sufficient size to allow statistical analysis . the development and utilization of 3 age - related definitions for pertussis can be expected to increase both the sensitivity and specificity in its diagnosis , which will result in the recognition of pertussis in all age groups , potentially leading to better control of pertussis .

existing clinical case definitions of pertussis are decades old and based largely on clinical presentation in infants and children , yet an increasing burden is borne by adolescents and adults who may manifest distinct signs / symptoms . therefore , a one - size - fits - all clinical case definition is no longer appropriate . seeking to improve pertussis diagnosis , the global pertussis initiative ( gpi ) developed an algorithm that delineates the signs / symptoms of pertussis most common to 3 age groups : 03 months , 4 months to 9 years , and 10 years . these case definitions are based on clinical presentation alone , but do include recommendations on laboratory diagnostics . until pertussis can be accurately diagnosed , its burden will remain underestimated , making the introduction of epidemiologically appropriate preventive strategies difficult . the proposed definitions are intended to be widely applicable and to encourage the expanded use of laboratory diagnostics . determination of their utility and their sensitivity and/or specificity versus existing case definitions is required .

INTRODUCTION SELECTED CURRENTLY USED CASE DEFINITIONS THE SENSITIVITY AND SPECIFICITY OF CLINICAL CASE DEFINITIONS CHALLENGES TO CURRENT CRITERIA AND THE DEVELOPMENT OF NEW CLINICAL DEFINITIONS OF PERTUSSIS HOW DO ADVANCES IN DIAGNOSTICS IMPACT CLINICAL PRACTICE? SUGGESTED CASE DEFINITIONS FOR PERTUSSIS

in a previous report , global pertussis initiative ( gpi ) participants described the difficulties in defining pertussis from a clinical perspective . most case definitions are supplemented with laboratory and epidemiologic data so that reports may be categorized as confirmed , probable , or suspect . for example , in vaccine efficacy trials , specificity is expected to be close to 100% . in the prevaccine era , pertussis was considered a disease of children , and all the present clinical case definitions reflect this bias . with the current awareness that pertussis is common in adolescents and adults and that disease manifestations may be different in older persons , it is apparent that the one - size - fits - all clinical pertussis case definition is no longer optimal . in addition , there is an increasing awareness that pertussis in early infancy has many unique characteristics that should be recognized in a separate case definition in order to improve recognition of disease in this population . in this communication , we provide background data relating to current case definitions and then propose age - stratified case definitions that we believe will increase diagnostic specificity without decreasing sensitivity . selected , currently used , clinical case definitions and additional laboratory and epidemiologic requirements are presented in table 1 . most primary clinical case definitions , such as those by the world health organization ( who ) , centers for disease control and prevention ( cdc ) , massachusetts department of health , european union ( eu ) , pan american health organization ( paho ) , and australian department of health and ageing , have in common a requirement for 2 weeks of cough . table 1.selected presently used pertussis case definitionsorganization / country , yearclinical criterialaboratory and epidemiologic criteriacommentwho , 2000a case diagnosed as pertussis by a physician , or a person with a cough lasting 2 weeks with 1 of the following symptoms : paroxysms ( ie , fits ) of coughinginspiratory whoopingposttussive vomiting ( ie , vomiting immediately after coughing ) without other apparent causeisolation of b. pertussis , or detection of genomic sequences by pcr , or positive paired serologycase classification : clinical case : a case that meets the clinical case definition , but is not laboratory confirmed . laboratory - confirmed case : a case that meets the clinical case definition and is laboratory confirmed.cste/cdc , 2010a cough illness lasting 2 weeks with 1 of the following : paroxysms of coughing , inspiratory whoop , or posttussive vomiting , without other apparent cause ( as reported by a health professional)isolation of b. pertussis from clinical specimen pcr positive for pertussiscase classification : probable : in the absence of a more likely diagnosis , a cough illness lasting 2 weeks , with 1 of the following symptoms : paroxysms of coughing orinspiratory whooporposttussive vomiting and absence of laboratory confirmation , andno epidemiologic linkage to a laboratory- confirmed case of pertussisconfirmed : acute cough illness of any duration , with isolation of b. pertussis from a clinical specimen , or cough illness lasting 2 weeks , with 1 of the following symptoms : paroxysms of coughing orinspiratory whoop orposttussive vomitingand 1 of the following : pcr positive for pertussis orcontact with a laboratory - confirmed case of pertussisfrance , 2009patient coughing 14 days with 1 or more of the following : whoopvomitingcyanosisapneapatient coughing 14 days with : positive pcr / culture>100 iu / ml of anti - pt antibodies > 3 year from vaccination or 100% change in the antibody titer between 2 serologies at 1-month intervalepidemiologically confirmed : patient coughing 7 days and in contact in the past 20 days with a biologically confirmed casecanada , 2009suspect case : one or more of the following , with no other known cause : paroxysmal cough of any durationcough with inspiratory whoopcough ending in vomiting or gagging , or associated with apnea probable case : cough lasting 2 weeks or longer in the absence of appropriate laboratory tests and not epidemiologically linked to a laboratory confirmed case and 1 of the following , with no other known cause : paroxysmal cough of any durationcough with inspiratory whoopcough ending in vomiting or gagging , or associated with apneaconfirmed case : laboratory confirmation of infection : isolation of b. pertussis from an appropriate clinical specimen ordetection of b. pertussis dna from an appropriate clinical specimen and 1 of the following : - cough lasting 2 weeks - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending with vomiting or gagging , or associated with apnea orepidemiologic link to a laboratory- confirmed case and 1 of the following for which there is no other known cause : - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending in vomiting or gagging , or associated with apneamassachusetts , 200919891992 : 1 week with paroxysms or posttussive vomiting from 1993 : cough 2 weeks with 1 of the following : paroxysms , whoop , or posttussive vomiting ( cdc definition)bacteriologic cases : positive culture ( or + dfa until 1992 ) , pcr added 2004 serologic case : positive single - serum anti - pertussis toxin antibody ( persons 11 years only ) + clinical case definition epidemiologically linked case : contact with a laboratory confirmed case + clinical case definitioneu , 2008cough 2 weeks with 1 of the following : paroxysmsinspiratory whoopingposttussive vomitingorany person diagnosed as pertussis by a physician or apnea episodes in infantsisolation of b. pertussis nucleic acids of b. pertussis b. pertussis specific antibody response epidemiologic link by human - to - human transmissionpossible case : any person with clinical criteria probable case : person with clinical criteria and epidemiologic link confirmed case : person meeting the clinical and laboratory criteriaaustralia , 2004coughing 2 weeks or paroxysms of coughing or inspiratory whoop or posttussive vomitingculture of b. pertussis pcr for b. pertussis seroconversion or significant increase of antibodies ( without recent vaccination ) single iga titer to whole cells detection of b. pertussis by dfaprobable case : any person with clinical criteria confirmed case : person meeting the clinical and laboratory criteria or epidemiologic linkabbreviations : cdc , centers for disease control and prevention ; cste , council of state and territorial epidemiologists ; dfa , direct fluorescent antibody ; eu , european union ; iga , immunoglobin a ; iu , international units ; pcr , polymerase chain reaction ; pt , pertussis toxin ; who , world health organization . selected presently used pertussis case definitions paroxysms ( ie , fits ) of coughing inspiratory whooping posttussive vomiting ( ie , vomiting immediately after coughing ) without other apparent cause paroxysms of coughing or inspiratory whoopor absence of laboratory confirmation , and no epidemiologic linkage to a laboratory- confirmed case of pertussis paroxysms of coughing or inspiratory whoop or pcr positive for pertussis or contact with a laboratory - confirmed case of pertussis > 100 iu / ml of anti - pt antibodies > 3 year from vaccination or 100% change in the antibody titer between 2 serologies at 1-month interval paroxysmal cough of any duration cough with inspiratory whoop cough ending in vomiting or gagging , or associated with apnea paroxysmal cough of any duration cough with inspiratory whoop cough ending in vomiting or gagging , or associated with apnea isolation of b. pertussis from an appropriate clinical specimen or detection of b. pertussis dna from an appropriate clinical specimen 1 of the following : - cough lasting 2 weeks - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending with vomiting or gagging , or associated with apnea or - cough lasting 2 weeks - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending with vomiting or gagging , or associated with apnea or epidemiologic link to a laboratory- confirmed case and 1 of the following for which there is no other known cause : - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending in vomiting or gagging , or associated with apnea - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending in vomiting or gagging , or associated with apnea inspiratory whooping abbreviations : cdc , centers for disease control and prevention ; cste , council of state and territorial epidemiologists ; dfa , direct fluorescent antibody ; eu , european union ; iga , immunoglobin a ; iu , international units ; pcr , polymerase chain reaction ; pt , pertussis toxin ; who , world health organization . the eu also accepts any physician 's diagnosis of pertussis and apnea as a clinically defining symptom in infants . almost all case definitions require laboratory or epidemiologic linkage data , and such data may affect whether the case is categorized as confirmed , probable , or possible . differences are also found concerning confirmation by serology : the cdc definition does not include serology , the who definition requires paired serology , and the eu definition elegantly compromises by requiring a b. pertussis specific antibody response . recently , ghanaie and associates studied the sensitivity and specificity of the who pertussis clinical case definition in 328 children aged 614 years with a persistent cough for 2 weeks . as the entry criterion for this study was cough of > 2 weeks duration , the mean duration of cough in the study population was 20 days , and because diagnosis was made by pcr without serologic study , it is likely that cases were missed . in children with a persistent cough that was characterized as paroxysmal , the sensitivity was 86% and the specificity was 23% ; persistent cough with posttussive vomiting had a sensitivity of 70% and a specificity of 61% ; persistent cough with whooping gave a sensitivity of 50% and a specificity of 74% . to obtain clinical case definition data in adolescents and adults , wang and harnden also used the data in the prospective pertussis surveillance study of strebel et al . evaluated 15 clinical case definitions for pertussis during community outbreaks and concluded that a definition of 14 days of cough was both sensitive ( 77%91% ) and specific ( 54%71% ) for monitoring culture - positive cases . however , in nonoutbreak situations , the use of their case definitions had low sensitivity . the present clinical case definitions of pertussis are inconsistent and are not used everywhere . furthermore , resource - rich and resource - limited countries have unique problems related to the control of pertussis and its diagnosis . however , in all situations , the true burden of pertussis is unknown and is significantly underestimated . in resource - rich countries , a major priority relates to the education , awareness , and recognition of pertussis in adolescents and adults and its transmission to infants [ 1517 ] . in order to improve the awareness and recognition of the disease in these populations , awareness of proper sampling techniques for obtaining nasopharyngeal specimens ( nasopharyngeal swabs , nasopharyngeal aspiration ) for culture and pcr , as well as the usefulness of single - serum serology in diagnosis should also be fostered . considerations related to adolescent and adult pertussis in resource - limited countries , pertussis burden is especially underestimated because of a number of factors , including misdiagnosis , lack of recognition , and absent requirements for notification . nevertheless , pertussis continues to be a serious health problem , especially among infants , in terms of both morbidity and mortality . in addition , because adolescent and adult pertussis is largely unrecognized , these age groups are not targeted for prevention and infected individuals are not treated , thereby facilitating spread of the disease in the community , including to vulnerable young infants . until healthcare professionals in both resource - rich and research - poor countries diagnose their adolescent and adult pertussis patients correctly , the burden of disease will continue to be significantly underestimated . without knowing that the disease predominantly occurs in this population , attempts to increase vaccine use in adolescents and adults are unlikely to be made . in persons with pertussis , the median time from cough onset to seeking medical care differs by age group . culture obtained during the first 3 weeks of cough has 100% specificity , but low sensitivity , ranging from 20% to 80% , when compared with pcr and/or serology . in general , other factors that may influence the sensitivity of culture are the type and quality of specimen , the type of transport media , and the duration of transport ( optimally within 48 hours ) . in general , by the time most adults seek medical care , the time windows for both culture and rt - pcr have passed ; therefore , serologic diagnosis should be the method of choice [ 18 , 19 ] . pt is unique to b. pertussis and is highly immunogenic ; therefore , it is the antigen that should be used for single serum diagnosis of b. pertussis cough illness . in europe , single - serum serology for the diagnosis of pertussis has been intensively studied in the netherlands , and commercial test kits with a variety of pertussis antigens and varying degrees of sensitivity and specificity are available . eu reference laboratories have recently suggested recommendations for the serologic diagnosis of pertussis ; these include mainly quantifying igg antibodies to pt and reporting results in international units / ml [ 32 , 33 ] . however , the tests with the greatest sensitivity and specificity are those that quantifiably measure igg and iga antibodies to pt ( personal clinical observation of one of the authors [ j. d. c. ] ) in recognition of the fact that the signs and symptoms of pertussis differ by age , we have tailored criteria for pertussis diagnosis in 3 different age cohorts ( 03 months , 4 months9 years , and 10 years ) . in figure 2 , clinical case definitions of pertussis for surveillance purposes are presented . figure 2.clinical case definition of pertussis for surveillance purposes . in resource - limited areas where pcr is not available , these case definitions are intended to : ( 1 ) be more specific and/or more sensitive than existing case definitions of pertussis ( which were developed more than 40 years ago and were primarily designed either for surveillance purposes or for vaccine efficacy studies ) , ( 2 ) be applicable to both resource - rich and resource - poor settings , ( 3 ) encourage the increased use of laboratory confirmation , and ( 4 ) increase the sensitivity and specificity of pertussis reporting . if a patient meets 1 or more of the criteria for pertussis diagnosis , the physician should treat the patient and report the case to the appropriate health agencies . general comments on the clinical presentation of pertussis and its laboratory diagnosis are presented in tables 4 and 5 , respectively . table 4.general comments on clinical presentation of pertussis pertussis should be increasingly suspected in patients who are afebrile with increasing cough duration and severity coryza is associated with illness onset and , in contrast with most viral respiratory infections , does not become purulent the key to identifying a paroxysmal cough is that the patient does not inhale until he has run out of breath ( possibly resulting in an inspiratory whoop) paroxysmal cough episodes are more disturbing to the patient at night among young infants , apnea and seizures may not be noted to occur with recognized paroxysms most infants with pertussis will have had a close exposure to an adolescent or adult ( usually a family member ) with a prolonged afebrile cough illness the cough in pertussis is not truly productive sweating episodes occur in adolescents and adults in time periods when coughing is not occurring table 5.general comments on laboratory diagnostics of pertussis pcr and culture are most useful in the first 3 weeks after illness onset serology should not be used to diagnose pertussis in patients < 1 year after inoculation with an acellular or whole - cell vaccine formulation igg anti - pt elisa is preferred to iga anti - pt testing because the iga response following infection is less common , and thus a negative iga anti - pt test should not be relied upon as diagnostic evidence of a pertussis infection the attendees strongly discouraged the use of dfa to detect b. pertussis . general comments on clinical presentation of pertussis general comments on laboratory diagnostics of pertussis abbreviations : dfa , direct fluorescent antibody ; elisa , enzyme - linked immunosorbent assay ; ig , immunoglobin ; pcr , polymerase chain reaction ; pt , pertussis toxin . in young infants , when these young infants are seen by physicians , they are thought to have a viral respiratory infection , and the parents are reassured . however , over the next day or two , the parents recognize the worsening of symptoms , but more often than not , the physicians do not ( based on author experience in california in 2010 [ j. d. c. ] ) . therefore , the presence of this triad would be expected to have high sensitivity and good specificity . in older children ( 4 months to 9 years ) , the presence of a worsening paroxysmal , nonproductive cough of 7 days duration in an afebrile child with coryza that has not become purulent as noted with current case definitions , the addition of whoop , apnea , and posttussive emesis will each increase specificity . in those persons 10 years of age , the same triad listed above for those 4 months to 9 years would also result in high sensitivity with good specificity . in addition , the notation of sweating episodes between paroxysms will significantly increase specificity . in dealing with adult patients adults will often say that the cough is productive , but on further questioning , it is apparent that they actually do not produce purulent sputum . the case definitions of pertussis delineated here should first be tested in clinical trials to determine their utility to the average clinician and then be compared with existing case definitions to determine whether they confer increased sensitivity and/or specificity . if the new case definitions appear promising , a prospective study should be conducted to evaluate the proposed diagnostic criteria in the 3 different age categories ( 03 months , 4 months to 9 years , 10 years ) . the development and utilization of 3 age - related definitions for pertussis can be expected to increase both the sensitivity and specificity in its diagnosis , which will result in the recognition of pertussis in all age groups , potentially leading to better control of pertussis .

the promise of diverse applications including optical data processing and biological imaging has stimulated much interest in organic nonlinear optical ( nlo ) materials.1 within this field , organotransition metal complexes offer intriguing possibilities for creating new multifunctional materials in which potentially useful optical behaviour is combined with the redox , magnetic and other properties characteristic of such compounds.2 as a means to enhance the prospects for molecular materials , approaches to modulating reversibly molecular nlo properties have attracted considerable attention recently.3 the first report of a very pronounced and reversible redox - switching of the first hyperpolarisability ( the origin of quadratic molecular nlo effects ) clearly demonstrated the potential significance of metal complexes in this area.4 this work has been extended to second harmonic generation ( shg ) in langmuir blodgett ( lb ) thin films,5 and various related solution and molecular - level theoretical studies involving both quadratic and cubic nlo effects have been described.6 although the switching of nlo responses via redox chemistry has attracted much attention , using other stimuli is also of great interest . light - induced ( photochromic ) molecular rearrangements have been explored relatively widely.3 , 7 however , the speed of switching accompanying such structural changes is often quite limited , and their effects on macroscopic structures may be substantial . populating transient electronic excited states should allow much faster modulation effects without significant structural changes . early cndo / s calculations on some simple dipolar molecules , such as 4-nitroaniline , indicated that responses can be increased in excited states,8 and these predictions were verified subsequently.9 various experimental studies with purely organic chromophores reveal similar behaviour , mostly focusing on second hyperpolarisabilities ,10 and attempts to determine excited - state values by hyper - rayleigh scattering ( hrs ) have been reported.11 however , such measurements are fraught with complications and hence there is currently no reliable experimental method by which such molecular - level excited - state responses can be determined . therefore , the establishment of accurate theoretical approaches is of significant interest , as a guide to empirical studies that will most likely focus on the switching of bulk effects like shg . some time ago , sakaguchi et al . noted a small photoinduced switching of shg at 295 nm from alternating and highly diluted lb films containing the amide - substituted [ ru(2,2-bpy)3 ] ( bpy = bipyridyl ) derivative 1 ( figure 1).12 this observation was attributed to changes in on mlct excitation . however , the ground - state ( gs ) complex shows an intense 2,2-bpy - based absorption near the shg wavelength , so excitation - induced changes in absorption may affect the shg signal . notably , very few other related experimental or theoretical studies with metal complexes have been reported to our knowledge ( and these concern only responses).13 a ruthenium complex ( 1 ) studied previously for ps timescale photoswitching of shg in lb films,12 and the complexes 24 considered in the present work . the [ ru(nh3)5 ] complexes that we have studied as redox - switchable nlo chromophores4 , 5 show intense mlct bands in the visible region . consequently , they show very large responses that compare favourably with those of purely organic chromophores with moderate -conjugation lengths.2i , 14 these systems are therefore highly attractive subjects for photoswitching studies , and we describe here theoretical investigations , which indicate that mlct excitations lead to large changes in molecular nlo responses . the use of time - dependent density functional theory ( td - dft ) and ab initio methods to predict hyperpolarisabilities of gs chromophores is now well developed,15 but relevant considerations of electronic excited states are restricted to polarisabilities and/or small molecules.16 complex 2 is well studied,17 and stark spectroscopy in a 1:1 glycerol / water glass has been used to derive a modest static first hyperpolarisability 0 ( < 10 esu ) for the salt [ bf4]3.18 we have used hrs in acetonitrile ( mecn ) solutions and stark measurements in butyronitrile ( prcn ) glasses to afford substantially larger 0 values ( hrs / stark , 10 esu ) of 246/240 for [ pf6]319 , 20 and 744/1092 for [ pf6]3.21 note that we consistently use the so - called t - convention in this work,22 whereas previous reports1921 use the b - convention ; to convert to the t - convention , we use t=2 b . in addition , all of our values refer to the dominant component along the long molecular axis , approximately parallel to the dipole moment . the pronounced increases in nlo response on moving along the series 24 are consistent with their steadily extending -conjugated structures . in order to determine the most appropriate theoretical method for treating ru ammine complexes , we have tried various approaches to model the mlct excited states . previous gas - phase dft calculations using the b3p86 functional with the lanl2dz basis set proved qualitatively useful , but lack quantitative accuracy.21 a similar basis set combination lanl2dz(ru)/6 - 31 g * or 6 - 31+g*(h , c , n , o ) has been used by inerbaev et al.,23 and in a recent investigation by zhang and champagne.24 here , we use a larger basis set ( lanl2tz(f ) ) for ru in dft calculations , as well as different larger basis sets for various high - level ab initio methods . we provide a more detailed account of the methodology in the computational methods . comparing the new computational results with the experimental data shows that td - dft with the hybrid functionals b3lyp , b3p86 or m06 yields very good values for the first dipole - allowed excited state ( fdaes ) of 3 , when mecn solvent is included via the polarisable continuum model ( pcm ; table 1 ) . selected simulated spectra are shown in figure 2 , together with the experimental spectra of complexes 3 and 4 as their pf6 salts . comparisons with data published previously23 , 24 show that although using a larger basis set for ru does not affect the qualitative picture of the excitation properties , some significant quantitative differences are observed . selected data calculated for complexes 24 by using dft and ab initio methods , together with previously reported measured data [ a ] molecules 2 and 3 were optimised with b3lyp/6 - 31g**/lanl2tz(f ) ( ru ) , whereas 6 - 31 g * was used for 4 . all optimisations were performed in the gas phase , except for 2 , which was optimised in the gas phase and in h2o . [ b ] mecn used as solvent for 3 and 4 , h2o used for 2 . [ h ] for [ clo4]3 in h2o ( a very weak nir band at max = 855 nm is observed also),17e [ pf6]3,19 and [ pf6]3;21 max values in mecn at room temperature ; directly corresponding fos values are unavailable , so those quoted are in prcn at 77 k ( only slight variations due to changing the solvent and temperature are expected).20 a ) normalised electronic absorption spectra of 2 ( simulated ) and 3 ( simulated and experimental ) in solution ; all the simulated spectra are convolutions of the computed b3lyp values with a gaussian function with =2000 cm . b ) experimental and simulated electronic absorption spectra of 4 ; both cam - b3lyp and b3lyp simulated spectra are shown . the experimental data were obtained with the complex salts [ pf6]3 or [ pf6]3 in mecn.19 , 21 the excitation into the fdaes consists for 3 and 4 nearly exclusively of the homolumo transition , whereas for 2 it is a homo1lumo transition . the orbitals , shown in figure 3 for b3lyp as an example , demonstrate clearly that in each case a charge transfer from ru to the organic ligand is involved , confirming the expected mlct character . to better quantify these transitions , recently for the semiquantitative analysis of photoinduced ct processes , which is based on the differences in electron density between the ground and excited states.25 figure 4 shows the spatially - resolved density differences upon excitation for the three complexes . this model also affords estimates of the transferred charge qct and the ct distance rct , which are 0.288 e/1.571 for 2 , 0.953 e/4.218 for 3 , and 0.978 e/5.799 for 4 , evaluated for b3lyp . to put these data into perspective , we note that the corresponding values for the prototypical ct molecule 4-nitroaniline in mecn are 0.62 e/2.72 .25c the calculated rct values agree relatively well with the effective ( localised ) electron - transfer distances rab calculated from 01ab / e , where 01ab is the dipole - moment change between the diabatic states involved in the mlct transition . based on stark spectroscopic measurements , respective rab values of 3.6 and 5.7 are determined for [ pf6]3 and [ pf6]3 in prcn glasses at 77 k.20 , 21 orbitals ( b3lyp ) involved in the main transition from the gs to the fdaes for 2 ( top ) , 3 ( middle ) and 4 ( bottom ) . plots showing difference electron density between the gs and the fdaes of 2 ( top ) , 3 ( middle ) and 4 ( bottom ) ; green denotes positive differences , whereas negative differences are in red ; isocontour values 0.001 . the predicted max values agree less well with those measured for 4 , and the observed blue shift in the mlct band on moving from 3 to 4 is not reproduced in solution . however , this blue shift is predicted by the gas - phase results , albeit with a decrease in the overall accuracy of the max values , reminiscent of previous gas - phase b3p86/lanl2dz calculations.21 our new calculations using b3lyp , b3p86 or m06 give rather large discrepancies between theory and experiment for 2 . nonetheless , it is gratifying that the results obtained for 3 by using these functionals are very similar to those from high - level restricted active - space scf second - order perturbation theory ( raspt2 ) with a very large basis set and large active space ( see the computational methods for details ) , both in solution and the gas - phase . the long - range corrected functionals ( lrcfs ) cam - b3lyp,26 lc-pbe27 and wb97xb28 give max predictions less accurate than those obtained when using b3lyp , b3p86 or m06 for all three complexes ( see also figure 2 b ) . these results are quite surprising because one of the reasons lrcfs were introduced was specifically to improve descriptions of charge - transfer excitations by standard dft functionals.2628 however , our results concur with other recent studies on organotransition metal compounds.29 for example , escudero and gonzlez29c found poor performance by cam - b3lyp and lc-pbe for mlct excitations in trans-[rucl2(2,2-bpy)(co)2 ] , when compared to experimental and raspt2 results , with more accurate predictions from hybrid functionals . the reasons for such unsatisfactory performance of lrcfs are unclear at present . although only gas - phase results are available for the fully ab initio and reasonably high - level resolution of identity approximate coupled - cluster ( ri - cc2 ) method , they differ substantially from those derived from dft and raspt2 , and notably overestimate max when compared with experiment for 2 . for 4 , ri - cc2 predicts two very close - lying transitions ( max=389 nm ) of comparable intensity , which is also not predicted by any other method . the relative failure of ri - cc2 may derive from the rather large values of the d1 diagnostic for the cc2 gs wavefunction ( 0.317 , 0.124 and 0.115 for 2 , 3 and 4 , respectively ) . values of d1 above 0.050 may indicate a large multi - configurational character of the gs , for which the single - reference method cc2 is not suitable.30 however , without taking into account the apparently large solvent effect , it is difficult to judge the performance of ri - cc2 with confidence . as a general point , we note that dft takes into account the non - dynamical correlation , although only partially and in a non - systematic way.31 this aspect may explain why the method performs better for complex 3 than for 2 , which according to the d1 diagnostic from cc2 may have stronger multi - configurational character . further tests using different optimised geometries show that the geometry does not have a large influence on max or fos . this observation validates the comparisons between the results of the raspt2 calculations for 3 , which for computational efficiency reasons used a geometry of cs symmetry , and the data obtained from the other methods that used a c1 symmetry . the relative geometry independence holds also for the nlo properties of 3 and 4 , but not for those of 2 . therefore , all of the properties of 2 were computed with the geometry optimised in the corresponding environment ( gas - phase or water solvent ) . considering that the nlo properties are very dependent on a good description of the excited - state manifold , it seems that , apart from highly accurate ( and computationally expensive ) multi - configurational methods , only the properties calculated with b3lyp , b3p86 or m06 may be at least approximately reliable . therefore , parameters for the gs and fdaes derived by using these functionals for the complexes embedded in a solvent continuum are collected in table 2 , together with cam - b3lyp results . all of the ( hyper)polarisabilities were computed by finite - field derivatives of the td - dft excited - state dipole - moments , and are thus static ( zero - frequency ) values . further data calculated for complexes 24 by using dft and raspt2 methods , together with previously reported measured data [ a ] all data calculated in h2o ( 2 ) or mecn ( 3 and 4 ) ; 3 and 4 optimised in vacuum , 2 optimised in h2o . [ b ] 2sa = two - state approximation ; 3sa = three - state approximation . calculated from 6 0iz(0i)/(e0i ) where 0i is the transition dipole - moment and e0i is the transition energy from the gs to the fdaes ( i=1 ) or sdaes ( i=2 ) ; n=1 ( 2 ) for 2sa ( 3sa ) ; an additional term for 3sa containing 12 was neglected because this property computed in the gas - phase is almost zero . the value for 4 is the total obtained by applying the 2sa to the two low - energy absorption bands separately ( the lowest energy band with mlct character involves the fdaes , whereas the other band has intraligand charge - transfer character and involves the sdaes).20 [ c ] data for [ bf4]3 taken from ref . 18 ; numbers in brackets indicate properties measured in ( or derived from data measured in ) prcn glasses . most of the fdaes properties were computed for the optimised gs geometries.32 nevertheless , in order to assess the possible effects of excited - state structural relaxation on the properties , an excited - state optimisation of 3 in the gas - phase was carried out , and properties were calculated also for the resulting geometry ( see the computational methods for details ) . the cam - b3lyp results are again clearly very different from those obtained with the other functionals . previously measured 01z and 01zz values were obtained by stark spectroscopy at 77 k in glassy prcn solution , and are thus not directly comparable to computed values in liquid mecn . nevertheless , the latter are of the right order of magnitude when using b3lyp , b3p86 or m06 . interestingly , zz is predicted to be smaller for the fdaes than for the gs for 2 and 3 , but the reverse is apparent for 4 when using b3lyp or m06.33 the calculations with b3lyp , b3p86 or m06 all predict the observed increases in 01z on moving along the series 24 , and the stark - based value of 8.8 au for 421 is closest to that obtained when using m06 . the gs first hyperpolarisability of complex 3 in the two - state approximation ( 2sa ) from raspt2(18,18 ) is very close to the value deduced from experimental hrs data ( 28 472 au).19 the corresponding values from the dft methods are considerably larger ; the reason for this can be traced back primarily to a substantially larger transition dipole - moment predicted by the dft methods , which is squared in the equation for 2sa , and is approximately 1.21.3-times larger than the raspt2 result . also , the dft calculations give slightly larger dipole - moment changes than does raspt2 . we find that the presence of diffuse functions in the basis set used to describe the first and second row atoms , which is generally of great importance for the nlo properties of the gs , has little influence on the nlo properties of the fdaes . the same can be said , to a lesser degree , of polarised functions , which generally are considered to be important for reliable descriptions of excited states . the basis set lan2tz(f ) for ru is about the minimum necessary for a reliable description of both the gs and fdaes properties . this last factor explains the main differences between our data and those obtained by inerbaev et al . and by zhang and champagne , who used the smaller basis set lan2dz.23 , 24 when considering the responses calculated by using b3lyp , b3p86 or m06 , these increase substantially ( as expected ) on increasing the -conjugation path - length along the series 24 , for both the gs and fdaes species ( table 2 ) . also , substantial enhancements are found for the fdaes with respect to the gs in each complex , with changes of similar magnitude predicted when using the three different functionals . however , the values calculated by using cam - b3lyp show less consistent trends . hence , although the gs ru - containing chromophores possess large nlo responses , the activity is even larger for the mlct excited states that formally contain ru coordinated to a reduced pyridyl ligand radical . the predicted excited - state enhancement of becomes more significant as the -conjugated system extends , being about threefold for 2 , approximately fivefold for 3 and about sevenfold for 4 . for 3 , geometry relaxation within the fdaes ( the s1 state ) leads to further considerable enhancement of , whereas increases slightly . the cubic nlo properties , denoted by the second hyperpolarisability , are generally more difficult to compute reliably with our method , and thus should be considered as more approximate . even so , they are also generally increased in the fdaes ( table 2 ) . however , unlike for , the results vary between the functionals b3lyp , b3p86 and m06 . using b3lyp or b3p86 yields enhancements of in each case , but these are much larger for 3 ( ca . in contrast , the calculations with m06 predict an excitation - induced increase in for 4 only ( ca . fourfold ) , whereas no change is evident for 3 and a decrease is found for 2 . unfortunately , no experimentally measured values are available for 24 to allow comparisons with theory , because these complexes are of interest primarily for their quadratic nlo properties . the general result that both and are larger in the fdaes is consistent with previous studies involving other types of chromophore.811 compared with previous computational studies on the electronic excitation and gs quadratic nlo properties of ru ammine complexes , we have used a larger basis set ( lanl2tz(f ) for ru ) in dft calculations . also , we have used different larger basis sets for various high - level ab initio methods . td - dft calculations with the hybrid functionals b3lyp , b3p86 or m06 and a mecn pcm yield very good agreement with the experimental spectrum for excitations to the fdaes of 3 . the same methods give a lower degree of matching with the experimental data for the shorter or longer complexes , underestimating max for 2 , but overestimating it for 4 . in each case , the calculations confirm the expected mlct character of the fdaes . a model developed by bahers et al . affords electron - transfer distances that agree well with those determined via stark spectroscopic measurements on [ pf6]3 and [ pf6]3 previously . notably , the results obtained for 3 by using b3lyp , b3p86 or m06 are very similar to those from raspt2 with a very large basis set and large active space , both in solution and the gas - phase . surprisingly , max values predicted by the long - range corrected functionals cam - b3lyp , lc-pbe and wb97xb are less accurate than those obtained with b3lyp , b3p86 or m06 for all three complexes . gas - phase results from the fully ab initio ri - cc2 method differ substantially from those derived from dft and raspt2 , possibly due to the relatively high multi - configurational character of the gs . considering both the gs and fdaes , polarisabilities and hyperpolarisabilities were computed by finite - field derivatives of the td - dft excited - state dipole - moments , by using the functionals b3lyp , b3p86 or m06 . the 01z and 01zz values are of magnitude similar to those measured previously by stark spectroscopy in the frozen solution state at 77 k. the gs 2sa value for 3 from raspt2(18,18 ) is very close to that derived experimentally by hrs , whereas the corresponding dft - based values are considerably larger , primarily due to substantially larger predicted 01 values . somewhat surprisingly , the presence of diffuse and polarised functions in the basis set used to describe the first and second row atoms has little influence on the nlo properties of the fdaes . as expected , the responses calculated by using b3lyp , b3p86 or m06 increase markedly as the -conjugation extends along the series 24 , for both the gs and fdaes species . also , substantial enhancements are found for the fdaes with respect to the gs in each complex , with the three different functionals predicting changes of similar magnitude . the excited - state enhancement of increases as the -conjugation extends , from approximately threefold for 2 to about sevenfold for 4 . although more approximate and of less interest for the complexes studied , the computed values also generally increase in the fdaes , but the results vary between the functionals b3lyp , b3p86 and m06 . in summary , state - of - the - art theoretical methods show that mlct excitation of ru ammine chromophores leads to large increases in molecular nlo responses . therefore , such complexes hold promise not only as redox - switchable species , but also for ultrafast photoswitching of bulk nlo effects in appropriate organised media , such as lb thin films . the molecules 2 and 3 were optimised with the 6 - 31 g * * basis set for chn and the lanl2tz(f ) ecp / basis set for ru at the dft level with the b3lyp functional . for 4 , the 6 - 31 g basis was used for h. molecule 2 was optimised in the gas - phase and in aqueous solution , by using the pcm , and the respective structures used for corresponding phase computations ; for 3 and 4 , gas - phase optimised structures were used for all calculations except for the raspt2 computations ( see below ) . excited - state optimisation of 3 in the gas - phase was carried out with three different basis sets for the atoms h , c , n , o : 6 - 31 g * * , 6 - 31+g * * and 6 - 31++g * * , while lanl2tz(f ) was applied to ru throughout . in order to be comparable with the other calculations , the properties were computed with the 6 - 31 g * * basis set and the pcm / mecn model . the results can be grouped into two different sets according to the basis sets underlying the excited - state optimisation . with the 6 - 31+g**-optimised structure , a small stokes shift , st , of 0.28 ev was obtained , with a large oscillator strength for the transition to s0 ( fos=0.43 ) . this is approximately in line with the results reported by zhang and champagne,24 ( st=0.25 ev , fos=0.31 ) , who apparently also used this basis set for the excited - state optimisation , although with lanl2dz for ru , the b3p86 functional and the pcm / mecn model already applied during the optimisation . however , contrary to their result of the relaxed state being s1 , we find it to be s3 , which is also the franck usually , such a highly excited state would relax into s1 by internal conversion before geometry relaxation is completed . for the 6 - 31 g * * and 6 - 31++g**-optimised structures , on the other hand , the relaxed state becomes s1 , showing large st values ( 1.2 and 1.1 ev for the 6 - 31 g * * and 6 - 31++g * * optimised structure , respectively ) and small fos values for dipolar transitions into s0 ( 0.05 for both structures ) , which is in reasonable agreement with the absence of luminescence observed experimentally . consequently , we used the 6 - 31g**-optimised structure to compute the excited - state properties . however , it should be noted that the optimisations of the second group were not fully successful in terms of the required criteria applied in gaussian 09 , even after about one hundred cycles . the best structures were finally used , where three of the four criteria were fulfilled . td - dft with several functionals was employed to compute transition moments and excited - state dipole moments , which in turn were used for finite - field numerical differentiations to obtain the hyperpolarisabilities in the fdaes . the dipole moments of the charged species are reported with respect to the centre of nuclear charge . the gs properties were calculated analytically or by finite - field differences from gs dipole moments . only the dominant long - axis component ( oriented in z - direction ) most of the pcm excited - state calculations were performed by using the state - specific description for the non - equilibrium solvation during a vertical excitation,34 although the differences when compared with the linear response non - equilibrium approximation35 were generally negligible . the transition energies were measured from the electronic gs minimum , and no vibrational averages were taken into account . it should be noted that the comparison of computed vertical excitation energies with the experimental absorption maxima is only an approximation condon factors ( overlap between the vibrational wavefunctions of ground and excited electronic states ) into account also . however , such an approach is computationally much more expensive,36 and has thus not been used here . the useable field strengths for the finite - field numerical differentiations had to be adapted for each molecule and functional individually : the lowest useful field strengths depended on the magnitude of the properties , while the high - field limit was set by field - induced state mixing and switching . the ri - cc2 calculations were carried out with turbomole.38 for the raspt2 calculations on complex 3 , its structure was first optimised at the pbe0/tzvp / mwb-28(ru ) level in cs symmetry with turbomole . although this structure is not a minimum , the symmetry was required to make the multi - configurational calculations manageable . tests with this structure at the dft level showed that the influence of the non - minimum structure on the properties of interest is minimal . state - averaged raspt2 calculations with extended ano - rcc basis sets , ( [ 8s 7p 6d 3f 2 g 1h ] ( ru ) , [ 4s 3p 2d 1f ] ( c , n ) , [ 3s 2p 1d ] ( h ) ) were then performed on this structure with molcas.39 extensive tests were performed to find the optimal active space . these led finally to the following partition : the ras2 space contains eight orbitals , five of ru 4d parentage , two of their bonding counterparts and the -orbital involved in the fdaes . single and double excitations were allowed out of ras1 and into ras3 , while all kinds of excitations were allowed in the ras2 space . for the three - state approximation of the first hyperpolarisability of 4 shown in table 2 , the excited - state dipole - moment between the fdaes and the second dipole - allowed excited state was computed with the dalton2011 package,40 in the gas phase . as the resulting value was very low , the corresponding term in the sum - over - states expression was neglected .

static excited - state polarisabilities and hyperpolarisabilities of three ruii ammine complexes are computed at the density functional theory ( dft ) and several correlated ab initio levels . most accurate modelling of the low energy electronic absorption spectrum is obtained with the hybrid functionals b3lyp , b3p86 or m06 for the complex [ ruii(nh3)5(meq+)]3 + ( meq+=n - methyl-4,4-bipyridinium , 3 ) in acetonitrile . the match with experimental data is less good for [ ruii(nh3)5l]3 + ( l = n - methylpyrazinium , 2 ; n - methyl-4-{e , e-4-(4-pyridyl)buta-1,3-dienyl}pyridinium , 4 ) . these calculations confirm that the first dipole- allowed excited state ( fdaes ) has metal - to - ligand charge - transfer ( mlct ) character . both the solution and gas - phase results obtained for 3 by using b3lyp , b3p86 or m06 are very similar to those from restricted active - space scf second - order perturbation theory ( raspt2 ) with a very large basis set and large active space . however , the time - dependent dft max predictions from the long - range corrected functionals cam - b3lyp , lc-pbe and wb97xb and also the fully ab initio resolution of identity approximate coupled - cluster method ( gas - phase only ) are less accurate for all three complexes . the ground state ( gs ) two - state approximation first hyperpolarisability 2sa for 3 from raspt2 is very close to that derived experimentally via hyper - rayleigh scattering , whereas the corresponding dft - based values are considerably larger . the responses calculated by using b3lyp , b3p86 or m06 increase markedly as the -conjugation extends on moving along the series 24 , for both the gs and fdaes species . all three functionals predict substantial fdaes enhancements for each complex , increasing with the -conjugation , up to about sevenfold for 4 . also , the computed second hyperpolarisabilities generally increase in the fdaes , but the results vary between the different functionals .

Introduction Results and Discussion Conclusion Computational Methods

the promise of diverse applications including optical data processing and biological imaging has stimulated much interest in organic nonlinear optical ( nlo ) materials.1 within this field , organotransition metal complexes offer intriguing possibilities for creating new multifunctional materials in which potentially useful optical behaviour is combined with the redox , magnetic and other properties characteristic of such compounds.2 as a means to enhance the prospects for molecular materials , approaches to modulating reversibly molecular nlo properties have attracted considerable attention recently.3 the first report of a very pronounced and reversible redox - switching of the first hyperpolarisability ( the origin of quadratic molecular nlo effects ) clearly demonstrated the potential significance of metal complexes in this area.4 this work has been extended to second harmonic generation ( shg ) in langmuir blodgett ( lb ) thin films,5 and various related solution and molecular - level theoretical studies involving both quadratic and cubic nlo effects have been described.6 although the switching of nlo responses via redox chemistry has attracted much attention , using other stimuli is also of great interest . early cndo / s calculations on some simple dipolar molecules , such as 4-nitroaniline , indicated that responses can be increased in excited states,8 and these predictions were verified subsequently.9 various experimental studies with purely organic chromophores reveal similar behaviour , mostly focusing on second hyperpolarisabilities ,10 and attempts to determine excited - state values by hyper - rayleigh scattering ( hrs ) have been reported.11 however , such measurements are fraught with complications and hence there is currently no reliable experimental method by which such molecular - level excited - state responses can be determined . however , the ground - state ( gs ) complex shows an intense 2,2-bpy - based absorption near the shg wavelength , so excitation - induced changes in absorption may affect the shg signal . the use of time - dependent density functional theory ( td - dft ) and ab initio methods to predict hyperpolarisabilities of gs chromophores is now well developed,15 but relevant considerations of electronic excited states are restricted to polarisabilities and/or small molecules.16 complex 2 is well studied,17 and stark spectroscopy in a 1:1 glycerol / water glass has been used to derive a modest static first hyperpolarisability 0 ( < 10 esu ) for the salt [ bf4]3.18 we have used hrs in acetonitrile ( mecn ) solutions and stark measurements in butyronitrile ( prcn ) glasses to afford substantially larger 0 values ( hrs / stark , 10 esu ) of 246/240 for [ pf6]319 , 20 and 744/1092 for [ pf6]3.21 note that we consistently use the so - called t - convention in this work,22 whereas previous reports1921 use the b - convention ; to convert to the t - convention , we use t=2 b . the pronounced increases in nlo response on moving along the series 24 are consistent with their steadily extending -conjugated structures . previous gas - phase dft calculations using the b3p86 functional with the lanl2dz basis set proved qualitatively useful , but lack quantitative accuracy.21 a similar basis set combination lanl2dz(ru)/6 - 31 g * or 6 - 31+g*(h , c , n , o ) has been used by inerbaev et al.,23 and in a recent investigation by zhang and champagne.24 here , we use a larger basis set ( lanl2tz(f ) ) for ru in dft calculations , as well as different larger basis sets for various high - level ab initio methods . comparing the new computational results with the experimental data shows that td - dft with the hybrid functionals b3lyp , b3p86 or m06 yields very good values for the first dipole - allowed excited state ( fdaes ) of 3 , when mecn solvent is included via the polarisable continuum model ( pcm ; table 1 ) . selected data calculated for complexes 24 by using dft and ab initio methods , together with previously reported measured data [ a ] molecules 2 and 3 were optimised with b3lyp/6 - 31g**/lanl2tz(f ) ( ru ) , whereas 6 - 31 g * was used for 4 . [ h ] for [ clo4]3 in h2o ( a very weak nir band at max = 855 nm is observed also),17e [ pf6]3,19 and [ pf6]3;21 max values in mecn at room temperature ; directly corresponding fos values are unavailable , so those quoted are in prcn at 77 k ( only slight variations due to changing the solvent and temperature are expected).20 a ) normalised electronic absorption spectra of 2 ( simulated ) and 3 ( simulated and experimental ) in solution ; all the simulated spectra are convolutions of the computed b3lyp values with a gaussian function with =2000 cm . the experimental data were obtained with the complex salts [ pf6]3 or [ pf6]3 in mecn.19 , 21 the excitation into the fdaes consists for 3 and 4 nearly exclusively of the homolumo transition , whereas for 2 it is a homo1lumo transition . to better quantify these transitions , recently for the semiquantitative analysis of photoinduced ct processes , which is based on the differences in electron density between the ground and excited states.25 figure 4 shows the spatially - resolved density differences upon excitation for the three complexes . to put these data into perspective , we note that the corresponding values for the prototypical ct molecule 4-nitroaniline in mecn are 0.62 e/2.72 .25c the calculated rct values agree relatively well with the effective ( localised ) electron - transfer distances rab calculated from 01ab / e , where 01ab is the dipole - moment change between the diabatic states involved in the mlct transition . based on stark spectroscopic measurements , respective rab values of 3.6 and 5.7 are determined for [ pf6]3 and [ pf6]3 in prcn glasses at 77 k.20 , 21 orbitals ( b3lyp ) involved in the main transition from the gs to the fdaes for 2 ( top ) , 3 ( middle ) and 4 ( bottom ) . plots showing difference electron density between the gs and the fdaes of 2 ( top ) , 3 ( middle ) and 4 ( bottom ) ; green denotes positive differences , whereas negative differences are in red ; isocontour values 0.001 . however , this blue shift is predicted by the gas - phase results , albeit with a decrease in the overall accuracy of the max values , reminiscent of previous gas - phase b3p86/lanl2dz calculations.21 our new calculations using b3lyp , b3p86 or m06 give rather large discrepancies between theory and experiment for 2 . nonetheless , it is gratifying that the results obtained for 3 by using these functionals are very similar to those from high - level restricted active - space scf second - order perturbation theory ( raspt2 ) with a very large basis set and large active space ( see the computational methods for details ) , both in solution and the gas - phase . the long - range corrected functionals ( lrcfs ) cam - b3lyp,26 lc-pbe27 and wb97xb28 give max predictions less accurate than those obtained when using b3lyp , b3p86 or m06 for all three complexes ( see also figure 2 b ) . these results are quite surprising because one of the reasons lrcfs were introduced was specifically to improve descriptions of charge - transfer excitations by standard dft functionals.2628 however , our results concur with other recent studies on organotransition metal compounds.29 for example , escudero and gonzlez29c found poor performance by cam - b3lyp and lc-pbe for mlct excitations in trans-[rucl2(2,2-bpy)(co)2 ] , when compared to experimental and raspt2 results , with more accurate predictions from hybrid functionals . although only gas - phase results are available for the fully ab initio and reasonably high - level resolution of identity approximate coupled - cluster ( ri - cc2 ) method , they differ substantially from those derived from dft and raspt2 , and notably overestimate max when compared with experiment for 2 . the relative failure of ri - cc2 may derive from the rather large values of the d1 diagnostic for the cc2 gs wavefunction ( 0.317 , 0.124 and 0.115 for 2 , 3 and 4 , respectively ) . values of d1 above 0.050 may indicate a large multi - configurational character of the gs , for which the single - reference method cc2 is not suitable.30 however , without taking into account the apparently large solvent effect , it is difficult to judge the performance of ri - cc2 with confidence . this observation validates the comparisons between the results of the raspt2 calculations for 3 , which for computational efficiency reasons used a geometry of cs symmetry , and the data obtained from the other methods that used a c1 symmetry . therefore , all of the properties of 2 were computed with the geometry optimised in the corresponding environment ( gas - phase or water solvent ) . considering that the nlo properties are very dependent on a good description of the excited - state manifold , it seems that , apart from highly accurate ( and computationally expensive ) multi - configurational methods , only the properties calculated with b3lyp , b3p86 or m06 may be at least approximately reliable . therefore , parameters for the gs and fdaes derived by using these functionals for the complexes embedded in a solvent continuum are collected in table 2 , together with cam - b3lyp results . calculated from 6 0iz(0i)/(e0i ) where 0i is the transition dipole - moment and e0i is the transition energy from the gs to the fdaes ( i=1 ) or sdaes ( i=2 ) ; n=1 ( 2 ) for 2sa ( 3sa ) ; an additional term for 3sa containing 12 was neglected because this property computed in the gas - phase is almost zero . the value for 4 is the total obtained by applying the 2sa to the two low - energy absorption bands separately ( the lowest energy band with mlct character involves the fdaes , whereas the other band has intraligand charge - transfer character and involves the sdaes).20 [ c ] data for [ bf4]3 taken from ref . most of the fdaes properties were computed for the optimised gs geometries.32 nevertheless , in order to assess the possible effects of excited - state structural relaxation on the properties , an excited - state optimisation of 3 in the gas - phase was carried out , and properties were calculated also for the resulting geometry ( see the computational methods for details ) . the cam - b3lyp results are again clearly very different from those obtained with the other functionals . nevertheless , the latter are of the right order of magnitude when using b3lyp , b3p86 or m06 . interestingly , zz is predicted to be smaller for the fdaes than for the gs for 2 and 3 , but the reverse is apparent for 4 when using b3lyp or m06.33 the calculations with b3lyp , b3p86 or m06 all predict the observed increases in 01z on moving along the series 24 , and the stark - based value of 8.8 au for 421 is closest to that obtained when using m06 . the gs first hyperpolarisability of complex 3 in the two - state approximation ( 2sa ) from raspt2(18,18 ) is very close to the value deduced from experimental hrs data ( 28 472 au).19 the corresponding values from the dft methods are considerably larger ; the reason for this can be traced back primarily to a substantially larger transition dipole - moment predicted by the dft methods , which is squared in the equation for 2sa , and is approximately 1.21.3-times larger than the raspt2 result . we find that the presence of diffuse functions in the basis set used to describe the first and second row atoms , which is generally of great importance for the nlo properties of the gs , has little influence on the nlo properties of the fdaes . the basis set lan2tz(f ) for ru is about the minimum necessary for a reliable description of both the gs and fdaes properties . and by zhang and champagne , who used the smaller basis set lan2dz.23 , 24 when considering the responses calculated by using b3lyp , b3p86 or m06 , these increase substantially ( as expected ) on increasing the -conjugation path - length along the series 24 , for both the gs and fdaes species ( table 2 ) . also , substantial enhancements are found for the fdaes with respect to the gs in each complex , with changes of similar magnitude predicted when using the three different functionals . however , the values calculated by using cam - b3lyp show less consistent trends . the predicted excited - state enhancement of becomes more significant as the -conjugated system extends , being about threefold for 2 , approximately fivefold for 3 and about sevenfold for 4 . however , unlike for , the results vary between the functionals b3lyp , b3p86 and m06 . the general result that both and are larger in the fdaes is consistent with previous studies involving other types of chromophore.811 compared with previous computational studies on the electronic excitation and gs quadratic nlo properties of ru ammine complexes , we have used a larger basis set ( lanl2tz(f ) for ru ) in dft calculations . td - dft calculations with the hybrid functionals b3lyp , b3p86 or m06 and a mecn pcm yield very good agreement with the experimental spectrum for excitations to the fdaes of 3 . the same methods give a lower degree of matching with the experimental data for the shorter or longer complexes , underestimating max for 2 , but overestimating it for 4 . in each case , the calculations confirm the expected mlct character of the fdaes . notably , the results obtained for 3 by using b3lyp , b3p86 or m06 are very similar to those from raspt2 with a very large basis set and large active space , both in solution and the gas - phase . surprisingly , max values predicted by the long - range corrected functionals cam - b3lyp , lc-pbe and wb97xb are less accurate than those obtained with b3lyp , b3p86 or m06 for all three complexes . gas - phase results from the fully ab initio ri - cc2 method differ substantially from those derived from dft and raspt2 , possibly due to the relatively high multi - configurational character of the gs . considering both the gs and fdaes , polarisabilities and hyperpolarisabilities were computed by finite - field derivatives of the td - dft excited - state dipole - moments , by using the functionals b3lyp , b3p86 or m06 . the 01z and 01zz values are of magnitude similar to those measured previously by stark spectroscopy in the frozen solution state at 77 k. the gs 2sa value for 3 from raspt2(18,18 ) is very close to that derived experimentally by hrs , whereas the corresponding dft - based values are considerably larger , primarily due to substantially larger predicted 01 values . somewhat surprisingly , the presence of diffuse and polarised functions in the basis set used to describe the first and second row atoms has little influence on the nlo properties of the fdaes . as expected , the responses calculated by using b3lyp , b3p86 or m06 increase markedly as the -conjugation extends along the series 24 , for both the gs and fdaes species . also , substantial enhancements are found for the fdaes with respect to the gs in each complex , with the three different functionals predicting changes of similar magnitude . the excited - state enhancement of increases as the -conjugation extends , from approximately threefold for 2 to about sevenfold for 4 . although more approximate and of less interest for the complexes studied , the computed values also generally increase in the fdaes , but the results vary between the functionals b3lyp , b3p86 and m06 . for 4 , the 6 - 31 g basis was used for h. molecule 2 was optimised in the gas - phase and in aqueous solution , by using the pcm , and the respective structures used for corresponding phase computations ; for 3 and 4 , gas - phase optimised structures were used for all calculations except for the raspt2 computations ( see below ) . excited - state optimisation of 3 in the gas - phase was carried out with three different basis sets for the atoms h , c , n , o : 6 - 31 g * * , 6 - 31+g * * and 6 - 31++g * * , while lanl2tz(f ) was applied to ru throughout . in order to be comparable with the other calculations , the properties were computed with the 6 - 31 g * * basis set and the pcm / mecn model . this is approximately in line with the results reported by zhang and champagne,24 ( st=0.25 ev , fos=0.31 ) , who apparently also used this basis set for the excited - state optimisation , although with lanl2dz for ru , the b3p86 functional and the pcm / mecn model already applied during the optimisation . however , contrary to their result of the relaxed state being s1 , we find it to be s3 , which is also the franck usually , such a highly excited state would relax into s1 by internal conversion before geometry relaxation is completed . for the 6 - 31 g * * and 6 - 31++g**-optimised structures , on the other hand , the relaxed state becomes s1 , showing large st values ( 1.2 and 1.1 ev for the 6 - 31 g * * and 6 - 31++g * * optimised structure , respectively ) and small fos values for dipolar transitions into s0 ( 0.05 for both structures ) , which is in reasonable agreement with the absence of luminescence observed experimentally . only the dominant long - axis component ( oriented in z - direction ) most of the pcm excited - state calculations were performed by using the state - specific description for the non - equilibrium solvation during a vertical excitation,34 although the differences when compared with the linear response non - equilibrium approximation35 were generally negligible . for the three - state approximation of the first hyperpolarisability of 4 shown in table 2 , the excited - state dipole - moment between the fdaes and the second dipole - allowed excited state was computed with the dalton2011 package,40 in the gas phase . as the resulting value was very low , the corresponding term in the sum - over - states expression was neglected .

imbalances in proinflammatory and anti - inflammatory immunomodulatory pathways can promote autoimmune responses that manifest as chronic inflammatory conditions . diarthrodial joints ( those with cartilage - capped surfaces , an intervening space filled with viscous fluid , and a synovium - lined capsule ) are one major target of autoimmune attack . the classic immune - mediated joint disease in humans is rheumatoid arthritis ( ra ) . the impact of this ailment on both individuals and society at large is immense . relative to healthy individuals , ra patients have three times greater direct healthcare costs and are also two times more likely to require hospitalization and ten times more likely to be disabled [ 1 , 2 ] . the exact etiology ( cause ) and pathogenesis ( mechanisms ) of autoimmune joint diseases are uncertain . current thinking is that the primary arthropathic immunological defects may include constitutive activation of immune surveillance cells resulting in persistent relative overproduction of proinflammatory [ 47 ] and proerosive [ 810 ] cytokines and abnormal recognition of self - antigens as nonself due to their similarity with a foreign protein [ 3 , 1113 ] . the nature of the immunoregulatory disturbance differs among individuals , a fact indicated by the divergent responses of ra patients to cytokine - specific biopharmaceutical inhibitors [ 7 , 14 ] . thus , ra is actually a syndrome in which a common set of structural changes is provoked by one or more of several cellular / molecular aberrations . multiple factors including age , gender ( hormonal status ) , genetic background , and environmental conditions influence the molecular events that regulate the onset and persistence of ra in people . various rodent models of immune - mediated arthritis ( rmia ) have become the standard means of evaluating hypothetical mechanisms of immune - mediated joint disease and for testing the comparative efficacy of novel antiarthritic drug candidates during preclinical development [ 16 , 17 ] . third , a subset of these rmia will be recommended as the most appropriate surrogates for human ra and a rationale given for this selection . fourth , procedures for the reliable production and assessment of the recommended rmia will be described . finally , practical principles that must be considered during rmia selection and experimental design during preclinical drug development will be defined . the larger joint size in nonrodent models such as rabbits [ 1820 ] or nonhuman primates [ 21 , 22 ] may be the more appropriate model for preclinical investigation for some purposes . nonetheless , this paper does not address analysis of immune - mediated joint disease in nonrodent models because they are used less commonly than rmia . many rmia have been evaluated during the past five decades as potential models for evaluating immune - mediated joint injury . some are suitable chiefly for evaluating cellular and molecular mechanisms of disease , while others may be employed to investigate both arthritis mechanisms and antiarthritic efficacy . the available rmia options may be categorized in several fashions , including by affected species ( rat , mouse , and guinea pig ) , disease type ( genetically engineered , induced , or spontaneous ) , and inciting agent ( e.g. , chemicals , collagen , or exogenous polysaccharides / proteins / proteoglycans ) . this section uses all these classification schemes to provide a brief overview of possible and preferred rmia . rats and mice are the most common rmia used for contemporary arthritis investigation and are the focus of the current review . guinea pigs are employed occasionally in immune - mediated arthritis research , primarily to explore basic mechanisms [ 18 , 2326 ] . first , their inexpensiveness , small size , and receptiveness to group housing substantially reduce research costs relative to studies in nonrodents . second , many different rodent stocks and inbred strains may be used to assess the impact of biological heterogeneity on arthritis progression . rodent strain - specific [ 27 , 28 ] and even substrain - specific genetic attributes as well as divergent immunological capacities [ 30 , 31 ] can modulate the extent and severity of immune - mediated diseases ; importantly , an analysis of conserved chromosomal homology among rats , mice , and humans suggests that arthritis susceptibility loci are highly conserved across these species [ 27 , 28 , 32 ] . third , modern techniques allow deliberate alteration of the rodent genome to evaluate molecular mechanisms that regulate immune - mediated joint disease ( e.g. , [ 3336 ] ) . fourth , procedures for initiating and evaluating rmia have been well characterized and may be undertaken with inexpensive laboratory equipment ( see below ) . fifth , as mice ( and rats ) are the main species used for immunological research , numerous complementary reagents ( e.g. , cytokines and anticytokine antibodies ) are readily available for these two species . finally , the animal models of ra with a well - proven track record for predicting whether or not novel antiarthritic agents might be useful in human patients are all performed in rodents [ 16 , 37 ] . the closest resemblance to ra among the induced rmia is the joint lesions in collagen - induced arthritis ( cia ) [ 38 , 39 ] . for each rodent species , susceptibility to immune - mediated arthritis is limited to certain strains and stocks . for rats , the inbred da and lewis strains are susceptible to arthritis induction ( figure 1 ) , while the inbred bn and f344 ( fisher ) strains are relatively resistant . arthritis - sensitive mice include b10.q , b10.riii , and dba/1 inbred strains [ 40 , 41 ] . susceptible rodent strains are not equally vulnerable to all arthritogenic agents . for example , the da rat develops aia after one subcutaneous injection of incomplete freund 's adjuvant ( ifa ) , while other rat strains are not sensitive to this very weak agent . furthermore , even within the sensitive da rat strain , arthritis susceptibility is not uniform , as different substrains exhibit varying propensities for developing common induced forms of immune - mediated arthritis . the same genetic manipulation may produce polyarthritis in one strain without causing lesions in another ; an example is the genetically engineered null mutation in interleukin-1 receptor antagonist ( il-1ra ) , where the knockout genotype yields a clinically prominent arthritis phenotype in balb / c mice but does not affect c57bl/6 mice . arthritis is more severe and prolonged if the inciting agent is a self - antigen , such as mouse type ii collagen , rather than an exogenous arthritogen [ 42 , 43 ] . these distinctions indicate that the suitability of each rmia should be reestablished each time it is imported into a new research facility and again on each occasion when the animal supplier is changed . three broad classes of rmia may be used to evaluate disease mechanisms and/or the potential efficacy of novel antiarthritic molecules . these categories are induced , genetically engineered , and spontaneous disease . in general , mechanistic studies may be undertaken using rmia from any of these classes , while efficacy studies performed during preclinical drug development usually employ one or more induced models ( sometimes supplemented with a well - chosen genetically engineered model ) . induced rmiathese models result from administration of an exogenous material and can be separated into several categories depending on the type of insult [ 44 , 45 ] . the first option , adjuvant - induced arthritis ( aia ) , results from intradermal administration of various oil - based chemicals . the traditional example is administration of heat - killed mycobacterium tuberculosis in ifa ( aia - myc ) , but comparable lesions result from introduction of various chemicals in ifa including avridine , heptadecane , lipoidal amine , pristane , or squalene , or by injection of ifa alone [ 46 , 47 ] . rats are susceptible to all these aia models , while mice are resistant to classic aia variants except for pristane . the second alternative , collagen - induced arthritis ( cia ) , is elicited reliably in both rats and mice by hyperimmunization with homologous or heterologous type ii collagen in ifa . the third option is injection of bacterial cell wall peptidoglycan ( polysaccharide ) fragments [ 50 , 51 ] . the classic example is streptococcal cell wall - induced arthritis ( scw ) , although other entities like lactobacillus or mycoplasma cell wall fragments , -glucan , and lipopolysaccharide are also arthritogenic [ 47 , 52 , 53 ] . the bacterial fragments may be injected locally ( i.e. , intra - articular ) or systemically . disease severity and progression in rats is greatest for aia , intermediate for cia , and least for scw ( figure 1 ) . adaptations of aia , cia , and to a lesser extent scw are the current workhorse rmia for preclinical drug development ( table 1).other induced rmia are used less frequently for product registration but still have considerable value for investigating mechanistic questions . the fourth induced rmia option is to administer an exogenous protein into the joint of an antigen - immunized animal to produce antigen - induced arthritis ( antia ) . in this model , initial subcutaneous immunization induces antibody production , after which subsequent intra - articular introduction of the antigen attracts antibodies into the joint . classic antigens for antia include bovine serum albumin , ovalbumin , horseradish peroxidase , and keyhole limpet hemocyanin [ 5456 ] . persistence within the synovium of many of these antigens , especially those with a cumulative positive charge , appears to be a major factor in the development of chronic synovitis . a fifth induced rmia , antibody - induced arthritis ( abia ) , arises following local ( intra - articular ) or systemic introduction of monoclonal antibodies ( typically a multiagent cocktail ) directed against type ii collagen or other self antigens that are highly expressed in joints [ 18 , 5861 ] . in this model , antibodies must enter the joint to encounter their antigen . these models typically present as a mild , acute lesion relative to the joint alterations produced by other inciting agents . a sixth alternative is to inject proinflammatory cytokines directly into a joint ( usually the tibiotarsal joint ( knee ) due to its large volume ) to induce an acute synovitis [ 62 , 63 ] . interestingly , il-1 and tnf- produce distinct , time - dependent patterns of acute arthritis in the rat knee following direct injection . thus , this rmia has utility not only to investigate proinflammatory mechanisms but also as a means of predicting potential schedules , relative potencies , and comparative efficacies of various inhibitors of cytokine blockers.two other induced rmia provoke subcutaneous lesions that serve as faux joints rather than immune - mediated disease of the rodent 's own diarthroidal joints . the first variant involves surgical implantation of human joint tissue normal or inflamed articular cartilage and/or synovium into scid ( severe combined immunodeficiency ) mice [ 6466 ] . the second option is introduction of sterile air into the subcutis to initiate a pouch granuloma , the wall of which exhibits many similarities to synovium [ 68 , 69 ] . subsequent injection of such molecules as carrageenan , -globulin , streptococcal cell wall fragments , or zymosan incites an inflammatory response in the pouch that morphologically resembles acute and chronic synovitis . both xenografts and air pouches can be employed to evaluate the efficacy of therapeutic agents on synovitis and cartilage degeneration [ 73 , 74 ] . advantages of these two rmia are their conceptual simplicity , their ability to incorporate tissue from normal and ra - affected human joints , and their large dimensions ( relative to the size of rodent joints ) . however , their main disadvantages are that these subcutaneous sites do not recapitulate the normal joint structure ( because they usually lack cartilage ) and function ( because they do not bear weight ) . these models result from administration of an exogenous material and can be separated into several categories depending on the type of insult [ 44 , 45 ] . the first option , adjuvant - induced arthritis ( aia ) , results from intradermal administration of various oil - based chemicals . the traditional example is administration of heat - killed mycobacterium tuberculosis in ifa ( aia - myc ) , but comparable lesions result from introduction of various chemicals in ifa including avridine , heptadecane , lipoidal amine , pristane , or squalene , or by injection of ifa alone [ 46 , 47 ] . rats are susceptible to all these aia models , while mice are resistant to classic aia variants except for pristane . the second alternative , collagen - induced arthritis ( cia ) , is elicited reliably in both rats and mice by hyperimmunization with homologous or heterologous type ii collagen in ifa . the third option is injection of bacterial cell wall peptidoglycan ( polysaccharide ) fragments [ 50 , 51 ] . the classic example is streptococcal cell wall - induced arthritis ( scw ) , although other entities like lactobacillus or mycoplasma cell wall fragments , -glucan , and lipopolysaccharide are also arthritogenic [ 47 , 52 , 53 ] . the bacterial fragments may be injected locally ( i.e. , intra - articular ) or systemically . disease severity and progression in rats is greatest for aia , intermediate for cia , and least for scw ( figure 1 ) . adaptations of aia , cia , and to a lesser extent scw are the current workhorse rmia for preclinical drug development ( table 1 ) . other induced rmia are used less frequently for product registration but still have considerable value for investigating mechanistic questions . the fourth induced rmia option is to administer an exogenous protein into the joint of an antigen - immunized animal to produce antigen - induced arthritis ( antia ) . in this model , initial subcutaneous immunization induces antibody production , after which subsequent intra - articular introduction of the antigen attracts antibodies into the joint . classic antigens for antia include bovine serum albumin , ovalbumin , horseradish peroxidase , and keyhole limpet hemocyanin [ 5456 ] . persistence within the synovium of many of these antigens , especially those with a cumulative positive charge , appears to be a major factor in the development of chronic synovitis . a fifth induced rmia , antibody - induced arthritis ( abia ) , arises following local ( intra - articular ) or systemic introduction of monoclonal antibodies ( typically a multiagent cocktail ) directed against type ii collagen or other self antigens that are highly expressed in joints [ 18 , 5861 ] . in this model , antibodies must enter the joint to encounter their antigen . these models typically present as a mild , acute lesion relative to the joint alterations produced by other inciting agents . a sixth alternative is to inject proinflammatory cytokines directly into a joint ( usually the tibiotarsal joint ( knee ) due to its large volume ) to induce an acute synovitis [ 62 , 63 ] . interestingly , il-1 and tnf- produce distinct , time - dependent patterns of acute arthritis in the rat knee following direct injection . thus , this rmia has utility not only to investigate proinflammatory mechanisms but also as a means of predicting potential schedules , relative potencies , and comparative efficacies of various inhibitors of cytokine blockers . two other induced rmia provoke subcutaneous lesions that serve as faux joints rather than immune - mediated disease of the rodent 's own diarthroidal joints . the first variant involves surgical implantation of human joint tissue normal or inflamed articular cartilage and/or synovium into scid ( severe combined immunodeficiency ) mice [ 6466 ] . the second option is introduction of sterile air into the subcutis to initiate a pouch granuloma , the wall of which exhibits many similarities to synovium [ 68 , 69 ] . subsequent injection of such molecules as carrageenan , -globulin , streptococcal cell wall fragments , or zymosan incites an inflammatory response in the pouch that morphologically resembles acute and chronic synovitis . both xenografts and air pouches can be employed to evaluate the efficacy of therapeutic agents on synovitis and cartilage degeneration [ 73 , 74 ] . advantages of these two rmia are their conceptual simplicity , their ability to incorporate tissue from normal and ra - affected human joints , and their large dimensions ( relative to the size of rodent joints ) . however , their main disadvantages are that these subcutaneous sites do not recapitulate the normal joint structure ( because they usually lack cartilage ) and function ( because they do not bear weight ) . genetically engineered rmiathese models have been deliberately constructed by gene targeting ( mice only ) or transgenic technology to overproduce , underexpress , or lack one or more immunoregulatory molecules ( ligands or receptors ) ( table 1 ) . in most instances , engineered rmia are generally used for basic experiments to explore proposed molecular mechanisms although they can be employed to evaluate the efficacy of therapeutic candidates designed to impact a particular immunoregulatory pathway . important transgenic mouse models of arthritis express human major histocompatibility complex ( mhc ) class ii allele hla - dr [ 35 , 75 , 76 ] or overexpress proinflammatory cytokines like tumor necrosis factor- ( tnf ; [ 34 , 77 ] ) or enzymes that degrade articular components ( e.g. , matrix metalloproteinases ( mmp ) ; ) or critical t lymphocyte receptors [ 36 , 79 ] . other significant mouse arthritis models have been constructed to lack endogenous cytokine inhibitors such as il-1ra [ 33 , 80 ] . in some instances , engineered mouse models of arthritis carry two or more genetic alterations ; a well - known example is the k / bxn mouse , which expresses both a human t - cell receptor transgene ( designated krn ) and the human mhc class ii molecule a(g7 ) [ 36 , 61 ] . the principal genetically engineered rat arthritis model carries transgenes for both the human mhc class i allele hla - b27 and human 2-microglobulin [ 81 , 82 ] and develops an immune - mediated joint disease which more closely resembles ankylosing spondylitis in humans rather than ra . defective function of all these molecules has been linked in humans to ra and ankylosing spondylitis [ 7 , 8385 ] . symmetrical polyarthritis in genetically engineered rodents develops at various times among the different models , with the span required for 100% penetrance ranging from 3 to 4 weeks of age in tnf-transgenic mice to 13 weeks of age in il-1ra knockout mice . the hock ( ankle ) joints appear to be the earliest and most commonly targeted sites in most models although other regions such as the interphalangeal ( toe ) and coxofemoral ( hip ) joints are attacked as well or even preferentially in some models . these models have been deliberately constructed by gene targeting ( mice only ) or transgenic technology to overproduce , underexpress , or lack one or more immunoregulatory molecules ( ligands or receptors ) ( table 1 ) . in most instances , engineered rmia are generally used for basic experiments to explore proposed molecular mechanisms although they can be employed to evaluate the efficacy of therapeutic candidates designed to impact a particular immunoregulatory pathway . important transgenic mouse models of arthritis express human major histocompatibility complex ( mhc ) class ii allele hla - dr [ 35 , 75 , 76 ] or overexpress proinflammatory cytokines like tumor necrosis factor- ( tnf ; [ 34 , 77 ] ) or enzymes that degrade articular components ( e.g. , matrix metalloproteinases ( mmp ) ; ) or critical t lymphocyte receptors [ 36 , 79 ] . other significant mouse arthritis models have been constructed to lack endogenous cytokine inhibitors such as il-1ra [ 33 , 80 ] . in some instances , engineered mouse models of arthritis carry two or more genetic alterations ; a well - known example is the k / bxn mouse , which expresses both a human t - cell receptor transgene ( designated krn ) and the human mhc class ii molecule a(g7 ) [ 36 , 61 ] . the principal genetically engineered rat arthritis model carries transgenes for both the human mhc class i allele hla - b27 and human 2-microglobulin [ 81 , 82 ] and develops an immune - mediated joint disease which more closely resembles ankylosing spondylitis in humans rather than ra . defective function of all these molecules has been linked in humans to ra and ankylosing spondylitis [ 7 , 8385 ] . symmetrical polyarthritis in genetically engineered rodents develops at various times among the different models , with the span required for 100% penetrance ranging from 3 to 4 weeks of age in tnf-transgenic mice to 13 weeks of age in il-1ra knockout mice . the hock ( ankle ) joints appear to be the earliest and most commonly targeted sites in most models although other regions such as the interphalangeal ( toe ) and coxofemoral ( hip ) joints are attacked as well or even preferentially in some models . spontaneous rmianaturally occurring animal models with immune - mediated joint disease resembling human ra are rare ( table 1 ) . the classic example is the mrl / mpj - lpr / lpr ( mrl / lpr ) mouse , which develops a systemic autoimmune disease that includes joint involvement . polyarthritis with pannus formation and cartilage degeneration primarily involving the hind limbs develops by five months of age in association with antibodies to type ii collagen [ 86 , 87 ] . onset of the condition is associated with several immunoregulatory deficiencies including macrophage activation [ 88 , 89 ] and altered production of various cytokines [ 90 , 91 ] . naturally occurring animal models with immune - mediated joint disease resembling human ra are rare ( table 1 ) . the classic example is the mrl / mpj - lpr / lpr ( mrl / lpr ) mouse , which develops a systemic autoimmune disease that includes joint involvement . polyarthritis with pannus formation and cartilage degeneration primarily involving the hind limbs develops by five months of age in association with antibodies to type ii collagen [ 86 , 87 ] . onset of the condition is associated with several immunoregulatory deficiencies including macrophage activation [ 88 , 89 ] and altered production of various cytokines [ 90 , 91 ] . all rmia have pathologic features that are reminiscent to some degree of the typical lesions observed in the human ra joint . the rmia resulting from systemic exposure to an arthritogen generally affect multiple joints and usually develop first in the hind paws [ 87 , 9294 ] . in contrast , targeted induction of rmia by direct injection of an agent into a single joint usually yields a monoarticular disease [ 6 , 41 , 62 ] . indeed , some researchers use the contralateral joint as an untreated control tissue although this practice is questionable since lesions also may be induced in the uninjected knee . the structural appearance of affected joints in rmia exhibits an overlapping spectrum of changes , the exact nature of which depends on both the inciting agent and the length of time over which arthritis has been allowed to progress . findings may be classified using structural effects ( inflammation , skeletal damage , and vascular changes ) or temporal criteria ( acute ( early ) and chronic ( late ) clinical stages ) . acute lesions [ 59 , 62 , 95 ] are characterized by substantial soft tissue edema , an extensive influx of neutrophils with lesser numbers of mononuclear leukocytes ( chiefly lymphocytes and macrophages ) , abundant extravasation of fibrin , and modest synovial hyperplasia and skeletal erosion . progression over time results in chronic lesions characterized by substantial synovial hyperplasia , production of fibrovascular tissue sheets ( pannus ) that extend from the synovium into the joint space , matrix degeneration in the articular cartilage , and extensive infiltration of perivascular soft tissues with a mixed inflammatory cell infiltrate in which lymphocytes , macrophages , and plasma cells predominate ; neutrophils , fibrin , and soft tissue edema are understated if present at all in such established lesions [ 8 , 93 , 94 , 96 ] . skeletal erosion begins one to two days after the paw swelling associated with acute synovitis develops [ 93 , 94 ] . left untreated , the extent of cartilage matrix degeneration ( specifically proteoglycan loss in the articular surface ) and bone attrition increases rapidly over time [ 8 , 93 , 94 , 98 ] . the widespread formation of osteophytes along the periosteal surface in some rmia ( aia > cia the bone marrow , including that in the cores of osteophytes , in some rmia ( aia cia > scw ) contains myriad inflammatory cells and activated osteoclasts early during disease . however , over time both these cell populations regress to be replaced by fibrous connective tissue or fat . vascular proliferation in the synovium and periarticular soft tissues is a prominent component of some rmia [ 100 , 101 ] . for example , arthritis in aia - myc in lewis rats reliably occurs early in the tibiotarsal and intertarsal joints ( hock ( or ankle ) ) of the hind paws and also in the femorotibial joints ( knee ) ( figure 2 ) but does not develop in the forepaws until much later . lesions in lewis rats with aia - la are evident in the knee but not in the ankle ( figure 2 ) or forepaws . in contrast , joints of both the fore paws and the hind paws are involved in lewis rats with cia . the degree of hind paw swelling is much greater for both aia models than it is for cia ( figure 3 ) or scw . as in ra , immune - mediated polyarthritis in rmia extra - articular structural changes observed in rat aia include autoimmune reactions at other sites ( e.g. , blood vessels , brain , and uvea of the eye ) , bone loss in the axial skeleton , bone marrow hyperplasia ( accompanied by multilineage leukocytosis ) , and reactive hyperplasia with enlargement of regional lymph nodes and spleen [ 10 , 94 , 103 ] . in contrast , the main extra - articular effects observed in rat cia are concomitant bone loss in the axial skeleton and reactive hyperplasia but not enlargement limited to the regional lymph nodes . significant systemic effects are not evident in rat scw . the pathologic presentation of rmia differs distinctly from that of human ra in several critical respects . first , rodents exhibit more prominent damage to the articular surface ( full - thickness cartilage matrix degeneration with later cartilage dissolution ; ) and adjacent bone ( ranging from partial erosion to complete penetration of the original cortex accompanied by exuberant formation of periosteal osteophytes ; [ 8 , 94 , 105 ] ) . these changes are evident even in those rmia in which pannus is a less prominent element of the joint lesion ( e.g. , aia - myc , acute scw ) . another difference is the accelerated progression of joint damage in rmia ( days to a few weeks ) relative to disease evolution in ra joints ( months to years ) . corollaries to this rapid advancement in rmia are that the nature of the inflammatory changes at the time of peak joint damage is subacute ( i.e. , includes a greater influx of neutrophils and more edema ) relative to ra , and that the severe destabilization of massively eroded joints in rmia leads to early and extensive ankylosis , which is rarely seen in human adult ra . finally , a much greater degree of osteoclast production is evident in affected joints in rmia . initial onset of synovitis in rmia results from innate immunity , but disease progression is a consequence of both cell - mediated ( th1-type ) and humoral ( antibody - based , or th2-type ) immune responses . the degree to which these systems regulate lesion evolution varies among models , but disease is most severe when both cellular and humoral branches are invoked . both innate immunity ( via early infiltration by neutrophils and later production of macrophages ) and acquired immunity ( through expansion of sensitized b- and t - lymphocyte lineages ) are involved . the initial intra - articular driving force in induced rmia appears to be deposition of exogenous antigens in synovial blood vessels except for cia , where the earliest event is thought to be production of anticollagen antibodies leading to immune complex deposition on and in the articular cartilage . all rmia as well as ra are driven by cell - mediated immunity , reflecting the activity of sensitized autoimmune t lymphocytes of the t - helper ( th ) phenotype . this dependence is shown by the ability to pass rat aia [ 11 , 108 , 109 ] as well as rat and mouse cia to nonimmunized ( naive ) recipients by transferring sensitized t - cells from affected donors and by the inability to induce aia or scw in athymic rats . a major function of the sensitized autoimmune cells is recognition of type ii collagen as a substrate . humoral immunity resulting from b - lymphocyte activity and plasma cell proliferation is a feature of many rmia and ra . anticollagen antibodies are found in cia [ 114 , 115 ] and in some [ 116 , 117 ] but not all aia variants , as well as in many ra patients [ 118 , 119 ] . indeed , activity of b - lymphocytes is absolutely required for cia induction as joint disease does not develop in b - cell - deficient mice . accumulation of immune complexes both in articular cartilage and in circulation is a common feature of many rmia [ 87 , 121 , 122 ] . introduction of anticollagen antibodies in the absence of sensitized lymphocytes can induce arthritis , although in the absence of t - helper cells to boost the immune response , the antibody - driven disease is transient and mild [ 114 , 123 ] ; this outcome suggests that the role of humoral immunity in rmia may be subsidiary to that played by the cell - mediated immune response . antibodies to type ii collagen are not observed in some rmia , such as bacterial cell wall - induced arthritis , and the presence of such antibodies in rmia does not automatically mean that they contribute significantly to joint destruction . the balance between cellular and humoral immunity in rmia , as in ra , is attributed to variations in cytokine expression patterns in immunoregulatory cells , particularly t - helper ( th ) lymphocytes . auto - immune diseases have been postulated to be dependent on th1 cells ( which regulate cell - mediated processes marshaled to counter tumor cells and intracellular pathogens ) more so than on th2 cells ( which generally drive humoral immune responses to vanquish extracellular organisms ) . expression of th1 versus th2 cytokines in aia changes over time and is subject to control by sensory innervation . overactivation of either th pathway can cause disease , and either pathway can downregulate the other . furthermore , other classes of th cells have been described , some of which play a role in autoimmune arthritis ( e.g. , th17 cells ) . while the original th1/th2 hypothesis of immune control was developed in mice , the recent literature reveals that cytokine patterns in rodent and human diseases seldom follow an exclusive th1-inducing or th2-inducing pattern . a complex web of chemokines and cytokines controls the immune response in joint tissue under normal circumstances . the balance between the functions of these molecules determines whether or not intra - articular inflammatory responses are transient and reparative or persistent and destructive . in general , immune - mediated joint disease results from overproduction of proinflammatory ( e.g. , il-1 , il-6 , il-17 , and tnf- ) and proerosive ( e.g. , receptor activator of nfb ligand ( rankl ) ) factors , hyperactivity of proinflammatory and proerosive signaling pathways , or a reduction in cytokines ( e.g. , il-4 , il-10 , and transforming growth factor- ( tgf- ) ) and soluble receptors ( e.g. , il-1ra , soluble tnf receptor ( stnfr ) ) that antagonize the proinflammatory response [ 127129 ] . abnormalities in the local ( intra - articular and/or periarticular ) or systemic ( circulating ) levels of such mediators have been reported in ra and many rmia , including aia , cia , and scw . the large number of mediators involved in regulating immune - mediated arthritis implies that one or a few molecules may serve as , early expression of which is the main upstream incident that induces all other events needed to launch and sustain arthritis . the master proinflammatory cytokines which appear to drive immune - mediated joint disease in ra and rmia are il-1 ( especially the inducible form ) and tnf- [ 132135 ] , possibly il-6 , and perhaps others . a good indicator of a master cytokine in a distinct rmia or human disease is superior antiarthritic efficacy by a cytokine inhibitor as exemplified for cia ( more il-1 dependent ) and aia ( more tnf dependent ) . however , although these master cytokines can act individually as arthritogens , they can also act together in synergistic fashion to potentiate joint inflammation ; for example , il-1 [ 4 , 137 ] and il-17 exhibit synergistic activity with tnf-. other cytokines such as il-12 , il-15 , il-17 , and il-18 [ 139141]as well as chemokines , such as chemokine ( c - c motif ) ligand 2 ( ccl2 ) and chemokine ( c - x - c motif ) ligand 8 ( cxcl8 ) , have also been implicated as additional participants in immune - mediated arthritis but are not thought to function as master cytokines , at least not in the immune - mediated joint diseases studied thus far . alterations in the balance between pro- and anti - inflammatory molecules occur locally and systemically in advance of the clinical onset of immune - mediated joint disease . in induced rmia , such changes can be demonstrated by four days after inoculation of the arthritogen , which is between five to ten days prior to initial joint swelling [ 27 , 93 , 94 ] . proinflammatory molecules required to initiate synovitis are upregulated earlier than proerosive mediators [ 93 , 94 ] ; this temporal sequence matches the evolution of structural changes within arthritic joints , where evidence of acute inflammation ( edema , leukocyte infiltration , and fibrin extravasation ) precedes visible damage to skeletal structures ( osteoclast proliferation , cartilage matrix degeneration , and skeletal erosion ) . the increase in local and systemic chemokine and cytokine levels is accompanied by enhanced production of other molecules that enhance inflammation ( e.g. , cyclooxygenase-2 and prostaglandin e2 ( pge2 ) ) , skeletal destruction ( e.g. , mmps ) , and vascular expansion ( e.g. , platelet - derived growth factor and vascular endothelial growth factor ) [ 27 , 144 ] . despite some overlap in their cytokine signatures , different rmia for example , rats with aia or cia both produce enhanced levels of il-1 , il-1 , and tgf- locally ( inside inflamed joints ) in conjunction with higher systemic levels of 1 acid glycoprotein , ccl2 , and tgf- prior to arthritis onset , and both models also exhibit higher intra - articular amounts of ccl2 , il-6 , pge2 , rankl , and tgf- with more circulating ccl2 , il-1 , il-1 , il-6 , and rankl [ 93 , 94 ] . in contrast , prior to arthritis onset , the pro - arthritic signature of male lewis rats with aia includes no mediator enhancement that was unique to joints but did have systemic increases in ccl2 , il-17 , tgf- , and tnf- , while female lewis rats with cia have locally higher levels of ccl2 and il-18 with systemic elevation of cxcl1 ( former designation : kc / gro ) . after clinical arthritis becomes evident , male rats with aia uniquely exhibit local enrichment for il-17 and tgf- in conjunction with systemic increases in il-17 , il-18 , and tnf- , whereas the distinctive profile in female rats with cia includes local augmentation of il-18 and cxcl1 with systemic amplification of il-1 . thus , systemic and local processes in immune - mediated arthritis are discrete processes in lewis rats , driven by multiple mediators with distinct spatiotemporal patterns of expression . hypothesis applies not only to events in the joint but also to immunological functions triggered throughout the individual . this latter premise is supported by the fact that the two major proinflammatory cytokines were confined to separate compartments in rmia ; il-1 is restricted chiefly to arthritic joints , while tnf- is limited mainly to the circulation [ 93 , 94 , 128 ] . both the hypothalamic - pituitary - gonadal ( hpg ) axis and the hypothalamic - pituitary - adrenal ( hpa ) axis are instrumental in regulating immune responsiveness , and thus the susceptibility to immune - mediated arthritis . adrenal ( glucocorticoids ) and gonadal ( androgens and estrogens ) hormones act as natural immune suppressors , so a deficiency of one or more of these molecules permits enhanced immune reactivity . females appear to rely more on the hpa axis while males seem to depend more on the hpg axis . the lapse in immune control by deficiencies in certain hormonal axes particularly enhances cell - mediated ( th1-type ) events . the impact of gender on the sensitivity to immune - mediated arthritis is readily apparent in the clinical setting . the incidence and severity of joint disease is higher in females for ra and most rmia [ 27 , 28 ] . exceptions to this female bias are abia and cia in mice and cia in rats , where males may exhibit a higher incidence and develop more severe disease than do females . administration of estradiol or castration of male rats results in a higher susceptibility to scw . for example , scw - susceptible lewis rats have small adrenal glands and a markedly impaired ability to release corticotropin and corticosterone ( products of the hpa axis ) relative to scw - resistant f344 animals . the hpa axis is also a player in aia in rats as well as cia in rats and mice . persistent production of stress hormones ( e.g. , cortisol ) and sympathetic system ( emergency ( fight or flight ) ) neurotransmitters , such as norepinephrine , boosts the baseline homeostatic state into a constant condition of relative proinflammatory readiness . despite this trend , the stress of handling has also been shown to reduce the sensitivity to cia induction , indicating that due care must be exercised to maintain identical husbandry practices to avoid confounding hormonal fluctuations among treatment groups . a reasonable practice in some settings will be to include untreated controls as well to ensure that a profound stress - induced deviation in the vehicle - treated cohort does not impede the ability to differentiate antiarthritic efficacy from handling stress . basic research may be pursued using any rmia , but where available a mouse model that has been genetically engineered to overexpress or lack a particular gene often provides the most straightforward means of testing a molecule - specific hypothesis . examples include the use of il-1ra knockout mice [ 33 , 80 ] or tnf - transgenic mice [ 34 , 77 , 151 , 152 ] to assess the impact of il-1 ( without interference by its endogenous soluble receptor , il-1ra ) or excessive tnf- on arthritis progression , respectively . for example , essentially 100% of animals induced to develop aia will actually present with acute disease by 9 days after initial inoculation with the arthritogen , and 100% of rats treated to produced chronic scw will undergo reactivation of residual disease within 1 to 2 days of reinjecting the arthritogen . such reliable induction permits treatment groups to be completely filled at one time . in contrast , the incidence of cia in lewis rats varies from 60% to 90% across laboratories , and arthritis develops over approximately a 5-day period usually starting 11 days after immunization . accordingly , treatment groups in cia studies must be enrolled over time , which complicates the treatment matrix of the study . both genetically engineered and spontaneous rmia also tend to require sporadic enrollment as the onset of disease may be spread over days to weeks . the third factor will be cost . genetically engineered and spontaneous disease models may be quite expensive . spontaneous arthritis in mrl / lpr mice typically does not strike until five or more months of age , which will increase the husbandry cost relative to shorter models . the most suitable rmia for preclinical development are the induced diseases that have been proven to predict the responsiveness of human ra patients : aia , cia , and scw [ 37 , 153 ] ( table 2 ) . the structural lesions in cia are more analogous to human ra [ 38 , 39 ] . however , aia has been used more extensively for pharmaceutical testing , so more data exist for cross - species comparison of antiarthritic efficacy . in our experience , all three of these models may be reliably elicited in rats , while mice develop cia but are relatively resistant to classic aia and scw [ 28 , 44 ] . as noted above , mouse cia exhibits a more variable disease pattern relative to the rat counterpart , and murine joints are very small . accordingly , we recommend that preclinical efficacy studies of antiarthritic agents in rmia be undertaken in rats . several other practical reasons recommend rat models of arthritis over mouse models if equivalent systems are available in both species ( e.g. , cia ) . one reason for this position is that rats are larger , so certain procedures are more readily accomplished in this species . ante mortem examples include intra - articular injections and blood sampling ( for biomarker analysis ) , while the most critical postmortem instance is tissue trimming ( to consistently orient joints for histologic sectioning ) . a second reason for preferring rats is that the distribution and extent of inflammatory changes in arthritic joints is more reproducible , typically developing first as a symmetric swelling of both hind paws before progressing to both fore paws [ 93 , 94 ] . in contrast , mice inoculated with an arthritogenic agent typically present first with modest swelling of only one fore paw or one hind paw and often only of a single interphalangeal ( toe ) joint . a third reason for favoring rats is that knowledge regarding the many genes that control induction of immune - mediated arthritis is better understood in this species [ 27 , 28 ] . potential disadvantages of using rat arthritis models include the larger specimen size , thereby requiring removal of the phalanges ( with loss of their joints to analysis ) and more cassettes for histologic processing and the more limited ability for genetic engineering ( especially gene targeting ( knockout ) ) in rats versus mice . these drawbacks are minor when compared to the benefits offered by the more reproducible and easily manipulated rat - based rmias . this section briefly summarizes the experimental procedures employed by our laboratory to reproducibly generate and analyze rmia for an industrial preclinical drug development program . the workhorse models are rat aia - myc , rat cia , and to a much lesser degree rat scw ( table 2 ) . these rmia were selected because of their widespread acceptance , and especially because of their utility as surrogates for predicting the response of ra patients to antiarthritic treatments [ 37 , 153 ] . novel molecules are tested first in aia - myc ( as rats develop arthritis in a highly predictable time frame and with a very uniform morphologic pattern ) and then in rat cia ( due to its greater degree of similarity to ra ) . drug candidates are tested in scw , and occasionally in mouse cia , only to provide more data for comparing the relative efficacy of lead candidates slated for human clinical trials ( table 3 ) . all in vivo rmia should be undertaken in accredited facilities ( e.g. , by the association for assessment and accreditation of laboratory animal care international ( aaalac ) ) in accordance with appropriate regulatory guidelines ( e.g. , guide for the care and use of laboratory animals ( the national academies press , washington , d.c . ) ) . humane endpoints and veterinary care needs should be clearly outlined in animal care and use protocols and approved by the institutional animal care and use committee in advance . in general , analgesics can not be given because their anti - inflammatory activities tend to inhibit arthritis induction and progression . instead , additional veterinary support such as fluid therapy , easier access to nutritional sources ( e.g. , food pellets placed at floor level ) , and/or supplemental cushioning may be provided during advanced stages of disease . the most humane practice , which is always followed in our laboratory , is to limit the length of rmia to the shortest possible time required to answer a given experimental question . this period is usually one week after the onset of clinical disease in our aia , cia , and scw models . adjuvant - induced arthritisthis model is produced in young adult ( 7- to 8-week - old ) , male lewis rats . animals are inoculated with a single intradermal injection at the tail base of heat - killed mycobacterium tuberculosis h37ra ( 0.5 mg ; difco laboratories , detroit , mi ) suspended in 0.05 ml paraffin oil ( crescent chemical co. , hauppauge , ny ) . animals are acclimated for one week and then randomly assigned to treatment groups ( n = 6 to 8) . this group size is used because interindividual variability in the initial disease severity and the day of onset is minimal . arthritis reliably develops in both hind paws of 100% of the animals on the 9th day after arthritogen inoculation .the course of rat aia - myc can be separated into three stages using a combination of macroscopic and microscopic findings . preclinical phase extends from the day of arthritogen administration until the day of disease onset ( which is designated study day 0 ) . the acute clinical phase , encompassing the peak of active disease , extends from study day 0 to study day 10 and is characterized by body weight loss , progressive inflammation and skeletal erosion in the hind paws and knees ( figure 2(c ) ) , and the onset of disease in the forepaws ( typically at study day 7 ) . phase represents all times beyond study day 11 , at which time lesions have stabilized . leukocyte and osteoclast numbers begin to regress by approximately four weeks after disease onset , presumably because the total loss of articular cartilage and extensive destruction of adjacent bone has removed the inciting antigen . this model is produced in young adult ( 7- to 8-week - old ) , male lewis rats . animals are inoculated with a single intradermal injection at the tail base of heat - killed mycobacterium tuberculosis h37ra ( 0.5 mg ; difco laboratories , detroit , mi ) suspended in 0.05 ml paraffin oil ( crescent chemical co. , hauppauge , ny ) . animals are acclimated for one week and then randomly assigned to treatment groups ( n = 6 to 8) . this group size is used because interindividual variability in the initial disease severity and the day of onset is minimal . arthritis reliably develops in both hind paws of 100% of the animals on the 9th day after arthritogen inoculation . the course of rat aia - myc can be separated into three stages using a combination of macroscopic and microscopic findings . preclinical phase extends from the day of arthritogen administration until the day of disease onset ( which is designated study day 0 ) . the acute clinical phase , encompassing the peak of active disease , extends from study day 0 to study day 10 and is characterized by body weight loss , progressive inflammation and skeletal erosion in the hind paws and knees ( figure 2(c ) ) , and the onset of disease in the forepaws ( typically at study day 7 ) . phase represents all times beyond study day 11 , at which time lesions have stabilized . leukocyte and osteoclast numbers begin to regress by approximately four weeks after disease onset , presumably because the total loss of articular cartilage and extensive destruction of adjacent bone has removed the inciting antigen . collagen - induced arthritisthis model is performed in young adult ( 7- to 8-week - old ) , female lewis rats . animals are immunized by intradermal injection of emulsified porcine type ii collagen ( chondrex , redmond , wa ) in ifa at ten different sites ( 100 l per site ) over the back ; other researchers use a lesser number of inoculations ( e.g. , four ) , which result in less aggressive joint involvement . the arthritogen is prepared by dissolving collagen ( 10 mg ) in 0.1 n acetic acid ( 5 ml ) two days prior to use while stirring on a rotating plate in the refrigerator . the collagen is then emulsified 1 : 1 with ifa ( difco laboratories ) , yielding a final concentration of 1 mg / ml , using an emulsification needle and glass syringes ( popper and sons , new hyde park , ny ) . animals are acclimated for one week and then randomly assigned to treatment groups ( n = 8) . arthritis develops in at least one hind paw , and usually both , in 80% to 100% of the animals between the 9th and the 11th day after injection of the arthritogen.the course of rat cia also can be separated into three stages using a combination of macroscopic and microscopic findings . preclinical phase extends from the day of collagen immunization until the day of disease onset in the hind paws ( designated study day 0 ) . the acute clinical phase , which encompasses the peak of active disease , extends from study day 0 to study day 14 and is characterized by body weight loss , progressive inflammation and skeletal erosion in the hind paws , and the onset of disease in the forepaws ( typically at study day 7 ) . phase represents all times beyond study day 14 , at which time lesions have plateaued in both forepaws and hind paws . this model is performed in young adult ( 7- to 8-week - old ) , female lewis rats . animals are immunized by intradermal injection of emulsified porcine type ii collagen ( chondrex , redmond , wa ) in ifa at ten different sites ( 100 l per site ) over the back ; other researchers use a lesser number of inoculations ( e.g. , four ) , which result in less aggressive joint involvement . the arthritogen is prepared by dissolving collagen ( 10 mg ) in 0.1 n acetic acid ( 5 ml ) two days prior to use while stirring on a rotating plate in the refrigerator . the collagen is then emulsified 1 : 1 with ifa ( difco laboratories ) , yielding a final concentration of 1 mg / ml , using an emulsification needle and glass syringes ( popper and sons , new hyde park , ny ) . animals are acclimated for one week and then randomly assigned to treatment groups ( n = 8) . arthritis develops in at least one hind paw , and usually both , in 80% to 100% of the animals between the 9th and the 11th day after injection of the arthritogen . the course of rat cia also can be separated into three stages using a combination of macroscopic and microscopic findings . preclinical phase extends from the day of collagen immunization until the day of disease onset in the hind paws ( designated study day 0 ) . the acute clinical phase , which encompasses the peak of active disease , extends from study day 0 to study day 14 and is characterized by body weight loss , progressive inflammation and skeletal erosion in the hind paws , and the onset of disease in the forepaws ( typically at study day 7 ) . the chronic clinical phase represents all times beyond study day 14 , at which time lesions have plateaued in both forepaws and hind paws . streptococcal cell wall - induced arthritisthis model is undertaken in young adult ( 7- to 8-week - old ) , female lewis rats using purified peptidoglycan - polysaccharide ( pg - ps ) cell wall polymers isolated from streptococcus pyogenes , group a , d58 strain ( pg - ps 100p ; lee laboratories , grayson , ga ) . the arthritogen is prepared by suspending pg - ps ( 0.09 ml , containing 600 g of rhamnose ) and phosphate - buffered saline ( pbs , 0.91 ml ) by sonication for 10 minutes , after which it is used immediately . rats are anesthetized deeply to permit direct intra - articular injection of the pg - ps suspension ( 10 l , containing 6 g of rhamnose ) into one tibiotarsal ( ankle ) joint per animal using a 30-gauge needle ; the intra - articular injection is repeated if the diameter of the induced hind paw does not equal or exceed 7.0 mm after 24 hours . animals are maintained for 4 weeks ( to allow acute inflammation to subside into chronic disease ) , after which they are randomized by ankle diameter into treatment groups ( n = 8) . arthritis is reactivated on day 29 or 30 after the initial intra - articular induction by intravenous injection of pg - ps / pbs suspension ( 35 l , containing about 200 g of rhamnose ) . arthritis develops in the injected hind paw in 90% to 100% of the animals between the 1st and the 2nd days after disease reactivation . paw swelling peaks at 72 hours after reactivation and then regresses rapidly over the next several days . alternatively , scw polyarthritis may be induced by parenteral administration ( e.g. , intraperitoneal ) of pg - ps , with the nature of the lesions depending on how long clinical and histopathologic analyses are delayed after pg - ps administration . to our knowledge , no detailed study has been published defining the progression of either acute or chronic scw in rats . this model is undertaken in young adult ( 7- to 8-week - old ) , female lewis rats using purified peptidoglycan - polysaccharide ( pg - ps ) cell wall polymers isolated from streptococcus pyogenes , group a , d58 strain ( pg - ps 100p ; lee laboratories , grayson , ga ) . the arthritogen is prepared by suspending pg - ps ( 0.09 ml , containing 600 g of rhamnose ) and phosphate - buffered saline ( pbs , 0.91 ml ) by sonication for 10 minutes , after which it is used immediately . rats are anesthetized deeply to permit direct intra - articular injection of the pg - ps suspension ( 10 l , containing 6 g of rhamnose ) into one tibiotarsal ( ankle ) joint per animal using a 30-gauge needle ; the intra - articular injection is repeated if the diameter of the induced hind paw does not equal or exceed 7.0 mm after 24 hours . animals are maintained for 4 weeks ( to allow acute inflammation to subside into chronic disease ) , after which they are randomized by ankle diameter into treatment groups ( n = 8) . arthritis is reactivated on day 29 or 30 after the initial intra - articular induction by intravenous injection of pg - ps / pbs suspension ( 35 l , containing about 200 g of rhamnose ) . arthritis develops in the injected hind paw in 90% to 100% of the animals between the 1st and the 2nd days after disease reactivation . paw swelling peaks at 72 hours after reactivation and then regresses rapidly over the next several days . alternatively , scw polyarthritis may be induced by parenteral administration ( e.g. , intraperitoneal ) of pg - ps , with the nature of the lesions depending on how long clinical and histopathologic analyses are delayed after pg - ps administration . to our knowledge , no detailed study has been published defining the progression of either acute or chronic scw in rats . the first is by tiers , where tier 1 includes routine screening procedures while tier 2 represents specialized methods that are undertaken only as needed to answer distinct questions . the second classification scheme is to divide the tests by the time frame during which they are performed ( i.e. , antemortem versus postmortem ) . measurements of total body weight and paw swelling may be taken repeatedly over time without inflicting any wound , and blood sampling to measure serum biomarkers is minimally invasive as long as blood collection is not excessive ( no more than 0.5% of body weight , or about 1 ml of blood from a 250 g rat ) . in contrast , histopathologic analysis requires that affected joints be removed and processed prior to evaluation . routine screening tests ( tier 1)these methods are used on all rmia in our laboratory and include total body weights , measurement of hind paw swelling , calculation of bone mineral density ( bmd ) loss , and histopathology . total body weight and paw swelling are evaluated daily from the day of disease onset throughout the treatment period ( and any recovery period ) to provide a graphic representation of disease progression . paw swelling is assessed by one of two techniques : plethysmography , which calculates the degree of paw volume expansion by computing the weight of fluid displaced by the swollen limb upon immersion in a water - filled beaker balanced on a commercial scale , or caliper measurements to define the diameter of the affected joint(s ) . plethysmography is best suited for massively swollen aia hind paws and calipers for more modestly swollen hind paws in cia and scw , but either technique may be applied successfully to any of these rat models.after necropsy , the hind paws are removed at the fur line ( just proximal to the hock ( ankle ) ) . one paw is stored in 70% ethanol at room temperature until dual x - ray absorptiometry ( dxa ) can be used to assess bmd loss . the degree of bmd reduction is dictated by the extent of leukocyte infiltration as osteoclast recruitment along bony surfaces is induced by numerous proinflammatory cytokines ; accordingly , bone dissolution is more severe in aia ( figure 4 ) , where leukocyte influx is more severe than in cia or scw . the other paw is fixed by immersion in either 70% ethanol ( to serve as a potential backup sample for dxa or a substrate for molecular pathology studies ) or either neutral buffered 10% formalin or zinc formalin ( to provide the best morphologic preservation of infiltrating leukocytes ) at room temperature for 72 to 96 hours . fixed specimens are decalcified in eight serial changes of a 1 : 4 mixture of 8 n formic acid and 1 n sodium formate for approximately a week ; more rapid decalcification may be achieved by using a 1 : 1 mixture , but delicate tissue antigens may be too degraded for subsequent molecular pathology analysis . the digits ( toes ) are then removed from the demineralized hind paws by cutting across the metatarsals ( the arch of the foot ) midway between the tip of the toes and the hock , after which the paw is divided longitudinally into approximately equal halves by cutting just lateral to the tibia and between the 2nd and the 3rd digits ( figure 5 ) . the two halves are placed into a single cassette and processed into paraffin using routine procedures . embedded specimens are faced to expose the distal tibia and entire talus , after which several serial 4- to 8-m - thick sections are cut.routine histopathologic analysis for tier 1 is performed using one to three slides . the first slide is stained with hematoxylin and eosin ( h&e ) for evaluation of general structural characteristics such as leukocyte infiltration , skeletal erosion , periosteal proliferation , and ( in cia only ) pannus production ( figure 6 ) . the second slide may be processed by indirect immunohistochemistry or in situ hybridization to detect the osteoclast marker cathepsin k . if desired , cathepsin k immunohistochemistry may be followed by an h&e counterstain to avoid the need for the h&e - stained slide ; this procedure may be automated for higher throughput . the third slide is stained with either safranin o or toluidine blue ( figure 6 ) to evaluate the degree of matrix integrity in articular cartilage . as the extent of inflammation increases , the articular cartilage loses matrix proteins and can no longer bind dye [ 8 , 156 ] . in practice , the loss of cartilage matrix is so advanced in chronic arthritis that this latter stain may be omitted if the disease has been present for more than five ( for aia ) to ten ( for cia ) days . histopathologic data for tier 1 screening is performed using semiquantitative grading scales ( normal joint , or minimal , mild , moderate , or marked disease ) , which can be rapidly gathered by an experienced pathologist ( 10 to 60 seconds per section for all stains , depending on the complexity of the lesion ) . these methods are used on all rmia in our laboratory and include total body weights , measurement of hind paw swelling , calculation of bone mineral density ( bmd ) loss , and histopathology . total body weight and paw swelling are evaluated daily from the day of disease onset throughout the treatment period ( and any recovery period ) to provide a graphic representation of disease progression . paw swelling is assessed by one of two techniques : plethysmography , which calculates the degree of paw volume expansion by computing the weight of fluid displaced by the swollen limb upon immersion in a water - filled beaker balanced on a commercial scale , or caliper measurements to define the diameter of the affected joint(s ) . plethysmography is best suited for massively swollen aia hind paws and calipers for more modestly swollen hind paws in cia and scw , but either technique may be applied successfully to any of these rat models . after necropsy , the hind paws are removed at the fur line ( just proximal to the hock ( ankle ) ) . one paw is stored in 70% ethanol at room temperature until dual x - ray absorptiometry ( dxa ) can be used to assess bmd loss . the degree of bmd reduction is dictated by the extent of leukocyte infiltration as osteoclast recruitment along bony surfaces is induced by numerous proinflammatory cytokines ; accordingly , bone dissolution is more severe in aia ( figure 4 ) , where leukocyte influx is more severe than in cia or scw . the other paw is fixed by immersion in either 70% ethanol ( to serve as a potential backup sample for dxa or a substrate for molecular pathology studies ) or either neutral buffered 10% formalin or zinc formalin ( to provide the best morphologic preservation of infiltrating leukocytes ) at room temperature for 72 to 96 hours . fixed specimens are decalcified in eight serial changes of a 1 : 4 mixture of 8 n formic acid and 1 n sodium formate for approximately a week ; more rapid decalcification may be achieved by using a 1 : 1 mixture , but delicate tissue antigens may be too degraded for subsequent molecular pathology analysis . the digits ( toes ) are then removed from the demineralized hind paws by cutting across the metatarsals ( the arch of the foot ) midway between the tip of the toes and the hock , after which the paw is divided longitudinally into approximately equal halves by cutting just lateral to the tibia and between the 2nd and the 3rd digits ( figure 5 ) . the two halves are placed into a single cassette and processed into paraffin using routine procedures . embedded specimens are faced to expose the distal tibia and entire talus , after which several serial 4- to 8-m - thick sections are cut . the first slide is stained with hematoxylin and eosin ( h&e ) for evaluation of general structural characteristics such as leukocyte infiltration , skeletal erosion , periosteal proliferation , and ( in cia only ) pannus production ( figure 6 ) . the second slide may be processed by indirect immunohistochemistry or in situ hybridization to detect the osteoclast marker cathepsin k . if desired , cathepsin k immunohistochemistry may be followed by an h&e counterstain to avoid the need for the h&e - stained slide ; this procedure may be automated for higher throughput . the third slide is stained with either safranin o or toluidine blue ( figure 6 ) to evaluate the degree of matrix integrity in articular cartilage . as the extent of inflammation increases , the articular cartilage loses matrix proteins and can no longer bind dye [ 8 , 156 ] . in practice , the loss of cartilage matrix is so advanced in chronic arthritis that this latter stain may be omitted if the disease has been present for more than five ( for aia ) to ten ( for cia ) days . histopathologic data for tier 1 screening is performed using semiquantitative grading scales ( normal joint , or minimal , mild , moderate , or marked disease ) , which can be rapidly gathered by an experienced pathologist ( 10 to 60 seconds per section for all stains , depending on the complexity of the lesion ) . specialized tests ( tier 2)the procedures used in our laboratory are applied in addition to , not instead of , the tier 1 methods . for example , a detailed examination of local and systemic events in rmia requires evaluation of numerous parameters other than involvement of joints in the distal limbs . whole blood may be collected for hematologic counts , or to harvest serum to measure circulating levels of biomarkers and immune proteins [ 93 , 94 ] . for comparison , unfixed joints may be homogenized to extract and quantify local biomarkers [ 93 , 94 ] . extra - articular tissues ( especially hematopoietic organs like bone marrow , lymph nodes , or spleen ) may be isolated to correlate tissue leukocyte numbers to circulating cell counts by flow cytometry or histopathology [ 93 , 94 ] or to permit evaluation of systemic bone loss at sites distant from affected joints ( e.g. , lumbar vertebrae ) . diseased joints may be imaged using conventional radiography , computed tomography [ 26 , 157 , 158 ] , or magnetic resonance imaging . paws may be harvested and split longitudinally while fresh using a circular , water - cooled diamond saw ( e.g. , isomet low speed model ; buehler , lake bluff , il ) to provide for more rapid penetration of fixative ; subsequent fixation time can be reduced substantially , thereby reducing the degradation of delicate antigens and nucleic acids while retaining good tissue morphology.the routine tier 1 semiquantitative histopathologic analysis may be augmented in tier 2 by supplementary endpoints . one common approach is to include additional special stains to demonstrate other constituents in the arthritic process , such as markers for blood vessels , different populations of leukocytes , or expression of various proinflammatory and proerosive mediators . another option is to examine lesions in other joints besides those of the affected paws . in this regard , preferred choices are the knee ( figure 7 ) and the interphalangeal ( toe ) joints . considerable care must be taken to ensure that the specimen is positioned correctly during trimming so that sections are oriented in the optimal plane ; for example , a frontal view is preferred for the knee to allow maximal scrutiny of the articular surface . a final variant is to procure quantitative data from histopathologic sections [ 101 , 102 ] . such special analyses require at least some extra time , and often a great deal of it , so should not be undertaken lightly in the course of preclinical development programs . the procedures used in our laboratory are applied in addition to , not instead of , the tier 1 methods . for example , a detailed examination of local and systemic events in rmia requires evaluation of numerous parameters other than involvement of joints in the distal limbs . whole blood may be collected for hematologic counts , or to harvest serum to measure circulating levels of biomarkers and immune proteins [ 93 , 94 ] . for comparison , unfixed joints may be homogenized to extract and quantify local biomarkers [ 93 , 94 ] . extra - articular tissues ( especially hematopoietic organs like bone marrow , lymph nodes , or spleen ) may be isolated to correlate tissue leukocyte numbers to circulating cell counts by flow cytometry or histopathology [ 93 , 94 ] or to permit evaluation of systemic bone loss at sites distant from affected joints ( e.g. , lumbar vertebrae ) . diseased joints may be imaged using conventional radiography , computed tomography [ 26 , 157 , 158 ] , or magnetic resonance imaging . paws may be harvested and split longitudinally while fresh using a circular , water - cooled diamond saw ( e.g. , isomet low speed model ; buehler , lake bluff , il ) to provide for more rapid penetration of fixative ; subsequent fixation time can be reduced substantially , thereby reducing the degradation of delicate antigens and nucleic acids while retaining good tissue morphology . the routine tier 1 semiquantitative histopathologic analysis may be augmented in tier 2 by supplementary endpoints . one common approach is to include additional special stains to demonstrate other constituents in the arthritic process , such as markers for blood vessels , different populations of leukocytes , or expression of various proinflammatory and proerosive mediators . another option is to examine lesions in other joints besides those of the affected paws . in this regard , preferred choices are the knee ( figure 7 ) and the interphalangeal ( toe ) joints . considerable care must be taken to ensure that the specimen is positioned correctly during trimming so that sections are oriented in the optimal plane ; for example , a frontal view is preferred for the knee to allow maximal scrutiny of the articular surface . a final variant is to procure quantitative data from histopathologic sections [ 101 , 102 ] . such special analyses require at least some extra time , and often a great deal of it , so should not be undertaken lightly in the course of preclinical development programs . the current section will briefly examine several practical principles that must be contemplated when using the induced rmia that we recommend above . failure to consider such points may delay the launch of studies , result in their premature termination , or require their repetition . time spent in optimizing rmia up front will save significant effort , money , and time when building a research program and generating the preclinical portion of a product registration package . in general , routine rodent arthritis studies should be performed in strains that are inherently sensitive to induction of immune - mediated joint disease . in our experience , the preferred wild - type strains are lewis rats , which exhibit an intermediate susceptibility to disease ( figure 1 ) , and dba/1 mice . each laboratory will have to validate that the animals from their supplier are sufficiently vulnerable to arthritis induction and will have to ensure that genetic drift does not alter the substrain 's sensitivity over time . as shown in figure 2 , rats with aia - la and aia - myc have different patterns of lesions and divergent dose responses to antiarthritic molecules . similarly , induction of cia in mice using the weak adjuvant ifa results in reduced susceptibility to arthritis relative to animals in which the carrier was complete freund 's adjuvant ( cfa ) . the source of adjuvant may impact the extent of disease as well ( figures 1 and 3 ) . the bacterial composition may play a large role in defining the sensitivity of various rodent strains to arthritogenic stimuli . for example , arthritis - resistant f344 rats become vulnerable to aia when housed under germfree conditions . in contrast , the germfree state prevents development of inflammatory diseases in joints ( and the intestinal tract ) of hla - b27-transgenic rats [ 81 , 82 ] . in our experience , preclinical development programs are best performed in rodent strains ( such as lewis rats ) , where an intermediate susceptibility to arthritis is compatible with the presence of a normal microbial complement , thereby avoiding the need for expensive germfree husbandry practices . in general , aggressive rmia are unresponsive to weak anti - inflammatory agents like nonsteroidal anti - inflammatory agents ( nsaids)although aia is easily inhibited with cyclooxygenase inhibitors but are sensitive to more potent molecules ( e.g. , anticytokine biologics , corticosteroids , and disease - modifying antirheumatic drugs ( dmards ) ) . less destructive rmia like cia and scw are more responsive to all classes of agents . in fact , each rmia embodies one distinct disease in one individual ( inbred strain ) as contrasted to human ra , which represents a continuum of disease expressions in an outbred population . thus , the heterogeneity of pharmacological responses in rmia reflects the heterogeneity of therapeutic success in ra . the impact of antiarthritic agents on immune - mediated arthritis is dependent on several factors . higher doses of antiarthritic molecules usually produce greater reductions in arthritis parameters than do lower doses . that said , the shape of the dose - response curve will not always be linear ( figure 2 ) . the production of proinflammatory mediators waxes and wanes [ 93 , 94 , 161 ] , so achieving a therapeutic effect is dependent on when therapeutic molecules are administered particularly when cytokines and chemokines are targeted with specific inhibitors . for example , cyclosporin a is an effective immunosuppressant that can significantly inhibit hind paw swelling in rat cia ( figure 8) . however , shifting the time frame over which cyclosporin a is delivered can even potentiate disease , resulting in an earlier onset or increased severity , or delay disease onset ( figure 8) . for example , osteoprotegerin ( opg ) is a soluble receptor for the proerosive ligand rankl . administration of opg to rats with aia - myc essentially halts bone erosions even in the face of severe joint inflammation but has very little impact on the inflammatory component of disease [ 8 , 99 , 156 ] . administration of these latter two agents blocks inflammation , and as a secondary consequence it prevents skeletal damage as well . thus , the nature of anticipated therapeutic benefit may dictate the design of the experiment and/or analysis . various rodent models of arthritis are the conventional means of evaluating hypothetical mechanisms of immune - mediated joint disease and the comparative efficacy of novel drug candidates with potential antiarthritic efficacy during preclinical development . the workhorse models are polyarthritides in rats and mice induced by injecting either bacterial ( especially mycobacterial ( aia - myc ) or streptococcal ( scw ) ) fragments or collagen type ii ( usually from a nonrodent mammalian source ( cia ) ) in adjuvant . efficacy is typically evaluated by a combination of semiquantitative and quantitative techniques including clinical measurements ( e.g. , paw volume and serum biomarker concentrations ) , noninvasive imaging ( e.g. , bone density analysis and computed tomography ) , and histopathology scores ( e.g. , lesion scores for inflammation , joint erosion , osteophyte production , and cartilage degradation ) . the extent and severity of arthritis depends on both the experimental methodology ( e.g. , inciting agent , adjuvant , number , and placement of sensitizing injections ) and individual physiologic parameters ( e.g. , age , gender , and genetics ) . similarly , the effectiveness of antiarthritic molecules varies with the nature of the agent , the therapeutic regimen ( e.g. , dose , route , and schedule of treatment ) , and the choice of rodent arthritis model . in our experience , rat models of aia , cia , and scw are preferred platforms for preclinical drug development because these rat systems are more reproducible among individuals and across studies than are corresponding mouse models . of these three rat models , aia is more severe but exhibits the most consistent lesions among study mates and across studies , while cia delivers joint lesions in rodents with a histopathologic appearance that better resembles that of the human rheumatoid arthritis joint . all the rat models are driven by relative overactivity of proinflammatory and proerosive signaling cascades , but the dominant cytokines differ among the models . as with human clinical experience , the efficacy of various antiarthritic molecules differs among rodent arthritis models , especially when the agent is a specific cytokine inhibitor .

rodent models of immune - mediated arthritis ( rmia ) are the conventional approach to evaluating mechanisms of inflammatory joint disease and the comparative efficacy of antiarthritic agents . rat adjuvant - induced ( aia ) , collagen - induced ( cia ) , and streptococcal cell wall - induced ( scw ) arthritides are preferred models of the joint pathology that occurs in human rheumatoid arthritis ( ra ) . lesions of aia are most severe and consistent ; structural and immunological changes of cia best resemble ra . lesion extent and severity in rmia depends on experimental methodology ( inciting agent , adjuvant , etc . ) and individual physiologic parameters ( age , genetics , hormonal status , etc . ) . the effectiveness of antiarthritic molecules varies with the agent , therapeutic regimen , and choice of rmia . all rmia are driven by overactivity of proinflammatory pathways , but the dominant molecules differ among the models . hence , as with the human clinical experience , the efficacy of various antiarthritic molecules differs among rmia , especially when the agent is a specific cytokine inhibitor .

1. Introduction 2. Objectives of the Review 3. Objective 1: Available Rodent Models of Immune-Mediated Arthritis (RMIA) 4. Objective 2: Comparative Biology of Rodent Models of Immune-Mediated Arthritis 5. Objective 3: Recommended Rodent Models of Immune-Mediated Arthritis for Use as Rheumatoid Arthritis Surrogates in Preclinical Drug Development 6. Objective 4: Suitable Procedures for Reproducible Production and Appropriate Assessment of Rat Models of Immune-Mediated Arthritis 7. Objective 5: Considerations in Model Selection and Experimental Design 8. Summary

the classic immune - mediated joint disease in humans is rheumatoid arthritis ( ra ) . multiple factors including age , gender ( hormonal status ) , genetic background , and environmental conditions influence the molecular events that regulate the onset and persistence of ra in people . various rodent models of immune - mediated arthritis ( rmia ) have become the standard means of evaluating hypothetical mechanisms of immune - mediated joint disease and for testing the comparative efficacy of novel antiarthritic drug candidates during preclinical development [ 16 , 17 ] . nonetheless , this paper does not address analysis of immune - mediated joint disease in nonrodent models because they are used less commonly than rmia . the available rmia options may be categorized in several fashions , including by affected species ( rat , mouse , and guinea pig ) , disease type ( genetically engineered , induced , or spontaneous ) , and inciting agent ( e.g. guinea pigs are employed occasionally in immune - mediated arthritis research , primarily to explore basic mechanisms [ 18 , 2326 ] . rodent strain - specific [ 27 , 28 ] and even substrain - specific genetic attributes as well as divergent immunological capacities [ 30 , 31 ] can modulate the extent and severity of immune - mediated diseases ; importantly , an analysis of conserved chromosomal homology among rats , mice , and humans suggests that arthritis susceptibility loci are highly conserved across these species [ 27 , 28 , 32 ] . third , modern techniques allow deliberate alteration of the rodent genome to evaluate molecular mechanisms that regulate immune - mediated joint disease ( e.g. fifth , as mice ( and rats ) are the main species used for immunological research , numerous complementary reagents ( e.g. finally , the animal models of ra with a well - proven track record for predicting whether or not novel antiarthritic agents might be useful in human patients are all performed in rodents [ 16 , 37 ] . the closest resemblance to ra among the induced rmia is the joint lesions in collagen - induced arthritis ( cia ) [ 38 , 39 ] . for each rodent species , susceptibility to immune - mediated arthritis is limited to certain strains and stocks . furthermore , even within the sensitive da rat strain , arthritis susceptibility is not uniform , as different substrains exhibit varying propensities for developing common induced forms of immune - mediated arthritis . arthritis is more severe and prolonged if the inciting agent is a self - antigen , such as mouse type ii collagen , rather than an exogenous arthritogen [ 42 , 43 ] . three broad classes of rmia may be used to evaluate disease mechanisms and/or the potential efficacy of novel antiarthritic molecules . the first option , adjuvant - induced arthritis ( aia ) , results from intradermal administration of various oil - based chemicals . the traditional example is administration of heat - killed mycobacterium tuberculosis in ifa ( aia - myc ) , but comparable lesions result from introduction of various chemicals in ifa including avridine , heptadecane , lipoidal amine , pristane , or squalene , or by injection of ifa alone [ 46 , 47 ] . the second alternative , collagen - induced arthritis ( cia ) , is elicited reliably in both rats and mice by hyperimmunization with homologous or heterologous type ii collagen in ifa . the classic example is streptococcal cell wall - induced arthritis ( scw ) , although other entities like lactobacillus or mycoplasma cell wall fragments , -glucan , and lipopolysaccharide are also arthritogenic [ 47 , 52 , 53 ] . adaptations of aia , cia , and to a lesser extent scw are the current workhorse rmia for preclinical drug development ( table 1).other induced rmia are used less frequently for product registration but still have considerable value for investigating mechanistic questions . the fourth induced rmia option is to administer an exogenous protein into the joint of an antigen - immunized animal to produce antigen - induced arthritis ( antia ) . a fifth induced rmia , antibody - induced arthritis ( abia ) , arises following local ( intra - articular ) or systemic introduction of monoclonal antibodies ( typically a multiagent cocktail ) directed against type ii collagen or other self antigens that are highly expressed in joints [ 18 , 5861 ] . thus , this rmia has utility not only to investigate proinflammatory mechanisms but also as a means of predicting potential schedules , relative potencies , and comparative efficacies of various inhibitors of cytokine blockers.two other induced rmia provoke subcutaneous lesions that serve as faux joints rather than immune - mediated disease of the rodent 's own diarthroidal joints . the first option , adjuvant - induced arthritis ( aia ) , results from intradermal administration of various oil - based chemicals . the traditional example is administration of heat - killed mycobacterium tuberculosis in ifa ( aia - myc ) , but comparable lesions result from introduction of various chemicals in ifa including avridine , heptadecane , lipoidal amine , pristane , or squalene , or by injection of ifa alone [ 46 , 47 ] . the second alternative , collagen - induced arthritis ( cia ) , is elicited reliably in both rats and mice by hyperimmunization with homologous or heterologous type ii collagen in ifa . the classic example is streptococcal cell wall - induced arthritis ( scw ) , although other entities like lactobacillus or mycoplasma cell wall fragments , -glucan , and lipopolysaccharide are also arthritogenic [ 47 , 52 , 53 ] . adaptations of aia , cia , and to a lesser extent scw are the current workhorse rmia for preclinical drug development ( table 1 ) . the fourth induced rmia option is to administer an exogenous protein into the joint of an antigen - immunized animal to produce antigen - induced arthritis ( antia ) . a fifth induced rmia , antibody - induced arthritis ( abia ) , arises following local ( intra - articular ) or systemic introduction of monoclonal antibodies ( typically a multiagent cocktail ) directed against type ii collagen or other self antigens that are highly expressed in joints [ 18 , 5861 ] . two other induced rmia provoke subcutaneous lesions that serve as faux joints rather than immune - mediated disease of the rodent 's own diarthroidal joints . in most instances , engineered rmia are generally used for basic experiments to explore proposed molecular mechanisms although they can be employed to evaluate the efficacy of therapeutic candidates designed to impact a particular immunoregulatory pathway . in some instances , engineered mouse models of arthritis carry two or more genetic alterations ; a well - known example is the k / bxn mouse , which expresses both a human t - cell receptor transgene ( designated krn ) and the human mhc class ii molecule a(g7 ) [ 36 , 61 ] . the principal genetically engineered rat arthritis model carries transgenes for both the human mhc class i allele hla - b27 and human 2-microglobulin [ 81 , 82 ] and develops an immune - mediated joint disease which more closely resembles ankylosing spondylitis in humans rather than ra . in most instances , engineered rmia are generally used for basic experiments to explore proposed molecular mechanisms although they can be employed to evaluate the efficacy of therapeutic candidates designed to impact a particular immunoregulatory pathway . in some instances , engineered mouse models of arthritis carry two or more genetic alterations ; a well - known example is the k / bxn mouse , which expresses both a human t - cell receptor transgene ( designated krn ) and the human mhc class ii molecule a(g7 ) [ 36 , 61 ] . the principal genetically engineered rat arthritis model carries transgenes for both the human mhc class i allele hla - b27 and human 2-microglobulin [ 81 , 82 ] and develops an immune - mediated joint disease which more closely resembles ankylosing spondylitis in humans rather than ra . spontaneous rmianaturally occurring animal models with immune - mediated joint disease resembling human ra are rare ( table 1 ) . naturally occurring animal models with immune - mediated joint disease resembling human ra are rare ( table 1 ) . all rmia have pathologic features that are reminiscent to some degree of the typical lesions observed in the human ra joint . the structural appearance of affected joints in rmia exhibits an overlapping spectrum of changes , the exact nature of which depends on both the inciting agent and the length of time over which arthritis has been allowed to progress . as in ra , immune - mediated polyarthritis in rmia extra - articular structural changes observed in rat aia include autoimmune reactions at other sites ( e.g. , blood vessels , brain , and uvea of the eye ) , bone loss in the axial skeleton , bone marrow hyperplasia ( accompanied by multilineage leukocytosis ) , and reactive hyperplasia with enlargement of regional lymph nodes and spleen [ 10 , 94 , 103 ] . these changes are evident even in those rmia in which pannus is a less prominent element of the joint lesion ( e.g. corollaries to this rapid advancement in rmia are that the nature of the inflammatory changes at the time of peak joint damage is subacute ( i.e. , includes a greater influx of neutrophils and more edema ) relative to ra , and that the severe destabilization of massively eroded joints in rmia leads to early and extensive ankylosis , which is rarely seen in human adult ra . initial onset of synovitis in rmia results from innate immunity , but disease progression is a consequence of both cell - mediated ( th1-type ) and humoral ( antibody - based , or th2-type ) immune responses . all rmia as well as ra are driven by cell - mediated immunity , reflecting the activity of sensitized autoimmune t lymphocytes of the t - helper ( th ) phenotype . introduction of anticollagen antibodies in the absence of sensitized lymphocytes can induce arthritis , although in the absence of t - helper cells to boost the immune response , the antibody - driven disease is transient and mild [ 114 , 123 ] ; this outcome suggests that the role of humoral immunity in rmia may be subsidiary to that played by the cell - mediated immune response . antibodies to type ii collagen are not observed in some rmia , such as bacterial cell wall - induced arthritis , and the presence of such antibodies in rmia does not automatically mean that they contribute significantly to joint destruction . the balance between cellular and humoral immunity in rmia , as in ra , is attributed to variations in cytokine expression patterns in immunoregulatory cells , particularly t - helper ( th ) lymphocytes . in general , immune - mediated joint disease results from overproduction of proinflammatory ( e.g. the large number of mediators involved in regulating immune - mediated arthritis implies that one or a few molecules may serve as , early expression of which is the main upstream incident that induces all other events needed to launch and sustain arthritis . the master proinflammatory cytokines which appear to drive immune - mediated joint disease in ra and rmia are il-1 ( especially the inducible form ) and tnf- [ 132135 ] , possibly il-6 , and perhaps others . other cytokines such as il-12 , il-15 , il-17 , and il-18 [ 139141]as well as chemokines , such as chemokine ( c - c motif ) ligand 2 ( ccl2 ) and chemokine ( c - x - c motif ) ligand 8 ( cxcl8 ) , have also been implicated as additional participants in immune - mediated arthritis but are not thought to function as master cytokines , at least not in the immune - mediated joint diseases studied thus far . alterations in the balance between pro- and anti - inflammatory molecules occur locally and systemically in advance of the clinical onset of immune - mediated joint disease . in contrast , prior to arthritis onset , the pro - arthritic signature of male lewis rats with aia includes no mediator enhancement that was unique to joints but did have systemic increases in ccl2 , il-17 , tgf- , and tnf- , while female lewis rats with cia have locally higher levels of ccl2 and il-18 with systemic elevation of cxcl1 ( former designation : kc / gro ) . thus , systemic and local processes in immune - mediated arthritis are discrete processes in lewis rats , driven by multiple mediators with distinct spatiotemporal patterns of expression . both the hypothalamic - pituitary - gonadal ( hpg ) axis and the hypothalamic - pituitary - adrenal ( hpa ) axis are instrumental in regulating immune responsiveness , and thus the susceptibility to immune - mediated arthritis . the impact of gender on the sensitivity to immune - mediated arthritis is readily apparent in the clinical setting . in contrast , the incidence of cia in lewis rats varies from 60% to 90% across laboratories , and arthritis develops over approximately a 5-day period usually starting 11 days after immunization . accordingly , we recommend that preclinical efficacy studies of antiarthritic agents in rmia be undertaken in rats . a third reason for favoring rats is that knowledge regarding the many genes that control induction of immune - mediated arthritis is better understood in this species [ 27 , 28 ] . potential disadvantages of using rat arthritis models include the larger specimen size , thereby requiring removal of the phalanges ( with loss of their joints to analysis ) and more cassettes for histologic processing and the more limited ability for genetic engineering ( especially gene targeting ( knockout ) ) in rats versus mice . drug candidates are tested in scw , and occasionally in mouse cia , only to provide more data for comparing the relative efficacy of lead candidates slated for human clinical trials ( table 3 ) . adjuvant - induced arthritisthis model is produced in young adult ( 7- to 8-week - old ) , male lewis rats . the acute clinical phase , encompassing the peak of active disease , extends from study day 0 to study day 10 and is characterized by body weight loss , progressive inflammation and skeletal erosion in the hind paws and knees ( figure 2(c ) ) , and the onset of disease in the forepaws ( typically at study day 7 ) . the acute clinical phase , encompassing the peak of active disease , extends from study day 0 to study day 10 and is characterized by body weight loss , progressive inflammation and skeletal erosion in the hind paws and knees ( figure 2(c ) ) , and the onset of disease in the forepaws ( typically at study day 7 ) . collagen - induced arthritisthis model is performed in young adult ( 7- to 8-week - old ) , female lewis rats . arthritis develops in at least one hind paw , and usually both , in 80% to 100% of the animals between the 9th and the 11th day after injection of the arthritogen.the course of rat cia also can be separated into three stages using a combination of macroscopic and microscopic findings . arthritis develops in at least one hind paw , and usually both , in 80% to 100% of the animals between the 9th and the 11th day after injection of the arthritogen . streptococcal cell wall - induced arthritisthis model is undertaken in young adult ( 7- to 8-week - old ) , female lewis rats using purified peptidoglycan - polysaccharide ( pg - ps ) cell wall polymers isolated from streptococcus pyogenes , group a , d58 strain ( pg - ps 100p ; lee laboratories , grayson , ga ) . the digits ( toes ) are then removed from the demineralized hind paws by cutting across the metatarsals ( the arch of the foot ) midway between the tip of the toes and the hock , after which the paw is divided longitudinally into approximately equal halves by cutting just lateral to the tibia and between the 2nd and the 3rd digits ( figure 5 ) . in practice , the loss of cartilage matrix is so advanced in chronic arthritis that this latter stain may be omitted if the disease has been present for more than five ( for aia ) to ten ( for cia ) days . the digits ( toes ) are then removed from the demineralized hind paws by cutting across the metatarsals ( the arch of the foot ) midway between the tip of the toes and the hock , after which the paw is divided longitudinally into approximately equal halves by cutting just lateral to the tibia and between the 2nd and the 3rd digits ( figure 5 ) . in practice , the loss of cartilage matrix is so advanced in chronic arthritis that this latter stain may be omitted if the disease has been present for more than five ( for aia ) to ten ( for cia ) days . in this regard , preferred choices are the knee ( figure 7 ) and the interphalangeal ( toe ) joints . in this regard , preferred choices are the knee ( figure 7 ) and the interphalangeal ( toe ) joints . in general , routine rodent arthritis studies should be performed in strains that are inherently sensitive to induction of immune - mediated joint disease . in our experience , the preferred wild - type strains are lewis rats , which exhibit an intermediate susceptibility to disease ( figure 1 ) , and dba/1 mice . in contrast , the germfree state prevents development of inflammatory diseases in joints ( and the intestinal tract ) of hla - b27-transgenic rats [ 81 , 82 ] . in our experience , preclinical development programs are best performed in rodent strains ( such as lewis rats ) , where an intermediate susceptibility to arthritis is compatible with the presence of a normal microbial complement , thereby avoiding the need for expensive germfree husbandry practices . the impact of antiarthritic agents on immune - mediated arthritis is dependent on several factors . various rodent models of arthritis are the conventional means of evaluating hypothetical mechanisms of immune - mediated joint disease and the comparative efficacy of novel drug candidates with potential antiarthritic efficacy during preclinical development . the workhorse models are polyarthritides in rats and mice induced by injecting either bacterial ( especially mycobacterial ( aia - myc ) or streptococcal ( scw ) ) fragments or collagen type ii ( usually from a nonrodent mammalian source ( cia ) ) in adjuvant . the extent and severity of arthritis depends on both the experimental methodology ( e.g. , inciting agent , adjuvant , number , and placement of sensitizing injections ) and individual physiologic parameters ( e.g. similarly , the effectiveness of antiarthritic molecules varies with the nature of the agent , the therapeutic regimen ( e.g. , dose , route , and schedule of treatment ) , and the choice of rodent arthritis model . in our experience , rat models of aia , cia , and scw are preferred platforms for preclinical drug development because these rat systems are more reproducible among individuals and across studies than are corresponding mouse models . of these three rat models , aia is more severe but exhibits the most consistent lesions among study mates and across studies , while cia delivers joint lesions in rodents with a histopathologic appearance that better resembles that of the human rheumatoid arthritis joint . all the rat models are driven by relative overactivity of proinflammatory and proerosive signaling cascades , but the dominant cytokines differ among the models . as with human clinical experience , the efficacy of various antiarthritic molecules differs among rodent arthritis models , especially when the agent is a specific cytokine inhibitor .

this is achieved by fine - tuning of all the processes governing gene expression , including transcription , splicing , mrna transport , rna stability , translation , protein stability and posttranslational modification . all the steps within this cascade of events are subjected to their own specific regulation , and contribute to generate a different composition of the proteome by modifying not only the levels but also the identity of the proteins present in the cell under specific conditions . the components that participate in these regulatory events are often engaged in the formation of macromolecular complexes . protein - protein as well as rna - protein interactions allow a compartmentalization of the factors needed to control gene expression . translation initiation can modify the proteome by altering the efficiency of translation as well as by enabling the synthesis of different forms of a protein from specific genes . the process of rna translation includes a series of sequential steps , known as initiation , elongation , termination and ribosome recycling . most of translational control is exerted at the initiation step , assisted by specific proteins designated translation initiation factors ( eifs ) . translation initiation of most eukaryotic mrnas commences with 5 end - dependent recruitment of the 43s complex ( that is composed of a 40s subunit bound to eif2-gtp / met - trnai , eif1a , eif1 and eif3 ) by eif4f recognition of the mgpppn ( cap ) at the 5 end of the mrna . in turn , the eif4f complex comprises eif4e that physically binds to the cap , eif4a that unwinds secondary structures in the 5 untranslated region ( 5utr ) and eif4 g , a scaffold protein that interacts with eif4e , eif4a and eif3 . aided by the helicase activity of eif4a and its cofactor eif4b , the 43s pre - initiation complex scans in 5 to 3 direction until an appropriate initiation codon is encountered . auxiliary factors , eif1 , eif2 and eif5 , help to identify the correct aug start codon , resulting in the formation of the 48s complex . eif5 induces hydrolysis of eif2-bound gtp , which is recycled to the active form by eif2b ( guanine nucleotide exchange factor ) . the poly(a ) tail present in the 3utr of most mrnas synergistically stimulates the efficiency of translation through recruitment of poly(a)-binding protein ( pabp ) , enabling its interaction with eif4f located at the mrna 5 end . finally , eif5b mediates joining of the 60s and 40s subunits , generating the 80s complex competent for protein synthesis . translation initiation , particularly in viral mrnas , can occur by an alternative mechanism driven by internal ribosome entry site ( ires ) elements , discovered near 20 years ago in two picornaviruses , encephalomyocarditis virus ( emcv ) and poliovirus ( pv ) [ 2 , 3 ] . these elements are cis - acting sequences that form secondary and tertiary rna structures and recruit the translation machinery to an internal position in the mrna , bypassing a large number of stable structural elements in the viral 5utr . hence , picornavirus ires - driven initiation is 5 end - independent and does not require eif4e to recruit the 40s subunit , in contrast to the cap - dependent initiation mechanism . the subsequent discovery of an ires element in hepatitis c virus ( hcv ) rna that was able to recruit 40s ribosomal subunits in the absence of eif4 g represented a major breakthrough in the translation field [ 5 , 6 ] . this finding opened the question of how iress differing in primary sequence , rna structure , and factor requirements , perform the same function . over the last two decades , the process of internal initiation has been found to be more general than originally thought , that is , it operates in other rna and dna viruses as well as in cellular mrnas . in all cases , iress direct translation of a subset of proteins when the excellent performance of iress , together with the fact that they are active in genetically engineered constructs , has been exploited to study how these diverse rna elements perform the same function . with the exception of one or more polypyrimidine tracts , no primary sequence conservation neither overall structural similarity is detected between picornavirus and hcv iress , strongly suggesting that different strategies may be used to recruit the ribosomal subunits . these strategies could be under the control of a distinct group of proteins specifically interacting with each of these regulatory elements . in this review understanding the role played by these ires trans - acting factors ( itafs ) may help to unravel the strategies employed by mrnas to capture the translation machinery internally . translation initiation of all picornavirus rnas is dependent on the ires located in the long 5utr ( figure 1(a ) ) . various structural elements in the 5utr region , which differ among picornavirus genera , control the viral replication cycle in concerted action with the 3utr [ 811 ] . the ires region spans about 450 nucleotides immediately upstream of the functional translation start codon of the polyprotein [ 2 , 3 , 12 , 13 ] . although less than 50% of primary sequence is conserved between different members of the picornavirus family , the similarity of their secondary structure allows their classification into four types , i to iv . type i includes enterovirus ( ev , pv , hrv ) , type ii , cardiovirus ( emcv ) and aphthovirus ( foot - and - mouth disease virus , fmdv ) , type iii , is used for hepatitis a virus ( hav ) , and the hcv - like ires conforms group iv . the acquisition of a proper structural organization is a key determinant of internal translation initiation driven by picornavirus ires [ 14 , 15 ] , and all viral ires in general [ 16 , 17 ] . this feature is well illustrated by mutational and structural studies conducted on the central domain ( termed 3 or i ) of type ii ires ( figure 1(a ) ) . this region is organized as a cruciform structure with phylogenetically conserved structural motifs that are essential for ires activity [ 18 , 19 ] and determine the tertiary structure of this region [ 2023 ] . picornavirus ires - driven translation initiation depends on the recognition of the ires by specific cellular proteins . iress belonging to types i and ii require eif4 g , eif4a , eif2 , eif3 and atp , but no eif4e , eif1 or eif1a to assemble 48s complexes in a reconstitution assay with purified components [ 13 , 24 , 25 ] . specific structural motifs in the stem - loops j - k - l ( or 4 - 5 ) provide the preferential binding site for eif4 g , eif4b and eif3 ( figure 1(a ) ) [ 2628 ] . however , interaction of these eifs is necessary but not sufficient to promote ires activity , demonstrating the essential function of domains 2 and 3 in ires function . the contribution of domains 2 and 3 to ires activity may consist in the acquisition of a proper rna structural organization , assisted by auxiliary proteins . along this line , while the requirement for eifs is well established , the itafs involved in picornavirus ires activity are still under study . here we review the rna - binding proteins ( rbps ) that can form ribonucleoprotein ( rnp ) complexes with iress modulating their efficiency of translation . picornavirus rnas differ on their ability to operate in the cell - free rabbit reticulocyte lysates ( rrl ) . early studies conducted on the pv rna , which was inactive in rrl , evidenced that its translation efficiency was greatly enhanced upon supplementation of the lysates with hela cell extracts . this difference was related to the requirement of factors that were missing or present in limiting amounts in rrl . hence , addition of hela cells soluble extract resulted in reconstitution of pv rna translation . in due course , these observations led to the discovery of auxiliary proteins behaving as ires trans - acting factors . most itafs described so far are well characterized rbps that contain rna - binding motifs organized in a modular structure [ 31 , 32 ] , as it also occur in proteins involved in rna processing and transport . however , modulation of ires activity by itafs is not well understood , and at least in part it is a controversial issue because , depending on the ires element , some proteins show a more stringent requirement than others do [ 33 , 34 ] . over the last decade , the interaction of rbps with picornavirus ires ( table 1 ) has been analyzed taking advantage of riboproteomic affinity methods . of interest , and confirming the validity of this methodology , proteins previously know to interact with iress by other methods this is the case of eif4b and eif3 , which were specifically identified bound to fmdv or hcv ires by mass spectrometry following rna affinity purification [ 3538 ] . soon after the discovery of picornavirus iress , a direct interaction of the polypyrimidine tract - binding protein ( ptb ) with emcv and fmdv iress was shown by uv - crosslinking [ 3941 ] , and later by rna foot - printing and hydroxyl radical probing . ptb is a multifunctional rbp with four rna recognition motifs ( rrm ) that belongs to the heterogeneous nuclear ribonucleoprotein ( hnrnp ) family . the rrm domains of ptb recognize u - rich loops on short stems and in general , u / c - rich sequences . however , it also performs critical roles in cellular processes pertaining rna metabolism , including polyadenylation , mrna stability and translation initiation . regarding its function as itaf , ptb stimulates picornavirus and retrovirus iress , but it represses translation initiation driven by the bip ires . as a consequence of their role as regulator of ires activity , itafs can mediate cell type specificity , and hence , determine viral spread . this property was brought about by the effects of the neural form of ptb ( nptb ) , that determines the neurovirulence of theiler 's murine encephalitis virus ( tmev ) , and by the double - stranded rna - binding protein 76 ( drbp76 , also termed nf90/nfar-1 ) , that forms a heterodimer with nf45 ( nuclear factor of activated t cells ) . the drbp76:nf45 heterodimer binds to the hrv2 ires and differs in subcellular distribution in neuronal and non - neuronal malignant cells , arresting hrv translation in neuronal cells but not in glioma [ 48 , 49 ] . picornavirus iress often contain more than one polypyrimidine tract located in distant domains at each end of the ires region . recent studies have shown that a single ptb molecule binds in a unique orientation to the emcv ires , with rrm1 - 2 contacting the 3 end , and rrm3 contacting the 5 end of the ires , thereby constraining the ires structure in a unique orientation . however , studies carried out on the fmdv ires raised the conclusion that rrm3 - 4 of ptb were bound in an oriented way to domain 2 and the ires 3 region , respectively . although the rrms involved in the ires - ptb interactions are significantly different between these two studies , both are consistent with a role of ptb in stabilizing the ires structure , thereby acting as a chaperone . itafs , as it is the case of ptb , do not act alone but in combination with various factors presumably contributing to explain the opposite effects on ires activity . hence , rbps interacting with different targets may result in different effects depending on the target and the other partners of the complex . for instance , two iress such as emcv and fmdv with apparent similar secondary structure but different primary sequence , exhibit different requirements in terms of functional rna - protein association . one example is ebp1 protein ( erbb-3-binding protein 1 ) , also known as proliferation - associated factor pa2g4 and itaf45 , identified interacting with domain 3 of fmdv ires in proteomic analysis . ebp1 cooperates with ptb to stimulate fmdv ires activity in reconstitution studies [ 13 , 51 ] , but its depletion does not produce any effect on emcv ires activity . this protein is expressed in proliferating cells during the s phase but not during cell cycle arrest consistent with the fact that fmdv ires is active in proliferating tissues . another example of a factor that mediates ires activity is unr ( upstream of n - ras ) , a cold - shock rbp that interacts with pabp1 and stimulates hrv and pv translation through its interaction with two distinct ires domains [ 53 , 54 ] . in support of the specific role of unr in internal initiation , ires activity of c - myc , apaf-1 , unr and pitslre cellular mrnas is differentially regulated depending on the unr - partners , hnrnp k / poly r(c)-binding protein pcbp1 - 2 , nptb , or hnrnp c1 - 2 , respectively [ 5658 ] . other example of rbp identified with iress is the constitutive heat shock protein hsc70 , although the possibility of an indirect binding can not be discarded . hsc70 forms part of rnp complexes that interact with au - rich elements in the 3utr of specific mrnas , enhancing their stability . in addition to ptb , several proteins implicated in rna splicing can function as itafs , suggesting the existence of a network of interactions between different gene expression processes . an illustrative example is the splicing factor srp20 that up - regulates pv ires - mediated translation via its interaction with pcbp2 . another example of an itaf involved in a different gene expression process is gemin5 that binds directly to fmdv and hcv ires , acting as a downregulator of translation . not surprisingly , gemin5 is associated with other factors in rnp complexes that perform rather different roles during gene expression control . gemin5 is the rna - binding factor of the survival of motor neurons ( smn ) complex , which assembles sm proteins on snrnas playing a critical role in the biogenesis of key components of the mrna splicing machinery , the small nuclear ribonucleoproteins ( snrnps ) . gemin5 is a nuclear protein , but it is predominantly located in the cell cytoplasm and it also appears to be present in p bodies . together , the conclusions derived from the study of multifunctional proteins such as ptb , pcbp2 , gemin5 and other itafs , suggest a novel mechanism for the coordinated regulation of translation initiation of a subset of mrnas bound by shuttling proteins such as hnrnps or splicing factors . in support of this , the splicing factor sf2/asf mediates post - splicing activities promoting translation initiation by suppressing the activity of 4e - bp and modulating the internal initiation of cellular mrnas . in fact , it has been suggested that some rbps might exert its function in translation control by binding to the ires of specific cellular mrnas during splicing complex assembly before nuclear export . this could be an additional layer of posttranscriptional regulation for proteins whose functions are important when cap - dependent translation is compromised . hnrnps are a family of proteins ( named from hnrnp a1 to hnrnp u ) with rna - binding and protein - protein binding motifs [ 31 , 66 ] . they have a nuclear localization associated with nascent rna polymerase ii transcripts and shuttle with the rna to the cytoplasm . the rgg rna - binding motif that mediates the interaction with rna as well as with other hnrnps was first described in hnrnp u , one of the factors identified by mass spectrometry interacting with the fmdv ires . both hnrnp u and gemin5 form part of a complex with eif4e , that may explain the down - regulatory role of gemin5 in cap - dependent translation by virtue of eif4e sequestration or its localization to p bodies . several members of the hnrnp family , hnrnp k , pcbp1 ( hnrnp e1 ) and pcbp2 ( hnrnp e2 ) , have been identified associated with various iress ( table 1 ) . these proteins have in common the kh rna binding domain first described in hnrnp k and , subsequently , in pcbp1 , 2 , 3 and 4 . hnrnp k is the most abundant member of the family of proteins that recognize poly(rc ) regions , and regulates transcription , rna turnover and translation [ 69 , 70 ] . proteins hnrnp k , pcbp1 and pcbp2 , together with daz-1 , have been identified associated to domain 3 of the fmdv ires . daz1 is a 3utr - binding protein that has been found bound to polysomes and stimulates translation initiation of polyadenylated mrna . pcbp2 interacts with a c - rich loop in stem - loop iv of pv , cvb3 and hrv iress and specifically stimulates their activity [ 7275 ] . in contrast , the activity of emcv and fmdv iress that also interact with pcbp2 was not modified by the addition of recombinant pcbp2 protein to depleted - rrl lysates , in agreement with the lack of effect of nucleotide substitutions in the c - rich loop of fmdv ires . pcbp2 performs a dual role in translation initiation and rna replication of the poliovirus genome through its interaction with different targets in the viral 5utr . furthermore , consistent with its relevance in ires function , pv ires competes out with the hav ires for pcbp2 binding . the balance between translation initiation and silencing depends on the cellular response to stress . indeed , many viruses regulate the assembly or disassembly of stress granules ( sgs ) modifying translation of host and virus - encoded mrnas . consistent with this observation , some rbps have been localized in sgs , as pabp1 , or cytoplasmic processing bodies ( pbs ) , as pcbp2 . thus , in response to stress signals including viral infection , these multifunctional proteins may perform distinct roles depending on their localization . the signaling factor ras - gtpase - activating protein ( g3bp ) that was identified interacting with the fmdv ires , belongs to a new family of rbps that link tyrosin kinase - mediated signals with rna metabolism . these cytoplasmic aggregates contain stalled translation preinitiation complexes thought to serve as sites of mrna storage during the cell stress response . g3bp has been found associated to the 3utr of hcv rna , and its depletion induced a reduction of both hcv rna and proteins , supporting the idea that it might be a component of hcv replicating complexes . interestingly , g3bp interacts with the transcriptional regulator gp1-anchored membrane protein ( gpiap1 ) also identified as an ires - binding protein . many itafs are predominantly nuclear proteins that localize to the cytoplasm in picornavirus - infected cells . nucleolin is a protein involved in rdna transcription , rrna maturation , ribosome assembly and nucleo - cytoplasmic transport , and is translocated into the cytoplasm following infection of cells with poliovirus . nucleolin interacts with hrv , fmdv and pv ires and stimulates pv ires - mediated translation in transfected cells overexpressing the full - length protein . during enterovirus ev71 infection , the nuclear protein fbp2 ( far upstream element ( fuse ) binding protein 2 ) was enriched in the cytoplasm where viral replication occurs , whereas in mock - infected cells fbp2 was localized in the nucleus . fbp2 is a kh protein that negatively regulates ev71 ires activity presumably through its capacity to compete with ptb binding . together with hnrnps , a group of proteins that are involved in gene silencing , transport , and stabilization ( eif2c , rna helicases ) have been identified in riboproteomic approaches bound to different iress ( table 1 ) . the recurrent identification of a subset of factors with different rna targets [ 91 , 92 ] points to the existence of a network of rnps with the potential for multiple levels of regulation . moreover , the modular structure of rbps that is at the basis of their capacity to recognize a large number of targets raises the possibility that binding to any particular rna could facilitate different sorts of regulation depending on the other protein partners and the cellular environment . initiation of protein synthesis in the positive - strand rna genome of hcv is also driven by an ires . the ires region spans 340 nucleotides and differs from picornavirus iress in rna structural organization and factor requirement . the hcv ires is organized in three conserved structural domains , termed ii , iii and iv ( figure 1(b ) ) that adopt a tertiary fold whose integrity is required for efficient protein synthesis . domain ii , which consists of a hairpin with basal and apical loops , is essential for hcv ires activity . this domain promotes eif5-induced gtp hydrolysis during 80s ribosome assembly and eif2/gdp release from the initiation complex . the basal portion of domain iii forms the core of the high - affinity interaction with the 40s subunit including a small stem - loop ( iiie ) and a pseudoknot ( iiif ) . in the absence of eifs , the hcv ires can establish a high - affinity interaction with ribosomal 40s subunits through the binding surface between subdomains iiiabc , iiief and iiid . despite the capacity to form binary complexes , localization of the met - trnai on the surface of the 40s subunit by eif2 is essential for translation , and eif3 significantly enhances formation of the 48s initiation complex interacting with the junction of subdomains iiiabc [ 5 , 99 ] . interaction of eif3 subunits with hcv ires has been analyzed by cryo - electron microscopy of the binary ires - eif3 complex and by mass spectrometry of ires - bound protein complexes [ 36 , 37 ] . however , under conditions of inactivation of eif2 by phosphorylation , the hcv ires can form a preinitiation complex in the presence of eif3 and eif5b , which is reminiscent of the bacterial - like initiation mode . the ribosomal proteins that participate in ires-40s interaction have been identified by different approaches as well , such as cross - linking studies [ 102104 ] and mass spectrometry . besides ribosomal proteins and eif3 subunits , the non - canonical factors rack1 and nucleolin were identified in native and ires-40s ribosomal complexes . rack1 functions as the receptor for activated protein kinase c , and regulates translation initiation by recruiting protein kinase c to the 40s subunit . it forms a stable complex with the 40s subunit , exposing the wd - repeats as a platform for interactions with other proteins to the ribosome . another non - canonical host factor , the insulin - like growth factor ii mrna - binding protein 1 ( igf2bp1 ) has been reported to associate with both the ires and the 3utr of hcv , and remarkably , to coimmunoprecipitate with eif3 and the 40s subunit . this result suggests that this factor may enhance hcv ires - dependent translation by recruiting the ribosomal subunits to a pseudo - circularized rna . in agreement with this possibility , a subset of the identified proteins , nf90/nf45 , also interact with the ends of the viral rna contributing to enhance viral rna replication . moreover , in support of the role of the 35 interactions in the control of gene expression in positive - strand rna viruses , stimulation of ires activity by the homologous 3utr has been shown in fmdv and hrv [ 10 , 11 ] , presumably mediated by functional bridges that bring together the rna ends by long - range rna - rna interactions . despite some controversy regarding the effect of ptb , most of the identified itafs regulate hcv ires activity in a positive manner [ 110 , 111 ] . la binds to pv , emcv and hcv ires stimulating translation [ 113115 ] , but it suppresses hav ires activity . nsap1 protein has a dual function in hcv life cycle participating in rna replication and enhancing ires - dependent translation through its binding to a - rich sequences in the core coding region . similar to nsap1 , hnrnp l interacts with the 3 border of the hcv ires in the core - coding sequence and it is required for ires - mediated translation . this protein is necessary for efficient translation of the cat-1 arginine / lysine transporter mrna during amino acid starvation . other hcv - interacting protein is hnrnp d that binds to the stem - loop ii and promotes translation . proteins of this family , hnrnp a / b 38 , have been identified interacting with diii of hcv ires . hnrnp a1 binds to the 5utr of ev71 and sindbis rna and is required for viral rna replication . this protein also mediates internal initiation of fgf-2 and apaf-1 mrnas [ 123 , 124 ] . in addition to direct rna - protein interactions , protein - protein association between rbps , such as hnrnp u or hnrnp a / b [ 125 , 126 ] during mrna transport can explain the identification of proteins belonging to the cytoskeleton machinery with fmdv and hcv ires [ 36 , 38 , 55 ] . protein - protein interactions may also explain the identification of glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) with hav ires . this protein competes with ptb for binding to stem - loop iiia , suppressing the ability of the hav 5utr to direct cap - independent translation . gapdh forms a macromolecular complex that binds to u - rich sequences in the 3utr of a selective group of cellular mrnas controlling their translation . however , as already mentioned for some factors , indirect interactions may be behind the identification of very abundant rbps , such as yb-1 , in riboproteomic studies . thus , the functional involvement of each factor as well as whether the binding is direct or mediated by another partner in the rnp complex , needs to be verified individually . a few proteins identified by mass spectrometry with a discrete domain of the hcv ires have been also identified in similar approaches interacting with the entire hcv ires , giving additional information about the binding site of the protein . this could be the case of rna helicase deah - box polypeptide 9 ( dhx9 ) or dead - box polypeptide 1 ( ddx1 ) . the ddx / dhx family of proteins play important roles in nucleic acid metabolism , including pre - mrna processing , ribosome biogenesis , rna turnover , rna export , translation , and association / dissociation of large rnp complexes . dhx9 recognizes a complex structure at the 5-utr of retrovirus mrna precursors , facilitating its association to polyribosomes . ddx1 , a dual interactor of hnrnp k and poly(a)-mrna , has been also identified bound to the 3utr of hcv suggesting a role for this protein in viral rna replication . in general , itafs are rna - binding proteins that shuttle between the nucleus and the cytoplasm . thus , a network of rna - protein and protein - protein interactions may assist to recruit the ires to the ribosome and possibly , to other cytoplasmic structures . rbps are key cellular components that control the temporal , spatial and functional dynamics of rna within the competitive cell environment . indeed , changes in the expression of rbps have profound implications for cellular physiology , affecting rna processes from pre - mrna splicing to protein translation . thereby , the composition of rnp complexes bound to the rna in a particular situation will determine the fate of the rna ( e.g. , stability , translatability , compartmentalization ) . in other words , binding of proteins , even those considered to be promiscuous , to a given rna could mediate specific regulation . in agreement with this , recent mass spectrometry identification of the proteins associated with argonaute proteins , the protein complex responsible for gene - silencing pathways guided by small rnas , revealed a common group of helicases , hnrnps and other rbps which are shared with rnp complexes involved in other cellular processes such as mrna transport , stabilization and translation . the observation that proteins with the potential for multiple levels of regulation can recognize various rna targets raises the possibility that protein - binding to specific rnas could facilitate different sorts of regulation depending on the other partners and the cellular environment . thus , elucidating the function of itafs will require a deep understanding of their rna targets , their protein partners , and their potential modifications . concerning the first issue , the recent advances in cross - linking immunoprecipitation and high - throughput sequencing appears to be a promising technique to help in this task . implementation of new proteomic approaches will continue to help in the second and third tasks . finally , regarding the modification of rbps in infected cells , understanding the effect of proteolytic cleavage of factors such as pcbp2 , ptb , pabp or g3bp [ 78 , 133 , 134 ] will need to be extended to newly identified itafs . all together , this will help to understand the integrated action of itafs on mrna targets . the rbps modulating picornavirus and hcv ires activity offer promising targets to combat these infectious pathogens . indeed , iress are ideal candidates to interfere with virus replication through direct ires - targeting or through itaf - targeting . in the first case , antiviral agents based on rna molecules aimed to disrupt the ires structure have been partially successful [ 135138 ] . in the second case , knowledge of the structural organization of itafs provided the basis to design antiviral therapy , as shown by a synthetic peptide derived from the rrm2 of la which acts as a dominant negative inhibitor of hcv rna translation . in the coming years , elucidation of the structural determinant of peptides derived from different itafs , interfering with the capacity of these proteins to generate protein - protein and rna - protein networks , will provide the basis for developing small peptidomimetic structures as potent anti - viral therapeutics .

translation initiation is a highly regulated process that exerts a strong influence on the posttranscriptional control of gene expression . two alternative mechanisms govern translation initiation in eukaryotic mrnas , the cap - dependent initiation mechanism operating in most mrnas , and the internal ribosome entry site ( ires)-dependent mechanism , first discovered in picornaviruses . ires elements are highly structured rna sequences that , in most instances , require specific proteins for recruitment of the translation machinery . some of these proteins are eukaryotic initiation factors . in addition , rna - binding proteins ( rbps ) play a key role in internal initiation control . rbps are pivotal regulators of gene expression in response to numerous stresses , including virus infection . this review discusses recent advances on riboproteomic approaches to identify ires transacting factors ( itafs ) and the relationship between rna - protein interaction and ires activity , highlighting the most relevant features on picornavirus and hepatitis c virus iress .

1. Translational Control of Gene Expression 2. Internal Initiation of Translation in Picornavirus RNAs 3. Internal Initiation of Translation in HCV RNA 4. Concluding Remarks

this is achieved by fine - tuning of all the processes governing gene expression , including transcription , splicing , mrna transport , rna stability , translation , protein stability and posttranslational modification . all the steps within this cascade of events are subjected to their own specific regulation , and contribute to generate a different composition of the proteome by modifying not only the levels but also the identity of the proteins present in the cell under specific conditions . protein - protein as well as rna - protein interactions allow a compartmentalization of the factors needed to control gene expression . translation initiation can modify the proteome by altering the efficiency of translation as well as by enabling the synthesis of different forms of a protein from specific genes . most of translational control is exerted at the initiation step , assisted by specific proteins designated translation initiation factors ( eifs ) . translation initiation of most eukaryotic mrnas commences with 5 end - dependent recruitment of the 43s complex ( that is composed of a 40s subunit bound to eif2-gtp / met - trnai , eif1a , eif1 and eif3 ) by eif4f recognition of the mgpppn ( cap ) at the 5 end of the mrna . in turn , the eif4f complex comprises eif4e that physically binds to the cap , eif4a that unwinds secondary structures in the 5 untranslated region ( 5utr ) and eif4 g , a scaffold protein that interacts with eif4e , eif4a and eif3 . aided by the helicase activity of eif4a and its cofactor eif4b , the 43s pre - initiation complex scans in 5 to 3 direction until an appropriate initiation codon is encountered . auxiliary factors , eif1 , eif2 and eif5 , help to identify the correct aug start codon , resulting in the formation of the 48s complex . the poly(a ) tail present in the 3utr of most mrnas synergistically stimulates the efficiency of translation through recruitment of poly(a)-binding protein ( pabp ) , enabling its interaction with eif4f located at the mrna 5 end . finally , eif5b mediates joining of the 60s and 40s subunits , generating the 80s complex competent for protein synthesis . translation initiation , particularly in viral mrnas , can occur by an alternative mechanism driven by internal ribosome entry site ( ires ) elements , discovered near 20 years ago in two picornaviruses , encephalomyocarditis virus ( emcv ) and poliovirus ( pv ) [ 2 , 3 ] . these elements are cis - acting sequences that form secondary and tertiary rna structures and recruit the translation machinery to an internal position in the mrna , bypassing a large number of stable structural elements in the viral 5utr . hence , picornavirus ires - driven initiation is 5 end - independent and does not require eif4e to recruit the 40s subunit , in contrast to the cap - dependent initiation mechanism . the subsequent discovery of an ires element in hepatitis c virus ( hcv ) rna that was able to recruit 40s ribosomal subunits in the absence of eif4 g represented a major breakthrough in the translation field [ 5 , 6 ] . this finding opened the question of how iress differing in primary sequence , rna structure , and factor requirements , perform the same function . over the last two decades , the process of internal initiation has been found to be more general than originally thought , that is , it operates in other rna and dna viruses as well as in cellular mrnas . these strategies could be under the control of a distinct group of proteins specifically interacting with each of these regulatory elements . in this review understanding the role played by these ires trans - acting factors ( itafs ) may help to unravel the strategies employed by mrnas to capture the translation machinery internally . translation initiation of all picornavirus rnas is dependent on the ires located in the long 5utr ( figure 1(a ) ) . although less than 50% of primary sequence is conserved between different members of the picornavirus family , the similarity of their secondary structure allows their classification into four types , i to iv . type i includes enterovirus ( ev , pv , hrv ) , type ii , cardiovirus ( emcv ) and aphthovirus ( foot - and - mouth disease virus , fmdv ) , type iii , is used for hepatitis a virus ( hav ) , and the hcv - like ires conforms group iv . the acquisition of a proper structural organization is a key determinant of internal translation initiation driven by picornavirus ires [ 14 , 15 ] , and all viral ires in general [ 16 , 17 ] . picornavirus ires - driven translation initiation depends on the recognition of the ires by specific cellular proteins . however , interaction of these eifs is necessary but not sufficient to promote ires activity , demonstrating the essential function of domains 2 and 3 in ires function . along this line , while the requirement for eifs is well established , the itafs involved in picornavirus ires activity are still under study . here we review the rna - binding proteins ( rbps ) that can form ribonucleoprotein ( rnp ) complexes with iress modulating their efficiency of translation . early studies conducted on the pv rna , which was inactive in rrl , evidenced that its translation efficiency was greatly enhanced upon supplementation of the lysates with hela cell extracts . most itafs described so far are well characterized rbps that contain rna - binding motifs organized in a modular structure [ 31 , 32 ] , as it also occur in proteins involved in rna processing and transport . however , modulation of ires activity by itafs is not well understood , and at least in part it is a controversial issue because , depending on the ires element , some proteins show a more stringent requirement than others do [ 33 , 34 ] . over the last decade , the interaction of rbps with picornavirus ires ( table 1 ) has been analyzed taking advantage of riboproteomic affinity methods . of interest , and confirming the validity of this methodology , proteins previously know to interact with iress by other methods this is the case of eif4b and eif3 , which were specifically identified bound to fmdv or hcv ires by mass spectrometry following rna affinity purification [ 3538 ] . soon after the discovery of picornavirus iress , a direct interaction of the polypyrimidine tract - binding protein ( ptb ) with emcv and fmdv iress was shown by uv - crosslinking [ 3941 ] , and later by rna foot - printing and hydroxyl radical probing . however , it also performs critical roles in cellular processes pertaining rna metabolism , including polyadenylation , mrna stability and translation initiation . regarding its function as itaf , ptb stimulates picornavirus and retrovirus iress , but it represses translation initiation driven by the bip ires . as a consequence of their role as regulator of ires activity , itafs can mediate cell type specificity , and hence , determine viral spread . this property was brought about by the effects of the neural form of ptb ( nptb ) , that determines the neurovirulence of theiler 's murine encephalitis virus ( tmev ) , and by the double - stranded rna - binding protein 76 ( drbp76 , also termed nf90/nfar-1 ) , that forms a heterodimer with nf45 ( nuclear factor of activated t cells ) . recent studies have shown that a single ptb molecule binds in a unique orientation to the emcv ires , with rrm1 - 2 contacting the 3 end , and rrm3 contacting the 5 end of the ires , thereby constraining the ires structure in a unique orientation . however , studies carried out on the fmdv ires raised the conclusion that rrm3 - 4 of ptb were bound in an oriented way to domain 2 and the ires 3 region , respectively . hence , rbps interacting with different targets may result in different effects depending on the target and the other partners of the complex . for instance , two iress such as emcv and fmdv with apparent similar secondary structure but different primary sequence , exhibit different requirements in terms of functional rna - protein association . in support of the specific role of unr in internal initiation , ires activity of c - myc , apaf-1 , unr and pitslre cellular mrnas is differentially regulated depending on the unr - partners , hnrnp k / poly r(c)-binding protein pcbp1 - 2 , nptb , or hnrnp c1 - 2 , respectively [ 5658 ] . in addition to ptb , several proteins implicated in rna splicing can function as itafs , suggesting the existence of a network of interactions between different gene expression processes . another example of an itaf involved in a different gene expression process is gemin5 that binds directly to fmdv and hcv ires , acting as a downregulator of translation . not surprisingly , gemin5 is associated with other factors in rnp complexes that perform rather different roles during gene expression control . gemin5 is the rna - binding factor of the survival of motor neurons ( smn ) complex , which assembles sm proteins on snrnas playing a critical role in the biogenesis of key components of the mrna splicing machinery , the small nuclear ribonucleoproteins ( snrnps ) . together , the conclusions derived from the study of multifunctional proteins such as ptb , pcbp2 , gemin5 and other itafs , suggest a novel mechanism for the coordinated regulation of translation initiation of a subset of mrnas bound by shuttling proteins such as hnrnps or splicing factors . in support of this , the splicing factor sf2/asf mediates post - splicing activities promoting translation initiation by suppressing the activity of 4e - bp and modulating the internal initiation of cellular mrnas . this could be an additional layer of posttranscriptional regulation for proteins whose functions are important when cap - dependent translation is compromised . hnrnps are a family of proteins ( named from hnrnp a1 to hnrnp u ) with rna - binding and protein - protein binding motifs [ 31 , 66 ] . the rgg rna - binding motif that mediates the interaction with rna as well as with other hnrnps was first described in hnrnp u , one of the factors identified by mass spectrometry interacting with the fmdv ires . both hnrnp u and gemin5 form part of a complex with eif4e , that may explain the down - regulatory role of gemin5 in cap - dependent translation by virtue of eif4e sequestration or its localization to p bodies . several members of the hnrnp family , hnrnp k , pcbp1 ( hnrnp e1 ) and pcbp2 ( hnrnp e2 ) , have been identified associated with various iress ( table 1 ) . these proteins have in common the kh rna binding domain first described in hnrnp k and , subsequently , in pcbp1 , 2 , 3 and 4 . hnrnp k is the most abundant member of the family of proteins that recognize poly(rc ) regions , and regulates transcription , rna turnover and translation [ 69 , 70 ] . proteins hnrnp k , pcbp1 and pcbp2 , together with daz-1 , have been identified associated to domain 3 of the fmdv ires . daz1 is a 3utr - binding protein that has been found bound to polysomes and stimulates translation initiation of polyadenylated mrna . in contrast , the activity of emcv and fmdv iress that also interact with pcbp2 was not modified by the addition of recombinant pcbp2 protein to depleted - rrl lysates , in agreement with the lack of effect of nucleotide substitutions in the c - rich loop of fmdv ires . pcbp2 performs a dual role in translation initiation and rna replication of the poliovirus genome through its interaction with different targets in the viral 5utr . the balance between translation initiation and silencing depends on the cellular response to stress . thus , in response to stress signals including viral infection , these multifunctional proteins may perform distinct roles depending on their localization . g3bp has been found associated to the 3utr of hcv rna , and its depletion induced a reduction of both hcv rna and proteins , supporting the idea that it might be a component of hcv replicating complexes . interestingly , g3bp interacts with the transcriptional regulator gp1-anchored membrane protein ( gpiap1 ) also identified as an ires - binding protein . nucleolin is a protein involved in rdna transcription , rrna maturation , ribosome assembly and nucleo - cytoplasmic transport , and is translocated into the cytoplasm following infection of cells with poliovirus . fbp2 is a kh protein that negatively regulates ev71 ires activity presumably through its capacity to compete with ptb binding . together with hnrnps , a group of proteins that are involved in gene silencing , transport , and stabilization ( eif2c , rna helicases ) have been identified in riboproteomic approaches bound to different iress ( table 1 ) . moreover , the modular structure of rbps that is at the basis of their capacity to recognize a large number of targets raises the possibility that binding to any particular rna could facilitate different sorts of regulation depending on the other protein partners and the cellular environment . the basal portion of domain iii forms the core of the high - affinity interaction with the 40s subunit including a small stem - loop ( iiie ) and a pseudoknot ( iiif ) . despite the capacity to form binary complexes , localization of the met - trnai on the surface of the 40s subunit by eif2 is essential for translation , and eif3 significantly enhances formation of the 48s initiation complex interacting with the junction of subdomains iiiabc [ 5 , 99 ] . interaction of eif3 subunits with hcv ires has been analyzed by cryo - electron microscopy of the binary ires - eif3 complex and by mass spectrometry of ires - bound protein complexes [ 36 , 37 ] . however , under conditions of inactivation of eif2 by phosphorylation , the hcv ires can form a preinitiation complex in the presence of eif3 and eif5b , which is reminiscent of the bacterial - like initiation mode . rack1 functions as the receptor for activated protein kinase c , and regulates translation initiation by recruiting protein kinase c to the 40s subunit . another non - canonical host factor , the insulin - like growth factor ii mrna - binding protein 1 ( igf2bp1 ) has been reported to associate with both the ires and the 3utr of hcv , and remarkably , to coimmunoprecipitate with eif3 and the 40s subunit . in agreement with this possibility , a subset of the identified proteins , nf90/nf45 , also interact with the ends of the viral rna contributing to enhance viral rna replication . moreover , in support of the role of the 35 interactions in the control of gene expression in positive - strand rna viruses , stimulation of ires activity by the homologous 3utr has been shown in fmdv and hrv [ 10 , 11 ] , presumably mediated by functional bridges that bring together the rna ends by long - range rna - rna interactions . despite some controversy regarding the effect of ptb , most of the identified itafs regulate hcv ires activity in a positive manner [ 110 , 111 ] . nsap1 protein has a dual function in hcv life cycle participating in rna replication and enhancing ires - dependent translation through its binding to a - rich sequences in the core coding region . in addition to direct rna - protein interactions , protein - protein association between rbps , such as hnrnp u or hnrnp a / b [ 125 , 126 ] during mrna transport can explain the identification of proteins belonging to the cytoskeleton machinery with fmdv and hcv ires [ 36 , 38 , 55 ] . protein - protein interactions may also explain the identification of glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) with hav ires . this protein competes with ptb for binding to stem - loop iiia , suppressing the ability of the hav 5utr to direct cap - independent translation . however , as already mentioned for some factors , indirect interactions may be behind the identification of very abundant rbps , such as yb-1 , in riboproteomic studies . the ddx / dhx family of proteins play important roles in nucleic acid metabolism , including pre - mrna processing , ribosome biogenesis , rna turnover , rna export , translation , and association / dissociation of large rnp complexes . in general , itafs are rna - binding proteins that shuttle between the nucleus and the cytoplasm . thus , a network of rna - protein and protein - protein interactions may assist to recruit the ires to the ribosome and possibly , to other cytoplasmic structures . rbps are key cellular components that control the temporal , spatial and functional dynamics of rna within the competitive cell environment . thereby , the composition of rnp complexes bound to the rna in a particular situation will determine the fate of the rna ( e.g. in agreement with this , recent mass spectrometry identification of the proteins associated with argonaute proteins , the protein complex responsible for gene - silencing pathways guided by small rnas , revealed a common group of helicases , hnrnps and other rbps which are shared with rnp complexes involved in other cellular processes such as mrna transport , stabilization and translation . the observation that proteins with the potential for multiple levels of regulation can recognize various rna targets raises the possibility that protein - binding to specific rnas could facilitate different sorts of regulation depending on the other partners and the cellular environment . concerning the first issue , the recent advances in cross - linking immunoprecipitation and high - throughput sequencing appears to be a promising technique to help in this task . the rbps modulating picornavirus and hcv ires activity offer promising targets to combat these infectious pathogens . in the second case , knowledge of the structural organization of itafs provided the basis to design antiviral therapy , as shown by a synthetic peptide derived from the rrm2 of la which acts as a dominant negative inhibitor of hcv rna translation . in the coming years , elucidation of the structural determinant of peptides derived from different itafs , interfering with the capacity of these proteins to generate protein - protein and rna - protein networks , will provide the basis for developing small peptidomimetic structures as potent anti - viral therapeutics .

childbirth , even though a normal physiological process has been associated with a number of risks , which may , in extreme cases , lead to loss of life . this concern was underscored by the millennium development goal 5 ( mdg ) which focused on improving maternal health services ( mhs ) to reduce morbidity , disability , and mortality due to pregnancy and delivery . worldwide , it has been estimated that 289,000 cases of maternal deaths were recorded in 2013 , indicating a raising trend by 2000 deaths compared to the figure obtained in 2011 , with 99 % of these deaths occurring in the developing countries . although it is the wealthiest nation in the central african sub - region , with an impressive economic growth since the end of the country s civil war in 2002 , the republic of angola has a higher life time risk of 1 in 39 women dying as a result of pregnancy and its related complications compared to neighboring republic of namibia with a life time risk of 1 in 160 . the current maternal mortality ratio ( mmr ) for angola was reported to be 460 per 100,000 live births in 2013 , which is still far above the desired mdg 5 goal of 300 per 100,000 live births by the year 2015 . the high mmr ( 460 per 100,000 live births ) is partly due to low utilization of maternal health services . for instance , between 2006 - 2013 , only 47% and 49% of pregnant women had at least four ante natal care visits as recommended by the world health organization ( who ) and delivered under the supervision of a skilled health worker respectively . more disturbing is the fact that only 32% of rural pregnant women were reported to have had their delivery supervised by a skilled health worker compared to 82% of their urban cohort . this scenario is further worsened by the fact that many pregnancies end up as stillbirths or neonatal deaths . one of the major reasons for sub - saharan african countries having the highest life time risk of dying due to pregnancy related issues and poor pregnancy outcomes such as still births and neonatal deaths is lack of systematic search for the root causes of these deaths as being done in the industrialized countries where confidential inquiries is the norm rather than an exception . unfortunately , the findings of these confidential inquiries in the developed countries are used to set out downstream public health intervention strategies in the developing nations notwithstanding their small sample sizes ( pregnancy related deaths are rare event in europe and north america ) and differences in terms of socio - economic , cultural and political systems including health care financing mechanisms between the developed and the underdeveloped countries . concerns about the lack of use of area - specific data have been referred to as inverse information and care law . this concern is legitimate in terms of the need for the utilization of local information in order to come up with the root causes that lead to poor utilization of maternal and child care services , which will subsequently guide local interventions at provincial / state and district levels . for instance , of the 2,500 articles on maternal mortality reviewed by gil - gonzlez and colleagues , and another 5,575 articles on maternal health services utilization and pregnancy outcomes reviewed by say and raine , and 54 articles on the effectiveness of interventions on maternal mortality in low income countries reviewed by burchette and mayhew , there was no single article from the republic of angola despite the country s high mmr of 460 per 100,000 live birth , and a life time risk of a woman dying from pregnancy and its related complications over 1000 times higher when compared to canada and scandinavian countries . this dearth of data on mhs utilization and maternal mortality in angola and many other high burden countries was further reported to have neither timely nor complete data on maternal deaths surveillance system . surveillance for maternal mortality was launched in 2004 with the aim not just to estimate the burden of the problem but , also to provide better insights on the why , how and where these deaths occur . nearly , a third of all districts in sub - saharan africa have not integrated maternal mortality among the immediate notifiable events / diseases in their integrated disease surveillance and response ( idsr ) program . furthermore , a review of the status of maternal mortality surveillance in 2012 showed that data on maternal deaths are lacking or incomplete in 48.9% of the 180 countries reviewed including angola . a recent review on status of implementation of maternal death surveillance and response among african countries has shown that the republic of angola was among the countries with no available data on maternal death surveillance and response . the country still relies on traditional non - electric surveillance information tools that focus on clinical causes of maternal deaths such as hemorrhage , ruptured uterus , sepsis , eclampsia etc . , which does not provide insights on what transpired at home , on the way to health facility and appropriateness of treatment received . these issues indicate the need for area specific studies in order to complement the weak surveillance system and also to guide interventions that are in context of the local issues which forms the objective of this study . independent autonomous decision making on reproductive health desires of women in many developing countries is defined not only by a woman s level of education or personal income but also by the norms in her community as dictated by cultural and religious beliefs . in order to identify the underlying root causes of utilization behavior and how they impact pregnancy outcomes , the anderson medical care utilization model was used as the theoretical framework of this study . the model is made up of three constructs namely : 1 ) the predisposing characteristics of women such as biological , cultural , social , and economic ; 2 ) the enabling characteristics such as health system and health care financing mechanisms ; and 3 ) the need characteristics which is the individual pregnant woman s perceived need to use modern health services and the perceptions of the health care worker . the constructs of the model are highly adaptable attested by its application in numerous research studies . this unique characteristic of the anderson s model was underscored by its use in various health.[18 - 20 ] and other social issues.[21 - 23 ] we examined the associations between women s bio - socio demographic and health system characteristic ( independent variables ) and pregnancy outcome ( mother is dead or alive ) as the outcome / dependent variables . this study will bridge the existing current gap of evidence - based information noted above and highlight areas that require further research in order to come up with interventions that are specific to local context in angola and in other sub - saharan african countries . kuando kubango is one of the 18 provinces of angola , situated in the southern part of the country . it has a common international boundary with the republic of namibia to the south and the republic of zambia to the southeast . it also shares local boundaries with moxico province to the north east , bie to the north , huila and cunene provinces to the west . it has an estimated population of 510 , 369 with 60% living in urban areas based on the 2014 census . it has a total of 102 health facilities out of which 10 provided obstetric services . the population of women in the reproductive age group is 104,342 with an estimated annual number of pregnancies of 24,843 based on the 2014 census . this study was conducted in the main regional referral center due to the availability of better skilled workers and diagnostic services in this center . all hospitals have a functional ambulance to facilitate the transfer of cases to the provincial maternity hospital . subsistence farming , petty trading , fishing , and hunting are the main occupations of the indigenes . the target population of this study was women that received obstetric services ( antenatal , delivery or post - natal ) in the provincial main referral maternity hospital menongue , kuando kubango province of angola , between 2010 and 2014 . it is basically the analysis of data on pregnancy , labor and puerperium from patient case notes and the delivery register for a period of five years ( 2010 - 2014 ) . only maternal deaths that meet the world health organization s ( who ) definition of maternal mortality were included in the study . according to who maternal mortality is the death of any woman while pregnant or within 42 days of termination of pregnancy , from any cause related to or aggravated by pregnancy or its management , irrespective of the duration and site of the pregnancy , but not from accidental or incidental causes . the data collection instruments were case files kept in the medical records department , labor ward ( delivery ) register , and a form designed to keep records for data extracted from each case file . the tool documented data on personal , and clinical information such as age , tribe , religion , marital status , place of domicile , parity , occupation of husband , occupation of cases , mode of admission ( self or referred ) , booking status , indication for admission , date of admission , duration of labor , interval between onset of labor and admission to hospital , place of delivery ( as indicated in the referral letter or case notes ) , previous obstetric operations , interval between admission and maternal death , cause of maternal death and date of death . maternal mortality ratio was calculated for each of the five years ( 2010 - 2014 ) and for the overall study period . the causes of death were examined under two heading viz : direct obstetric causes and indirect non obstetric medical causes . cross tabulations of variables , the chi square test of association and fisher exact test were conducted with the level of statistical significance set at p<0.05 at 95% confidence interval . kuando kubango is one of the 18 provinces of angola , situated in the southern part of the country . it has a common international boundary with the republic of namibia to the south and the republic of zambia to the southeast . it also shares local boundaries with moxico province to the north east , bie to the north , huila and cunene provinces to the west . it has an estimated population of 510 , 369 with 60% living in urban areas based on the 2014 census . it has a total of 102 health facilities out of which 10 provided obstetric services . the population of women in the reproductive age group is 104,342 with an estimated annual number of pregnancies of 24,843 based on the 2014 census . this study was conducted in the main regional referral center due to the availability of better skilled workers and diagnostic services in this center . all hospitals have a functional ambulance to facilitate the transfer of cases to the provincial maternity hospital . subsistence farming , petty trading , fishing , and hunting are the main occupations of the indigenes . the target population of this study was women that received obstetric services ( antenatal , delivery or post - natal ) in the provincial main referral maternity hospital menongue , kuando kubango province of angola , between 2010 and 2014 . it is basically the analysis of data on pregnancy , labor and puerperium from patient case notes and the delivery register for a period of five years ( 2010 - 2014 ) . only maternal deaths that meet the world health organization s ( who ) definition of maternal mortality were included in the study . according to who maternal mortality is the death of any woman while pregnant or within 42 days of termination of pregnancy , from any cause related to or aggravated by pregnancy or its management , irrespective of the duration and site of the pregnancy , but not from accidental or incidental causes . the data collection instruments were case files kept in the medical records department , labor ward ( delivery ) register , and a form designed to keep records for data extracted from each case file . the tool documented data on personal , and clinical information such as age , tribe , religion , marital status , place of domicile , parity , occupation of husband , occupation of cases , mode of admission ( self or referred ) , booking status , indication for admission , date of admission , duration of labor , interval between onset of labor and admission to hospital , place of delivery ( as indicated in the referral letter or case notes ) , previous obstetric operations , interval between admission and maternal death , cause of maternal death and date of death . maternal mortality ratio was calculated for each of the five years ( 2010 - 2014 ) and for the overall study period . the causes of death were examined under two heading viz : direct obstetric causes and indirect non obstetric medical causes . cross tabulations of variables , the chi square test of association and fisher exact test were conducted with the level of statistical significance set at p<0.05 at 95% confidence interval . during the period under study ( 2010 - 2014 ) , 131 maternal deaths were recorded , out of 7,158 live births , giving a maternal mortality ratio ( mmr ) of 1830 per 100,000 deliveries . the annual and overall maternal mortality ratio the denominator of the former ( ratio ) is live births while the later ( rate ) is women in the reproductive age group 15 - 49 years . in this study we used live births as the denominator to calculate the annual and overall mortality ratio is shown in table 1 . annual distribution of maternal mortality ratio the peak incidence of mmr was recorded in 2012 with mmr of 2,487 per 100,000 live births ( figure 1 ) . the overall trend although decreasing between 2013 and 2014 , however , the 2014 mmr of 1,757 per 100,000 live births is still higher than the figure of 1,360 per 100,000 live births recorded in 2010 ( figure 1 ) . trend of mmr per 100,000 live births , 2010 - 2014 , menongue , kuando kubango province out of the 131 maternal deaths , 31 ( 24% ) of these cases had inadequate documentation on working diagnosis , presenting complain , history of presenting complain and or management outline and therefore were not included in analysis of direct and indirect causes of maternal death . of the remaining 100 deaths that had information on diagnoses , 51 ( 51% ) and 49 ( 49% ) were as a result of direct and indirect causes respectively ( table 2 ) . the three leading direct causes of maternal deaths are hemorrhage ( 15% ) , puerperal sepsis ( 13% ) and eclampsia ( 11% ) which combined accounted for 39% of all deaths ( table 2 ) . proportion of direct obstetric and indirect non - obstetric causes of deaths , 2010 - 2014 , kuando kubango , angola indirect non - obstetric medical causes , accounted for 49% of all maternal deaths . malaria in pregnancy ( 14% ) , anemic heart failure ( 13% ) and pneumonia ( 5% ) were the leading cause of indirect non obstetric medical causes of maternal death , accounting for 32% of all deaths ( table 2 ) . the age distribution of maternal deaths indicated that more than half of all deaths were accounted by women between the ages of 15 - 19 and those > 35 years with age specific mmr ( asmmr ) of 1,058 and 1,354 per 100,000 live births respectively . women between the ages of 20 - 34 years had the lowest asmmr of 876 per 100,000 live births . however , the difference is not statistically significant ( =4.572 ; df 2 ; p>0.05 ) ( table 3 ) . pregnancy outcomes in relation to some bio - socio - demographic factors among 12,573 deliveries , 2010 - 2014 , kuando kubango , angola fisher exact test with yates correction one hundred and seventeen out of the 131 cases of deaths were from comuna sede of menongue district accounting for 89% of all deaths recorded during the review period . additionally , caiundo and missombo comunas of menongue district accounted for another 2.7% indicating that about 93% of all deaths were from menongue district . the districts of cuito cuanavale and cuchi accounted for only 3.8% and 1.5% of all deaths respectively . women living in rural areas accounted for 96.2% of all deaths and the difference in the place of domicile ( rural versus urban ) was significantly associated with maternal deaths ( p < 0.0001 ) ( table 3 ) . distance to the nearest health facility that provides maternal health services ( mhs ) was significantly associated maternal mortality ( p < 0.0001 ) , with women who responded that distance is not an obstacle accounting for only 30% of all maternal deaths during the period under review ( table 3 ) . out of the 131 maternal deaths , 52 ( 40% ) occurred within the first 24 hours of been admitted in the hospital , 29 ( 22% ) deaths occurred 24 - 48 hours after admission , while 47 ( 35.6% ) occurred between 2 to 11 days after admission . the maternity hospital has two medical doctors in 2010 which increased to four in 2014 . with an estimated 62 , 736 women in the reproductive age group and 14,937 annual pregnancies for menongue district where the hospital is located , the ratio of midwife per 1000 women in the reproductive age group for menongue district is 1 per 2,134 pregnancies per annum . the study hospital has no ultrasound services that could aid in gynecological and obstetric diagnoses . the mean mmr of 1830 per 100,000 live births recorded in this study is five times higher than the desired mdg 5 goal of 300 per 100,000 live births by the year 2015 , and four times higher than the who estimate for angola . as earlier pointed out above , there is a general lack of published journal articles on maternal mortality ( hospital or community based ) in angola . the official report by the health authorities of cabinda province of angola indicated in 2012 the mmr was 234.3/100,000 live births . however , a review of records in all the 10 health facilities that provide obstetric care in cabinda province by rodrigues ( 2013 ) concluded that the provincial official mmr is an under estimation and that the magnitude of the problem is unknown because data is generally incomplete or not available in all the 10 health facilities . however , when compared with published studies ( 2010 - 2015 ) from central africa region to which angola belongs , the mmr of this study ( 1,830 per 100,000 ) was higher than other hospital based study in cameroon ( 287.57100 , 000 ) . the huge difference in mmr between our finding and the cameroonian study may partially be due to the fact that more than half of all the cases of maternal mortality in our study area was among women who were less than 20 years or more than 35 years and presented with complicated pregnancies that resulted in maternal deaths moreover , the difference in mmr could also be as a result of a general lack of high skilled medical doctors who have not yet specialized in obstetrics and the lack of basic obstetric diagnostic equipment s like ultrasound machine which might have limit the quality of case management in our study area compared to the cameroonian study that was conducted in a tertiary , research and training hospital and therefore more capable to deal with high risk pregnancies . when compared with hospital based studies that were conducted between 2010 - 2015 from other regions of africa , a high level of mmr was also reported from nigeria , west africa , ranging from 866 to 1,791 per 100 , 0000 live births in the southern and northern parts of nigeria respectively . however , lower figures of between 124 and 492 per 100,000 live births was reported east african countries - kenya and tanzania respectively . it is important to note that since 2010 , tanzania and kenya were reported to have made significant progress in the implementation of emergency obstetric care and maternal death surveillance and response compared to angola and therefore may partly account for their lower mmr . similar disparity was observed in hospital based studies among south east asian countries with nepal and pakistan both having lower mmr of 357 and 1007 per 100,000 live births respectively compared to the 1,830 per 100,000 live births reported in this study . however , when compared with the mmr from northern europe and north america , the figure obtained in this study is 167 times higher and hence , reinforces the huge disparity in population health outcome between developed and developing countries . this is driven , in part , by differences in socioeconomic , cultural and political development of these developed countries compared to under developed countries like angola . overall , the mmr obtained in this series is far higher than angola s 2014 estimated mmr of 460 per 100,000 live births by the who . this might not be unrelated to the fact that , the data used in this study were largely incomplete with more than a third of all cases of maternal deaths lacking information on the working diagnosis or clinical management and hence could be a marker for possible under estimation . the possibility of under estimation is further reinforced by the reports on the status of implementation of maternal death surveillance and response which rated it to be low in angola . the dearth of published information on maternal deaths in angola , which could have provided additional information on the state of maternal mortality , is also a big challenge . for instance , extensive systematic reviews of over 7000 articles on maternal mortality , reproductive health and female autonomy , there was no single article from angola , despite having a higher than the desired mdg 5 goal of 300 per 100,000 live births by the year 2015 . although , hospital based studies are likely to limited by misrepresentation or selection bias , we believe that the likelihood of under estimation can not be easily dismissed without conducting large scale community based quantitative and qualitative studies . the available data for this study were characterized by the lack of basic information on some or all of the following variables such as parity , birth order , booking status and number of ante natal care visits in majority of the cases . this was further compounded by none of the cases have information on well - established drivers of utilization of maternal health services such as the level of education , income , religion , ethnicity , and occupation as was reported by several studies.[39 - 41 ] the lack of information on these variables makes it difficult to estimate the risk of dying from pregnancy and its related complications based on parity , education , income , and religion , which invariably compromises the appropriateness and quality of any population based public health intervention that is currently being implemented in the province . furthermore , although a maternal mortality review committee was established , however , during the period under review ( 2010 - 2014 ) , not a single review meeting was held . this further reinforces the likelihood of the current plans directed to address maternal deaths are not data driven and could be contributory to the high mmr recorded in this study . the causes of direct obstetric death ( table 2 ) in this study were the same to those reported from various regions of the developing countries . however , although , literature on direct obstetric causes of maternal death was reported to account for 80% of all maternal deaths in the developing countries , however , a much lower figure ( 51% ) was recorded in this study . the reason been largely attributable to the poor documentation with at least a quarter of cases had no working diagnosis . nevertheless , hemorrhage , puerperal sepsis , eclampsia , and ruptured uterus were the major causes of direct deaths as was similarly reported from several developing countries.[42 - 44 ] the high proportion of these causes in this study may be because many of the cases were teenagers ( 37.8% ) and had delivered at home ( 27.2% ) before seeking help in a health facility . thus , teenage pregnancy , lack of prenatal care and delay in seeking early medical intervention increase the risk of ruptured uterus due cephalo - pelvic disproportion / malpresentation , and undiagnosed preeclampsia that resulted in eclampsia , hemorrhage and sepsis due to unhygienic condition associated with home delivery . first , this study is essentially hospital based and therefore , estimates calculated , tend to be very high . thus , women who died or deliver in this hospital are not the true representation of the entire country . second , patients referred to this hospital , are high risk women who had presented themselves for prenatal care at other health institutions , but were referred here for delivery . this means that , among the women giving birth in these hospitals , there will be a disproportionate number of women with obstetric complication and women who die here which may partly explain the high mmr recorded . third , a large proportion of women who died might have been admitted as an emergency and in moribund condition ; but whose deaths will swell the number of total hospital deaths . first , this study is essentially hospital based and therefore , estimates calculated , tend to be very high . thus , women who died or deliver in this hospital are not the true representation of the entire country . second , patients referred to this hospital , are high risk women who had presented themselves for prenatal care at other health institutions , but were referred here for delivery . this means that , among the women giving birth in these hospitals , there will be a disproportionate number of women with obstetric complication and women who die here which may partly explain the high mmr recorded . third , a large proportion of women who died might have been admitted as an emergency and in moribund condition ; but whose deaths will swell the number of total hospital deaths . with an mmr of 1,830 per 100,000 live births , the scourge of pregnancy and childbearing are enormous in this environment . this study demonstrated that , mmr is higher than the estimates by who and the province is not likely to achieve the desired mdg 5 target of mmr 300 per 100,000 live births . although , hospital based studies suffer from berksonian bias , however , the lack of periodic reviews or community based surveys made this study useful by highlighting the lack of documentation of basic information that constituted the who framework on social determinants of disparities in population health , and documentation is the starting point of evidence based planning . hence , the result of the study is an indictment on the policy standard operating procedures for maternal health services and the likelihood of using plans that are not data driven . the fact that the hospital operates without an ultrasound machine to enhance appropriate diagnosis and management of cases , further underscores the likelihood of minimal impact of clinical intervention to avert maternal deaths . this study also sheds more light on the need for operational and community based quantitative and qualitative studies , in order to provide better insights on what operates in the community from the time when danger signs of pregnancy are recognized at household level to the time when appropriate management has commence in a health facility . such approach will provide basis for the development of holistic multi - prong evidence based intervention that will improve the quality of service with subsequent reduction in mmr . based on the findings above , the following recommendations were made : measure progress , by way of provision of funds for research and evaluation , so that lapses will be easily detected and corrected through policy formulation . this process should include review of all cases of maternal death by the maternal mortality review committee . findings should be use to come up with minimal documentation required , development of standard operating procedures and the creation of a simple microsoft excel data based . there is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality.discourage teenage marriage and early childbearing . this should be through community mobilization ; giving them basic information about pregnancy and childbearing . another approach is to provide compulsory formal education up to secondary school level for all children . the overall effect is the modification of the young woman s behavior towards health and ultimately enhance female autonomy and empowerment . measure progress , by way of provision of funds for research and evaluation , so that lapses will be easily detected and corrected through policy formulation . this process should include review of all cases of maternal death by the maternal mortality review committee . findings should be use to come up with minimal documentation required , development of standard operating procedures and the creation of a simple microsoft excel data based . there is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality . this should be through community mobilization ; giving them basic information about pregnancy and childbearing . another approach is to provide compulsory formal education up to secondary school level for all children . the overall effect is the modification of the young woman s behavior towards health and ultimately enhance female autonomy and empowerment . mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000.there is the lack of documentation of basic information on social determinants of disparities in population health outcome , inadequate staff and none availability of diagnostic equipment like ultrasound.there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death . mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000 . there is the lack of documentation of basic information on social determinants of disparities in population health outcome , inadequate staff and none availability of diagnostic equipment like ultrasound . there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death . based on the findings above , the following recommendations were made : measure progress , by way of provision of funds for research and evaluation , so that lapses will be easily detected and corrected through policy formulation . this process should include review of all cases of maternal death by the maternal mortality review committee . findings should be use to come up with minimal documentation required , development of standard operating procedures and the creation of a simple microsoft excel data based . there is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality.discourage teenage marriage and early childbearing . this should be through community mobilization ; giving them basic information about pregnancy and childbearing . another approach is to provide compulsory formal education up to secondary school level for all children . the overall effect is the modification of the young woman s behavior towards health and ultimately enhance female autonomy and empowerment . measure progress , by way of provision of funds for research and evaluation , so that lapses will be easily detected and corrected through policy formulation . this process should include review of all cases of maternal death by the maternal mortality review committee . findings should be use to come up with minimal documentation required , development of standard operating procedures and the creation of a simple microsoft excel data based . there is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality . this should be through community mobilization ; giving them basic information about pregnancy and childbearing . another approach is to provide compulsory formal education up to secondary school level for all children . the overall effect is the modification of the young woman s behavior towards health and ultimately enhance female autonomy and empowerment . mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000.there is the lack of documentation of basic information on social determinants of disparities in population health outcome , inadequate staff and none availability of diagnostic equipment like ultrasound.there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death . mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000 . there is the lack of documentation of basic information on social determinants of disparities in population health outcome , inadequate staff and none availability of diagnostic equipment like ultrasound . there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death .

background : increasing global health efforts have focused on preventing pregnancy - related maternal deaths , but the factors that contribute to maternal deaths in specific high - burden nations are poorly understood . the aim of this study was to identify factors that influence the occurrence of maternal deaths in a regional maternity hospital in kuando kubango province of angola.methods:the study was a retrospective cross - sectional analysis of case notes of all maternal deaths and deliveries that were recorded from 2010 to 2014 . the information collected included data on pregnancy , labor and post - natal period retrieved from case notes and the delivery register.results:during the period under study , a total of 7,158 live births were conducted out of which 131 resulted in maternal death with an overall maternal mortality ratio of 1,830 per 100,000 live births . the causes of death and their importance was relatively similar over the period reviewed . the direct obstetric causes accounted for 51% of all deaths . the major causes were hemorrhage ( 15% ) , puerperal sepsis ( 13% ) , eclampsia ( 11% ) and ruptured uterus ( 10% ) . in addition , indirect non - obstetric medical causes such as malaria , anemia , hepatitis , aids and cardiovascular diseases accounted for 49% of all maternal deaths . there is poor documentation of personal data and clinical case management of cases . the factors of mutual instability of statistical significance associated with maternal death are : place of domicile ( p=0.0001 ) and distance to the hospital ( p=0.0001).conclusion and global health implication : the study demonstrated that the mmr in maternity hospital is very high and is higher than the who 2014 estimates and the province is yet to achieve the desired mdg 5 target by the end of 2015 . a reversal of the present state requires data driven planning in order to improve access and use of maternal health services ( mhs ) and ultimately lower the number of pregnancy - related maternal deaths .

Introduction Methodology Study area Study population Study design Data analysis Results Discussion Limitation of the study Conclusions and Global Health Implication Recommendations

this concern was underscored by the millennium development goal 5 ( mdg ) which focused on improving maternal health services ( mhs ) to reduce morbidity , disability , and mortality due to pregnancy and delivery . worldwide , it has been estimated that 289,000 cases of maternal deaths were recorded in 2013 , indicating a raising trend by 2000 deaths compared to the figure obtained in 2011 , with 99 % of these deaths occurring in the developing countries . although it is the wealthiest nation in the central african sub - region , with an impressive economic growth since the end of the country s civil war in 2002 , the republic of angola has a higher life time risk of 1 in 39 women dying as a result of pregnancy and its related complications compared to neighboring republic of namibia with a life time risk of 1 in 160 . the current maternal mortality ratio ( mmr ) for angola was reported to be 460 per 100,000 live births in 2013 , which is still far above the desired mdg 5 goal of 300 per 100,000 live births by the year 2015 . the high mmr ( 460 per 100,000 live births ) is partly due to low utilization of maternal health services . for instance , between 2006 - 2013 , only 47% and 49% of pregnant women had at least four ante natal care visits as recommended by the world health organization ( who ) and delivered under the supervision of a skilled health worker respectively . one of the major reasons for sub - saharan african countries having the highest life time risk of dying due to pregnancy related issues and poor pregnancy outcomes such as still births and neonatal deaths is lack of systematic search for the root causes of these deaths as being done in the industrialized countries where confidential inquiries is the norm rather than an exception . this concern is legitimate in terms of the need for the utilization of local information in order to come up with the root causes that lead to poor utilization of maternal and child care services , which will subsequently guide local interventions at provincial / state and district levels . for instance , of the 2,500 articles on maternal mortality reviewed by gil - gonzlez and colleagues , and another 5,575 articles on maternal health services utilization and pregnancy outcomes reviewed by say and raine , and 54 articles on the effectiveness of interventions on maternal mortality in low income countries reviewed by burchette and mayhew , there was no single article from the republic of angola despite the country s high mmr of 460 per 100,000 live birth , and a life time risk of a woman dying from pregnancy and its related complications over 1000 times higher when compared to canada and scandinavian countries . furthermore , a review of the status of maternal mortality surveillance in 2012 showed that data on maternal deaths are lacking or incomplete in 48.9% of the 180 countries reviewed including angola . a recent review on status of implementation of maternal death surveillance and response among african countries has shown that the republic of angola was among the countries with no available data on maternal death surveillance and response . the country still relies on traditional non - electric surveillance information tools that focus on clinical causes of maternal deaths such as hemorrhage , ruptured uterus , sepsis , eclampsia etc . these issues indicate the need for area specific studies in order to complement the weak surveillance system and also to guide interventions that are in context of the local issues which forms the objective of this study . in order to identify the underlying root causes of utilization behavior and how they impact pregnancy outcomes , the anderson medical care utilization model was used as the theoretical framework of this study . the model is made up of three constructs namely : 1 ) the predisposing characteristics of women such as biological , cultural , social , and economic ; 2 ) the enabling characteristics such as health system and health care financing mechanisms ; and 3 ) the need characteristics which is the individual pregnant woman s perceived need to use modern health services and the perceptions of the health care worker . it has a total of 102 health facilities out of which 10 provided obstetric services . the target population of this study was women that received obstetric services ( antenatal , delivery or post - natal ) in the provincial main referral maternity hospital menongue , kuando kubango province of angola , between 2010 and 2014 . it is basically the analysis of data on pregnancy , labor and puerperium from patient case notes and the delivery register for a period of five years ( 2010 - 2014 ) . only maternal deaths that meet the world health organization s ( who ) definition of maternal mortality were included in the study . according to who maternal mortality is the death of any woman while pregnant or within 42 days of termination of pregnancy , from any cause related to or aggravated by pregnancy or its management , irrespective of the duration and site of the pregnancy , but not from accidental or incidental causes . the tool documented data on personal , and clinical information such as age , tribe , religion , marital status , place of domicile , parity , occupation of husband , occupation of cases , mode of admission ( self or referred ) , booking status , indication for admission , date of admission , duration of labor , interval between onset of labor and admission to hospital , place of delivery ( as indicated in the referral letter or case notes ) , previous obstetric operations , interval between admission and maternal death , cause of maternal death and date of death . maternal mortality ratio was calculated for each of the five years ( 2010 - 2014 ) and for the overall study period . the causes of death were examined under two heading viz : direct obstetric causes and indirect non obstetric medical causes . it has a total of 102 health facilities out of which 10 provided obstetric services . all hospitals have a functional ambulance to facilitate the transfer of cases to the provincial maternity hospital . the target population of this study was women that received obstetric services ( antenatal , delivery or post - natal ) in the provincial main referral maternity hospital menongue , kuando kubango province of angola , between 2010 and 2014 . it is basically the analysis of data on pregnancy , labor and puerperium from patient case notes and the delivery register for a period of five years ( 2010 - 2014 ) . only maternal deaths that meet the world health organization s ( who ) definition of maternal mortality were included in the study . according to who maternal mortality is the death of any woman while pregnant or within 42 days of termination of pregnancy , from any cause related to or aggravated by pregnancy or its management , irrespective of the duration and site of the pregnancy , but not from accidental or incidental causes . the tool documented data on personal , and clinical information such as age , tribe , religion , marital status , place of domicile , parity , occupation of husband , occupation of cases , mode of admission ( self or referred ) , booking status , indication for admission , date of admission , duration of labor , interval between onset of labor and admission to hospital , place of delivery ( as indicated in the referral letter or case notes ) , previous obstetric operations , interval between admission and maternal death , cause of maternal death and date of death . maternal mortality ratio was calculated for each of the five years ( 2010 - 2014 ) and for the overall study period . the causes of death were examined under two heading viz : direct obstetric causes and indirect non obstetric medical causes . during the period under study ( 2010 - 2014 ) , 131 maternal deaths were recorded , out of 7,158 live births , giving a maternal mortality ratio ( mmr ) of 1830 per 100,000 deliveries . the annual and overall maternal mortality ratio the denominator of the former ( ratio ) is live births while the later ( rate ) is women in the reproductive age group 15 - 49 years . annual distribution of maternal mortality ratio the peak incidence of mmr was recorded in 2012 with mmr of 2,487 per 100,000 live births ( figure 1 ) . the overall trend although decreasing between 2013 and 2014 , however , the 2014 mmr of 1,757 per 100,000 live births is still higher than the figure of 1,360 per 100,000 live births recorded in 2010 ( figure 1 ) . trend of mmr per 100,000 live births , 2010 - 2014 , menongue , kuando kubango province out of the 131 maternal deaths , 31 ( 24% ) of these cases had inadequate documentation on working diagnosis , presenting complain , history of presenting complain and or management outline and therefore were not included in analysis of direct and indirect causes of maternal death . the three leading direct causes of maternal deaths are hemorrhage ( 15% ) , puerperal sepsis ( 13% ) and eclampsia ( 11% ) which combined accounted for 39% of all deaths ( table 2 ) . proportion of direct obstetric and indirect non - obstetric causes of deaths , 2010 - 2014 , kuando kubango , angola indirect non - obstetric medical causes , accounted for 49% of all maternal deaths . malaria in pregnancy ( 14% ) , anemic heart failure ( 13% ) and pneumonia ( 5% ) were the leading cause of indirect non obstetric medical causes of maternal death , accounting for 32% of all deaths ( table 2 ) . the age distribution of maternal deaths indicated that more than half of all deaths were accounted by women between the ages of 15 - 19 and those > 35 years with age specific mmr ( asmmr ) of 1,058 and 1,354 per 100,000 live births respectively . pregnancy outcomes in relation to some bio - socio - demographic factors among 12,573 deliveries , 2010 - 2014 , kuando kubango , angola fisher exact test with yates correction one hundred and seventeen out of the 131 cases of deaths were from comuna sede of menongue district accounting for 89% of all deaths recorded during the review period . additionally , caiundo and missombo comunas of menongue district accounted for another 2.7% indicating that about 93% of all deaths were from menongue district . the districts of cuito cuanavale and cuchi accounted for only 3.8% and 1.5% of all deaths respectively . women living in rural areas accounted for 96.2% of all deaths and the difference in the place of domicile ( rural versus urban ) was significantly associated with maternal deaths ( p < 0.0001 ) ( table 3 ) . distance to the nearest health facility that provides maternal health services ( mhs ) was significantly associated maternal mortality ( p < 0.0001 ) , with women who responded that distance is not an obstacle accounting for only 30% of all maternal deaths during the period under review ( table 3 ) . out of the 131 maternal deaths , 52 ( 40% ) occurred within the first 24 hours of been admitted in the hospital , 29 ( 22% ) deaths occurred 24 - 48 hours after admission , while 47 ( 35.6% ) occurred between 2 to 11 days after admission . with an estimated 62 , 736 women in the reproductive age group and 14,937 annual pregnancies for menongue district where the hospital is located , the ratio of midwife per 1000 women in the reproductive age group for menongue district is 1 per 2,134 pregnancies per annum . the mean mmr of 1830 per 100,000 live births recorded in this study is five times higher than the desired mdg 5 goal of 300 per 100,000 live births by the year 2015 , and four times higher than the who estimate for angola . as earlier pointed out above , there is a general lack of published journal articles on maternal mortality ( hospital or community based ) in angola . the official report by the health authorities of cabinda province of angola indicated in 2012 the mmr was 234.3/100,000 live births . however , a review of records in all the 10 health facilities that provide obstetric care in cabinda province by rodrigues ( 2013 ) concluded that the provincial official mmr is an under estimation and that the magnitude of the problem is unknown because data is generally incomplete or not available in all the 10 health facilities . however , when compared with published studies ( 2010 - 2015 ) from central africa region to which angola belongs , the mmr of this study ( 1,830 per 100,000 ) was higher than other hospital based study in cameroon ( 287.57100 , 000 ) . the huge difference in mmr between our finding and the cameroonian study may partially be due to the fact that more than half of all the cases of maternal mortality in our study area was among women who were less than 20 years or more than 35 years and presented with complicated pregnancies that resulted in maternal deaths moreover , the difference in mmr could also be as a result of a general lack of high skilled medical doctors who have not yet specialized in obstetrics and the lack of basic obstetric diagnostic equipment s like ultrasound machine which might have limit the quality of case management in our study area compared to the cameroonian study that was conducted in a tertiary , research and training hospital and therefore more capable to deal with high risk pregnancies . when compared with hospital based studies that were conducted between 2010 - 2015 from other regions of africa , a high level of mmr was also reported from nigeria , west africa , ranging from 866 to 1,791 per 100 , 0000 live births in the southern and northern parts of nigeria respectively . similar disparity was observed in hospital based studies among south east asian countries with nepal and pakistan both having lower mmr of 357 and 1007 per 100,000 live births respectively compared to the 1,830 per 100,000 live births reported in this study . overall , the mmr obtained in this series is far higher than angola s 2014 estimated mmr of 460 per 100,000 live births by the who . this might not be unrelated to the fact that , the data used in this study were largely incomplete with more than a third of all cases of maternal deaths lacking information on the working diagnosis or clinical management and hence could be a marker for possible under estimation . the dearth of published information on maternal deaths in angola , which could have provided additional information on the state of maternal mortality , is also a big challenge . for instance , extensive systematic reviews of over 7000 articles on maternal mortality , reproductive health and female autonomy , there was no single article from angola , despite having a higher than the desired mdg 5 goal of 300 per 100,000 live births by the year 2015 . the available data for this study were characterized by the lack of basic information on some or all of the following variables such as parity , birth order , booking status and number of ante natal care visits in majority of the cases . this was further compounded by none of the cases have information on well - established drivers of utilization of maternal health services such as the level of education , income , religion , ethnicity , and occupation as was reported by several studies. furthermore , although a maternal mortality review committee was established , however , during the period under review ( 2010 - 2014 ) , not a single review meeting was held . this further reinforces the likelihood of the current plans directed to address maternal deaths are not data driven and could be contributory to the high mmr recorded in this study . the causes of direct obstetric death ( table 2 ) in this study were the same to those reported from various regions of the developing countries . however , although , literature on direct obstetric causes of maternal death was reported to account for 80% of all maternal deaths in the developing countries , however , a much lower figure ( 51% ) was recorded in this study . nevertheless , hemorrhage , puerperal sepsis , eclampsia , and ruptured uterus were the major causes of direct deaths as was similarly reported from several developing countries. [42 - 44 ] the high proportion of these causes in this study may be because many of the cases were teenagers ( 37.8% ) and had delivered at home ( 27.2% ) before seeking help in a health facility . thus , teenage pregnancy , lack of prenatal care and delay in seeking early medical intervention increase the risk of ruptured uterus due cephalo - pelvic disproportion / malpresentation , and undiagnosed preeclampsia that resulted in eclampsia , hemorrhage and sepsis due to unhygienic condition associated with home delivery . third , a large proportion of women who died might have been admitted as an emergency and in moribund condition ; but whose deaths will swell the number of total hospital deaths . with an mmr of 1,830 per 100,000 live births , the scourge of pregnancy and childbearing are enormous in this environment . this study demonstrated that , mmr is higher than the estimates by who and the province is not likely to achieve the desired mdg 5 target of mmr 300 per 100,000 live births . hence , the result of the study is an indictment on the policy standard operating procedures for maternal health services and the likelihood of using plans that are not data driven . the fact that the hospital operates without an ultrasound machine to enhance appropriate diagnosis and management of cases , further underscores the likelihood of minimal impact of clinical intervention to avert maternal deaths . this study also sheds more light on the need for operational and community based quantitative and qualitative studies , in order to provide better insights on what operates in the community from the time when danger signs of pregnancy are recognized at household level to the time when appropriate management has commence in a health facility . this process should include review of all cases of maternal death by the maternal mortality review committee . there is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality.discourage teenage marriage and early childbearing . this process should include review of all cases of maternal death by the maternal mortality review committee . there is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality . mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000.there is the lack of documentation of basic information on social determinants of disparities in population health outcome , inadequate staff and none availability of diagnostic equipment like ultrasound.there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death . mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000 . there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death . this process should include review of all cases of maternal death by the maternal mortality review committee . there is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality.discourage teenage marriage and early childbearing . this process should include review of all cases of maternal death by the maternal mortality review committee . there is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality . mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000.there is the lack of documentation of basic information on social determinants of disparities in population health outcome , inadequate staff and none availability of diagnostic equipment like ultrasound.there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death . mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000 . there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death .

tissue engineering attempts to generate new living tissues through the use of engineering principles and biological sciences . there are many different techniques and methodologies used to generate these new tissues ( fig . 1 ) , which have progressed beyond contemporary structural design . traditionally , when constructing a building , the process begins with the designer using a protractor , straight edge and compass to produce a sketch that will be translated to computer aided design ( cad ) software for blueprint production . however , in nature , one rarely sees right angles and straight edges . in the human body the curved surfaces on the exterior of the body result in one 's identity ( e.g. facial mapping and finger prints ) . internally , geometric features result in proper joint load distributions in the hip , knee and ankle . blood flow in a beating heart is properly restricted by the size and behaviour of leaflet valves . larger organs , such as the liver , have highly organized circulating systems necessary to deliver oxygenated blood through the larger structure . replicating the complex geometries in naturally occurring structures in the body will require more than protractor and compass . to this end , the development of high - resolution imaging techniques combined with biomaterials processing technology has given rise to the field of image - guided tissue engineering . image guided tissue engineering process tree . typically , imaging modalities such as magnetic resonance imaging ( mri ) and computed tomography ( ct ) have been used as diagnostic tools to visualize the body and develop treatment strategies . treatment strategies include choosing the type of implant , designing a patient specific implant / prosthetic or perhaps using medical imaging data to guide implantation of a device . medical imaging can be used not only for prosthetic designs , but can serve as templates for organ scaffold construction . medically , there exists a large need to provide alternatives for cadaveric allo - grafts , autografts and prosthetic implantations . for example in orthopaedic surgery , the number of patients receiving total hip and knee replacements in 1995 totalled 457,000 in the united states alone and is expected to double by the year 2025 . although the number of patients affected is smaller , those awaiting liver transplant had a death rate of 8.3% in 1999 . similarly , patients awaiting a heart transplant have a 6-month mortality rate of 2470% . facial reconstruction , though less life threatening , represents a cornerstone that interfaces cosmetic and reconstructive surgery to restore both functionality and aesthetic properties important to one 's quality of life . internally transplanted tissues need to fit into the desired space and conform to the surrounding tissues . as a result fit the recipient whether it is a liver , heart , meniscus , or flap of skin . in addition to function , external tissue transplants require appearance to be taken into consideration as well . however , aesthetic appearance becomes a secondary objective to functionality and restoration of health , because no established treatment exists that meets all other primary criteria to prevent rejection , chronic pain and decrease mortality . indeed some of the most exciting applications of tissue engineered ( te ) technology have involved replication of anatomic geometry . some early examples in the field of tissue engineering have been successful in forming cartilage in the shape of a human ear , producing a bone - cartilage composite shaped as a mandibular joint , generation of a distal phalanx for thumb reconstruction and anatomically shaped menisci for the knee . in these cases , these initial studies , although very important , are unlikely to be implemented on a wide scale for generating patient specific geometry on a case by case basis . an obvious solution would be using medical imaging to obtain the necessary information on the patient 's specific anatomical needs . this article will present a brief review of the current methods used to replicate the complex tissues in the body . anatomical geometries can be extracted from any medical imaging modality capable of rendering a 3d image , such as angiography , fluoroscopy , mammography , mri , ct , ct , stereophotogrammetry ( 3d photogrammy ) and ultrasound . although there exists a large selection of imaging modalities from which to choose , mri and ct are the most widely used to visualize cardiovascular , musculoskeletal , neural and dental tissues . however each imaging technique may present distinct advantages for a specific application of tissue replacement . mri can readily register bone and soft tissues and has scan volumes that can range from as large as the human body to small precision scans that image the wrist and knee ( table 1 ) . scan times for an mri range from 5 to 40 min . with resolutions that increase with both scan time and magnetic coil strength . resolutions for a 3 t mri have been reported as high as 250 m 250 m 0.5 mm . scan time can be reduced with the use of higher powered magnetic fields , but human beings are rarely exposed to fields greater than 3 tesla ( t ) . exposure to a 7 t mr coil can cause higher incidence of discomfort and sensations of vertigo than lower strength mr coils . although mri scans are preferred over ct because there is no radiation exposure , it is important to note that there is a sizable percentage of the population that experiences uncomfortable anxiety and claustrophobia when having a full body mri ( table 1 ) . image modality characteristics = other tissues can be imaged with the aid of contrast agents . specifications for mri , ct , and [ ct provided by siemens medical solutions usa , inc . malvern , pa , usa and ge healthcare , formerly evs corporation , ontario , canada . 3d digital photogrammy specifications provided by 3dmd , atlanta , ga , usa and ultrasound specifications provided by elliott and thrush . ct scans can generate higher resolution images than mri ( 0.240.3 mm ) , but can only image bone without the use of contrast agents ( table 1 ) . three - dimensional models are more readily generated from ct scans with little to no manual editing , where as mri requires many manual techniques to acquire the geometry . scan times are much shorter for ct than for mri , but this imaging technique requires the use of ionizing radiation . this presents a minimal but finite risk to individual patients , but collectively a much bigger risk to larger patient population . ct has ultra high resolution ( 1200 m ) , but is limited by the volume in which it can scan ( table 1 ) . due to the volume limitation of ct , it can not be considered non - invasive for animals larger than mice . also , ct , like ct , will not readily register soft tissues in the absence of contrast agents , which may alter tissue structure or geometry . ultrasound can readily image most tissue and does not use ionizing radiation or require a person to be in an enclosed area . although scan times for ultrasound are short , it is limited in the resolution quality it can provide ( 1 1.5 0.2 mm ) . typical volumes that are scanned via ultrasound include small structures such as blood vessels to large ones such as neonatal infants ( table 1 ) . three - dimensional digital photogrammy can obtain high - resolution images ( 150 m ) in less than a minute ( table 1 ) . three - dimensional photogrammy is primarily used for external structures it is done in an open area so patients do not have to worry about the claustrophobia that is common to mri . further , there is no ionizing radiation associated with 3d digital photogrammy , unlike ct or ct . the process for selecting the most appropriate imaging method is tightly coupled to the target tissue . for example , if the desire is to obtain medical imaging data from a patient to generate a femoral head , meniscus , or heart leaflet valve , three very different approaches would be used . in the case of the femoral head , although ct would provide the highest resolution image of the boney structure , it does not image cartilage or soft tissues readily . ct would not be used because the femoral head is too large to fit into current scanning devices . an mri scan , on the other hand , could be used to obtain both the articular surface and boney structure without contrast agents . in the case of the meniscus , high - resolution images of the meniscus can be obtained via mri by increasing the scan time . however , increased scan time increases cost and becomes a compliancy issue for the patient . the longer the patient is required to remain still during the scan the higher the probability of geometry artefact due to movement . the alternative would be to excise the tissue from the joint , soak it in a contrast agent to allow for ct scanning . it is important to note that mri can acquire geometries under loaded conditions whereas ct may have altered geometry due to being soaked in a contrast agent . in the case of the heart valve , mri and ct both require contrast agents to visualize the inner workings of the heart and have similar image resolutions . due to the high radiation exposure needed to perform a ct scan of the heart and the high expense associated with mri usage , echocardiography ( cardiac ultrasound ) is becoming a more widely used non - invasive method to obtain 3d geometric models of mitral valves [ 13 , 14 ] . however , to maximize resolution , the valve can still be excised , soaked in a contrast agent and scanned viact . generating anatomically shaped engineered tissues does not require medical images . as mentioned earlier , many early te efforts to generate anatomically shaped constructs used impression moulds [ 6 , 7 , 1518 ] to serve as negative templates . the paradigm shift to using medical images for cad design has only very recently been established . there are multiple methods to replicate anatomical shape through injection moulding or different rapid prototyping techniques and for each method there exists an even larger choice of biomaterials to use as a scaffold . choice of scaffold will dictate the design and fabrication process of the engineered tissue , which is driven by the application and tissue one is trying to generate . here we will briefly take a look at some promising results across a number of different engineered tissues . as stated above , scaffold choice has a major role in guiding the fabrication process of generating te constructs . many traditional scaffold materials ( e.g. polyglycolic acid fibres [ pga ] , polylactic acid [ pla ] , polycaprolattone [ pcl ] ) require processing at high temperatures or in organic solvents to control shape . as such cells can not be introduced until the scaffold has cooled and solvents have been removed . in contrast , materials such as hydro - gels undergo phase transitions that enable maintenance of cell viability during gel formation . as such , cells can be introduced to these materials prior to moulding . initial efforts in cartilage tissue engineering used acellular scaffolds and began with the simple geometries in the shape of triangles , rectangles and cylinders . more complicated geometries were also achieved , such as a human ear using a synthetic non - woven mesh composed of pga . the pga mesh was moulded into desired geometries through the use of plaster prosthetic mould , cells were then later seeded onto pga scaffolds and allowed to culture subcutaneously in nude mice [ 6 , 17 ] . similarly , bone te requires scaffolds with a high rigidity that emulates the physical properties of native bone . the processes involved in bone scaffold formation are often unfavourable for cell viability and therefore seeding of these constructs occurred after they were constructed . one such study successfully te phalanges and small joints through the use of pga and pla . the seeding of acelluar scaffolds has also been applied to engineered cardiovascular tissue such as blood vessels and heart valves . in one promising study , pcl was electro - spun into the shape of a trileaflet valve using a custom designed aluminium template modelled after native tissue before being seeded with cardiac cells for in vitro culture . although seeding cells after scaffold generation has produced promising results , this methodology is very time consuming and does not ensure equal cell distribution throughout the scaffold . a more efficient approach would be to seed scaffolds before they are formed , though this would require biomaterials with a non - toxic liquid phase that maintain viability during the solidification or gelation process . biomaterials that allow this approach include , but are not limited to , alginate , agarose , chitosan , collagen gel , fibrin glue and poly(lactide - co - glycolide ) ( plg ) . the algi - nate - cell solution was crossed - linked with caso4 and injected into silastic impression moulds of chin and nose implants for facial reconstruction . using various cell seeding densities they were able to culture these implants in the back of nude mice for 30 weeks and maintain both shape and cell viability . uniform cell distribution becomes more critical when generating injection moulds of larger constructs , such as the mandible for craniofacial reconstruction [ 15 , 18 ] or the meniscus of the knee . seeding the scaffold while it is liquid enhances homogeneity of cell distribution upon initial construct formation . cad - based injection moulds have been used to design a wide array of geometries from very small volume structures such as tympanic membrane patches ( 3 l ) , and engineered heart valves ( 1 ml ) , to larger sized tissues such as the meniscus ( 25 ml ) . injection moulding techniques , although not optimal for multi - material constructs , can be altered to generate more complex tissues . a prime example is the production of an anatomically shaped osteochondral construct based on stereophotogrammetry data via injection moulding . patellar shaped composites were made possible through computer numerical control ( cnc ) milling of demarrowed bone blocks that fit into a mould allowing for injection of cell seeded agarose resulting in partially integrated bone plugs . another composite injection moulding study by mizuno et al . produced both a multi - material and multi - cellular te intervertebral disc [ 24 , 25 ] . the intervertebral disc was composed of an annulus fibrosus made from pla / pga scaffold and a nucleus pulposus made from calcium cross - linked alginate that was injected into the centre void of the pla / pga scaffold . each region was composed of its respective cell type and exhibited both biochemical and mechanical properties similar to that of native tissue [ 24 , 25 ] . one of the most recent advances in generating patient specific implants via injection moulding were achieved using alginate and meniscal fibrochondrocytes from bovine knees . the geometry was obtained using both mri and ct scans of sheep knees and used to produce cad moulds that were 3d printed out of acryloni - trile butadiene styrene ( abs ) plastic . alginate - cell solution was cross - linked with caso4 and cultured for up to 8 weeks in vitro . anatomical shape was retained and constructs had both mechanical and biochemical properties similar to that of native tissues ( fig . future efforts are now focusing on stimulating extracellular matrix ( ecm ) production as well as evaluation of geometric fidelity based on imaging type and time in culture . ( a ) an injection moulded menisci derived from a ct scan and fibrochondrocyte seeded alginate after 8 weeks of in vitro culture . ( b ) medical grade pcl composite formed via fused deposition modelling ( image provided by dr . ( c ) chondrocyte seeded alginate micro - channel network with 50 50 m channels spaced 100 m apart . ( d ) cartilagenous disc 1 cm in diameter composed of plg micro - beads seeded with chon - drocytes after 8 weeks of in vitro culture . the basis for this technique is to produce usable scaffolds in a short time scale ( i.e. hours to days ) . solid freeform fabrication ( sff ) and 3d printing are two of the more popular rapid prototyping techniques that are capable of generating multi - material and multi - cellular anatomical constructs . hutmacher and cool have nicely reviewed applications of sff on bone tissue engineering in this journal ( fig . fabrication techniques and the various biomaterials used for cell seeded scaffolds and acellular scaffolds as well as multi - cell / material capability and current resolution capabilities most bone te methods involve seeding of acellular constructs or insertion of acellular implants with the expectation of cellular ingrowth in vivo . some successful studies include the use of porous coral in the shape of a distal phalanx seeded with periosteal cells for thumb reconstruction , 3d printing brushite implants and a cranial segment using tricalcium phosphate ( tcp ) and tetracalcium phosphate respectively . used localized gene therapy to increase and localize cellular and tissue ingrowth using an sff polypropylene fumarate / tcp composite that provided a stable matrix that could be matched to specific patient defect geometry . ( princeton , nj , usa ) produced osteochondral composites using tcp combined with either plg or pla for the chon - dral surface . the composite structure exhibited region specific mechanical properties and integration between the two biomateri - als making it suitable for implantation . therics , inc . also has a number of other tcp based therapeutic products that are currently undergoing clinical studies . sff techniques are able to produce patient specific scaffolds that can be modified to increase and guide cellular in growth through variation of surface roughness , chemically bonded growth factors , and altered scaffold porosity . for more heterogeneous tissues , such as the meniscus , heart valve and liver , control over spatial and temporal differences in cell type / morphology and mechanical properties is necessary . achieving structures that have the necessary cell distributions and biomechanical properties is a major challenge . cytoscribing , as termed by klebe involved alternating deposition of layers of cells and materials to generate 2d and 3d tissues . klebe established this technique using a variety of different cell types from different species and bound them to substrates using fibronectin that was deposited via hewlett packard graphics plotter of ink jet printer . an excellent example of this is by cohen et al . via sff using alginate and chon - drocytes . the work established the ability to print cell seeded alginate using different materials ( i.e. two different grades of alginate ) and in different structurally sound shapes including a disc , crescent and meniscus based on ct data with printing resolution of 270 m ( table 2 ) . rapid prototyping has also been used in the fabrication of 3d hepatic tissues with complex internal microstructure . constructs were generated using both multi - cell and multi - material as means to improve nutrient transport . cell printing efforts by chang et al . have evaluated cell viability of hepg2 cells based on dispensing pressure and nozzle diameter with calcium cross - linked alginate and combined these sff techniques with lithography methods to generate 3d microorgans . the microorgans had vascular networks serving as pharma - cokinetic models and were able to replicate consistent prints with 250 m resolution ( table 2 ) . the transport of solutes and removal of waste products is a large concern in te , especially when trying to engineer large volume tissues or engineering organs like the liver . in the body this solute transport is accomplished primarily by the vascular system , which is effectively a network of perfused micro channels . preliminary studies using a polydimethylsiroxane ( pdms ) substrate established the efficacy of this technique using both hepatocytes and endothe - lial cells . other biomaterials used in lithography te efforts include polyvinyl alcohol ( pva ) with fibroblasts , pcl and plg with vascular smooth muscle cells , peg with osteoblasts and embryonic stem cells , matrigel with epithelial cells and fibroblasts , as well as collagen and agarose with fibroblasts . other work done by khademhosseini et al . generated 3d micropatterned substrates consisting of hyaluronic acid and fibronectin seeded with cardiomyocytes , which aligned along the interface between the scaffold and glass substrate . recent innovative studies using chondrocytes seeded in alginate have shown great promise in their ability to generate various micro - fluidic patterns via laminated sheets with sealed channels as small as 25 25 m [ 44 , 45 ] ( table 2 ) . after 4 weeks in culture , laminated sheets integrated well with no visible interface where two sheets were bonded together ( fig . this work by choi and coworkers really demonstrates the resolution of image based te and can be implemented to produce larger volume constructs that not only have a custom circulation network , but a network that can be controlled spatially with gradients of nutrients , growth factors and region - specific flow rates [ 4446 ] . the deposition of micro - particles or micro - beads to alter surface properties or to build up structures is known as sintering . sintering has become a valuable fabrication technique that allows designation of specific localized properties that control for porosity , surface chemistry and mechanical properties . most sintering efforts have focused on its application to bone te through the use of pva , hydroxyapatite ( ha ) , tcp and plg . studies have shown improved osteoblast cell growth throughout the sintered matrix . other works done with plg and its application to cartilage tissue engineering have shown its ability to be used as a mouldable scaffold capable of cellular proliferation and infiltration in vivo ( fig . the use of sintering cell seeded plg micro - beads in combination with free chondrocytes can be used to address focal defects in vivo . furthermore , integrating the use of image guided tissue engineering bead - cell mixtures can be deposited to repair articular surfaces to their original geometry before injury . the repair resolution of this technique is only limited by the consistency and size of the micro particles / bead , which can range from 40600 m [ 4751 ] ( table 2 ) . as stated above , scaffold choice has a major role in guiding the fabrication process of generating te constructs . many traditional scaffold materials ( e.g. polyglycolic acid fibres [ pga ] , polylactic acid [ pla ] , polycaprolattone [ pcl ] ) require processing at high temperatures or in organic solvents to control shape . as such cells can not be introduced until the scaffold has cooled and solvents have been removed . in contrast , materials such as hydro - gels undergo phase transitions that enable maintenance of cell viability during gel formation . as such , cells can be introduced to these materials prior to moulding . initial efforts in cartilage tissue engineering used acellular scaffolds and began with the simple geometries in the shape of triangles , rectangles and cylinders . more complicated geometries were also achieved , such as a human ear using a synthetic non - woven mesh composed of pga . the pga mesh was moulded into desired geometries through the use of plaster prosthetic mould , cells were then later seeded onto pga scaffolds and allowed to culture subcutaneously in nude mice [ 6 , 17 ] . similarly , bone te requires scaffolds with a high rigidity that emulates the physical properties of native bone . the processes involved in bone scaffold formation are often unfavourable for cell viability and therefore seeding of these constructs occurred after they were constructed . one such study successfully te phalanges and small joints through the use of pga and pla . the seeding of acelluar scaffolds has also been applied to engineered cardiovascular tissue such as blood vessels and heart valves . in one promising study , pcl was electro - spun into the shape of a trileaflet valve using a custom designed aluminium template modelled after native tissue before being seeded with cardiac cells for in vitro culture . although seeding cells after scaffold generation has produced promising results , this methodology is very time consuming and does not ensure equal cell distribution throughout the scaffold . a more efficient approach would be to seed scaffolds before they are formed , though this would require biomaterials with a non - toxic liquid phase that maintain viability during the solidification or gelation process . biomaterials that allow this approach include , but are not limited to , alginate , agarose , chitosan , collagen gel , fibrin glue and poly(lactide - co - glycolide ) ( plg ) . the algi - nate - cell solution was crossed - linked with caso4 and injected into silastic impression moulds of chin and nose implants for facial reconstruction . using various cell seeding densities they were able to culture these implants in the back of nude mice for 30 weeks and maintain both shape and cell viability . uniform cell distribution becomes more critical when generating injection moulds of larger constructs , such as the mandible for craniofacial reconstruction [ 15 , 18 ] or the meniscus of the knee . seeding the scaffold while it is liquid enhances homogeneity of cell distribution upon initial construct formation . cad - based injection moulds have been used to design a wide array of geometries from very small volume structures such as tympanic membrane patches ( 3 l ) , and engineered heart valves ( 1 ml ) , to larger sized tissues such as the meniscus ( 25 ml ) . the resolution for injection moulding injection moulding techniques , although not optimal for multi - material constructs , can be altered to generate more complex tissues . a prime example is the production of an anatomically shaped osteochondral construct based on stereophotogrammetry data via injection moulding . patellar shaped composites were made possible through computer numerical control ( cnc ) milling of demarrowed bone blocks that fit into a mould allowing for injection of cell seeded agarose resulting in partially integrated bone plugs . another composite injection moulding study by mizuno et al . produced both a multi - material and multi - cellular te intervertebral disc [ 24 , 25 ] . the intervertebral disc was composed of an annulus fibrosus made from pla / pga scaffold and a nucleus pulposus made from calcium cross - linked alginate that was injected into the centre void of the pla / pga scaffold . each region was composed of its respective cell type and exhibited both biochemical and mechanical properties similar to that of native tissue [ 24 , 25 ] . one of the most recent advances in generating patient specific implants via injection moulding were achieved using alginate and meniscal fibrochondrocytes from bovine knees . the geometry was obtained using both mri and ct scans of sheep knees and used to produce cad moulds that were 3d printed out of acryloni - trile butadiene styrene ( abs ) plastic . alginate - cell solution was cross - linked with caso4 and cultured for up to 8 weeks in vitro . anatomical shape was retained and constructs had both mechanical and biochemical properties similar to that of native tissues ( fig . future efforts are now focusing on stimulating extracellular matrix ( ecm ) production as well as evaluation of geometric fidelity based on imaging type and time in culture . ( a ) an injection moulded menisci derived from a ct scan and fibrochondrocyte seeded alginate after 8 weeks of in vitro culture . ( b ) medical grade pcl composite formed via fused deposition modelling ( image provided by dr . ( c ) chondrocyte seeded alginate micro - channel network with 50 50 m channels spaced 100 m apart . ( d ) cartilagenous disc 1 cm in diameter composed of plg micro - beads seeded with chon - drocytes after 8 weeks of in vitro culture . the basis for this technique is to produce usable scaffolds in a short time scale ( i.e. hours to days ) . solid freeform fabrication ( sff ) and 3d printing are two of the more popular rapid prototyping techniques that are capable of generating multi - material and multi - cellular anatomical constructs . hutmacher and cool have nicely reviewed applications of sff on bone tissue engineering in this journal ( fig . fabrication techniques and the various biomaterials used for cell seeded scaffolds and acellular scaffolds as well as multi - cell / material capability and current resolution capabilities most bone te methods involve seeding of acellular constructs or insertion of acellular implants with the expectation of cellular ingrowth in vivo . some successful studies include the use of porous coral in the shape of a distal phalanx seeded with periosteal cells for thumb reconstruction , 3d printing brushite implants and a cranial segment using tricalcium phosphate ( tcp ) and tetracalcium phosphate respectively . used localized gene therapy to increase and localize cellular and tissue ingrowth using an sff polypropylene fumarate / tcp composite that provided a stable matrix that could be matched to specific patient defect geometry . ( princeton , nj , usa ) produced osteochondral composites using tcp combined with either plg or pla for the chon - dral surface . the composite structure exhibited region specific mechanical properties and integration between the two biomateri - als making it suitable for implantation . therics , inc . also has a number of other tcp based therapeutic products that are currently undergoing clinical studies . sff techniques are able to produce patient specific scaffolds that can be modified to increase and guide cellular in growth through variation of surface roughness , chemically bonded growth factors , and altered scaffold porosity . for more heterogeneous tissues , such as the meniscus , heart valve and liver , control over spatial and temporal differences in cell type / morphology and mechanical properties is necessary . achieving structures that have the necessary cell distributions and biomechanical properties is a major challenge . cytoscribing , as termed by klebe involved alternating deposition of layers of cells and materials to generate 2d and 3d tissues . klebe established this technique using a variety of different cell types from different species and bound them to substrates using fibronectin that was deposited via hewlett packard graphics plotter of ink jet printer . an excellent example of this is by cohen et al . via sff using alginate and chon - drocytes . the work established the ability to print cell seeded alginate using different materials ( i.e. two different grades of alginate ) and in different structurally sound shapes including a disc , crescent and meniscus based on ct data with printing resolution of 270 m ( table 2 ) . rapid prototyping has also been used in the fabrication of 3d hepatic tissues with complex internal microstructure . constructs were generated using both multi - cell and multi - material as means to improve nutrient transport . have evaluated cell viability of hepg2 cells based on dispensing pressure and nozzle diameter with calcium cross - linked alginate and combined these sff techniques with lithography methods to generate 3d microorgans . the microorgans had vascular networks serving as pharma - cokinetic models and were able to replicate consistent prints with 250 m resolution ( table 2 ) . the transport of solutes and removal of waste products is a large concern in te , especially when trying to engineer large volume tissues or engineering organs like the liver . in the body this solute transport is accomplished primarily by the vascular system , which is effectively a network of perfused micro channels . preliminary studies using a polydimethylsiroxane ( pdms ) substrate established the efficacy of this technique using both hepatocytes and endothe - lial cells . other biomaterials used in lithography te efforts include polyvinyl alcohol ( pva ) with fibroblasts , pcl and plg with vascular smooth muscle cells , peg with osteoblasts and embryonic stem cells , matrigel with epithelial cells and fibroblasts , as well as collagen and agarose with fibroblasts . other work done by khademhosseini et al . generated 3d micropatterned substrates consisting of hyaluronic acid and fibronectin seeded with cardiomyocytes , which aligned along the interface between the scaffold and glass substrate . recent innovative studies using chondrocytes seeded in alginate have shown great promise in their ability to generate various micro - fluidic patterns via laminated sheets with sealed channels as small as 25 25 m [ 44 , 45 ] ( table 2 ) . after 4 weeks in culture , laminated sheets integrated well with no visible interface where two sheets were bonded together ( fig . this work by choi and coworkers really demonstrates the resolution of image based te and can be implemented to produce larger volume constructs that not only have a custom circulation network , but a network that can be controlled spatially with gradients of nutrients , growth factors and region - specific flow rates [ 4446 ] . the deposition of micro - particles or micro - beads to alter surface properties or to build up structures is known as sintering . sintering has become a valuable fabrication technique that allows designation of specific localized properties that control for porosity , surface chemistry and mechanical properties . most sintering efforts have focused on its application to bone te through the use of pva , hydroxyapatite ( ha ) , tcp and plg . studies have shown improved osteoblast cell growth throughout the sintered matrix . other works done with plg and its application to cartilage tissue engineering have shown its ability to be used as a mouldable scaffold capable of cellular proliferation and infiltration in vivo ( fig . the use of sintering cell seeded plg micro - beads in combination with free chondrocytes can be used to address focal defects in vivo . furthermore , integrating the use of image guided tissue engineering bead - cell mixtures can be deposited to repair articular surfaces to their original geometry before injury . the repair resolution of this technique is only limited by the consistency and size of the micro particles / bead , which can range from 40600 m [ 4751 ] ( table 2 ) image guided tissue engineering shows great promise for the generation of patient specific engineered tissues . ct and mri can provide adequate templates for custom , patient - specific implants . other imaging modalities do hold promise but have yet to be established . although most image based efforts have focused on musculoskeletal tissues , image - based templates are starting to be used for cardiovascular models and small scale micro - vascular channels for hepatic tissues via cad . the methods for generating these constructs vary greatly depending on the scale , tissue type and biomaterial . there exists the possibility to not only generate constructs that mimic the gross anatomy , but also generate proper substructure and networks of the desired tissue . both injection moulding and sff techniques can generate anatomically shaped te constructs that appear to have high geometric fidelity . a major challenge to all who work on image - guided tissue engineering lies in the lack of methods to quantify shape fidelity of fabricated implants . similarly , there is essentially no data describing how shape fidelity is maintained throughout culture whether in vivo or in vitro . these issues are complicated by the fact that there is still no established technique for evaluating shape fidelity of anatomically shaped te constructs . the topic of shape fidelity is still in dire need of further investigation , because for many of these complex shaped tissues such as the meniscus [ 5254 ] or heart halve [ 55 , 56 ] critical dimensions and tolerance levels for implantation are still being debated . it is clear that medical imaging is an excellent tool to quantitatively define the geometry of structures especially in situ , such as the meniscus or heart valve . now with new advances in medical imaging techniques , location specific microstructure can be extracted as well . three - dimensional printing can provide the ability to create tissue - specific properties that vary with location within the tissue / organ ( i.e. cell type , mechanical properties , porosity , etc . ) , which would otherwise not be possible with injection moulding . spatial properties can be gathered from medical images to aid in the construction of engineered tissues . mri and ct have been used to look at gag concentration in cartilage , ct to look at bone density and trabecular architecture , second harmonic generation microscopy to look at collagen fibre orientation and density . combining imaging data techniques with rapid prototyping could allow generation of anatomical structures in situ with region specific microstructure similar to that of native tissues . imaging tools and fabrication techniques have enhanced fabrication of engineered constructs , but on the list of tissue engineering goals this seems to be only the tip of the iceberg . how exactly does one go from a newly fabricated construct and produce engineered tissues ready for implantation ? even without considering shape fidelity , quality control for te implants for dynamically loaded tissues such as the heart valve or meniscus , complicated geometry often results in complicated mechanics . for years , medical imaging has been used to extract geometries of bones , muscles , and cartilage to develop constitutive models to better describe the inner workings of joints in the body through finite element modelling ( fem ) . medical imaging combined with fem will continue to play a major role in assessing the functionality and durability of engineered tissues . as new knowledge is acquired about in vivo behaviour through fem simulations , engineered tissues can be specifically conditioned in vitro to withstand these stresses . the idea of in vitro conditioning is becoming more and more popular not only for engineered tissues such as tendon , heart valve , bone and cartilage , but for cadaveric explants as well [ 65 , 66 ] . exposure to limited in vivo like stimuli in a reduced or gradual manner has shown to be beneficial to cells and resulted in increased ecm formation as well as corresponding improvements in mechanical behaviour . optimal in vitro conditioning settings have yet to be elucidated , but as it stands now the time scale for generating functional tissues is lengthy . nonetheless image - guided tissue engineering is still likely a very valuable tool for generating patient specific tissues and organs . challenges still lie in the ability to integrate these techniques to engineer large volume tissues with micro - vasculature and generate proper ecm organization and alignment . these techniques in combination with in vitro conditioning

replication of anatomic shape is a significant challenge in developing implants for regenerative medicine . this has lead to significant interest in using medical imaging techniques such as magnetic resonance imaging and computed tomography to design tissue engineered constructs . implementation of medical imaging and computer aided design in combination with technologies for rapid prototyping of living implants enables the generation of highly reproducible constructs with spatial resolution up to 25 m . in this paper , we review the medical imaging modalities available and a paradigm for choosing a particular imaging technique . we also present fabrication techniques and methodologies for producing cellular engineered constructs . finally , we comment on future challenges involved with image guided tissue engineering and efforts to generate engineered constructs ready for implantation .

Introduction Imaging techniques Fabrication techniques Injection moulding Rapid prototyping Lithography Sintering Conclusions

tissue engineering attempts to generate new living tissues through the use of engineering principles and biological sciences . there are many different techniques and methodologies used to generate these new tissues ( fig . traditionally , when constructing a building , the process begins with the designer using a protractor , straight edge and compass to produce a sketch that will be translated to computer aided design ( cad ) software for blueprint production . in the human body the curved surfaces on the exterior of the body result in one 's identity ( e.g. larger organs , such as the liver , have highly organized circulating systems necessary to deliver oxygenated blood through the larger structure . to this end , the development of high - resolution imaging techniques combined with biomaterials processing technology has given rise to the field of image - guided tissue engineering . image guided tissue engineering process tree . typically , imaging modalities such as magnetic resonance imaging ( mri ) and computed tomography ( ct ) have been used as diagnostic tools to visualize the body and develop treatment strategies . treatment strategies include choosing the type of implant , designing a patient specific implant / prosthetic or perhaps using medical imaging data to guide implantation of a device . medical imaging can be used not only for prosthetic designs , but can serve as templates for organ scaffold construction . similarly , patients awaiting a heart transplant have a 6-month mortality rate of 2470% . as a result fit the recipient whether it is a liver , heart , meniscus , or flap of skin . indeed some of the most exciting applications of tissue engineered ( te ) technology have involved replication of anatomic geometry . some early examples in the field of tissue engineering have been successful in forming cartilage in the shape of a human ear , producing a bone - cartilage composite shaped as a mandibular joint , generation of a distal phalanx for thumb reconstruction and anatomically shaped menisci for the knee . an obvious solution would be using medical imaging to obtain the necessary information on the patient 's specific anatomical needs . anatomical geometries can be extracted from any medical imaging modality capable of rendering a 3d image , such as angiography , fluoroscopy , mammography , mri , ct , ct , stereophotogrammetry ( 3d photogrammy ) and ultrasound . although there exists a large selection of imaging modalities from which to choose , mri and ct are the most widely used to visualize cardiovascular , musculoskeletal , neural and dental tissues . however each imaging technique may present distinct advantages for a specific application of tissue replacement . although mri scans are preferred over ct because there is no radiation exposure , it is important to note that there is a sizable percentage of the population that experiences uncomfortable anxiety and claustrophobia when having a full body mri ( table 1 ) . scan times are much shorter for ct than for mri , but this imaging technique requires the use of ionizing radiation . ultrasound can readily image most tissue and does not use ionizing radiation or require a person to be in an enclosed area . typical volumes that are scanned via ultrasound include small structures such as blood vessels to large ones such as neonatal infants ( table 1 ) . three - dimensional photogrammy is primarily used for external structures it is done in an open area so patients do not have to worry about the claustrophobia that is common to mri . for example , if the desire is to obtain medical imaging data from a patient to generate a femoral head , meniscus , or heart leaflet valve , three very different approaches would be used . however , increased scan time increases cost and becomes a compliancy issue for the patient . the longer the patient is required to remain still during the scan the higher the probability of geometry artefact due to movement . due to the high radiation exposure needed to perform a ct scan of the heart and the high expense associated with mri usage , echocardiography ( cardiac ultrasound ) is becoming a more widely used non - invasive method to obtain 3d geometric models of mitral valves [ 13 , 14 ] . as mentioned earlier , many early te efforts to generate anatomically shaped constructs used impression moulds [ 6 , 7 , 1518 ] to serve as negative templates . the paradigm shift to using medical images for cad design has only very recently been established . there are multiple methods to replicate anatomical shape through injection moulding or different rapid prototyping techniques and for each method there exists an even larger choice of biomaterials to use as a scaffold . choice of scaffold will dictate the design and fabrication process of the engineered tissue , which is driven by the application and tissue one is trying to generate . polyglycolic acid fibres [ pga ] , polylactic acid [ pla ] , polycaprolattone [ pcl ] ) require processing at high temperatures or in organic solvents to control shape . in contrast , materials such as hydro - gels undergo phase transitions that enable maintenance of cell viability during gel formation . as such , cells can be introduced to these materials prior to moulding . initial efforts in cartilage tissue engineering used acellular scaffolds and began with the simple geometries in the shape of triangles , rectangles and cylinders . more complicated geometries were also achieved , such as a human ear using a synthetic non - woven mesh composed of pga . the pga mesh was moulded into desired geometries through the use of plaster prosthetic mould , cells were then later seeded onto pga scaffolds and allowed to culture subcutaneously in nude mice [ 6 , 17 ] . similarly , bone te requires scaffolds with a high rigidity that emulates the physical properties of native bone . the seeding of acelluar scaffolds has also been applied to engineered cardiovascular tissue such as blood vessels and heart valves . although seeding cells after scaffold generation has produced promising results , this methodology is very time consuming and does not ensure equal cell distribution throughout the scaffold . a more efficient approach would be to seed scaffolds before they are formed , though this would require biomaterials with a non - toxic liquid phase that maintain viability during the solidification or gelation process . the algi - nate - cell solution was crossed - linked with caso4 and injected into silastic impression moulds of chin and nose implants for facial reconstruction . using various cell seeding densities they were able to culture these implants in the back of nude mice for 30 weeks and maintain both shape and cell viability . uniform cell distribution becomes more critical when generating injection moulds of larger constructs , such as the mandible for craniofacial reconstruction [ 15 , 18 ] or the meniscus of the knee . cad - based injection moulds have been used to design a wide array of geometries from very small volume structures such as tympanic membrane patches ( 3 l ) , and engineered heart valves ( 1 ml ) , to larger sized tissues such as the meniscus ( 25 ml ) . injection moulding techniques , although not optimal for multi - material constructs , can be altered to generate more complex tissues . the intervertebral disc was composed of an annulus fibrosus made from pla / pga scaffold and a nucleus pulposus made from calcium cross - linked alginate that was injected into the centre void of the pla / pga scaffold . alginate - cell solution was cross - linked with caso4 and cultured for up to 8 weeks in vitro . anatomical shape was retained and constructs had both mechanical and biochemical properties similar to that of native tissues ( fig . ( c ) chondrocyte seeded alginate micro - channel network with 50 50 m channels spaced 100 m apart . the basis for this technique is to produce usable scaffolds in a short time scale ( i.e. solid freeform fabrication ( sff ) and 3d printing are two of the more popular rapid prototyping techniques that are capable of generating multi - material and multi - cellular anatomical constructs . hutmacher and cool have nicely reviewed applications of sff on bone tissue engineering in this journal ( fig . fabrication techniques and the various biomaterials used for cell seeded scaffolds and acellular scaffolds as well as multi - cell / material capability and current resolution capabilities most bone te methods involve seeding of acellular constructs or insertion of acellular implants with the expectation of cellular ingrowth in vivo . some successful studies include the use of porous coral in the shape of a distal phalanx seeded with periosteal cells for thumb reconstruction , 3d printing brushite implants and a cranial segment using tricalcium phosphate ( tcp ) and tetracalcium phosphate respectively . ( princeton , nj , usa ) produced osteochondral composites using tcp combined with either plg or pla for the chon - dral surface . the composite structure exhibited region specific mechanical properties and integration between the two biomateri - als making it suitable for implantation . also has a number of other tcp based therapeutic products that are currently undergoing clinical studies . for more heterogeneous tissues , such as the meniscus , heart valve and liver , control over spatial and temporal differences in cell type / morphology and mechanical properties is necessary . achieving structures that have the necessary cell distributions and biomechanical properties is a major challenge . cytoscribing , as termed by klebe involved alternating deposition of layers of cells and materials to generate 2d and 3d tissues . rapid prototyping has also been used in the fabrication of 3d hepatic tissues with complex internal microstructure . have evaluated cell viability of hepg2 cells based on dispensing pressure and nozzle diameter with calcium cross - linked alginate and combined these sff techniques with lithography methods to generate 3d microorgans . the transport of solutes and removal of waste products is a large concern in te , especially when trying to engineer large volume tissues or engineering organs like the liver . other work done by khademhosseini et al . recent innovative studies using chondrocytes seeded in alginate have shown great promise in their ability to generate various micro - fluidic patterns via laminated sheets with sealed channels as small as 25 25 m [ 44 , 45 ] ( table 2 ) . after 4 weeks in culture , laminated sheets integrated well with no visible interface where two sheets were bonded together ( fig . sintering has become a valuable fabrication technique that allows designation of specific localized properties that control for porosity , surface chemistry and mechanical properties . studies have shown improved osteoblast cell growth throughout the sintered matrix . other works done with plg and its application to cartilage tissue engineering have shown its ability to be used as a mouldable scaffold capable of cellular proliferation and infiltration in vivo ( fig . the use of sintering cell seeded plg micro - beads in combination with free chondrocytes can be used to address focal defects in vivo . furthermore , integrating the use of image guided tissue engineering bead - cell mixtures can be deposited to repair articular surfaces to their original geometry before injury . in contrast , materials such as hydro - gels undergo phase transitions that enable maintenance of cell viability during gel formation . initial efforts in cartilage tissue engineering used acellular scaffolds and began with the simple geometries in the shape of triangles , rectangles and cylinders . more complicated geometries were also achieved , such as a human ear using a synthetic non - woven mesh composed of pga . the seeding of acelluar scaffolds has also been applied to engineered cardiovascular tissue such as blood vessels and heart valves . the algi - nate - cell solution was crossed - linked with caso4 and injected into silastic impression moulds of chin and nose implants for facial reconstruction . uniform cell distribution becomes more critical when generating injection moulds of larger constructs , such as the mandible for craniofacial reconstruction [ 15 , 18 ] or the meniscus of the knee . cad - based injection moulds have been used to design a wide array of geometries from very small volume structures such as tympanic membrane patches ( 3 l ) , and engineered heart valves ( 1 ml ) , to larger sized tissues such as the meniscus ( 25 ml ) . the resolution for injection moulding injection moulding techniques , although not optimal for multi - material constructs , can be altered to generate more complex tissues . patellar shaped composites were made possible through computer numerical control ( cnc ) milling of demarrowed bone blocks that fit into a mould allowing for injection of cell seeded agarose resulting in partially integrated bone plugs . the intervertebral disc was composed of an annulus fibrosus made from pla / pga scaffold and a nucleus pulposus made from calcium cross - linked alginate that was injected into the centre void of the pla / pga scaffold . one of the most recent advances in generating patient specific implants via injection moulding were achieved using alginate and meniscal fibrochondrocytes from bovine knees . alginate - cell solution was cross - linked with caso4 and cultured for up to 8 weeks in vitro . ( c ) chondrocyte seeded alginate micro - channel network with 50 50 m channels spaced 100 m apart . ( d ) cartilagenous disc 1 cm in diameter composed of plg micro - beads seeded with chon - drocytes after 8 weeks of in vitro culture . solid freeform fabrication ( sff ) and 3d printing are two of the more popular rapid prototyping techniques that are capable of generating multi - material and multi - cellular anatomical constructs . hutmacher and cool have nicely reviewed applications of sff on bone tissue engineering in this journal ( fig . fabrication techniques and the various biomaterials used for cell seeded scaffolds and acellular scaffolds as well as multi - cell / material capability and current resolution capabilities most bone te methods involve seeding of acellular constructs or insertion of acellular implants with the expectation of cellular ingrowth in vivo . some successful studies include the use of porous coral in the shape of a distal phalanx seeded with periosteal cells for thumb reconstruction , 3d printing brushite implants and a cranial segment using tricalcium phosphate ( tcp ) and tetracalcium phosphate respectively . used localized gene therapy to increase and localize cellular and tissue ingrowth using an sff polypropylene fumarate / tcp composite that provided a stable matrix that could be matched to specific patient defect geometry . ( princeton , nj , usa ) produced osteochondral composites using tcp combined with either plg or pla for the chon - dral surface . the composite structure exhibited region specific mechanical properties and integration between the two biomateri - als making it suitable for implantation . for more heterogeneous tissues , such as the meniscus , heart valve and liver , control over spatial and temporal differences in cell type / morphology and mechanical properties is necessary . achieving structures that have the necessary cell distributions and biomechanical properties is a major challenge . cytoscribing , as termed by klebe involved alternating deposition of layers of cells and materials to generate 2d and 3d tissues . an excellent example of this is by cohen et al . rapid prototyping has also been used in the fabrication of 3d hepatic tissues with complex internal microstructure . have evaluated cell viability of hepg2 cells based on dispensing pressure and nozzle diameter with calcium cross - linked alginate and combined these sff techniques with lithography methods to generate 3d microorgans . the transport of solutes and removal of waste products is a large concern in te , especially when trying to engineer large volume tissues or engineering organs like the liver . recent innovative studies using chondrocytes seeded in alginate have shown great promise in their ability to generate various micro - fluidic patterns via laminated sheets with sealed channels as small as 25 25 m [ 44 , 45 ] ( table 2 ) . this work by choi and coworkers really demonstrates the resolution of image based te and can be implemented to produce larger volume constructs that not only have a custom circulation network , but a network that can be controlled spatially with gradients of nutrients , growth factors and region - specific flow rates [ 4446 ] . other works done with plg and its application to cartilage tissue engineering have shown its ability to be used as a mouldable scaffold capable of cellular proliferation and infiltration in vivo ( fig . the use of sintering cell seeded plg micro - beads in combination with free chondrocytes can be used to address focal defects in vivo . furthermore , integrating the use of image guided tissue engineering bead - cell mixtures can be deposited to repair articular surfaces to their original geometry before injury . the repair resolution of this technique is only limited by the consistency and size of the micro particles / bead , which can range from 40600 m [ 4751 ] ( table 2 ) image guided tissue engineering shows great promise for the generation of patient specific engineered tissues . ct and mri can provide adequate templates for custom , patient - specific implants . other imaging modalities do hold promise but have yet to be established . although most image based efforts have focused on musculoskeletal tissues , image - based templates are starting to be used for cardiovascular models and small scale micro - vascular channels for hepatic tissues via cad . the methods for generating these constructs vary greatly depending on the scale , tissue type and biomaterial . both injection moulding and sff techniques can generate anatomically shaped te constructs that appear to have high geometric fidelity . a major challenge to all who work on image - guided tissue engineering lies in the lack of methods to quantify shape fidelity of fabricated implants . the topic of shape fidelity is still in dire need of further investigation , because for many of these complex shaped tissues such as the meniscus [ 5254 ] or heart halve [ 55 , 56 ] critical dimensions and tolerance levels for implantation are still being debated . it is clear that medical imaging is an excellent tool to quantitatively define the geometry of structures especially in situ , such as the meniscus or heart valve . now with new advances in medical imaging techniques , location specific microstructure can be extracted as well . combining imaging data techniques with rapid prototyping could allow generation of anatomical structures in situ with region specific microstructure similar to that of native tissues . imaging tools and fabrication techniques have enhanced fabrication of engineered constructs , but on the list of tissue engineering goals this seems to be only the tip of the iceberg . how exactly does one go from a newly fabricated construct and produce engineered tissues ready for implantation ? even without considering shape fidelity , quality control for te implants for dynamically loaded tissues such as the heart valve or meniscus , complicated geometry often results in complicated mechanics . for years , medical imaging has been used to extract geometries of bones , muscles , and cartilage to develop constitutive models to better describe the inner workings of joints in the body through finite element modelling ( fem ) . medical imaging combined with fem will continue to play a major role in assessing the functionality and durability of engineered tissues . the idea of in vitro conditioning is becoming more and more popular not only for engineered tissues such as tendon , heart valve , bone and cartilage , but for cadaveric explants as well [ 65 , 66 ] . nonetheless image - guided tissue engineering is still likely a very valuable tool for generating patient specific tissues and organs . challenges still lie in the ability to integrate these techniques to engineer large volume tissues with micro - vasculature and generate proper ecm organization and alignment . these techniques in combination with in vitro conditioning

the study was conducted in 28 undergraduate students ( 18 to 24 years , mean age 20.9 ; 10 males ) . stimuli were created at 44.1-khz sampling rate ( 16-bit resolution ) , delivered via external soundcard ( edirol ua-4fx ) and closed headphones ( sennheiser hd 265 linear ) at approximately 80 db rms sound pressure level . all stimuli were composed of 200-hz pure tones ( 100ms , including 20ms ramps ) . any task - specific roving applied to the stimuli was balanced and fixed across subjects . the order of stimuli was determined pseudo - randomly , and subjects indicated the perceived target position by corresponding number - key press . target - to - reference differences were supra - threshold initially and adaptively controlled following a two - down - one - up algorithm ( levitt , 1971 ) , using a larger step size before the fourth reversal and a smaller one thereafter . thresholds were estimated as the mean of the last six reversals ( 70.9% correct ) . the single - interval task ( 50 trials ; grube , cooper , chinnery , & griffiths , 2010 ) required subjects to indicate which of two time intervals marked by pairs of tones was longer . the silent interval within the target was longer by 90% of the reference 's initially , and adaptively changed in steps of 12% and 6% . the regularity task ( see figure 1b ; 40 trials ; grube et al . , 2010 ) sequences were based on an underlying regular beat of 400-ms inter - onset - interval . the reference had an average irregularity of 30% , due to shortening or lengthening of individual intervals by 15 to 45% each , rendering the beat imperceptible ( madison & merker , 2002 ) . the target had 0% irregularity initially , adaptively adjusted in steps of 4% and 2.5% . ( a ) single time - interval duration discrimination , using pairs of tones and a reference inter - onset - interval of 300 to 600 ms ; ( b ) regularity detection , using sequences of 11 tones and an underlying beat of 400 ms inter - onset - interval ; ( c ) metrical pattern discrimination , using a reference sequence with a metrical beat of four and an underlying beat of 180 to 220 ms ; ( d ) tempo contour detection , using sequences of 11 tones with a succession inter - onset - intervals controlled by an algorithm for scaling noise . depicted the metrical pattern task ( see figure 1c ; 40 trials ; grube et al . , 2010 ) required subjects to indicate which of three rhythmic sequences was different . the reference had a metrical beat of four induced by the temporal spacing of seven tones ( for more details see grube & griffiths , 2009 ) . the target ( second or third ) had a change in timing such that the long intervals were not multiples of the underlying beat ( 180 to 220ms ) . the change in long intervals ( lengthening or shortening ; balanced across the sequence ) was 65% initially , adaptively adjusted in steps of 12% and 6% . the tempo - contour detection task ( see figure 1d ; 40 trials ) required subjects to identify the target with a more gradual change in tempo compared to a reference with randomly changing tempo ( see figure 1d ) . the tempo contours were created using an algorithm for scaling noise ( 1/f ) to compose sequences of varying inter - onset - interval duration ; the same algorithm we previously used in the creation of pitch sequences with varying degree of melodic contour , with n = 0 creating a random sequence , and increasingly higher values creating increasingly more ordered contours ( overath et al . , 2007 ) . the reference n = 0 , the target an initial value of 2.1 , adaptively adjusted in steps of 0.3 and 0.1 . five phonological language and literacy tests were used , which were all standardized tests with the exception of ( 4 ) : ( 1 ) irregular word reading , i.e. , words with atypical grapheme to phoneme translations , combining the nart ( nelson & willison , 1991 ) , wrat ( wilkinson , 1993 ) , and wtar ( wechsler , 2001 ) ( 118 items in total ) ; ( 2 ) non - word reading based on olson , forsberg , & wise ( 1994 ) , as used by foxton et al . ( 2003 ) but extended to contain 120 items ( 30 each with two , three , or four syllables in randomized order ) ; ( 3 ) word / non - word reading from the palpa ( kay , coltheart , & lesser , 1992 ; 160 items ) ; ( 4 ) reading of a difficult english poem containing 800 examples of irregular spelling and pronunciation , in misleading order ( the chaos , gerard nolst trenit ) ; ( 5 ) digit span ( wais , 32 items ) . the reading tasks were scored as the number of errors ( incorrect items ) ; digit - span was scored as the number of correct items . non - verbal intellectual skill was measured using a shortened version of raven 's advanced progressive matrices ( raven , court , & raven , 1988 ) , scored as the number of items correct ( 18 items ; 20 min ) . correlation analysis used spearman 's rho , due to significant deviations from normal distribution ( see table 1 ; kolmogorov - smirnoff test ) . the one - tailed version was used based on expecting a positive correlation between better performance on language and auditory tasks , with a significance limit of p < .05 and bonferroni - correction for n = 4 , i.e. , the number of correlations carried out for each language - based measure . a principal component analyses ( pca ) was carried out on the five language - based measures , to extract an underlying principle mechanism and test for correlation with auditory skill . pca decomposes the data matrix x of size ( n m ) , with n being the number of observations ( subjects ) and m the number of variables ( measures ) : where t is the scores matrix of size ( n d ) with d ( m ) being the number of components retained in the pca model , p the loading matrix ( size m d ) and e an error matrix . descriptive statistics for the four auditory timing tasks : single - interval timing , metrical pattern discrimination ; tempo contour detection ; and the five phonological tasks : irregular word reading , word / non - word reading , non - word reading , poem reading , digit span . we report median values and mean absolute deviation ( mad ) , minimum and maximum , due to a number of deviations from normal distributions , marked by an asterisk next to the median * ( lilliefors kolmogorov - smirnoff test , p < .05 ) . note that regularity detection , digit span and raven 's scores are the only measures featuring larger values corresponding to better performance ; all others feature smaller values for better performance ( auditory : thresholds ; phonological and literacy : number or incorrect items ) correlations were analyzed between auditory timing measures and both the raw language and literacy scores and a composite measure : the first principal component of phonological skill ( p - pc1 ) . p - pci explained 65% of the variance with equal loadings across all five tests . in support of our hypothesis , two of the three measures of rhythmic sequence processing regularity detection and metrical pattern processing correlated most consistently with the test - specific language - based measures and the p - pc1 . regularity detection correlated significantly with irregular word and non - word reading before and after bonferroni correction , with poem reading and digit span before but not after bonferroni correction , and borderline signficantly with word / non - word reading . metrical pattern discrimination correlated significantly with the reading of the difficult poem before and after bonferroni correction , and with irregular - word and non - word reading before but not after . in contrast , no single significant correlation was found between the contour detection task and the language - related measures . single - interval timing correlated significantly only with irregular word and the difficult poem reading and p - pc1 , but only before bonferroni correction . the average rho values across the five measures of phonological language and literacy skill were : .4 for metrical pattern discrimination ; .39 for regularity detection ; .03 ; .22 for single - interval timing ; .03 for tempo contour detection . correlations between auditory timing and phonological language and literacy measures . listed are spearman 's rho correlation coefficients and corresponding p values ( rho / p , 1-tailed ) for task - specific measures and the first principle phonological component ( p - pc1 ) before and after partialling out non - verbal intelligence ( first and second row within each cell ) . significance level was p < .05 ; marked in bold are those that survive bonferroni - correction , in brackets those that were not significant , grayed out those that had a correlation coefficient < .22 , i.e. , explaining less than 5% of variance , and/or a p value > .2 . note that sign of scores was reversed for regularity detection and digit span ( see table 1 ) , so that smaller ( more negative ) values represented better performance for all variables and that positive correlation coefficients would throughout denote correlations in the hypothesized direction in order to test for a possible confounding effect of non - verbal intellectual skill , we assessed univariate , one - tailed correlations with the raven 's score . of the four auditory timing measures , regularity and contour detection showed a trend of a positive correlation ( regularity : rho = .24 , p = .103 ; tempo contour : rho = .35 , p = .036 ) , whilst the single - interval and metrical timing did not ( single time - interval : rho = .14 , p = .766 ; metrical pattern : rho = .04 , p = .586 ) . the language - based measures showed no positive correlation with the raven 's score , but negative trends instead ( irregular words : rho = .18 , p = .824 ; words / non - words : rho = .17 , p = .917 ; non - words : rho = .31 , p = .948 ; difficult poem : rho = .02 , p = .459 ; digit span : rho = .21 , p = .856 ) . partial correlations between the auditory and language - related measures of phonological language and literacy skill had no discernible effect compared to the orginal outcomes presented in table 2 , except a general increase in correlations for the detection of regularity . average correlation coefficients after partialling out non - verbal intellectual skill were : .45 for regularity detection ; .38 for metrical pattern discrimination ; .20 for single - interval timing ; .09 for tempo contour detection . the partial , task - specific correlation coefficients for regularity detection were : irregular word reading , rho = .52 ( p = .003 , surviving bonferroni correction ) ; word / non - word reading , rho = .36 ( p = .031 ) ; non - word reading , rho = .55 ( p = .002 , surviving bonferroni correction ) ; poem reading , rho = .41 ( p = .017 ) ; digit span , rho = .43 ( p = .012 , surviving bonferroni correction ) . the correlation between regularity detection and p - pc1 explained 26% of the variance before , and 36% after partialling out non - verbal intellectual skill . the correlation between metrical pattern discrimination and p - pc1 explained 31% of the variance , with no change due to partialling out non - verbal intellectual skill . the study was conducted in 28 undergraduate students ( 18 to 24 years , mean age 20.9 ; 10 males ) . stimuli were created at 44.1-khz sampling rate ( 16-bit resolution ) , delivered via external soundcard ( edirol ua-4fx ) and closed headphones ( sennheiser hd 265 linear ) at approximately 80 db rms sound pressure level . all stimuli were composed of 200-hz pure tones ( 100ms , including 20ms ramps ) . any task - specific roving applied to the stimuli was balanced and fixed across subjects . the order of stimuli was determined pseudo - randomly , and subjects indicated the perceived target position by corresponding number - key press . target - to - reference differences were supra - threshold initially and adaptively controlled following a two - down - one - up algorithm ( levitt , 1971 ) , using a larger step size before the fourth reversal and a smaller one thereafter . thresholds were estimated as the mean of the last six reversals ( 70.9% correct ) . inter - stimulus and inter - trial intervals were 1500ms . the single - interval task ( 50 trials ; grube , cooper , chinnery , & griffiths , 2010 ) required subjects to indicate which of two time intervals marked by pairs of tones was longer . the silent interval within the target was longer by 90% of the reference 's initially , and adaptively changed in steps of 12% and 6% . the regularity task ( see figure 1b ; 40 trials ; grube et al . , 2010 ) sequences were based on an underlying regular beat of 400-ms inter - onset - interval . the reference had an average irregularity of 30% , due to shortening or lengthening of individual intervals by 15 to 45% each , rendering the beat imperceptible ( madison & merker , 2002 ) . the target had 0% irregularity initially , adaptively adjusted in steps of 4% and 2.5% . ( a ) single time - interval duration discrimination , using pairs of tones and a reference inter - onset - interval of 300 to 600 ms ; ( b ) regularity detection , using sequences of 11 tones and an underlying beat of 400 ms inter - onset - interval ; ( c ) metrical pattern discrimination , using a reference sequence with a metrical beat of four and an underlying beat of 180 to 220 ms ; ( d ) tempo contour detection , using sequences of 11 tones with a succession inter - onset - intervals controlled by an algorithm for scaling noise . depicted horizontal lines : tones ( 200 hz ; 100 ms ) . the metrical pattern task ( see figure 1c ; 40 trials ; grube et al . , 2010 ) required subjects to indicate which of three rhythmic sequences was different . the reference had a metrical beat of four induced by the temporal spacing of seven tones ( for more details see grube & griffiths , 2009 ) . the target ( second or third ) had a change in timing such that the long intervals were not multiples of the underlying beat ( 180 to 220ms ) . the change in long intervals ( lengthening or shortening ; balanced across the sequence ) was 65% initially , adaptively adjusted in steps of 12% and 6% . the tempo - contour detection task ( see figure 1d ; 40 trials ) required subjects to identify the target with a more gradual change in tempo compared to a reference with randomly changing tempo ( see figure 1d ) . the tempo contours were created using an algorithm for scaling noise ( 1/f ) to compose sequences of varying inter - onset - interval duration ; the same algorithm we previously used in the creation of pitch sequences with varying degree of melodic contour , with n = 0 creating a random sequence , and increasingly higher values creating increasingly more ordered contours ( overath et al . , 2007 ) . the reference n = 0 , the target an initial value of 2.1 , adaptively adjusted in steps of 0.3 and 0.1 . five phonological language and literacy tests were used , which were all standardized tests with the exception of ( 4 ) : ( 1 ) irregular word reading , i.e. , words with atypical grapheme to phoneme translations , combining the nart ( nelson & willison , 1991 ) , wrat ( wilkinson , 1993 ) , and wtar ( wechsler , 2001 ) ( 118 items in total ) ; ( 2 ) non - word reading based on olson , forsberg , & wise ( 1994 ) , as used by foxton et al . ( 2003 ) but extended to contain 120 items ( 30 each with two , three , or four syllables in randomized order ) ; ( 3 ) word / non - word reading from the palpa ( kay , coltheart , & lesser , 1992 ; 160 items ) ; ( 4 ) reading of a difficult english poem containing 800 examples of irregular spelling and pronunciation , in misleading order ( the chaos , gerard nolst trenit ) ; ( 5 ) digit span ( wais , 32 items ) . the reading tasks were scored as the number of errors ( incorrect items ) ; digit - span was scored as the number of correct items . non - verbal intellectual skill was measured using a shortened version of raven 's advanced progressive matrices ( raven , court , & raven , 1988 ) , scored as the number of items correct ( 18 items ; 20 min ) . correlation analysis used spearman 's rho , due to significant deviations from normal distribution ( see table 1 ; kolmogorov - smirnoff test ) . the one - tailed version was used based on expecting a positive correlation between better performance on language and auditory tasks , with a significance limit of p < .05 and bonferroni - correction for n = 4 , i.e. , the number of correlations carried out for each language - based measure . a principal component analyses ( pca ) was carried out on the five language - based measures , to extract an underlying principle mechanism and test for correlation with auditory skill . pca decomposes the data matrix x of size ( n m ) , with n being the number of observations ( subjects ) and m the number of variables ( measures ) : where t is the scores matrix of size ( n d ) with d ( m ) being the number of components retained in the pca model , p the loading matrix ( size m d ) and e an error matrix . descriptive statistics for the four auditory timing tasks : single - interval timing , metrical pattern discrimination ; tempo contour detection ; and the five phonological tasks : irregular word reading , word / non - word reading , non - word reading , poem reading , digit span . we report median values and mean absolute deviation ( mad ) , minimum and maximum , due to a number of deviations from normal distributions , marked by an asterisk next to the median * ( lilliefors kolmogorov - smirnoff test , p < .05 ) . note that regularity detection , digit span and raven 's scores are the only measures featuring larger values corresponding to better performance ; all others feature smaller values for better performance ( auditory : thresholds ; phonological and literacy : number or incorrect items ) correlations were analyzed between auditory timing measures and both the raw language and literacy scores and a composite measure : the first principal component of phonological skill ( p - pc1 ) . p - pci explained 65% of the variance with equal loadings across all five tests . in support of our hypothesis , two of the three measures of rhythmic sequence processing regularity detection and metrical pattern processing correlated most consistently with the test - specific language - based measures and the p - pc1 . regularity detection correlated significantly with irregular word and non - word reading before and after bonferroni correction , with poem reading and digit span before but not after bonferroni correction , and borderline signficantly with word / non - word reading . metrical pattern discrimination correlated significantly with the reading of the difficult poem before and after bonferroni correction , and with irregular - word and non - word reading before but not after . in contrast , no single significant correlation was found between the contour detection task and the language - related measures . single - interval timing correlated significantly only with irregular word and the difficult poem reading and p - pc1 , but only before bonferroni correction . the average rho values across the five measures of phonological language and literacy skill were : .4 for metrical pattern discrimination ; .39 for regularity detection ; .03 ; .22 for single - interval timing ; .03 for tempo contour detection . correlations between auditory timing and phonological language and literacy measures . listed are spearman 's rho correlation coefficients and corresponding p values ( rho / p , 1-tailed ) for task - specific measures and the first principle phonological component ( p - pc1 ) before and after partialling out non - verbal intelligence ( first and second row within each cell ) . significance level was p < .05 ; marked in bold are those that survive bonferroni - correction , in brackets those that were not significant , grayed out those that had a correlation coefficient < .22 , i.e. , explaining less than 5% of variance , and/or a p value > .2 . note that sign of scores was reversed for regularity detection and digit span ( see table 1 ) , so that smaller ( more negative ) values represented better performance for all variables and that positive correlation coefficients would throughout denote correlations in the hypothesized direction in order to test for a possible confounding effect of non - verbal intellectual skill , we assessed univariate , one - tailed correlations with the raven 's score . of the four auditory timing measures , regularity and contour detection showed a trend of a positive correlation ( regularity : rho = .24 , p = .103 ; tempo contour : rho = .35 , p = .036 ) , whilst the single - interval and metrical timing did not ( single time - interval : rho = .14 , p = .766 ; metrical pattern : rho = .04 , p = .586 ) . the language - based measures showed no positive correlation with the raven 's score , but negative trends instead ( irregular words : rho = .18 , p = .824 ; words / non - words : rho = .17 , p = .917 ; non - words : rho = .31 , p = .948 ; difficult poem : rho = .02 , p = .459 ; digit span : rho = .21 , p = .856 ) . partial correlations between the auditory and language - related measures of phonological language and literacy skill had no discernible effect compared to the orginal outcomes presented in table 2 , except a general increase in correlations for the detection of regularity . average correlation coefficients after partialling out non - verbal intellectual skill were : .45 for regularity detection ; .38 for metrical pattern discrimination ; .20 for single - interval timing ; .09 for tempo contour detection . the partial , task - specific correlation coefficients for regularity detection were : irregular word reading , rho = .52 ( p = .003 , surviving bonferroni correction ) ; word / non - word reading , rho = .36 ( p = .031 ) ; non - word reading , rho = .55 ( p = .002 , surviving bonferroni correction ) ; poem reading , rho = .41 ( p = .017 ) ; digit span , rho = .43 ( p = .012 , surviving bonferroni correction ) . the correlation between regularity detection and p - pc1 explained 26% of the variance before , and 36% after partialling out non - verbal intellectual skill . the correlation between metrical pattern discrimination and p - pc1 explained 31% of the variance , with no change due to partialling out non - verbal intellectual skill . the data show a significant relationship between phonological language and literacy skill and both the auditory cognitive ability to analyze temporal , beat - based regularity and metrical patterns in young adults . the data suggests a role for of the analysis of event structure over time in speech and language processing , at the hundreds - of - milliseconds level over a few seconds at a time . the findings support the existence of a shared mechanism of beat - based regularity processing supporting rhythm perception as well as speech and language skills : the use of temporal regularities as a scaffolding to structure input and output in time . the strongest correlation was observed between the metrical beat and the poem , a special instance of the feeling of the ( musical ) beat leading us to synchronize our movements with it . the causality of the synergistic effects between auditory , language , and musical skills remains to be further explored ( c.f . strait et al . , 2011 ; strait , o'connell , parbery - clark , & kraus , 2013 ) . the lack of correlation for the analysis of a gradually changing tempo contour , despite comparable range of inter - onset - interval durations , sequence length , and complexity and their relevance to music ( levitin , chordia , & menon , 2012 ) , supports the interpretation that the ability to analyze specifically those temporal structures with a regular , or quasi - regular beat is relevant to speech and language skills . this finding is consistent with recent models of oscillatory brain reflecting the suprasegmental analysis of speech engaged by its quasi - rhythmic structure ( abrams , nicol , zecker , & kraus , 2009 ; giraud & poeppel , 2012 ; luo & poeppel , 2007 ; morillon et al . , 2010 ) . activity at frequencies in the theta range ( 38hz ) , is assumed as the master oscillation processing the equivalent modulation frequencies , the most prominent energy fluctuations in speech corresponding to the syllable level ( ghitza , 2013 ; ghitza , giraud , & poeppel , 2012 ) , whilst delta oscillations ( 13hz ) would relate to the next higher level , i.e. , that of stress timing . our beat - based regularity and metrical tasks tap directly into the generic , underlying mechanisms of temporal regularity processing at these frequencies , likely achieved by one common subsystem of beat - based timing ( grube et al . , 2010 ; teki , grube , & griffiths , 2012 ) . the present data demonstrate a clear correlation between the processing of beat - based temporal regularities in generic , pre - phonemic auditory stimuli and language skill in early adulthood , whilst no corresponding correlations were found in our work in a large cohort of 11-year - olds ( grube et al . , 2012 ) . the data available support a link between higher levels of rhythmic complexity and language skill emerging in early adulthood , warranting further testing of the causality of this link .

this work tests the hypothesis that language skill depends on the ability to incorporate streams of sound into an accurate temporal framework . we tested the ability of young english - speaking adults to process single time intervals and rhythmic sequences of such intervals , hypothesized to be relevant to the analysis of the temporal structure of language . the data implicate a specific role for the ability to process beat - based temporal regularities in phonological language and literacy skill .

METHODS Participants Auditory timing tasks Tests of language, literacy, and intellectual skill Statistical data analysis RESULTS GENERAL DISCUSSION

stimuli were created at 44.1-khz sampling rate ( 16-bit resolution ) , delivered via external soundcard ( edirol ua-4fx ) and closed headphones ( sennheiser hd 265 linear ) at approximately 80 db rms sound pressure level . all stimuli were composed of 200-hz pure tones ( 100ms , including 20ms ramps ) . any task - specific roving applied to the stimuli was balanced and fixed across subjects . thresholds were estimated as the mean of the last six reversals ( 70.9% correct ) . the single - interval task ( 50 trials ; grube , cooper , chinnery , & griffiths , 2010 ) required subjects to indicate which of two time intervals marked by pairs of tones was longer . the silent interval within the target was longer by 90% of the reference 's initially , and adaptively changed in steps of 12% and 6% . the reference had an average irregularity of 30% , due to shortening or lengthening of individual intervals by 15 to 45% each , rendering the beat imperceptible ( madison & merker , 2002 ) . ( a ) single time - interval duration discrimination , using pairs of tones and a reference inter - onset - interval of 300 to 600 ms ; ( b ) regularity detection , using sequences of 11 tones and an underlying beat of 400 ms inter - onset - interval ; ( c ) metrical pattern discrimination , using a reference sequence with a metrical beat of four and an underlying beat of 180 to 220 ms ; ( d ) tempo contour detection , using sequences of 11 tones with a succession inter - onset - intervals controlled by an algorithm for scaling noise . , 2010 ) required subjects to indicate which of three rhythmic sequences was different . the reference had a metrical beat of four induced by the temporal spacing of seven tones ( for more details see grube & griffiths , 2009 ) . the target ( second or third ) had a change in timing such that the long intervals were not multiples of the underlying beat ( 180 to 220ms ) . the change in long intervals ( lengthening or shortening ; balanced across the sequence ) was 65% initially , adaptively adjusted in steps of 12% and 6% . the tempo contours were created using an algorithm for scaling noise ( 1/f ) to compose sequences of varying inter - onset - interval duration ; the same algorithm we previously used in the creation of pitch sequences with varying degree of melodic contour , with n = 0 creating a random sequence , and increasingly higher values creating increasingly more ordered contours ( overath et al . , 2007 ) . five phonological language and literacy tests were used , which were all standardized tests with the exception of ( 4 ) : ( 1 ) irregular word reading , i.e. ( 2003 ) but extended to contain 120 items ( 30 each with two , three , or four syllables in randomized order ) ; ( 3 ) word / non - word reading from the palpa ( kay , coltheart , & lesser , 1992 ; 160 items ) ; ( 4 ) reading of a difficult english poem containing 800 examples of irregular spelling and pronunciation , in misleading order ( the chaos , gerard nolst trenit ) ; ( 5 ) digit span ( wais , 32 items ) . non - verbal intellectual skill was measured using a shortened version of raven 's advanced progressive matrices ( raven , court , & raven , 1988 ) , scored as the number of items correct ( 18 items ; 20 min ) . correlation analysis used spearman 's rho , due to significant deviations from normal distribution ( see table 1 ; kolmogorov - smirnoff test ) . the one - tailed version was used based on expecting a positive correlation between better performance on language and auditory tasks , with a significance limit of p < .05 and bonferroni - correction for n = 4 , i.e. , the number of correlations carried out for each language - based measure . a principal component analyses ( pca ) was carried out on the five language - based measures , to extract an underlying principle mechanism and test for correlation with auditory skill . pca decomposes the data matrix x of size ( n m ) , with n being the number of observations ( subjects ) and m the number of variables ( measures ) : where t is the scores matrix of size ( n d ) with d ( m ) being the number of components retained in the pca model , p the loading matrix ( size m d ) and e an error matrix . descriptive statistics for the four auditory timing tasks : single - interval timing , metrical pattern discrimination ; tempo contour detection ; and the five phonological tasks : irregular word reading , word / non - word reading , non - word reading , poem reading , digit span . we report median values and mean absolute deviation ( mad ) , minimum and maximum , due to a number of deviations from normal distributions , marked by an asterisk next to the median * ( lilliefors kolmogorov - smirnoff test , p < .05 ) . note that regularity detection , digit span and raven 's scores are the only measures featuring larger values corresponding to better performance ; all others feature smaller values for better performance ( auditory : thresholds ; phonological and literacy : number or incorrect items ) correlations were analyzed between auditory timing measures and both the raw language and literacy scores and a composite measure : the first principal component of phonological skill ( p - pc1 ) . p - pci explained 65% of the variance with equal loadings across all five tests . in support of our hypothesis , two of the three measures of rhythmic sequence processing regularity detection and metrical pattern processing correlated most consistently with the test - specific language - based measures and the p - pc1 . regularity detection correlated significantly with irregular word and non - word reading before and after bonferroni correction , with poem reading and digit span before but not after bonferroni correction , and borderline signficantly with word / non - word reading . metrical pattern discrimination correlated significantly with the reading of the difficult poem before and after bonferroni correction , and with irregular - word and non - word reading before but not after . in contrast , no single significant correlation was found between the contour detection task and the language - related measures . the average rho values across the five measures of phonological language and literacy skill were : .4 for metrical pattern discrimination ; .39 for regularity detection ; .03 ; .22 for single - interval timing ; .03 for tempo contour detection . correlations between auditory timing and phonological language and literacy measures . , explaining less than 5% of variance , and/or a p value > .2 . note that sign of scores was reversed for regularity detection and digit span ( see table 1 ) , so that smaller ( more negative ) values represented better performance for all variables and that positive correlation coefficients would throughout denote correlations in the hypothesized direction in order to test for a possible confounding effect of non - verbal intellectual skill , we assessed univariate , one - tailed correlations with the raven 's score . of the four auditory timing measures , regularity and contour detection showed a trend of a positive correlation ( regularity : rho = .24 , p = .103 ; tempo contour : rho = .35 , p = .036 ) , whilst the single - interval and metrical timing did not ( single time - interval : rho = .14 , p = .766 ; metrical pattern : rho = .04 , p = .586 ) . the language - based measures showed no positive correlation with the raven 's score , but negative trends instead ( irregular words : rho = .18 , p = .824 ; words / non - words : rho = .17 , p = .917 ; non - words : rho = .31 , p = .948 ; difficult poem : rho = .02 , p = .459 ; digit span : rho = .21 , p = .856 ) . partial correlations between the auditory and language - related measures of phonological language and literacy skill had no discernible effect compared to the orginal outcomes presented in table 2 , except a general increase in correlations for the detection of regularity . the correlation between regularity detection and p - pc1 explained 26% of the variance before , and 36% after partialling out non - verbal intellectual skill . the correlation between metrical pattern discrimination and p - pc1 explained 31% of the variance , with no change due to partialling out non - verbal intellectual skill . the study was conducted in 28 undergraduate students ( 18 to 24 years , mean age 20.9 ; 10 males ) . any task - specific roving applied to the stimuli was balanced and fixed across subjects . the order of stimuli was determined pseudo - randomly , and subjects indicated the perceived target position by corresponding number - key press . target - to - reference differences were supra - threshold initially and adaptively controlled following a two - down - one - up algorithm ( levitt , 1971 ) , using a larger step size before the fourth reversal and a smaller one thereafter . thresholds were estimated as the mean of the last six reversals ( 70.9% correct ) . the single - interval task ( 50 trials ; grube , cooper , chinnery , & griffiths , 2010 ) required subjects to indicate which of two time intervals marked by pairs of tones was longer . the silent interval within the target was longer by 90% of the reference 's initially , and adaptively changed in steps of 12% and 6% . the target had 0% irregularity initially , adaptively adjusted in steps of 4% and 2.5% . ( a ) single time - interval duration discrimination , using pairs of tones and a reference inter - onset - interval of 300 to 600 ms ; ( b ) regularity detection , using sequences of 11 tones and an underlying beat of 400 ms inter - onset - interval ; ( c ) metrical pattern discrimination , using a reference sequence with a metrical beat of four and an underlying beat of 180 to 220 ms ; ( d ) tempo contour detection , using sequences of 11 tones with a succession inter - onset - intervals controlled by an algorithm for scaling noise . the metrical pattern task ( see figure 1c ; 40 trials ; grube et al . , 2010 ) required subjects to indicate which of three rhythmic sequences was different . the reference had a metrical beat of four induced by the temporal spacing of seven tones ( for more details see grube & griffiths , 2009 ) . the target ( second or third ) had a change in timing such that the long intervals were not multiples of the underlying beat ( 180 to 220ms ) . the change in long intervals ( lengthening or shortening ; balanced across the sequence ) was 65% initially , adaptively adjusted in steps of 12% and 6% . the tempo - contour detection task ( see figure 1d ; 40 trials ) required subjects to identify the target with a more gradual change in tempo compared to a reference with randomly changing tempo ( see figure 1d ) . the tempo contours were created using an algorithm for scaling noise ( 1/f ) to compose sequences of varying inter - onset - interval duration ; the same algorithm we previously used in the creation of pitch sequences with varying degree of melodic contour , with n = 0 creating a random sequence , and increasingly higher values creating increasingly more ordered contours ( overath et al . five phonological language and literacy tests were used , which were all standardized tests with the exception of ( 4 ) : ( 1 ) irregular word reading , i.e. , words with atypical grapheme to phoneme translations , combining the nart ( nelson & willison , 1991 ) , wrat ( wilkinson , 1993 ) , and wtar ( wechsler , 2001 ) ( 118 items in total ) ; ( 2 ) non - word reading based on olson , forsberg , & wise ( 1994 ) , as used by foxton et al . the reading tasks were scored as the number of errors ( incorrect items ) ; digit - span was scored as the number of correct items . the one - tailed version was used based on expecting a positive correlation between better performance on language and auditory tasks , with a significance limit of p < .05 and bonferroni - correction for n = 4 , i.e. , the number of correlations carried out for each language - based measure . a principal component analyses ( pca ) was carried out on the five language - based measures , to extract an underlying principle mechanism and test for correlation with auditory skill . pca decomposes the data matrix x of size ( n m ) , with n being the number of observations ( subjects ) and m the number of variables ( measures ) : where t is the scores matrix of size ( n d ) with d ( m ) being the number of components retained in the pca model , p the loading matrix ( size m d ) and e an error matrix . descriptive statistics for the four auditory timing tasks : single - interval timing , metrical pattern discrimination ; tempo contour detection ; and the five phonological tasks : irregular word reading , word / non - word reading , non - word reading , poem reading , digit span . we report median values and mean absolute deviation ( mad ) , minimum and maximum , due to a number of deviations from normal distributions , marked by an asterisk next to the median * ( lilliefors kolmogorov - smirnoff test , p < .05 ) . note that regularity detection , digit span and raven 's scores are the only measures featuring larger values corresponding to better performance ; all others feature smaller values for better performance ( auditory : thresholds ; phonological and literacy : number or incorrect items ) correlations were analyzed between auditory timing measures and both the raw language and literacy scores and a composite measure : the first principal component of phonological skill ( p - pc1 ) . p - pci explained 65% of the variance with equal loadings across all five tests . in support of our hypothesis , two of the three measures of rhythmic sequence processing regularity detection and metrical pattern processing correlated most consistently with the test - specific language - based measures and the p - pc1 . metrical pattern discrimination correlated significantly with the reading of the difficult poem before and after bonferroni correction , and with irregular - word and non - word reading before but not after . in contrast , no single significant correlation was found between the contour detection task and the language - related measures . single - interval timing correlated significantly only with irregular word and the difficult poem reading and p - pc1 , but only before bonferroni correction . the average rho values across the five measures of phonological language and literacy skill were : .4 for metrical pattern discrimination ; .39 for regularity detection ; .03 ; .22 for single - interval timing ; .03 for tempo contour detection . correlations between auditory timing and phonological language and literacy measures . , explaining less than 5% of variance , and/or a p value > .2 . of the four auditory timing measures , regularity and contour detection showed a trend of a positive correlation ( regularity : rho = .24 , p = .103 ; tempo contour : rho = .35 , p = .036 ) , whilst the single - interval and metrical timing did not ( single time - interval : rho = .14 , p = .766 ; metrical pattern : rho = .04 , p = .586 ) . the language - based measures showed no positive correlation with the raven 's score , but negative trends instead ( irregular words : rho = .18 , p = .824 ; words / non - words : rho = .17 , p = .917 ; non - words : rho = .31 , p = .948 ; difficult poem : rho = .02 , p = .459 ; digit span : rho = .21 , p = .856 ) . partial correlations between the auditory and language - related measures of phonological language and literacy skill had no discernible effect compared to the orginal outcomes presented in table 2 , except a general increase in correlations for the detection of regularity . the partial , task - specific correlation coefficients for regularity detection were : irregular word reading , rho = .52 ( p = .003 , surviving bonferroni correction ) ; word / non - word reading , rho = .36 ( p = .031 ) ; non - word reading , rho = .55 ( p = .002 , surviving bonferroni correction ) ; poem reading , rho = .41 ( p = .017 ) ; digit span , rho = .43 ( p = .012 , surviving bonferroni correction ) . the correlation between regularity detection and p - pc1 explained 26% of the variance before , and 36% after partialling out non - verbal intellectual skill . the correlation between metrical pattern discrimination and p - pc1 explained 31% of the variance , with no change due to partialling out non - verbal intellectual skill . the data show a significant relationship between phonological language and literacy skill and both the auditory cognitive ability to analyze temporal , beat - based regularity and metrical patterns in young adults . the data suggests a role for of the analysis of event structure over time in speech and language processing , at the hundreds - of - milliseconds level over a few seconds at a time . the findings support the existence of a shared mechanism of beat - based regularity processing supporting rhythm perception as well as speech and language skills : the use of temporal regularities as a scaffolding to structure input and output in time . the strongest correlation was observed between the metrical beat and the poem , a special instance of the feeling of the ( musical ) beat leading us to synchronize our movements with it . the causality of the synergistic effects between auditory , language , and musical skills remains to be further explored ( c.f . strait et al . the lack of correlation for the analysis of a gradually changing tempo contour , despite comparable range of inter - onset - interval durations , sequence length , and complexity and their relevance to music ( levitin , chordia , & menon , 2012 ) , supports the interpretation that the ability to analyze specifically those temporal structures with a regular , or quasi - regular beat is relevant to speech and language skills . this finding is consistent with recent models of oscillatory brain reflecting the suprasegmental analysis of speech engaged by its quasi - rhythmic structure ( abrams , nicol , zecker , & kraus , 2009 ; giraud & poeppel , 2012 ; luo & poeppel , 2007 ; morillon et al . activity at frequencies in the theta range ( 38hz ) , is assumed as the master oscillation processing the equivalent modulation frequencies , the most prominent energy fluctuations in speech corresponding to the syllable level ( ghitza , 2013 ; ghitza , giraud , & poeppel , 2012 ) , whilst delta oscillations ( 13hz ) would relate to the next higher level , i.e. our beat - based regularity and metrical tasks tap directly into the generic , underlying mechanisms of temporal regularity processing at these frequencies , likely achieved by one common subsystem of beat - based timing ( grube et al . the present data demonstrate a clear correlation between the processing of beat - based temporal regularities in generic , pre - phonemic auditory stimuli and language skill in early adulthood , whilst no corresponding correlations were found in our work in a large cohort of 11-year - olds ( grube et al . the data available support a link between higher levels of rhythmic complexity and language skill emerging in early adulthood , warranting further testing of the causality of this link .

life expectancy has extended worldwide and a growing number of patients with multiple comorbidities including ischemic heart diseases and cancer have undergone surgeries . consequently , postoperative cardiovascular complications are expected to increase,1 and perioperative acute myocardial infarction ( ami ) has become a major health concern.2,3 it is well known that surgery induces a stress response , its extent is directly dependent on the magnitude of tissue destruction , and may be modified by the type of perioperative analgesia used . this stress response can lead to an increase in heart rate ( hr ) and blood pressure , which can precipitate episodes of myocardial ischemia.4 perioperative myocardial infarction ( pmi ) is one of the most important predictors of short- and long - term morbidity and mortality associated with noncardiac surgery.58 prevention of pmi is thus a prerequisite for the improvement in overall postoperative outcome . thoracic epidural anesthesia ( tea ) has been established as a cornerstone in perioperative care after thoracic and major abdominal surgery providing most effective analgesia.9,10 beyond its analgesic properties , tea s effects on postoperative neurohumoral stress response , cardiovascular pathophysiology , and intestinal dysfunction have been in the focus of both clinical and experimental investigations for years.1115 the aim of the study was to test whether epidural analgesia added to a general anesthetic , compared with systemic , opioid - based standard care analgesia in ischemic patients undergoing major abdominal cancer surgery , provided any reduction in adverse cardiac events . this study was approved by the local ethics committee of the south egypt cancer institute , assiut university , assiut , egypt . after obtaining written informed consent from each patient , 120 adult patients , complaining of coronary artery disease ( cad ) , classified as american society of anesthesiologists grade ii and iii and new york heart association class ii and iii , scheduled for elective major abdominal cancer surgery were consecutively enrolled . patients with coagulopathy , active neurological disease , cutaneous disorders at the epidural insertion site , and allergy to the studied medications were excluded from the study . every patient was evaluated by a cardiologist and anesthesiologist for medical history , physical examination , electrocardiography ( ecg ) , and echocardiography . anti - ischemic and antihypertensive drugs were continued during the perioperative period , including the morning of surgery ; however , angiotensin - converting enzyme inhibitors , diuretics , and calcium channel blockers were suspended the day before surgery . the day before surgery , all patients were taught how to evaluate their own pain intensity using the visual analog scale ( vas ) , scored from 0 to 10 ( where 0= no pain and 10= worst pain imaginable ) and how to use the patient controlled analgesia ( pca ) device ( abbott laboratories , north chicago , il , usa ) . each patient was given oral ranitidine tablet , 50 mg and lorazepam tablet , 3 mg on the night of surgery . patients were randomly assigned into two groups , 60 patients each , by using opaque sealed envelopes containing a computer generated randomization schedule ; the opaque envelopes were sequentially numbered and were opened before application of anesthetic plan . in the patient controlled intravenous analgesia ( pcia ) group ( n=60 ) , patients received intraoperative analgesia with intravenous fentanyl bolus dose , 0.5 g / kg , followed by continuous infusion of 1 g / kg / h till the end of surgery . postoperative analgesia consisted of intravenous fentanyl pca , 10 g / ml , background infusion 2 ml / h , bolus dose 3 ml and lockout interval 15 min . in the patient controlled epidural analgesia ( pcea ) group ( n=60 ) , where patients received pcea in conjunction with ga , intraoperative analgesia was started before skin incision by epidural bolus dose of 0.1 ml / kg of 0.125% bupivacaine / fentanyl 10 g / ml , followed by continuous infusion of 0.1 ml / kg / h of 0.125% bupivacaine / fentanyl 5 postoperative analgesia was provided through pcea for 72 h postoperatively ( background infusion of 0.1 ml / kg / h of 0.125% bupivacaine / fentanyl 3 g / ml , bolus dose of 3 ml , lockout interval was set at 20 min ) . before induction of ga and under strict aseptic precautions , thoracic epidural catheter was inserted using a 16 gauge , tuohy epidural needle by a paramedian approach . skin at insertion site was anesthetized by 3 ml of lidocaine 1% , the epidural space was identified by the loss of resistance technique , the catheter was introduced ~24 cm into the epidural space , and epidural test dose of 3 ml of lidocaine 2% with 1:200,000 adrenaline was injected to confirm its position . the epidural was loaded with 0.1 ml / kg of 0.125% bupivacaine / fentanyl 10 g / ml to obtain t4 sensory level ; if the injected dose was not enough to achieve t4 , another dose of 0.05 ml / kg was injected . after preoxygenation for 3 min , anesthesia was induced with iv propofol ( 1.5 mg / kg ) and fentanyl 2 g / kg . anesthesia was maintained by isoflurane 11.5 minimum alveolar concentration ( mac ) ; cisatracurium 0.03 mg / kg was administered when indicated . fentanyl 0.5 g / kg was given to maintain hr and blood pressure within 20% of the basal value . patients were mechanically ventilated to maintain end tidal co2 between 35 and 40 mmhg . the inspired oxygen fraction ( fio2 ) was 0.5 using oxygen - and - air mixtures . at the end of surgery , neuromuscular block was antagonized in all patients with neostigmine 0.05 mg / kg and atropine 0.02 mg / kg and finally the patients were extubated in the operating room . hypotension was determined as systolic blood pressure < 85 mmhg and was managed with iv ephedrine 0.1 mg / kg . bradycardia was determined as hr slower than 50 beats / min and was taken care of by atropine 0.01 mg / kg . all patients were admitted to the surgical intensive care unit ( icu ) , and were followed up for 2 weeks by the following observations : 12-lead ecg was recorded daily and if there was any suspicion of ischemic attacks.vital signs were recorded every 1 h in the icu.echocardiography was requested if there ecg findings ( on continuous monitoring ) suggested ischemic episodes or if the patient s complaint was consistent with angina . ( all ecgs and echocardiography were analyzed by a consultant cardiologist who was blinded to the patients condition.)vas was recorded every 4 h for 3 days postoperatively.venous blood samples for troponin i measurement were withdrawn routinely every day and at any time if there were ecg findings suggestive of ischemia . echocardiography was requested if there ecg findings ( on continuous monitoring ) suggested ischemic episodes or if the patient s complaint was consistent with angina . ( all ecgs and echocardiography were analyzed by a consultant cardiologist who was blinded to the patients condition . ) venous blood samples for troponin i measurement were withdrawn routinely every day and at any time if there were ecg findings suggestive of ischemia . the primary endpoint was the overall occurrence of adverse cardiac events that include new ecg findings suggestive of ischemia such as new st segment changes , new pathologic q wave , or new t wave inversion;new echocardiographic findings suggestive of ischemia ( new regional wall motion abnormalities);new critical arrhythmia , such as atrial flutter and fibrillation , second or third degree heart block , and any other arrhythmia affecting the hemodynamics;postoperative myocardial infarction diagnosed clinically , and by ecg and echocardiography , and in conjunction with cardiac troponin i , level > 0.23 ng / ml was considered the cut - point for diagnosis of myocardial injury ( mi);nonfatal cardiac arrest;heart failure , diagnosed clinically ( new in - hospital signs or symptoms of dyspnea , orthopnea , paroxysmal nocturnal dyspnea , increased jugular venous pressure , pulmonary rales on physical examination ) and by measuring b - type natriuretic peptide ( bnp ) level ( the decision cut - point of bnp level for the diagnosis of heart failure was identical to that of 100 pg / ml).16 new ecg findings suggestive of ischemia such as new st segment changes , new pathologic q wave , or new t wave inversion ; new echocardiographic findings suggestive of ischemia ( new regional wall motion abnormalities ) ; new critical arrhythmia , such as atrial flutter and fibrillation , second or third degree heart block , and any other arrhythmia affecting the hemodynamics ; postoperative myocardial infarction diagnosed clinically , and by ecg and echocardiography , and in conjunction with cardiac troponin i , level > 0.23 ng / ml was considered the cut - point for diagnosis of myocardial injury ( mi ) ; nonfatal cardiac arrest ; heart failure , diagnosed clinically ( new in - hospital signs or symptoms of dyspnea , orthopnea , paroxysmal nocturnal dyspnea , increased jugular venous pressure , pulmonary rales on physical examination ) and by measuring b - type natriuretic peptide ( bnp ) level ( the decision cut - point of bnp level for the diagnosis of heart failure was identical to that of 100 pg / ml).16 the secondary endpoints were intensity of pain measured by vas ( resting and dynamic);occurrence of other systems adverse events and complications;all in - hospital 30 days mortality . intensity of pain measured by vas ( resting and dynamic ) ; occurrence of other systems adverse events and complications ; all in - hospital 30 days mortality . blood samples for troponins i and plasma bnp levels were collected in non - pyrogenic , sterile falcon tubes . troponin i and bnp were measured by a newly developed high - sensitive elecsys analyzer ( fully automated enzyme linked immunosorbent assay [ elisa ] evolis ; bio - rad laboratories inc . bnp kit uses competitive elisa as the method while the cardiac - specific troponin i ( ctni ) elisa test is based on the principle of a solid phase elisa . statistical analysis was carried out on a personal computer using statistical package for the social sciences ( spss ) version 20 software . the sample size included all eligible patients admitted to the institute from august 2014 to august 2016 who were consecutively enrolled . independent samples student s t - test was used for comparison between two independent groups ( pcia and pcea ) . skewed data were presented as median ( interquartile range ) and differences between the two groups were compared nonparametrically using mann before induction of ga and under strict aseptic precautions , thoracic epidural catheter was inserted using a 16 gauge , tuohy epidural needle by a paramedian approach . skin at insertion site was anesthetized by 3 ml of lidocaine 1% , the epidural space was identified by the loss of resistance technique , the catheter was introduced ~24 cm into the epidural space , and epidural test dose of 3 ml of lidocaine 2% with 1:200,000 adrenaline was injected to confirm its position . the epidural was loaded with 0.1 ml / kg of 0.125% bupivacaine / fentanyl 10 g / ml to obtain t4 sensory level ; if the injected dose was not enough to achieve t4 , another dose of 0.05 ml / kg was injected . after preoxygenation for 3 min , anesthesia was induced with iv propofol ( 1.5 mg / kg ) and fentanyl 2 g / kg . anesthesia was maintained by isoflurane 11.5 minimum alveolar concentration ( mac ) ; cisatracurium 0.03 mg / kg was administered when indicated . fentanyl 0.5 g / kg was given to maintain hr and blood pressure within 20% of the basal value . patients were mechanically ventilated to maintain end tidal co2 between 35 and 40 mmhg . the inspired oxygen fraction ( fio2 ) was 0.5 using oxygen - and - air mixtures . at the end of surgery , neuromuscular block was antagonized in all patients with neostigmine 0.05 mg / kg and atropine 0.02 mg / kg and finally the patients were extubated in the operating room . hypotension was determined as systolic blood pressure < 85 mmhg and was managed with iv ephedrine 0.1 mg / kg . bradycardia was determined as hr slower than 50 beats / min and was taken care of by atropine 0.01 mg / kg . all patients were admitted to the surgical intensive care unit ( icu ) , and were followed up for 2 weeks by the following observations : 12-lead ecg was recorded daily and if there was any suspicion of ischemic attacks.vital signs were recorded every 1 h in the icu.echocardiography was requested if there ecg findings ( on continuous monitoring ) suggested ischemic episodes or if the patient s complaint was consistent with angina . ( all ecgs and echocardiography were analyzed by a consultant cardiologist who was blinded to the patients condition.)vas was recorded every 4 h for 3 days postoperatively.venous blood samples for troponin i measurement were withdrawn routinely every day and at any time if there were ecg findings suggestive of ischemia . echocardiography was requested if there ecg findings ( on continuous monitoring ) suggested ischemic episodes or if the patient s complaint was consistent with angina . ( all ecgs and echocardiography were analyzed by a consultant cardiologist who was blinded to the patients condition . ) venous blood samples for troponin i measurement were withdrawn routinely every day and at any time if there were ecg findings suggestive of ischemia . the primary endpoint was the overall occurrence of adverse cardiac events that include new ecg findings suggestive of ischemia such as new st segment changes , new pathologic q wave , or new t wave inversion;new echocardiographic findings suggestive of ischemia ( new regional wall motion abnormalities);new critical arrhythmia , such as atrial flutter and fibrillation , second or third degree heart block , and any other arrhythmia affecting the hemodynamics;postoperative myocardial infarction diagnosed clinically , and by ecg and echocardiography , and in conjunction with cardiac troponin i , level > 0.23 ng / ml was considered the cut - point for diagnosis of myocardial injury ( mi);nonfatal cardiac arrest;heart failure , diagnosed clinically ( new in - hospital signs or symptoms of dyspnea , orthopnea , paroxysmal nocturnal dyspnea , increased jugular venous pressure , pulmonary rales on physical examination ) and by measuring b - type natriuretic peptide ( bnp ) level ( the decision cut - point of bnp level for the diagnosis of heart failure was identical to that of 100 pg / ml).16 new ecg findings suggestive of ischemia such as new st segment changes , new pathologic q wave , or new t wave inversion ; new echocardiographic findings suggestive of ischemia ( new regional wall motion abnormalities ) ; new critical arrhythmia , such as atrial flutter and fibrillation , second or third degree heart block , and any other arrhythmia affecting the hemodynamics ; postoperative myocardial infarction diagnosed clinically , and by ecg and echocardiography , and in conjunction with cardiac troponin i , level > 0.23 ng / ml was considered the cut - point for diagnosis of myocardial injury ( mi ) ; nonfatal cardiac arrest ; heart failure , diagnosed clinically ( new in - hospital signs or symptoms of dyspnea , orthopnea , paroxysmal nocturnal dyspnea , increased jugular venous pressure , pulmonary rales on physical examination ) and by measuring b - type natriuretic peptide ( bnp ) level ( the decision cut - point of bnp level for the diagnosis of heart failure was identical to that of 100 pg / ml).16 the secondary endpoints were intensity of pain measured by vas ( resting and dynamic);occurrence of other systems adverse events and complications;all in - hospital 30 days mortality . intensity of pain measured by vas ( resting and dynamic ) ; occurrence of other systems adverse events and complications ; all in - hospital 30 days mortality . blood samples for troponins i and plasma bnp levels were collected in non - pyrogenic , sterile falcon tubes . troponin i and bnp were measured by a newly developed high - sensitive elecsys analyzer ( fully automated enzyme linked immunosorbent assay [ elisa ] evolis ; bio - rad laboratories inc . , hercules , ca , usa ) . bnp kit uses competitive elisa as the method while the cardiac - specific troponin i ( ctni ) elisa test is based on the principle of a solid phase elisa . statistical analysis was carried out on a personal computer using statistical package for the social sciences ( spss ) version 20 software . the sample size included all eligible patients admitted to the institute from august 2014 to august 2016 who were consecutively enrolled . independent samples student s t - test was used for comparison between two independent groups ( pcia and pcea ) . skewed data were presented as median ( interquartile range ) and differences between the two groups were compared nonparametrically using mann the demographic data and the characteristics of the patients were similar between groups ( table 1 ) . there was a significant decrease in overall adverse cardiac events ( myocardial injury , ventricular and atrial arrhythmia , angina , heart failure and nonfatal cardiac arrest ) in pcea group in comparison to pcia group ( table 2 ) . the level of troponin i was significantly higher in group pcea in comparison to group pcia at all measured time points ( p<0.038 ) ( figure 3 ) . the number of patients with increased troponin level was higher in pcia group ( figure 4 ) . regarding post - operative pain , the vas pain score at rest was similar between groups ( table 3 ) , while the vas pain score during movement was significantly decreased in pcea group in comparison to pcia group at all measured time points ( p<0.04 ) ( figure 2 ) . regarding perioperative hemodynamics , there was a significant reduction in intra - operative map and heart rate in pcea group in comparison to pcia group at most of measured time points while there was no significant reduction in postoperative map and heart rate in the second and third post - operative days ( table 4 ) . the incidence of other postoperative complications such as dvt , pneumonia and in hospital mortality were decreased in pcea group ( table 2 ) . the present study showed that perioperative thoracic epidural analgesia in patients suffering from cad subjected to major abdominal cancer surgery reduced significantly postoperative major adverse cardiac events in comparison with perioperative iv analgesia ( table 2 ) . moreover , the intensity of pain during movement was significantly decreased in pcea group in comparison to pcia group . the choice of 72 h as the period for pca ( either iv or epidural ) because of the large proportion of clinically unrecognized ami is related to the fact that most ami occur during the early postoperative period.17 in agreement with the present study , previous study showed that cardiac morbidity was lower among patients undergoing major vascular surgery after the administration of ga combined with postoperative epidural analgesia compared to the administration of ga alone and postoperative systemic opioid analgesia.18 master trial showed a significant reduction in pmi with thoracic epidural catheters in comparison with control groups among 11 randomized studies involving 1,173 patients . their inclusion criteria demanded that epidural analgesia should continue for at least 24 h after surgery , but their article does not state how they accounted for mortality among those patients randomized to the epidural group who may have died within the first 24 h.19 the cochrane study in 2016 concluded from a review of 15 clinical trials that epidural analgesia provides better pain management than systemic opioids . it significantly reduces the number of people who suffer heart damage , time to return of unassisted respiration , gastrointestinal bleeding , and icu length of stay . no difference was found in death rates at 30 days.20 a study was conducted by mohamed et al in the same institute to observe 60 ischemic patients , assigned into two groups , who underwent elective major abdominal cancer surgery ; 30 patients receiving ga ( g1 ) and the others receiving combined general and epidural anesthesia ( g2 ) . they concluded that lumbar epidural anesthesia combined with general anesthesia in high - risk patients with ischemic heart disease undergoing major abdominal cancer surgery provided better pain relief , and ischemic cardiac events were similar in both groups.22 according to moltner , dysrhythmias are common complications in the immediate postoperative period , even more common after upper abdominal and thoracic surgeries.22 scott et al presented the first randomized evaluation of the impact of perioperative tea on outcome in a large series of 400 patients with normal ventricular function undergoing coronary artery bypass grafting , wherein epidural catheters were placed immediately before surgery . there was a reduction in the incidence of supraventricular arrhythmias.23 in agreement with the present analysis , a study conducted by giroban et al registered dysrhythmias in the postoperative period of 20% of 185 patients undergoing thoracoabdominal surgeries.24 the occurrence of arrhythmias can be explained by many factors such as preexisting cardiac pathology , intraoperative events , and arrhythmia triggers . autonomic imbalance after operation has been implicated as a possible trigger , and is thought to be characterized by increased sympathetic tone and lower vagal tone.25 in this study , pcea resulted in a better optimization of hr and mean arterial pressure during the intra and postoperative period in comparison with the pcia group . this result showed the advantage of tea over iv pca by means of decreased hr and improved coronary blood flow . consistent with these results , kessler et al compared hr between patients who received ga together with tea ( group1 ) and those who received only ga ( group2 ) during coronary artery bypass surgery performed on a beating heart and reported that the hr in group 1 was lower than preoperative values , during sternotomy and anastomosis compared to group 2 . in that study , iv esmolol was administered in the group that received ga because of a high hr.26 berendes et al and fillinger et al , however , reported contradictory results as they did not observe a difference in hemodynamic findings between the control group and the tea treatment group when they studied tea in patients undergoing coronary artery bypass grafting.27,28 all the above studies resulted in low morbidity and mortality in patients receiving tea and this reflected on hospital icu stay . in contrast , the present study showed no significant difference with regard to icu and hospital stay between the two groups . this is consistent with the observations of kessler et al who found no differences in icu and hospital stay between the tea and ga groups.29 in contrast , priestly et al found no difference in troponin levels between ga alone and ga plus high tea groups.30 tea modifies the electrical activity of the heart in addition to ventricular function and wall motion . improvements in regional blood flow and reduction of major determinants of cardiac oxygen consumption lead to less severe ischemic injury.31 large coronary epicardial arteries and coronary arterioles are densely innervated by sympathetic adrenergic nerve fibers . cardiac sympathetic stimulation results in vasoconstriction of both normal and diseased coronary arteries in animals and in humans.32,33 in a canine model of experimentally induced cardiac ischemia , cardiac sympathectomy by tea has been shown to increase regional cardiac blood flow , and redistribute coronary blood flow in favor of the endocardium in both normal and diseased areas.31 davis et al observed favorable alteration in myocardial oxygen supply demand ratio.34 in patients with severe cad , tea relieved angina and improved myocardial oxygen supply by lowering systolic blood pressure and hr as well as pulmonary capillary wedge pressure with no significant improvement in coronary perfusion pressure.35,36 cardioselective epidural blocks can increase the luminal diameter of stenosed segments of epicardial coronary arteries without affecting the diameter of non - stenosed segments.37 small sample size that may hinder providing well - drawn results with smaller statistical error and better conclusions with shorter duration follow - up period . small sample size that may hinder providing well - drawn results with smaller statistical error and better conclusions with shorter duration follow - up period . perioperative thoracic epidural analgesia in patients suffering from cad subjected to major abdominal cancer surgery reduced significantly postoperative major adverse cardiac events with better pain control in comparison with perioperative iv analgesia .

background and objectivesmajor abdominal cancer surgeries are associated with significant perioperative mortality and morbidity due to myocardial ischemia and infarction . this study examined the effect of perioperative patient controlled epidural analgesia ( pcea ) on occurrence of ischemic cardiac injury in ischemic patients undergoing major abdominal cancer surgery.patients and methodsone hundred and twenty patients ( american society of anesthesiologists grade ii and iii ) of either sex were scheduled for elective upper gastrointestinal cancer surgeries . patients were allocated randomly into two groups ( 60 patients each ) to receive , besides general anesthesia : continuous intra and postoperative intravenous ( iv ) infusion with fentanyl for 72 h postoperatively ( patient controlled intravenous analgesia [ pcia ] group ) or continuous intra and postoperative epidural infusion with bupivacaine 0.125% and fentanyl ( pcea group ) for 72 h postoperatively . perioperative hemodynamics were recorded . postoperative pain was assessed over 72 h using visual analog scale ( vas ) . all patients were screened for occurrence of myocardial injury ( mi ) by electrocardiography , echocardiography , and cardiac troponin i serum level . other postoperative complications as arrhythmia , deep venous thrombosis ( dvt ) , pulmonary embolism , pneumonia , and death were recorded.resultsthere was a significant reduction in overall adverse cardiac events ( myocardial injury , arrhythmias , angina , heart failure and nonfatal cardiac arrest ) in pcea group in comparison to pcia group . also , there was a significant reduction in dynamic vas pain score in group pcea in comparison to pcia at all measured time points . regarding perioperative hemodynamics , there was a significant reduction in intra - operative mean arterial pressure ( map ) ; and heart rate in pcea group in comparison to pcia group at most of measured time points while there was not a significant reduction in postoperative map and heart rate in the second and third postoperative days . the incidence of other postoperative complications such as dvt , pneumonia and in hospital mortality were decreased in pcea group.conclusionperioperative thoracic epidural analgesia in patients suffering from coronary artery disease subjected to major abdominal cancer surgery reduced significantly postoperative major adverse cardiac events with better pain control in comparison with perioperative iv analgesia .

Introduction Patients and methods The technique of thoracic epidural GA Technique of measurement Statistical analysis Results Discussion Study limitations Conclusion

consequently , postoperative cardiovascular complications are expected to increase,1 and perioperative acute myocardial infarction ( ami ) has become a major health concern.2,3 it is well known that surgery induces a stress response , its extent is directly dependent on the magnitude of tissue destruction , and may be modified by the type of perioperative analgesia used . this stress response can lead to an increase in heart rate ( hr ) and blood pressure , which can precipitate episodes of myocardial ischemia.4 perioperative myocardial infarction ( pmi ) is one of the most important predictors of short- and long - term morbidity and mortality associated with noncardiac surgery.58 prevention of pmi is thus a prerequisite for the improvement in overall postoperative outcome . thoracic epidural anesthesia ( tea ) has been established as a cornerstone in perioperative care after thoracic and major abdominal surgery providing most effective analgesia.9,10 beyond its analgesic properties , tea s effects on postoperative neurohumoral stress response , cardiovascular pathophysiology , and intestinal dysfunction have been in the focus of both clinical and experimental investigations for years.1115 the aim of the study was to test whether epidural analgesia added to a general anesthetic , compared with systemic , opioid - based standard care analgesia in ischemic patients undergoing major abdominal cancer surgery , provided any reduction in adverse cardiac events . after obtaining written informed consent from each patient , 120 adult patients , complaining of coronary artery disease ( cad ) , classified as american society of anesthesiologists grade ii and iii and new york heart association class ii and iii , scheduled for elective major abdominal cancer surgery were consecutively enrolled . patients with coagulopathy , active neurological disease , cutaneous disorders at the epidural insertion site , and allergy to the studied medications were excluded from the study . every patient was evaluated by a cardiologist and anesthesiologist for medical history , physical examination , electrocardiography ( ecg ) , and echocardiography . the day before surgery , all patients were taught how to evaluate their own pain intensity using the visual analog scale ( vas ) , scored from 0 to 10 ( where 0= no pain and 10= worst pain imaginable ) and how to use the patient controlled analgesia ( pca ) device ( abbott laboratories , north chicago , il , usa ) . patients were randomly assigned into two groups , 60 patients each , by using opaque sealed envelopes containing a computer generated randomization schedule ; the opaque envelopes were sequentially numbered and were opened before application of anesthetic plan . in the patient controlled intravenous analgesia ( pcia ) group ( n=60 ) , patients received intraoperative analgesia with intravenous fentanyl bolus dose , 0.5 g / kg , followed by continuous infusion of 1 g / kg / h till the end of surgery . in the patient controlled epidural analgesia ( pcea ) group ( n=60 ) , where patients received pcea in conjunction with ga , intraoperative analgesia was started before skin incision by epidural bolus dose of 0.1 ml / kg of 0.125% bupivacaine / fentanyl 10 g / ml , followed by continuous infusion of 0.1 ml / kg / h of 0.125% bupivacaine / fentanyl 5 postoperative analgesia was provided through pcea for 72 h postoperatively ( background infusion of 0.1 ml / kg / h of 0.125% bupivacaine / fentanyl 3 g / ml , bolus dose of 3 ml , lockout interval was set at 20 min ) . before induction of ga and under strict aseptic precautions , thoracic epidural catheter was inserted using a 16 gauge , tuohy epidural needle by a paramedian approach . the epidural was loaded with 0.1 ml / kg of 0.125% bupivacaine / fentanyl 10 g / ml to obtain t4 sensory level ; if the injected dose was not enough to achieve t4 , another dose of 0.05 ml / kg was injected . after preoxygenation for 3 min , anesthesia was induced with iv propofol ( 1.5 mg / kg ) and fentanyl 2 g / kg . anesthesia was maintained by isoflurane 11.5 minimum alveolar concentration ( mac ) ; cisatracurium 0.03 mg / kg was administered when indicated . patients were mechanically ventilated to maintain end tidal co2 between 35 and 40 mmhg . at the end of surgery , neuromuscular block was antagonized in all patients with neostigmine 0.05 mg / kg and atropine 0.02 mg / kg and finally the patients were extubated in the operating room . all patients were admitted to the surgical intensive care unit ( icu ) , and were followed up for 2 weeks by the following observations : 12-lead ecg was recorded daily and if there was any suspicion of ischemic attacks.vital signs were recorded every 1 h in the icu.echocardiography was requested if there ecg findings ( on continuous monitoring ) suggested ischemic episodes or if the patient s complaint was consistent with angina . venous blood samples for troponin i measurement were withdrawn routinely every day and at any time if there were ecg findings suggestive of ischemia . the primary endpoint was the overall occurrence of adverse cardiac events that include new ecg findings suggestive of ischemia such as new st segment changes , new pathologic q wave , or new t wave inversion;new echocardiographic findings suggestive of ischemia ( new regional wall motion abnormalities);new critical arrhythmia , such as atrial flutter and fibrillation , second or third degree heart block , and any other arrhythmia affecting the hemodynamics;postoperative myocardial infarction diagnosed clinically , and by ecg and echocardiography , and in conjunction with cardiac troponin i , level > 0.23 ng / ml was considered the cut - point for diagnosis of myocardial injury ( mi);nonfatal cardiac arrest;heart failure , diagnosed clinically ( new in - hospital signs or symptoms of dyspnea , orthopnea , paroxysmal nocturnal dyspnea , increased jugular venous pressure , pulmonary rales on physical examination ) and by measuring b - type natriuretic peptide ( bnp ) level ( the decision cut - point of bnp level for the diagnosis of heart failure was identical to that of 100 pg / ml).16 new ecg findings suggestive of ischemia such as new st segment changes , new pathologic q wave , or new t wave inversion ; new echocardiographic findings suggestive of ischemia ( new regional wall motion abnormalities ) ; new critical arrhythmia , such as atrial flutter and fibrillation , second or third degree heart block , and any other arrhythmia affecting the hemodynamics ; postoperative myocardial infarction diagnosed clinically , and by ecg and echocardiography , and in conjunction with cardiac troponin i , level > 0.23 ng / ml was considered the cut - point for diagnosis of myocardial injury ( mi ) ; nonfatal cardiac arrest ; heart failure , diagnosed clinically ( new in - hospital signs or symptoms of dyspnea , orthopnea , paroxysmal nocturnal dyspnea , increased jugular venous pressure , pulmonary rales on physical examination ) and by measuring b - type natriuretic peptide ( bnp ) level ( the decision cut - point of bnp level for the diagnosis of heart failure was identical to that of 100 pg / ml).16 the secondary endpoints were intensity of pain measured by vas ( resting and dynamic);occurrence of other systems adverse events and complications;all in - hospital 30 days mortality . intensity of pain measured by vas ( resting and dynamic ) ; occurrence of other systems adverse events and complications ; all in - hospital 30 days mortality . troponin i and bnp were measured by a newly developed high - sensitive elecsys analyzer ( fully automated enzyme linked immunosorbent assay [ elisa ] evolis ; bio - rad laboratories inc . bnp kit uses competitive elisa as the method while the cardiac - specific troponin i ( ctni ) elisa test is based on the principle of a solid phase elisa . independent samples student s t - test was used for comparison between two independent groups ( pcia and pcea ) . skewed data were presented as median ( interquartile range ) and differences between the two groups were compared nonparametrically using mann before induction of ga and under strict aseptic precautions , thoracic epidural catheter was inserted using a 16 gauge , tuohy epidural needle by a paramedian approach . the epidural was loaded with 0.1 ml / kg of 0.125% bupivacaine / fentanyl 10 g / ml to obtain t4 sensory level ; if the injected dose was not enough to achieve t4 , another dose of 0.05 ml / kg was injected . after preoxygenation for 3 min , anesthesia was induced with iv propofol ( 1.5 mg / kg ) and fentanyl 2 g / kg . at the end of surgery , neuromuscular block was antagonized in all patients with neostigmine 0.05 mg / kg and atropine 0.02 mg / kg and finally the patients were extubated in the operating room . all patients were admitted to the surgical intensive care unit ( icu ) , and were followed up for 2 weeks by the following observations : 12-lead ecg was recorded daily and if there was any suspicion of ischemic attacks.vital signs were recorded every 1 h in the icu.echocardiography was requested if there ecg findings ( on continuous monitoring ) suggested ischemic episodes or if the patient s complaint was consistent with angina . venous blood samples for troponin i measurement were withdrawn routinely every day and at any time if there were ecg findings suggestive of ischemia . the primary endpoint was the overall occurrence of adverse cardiac events that include new ecg findings suggestive of ischemia such as new st segment changes , new pathologic q wave , or new t wave inversion;new echocardiographic findings suggestive of ischemia ( new regional wall motion abnormalities);new critical arrhythmia , such as atrial flutter and fibrillation , second or third degree heart block , and any other arrhythmia affecting the hemodynamics;postoperative myocardial infarction diagnosed clinically , and by ecg and echocardiography , and in conjunction with cardiac troponin i , level > 0.23 ng / ml was considered the cut - point for diagnosis of myocardial injury ( mi);nonfatal cardiac arrest;heart failure , diagnosed clinically ( new in - hospital signs or symptoms of dyspnea , orthopnea , paroxysmal nocturnal dyspnea , increased jugular venous pressure , pulmonary rales on physical examination ) and by measuring b - type natriuretic peptide ( bnp ) level ( the decision cut - point of bnp level for the diagnosis of heart failure was identical to that of 100 pg / ml).16 new ecg findings suggestive of ischemia such as new st segment changes , new pathologic q wave , or new t wave inversion ; new echocardiographic findings suggestive of ischemia ( new regional wall motion abnormalities ) ; new critical arrhythmia , such as atrial flutter and fibrillation , second or third degree heart block , and any other arrhythmia affecting the hemodynamics ; postoperative myocardial infarction diagnosed clinically , and by ecg and echocardiography , and in conjunction with cardiac troponin i , level > 0.23 ng / ml was considered the cut - point for diagnosis of myocardial injury ( mi ) ; nonfatal cardiac arrest ; heart failure , diagnosed clinically ( new in - hospital signs or symptoms of dyspnea , orthopnea , paroxysmal nocturnal dyspnea , increased jugular venous pressure , pulmonary rales on physical examination ) and by measuring b - type natriuretic peptide ( bnp ) level ( the decision cut - point of bnp level for the diagnosis of heart failure was identical to that of 100 pg / ml).16 the secondary endpoints were intensity of pain measured by vas ( resting and dynamic);occurrence of other systems adverse events and complications;all in - hospital 30 days mortality . intensity of pain measured by vas ( resting and dynamic ) ; occurrence of other systems adverse events and complications ; all in - hospital 30 days mortality . bnp kit uses competitive elisa as the method while the cardiac - specific troponin i ( ctni ) elisa test is based on the principle of a solid phase elisa . independent samples student s t - test was used for comparison between two independent groups ( pcia and pcea ) . skewed data were presented as median ( interquartile range ) and differences between the two groups were compared nonparametrically using mann the demographic data and the characteristics of the patients were similar between groups ( table 1 ) . there was a significant decrease in overall adverse cardiac events ( myocardial injury , ventricular and atrial arrhythmia , angina , heart failure and nonfatal cardiac arrest ) in pcea group in comparison to pcia group ( table 2 ) . the level of troponin i was significantly higher in group pcea in comparison to group pcia at all measured time points ( p<0.038 ) ( figure 3 ) . regarding post - operative pain , the vas pain score at rest was similar between groups ( table 3 ) , while the vas pain score during movement was significantly decreased in pcea group in comparison to pcia group at all measured time points ( p<0.04 ) ( figure 2 ) . regarding perioperative hemodynamics , there was a significant reduction in intra - operative map and heart rate in pcea group in comparison to pcia group at most of measured time points while there was no significant reduction in postoperative map and heart rate in the second and third post - operative days ( table 4 ) . the incidence of other postoperative complications such as dvt , pneumonia and in hospital mortality were decreased in pcea group ( table 2 ) . the present study showed that perioperative thoracic epidural analgesia in patients suffering from cad subjected to major abdominal cancer surgery reduced significantly postoperative major adverse cardiac events in comparison with perioperative iv analgesia ( table 2 ) . moreover , the intensity of pain during movement was significantly decreased in pcea group in comparison to pcia group . the choice of 72 h as the period for pca ( either iv or epidural ) because of the large proportion of clinically unrecognized ami is related to the fact that most ami occur during the early postoperative period.17 in agreement with the present study , previous study showed that cardiac morbidity was lower among patients undergoing major vascular surgery after the administration of ga combined with postoperative epidural analgesia compared to the administration of ga alone and postoperative systemic opioid analgesia.18 master trial showed a significant reduction in pmi with thoracic epidural catheters in comparison with control groups among 11 randomized studies involving 1,173 patients . their inclusion criteria demanded that epidural analgesia should continue for at least 24 h after surgery , but their article does not state how they accounted for mortality among those patients randomized to the epidural group who may have died within the first 24 h.19 the cochrane study in 2016 concluded from a review of 15 clinical trials that epidural analgesia provides better pain management than systemic opioids . no difference was found in death rates at 30 days.20 a study was conducted by mohamed et al in the same institute to observe 60 ischemic patients , assigned into two groups , who underwent elective major abdominal cancer surgery ; 30 patients receiving ga ( g1 ) and the others receiving combined general and epidural anesthesia ( g2 ) . they concluded that lumbar epidural anesthesia combined with general anesthesia in high - risk patients with ischemic heart disease undergoing major abdominal cancer surgery provided better pain relief , and ischemic cardiac events were similar in both groups.22 according to moltner , dysrhythmias are common complications in the immediate postoperative period , even more common after upper abdominal and thoracic surgeries.22 scott et al presented the first randomized evaluation of the impact of perioperative tea on outcome in a large series of 400 patients with normal ventricular function undergoing coronary artery bypass grafting , wherein epidural catheters were placed immediately before surgery . there was a reduction in the incidence of supraventricular arrhythmias.23 in agreement with the present analysis , a study conducted by giroban et al registered dysrhythmias in the postoperative period of 20% of 185 patients undergoing thoracoabdominal surgeries.24 the occurrence of arrhythmias can be explained by many factors such as preexisting cardiac pathology , intraoperative events , and arrhythmia triggers . autonomic imbalance after operation has been implicated as a possible trigger , and is thought to be characterized by increased sympathetic tone and lower vagal tone.25 in this study , pcea resulted in a better optimization of hr and mean arterial pressure during the intra and postoperative period in comparison with the pcia group . consistent with these results , kessler et al compared hr between patients who received ga together with tea ( group1 ) and those who received only ga ( group2 ) during coronary artery bypass surgery performed on a beating heart and reported that the hr in group 1 was lower than preoperative values , during sternotomy and anastomosis compared to group 2 . in that study , iv esmolol was administered in the group that received ga because of a high hr.26 berendes et al and fillinger et al , however , reported contradictory results as they did not observe a difference in hemodynamic findings between the control group and the tea treatment group when they studied tea in patients undergoing coronary artery bypass grafting.27,28 all the above studies resulted in low morbidity and mortality in patients receiving tea and this reflected on hospital icu stay . in contrast , the present study showed no significant difference with regard to icu and hospital stay between the two groups . cardiac sympathetic stimulation results in vasoconstriction of both normal and diseased coronary arteries in animals and in humans.32,33 in a canine model of experimentally induced cardiac ischemia , cardiac sympathectomy by tea has been shown to increase regional cardiac blood flow , and redistribute coronary blood flow in favor of the endocardium in both normal and diseased areas.31 davis et al observed favorable alteration in myocardial oxygen supply demand ratio.34 in patients with severe cad , tea relieved angina and improved myocardial oxygen supply by lowering systolic blood pressure and hr as well as pulmonary capillary wedge pressure with no significant improvement in coronary perfusion pressure.35,36 cardioselective epidural blocks can increase the luminal diameter of stenosed segments of epicardial coronary arteries without affecting the diameter of non - stenosed segments.37 small sample size that may hinder providing well - drawn results with smaller statistical error and better conclusions with shorter duration follow - up period . perioperative thoracic epidural analgesia in patients suffering from cad subjected to major abdominal cancer surgery reduced significantly postoperative major adverse cardiac events with better pain control in comparison with perioperative iv analgesia .

briefly , diagnosis of type 1 diabetes was made according to criteria by the australasian pediatric endocrine group diabetes register and national guidelines . baseline assessment and retinal photography was performed at the initial visit during the recruitment period . of 1,159 participants who had retinal photographs taken at their baseline visits , we excluded those with ungradable photographs ( n = 215 , 18.6% ) , due to either poor quality of retinal photographs or having less than four big vessels that could be traced by the computer - assisted program , leaving 944 ( 81.4% ) participants included in analyses for this report . key diabetes - related characteristics assessed included pubertal stage , duration of diabetes , and levels of a1c , cholesterol , and blood pressure , which were collected at the baseline visits via interviews , clinical examinations , and laboratory investigations following standardized protocols ( 1 ) . bmi was calculated by dividing subject 's weight ( in kilograms ) with the square of height ( in meters ) . systolic ( sbp ) and diastolic ( dbp ) blood pressure were measured after resting for 5 min , sitting in an upright position using a standard sphygmomanometer with an appropriately sized cuff . a1c and total plasma cholesterol levels were measured following standardized laboratory procedures ( 1 ) . retinal photography was performed according to a standardized protocol , as detailed elsewhere ( 1 ) . briefly , stereoscopic retinal photographs in seven standard fields of the early treatment diabetic retinopathy study ( etdrs ) were taken on film from both eyes after pupil dilation , using a topcon fundus camera ( trc 50-vt ; tokyo optical , tokyo , japan ) ( 1 ) . retinopathy was assessed by an ophthalmologist and defined as present when any microaneurysm / retinal hemorrhage was found in either eye ( 1 ) . for measuring retinal microvascular geometric properties , right - eye retinal photographs of each patient were digitized and analyses were performed using a semiautomated computer - assisted image program ( singapore i vessel assessment or siva , singapore ) . retinal photographs that were centered on the optic disc were viewed on two 19-inch monitors with a resolution of 1,280 1,024 . for each retinal photograph , a trained grader , masked to participants ' identities , applied the program to measure retinal microvascular geometric parameters within a concentric zone between the optic disc margin and two optic disc diameters away from the optic disc margin . the grader allowed the software to detect the center of the optic disc and divided the region into three subzones ( a , b , and c ) surrounding the optic disc , each zone corresponding to 0.5 , 1.0 , and 2.0 optic disc diameters away from the optic disc margin , respectively . once the optic disc and the three concentric subzones were considered appropriately located , the grader executed the program to trace all vessels . however , the grader checked each graded image to see if all arterioles and venules were correctly identified , based on information of parent vessels , crossing between arterioles and venules and the color of the vessels . graders were trained and tested for his / her ability in identifying arterioles and venules by a senior grader . during the grading process of this project , 100 randomly selected images were used to test intra- and intergrader reliabilities in classification of arterioles and venules , with value 0.950.99 . images were considered poor quality if they were blurred or had an incomplete representation of zone c and as ungradable if there were less than four gradable large arterioles or venules . the software combined the individual measurement into summary indexes of tortuosity , branching angles , optimality deviation , and ldr for arterioles and venules separately , as described below : vessel tortuosity ( fig . 1 ) reflects the shape of the vessel and is expressed as a curvature tortuosity index ( 4 ) , calculated from the integral of the total squared curvature along the path of the vessel divided by the total arc length . increased tortuosity has been linked with hypertension ( 8) and diabetic retinopathy ( 9 ) , while decreased tortuosity has been associated with ischemic heart disease related death ( 10 ) , ageing , and hypertension ( c. y. cheung , e. lamoureux , l. xu , w. hsu , m. l. lee , q. p. lau , j. j. wang , p. mitchell , t. y. wong).branching angle represents ( in degrees ) the angle between two daughter vessels ( 5 ) and is thought to be related to blood flow efficiency , energy cost of bulk flow , and diffusion distance ( 11 ) . ( 5 ) proposed that the optimal value for the branching angle is 75 degrees ( fig . 1 ) , and increased angles have been related to decreased blood flow ( 12 ) , while decreased angles are associated with ageing and hypertension ( 11).optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0 , where d0 , d1 , and d2 are diameters of the parent , larger , and smaller daughter vessels , respectively ( 11 ) . the greater the value of x , the larger the daughter arterioles are relative to the parent vessel . junctional exponent provides an index of caliber sizes of two daughter vessels relative to the parent vessel and is considered to represent an optimality state of microvascular networks ( 5 ) . it has been proposed that in an optimal state , the value of junctional exponent is three ( 5 ) , and optimality deviation represents the deviation from this value.ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch ( 6 ) . ldr is a measure of diameter changes that are independent of refractive magnification power of the eye ( 6 ) . 1 ) reflects the shape of the vessel and is expressed as a curvature tortuosity index ( 4 ) , calculated from the integral of the total squared curvature along the path of the vessel divided by the total arc length . normal vessels are generally straight and smooth ( 4 ) . increased tortuosity has been linked with hypertension ( 8) and diabetic retinopathy ( 9 ) , while decreased tortuosity has been associated with ischemic heart disease related death ( 10 ) , ageing , and hypertension ( c. y. cheung , e. lamoureux , l. xu , w. hsu , m. l. lee , q. p. lau , j. j. wang , p. mitchell , t. y. wong ) . branching angle represents ( in degrees ) the angle between two daughter vessels ( 5 ) and is thought to be related to blood flow efficiency , energy cost of bulk flow , and diffusion distance ( 11 ) . ( 5 ) proposed that the optimal value for the branching angle is 75 degrees ( fig . 1 ) , and increased angles have been related to decreased blood flow ( 12 ) , while decreased angles are associated with ageing and hypertension ( 11 ) . optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0 , where d0 , d1 , and d2 are diameters of the parent , larger , and smaller daughter vessels , respectively ( 11 ) . the greater the value of x , the larger the daughter arterioles are relative to the parent vessel . junctional exponent provides an index of caliber sizes of two daughter vessels relative to the parent vessel and is considered to represent an optimality state of microvascular networks ( 5 ) . it has been proposed that in an optimal state , the value of junctional exponent is three ( 5 ) , and optimality deviation represents the deviation from this value . ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch ( 6 ) . ldr is a measure of diameter changes that are independent of refractive magnification power of the eye ( 6 ) . measures of retinal vessel tortuosity , branching angle , and optimality deviation . a comparison of optimal ( a ) and less optimal ( b ) arrangement of arteriolar geometry . a : tortuosity of 0.00 , branching angle of 72 , and optimality deviation of 0.16 . b : tortuosity of 47.2 10 , branching angle of 130 , and optimality deviation of 0.61 . a high - quality color representation of this image is available online . all of statistical procedures were performed using intercooled stata 10.1 for windows ( statacorp , college station , tx ) . retinal microvascular geometry ( tortuosity , branching angle , optimality deviation , and ldr ) of the right eye of each patient were used as dependent , continuous variables . we used ancova to compare mean retinal vessel geometric parameters by age - group ( 1214 and 1420 years ) , sex ( female and male ) , pubertal stage ( stage 1 to stage 5 ) , bmi levels ( 18.0 , 18.121.0 , 21.125.0 , and 25.1 kg / m ) , sbp ( 120 vs. 120 mmhg ) , cholesterol ( 3.7 , 3.84.3 , 4.44.8 , and 4.9 mmol / l ) , duration of diabetes ( 5.0 , 5.110.0 , and 10.1 years ) , and a1c ( 8.5 vs. > 8.5% ) ( 1 ) ( see supplementary tables in the online appendix , available at http://care.diabetesjournals.org/cgi/content/full/dc10-0055/dc1 ) . multiple linear regression models were subsequently used to examine the independent determinants of retinal microvascular geometric parameters as continuous variables . we constructed three models : model 1 included age and sex ; model 2 additionally included bmi , sbp , cholesterol , duration of diabetes , a1c ( all assessed as per sd change ) , presence of retinopathy , and retinal arteriolar caliber ( in models for arteriolar geometric parameters ) or venular caliber ( in models for venular geometric parameters ) ; and model 3 included all variables in model 2 after excluding subjects with diabetic retinopathy . finally , sensitivity analyses were performed by including and excluding subjects with poor - quality images . key diabetes - related characteristics assessed included pubertal stage , duration of diabetes , and levels of a1c , cholesterol , and blood pressure , which were collected at the baseline visits via interviews , clinical examinations , and laboratory investigations following standardized protocols ( 1 ) . bmi was calculated by dividing subject 's weight ( in kilograms ) with the square of height ( in meters ) . systolic ( sbp ) and diastolic ( dbp ) blood pressure were measured after resting for 5 min , sitting in an upright position using a standard sphygmomanometer with an appropriately sized cuff . a1c and total plasma cholesterol levels were measured following standardized laboratory procedures ( 1 ) . retinal photography was performed according to a standardized protocol , as detailed elsewhere ( 1 ) . briefly , stereoscopic retinal photographs in seven standard fields of the early treatment diabetic retinopathy study ( etdrs ) were taken on film from both eyes after pupil dilation , using a topcon fundus camera ( trc 50-vt ; tokyo optical , tokyo , japan ) ( 1 ) . retinopathy was assessed by an ophthalmologist and defined as present when any microaneurysm / retinal hemorrhage was found in either eye ( 1 ) . for measuring retinal microvascular geometric properties , right - eye retinal photographs of each patient were digitized and analyses were performed using a semiautomated computer - assisted image program ( singapore i vessel assessment or siva , singapore ) . retinal photographs that were centered on the optic disc were viewed on two 19-inch monitors with a resolution of 1,280 1,024 . for each retinal photograph , a trained grader , masked to participants ' identities , applied the program to measure retinal microvascular geometric parameters within a concentric zone between the optic disc margin and two optic disc diameters away from the optic disc margin . the grader allowed the software to detect the center of the optic disc and divided the region into three subzones ( a , b , and c ) surrounding the optic disc , each zone corresponding to 0.5 , 1.0 , and 2.0 optic disc diameters away from the optic disc margin , respectively . once the optic disc and the three concentric subzones were considered appropriately located , the grader executed the program to trace all vessels . however , the grader checked each graded image to see if all arterioles and venules were correctly identified , based on information of parent vessels , crossing between arterioles and venules and the color of the vessels . graders were trained and tested for his / her ability in identifying arterioles and venules by a senior grader . during the grading process of this project , 100 randomly selected images were used to test intra- and intergrader reliabilities in classification of arterioles and venules , with value 0.950.99 . images were considered poor quality if they were blurred or had an incomplete representation of zone c and as ungradable if there were less than four gradable large arterioles or venules . the software combined the individual measurement into summary indexes of tortuosity , branching angles , optimality deviation , and ldr for arterioles and venules separately , as described below : vessel tortuosity ( fig . 1 ) reflects the shape of the vessel and is expressed as a curvature tortuosity index ( 4 ) , calculated from the integral of the total squared curvature along the path of the vessel divided by the total arc length . normal vessels are generally straight and smooth ( 4 ) . increased tortuosity has been linked with hypertension ( 8) and diabetic retinopathy ( 9 ) , while decreased tortuosity has been associated with ischemic heart disease related death ( 10 ) , ageing , and hypertension ( c. y. cheung , e. lamoureux , l. xu , w. hsu , m. l. lee , q. p. lau , j. j. wang , p. mitchell , t. y. wong).branching angle represents ( in degrees ) the angle between two daughter vessels ( 5 ) and is thought to be related to blood flow efficiency , energy cost of bulk flow , and diffusion distance ( 11 ) . ( 5 ) proposed that the optimal value for the branching angle is 75 degrees ( fig . 1 ) , and increased angles have been related to decreased blood flow ( 12 ) , while decreased angles are associated with ageing and hypertension ( 11).optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0 , where d0 , d1 , and d2 are diameters of the parent , larger , and smaller daughter vessels , respectively ( 11 ) . the greater the value of x , the larger the daughter arterioles are relative to the parent vessel . junctional exponent provides an index of caliber sizes of two daughter vessels relative to the parent vessel and is considered to represent an optimality state of microvascular networks ( 5 ) . it has been proposed that in an optimal state , the value of junctional exponent is three ( 5 ) , and optimality deviation represents the deviation from this value.ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch ( 6 ) . ldr is a measure of diameter changes that are independent of refractive magnification power of the eye ( 6 ) . 1 ) reflects the shape of the vessel and is expressed as a curvature tortuosity index ( 4 ) , calculated from the integral of the total squared curvature along the path of the vessel divided by the total arc length . normal vessels are generally straight and smooth ( 4 ) . increased tortuosity has been linked with hypertension ( 8) and diabetic retinopathy ( 9 ) , while decreased tortuosity has been associated with ischemic heart disease related death ( 10 ) , ageing , and hypertension ( c. y. cheung , e. lamoureux , l. xu , w. hsu , m. l. lee , q. p. lau , j. j. wang , p. mitchell , t. y. wong ) . branching angle represents ( in degrees ) the angle between two daughter vessels ( 5 ) and is thought to be related to blood flow efficiency , energy cost of bulk flow , and diffusion distance ( 11 ) . ( 5 ) proposed that the optimal value for the branching angle is 75 degrees ( fig . 1 ) , and increased angles have been related to decreased blood flow ( 12 ) , while decreased angles are associated with ageing and hypertension ( 11 ) . optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0 , where d0 , d1 , and d2 are diameters of the parent , larger , and smaller daughter vessels , respectively ( 11 ) . the greater the value of x , the larger the daughter arterioles are relative to the parent vessel . junctional exponent provides an index of caliber sizes of two daughter vessels relative to the parent vessel and is considered to represent an optimality state of microvascular networks ( 5 ) . it has been proposed that in an optimal state , the value of junctional exponent is three ( 5 ) , and optimality deviation represents the deviation from this value . ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch ( 6 ) . ldr is a measure of diameter changes that are independent of refractive magnification power of the eye ( 6 ) . a comparison of optimal ( a ) and less optimal ( b ) arrangement of arteriolar geometry . a : tortuosity of 0.00 , branching angle of 72 , and optimality deviation of 0.16 . b : tortuosity of 47.2 10 , branching angle of 130 , and optimality deviation of 0.61 . a high - quality color representation of this image is available online . all of statistical procedures were performed using intercooled stata 10.1 for windows ( statacorp , college station , tx ) . retinal microvascular geometry ( tortuosity , branching angle , optimality deviation , and ldr ) of the right eye of each patient were used as dependent , continuous variables . we used ancova to compare mean retinal vessel geometric parameters by age - group ( 1214 and 1420 years ) , sex ( female and male ) , pubertal stage ( stage 1 to stage 5 ) , bmi levels ( 18.0 , 18.121.0 , 21.125.0 , and 25.1 kg / m ) , sbp ( 120 vs. 120 mmhg ) , cholesterol ( 3.7 , 3.84.3 , 4.44.8 , and 4.9 mmol / l ) , duration of diabetes ( 5.0 , 5.110.0 , and 10.1 years ) , and a1c ( 8.5 vs. > 8.5% ) ( 1 ) ( see supplementary tables in the online appendix , available at http://care.diabetesjournals.org/cgi/content/full/dc10-0055/dc1 ) . multiple linear regression models were subsequently used to examine the independent determinants of retinal microvascular geometric parameters as continuous variables . we constructed three models : model 1 included age and sex ; model 2 additionally included bmi , sbp , cholesterol , duration of diabetes , a1c ( all assessed as per sd change ) , presence of retinopathy , and retinal arteriolar caliber ( in models for arteriolar geometric parameters ) or venular caliber ( in models for venular geometric parameters ) ; and model 3 included all variables in model 2 after excluding subjects with diabetic retinopathy . finally , sensitivity analyses were performed by including and excluding subjects with poor - quality images . of 944 patients in this study , 170 ( 14.7% ) had retinopathy ( all classified as mild nonproliferative retinopathy ) . the baseline characteristics of participants with and without gradable right - eye retinal photographs are shown in table 1 and retinal microvascular measurements are shown in table 2 . baseline characteristics , according to the gradability of retinal photograph data are age and sex - adjusted means se or % . retinal microvascular parameters in 944 children and adolescents aged 1220 years with type 1 diabetes data are means sd or median ( interquartile range ) . table 3 shows that older age was associated with decreased arteriolar and venular tortuosity , and female patients on average had larger arteriolar branching angle than male patients . after adjusting for age , sex , sbp , cholesterol level , duration of diabetes , a1c level , retinopathy , and vessel caliber , the following associations were evident : 1 ) increasing diabetes duration was associated with increased arteriolar branching angle ( p 0.002 ) and increased optimality deviation of arterioles ( p = 0.014 ) , 2 ) high a1c ( > 8.5 vs. 8.5% , ) was associated with increased arteriolar tortuosity ( p = 0.018 ) , 3 ) increasing sbp was associated with decreasing arteriolar ldr ( p = 0.001 ) and nonsignificantly decreasing venular ldr ( 0.05 < p < 0.10 ) , and 4 ) increasing total cholesterol level was associated with increasing arteriolar ldr ( p = 0.014 ) and decreasing optimality deviation of venules ( p = 0.07 ) . the observed significant associations largely remained after excluding eyes with retinopathy ( n = 170 , model 3 ) ( table 3 ) . associations of baseline and diabetes - related factors with retinal microvascular geometry * data are mean difference ( 95% ci ) of retinal parameters per sd increase in duration ( 3.3 years ) or 8.5 vs. > 8.5% a1c level and per 1-sd increase in sbp ( 12 mmhg ) , cholesterol ( 0.9 mmol / l ) , and bmi ( 3.5 kg / m ) using linear regression models . p , p value of coefficient adjusted for age and sex ; p , p value of coefficient adjusted for age , sex , sbp , cholesterol , duration , a1c , retinopathy , and retinal vessel caliber ; p , p value of coefficient , adjusted for age , sex , sbp , cholesterol , duration , a1c , and retinal vessel caliber , excluding subjects with retinopathy . there were no significant interactions between age , sex , blood pressure , bmi , cholesterol , duration of diabetes , and a1c level . analyses after excluding subjects with relatively poor image quality ( n = 129 , 11.1% ) showed that these associations remained significant in 815 ( 70.3% ) patients with good - quality retinal images ( data not shown ) . in young type 1 diabetes , we showed that variations in retinal microvascular geometric characteristics were associated with key diabetes - related risk factors , including longer diabetes duration and higher a1c , blood pressure , and cholesterol levels . the principal - specific findings are the following : longer duration of diabetes was associated with larger arteriolar branching angles and increasing deviation of optimality , higher a1c was associated with more tortuous arterioles , increasing sbp level was associated with smaller arteriolar and venular ldr , and increasing cholesterol level was associated with increasing arteriolar ldr and decreasing venular optimality deviation . importantly , we showed that these associations were present even in subjects without mild retinopathy signs , suggesting that early retinal microvascular alternations are present well before the onset of clinical microvascular complications in young patients with type 1 diabetes . most previous studies that have examined retinal microvascular geometric parameters have been conducted in nondiabetic populations , including healthy subjects ( 13 ) , subjects with hypertension ( 11 ) , or those with coronary heart disease ( 10 ) . to the best of our knowledge , there has been no study reporting retinal microvascular geometric characteristics in young type 1 diabetes . our findings that longer duration of diabetes was associated with larger arteriolar branching angle and increasing deviation from optimality of arterioles is biologically plausible and may provide clues to early microvascular alterations in type 1 diabetes . an optimal branching angle is associated with greater efficiency in blood flow with lower energy spent ( 5,7 ) . such efficiency reduces when the branching angle is too large or when the size of daughter vessel is too large or too small relative to its parent vessel ( 5,7 ) . ( 5 ) have shown that departure from normal or increased deviation from optimality could result in increased workload and energy loss in maintaining the blood circulation . branching angle and junctional exponent have been found to be impaired in atherosclerosis ( 14 ) , altered blood flow ( 12 ) , and endothelial dysfunction ( 15 ) . moreover , chapman et al . ( 16 ) demonstrated that branching angle was likely to increase in response to less oxygen saturation . thus , our finding of associations of longer diabetes duration with larger branching angle and higher optimality deviation may reflect alterations in blood flow ( 17 ) , endothelial dysfunction ( 18 ) , and attenuation in oxygen saturation ( 16 ) . the sex difference in arteriolar branching angle , with female subjects having larger arteriolar branching angles than male subjects , could also explain our earlier observations that female patients with type 1 diabetes have greater risk for diabetic microvascular complications compared with male patients of the same age ( 1,3 ) . diabetes is known to be associated with increased shear stress and impaired microvascular endothelium ( 2 ) . previously , increased a1c level had been shown to have a role in reduced endothelial function in people with type 1 diabetes ( 19 ) . increased vessel tortuosity has been documented to be related to increased angiogenesis ( 20 ) and endothelial dysfunction ( 10 ) . we did not find a linear association between a1c levels and arteriolar tortuosity , but we observed a significant difference in tortuosity between patients with a1c 8.5 vs. 8.5% . there have been studies showing that different cutoffs of a1c levels identify people with diabetes at high risk of microvascular injury ( 21,22 ) , although there is no universal consensus on these cutoffs ( 22 ) . we have previously reported that wider arteriolar caliber is associated with an increased risk of diabetic retinopathy in both type 1 and type 2 diabetes ( 3,23 ) . thus , the finding of an association of higher blood pressure levels with increasing arteriolar ldr ( reflecting wider arteriolar caliber ) is consistent with the influence of diabetes and blood pressure on autoregulatory processes in small blood vessels . there have been few studies on cholesterol levels on microvascular structure , with most previous studies reporting associations between cholesterol level and retinal microvascular changes in nondiabetic populations showing inconsistent results ( 24,25 ) . therefore , our findings that higher total cholesterol levels was associated with changes in arteriolar ldr and venular optimality deviation suggests that lipids may also have an influence on microvasculature in young type 1 diabetes . first , our study includes a large cohort of young patients with type 1 diabetes , with a participation rate of > 80% . second , our study included an objective quantitative measurement of the geometry of retinal microvasculature using computer programs . the cross - sectional nature of our study implies that longitudinal studies are needed to determine the temporal sequence of these associations . a number of images ( 18.6% ) were ungradable and thus excluded from the analysis . however , baseline characteristics and diabetes - related risk factors were largely similar in participants with and without gradable images . therefore , exclusion of participants with ungradeable images did not substantially influence our findings . in addition , there are several potential sources of measurement errors in assessing branching / bifurcation angles , particularly in subjects with poor retinal image quality , given that the computer software needs some manual intervention . however , as the measurement errors are random , these study findings are unlikely to be altered substantially by random errors . in summary , our study demonstrated that in young type 1 diabetes , subtle alterations in retinal microvascular geometric parameters are associated with key diabetes - related characteristics , including duration of diabetes and a1c and blood pressure levels . these findings were present in those without any signs of retinopathy , suggesting an effect of these risk factors on the microcirculation prior to development of clinical complications . some of the observed associations are related to known effects of diabetes on microvasculature ; for other associations , the underlying mechanisms remain to be determined . nonetheless , these data support the concept that retinal microvascular geometric changes might represent a novel marker indicative of early diabetes - related microvascular injury in patients with type 1 diabetes . further studies are warrant to determine if these early retinal microvascular geometric alterations can predict the subsequent development of overt microvascular complications .

objectiveto describe retinal microvascular geometric parameters in young patients with type 1 diabetes.research design and methodspatients with type 1 diabetes ( aged 1220 years ) had clinical assessments and retinal photography following standardized protocol at a tertiary - care hospital in sydney . retinal microvascular geometry , including arteriolar and venular tortuosity , branching angles , optimality deviation , and length - to - diameter ratio ( ldr ) , were measured from digitized photographs . associations of these geometric characteristics with diabetes duration , a1c level , systolic blood pressure ( sbp ) , and other risk factors were assessed.resultsof 1,159 patients enrolled , 944 ( 81.4% ) had gradable photographs and 170 ( 14.7% ) had retinopathy . older age was associated with decreased arteriolar ( p = 0.024 ) and venular ( p = 0.002 ) tortuosity , and female subjects had larger arteriolar branching angle than male subjects ( p = 0.03 ) . after adjusting for age and sex , longer diabetes duration was associated with larger arteriolar branching angle ( p 0.001 ) and increased arteriolar optimality deviation ( p = 0.018 ) , higher a1c was associated with increased arteriolar tortuosity ( > 8.5 vs. 8.5% , p = 0.008 ) , higher sbp was associated with decreased arteriolar ldr ( p = 0.002 ) , and higher total cholesterol levels were associated with increased arteriolar ldr ( p = 0.044 ) and decreased venular optimality deviation ( p = 0.044 ) . these associations remained after controlling for a1c , retinal vessel caliber , and retinopathy status and were seen in subjects without retinopathy.conclusionskey diabetes - related factors affect retinal microvascular geometry in young type 1 diabetes , even in those without evidence of retinopathy . these early retinal alterations may be markers of diabetes microvascular complications .

RESEARCH DESIGN AND METHODS Risk factors assessment Retinal photography and retinal image analysis Statistical analysis RESULTS CONCLUSIONS Supplementary Material

briefly , diagnosis of type 1 diabetes was made according to criteria by the australasian pediatric endocrine group diabetes register and national guidelines . of 1,159 participants who had retinal photographs taken at their baseline visits , we excluded those with ungradable photographs ( n = 215 , 18.6% ) , due to either poor quality of retinal photographs or having less than four big vessels that could be traced by the computer - assisted program , leaving 944 ( 81.4% ) participants included in analyses for this report . key diabetes - related characteristics assessed included pubertal stage , duration of diabetes , and levels of a1c , cholesterol , and blood pressure , which were collected at the baseline visits via interviews , clinical examinations , and laboratory investigations following standardized protocols ( 1 ) . systolic ( sbp ) and diastolic ( dbp ) blood pressure were measured after resting for 5 min , sitting in an upright position using a standard sphygmomanometer with an appropriately sized cuff . a1c and total plasma cholesterol levels were measured following standardized laboratory procedures ( 1 ) . for measuring retinal microvascular geometric properties , right - eye retinal photographs of each patient were digitized and analyses were performed using a semiautomated computer - assisted image program ( singapore i vessel assessment or siva , singapore ) . for each retinal photograph , a trained grader , masked to participants ' identities , applied the program to measure retinal microvascular geometric parameters within a concentric zone between the optic disc margin and two optic disc diameters away from the optic disc margin . the software combined the individual measurement into summary indexes of tortuosity , branching angles , optimality deviation , and ldr for arterioles and venules separately , as described below : vessel tortuosity ( fig . increased tortuosity has been linked with hypertension ( 8) and diabetic retinopathy ( 9 ) , while decreased tortuosity has been associated with ischemic heart disease related death ( 10 ) , ageing , and hypertension ( c. y. cheung , e. lamoureux , l. xu , w. hsu , m. l. lee , q. p. lau , j. j. wang , p. mitchell , t. y. wong).branching angle represents ( in degrees ) the angle between two daughter vessels ( 5 ) and is thought to be related to blood flow efficiency , energy cost of bulk flow , and diffusion distance ( 11 ) . 1 ) , and increased angles have been related to decreased blood flow ( 12 ) , while decreased angles are associated with ageing and hypertension ( 11).optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0 , where d0 , d1 , and d2 are diameters of the parent , larger , and smaller daughter vessels , respectively ( 11 ) . it has been proposed that in an optimal state , the value of junctional exponent is three ( 5 ) , and optimality deviation represents the deviation from this value.ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch ( 6 ) . increased tortuosity has been linked with hypertension ( 8) and diabetic retinopathy ( 9 ) , while decreased tortuosity has been associated with ischemic heart disease related death ( 10 ) , ageing , and hypertension ( c. y. cheung , e. lamoureux , l. xu , w. hsu , m. l. lee , q. p. lau , j. j. wang , p. mitchell , t. y. wong ) . branching angle represents ( in degrees ) the angle between two daughter vessels ( 5 ) and is thought to be related to blood flow efficiency , energy cost of bulk flow , and diffusion distance ( 11 ) . 1 ) , and increased angles have been related to decreased blood flow ( 12 ) , while decreased angles are associated with ageing and hypertension ( 11 ) . optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0 , where d0 , d1 , and d2 are diameters of the parent , larger , and smaller daughter vessels , respectively ( 11 ) . it has been proposed that in an optimal state , the value of junctional exponent is three ( 5 ) , and optimality deviation represents the deviation from this value . measures of retinal vessel tortuosity , branching angle , and optimality deviation . a : tortuosity of 0.00 , branching angle of 72 , and optimality deviation of 0.16 . b : tortuosity of 47.2 10 , branching angle of 130 , and optimality deviation of 0.61 . retinal microvascular geometry ( tortuosity , branching angle , optimality deviation , and ldr ) of the right eye of each patient were used as dependent , continuous variables . we used ancova to compare mean retinal vessel geometric parameters by age - group ( 1214 and 1420 years ) , sex ( female and male ) , pubertal stage ( stage 1 to stage 5 ) , bmi levels ( 18.0 , 18.121.0 , 21.125.0 , and 25.1 kg / m ) , sbp ( 120 vs. 120 mmhg ) , cholesterol ( 3.7 , 3.84.3 , 4.44.8 , and 4.9 mmol / l ) , duration of diabetes ( 5.0 , 5.110.0 , and 10.1 years ) , and a1c ( 8.5 vs. > 8.5% ) ( 1 ) ( see supplementary tables in the online appendix , available at http://care.diabetesjournals.org/cgi/content/full/dc10-0055/dc1 ) . multiple linear regression models were subsequently used to examine the independent determinants of retinal microvascular geometric parameters as continuous variables . we constructed three models : model 1 included age and sex ; model 2 additionally included bmi , sbp , cholesterol , duration of diabetes , a1c ( all assessed as per sd change ) , presence of retinopathy , and retinal arteriolar caliber ( in models for arteriolar geometric parameters ) or venular caliber ( in models for venular geometric parameters ) ; and model 3 included all variables in model 2 after excluding subjects with diabetic retinopathy . key diabetes - related characteristics assessed included pubertal stage , duration of diabetes , and levels of a1c , cholesterol , and blood pressure , which were collected at the baseline visits via interviews , clinical examinations , and laboratory investigations following standardized protocols ( 1 ) . systolic ( sbp ) and diastolic ( dbp ) blood pressure were measured after resting for 5 min , sitting in an upright position using a standard sphygmomanometer with an appropriately sized cuff . a1c and total plasma cholesterol levels were measured following standardized laboratory procedures ( 1 ) . for measuring retinal microvascular geometric properties , right - eye retinal photographs of each patient were digitized and analyses were performed using a semiautomated computer - assisted image program ( singapore i vessel assessment or siva , singapore ) . for each retinal photograph , a trained grader , masked to participants ' identities , applied the program to measure retinal microvascular geometric parameters within a concentric zone between the optic disc margin and two optic disc diameters away from the optic disc margin . the software combined the individual measurement into summary indexes of tortuosity , branching angles , optimality deviation , and ldr for arterioles and venules separately , as described below : vessel tortuosity ( fig . increased tortuosity has been linked with hypertension ( 8) and diabetic retinopathy ( 9 ) , while decreased tortuosity has been associated with ischemic heart disease related death ( 10 ) , ageing , and hypertension ( c. y. cheung , e. lamoureux , l. xu , w. hsu , m. l. lee , q. p. lau , j. j. wang , p. mitchell , t. y. wong).branching angle represents ( in degrees ) the angle between two daughter vessels ( 5 ) and is thought to be related to blood flow efficiency , energy cost of bulk flow , and diffusion distance ( 11 ) . 1 ) , and increased angles have been related to decreased blood flow ( 12 ) , while decreased angles are associated with ageing and hypertension ( 11).optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0 , where d0 , d1 , and d2 are diameters of the parent , larger , and smaller daughter vessels , respectively ( 11 ) . it has been proposed that in an optimal state , the value of junctional exponent is three ( 5 ) , and optimality deviation represents the deviation from this value.ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch ( 6 ) . increased tortuosity has been linked with hypertension ( 8) and diabetic retinopathy ( 9 ) , while decreased tortuosity has been associated with ischemic heart disease related death ( 10 ) , ageing , and hypertension ( c. y. cheung , e. lamoureux , l. xu , w. hsu , m. l. lee , q. p. lau , j. j. wang , p. mitchell , t. y. wong ) . branching angle represents ( in degrees ) the angle between two daughter vessels ( 5 ) and is thought to be related to blood flow efficiency , energy cost of bulk flow , and diffusion distance ( 11 ) . 1 ) , and increased angles have been related to decreased blood flow ( 12 ) , while decreased angles are associated with ageing and hypertension ( 11 ) . it has been proposed that in an optimal state , the value of junctional exponent is three ( 5 ) , and optimality deviation represents the deviation from this value . a : tortuosity of 0.00 , branching angle of 72 , and optimality deviation of 0.16 . b : tortuosity of 47.2 10 , branching angle of 130 , and optimality deviation of 0.61 . retinal microvascular geometry ( tortuosity , branching angle , optimality deviation , and ldr ) of the right eye of each patient were used as dependent , continuous variables . we used ancova to compare mean retinal vessel geometric parameters by age - group ( 1214 and 1420 years ) , sex ( female and male ) , pubertal stage ( stage 1 to stage 5 ) , bmi levels ( 18.0 , 18.121.0 , 21.125.0 , and 25.1 kg / m ) , sbp ( 120 vs. 120 mmhg ) , cholesterol ( 3.7 , 3.84.3 , 4.44.8 , and 4.9 mmol / l ) , duration of diabetes ( 5.0 , 5.110.0 , and 10.1 years ) , and a1c ( 8.5 vs. > 8.5% ) ( 1 ) ( see supplementary tables in the online appendix , available at http://care.diabetesjournals.org/cgi/content/full/dc10-0055/dc1 ) . multiple linear regression models were subsequently used to examine the independent determinants of retinal microvascular geometric parameters as continuous variables . we constructed three models : model 1 included age and sex ; model 2 additionally included bmi , sbp , cholesterol , duration of diabetes , a1c ( all assessed as per sd change ) , presence of retinopathy , and retinal arteriolar caliber ( in models for arteriolar geometric parameters ) or venular caliber ( in models for venular geometric parameters ) ; and model 3 included all variables in model 2 after excluding subjects with diabetic retinopathy . of 944 patients in this study , 170 ( 14.7% ) had retinopathy ( all classified as mild nonproliferative retinopathy ) . the baseline characteristics of participants with and without gradable right - eye retinal photographs are shown in table 1 and retinal microvascular measurements are shown in table 2 . baseline characteristics , according to the gradability of retinal photograph data are age and sex - adjusted means se or % . retinal microvascular parameters in 944 children and adolescents aged 1220 years with type 1 diabetes data are means sd or median ( interquartile range ) . table 3 shows that older age was associated with decreased arteriolar and venular tortuosity , and female patients on average had larger arteriolar branching angle than male patients . after adjusting for age , sex , sbp , cholesterol level , duration of diabetes , a1c level , retinopathy , and vessel caliber , the following associations were evident : 1 ) increasing diabetes duration was associated with increased arteriolar branching angle ( p 0.002 ) and increased optimality deviation of arterioles ( p = 0.014 ) , 2 ) high a1c ( > 8.5 vs. 8.5% , ) was associated with increased arteriolar tortuosity ( p = 0.018 ) , 3 ) increasing sbp was associated with decreasing arteriolar ldr ( p = 0.001 ) and nonsignificantly decreasing venular ldr ( 0.05 < p < 0.10 ) , and 4 ) increasing total cholesterol level was associated with increasing arteriolar ldr ( p = 0.014 ) and decreasing optimality deviation of venules ( p = 0.07 ) . associations of baseline and diabetes - related factors with retinal microvascular geometry * data are mean difference ( 95% ci ) of retinal parameters per sd increase in duration ( 3.3 years ) or 8.5 vs. > 8.5% a1c level and per 1-sd increase in sbp ( 12 mmhg ) , cholesterol ( 0.9 mmol / l ) , and bmi ( 3.5 kg / m ) using linear regression models . p , p value of coefficient adjusted for age and sex ; p , p value of coefficient adjusted for age , sex , sbp , cholesterol , duration , a1c , retinopathy , and retinal vessel caliber ; p , p value of coefficient , adjusted for age , sex , sbp , cholesterol , duration , a1c , and retinal vessel caliber , excluding subjects with retinopathy . there were no significant interactions between age , sex , blood pressure , bmi , cholesterol , duration of diabetes , and a1c level . analyses after excluding subjects with relatively poor image quality ( n = 129 , 11.1% ) showed that these associations remained significant in 815 ( 70.3% ) patients with good - quality retinal images ( data not shown ) . in young type 1 diabetes , we showed that variations in retinal microvascular geometric characteristics were associated with key diabetes - related risk factors , including longer diabetes duration and higher a1c , blood pressure , and cholesterol levels . the principal - specific findings are the following : longer duration of diabetes was associated with larger arteriolar branching angles and increasing deviation of optimality , higher a1c was associated with more tortuous arterioles , increasing sbp level was associated with smaller arteriolar and venular ldr , and increasing cholesterol level was associated with increasing arteriolar ldr and decreasing venular optimality deviation . importantly , we showed that these associations were present even in subjects without mild retinopathy signs , suggesting that early retinal microvascular alternations are present well before the onset of clinical microvascular complications in young patients with type 1 diabetes . most previous studies that have examined retinal microvascular geometric parameters have been conducted in nondiabetic populations , including healthy subjects ( 13 ) , subjects with hypertension ( 11 ) , or those with coronary heart disease ( 10 ) . to the best of our knowledge , there has been no study reporting retinal microvascular geometric characteristics in young type 1 diabetes . our findings that longer duration of diabetes was associated with larger arteriolar branching angle and increasing deviation from optimality of arterioles is biologically plausible and may provide clues to early microvascular alterations in type 1 diabetes . branching angle and junctional exponent have been found to be impaired in atherosclerosis ( 14 ) , altered blood flow ( 12 ) , and endothelial dysfunction ( 15 ) . thus , our finding of associations of longer diabetes duration with larger branching angle and higher optimality deviation may reflect alterations in blood flow ( 17 ) , endothelial dysfunction ( 18 ) , and attenuation in oxygen saturation ( 16 ) . the sex difference in arteriolar branching angle , with female subjects having larger arteriolar branching angles than male subjects , could also explain our earlier observations that female patients with type 1 diabetes have greater risk for diabetic microvascular complications compared with male patients of the same age ( 1,3 ) . diabetes is known to be associated with increased shear stress and impaired microvascular endothelium ( 2 ) . previously , increased a1c level had been shown to have a role in reduced endothelial function in people with type 1 diabetes ( 19 ) . we did not find a linear association between a1c levels and arteriolar tortuosity , but we observed a significant difference in tortuosity between patients with a1c 8.5 vs. 8.5% . there have been studies showing that different cutoffs of a1c levels identify people with diabetes at high risk of microvascular injury ( 21,22 ) , although there is no universal consensus on these cutoffs ( 22 ) . we have previously reported that wider arteriolar caliber is associated with an increased risk of diabetic retinopathy in both type 1 and type 2 diabetes ( 3,23 ) . thus , the finding of an association of higher blood pressure levels with increasing arteriolar ldr ( reflecting wider arteriolar caliber ) is consistent with the influence of diabetes and blood pressure on autoregulatory processes in small blood vessels . there have been few studies on cholesterol levels on microvascular structure , with most previous studies reporting associations between cholesterol level and retinal microvascular changes in nondiabetic populations showing inconsistent results ( 24,25 ) . therefore , our findings that higher total cholesterol levels was associated with changes in arteriolar ldr and venular optimality deviation suggests that lipids may also have an influence on microvasculature in young type 1 diabetes . first , our study includes a large cohort of young patients with type 1 diabetes , with a participation rate of > 80% . the cross - sectional nature of our study implies that longitudinal studies are needed to determine the temporal sequence of these associations . however , baseline characteristics and diabetes - related risk factors were largely similar in participants with and without gradable images . in summary , our study demonstrated that in young type 1 diabetes , subtle alterations in retinal microvascular geometric parameters are associated with key diabetes - related characteristics , including duration of diabetes and a1c and blood pressure levels . these findings were present in those without any signs of retinopathy , suggesting an effect of these risk factors on the microcirculation prior to development of clinical complications . nonetheless , these data support the concept that retinal microvascular geometric changes might represent a novel marker indicative of early diabetes - related microvascular injury in patients with type 1 diabetes . further studies are warrant to determine if these early retinal microvascular geometric alterations can predict the subsequent development of overt microvascular complications .

conformational changes in a molecule are implicated in many biological processes . for example , during transcription rna polymerase recognizes and binds to a promotor region on double - stranded dna ( dsdna ) and then unwinds it , dissociating 17 bp in the process ( 1 ) . this transition from dsdna to single - stranded dna ( ssdna ) , called the helix - coil transition , is vital to biology . single molecule force spectroscopy ( smfs ) has been used to analyze the process , revealing a dichotomy in the forces required to induce the transition . the force required to dissociate base pairs is different depending on whether the dna is unzipped by pulling parallel to the bases , or stretched by pulling transverse to the base - pairs . depending on the sequence , about funzip = 1030 pn of force is required to unzip dsdna ( 28 ) . on the other hand , when a single molecule of dsdna is stretched beyond its contour length , it undergoes a cooperative transition near fstr = 6070 pn in which the dna doubles ( 1.7 ) in length ( 912 ) . this overstretching transition has been interpreted as force - induced melting in which the two dna strands break apart and unwind ( 9 ) . synthetic nanopores can be used to accomplish both types of measurements because large forces can be applied using the electric field in the pore , and because the diameter of the pore can be controlled with sub - nanometer precision . smfs is usually accomplished using the tip of an afm cantilever or an optical tweezers , and generally involves a molecule under test that is tethered at one end to a force probe and anchored at the other to a surface or another molecule so that the load is variable ( 1 ) . but throughput ( measurements / second ) is abysmal , which usually forestalls a direct comparison with ensemble - averaged measurements . instead of these tools , we use a nanopore in a silicon nitride membrane . both synthetic nanopores like this and proteinaceous pores in a lipid bilayer have been used before for force spectroscopy ( 10,1318 ) . this strategy does not require a molecular tether or anchor , and the loading rate or the force can be held constant and range up to 1 nn / ns , while maintaining high throughput ( 1000 molecules / s ) . what is more important , a synthetic nanopore affords us the opportunity to constrain the transverse motion of the molecule on a sub - nanometer scale through control of the diameter ( 19 ) . we have previously explored the electromechanical properties of dna using the electric field to force single molecules to translocate across a silicon nitride membrane through a pore ( 10,13 ) . when a voltage is applied in a bi - cell across a membrane with a pore in it , polyanionic dna immersed in electrolyte at the cathode diffuses toward the anode and the force due to the field acting on the strand impels dna to bend and stretch within the pore . we have shown that for low electric fields e < 0.2 mv / cm = 200 mv/10 nm , ssdna can permeate pores with diameters 1.0 nm , while dsdna only permeates pores with diameters 2.5 nm . for pores < 2.5 nm in diameter , there is a threshold for permeation of dsdna that depends on the electric field and ph . molecular dynamics indicates that the field threshold originates from a stretching transition in dna that occurs under the force gradient in a nanopore . in this report , using hairpin dna ( hpdna ) instead of dsdna , we observe a threshold voltage for translocation of the molecule through the synthetic pore that depends sensitively on the pore diameter and the secondary structure of the dna . nucleic acid hairpins or stem - loops , formed from self - complementary sequences , are found regularly in dna and rna secondary structure ( 1 ) . the structure of a hairpin is not static , however ; there is a folded ( closed ) conformation and unfolded ( open or melted ) state ( 2023 ) . the folded ( closed ) conformation is characterized by a low enthalpy due to base pairing in the stem , while the open state has high entropy due to the large number of configurations available to ssdna . we find that the threshold voltage to induce a translocation of hpdna through a pore with a diameter 1.5 < d < 2.3 nm is v > 1.5 v , corresponding to the force required to stretch the stem of the hairpin , according to molecular dynamic simulations . on the other hand , for pores with diameters 1.0 < d < 1.5 nm , the threshold collapses to v < 0.5 v because the stem unzips with a lower force . these data indicate that a synthetic nanopore can be used like a molecular gate to discriminate between the secondary structures in dna . this effort demands synthetic pores ranging from 1 to 2 nm , comparable in diameter with hpdna the smallest synthetics ever made . the experimental procedures used to fabricate and characterize the synthetic nanopores are described in detail elsewhere ( 19 ) . we started by measuring the thickness of the si3n4 membranes using electron energy loss spectrum ( eels ) and tilted scanning transmission electron microscopy ( stem ) . membranes that were nominally 10 nm showed a range of thickness from 9 to 15 nm . a single nanopore is created in a membrane by stimulated decomposition and sputtering using a tightly focused electron beam in a jeol 2010f stem operating at 200 kev . figure 1a shows transmission electron micrographs ( tem ) of three pores : 0.8 1.0 0.2 nm ( red ) ; 1.2 1.4 0.2 nm ( blue ) ; and 2.1 2.2 0.2 nm ( black ) diameter the shot noise observed in the area identified as the pore is indicative of perfect transmission of the electron beam through the membrane . although it is not unique , a simple model for the 3d structure of the pore consists of two intersecting cones each with a cone angle that ranges from 10 to 20 ( 19 ) . figure 1.characterization of synthetic nanopores . ( a ) tem micrographs taken at a tilt angle of 0 of three nanopores : 0.8 1.0 nm ( red ) , 1.2 1.4 nm ( blue ) and 2.1 2.2 nm ( black ) diameter , sputtered with a tightly focused high - energy electron beam in nominally 10 nm thick si3n4 membranes . the shot noise observed in the area identified as the pore is indicative of perfect transmission of the electron beam through the membrane . ( b ) i v characteristics of the nanopores shown in ( a ) taken in 1 m kcl solution . line fits yield conductances of 1.13 0.03 ns , 1.98 0.03 ns and 3.20 0.03 ns for the three pores , respectively . ( c ) the electrolytic current through the 1.2 1.4 nm pore shown in figure as a function of time with the membrane voltage at v = 0.5 v. the open pore current at this voltage is 0.9 na ; the transients in the current are associated with 150 hpdna interacting with the pore . long duration transients > 10 s and transient currents greater than the open pore current > i0 are observed . the variations in current associated with the transient are above the noise level associated the measurement system ( red ) . ( a ) tem micrographs taken at a tilt angle of 0 of three nanopores : 0.8 1.0 nm ( red ) , 1.2 1.4 nm ( blue ) and 2.1 2.2 nm ( black ) diameter , sputtered with a tightly focused high - energy electron beam in nominally 10 nm thick si3n4 membranes . the shot noise observed in the area identified as the pore is indicative of perfect transmission of the electron beam through the membrane . ( b ) i v characteristics of the nanopores shown in ( a ) taken in 1 m kcl solution . line fits yield conductances of 1.13 0.03 ns , 1.98 0.03 ns and 3.20 0.03 ns for the three pores , respectively . ( c ) the electrolytic current through the 1.2 1.4 nm pore shown in figure as a function of time with the membrane voltage at v = 0.5 v. the open pore current at this voltage is 0.9 na ; the transients in the current are associated with 150 hpdna interacting with the pore . long duration transients > 10 s and transient currents greater than the open pore current > i0 are observed . the variations in current associated with the transient are above the noise level associated the measurement system ( red ) . to further characterize the pore , we measured the electrolytic current as a function of the electrochemical potential applied across the membrane . membranes including these pores were mounted in a membrane transport bi - cell made from acrylic using silicone o - rings coated with pdms to seal the membrane to the acrylic ( with > 5.0 0.2 t resistance ) . the membrane separates two reservoirs in the bi - cell : a 40 l volume on the cis - side ; and a 15 ml volume on the trans - side . each reservoir contains microfiltered and buffered ( 10 mm tris , ph 8.0 ) 1 m kcl and a ag / agcl electrode that is connected to an axopatch 200b amplifier used in resistive feedback mode . this electrolyte concentration was chosen primarily to minimize the secondary structure in the hairpin ; increase the closing rate for the loop ( 20 ) ; and economize on the computer time required for simulation of the results . labview software is used to measure the electrolytic current at 23.5 1c and to apply voltages in range of 5 v. with a voltage applied across the membrane , we observed an electrolytic current through the pore . as illustrated by figure 1b , the i v characteristics were approximately linear line fits to the data indicate conductances : 1.13 0.03 ns , 1.98 0.03 ns and 3.20 0.03 ns , for the three pores shown in figure 1a , respectively . but it is apparent that the conductances do not scale linearly with the cross - section of the pore inferred from tem . we have analyzed similar measurements using molecular dynamics to estimate the ion mobility , and then solved coupled poisson nernst planck and the stokes equations self - consistently for the ion concentration , velocity and electrical potential . we find that the measurements are consistent with the presence of a fixed negative charge in the pore and a reduction of the ion mobility due to the fixed charge and the ion proximity to the pore wall ( 19 ) . v characteristic is not always strictly linear either as evident from the deviations from the lines fit through the data at low voltage . nonlinearities and rectifying behavior have been attributed by siwy ( 24,25 ) to asymmetry in the pore geometry with an excess of fixed surface charge in the lumen . after characterizing electrolytic transport through the pore , a solution containing hpdna was injected at the ( negative ) ag / agcl cathode and the current through the pore was measured . we used polyacrylamide gel electrophoresis ( page ) purified hairpins ( idt , ames , ia , usa ) at a concentration of 0.1 g/l buffered in 10 mm tris , 1 m kcl , ph 8.0 solution . to form the hairpin , the solution was first heated to 75c for 10 min and then quenched to 4c . the hairpins used in these experiments were designed to satisfy specifications on stability and persistence length in the loop , while avoiding undesirable secondary structures . bulk measurements such as calorimetry and melting assays reveal some of the rules governing the stability of hairpins ( 12,14 ) . among other things , the stability is affected by : the base composition of the paired region ; the length and constituency of the loop ; and the number of mismatches or bubbles the stem - loop contains . c pairings are more stable than a t because of an extra hydrogen bond . loops smaller than 3 bases are sterically forbidden , while larger loops with no secondary structure are also unstable the optimal loop tends to be 410 bp long . a hairpin loop consisting of adenosine repeats leads to lower rates and higher activation energies associated with closing ( 23 ) , which is undesirable for these experiments . poly(a ) adds +0.5 kcalmolbase due to the enthalpy due to base stacking , while poly(t ) is purely entropic ( 23 , 26 ) . on the other hand , we are motivated to use a poly(a ) overhang to control the secondary structure . it is well known that poly(a ) exists in single or double helical form in aqueous solution depending on ph ( 27 ) , and the single helical structure prevails for neutral or alkaline ph used in these experiments . all of these considerations along with the requirements for qpcr , prompted us to investigate two types of hpdna : the first 150-mer hairpin has a single stranded overhang ( 50-mer poly - da ) of dna , and a 12 bp long stem containing an intervening 76-base loop with the following sequence ( the self - complementary parts are indicated in bold italic ) : 5-gctctgttgc ttgggcgcgt tatttatcgg agttgcagtt gcgcccgcga acgacattta taatgaacgt gaattgctca acagtatgaa gcaacagagc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa -3. this hairpin design yields a stable hairpin ( g = 19.3 kcal / mol = 32 kbt ) with a flexible loop with some secondary structure [ http://frontend.bioinfo.rpi.edu/applications/mfold/ , ( 28,29 ) ] . we expect the dna in the loop to show a very short persistence length ( < 2 nm ) making it flexible enough to penetrate to the high electric field deep in the lumen of the pore.the second 110-mer hairpin has a single - stranded overhang consisting primarily of a 50-mer poly da strand of dna , and a stem 10 bp long containing an intervening 6 base loop with the following sequence ( the self - complementary parts are indicated in bold italic ) : 5-gctctgttgc tctctcgcaa cagagcatga acg tga aaag gtctacagta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa aaaaaa aaaaaaaaa gaa tcg cag tg-3. this hairpin design yields a loop - stem that is less stable ( g = 11.6 kcal / mol = 19 kbt ) than its 150-mer counterpart with a 6 bp loop that frustrates the formation of secondary structures [ http://frontend.bioinfo.rpi.edu/applications/mfold/ , ( 28,29 ) ] . the first 150-mer hairpin has a single stranded overhang ( 50-mer poly - da ) of dna , and a 12 bp long stem containing an intervening 76-base loop with the following sequence ( the self - complementary parts are indicated in bold italic ) : 5-gctctgttgc ttgggcgcgt tatttatcgg agttgcagtt gcgcccgcga acgacattta taatgaacgt gaattgctca acagtatgaa gcaacagagc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa -3. this hairpin design yields a stable hairpin ( g = 19.3 kcal / mol = 32 kbt ) with a flexible loop with some secondary structure [ http://frontend.bioinfo.rpi.edu/applications/mfold/ , ( 28,29 ) ] . we expect the dna in the loop to show a very short persistence length ( < 2 nm ) making it flexible enough to penetrate to the high electric field deep in the lumen of the pore . the second 110-mer hairpin has a single - stranded overhang consisting primarily of a 50-mer poly da strand of dna , and a stem 10 bp long containing an intervening 6 base loop with the following sequence ( the self - complementary parts are indicated in bold italic ) : 5-gctctgttgc tctctcgcaa cagagcatga acg tga aaag gtctacagta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa aaaaaa aaaaaaaaa gaa tcg cag tg-3. this hairpin design yields a loop - stem that is less stable ( g = 11.6 kcal / mol = 19 kbt ) than its 150-mer counterpart with a 6 bp loop that frustrates the formation of secondary structures [ http://frontend.bioinfo.rpi.edu/applications/mfold/ , ( 28,29 ) ] . associated with these hairpins interacting with a pore , we often observed transients superimposed on the open pore electrolytic current . figure 1c shows a transient associated with the first 150-mer hpdna interacting with the 1.2 1.4 nm pore shown in figure 1a . we do not observe transients in the open pore current without dna at the negative electrode ( as indicated by the red trace in the same figure taken at 0.25 v without any hpdna at the negative electrode ) ( 30 ) . it is apparent that the observed current transients as well as features observed during the event are easily resolved from the electronic noise ( red trace ) , but the narrow bandwidth ( 10100 khz ) of the current amplifier coupled with the capacitance of the membrane we used for these measurements precludes the observation of transients shorter than 10100 s . because of the limited bandwidth , to determine if dna injected at the cathode permeates the membrane through the pore , we analyzed the extract taken from the anode using pcr analyzed by gel electrophoresis along with quantitative pcr ( qpcr ) . the dna was concentrated with an amicon ultra-4 centrifugal filter ( millipore , bedford , ma , usa ) , and the buffer was exchanged to water on the same filter . the pcr reagent system was obtained from invitrogen ( carlsbad , ca , usa ) , the sequence specific primers were synthesized by idt ( ames , ia , usa ) . after pcr , the amplified dna was analyzed by gel electrophoresis run on a 2.0% agarose gel at room temperature . two pcr primers were designed to amplify a 66 bp region within a 150 base target sequence . a taqman probe was designed to map to an 18-base segment within this 66 bp target sequence and labeled with an fam reporter dye at the 5-end and with a black hole quencher dye at the 3-end . to complement the experimental work , we also simulated the measurements using two sets of molecular dynamics simulations . in the first set , a uniform electric field is applied to a pore in a 20.0 nm - thick si3n4 membrane containing a hpdna molecule to study the dependence of the electrolytic current on the molecule 's conformation . in the second set of simulations , the effect of the pore geometry on the pathway of the helix - coil transition is probed by pulling the hpdna through four different pores in 2.3 nm - thick si3n4 membranes . for the first set of simulations , a microscopic model of a si3n4 pore was built by replicating a unit cell of the -si3n4 crystal ( 31 ) to form a prism with a hexagonal cross - section of 50.1 nm in the xy - plane and thickness of 20.0 nm along the z - axis . we have shown previously ( 10 ) that the electric fields within pores of different length fall along the same curve when the position is expressed in units of the pore length . so , we chose a pore length of 20.0 nm to serve as a model system for nanopores in the 1030 nm - length range . by removing silicon and nitrogen atoms , a double - cone pore was formed in the membrane with a diameter given by d(z ) = d0 + 2ztan( ) , where z = 0 is the center of the prism , d0 = 2.0 nm is the constriction diameter , and = 10 is the angle that the cones make with the z - axis . a hpdna model with a 50-base poly(da ) coil , a 10 bp stem , and a 6-base loop was generated with the following sequence ( the self - complementary parts are indicated in bold italic ) : 5-a50cgagacaacgctctctcgttgtctcg-3. the si3n4 structure was merged with the hpdna model in four conformations ( figure 3 ) by rigid - body transformations and mapping of the molecule 's coil to a smooth spline curve . the resulting five structures , four containing hpdna and one only an empty pore , were then solvated in a volume of pre - equilibrated tip3p water molecules . potassium and chloride ions were added , corresponding to a concentration of 1.0 m kcl . for the second set of simulations four si3n4 membranes were generated in the same way as for the electrolytic current simulations except that the thickness of the hexagonal prism was 2.3 nm . this thickness was chosen because it allowed for the greatest efficiency of the computation , while maintaining the geometry of the pore near the constriction . pores were formed in each in the shape of a conic frustum with the smallest diameter at z = 1.15 nm and the largest at z = + 1.15 nm . the diameters of the four pores respectively varied from 1.0 to 1.8 nm , 1.3 to 2.1 nm , 1.4 to 2.2 nm and 1.6 to 2.4 nm . the hpdna model had the same sequence as above except that the coil had been trimmed to just four adenine bases . the model was placed within each pore , with the helix above the si3n4 membrane and the coil penetrating the pore ( figure 6 ) . a 1.0 m kcl solution was added to each system as before , yielding about 50 000 atoms . all systems constructed underwent 2000 steps of energy minimization followed by gradual heating from 0 to 295k in 2 ps . each system was equilibrated in the npt ensemble ( i.e. with particle number n , pressure p and temperature t fixed ) for 0.5 ns . our md simulations were performed using the program namd2 ( 32 ) , periodic boundary conditions , particle mesh ewald full electrostatics and multiple time stepping ( 33 ) , the amber ( 34 ) force field describing dna , water and ions , and a custom force field ( 10 ) describing the si3n4 membrane . the integration time step chosen was 1 fs . the equilibration in the npt ensemble was performed using the nos hoover langevin piston pressure control ( 35 ) to obtain a pressure of 1.00 atm and langevin dynamics to maintain a temperature of 295k . for the first set of simulations those to determine the electrolytic current a uniform external electric field was applied to produce a 4 v voltage drop along the z - axis of the systems . the simulations were performed at fixed volume and with langevin dynamics applied only to the si3n4 membrane to maintain a temperature of 295k despite heating due to current flow . each of the five systems was simulated for more than 15 ns to obtain a steady current . the second set of simulations used steered molecular dynamics ( 36 ) to pull the hpdna through the pore . the phosphate on the 5 end of the hpdna was attached by a harmonic spring to a dummy atom that moved in the negative z - direction at a rate of 1.0 nm / ns . the simulations were performed in the nvt ensemble with the temperature and volume fixed in the same way as for the first set of simulations . we measured the permeability of hpdna through nanometer diameter pores in nominally 10 nm thick si3n4 membranes as a function of the applied voltage . figure 2 delineates the variety of current transients observed when the 150-mer hpdna interacts with the 1.2 1.4 0.2 nm pore ( blue ) shown in figure 1a over the range of bias voltages used in these experiments ( which corresponds to voltages above and below threshold ) . the long duration of the current transients is extraordinary , even though they represent only a fraction of those observed during a typical measurement . we previously reported that field - driven translocations of the ssdna cause temporary blockades of the open current i0 through the pore lasting for only few milliseconds ( 30 ) . for example , the translocation velocity of unbound dna through a large diameter > 5 nm synthetic pore is estimated to be > 1 bp / ns at these voltages ( 31,37,38 ) implying that the duration of a translocation is < 0.1 s for 100 bp dna . in contrast , we observe transients with a duration > 10 s associated with the hpdna , which is unprecedented for a synthetic pore . ( a ) the electrolytic current through the 1.2 1.4 nm pore shown in figure 1a as a function of time with the membrane voltage at v = 0.5 v. the open pore current at this voltage is 0.9 na ( red ) ; the transients in the current are associated with 150-mer hpdna interacting with the pore ( blue ) . long - duration transients > 10 s and transient currents greater than the open pore current > i0 are observed . ( b ) , ( c ) and ( d ) show representative currents trace taken from the same pore at observed v = 1.0 v , 1.5 v and 2.0 v , respectively . the same features , which we associate with the secondary structure in the hairpin , are observed above and below threshold . ( a ) the electrolytic current through the 1.2 1.4 nm pore shown in figure 1a as a function of time with the membrane voltage at v = 0.5 v. the open pore current at this voltage is 0.9 na ( red ) ; the transients in the current are associated with 150-mer hpdna interacting with the pore ( blue ) . long - duration transients > 10 s and transient currents greater than the open pore current > i0 are observed . ( b ) , ( c ) and ( d ) show representative currents trace taken from the same pore at observed v = 1.0 v , 1.5 v and 2.0 v , respectively . the same features , which we associate with the secondary structure in the hairpin , are observed above and below threshold . for an interval this long ( > 10 s ) , it is possible that more than one hairpin interacts with the pore at the same time at low voltage . the capture rate of a perfectly absorbing hemisphere of radius r with a diffusive flux impinging on it is given by : 1/ = 2c0dr , where c0 10 cm ( 300 nm ) is the hairpin concentration and d10 cm / s is the diffusion coefficient of the hairpin in electrolyte . the capture radius is proportional to the distance at which the electrophoretic drift velocity is comparable to the average diffusion velocity : i.e. r cod e/4d , where is the mobility and e is the maximum electric field in the pore ( 39 ) . < 100 ms for voltages between 100 mv and 1 v , which is consistent with the ( upper bound ) estimate of the inter - arrival time extracted from observations of the current transients 1.8 s at 500 mv . therefore , the duration of these events may represent an extended residence time of one or more hairpins over the pore that terminates either through translocation or with the hairpin(s ) uneventfully exiting the pore due to thermal agitation . figure 2 also shows transients with a current greater than the open pore current , i0 . the red trace shows the typical open pore current , i0 = 0.92 na , at v = 0.5 v. apparently , the current can exceed i0 ( represented by the dotted line in figure 2a d ) beyond the noise in the measurement for an extended duration . currents > i0 have been observed before ( 16,30,31,40 ) and attributed to a local enrichment of electrolyte near the pore due to counter - ions responding to the molecular charge , but not at the 1 m kcl concentration used in these experiments . with pores this small , previous experience ( 30,31 ) indicates that a current transient can not be unequivocally interpreted simply as the translocation of a hairpin across the membrane as it is in -hemolysin , for example . instead , we hypothesize that the transients are caused by hairpin molecules modulating the current through the pore , and that the modulation depends on the molecular configuration relative to the pore . in support of this hypothesis , molecular dynamics reveals that the pore current is a function of the position and configuration of a hairpin . the results of md simulations shown in figure 3 represent the dependence of the pore current on the configuration of the hairpin over a pore with a 1.9 2.1 nm cross - section in a 20 nm thick nitride membrane with an applied transmembrane bias of 4 v. each steady - state current value shown was obtained by averaging the current in the corresponding md trajectory ( > 12 ns ) from t = 7 ns to the end of the simulation , except for the system shown in figure 3c where the averaging began at 6 ns . notice that the open pore current shown in figure 3a is i0 = 5.81 0.08 na , but with the hairpin in the vicinity of the pore , the electrolytic current changes dramatically depending on the relative position . for example , in figure 3b the hairpin blockades the pore resulting in a minimal current of 2.88 0.09 na , a blockade of about i / i0 = 50% . also notice that in figure 3d , the hairpin blocks the entrance to the pore , yet the pore current assumes a value close to the open pore current . and finally , in figure 3e , we see that the current through the pore can exceed the open pore value ( by i / i0 = + 13% ) , indicating that a positive current transient is possible even when a hairpin assumes this position near the entrance to the pore on the cis - side of the membrane . figure 3.molecular dynamics simulations showing the dependence of the pore current on the configuration of the hairpin over the pore . note that the thickness of the membrane is 20 nm and the pore diameter is 2 nm , both larger than the corresponding values used in the experiments shown in figure 2 . ( a ) the open pore current is i0 = 5.8 1 0.08 na corresponding to an applied voltage of 4 v. ( b e ) show the variation in the pore current , i , with molecular configuration . notice in ( b ) that a blockade of the pore results in a minimal current of 2.88 0.09 na through the pore . also notice that in ( d ) the hairpin blocks the entrance to the pore , yet the pore current still assumes a value close to the open pore current . finally , in ( e ) see that the current through the pore exceeds the open pore current value . molecular dynamics simulations showing the dependence of the pore current on the configuration of the hairpin over the pore . note that the thickness of the membrane is 20 nm and the pore diameter is 2 nm , both larger than the corresponding values used in the experiments shown in figure 2 . ( a ) the open pore current is i0 = 5.8 1 0.08 na corresponding to an applied voltage of 4 v. ( b e ) show the variation in the pore current , i , with molecular configuration . notice in ( b ) that a blockade of the pore results in a minimal current of 2.88 0.09 na through the pore . also notice that in ( d ) the hairpin blocks the entrance to the pore , yet the pore current still assumes a value close to the open pore current . finally , in ( e ) see that the current through the pore exceeds the open pore current value . neither the enhancement of the current over i0 represented in figure 3e or the blockade current shown in figure 3b match the corresponding experimental values indicated in figure 2 , but they are only supposed to be representative of a 2 nm pore in a 20 nm membrane . generally , our simulations indicate that the ion concentration in the region on the cis side of the pore within 6 nm of the constriction is less than in the bulk , dropping below 0.2 m for k and 0.5 m for cl for a 1 m kcl solution , which is consistent with the results of folgea et al . ( 41 ) dilution of the electrolyte concentration in the vicinity of the pore makes current enhancement more likely by increasing the disparity with the molecular charge . an enhancement of 100% suggests that ion concentration may be depleted more than indicated in the simulation or that more than one dna molecule is in the vicinity of the pore . and since we measure a smaller pore diameter than we actually simulate , we expect a larger percent blockade current in the experimental data . due to the dependence on the molecular configuration relative to the pore and the limited bandwidth of the measurement , a current transient is not an unambiguous signature of a translocation across the membrane through the pore . so , to establish unequivocally if hpdna injected at the cathode permeates the membrane through the pore , the dna near the anode was analyzed using qpcr along with pcr analyzed by gel electrophoresis . along with each gel array , we also ran control experiments containing no dna , a molecular weight ( mw ) reference denoted as 100 bp ladder , which contains a spread of dna mw consisting of 15 blunt - ended fragments between 100 and 1500 bp in multiples of 100 bp is also included , and various dilutions of dna ranging from 10 to 100 000 molecules per batch to test the copy number . we find that a synthetic nanopore acts like a molecular gate that discriminates between dna according to the secondary structure . the gel arrays shown in figure 4b d illustrate the permeation of dsdna , and the 150-mer and 110-mer hpdna through the 2 nm ( 2.0 nm 1.9 nm ) diameter pore pictured in figure 4a . each lane in the gel array is denoted by the corresponding voltage applied across the membrane . in particular , the fluorescent bands in figure 4b indicate that 110-mer hpdna is collected at the positive electrode only for v > 2.50 v ; below this threshold voltage the translocation of 110-mer hpdna across the membrane can not be detected . on the other hand , the 150-mer hpdna , which is nominally more stable , permeates the same pore for v > 2.00 v. in contrast , the stiffer 105 bp dsdna permeates the same pore only for v > 3.0 v , in correspondence with previously reported measurements obtained on a similar pore ( 10,13 ) . that earlier work attributed this threshold to the stretching transition in dsdna ( 11,12 ) . so , apparently the threshold voltage is not a measure of molecular stability alone . rather , the more flexibility or disorder in the loop ( a ) a transmission electron micrograph of a 2.0 1.9 nm pore in a nominally 10 nm thick nitride membrane . ( b ) a gel array indicating that 110-mer hpdna ( 10 bp stem with a 6 bp loop ) permeates the pore only for voltages v > 2.5 v. ( c ) a gel array indicating that 150-mer hpdna ( 12 bp stem with a 76 bp loop ) permeates the same pore only for voltages v > 2.0 v. and ( d ) a gel array indicating that 105 bp dsdna permeates the pore only for voltages v > 3.0 v. a synthetic nanopore is like a molecular gate . ( a ) a transmission electron micrograph of a 2.0 1.9 nm pore in a nominally 10 nm thick nitride membrane . ( b ) a gel array indicating that 110-mer hpdna ( 10 bp stem with a 6 bp loop ) permeates the pore only for voltages v > 2.5 v. ( c ) a gel array indicating that 150-mer hpdna ( 12 bp stem with a 76 bp loop ) permeates the same pore only for voltages v > 2.0 v. and ( d ) a gel array indicating that 105 bp dsdna permeates the pore only for voltages v > 3.0 v. we systematically investigated hpdna permeability through a membrane as a function of the pore diameter , using diameters small enough to forestall the translocation of dsdna , while at the same time allowing ssdna to easily permeate the membrane through the pore ( i.e. d < 2.4 ) ( 10,13 ) . the gel arrays shown in figure 5a unambiguously illustrate the permeation of the 150-mer hpdna through the three pores shown in figure 1a . figure 5a shows 150-mer hpdna that is collected at the positive electrode ; each lane in the gel array is denoted by the corresponding voltage applied across the membrane . we observe a threshold voltage for the translocation of 150-mer hpdna across the membrane through the pore that depends on the pore diameter . in particular , the fluorescent bands indicate that hpdna translocates only for v 0.5 v for a 1 nm diameter pore , while for v < 0.5 v only the unreacted primers are found in solution . according to the summary shown in figure 5c , for the 1.4 and 2.2 nm pores , the threshold is about v 1.5 v. the largest threshold value , v = 2.25 v , is found for pores with one axis ranging from 1.5 nm < d < 2 nm . apparently , hpdna can be forced through pores with a diameter > 1.5 nm only if the voltage is v > 1.5 v. figure 5.threshold voltage for hairpin translocation depends on the pore diameter . ( a ) gel arrays containing eight horizontal lanes with bands indicating 150-mer hpdna found as a function of the bias voltage , v , in a bi - cell corresponding to the three pores shown in figure 1a . the 150 hpdna permeates the 2.2 nm pore only for v > 1.60 v ; hpdna permeates the 1.4 nm pore only for v > 1.50 v ; and hpdna permeates the 1 nm pore only for v > 0.5 v. for reference , a 100 bp ladder is shown in the top lane of each gel . the ( + ) lane shown for the 2.2 nm pore is a positive control showing the dna at the negative electrode in the bi - cell . notice that there are two bands : one corresponding to unamplified dna and another showing an amplified portion of the original hairpin . ( b ) qpcr results indicate the number of 150-mer hpdna ( hp150 ) copies that permeate the membrane through the pores in ( a ) and are subsequently found at the positive ( + ) electrode in a bi - cell as a function of the voltage across the membrane . ( c ) a summary of the dependence of the threshold for permeation of 150 hp on the easy axis of the pore . the threshold for d 1.6 nm is supposed to be associated with the stem of the hairpin stretching in the pore to facilitate the translocation , while the collapse of the threshold when d < 1.5 nm is supposed to be due to unzipping of the hairpin . ( a ) gel arrays containing eight horizontal lanes with bands indicating 150-mer hpdna found as a function of the bias voltage , v , in a bi - cell corresponding to the three pores shown in figure 1a . the 150 hpdna permeates the 2.2 nm pore only for v > 1.60 v ; hpdna permeates the 1.4 nm pore only for v > 1.50 v ; and hpdna permeates the 1 nm pore only for v > 0.5 v. for reference , a 100 bp ladder is shown in the top lane of each gel . the ( + ) lane shown for the 2.2 nm pore is a positive control showing the dna at the negative electrode in the bi - cell . notice that there are two bands : one corresponding to unamplified dna and another showing an amplified portion of the original hairpin . ( b ) qpcr results indicate the number of 150-mer hpdna ( hp150 ) copies that permeate the membrane through the pores in ( a ) and are subsequently found at the positive ( + ) electrode in a bi - cell as a function of the voltage across the membrane . ( c ) a summary of the dependence of the threshold for permeation of 150 hp on the easy axis of the pore . the threshold for d 1.6 nm is supposed to be associated with the stem of the hairpin stretching in the pore to facilitate the translocation , while the collapse of the threshold when d < 1.5 nm is supposed to be due to unzipping of the hairpin . a crude estimate for the number of dna copies that permeated the pore was determined through a comparison of the measured fluorescent intensity with controlled dilutions ( data not shown ) . accordingly , in figure 5a we estimate that about < 10 copies permeated the pore for v < 2.25 v. the number of dna copies that permeated the pore was also determined by qpcr . in correspondence with the thresholds obtained from the gels , the qpcr data shown in figure 5b exhibits a consistently lower value . for example , the threshold voltage for the 1.4 nm pore is v < 2 v , while the gel shows v = 2.25 v. the relatively lower threshold voltage can be attributed to the higher sensitivity of qpcr . figure 5c summarizes the dependence of the threshold voltage inferred from electrophoresis on the pore diameter . we assume that the threshold voltage corresponds to the minimum force required to impel a hairpin through the pore . assuming that the stem frustrates the permeation of hpdna , a large force corresponding to the change in free energy , g , of the helix - coil transition will be required to dissociate the bases and induce the translocation of dna . the data of figure 5c indicate the threshold voltage collapses from 2 to 0.5 v as the pore size decreases from 1.5 to 1 nm , indicative of a dramatic change ( 4 ) in the force required to induce the helix - coil transition . assuming that a pore diameter d < 1.5 nm excludes dsdna and precludes stretching , we attribute this change in threshold to the difference between stretching and unzipping dna . to assess the influence of pore geometry on the pathways for the helix - coil transition , we performed steered molecular dynamics simulations in the coil - first orientation , where the phosphate on the end of the coil was pulled at a constant velocity of 1.0 nm / ns . as illustrated in figure 6 , the mechanics of a hairpin permeating a pore depend dramatically on the diameter . while in all cases the helical structure disappears , the final conformation for the smallest pore ( figure 6a ) is much different than the final conformation for the largest pore ( figure 6d ) . in the case of the smallest pore , with a 1.8 nm diameter opening , ( figure 6a ) , the helix - coil transition proceeded through the unzipping pathway . in contrast , for a pore with 2.4 nm opening ( figure 6d ) , the helix was able to pass some distance into the pore . as the pore diameter decreased along its axis , the double helical structure could no longer be maintained and transition occurred by stretching and distortion . figure 6.snapshots from steered molecular dynamics simulations of the helix - coil transition of dna in a synthetic pore . in all cases , the phosphate of the 5 terminal base on the single - stranded coil portion is pulled downward at a rate of 1 nm / ns . the portion initially forming the single - stranded coil is shown in blue , while the two portions with complementary sequences , initially forming a double - stranded helix , are shown in yellow and red , respectively . note that the membrane thickness was chosen to increase the computational efficiency and is less than that in the experiments . nevertheless , the simulation results can be used to interpret experiment , as in these simulations the dna translocation was induced by applying an external mechanical force , not a transmembrane potential . in the two smallest pores , ( a ) with a diameter from 1.0 to 1.8 nm and ( b ) with a diameter from 1.3 to 2.1 nm , the helix - coil transition occurs through unzipping of the bases . for the largest two pores , ( c ) with a diameter from 1.4 to 2.2 nm and ( d ) with a diameter 1.62.4 nm , the helix - coil transition proceeds by stretching of the backbone . snapshots from steered molecular dynamics simulations of the helix - coil transition of dna in a synthetic pore . in all cases , the phosphate of the 5 terminal base on the single - stranded coil portion is pulled downward at a rate of 1 nm / ns . the portion initially forming the single - stranded coil is shown in blue , while the two portions with complementary sequences , initially forming a double - stranded helix , are shown in yellow and red , respectively . note that the membrane thickness was chosen to increase the computational efficiency and is less than that in the experiments . nevertheless , the simulation results can be used to interpret experiment , as in these simulations the dna translocation was induced by applying an external mechanical force , not a transmembrane potential . in the two smallest pores , ( a ) with a diameter from 1.0 to 1.8 nm and ( b ) with a diameter from 1.3 to 2.1 nm , the helix - coil transition occurs through unzipping of the bases . for the largest two pores , ( c ) with a diameter from 1.4 to 2.2 nm and ( d ) with a diameter 1.62.4 nm , the helix - coil transition proceeds by stretching of the backbone . ostensibly , the force required to dissociate base - pairs is different depending on whether the dna is unzipped by pulling parallel to the bases or stretched by pulling transverse to the base - pairs . it takes less force to unzip dna than to stretch the backbone . with the hpdna 's conformation unconstrained by the walls of the pore , the bases can rotate so that much of the force is applied along the axis of the hydrogen bonds connecting the bases . however , if the hairpin penetrates deep within the pore , only a small portion of the force is directed along the axis of the hydrogen bonds . we attribute the sharp threshold voltage for permeation of dna to the distribution of the electrostatic potential within the pore . we have previously shown that the potential drops abruptly near the pore 's constriction whether or not there is dna in the constriction ( 10,13 ) . we find that for a pore containing only electrolyte and no dna , the variation of the electrostatic potential across the membrane follows the rule ( 10 ) : v(z)/vbias = ( 1/)arctan(b(zz0)/lmem ) , where the z0 represents the center of the membrane , lmem is the membrane thickness , and the geometrical factor b 9.4 . the force on the molecule can be determined by differentiating the potential distribution along the axis of symmetry of the pore . thus , the force on an elementary test charge e = 1.602 10c is estimated to be : f(z ) = e(bvbias/ lmem ) 1/(1 + ( b(z - z0)/lmem ) , which scales with the transmembrane potential and increases rapidly near the center of the membrane . and the force on a test charge near the center of the membrane is about : f(z ) e(bvbias / lmem ) . we can estimate the energy required to stretch a hairpin from the corresponding force , fstr , and the change in the distance between bases that develops when the molecule is stretched , xstr ( 5,6 ) . according to our simulations , when dna is stretched , the final separation can be as much as x 0.57 nm for each base , while the equilibrium length of the paired region per base pair is 0.28 nm , so that the change in the separation due to stretching must be about xstr 0.3 nm . in simulations , 0.35 nm is the equilibrium distance between bases . the length of 0.28 nm / pair reflects the equilibrium end - to - end length of the 10-bp hairpin , which is smaller than 0.35 nm / pair due to curvature of the duplex region . therefore , the maximum energy required to stretch the leading edge of the stem is approximately : g = ( q*/e ) fstr xstr 1.5 10 j 49 kb t where q * is the effective charge in the pore . the effective charge at the leading edge of the stem could be as high as q * = 2 , but recently , using a direct measurement of the force on a single dna molecule , keyser et al . ( 18 ) estimated the effective charge for dna in synthetic nanopores ranging in diameter from 6 to 15 nm in electrolyte ranging from 0.2 to 1 m kcl to be 0.5 0.05e per base pair equivalent . and so , depending on the effective charge in the pore the free energy could range over 12kbt < g 49kbt , which is comparable to the loop formation energy g 32kbt . finally , in contrast to the -hemolysin , hpdna can enter a synthetic , solid - state nanopore in either the coil- or loop - first orientation , which could affect our estimate of the threshold and enthalpy . preliminary results indicate that a hairpin with either orientation can squeeze through a nanopore that is narrower than a dna double helix provided that the driving field is high enough . in summary , we observe a threshold voltage for translocation of the hairpin through the pore that depends sensitively on the diameter and the secondary structure of the dna . for a diameter 1.5 < d < 2.3 nm the threshold corresponds to the force required to stretch the stem of the hairpin , while for 1.0 < d < 1.5 nm , the threshold collapses because the stem unzips with a lower force than required for stretching . in related work , we have observed a threshold voltage for the rupture of the bond between a restriction enzyme and dna that can be used to discriminate single nucleotide polymorphisms ( 16 ) . although speculative , it seems likely that the threshold for a hairpin to permeate the pore is related to the free energy and molecular stability , but an unambiguous interpretation requires knowledge of the molecular configuration in the pore : i.e. whether the molecule enters oriented coil- or loop - first . this information could be recovered through force spectroscopy studies of the translocation of hairpins one at a time through the pore , but first we have to sort out the relationship between the current transients and the configuration of the molecule in the pore .

we have explored the electromechanical properties of dna by using an electric field to force single hairpin molecules to translocate through a synthetic pore in a silicon nitride membrane . we observe a threshold voltage for translocation of the hairpin through the pore that depends sensitively on the diameter and the secondary structure of the dna . the threshold for a diameter 1.5 < d < 2.3 nm is v > 1.5 v , which corresponds to the force required to stretch the stem of the hairpin , according to molecular dynamics simulations . on the other hand , for 1.0 < d < 1.5 nm , the threshold voltage collapses to v < 0.5 v because the stem unzips with a lower force than required for stretching . the data indicate that a synthetic nanopore can be used like a molecular gate to discriminate between the secondary structures in dna .

INTRODUCTION EXPERIMENTAL AND COMPUTATION METHODS RESULTS AND DISCUSSION

the force required to dissociate base pairs is different depending on whether the dna is unzipped by pulling parallel to the bases , or stretched by pulling transverse to the base - pairs . on the other hand , when a single molecule of dsdna is stretched beyond its contour length , it undergoes a cooperative transition near fstr = 6070 pn in which the dna doubles ( 1.7 ) in length ( 912 ) . synthetic nanopores can be used to accomplish both types of measurements because large forces can be applied using the electric field in the pore , and because the diameter of the pore can be controlled with sub - nanometer precision . instead of these tools , we use a nanopore in a silicon nitride membrane . we have previously explored the electromechanical properties of dna using the electric field to force single molecules to translocate across a silicon nitride membrane through a pore ( 10,13 ) . when a voltage is applied in a bi - cell across a membrane with a pore in it , polyanionic dna immersed in electrolyte at the cathode diffuses toward the anode and the force due to the field acting on the strand impels dna to bend and stretch within the pore . we have shown that for low electric fields e < 0.2 mv / cm = 200 mv/10 nm , ssdna can permeate pores with diameters 1.0 nm , while dsdna only permeates pores with diameters 2.5 nm . for pores < 2.5 nm in diameter , there is a threshold for permeation of dsdna that depends on the electric field and ph . in this report , using hairpin dna ( hpdna ) instead of dsdna , we observe a threshold voltage for translocation of the molecule through the synthetic pore that depends sensitively on the pore diameter and the secondary structure of the dna . we find that the threshold voltage to induce a translocation of hpdna through a pore with a diameter 1.5 < d < 2.3 nm is v > 1.5 v , corresponding to the force required to stretch the stem of the hairpin , according to molecular dynamic simulations . on the other hand , for pores with diameters 1.0 < d < 1.5 nm , the threshold collapses to v < 0.5 v because the stem unzips with a lower force . these data indicate that a synthetic nanopore can be used like a molecular gate to discriminate between the secondary structures in dna . this electrolyte concentration was chosen primarily to minimize the secondary structure in the hairpin ; increase the closing rate for the loop ( 20 ) ; and economize on the computer time required for simulation of the results . labview software is used to measure the electrolytic current at 23.5 1c and to apply voltages in range of 5 v. with a voltage applied across the membrane , we observed an electrolytic current through the pore . as illustrated by figure 1b , the i v characteristics were approximately linear line fits to the data indicate conductances : 1.13 0.03 ns , 1.98 0.03 ns and 3.20 0.03 ns , for the three pores shown in figure 1a , respectively . among other things , the stability is affected by : the base composition of the paired region ; the length and constituency of the loop ; and the number of mismatches or bubbles the stem - loop contains . on the other hand , we are motivated to use a poly(a ) overhang to control the secondary structure . we expect the dna in the loop to show a very short persistence length ( < 2 nm ) making it flexible enough to penetrate to the high electric field deep in the lumen of the pore.the second 110-mer hairpin has a single - stranded overhang consisting primarily of a 50-mer poly da strand of dna , and a stem 10 bp long containing an intervening 6 base loop with the following sequence ( the self - complementary parts are indicated in bold italic ) : 5-gctctgttgc tctctcgcaa cagagcatga acg tga aaag gtctacagta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa aaaaaa aaaaaaaaa gaa tcg cag tg-3. we expect the dna in the loop to show a very short persistence length ( < 2 nm ) making it flexible enough to penetrate to the high electric field deep in the lumen of the pore . in the first set , a uniform electric field is applied to a pore in a 20.0 nm - thick si3n4 membrane containing a hpdna molecule to study the dependence of the electrolytic current on the molecule 's conformation . in the second set of simulations , the effect of the pore geometry on the pathway of the helix - coil transition is probed by pulling the hpdna through four different pores in 2.3 nm - thick si3n4 membranes . for example , the translocation velocity of unbound dna through a large diameter > 5 nm synthetic pore is estimated to be > 1 bp / ns at these voltages ( 31,37,38 ) implying that the duration of a translocation is < 0.1 s for 100 bp dna . in contrast , we observe transients with a duration > 10 s associated with the hpdna , which is unprecedented for a synthetic pore . ( b ) , ( c ) and ( d ) show representative currents trace taken from the same pore at observed v = 1.0 v , 1.5 v and 2.0 v , respectively . the same features , which we associate with the secondary structure in the hairpin , are observed above and below threshold . ( a ) the electrolytic current through the 1.2 1.4 nm pore shown in figure 1a as a function of time with the membrane voltage at v = 0.5 v. the open pore current at this voltage is 0.9 na ( red ) ; the transients in the current are associated with 150-mer hpdna interacting with the pore ( blue ) . the same features , which we associate with the secondary structure in the hairpin , are observed above and below threshold . instead , we hypothesize that the transients are caused by hairpin molecules modulating the current through the pore , and that the modulation depends on the molecular configuration relative to the pore . the results of md simulations shown in figure 3 represent the dependence of the pore current on the configuration of the hairpin over a pore with a 1.9 2.1 nm cross - section in a 20 nm thick nitride membrane with an applied transmembrane bias of 4 v. each steady - state current value shown was obtained by averaging the current in the corresponding md trajectory ( > 12 ns ) from t = 7 ns to the end of the simulation , except for the system shown in figure 3c where the averaging began at 6 ns . notice that the open pore current shown in figure 3a is i0 = 5.81 0.08 na , but with the hairpin in the vicinity of the pore , the electrolytic current changes dramatically depending on the relative position . and finally , in figure 3e , we see that the current through the pore can exceed the open pore value ( by i / i0 = + 13% ) , indicating that a positive current transient is possible even when a hairpin assumes this position near the entrance to the pore on the cis - side of the membrane . figure 3.molecular dynamics simulations showing the dependence of the pore current on the configuration of the hairpin over the pore . note that the thickness of the membrane is 20 nm and the pore diameter is 2 nm , both larger than the corresponding values used in the experiments shown in figure 2 . notice in ( b ) that a blockade of the pore results in a minimal current of 2.88 0.09 na through the pore . molecular dynamics simulations showing the dependence of the pore current on the configuration of the hairpin over the pore . notice in ( b ) that a blockade of the pore results in a minimal current of 2.88 0.09 na through the pore . generally , our simulations indicate that the ion concentration in the region on the cis side of the pore within 6 nm of the constriction is less than in the bulk , dropping below 0.2 m for k and 0.5 m for cl for a 1 m kcl solution , which is consistent with the results of folgea et al . due to the dependence on the molecular configuration relative to the pore and the limited bandwidth of the measurement , a current transient is not an unambiguous signature of a translocation across the membrane through the pore . we find that a synthetic nanopore acts like a molecular gate that discriminates between dna according to the secondary structure . in particular , the fluorescent bands in figure 4b indicate that 110-mer hpdna is collected at the positive electrode only for v > 2.50 v ; below this threshold voltage the translocation of 110-mer hpdna across the membrane can not be detected . on the other hand , the 150-mer hpdna , which is nominally more stable , permeates the same pore for v > 2.00 v. in contrast , the stiffer 105 bp dsdna permeates the same pore only for v > 3.0 v , in correspondence with previously reported measurements obtained on a similar pore ( 10,13 ) . ( b ) a gel array indicating that 110-mer hpdna ( 10 bp stem with a 6 bp loop ) permeates the pore only for voltages v > 2.5 v. ( c ) a gel array indicating that 150-mer hpdna ( 12 bp stem with a 76 bp loop ) permeates the same pore only for voltages v > 2.0 v. and ( d ) a gel array indicating that 105 bp dsdna permeates the pore only for voltages v > 3.0 v. a synthetic nanopore is like a molecular gate . ( b ) a gel array indicating that 110-mer hpdna ( 10 bp stem with a 6 bp loop ) permeates the pore only for voltages v > 2.5 v. ( c ) a gel array indicating that 150-mer hpdna ( 12 bp stem with a 76 bp loop ) permeates the same pore only for voltages v > 2.0 v. and ( d ) a gel array indicating that 105 bp dsdna permeates the pore only for voltages v > 3.0 v. we systematically investigated hpdna permeability through a membrane as a function of the pore diameter , using diameters small enough to forestall the translocation of dsdna , while at the same time allowing ssdna to easily permeate the membrane through the pore ( i.e. we observe a threshold voltage for the translocation of 150-mer hpdna across the membrane through the pore that depends on the pore diameter . in particular , the fluorescent bands indicate that hpdna translocates only for v 0.5 v for a 1 nm diameter pore , while for v < 0.5 v only the unreacted primers are found in solution . according to the summary shown in figure 5c , for the 1.4 and 2.2 nm pores , the threshold is about v 1.5 v. the largest threshold value , v = 2.25 v , is found for pores with one axis ranging from 1.5 nm < d < 2 nm . apparently , hpdna can be forced through pores with a diameter > 1.5 nm only if the voltage is v > 1.5 v. figure 5.threshold voltage for hairpin translocation depends on the pore diameter . ( a ) gel arrays containing eight horizontal lanes with bands indicating 150-mer hpdna found as a function of the bias voltage , v , in a bi - cell corresponding to the three pores shown in figure 1a . the 150 hpdna permeates the 2.2 nm pore only for v > 1.60 v ; hpdna permeates the 1.4 nm pore only for v > 1.50 v ; and hpdna permeates the 1 nm pore only for v > 0.5 v. for reference , a 100 bp ladder is shown in the top lane of each gel . ( c ) a summary of the dependence of the threshold for permeation of 150 hp on the easy axis of the pore . the threshold for d 1.6 nm is supposed to be associated with the stem of the hairpin stretching in the pore to facilitate the translocation , while the collapse of the threshold when d < 1.5 nm is supposed to be due to unzipping of the hairpin . ( c ) a summary of the dependence of the threshold for permeation of 150 hp on the easy axis of the pore . the threshold for d 1.6 nm is supposed to be associated with the stem of the hairpin stretching in the pore to facilitate the translocation , while the collapse of the threshold when d < 1.5 nm is supposed to be due to unzipping of the hairpin . a crude estimate for the number of dna copies that permeated the pore was determined through a comparison of the measured fluorescent intensity with controlled dilutions ( data not shown ) . accordingly , in figure 5a we estimate that about < 10 copies permeated the pore for v < 2.25 v. the number of dna copies that permeated the pore was also determined by qpcr . for example , the threshold voltage for the 1.4 nm pore is v < 2 v , while the gel shows v = 2.25 v. the relatively lower threshold voltage can be attributed to the higher sensitivity of qpcr . figure 5c summarizes the dependence of the threshold voltage inferred from electrophoresis on the pore diameter . we assume that the threshold voltage corresponds to the minimum force required to impel a hairpin through the pore . assuming that the stem frustrates the permeation of hpdna , a large force corresponding to the change in free energy , g , of the helix - coil transition will be required to dissociate the bases and induce the translocation of dna . the data of figure 5c indicate the threshold voltage collapses from 2 to 0.5 v as the pore size decreases from 1.5 to 1 nm , indicative of a dramatic change ( 4 ) in the force required to induce the helix - coil transition . assuming that a pore diameter d < 1.5 nm excludes dsdna and precludes stretching , we attribute this change in threshold to the difference between stretching and unzipping dna . to assess the influence of pore geometry on the pathways for the helix - coil transition , we performed steered molecular dynamics simulations in the coil - first orientation , where the phosphate on the end of the coil was pulled at a constant velocity of 1.0 nm / ns . in the case of the smallest pore , with a 1.8 nm diameter opening , ( figure 6a ) , the helix - coil transition proceeded through the unzipping pathway . figure 6.snapshots from steered molecular dynamics simulations of the helix - coil transition of dna in a synthetic pore . in the two smallest pores , ( a ) with a diameter from 1.0 to 1.8 nm and ( b ) with a diameter from 1.3 to 2.1 nm , the helix - coil transition occurs through unzipping of the bases . for the largest two pores , ( c ) with a diameter from 1.4 to 2.2 nm and ( d ) with a diameter 1.62.4 nm , the helix - coil transition proceeds by stretching of the backbone . snapshots from steered molecular dynamics simulations of the helix - coil transition of dna in a synthetic pore . nevertheless , the simulation results can be used to interpret experiment , as in these simulations the dna translocation was induced by applying an external mechanical force , not a transmembrane potential . in the two smallest pores , ( a ) with a diameter from 1.0 to 1.8 nm and ( b ) with a diameter from 1.3 to 2.1 nm , the helix - coil transition occurs through unzipping of the bases . for the largest two pores , ( c ) with a diameter from 1.4 to 2.2 nm and ( d ) with a diameter 1.62.4 nm , the helix - coil transition proceeds by stretching of the backbone . ostensibly , the force required to dissociate base - pairs is different depending on whether the dna is unzipped by pulling parallel to the bases or stretched by pulling transverse to the base - pairs . with the hpdna 's conformation unconstrained by the walls of the pore , the bases can rotate so that much of the force is applied along the axis of the hydrogen bonds connecting the bases . we attribute the sharp threshold voltage for permeation of dna to the distribution of the electrostatic potential within the pore . we find that for a pore containing only electrolyte and no dna , the variation of the electrostatic potential across the membrane follows the rule ( 10 ) : v(z)/vbias = ( 1/)arctan(b(zz0)/lmem ) , where the z0 represents the center of the membrane , lmem is the membrane thickness , and the geometrical factor b 9.4 . the force on the molecule can be determined by differentiating the potential distribution along the axis of symmetry of the pore . according to our simulations , when dna is stretched , the final separation can be as much as x 0.57 nm for each base , while the equilibrium length of the paired region per base pair is 0.28 nm , so that the change in the separation due to stretching must be about xstr 0.3 nm . therefore , the maximum energy required to stretch the leading edge of the stem is approximately : g = ( q*/e ) fstr xstr 1.5 10 j 49 kb t where q * is the effective charge in the pore . and so , depending on the effective charge in the pore the free energy could range over 12kbt < g 49kbt , which is comparable to the loop formation energy g 32kbt . finally , in contrast to the -hemolysin , hpdna can enter a synthetic , solid - state nanopore in either the coil- or loop - first orientation , which could affect our estimate of the threshold and enthalpy . in summary , we observe a threshold voltage for translocation of the hairpin through the pore that depends sensitively on the diameter and the secondary structure of the dna . for a diameter 1.5 < d < 2.3 nm the threshold corresponds to the force required to stretch the stem of the hairpin , while for 1.0 < d < 1.5 nm , the threshold collapses because the stem unzips with a lower force than required for stretching . in related work , we have observed a threshold voltage for the rupture of the bond between a restriction enzyme and dna that can be used to discriminate single nucleotide polymorphisms ( 16 ) . although speculative , it seems likely that the threshold for a hairpin to permeate the pore is related to the free energy and molecular stability , but an unambiguous interpretation requires knowledge of the molecular configuration in the pore : i.e. this information could be recovered through force spectroscopy studies of the translocation of hairpins one at a time through the pore , but first we have to sort out the relationship between the current transients and the configuration of the molecule in the pore .

conventional periodontal regenerative therapies such as bone replacement grafts and guided tissue regeneration have not been able to achieve complete and predictable periodontal regeneration . in spite of allowing progenitor cells to selectively migrate and differentiate as in guided tissue regeneration , the possible reason for this could be attributed to the mechanical , physical and chemical changes that take place in the cementum during the progression of periodontitis . current research has focused on regeneration of acellular extrinsic fiber cementum through functional tissue engineering and , more importantly , the biomimetic approaches . according to the concepts of functional tissue engineering , the cells can sense and respond to mechanical factors and various other environmental cues of the substrate . in addition to this , the regeneration of lost tissue can also be achieved by biomimicking the physical and mechanical properties of the tissue , which serves as a scaffold in nature . understanding the nanostructure of cementum may aid in designing a biomimetic scaffold that will match with the mechanical properties of the root surface . this will provide a favorable micromechanical environment for progenitor cells and for successful regeneration of acellular extrinsic fiber cementum . the effect of the physical properties of the extracellular matrix on cell differentiation and proliferation has been well documented . apart from the components of the cementum matrix , the local microenvironment of the extracellular matrix also plays a major role in periodontal regeneration . in addition to this , various studies have reported the importance of the mechanical properties of the matrix in directing stem cell differentiation . the cementum undergoes numerous physical , chemical , structural and cytotoxic alterations during the pathogenesis of periodontal disease . during the early stage of periodontitis , the acellular extrinsic fiber cementum gets irreversibly damaged as it is found in the cervical third of the root . although various studies have reported the cemental changes that take place during the progression of periodontal disease , the mechanical properties of the cementum are not completely understood . the mechanical properties of the cementum that are commonly analyzed are hardness , modulus of elasticity and surface roughness . moreover , various studies have demonstrated the significance of analyzing the substrate elasticity and its impact on hematopoietic stem cell and progenitor migration and adhesion . it is now well understood from the available data that the mechanical integrity of a tissue is predominantly a function of its nanostructure . although the mechanical properties of the cementum have been estimated on macro- and microscales , the nanostructure of the cementum has not yet been characterized in chronic periodontitis in detail . the aim of this study is to assess and compare the nanomechanical properties of acellular extrinsic fiber cementum at the cervical third of the root in health and in chronic periodontitis . the study protocol was approved by the institutional ethics committee , sri ramachandra university , chennai . a total of 20 teeth were collected from 12 subjects reporting to the outpatient department , department of periodontics . the healthy teeth ( n = 10 ) were collected from six individuals with age ranging from 30 to 40 years for whom orthodontic extractions were indicated . the criteria for selecting healthy teeth samples included absence of dental caries , absence of bleeding on probing and probing depth / attachment loss and no radiographic evidence of bone loss . periodontally diseased teeth samples ( n = 10 ) were collected from six patients with age ranging from 30 to 40 years diagnosed with generalized severe chronic periodontitis . tooth type distribution in the healthy teeth group and diseased teeth group are presented in table 1 . periodontally compromised teeth were selected if the probing depth and attachment loss was more than 5 mm with radiographic evidence of bone loss up to the apical third . distribution of tooth types between the healthy and the diseased group the exclusion criteria were as follows : presence of gingival recession around the selected teeth , cigarette / tobacco smoking habit , patients who have undergone periodontal treatment in the last 5 years , patients who have taken any antibiotics for the past 3 months , presence of any systemic disease , root caries , fractured teeth and non - vital teeth and pregnant or lactating women . before nanoindentation , the topography of the sputter - coated outer surface of two healthy and two diseased transverse sections of 3 - 5-mm - thick cervical third cementum sections [ figures 1a , b and 2 ] were characterized using fei quanta feg 200 high - resolution scanning electron microscopy ( sem ) at various magnifications ranging from 6500 to 400 . these samples were not used further for characterizing nanomechanical properties as they were sputter coated with gold . the sections were placed on appropriate stubs by fixing them using a double - sided adhesive . the stubs with the sections on top were placed inside the apparatus that was later maintained at a low vacuum of 0.97 torr throughout the analysis . the specimen sections were examined using an electron energy of 20 kev to obtain the micrographs . the depth - sensing nanoindentation technique requires the sample 's surface to be flat and even . because the outer surface of the root is convex , the transverse sections of cementum were characterized for the nanomechanical properties . briefly , the freshly extracted teeth were cleaned and disinfected and the periodontal ligament fragments were removed by using milton 's solution ( 1% sodium hypochlorite ) for 10 min . the crown of the teeth was transversely sectioned at the level of the cement - enamel junction [ figures 1a , b and 2 ] . the cervical third of the root was sectioned transversely to obtain 5-mm - thick sections [ figure 1a ] using a diamond wafering blade on a low - speed cutter under wet conditions . the cervical third sections were stored in deionized water at an ambient temperature of 23 2c in a polyethylene container until further analysis . the transverse sections of the cervical third of root [ figures 1b and 2 ] were embedded in epoxy resin and the resin blocks were trimmed and polished using a basic metallography polishing technique . ( a ) ( color online ) transverse section of the cervical third cementum after decoronation and ( b ) transverse section of the cervical third cementum before embedding in resin schematic of the sectioning of the cervical third of the root and the cementum surface characterized using nanoindentation and scanning electron microscopy the specimens were polished sequentially using sic grit papers ( 200 - 1000 sizes ) , then fine polishing using diamond suspension slurries ( 9 m , 6 m , 3 m and 1 m ) on a polishing cloth . the final polishing process was carried out with colloidal silica suspension ( ops ) 0.25 m for 2 min at a speed of 200 rpm . the specimens were ultrasonificated in deionized water for 10 min between each level of polishing before proceeding to the next level of polishing to remove any abrasives and they were air dried for 3 s prior to mounting the specimens in the nanoindentation specimen holder . the local elastic and plastic properties of the cementum were investigated by performing nanoindentation experiments with a three - sided berkovich - type diamond indenter . these tests were conducted under dry conditions with a csm nanoindenter ( peseux , switzerland ) [ figure 3 ] ; during indentation , a load - displacement curve was recorded , from which the contact area , hardness and elastic modulus were calculated using the oliver and pharr method . the depth calibration to identify the surface to indent was carried out before the nanoindentation process . the determination of elastic modulus from the elastic recovery of the material by measuring the contact stiffness s ( = dp / dh ) has been achieved by the controlled unloading after indentation . the hardness h and the young 's modulus e were calculated from the following fundamental relations : csm nanoindenter apparatus where p is the load and a is the projected contact area at that load , and where er is the reduced elastic modulus and is a constant that depends on the geometry of the indenter . a reduced modulus er is used in equation ( 2 ) to account for the fact that elastic displacements occur in both the indenter and the sample . the elastic modulus of the test material , e , is calculated from er using where n is the poisson 's ratio for the test material and ei and vr are the elastic modulus and poisson 's ratio , respectively , of the indenter . for diamond , the elastic constant ei = 1141 gpa and poisson 's ratio vr the test zone , maximum force , number of indentations and distance between indentations were programmed into the computer . ten nanoindentations were performed per sample , adding up to 100 nanoindentations in each group . their locations were selected midway between the cement - dentinal junction and the peripheral cementum to avoid the resin , residual calculus and the cement - dentinal junction [ figure 2 ] . the optical microscopic image [ figure 4 ] shows a typical indentation on the cementum ( black arrow ) , with some striations on the dentin . optical microscope image of the transverse sections of the cervical third cementum open arrow - dentin , white arrow - resin , black arrow showing the impression of the indent in the middle portion of the cervical cementum load / unload cycle parameters used during nanoindentation all the statistical analysis was performed using spss statistical software ( version 17.0 ) . the mean and standard deviation of the test parameters was estimated for the test and healthy control samples . the intergroup comparison was carried out using a non - parametric test ( mann - whitney test ) , and the difference was considered to be statistically significant if the p value was less than 0.05 . the study protocol was approved by the institutional ethics committee , sri ramachandra university , chennai . a total of 20 teeth were collected from 12 subjects reporting to the outpatient department , department of periodontics . the healthy teeth ( n = 10 ) were collected from six individuals with age ranging from 30 to 40 years for whom orthodontic extractions were indicated . the criteria for selecting healthy teeth samples included absence of dental caries , absence of bleeding on probing and probing depth / attachment loss and no radiographic evidence of bone loss . periodontally diseased teeth samples ( n = 10 ) were collected from six patients with age ranging from 30 to 40 years diagnosed with generalized severe chronic periodontitis . tooth type distribution in the healthy teeth group and diseased teeth group are presented in table 1 . periodontally compromised teeth were selected if the probing depth and attachment loss was more than 5 mm with radiographic evidence of bone loss up to the apical third . distribution of tooth types between the healthy and the diseased group the exclusion criteria were as follows : presence of gingival recession around the selected teeth , cigarette / tobacco smoking habit , patients who have undergone periodontal treatment in the last 5 years , patients who have taken any antibiotics for the past 3 months , presence of any systemic disease , root caries , fractured teeth and non - vital teeth and pregnant or lactating women . before nanoindentation , the topography of the sputter - coated outer surface of two healthy and two diseased transverse sections of 3 - 5-mm - thick cervical third cementum sections [ figures 1a , b and 2 ] were characterized using fei quanta feg 200 high - resolution scanning electron microscopy ( sem ) at various magnifications ranging from 6500 to 400 . these samples were not used further for characterizing nanomechanical properties as they were sputter coated with gold . the sections were placed on appropriate stubs by fixing them using a double - sided adhesive . the stubs with the sections on top were placed inside the apparatus that was later maintained at a low vacuum of 0.97 torr throughout the analysis . the specimen sections were examined using an electron energy of 20 kev to obtain the micrographs . the depth - sensing nanoindentation technique requires the sample 's surface to be flat and even . because the outer surface of the root is convex , the transverse sections of cementum were characterized for the nanomechanical properties . briefly , the freshly extracted teeth were cleaned and disinfected and the periodontal ligament fragments were removed by using milton 's solution ( 1% sodium hypochlorite ) for 10 min . the crown of the teeth was transversely sectioned at the level of the cement - enamel junction [ figures 1a , b and 2 ] . the cervical third of the root was sectioned transversely to obtain 5-mm - thick sections [ figure 1a ] using a diamond wafering blade on a low - speed cutter under wet conditions . the cervical third sections were stored in deionized water at an ambient temperature of 23 2c in a polyethylene container until further analysis . the transverse sections of the cervical third of root [ figures 1b and 2 ] were embedded in epoxy resin and the resin blocks were trimmed and polished using a basic metallography polishing technique . ( a ) ( color online ) transverse section of the cervical third cementum after decoronation and ( b ) transverse section of the cervical third cementum before embedding in resin schematic of the sectioning of the cervical third of the root and the cementum surface characterized using nanoindentation and scanning electron microscopy the specimens were polished sequentially using sic grit papers ( 200 - 1000 sizes ) , then fine polishing using diamond suspension slurries ( 9 m , 6 m , 3 m and 1 m ) on a polishing cloth . the final polishing process was carried out with colloidal silica suspension ( ops ) 0.25 m for 2 min at a speed of 200 rpm . the specimens were ultrasonificated in deionized water for 10 min between each level of polishing before proceeding to the next level of polishing to remove any abrasives and they were air dried for 3 s prior to mounting the specimens in the nanoindentation specimen holder . the local elastic and plastic properties of the cementum were investigated by performing nanoindentation experiments with a three - sided berkovich - type diamond indenter . these tests were conducted under dry conditions with a csm nanoindenter ( peseux , switzerland ) [ figure 3 ] ; during indentation , a load - displacement curve was recorded , from which the contact area , hardness and elastic modulus were calculated using the oliver and pharr method . the depth calibration to identify the surface to indent was carried out before the nanoindentation process . the determination of elastic modulus from the elastic recovery of the material by measuring the contact stiffness s ( = dp / dh ) has been achieved by the controlled unloading after indentation . the hardness h and the young 's modulus e were calculated from the following fundamental relations : csm nanoindenter apparatus where p is the load and a is the projected contact area at that load , and where er is the reduced elastic modulus and is a constant that depends on the geometry of the indenter . a reduced modulus er is used in equation ( 2 ) to account for the fact that elastic displacements occur in both the indenter and the sample . the elastic modulus of the test material , e , is calculated from er using where n is the poisson 's ratio for the test material and ei and vr are the elastic modulus and poisson 's ratio , respectively , of the indenter . for diamond , the elastic constant ei = 1141 gpa and poisson 's ratio vr , maximum force , number of indentations and distance between indentations were programmed into the computer . ten nanoindentations were performed per sample , adding up to 100 nanoindentations in each group . their locations were selected midway between the cement - dentinal junction and the peripheral cementum to avoid the resin , residual calculus and the cement - dentinal junction [ figure 2 ] . the optical microscopic image [ figure 4 ] shows a typical indentation on the cementum ( black arrow ) , with some striations on the dentin . optical microscope image of the transverse sections of the cervical third cementum open arrow - dentin , white arrow - resin , black arrow showing the impression of the indent in the middle portion of the cervical cementum load / unload cycle parameters used during nanoindentation the mean and standard deviation of the test parameters was estimated for the test and healthy control samples . the intergroup comparison was carried out using a non - parametric test ( mann - whitney test ) , and the difference was considered to be statistically significant if the p value was less than 0.05 . the sem micrographs of the morphology of the healthy outer surface of the cervical third cementum sections are shown in figure 5 and those of the diseased sections are shown in figure 6 . sem characterization revealed the presence of mineralized collagen fibers in the healthy cementum , which were more predominant when compared with the diseased cementum . the sem micrographs of the diseased cementum showed areas of foreign bodies that could be deposits of calculus . scanning electron microscopy micrograph of the outer surface of the healthy cervical third cementum ( at 6011 magnification ) scanning electron microscopy micrograph of the outer surface of the diseased cervical third cementum section ( at 6011 magnification ) typical indentation profiles of the healthy and diseased cementum cervical sections are shown in figure 7 . these profiles clearly show a penetration depth difference between the healthy and diseased for the same indentation load . ( color online ) typical nanoindentation profiles of healthy and diseased cervical third cementum the results showed that the hardness values varied between 0.546 and 1.124 gpa for the healthy cementum sections and , for the diseased cementum sections , the values ranged from 0.273 to 0.726 gpa . the mean hardness of the diseased cementum was significantly lower compared with the healthy cementum ( p < 0.05 ) [ table 3 ] . the mean hardness values for the cementum of the healthy samples and the diseased cementum sample are shown in figure 8 . nanomechanical properties of the healthy and the diseased cementum comparison of hardness values of healthy and diseased cervical third cementum ( n = 10 ) the modulus of elasticity of the healthy cementum was observed to be between 12.981 and 21.912 gpa and that of the diseased cementum varied between 6.781 and 13.443 gpa [ figure 5 ] . the difference between the mean modulus of elasticity of the healthy and diseased cementum [ table 3 ] was statistically significant ( p < 0.05 ) . the mean modulus of elasticity values for the cementum of the healthy samples and the diseased cementum samples are shown in figure 9 . comparison of modulus of elasticity values of healthy and diseased cervical third cementum ( n = 10 ) the sem micrographs of the morphology of the healthy outer surface of the cervical third cementum sections are shown in figure 5 and those of the diseased sections are shown in figure 6 . sem characterization revealed the presence of mineralized collagen fibers in the healthy cementum , which were more predominant when compared with the diseased cementum . the sem micrographs of the diseased cementum showed areas of foreign bodies that could be deposits of calculus . scanning electron microscopy micrograph of the outer surface of the healthy cervical third cementum ( at 6011 magnification ) scanning electron microscopy micrograph of the outer surface of the diseased cervical third cementum section ( at 6011 magnification ) typical indentation profiles of the healthy and diseased cementum cervical sections are shown in figure 7 . these profiles clearly show a penetration depth difference between the healthy and diseased for the same indentation load . ( color online ) typical nanoindentation profiles of healthy and diseased cervical third cementum the results showed that the hardness values varied between 0.546 and 1.124 gpa for the healthy cementum sections and , for the diseased cementum sections , the values ranged from 0.273 to 0.726 gpa . the mean hardness of the diseased cementum was significantly lower compared with the healthy cementum ( p < 0.05 ) [ table 3 ] . the mean hardness values for the cementum of the healthy samples and the diseased cementum sample are shown in figure 8 . nanomechanical properties of the healthy and the diseased cementum comparison of hardness values of healthy and diseased cervical third cementum ( n = 10 ) the modulus of elasticity of the healthy cementum was observed to be between 12.981 and 21.912 gpa and that of the diseased cementum varied between 6.781 and 13.443 gpa [ figure 5 ] . the difference between the mean modulus of elasticity of the healthy and diseased cementum [ table 3 ] was statistically significant ( p < 0.05 ) . the mean modulus of elasticity values for the cementum of the healthy samples and the diseased cementum samples are shown in figure 9 . comparison of modulus of elasticity values of healthy and diseased cervical third cementum ( n = 10 ) the results showed that the hardness values varied between 0.546 and 1.124 gpa for the healthy cementum sections and , for the diseased cementum sections , the values ranged from 0.273 to 0.726 gpa . the mean hardness of the diseased cementum was significantly lower compared with the healthy cementum ( p < 0.05 ) [ table 3 ] . the mean hardness values for the cementum of the healthy samples and the diseased cementum sample are shown in figure 8 . nanomechanical properties of the healthy and the diseased cementum comparison of hardness values of healthy and diseased cervical third cementum ( n = 10 ) the modulus of elasticity of the healthy cementum was observed to be between 12.981 and 21.912 gpa and that of the diseased cementum varied between 6.781 and 13.443 gpa [ figure 5 ] . the difference between the mean modulus of elasticity of the healthy and diseased cementum [ table 3 ] was statistically significant ( p < 0.05 ) . the mean modulus of elasticity values for the cementum of the healthy samples and the diseased cementum samples are shown in figure 9 . comparison of modulus of elasticity values of healthy and diseased cervical third cementum ( n = 10 ) even though regeneration of alveolar bone can be accomplished by the presently available therapeutic modalities , the regeneration of the acellular extrinsic fiber cementum still remains elusive . functional tissue engineering strategies using scaffolds , cells and growth factors may offer more predictable avenues for periodontal regeneration . an important principle in functional tissue engineering is the determination of the biomechanical properties of the native tissue in health and in diseased conditions . in the case of periodontal tissue engineering , this information can be obtained by analyzing the mechanical properties of both the healthy and the diseased root surface . this study was undertaken to investigate the effect of chronic periodontitis on the nanomechanical properties of the cervical third of the cementum within a periodontal pocket environment . in order to evaluate the changes in the morphology of the diseased cementum the topography of the outer surface of the healthy and diseased transverse sections of the cervical third of the cementum was characterized using high - resolution sem . sem revealed morphological changes in the outer surface of the diseased cementum such as the absence of mineralized cemental collagen fibers that were a predominant feature in the healthy cementum sections . however , these samples were not characterized for nanoindentation as the sputtering may itself influence the nanomechanical properties . hence , it was not possible to correlate the finding of sem micrographs with nanoindentation measurements as both these analyses were performed on two different cementum surfaces . previous studies have estimated hardness and modulus of elasticity of healthy cementum . to the best our knowledge , this is the first study comparing the hardness and modulus of elasticity between healthy and diseased transverse sections of the cervical third cementum using depth - sensing nanoindentation techniques . the results of our study revealed a statistically significant difference in the hardness of the diseased cementum as compared with the healthy cementum ( p < 0.05 ) . this decrease in hardness of the diseased cementum could be due to the softening of the cementum induced by demineralization by organic acids of inflammatory exudates and resorption of collagen and protein polysaccharide matrix via enzyme activities within the confines of the periodontal pocket . in order to avoid the hypermineralized layer of cementum that forms when the root surface becomes exposed to the oral cavity due to gingival recession , periodontally compromised teeth with absence of gingival recession another important factor that must be taken into account is the total duration each of these diseased cementum sections had been exposed to the periodontal pocket environment since the onset of the periodontitis . as it was difficult to obtain this information , it could be considered as a limitation of this study . there was a statistically significant ( p < 0.05 ) difference in the modulus of elasticity of the diseased cervical third cementum as compared with the healthy cementum . this could be due to the organic acids and enzymes of inflammatory exudates within the periodontal pocket that result in dissolution of mineral contents and proteolytic breakdown of collagen fibers . the results of our study are in agreement with various other studies that have characterized similar cemental sections from healthy teeth under similar conditions . in the study by gungormus et al . , a cementum - like biomineralized microlayer was constructed using amelogenin - derived peptides and the authors compared the mechanical properties , namely modulus of elasticity and hardness using nanoindentation , with that of the native cementum . the hardness and modulus of elasticity values for the native healthy cementum obtained in their study are in accordance to the values observed in our study . nanoindentation has been used in dentistry to evaluate the mechanical properties of the dental hard tissues for the past two decades . it has been used in the field of endodontics to determine the nanomechanical properties of endodontically treated teeth and carious human teeth , in implantology to study the elasticity of the alveolar bone near the dental implant and in orthodontics . in the field of periodontics , nanoindentation has been used to determine the nanomechanical properties of the cement - dentinal junction , cementum in ank / ank mutant mouse bone - periodontal ligament and cementum complex . this technique was utilized in our study as it is more accurate when compared with other conventional mechanical tests . in addition , it allows the measurement of the mechanical properties of a very small selected region of the cementum . although there are few reports in the literature that have assessed the hardness of the cementum , most of these studies have used micromechanical testing techniques . evaluation of mechanical properties using microindentation found no statistically significant difference between microhardness of the cementum in teeth with and without periodontal involvement . also , healthy human dental cementum of premolar teeth analyzed using microindentation showed no significant differences in the hardness and elastic modulus of the cementum between the buccal and the lingual surfaces or between the upper and the lower teeth the importance of cervical third of the cementum is that it contains acellular extrinsic fiber cementum and its regeneration is considered to be the gold standard for periodontal regeneration . because predictable regeneration of new acellular extrinsic fiber cementum on a diseased root surface is yet to be achieved , current research on periodontal regeneration has focused on inducing the formation of an acellular extrinsic fiber cementum . however , the cementum formed after treatment with guided tissue regeneration , bone grafts and a derivative of enamel matrix proteins is of the cellular type . the local environmental factors , especially the substrate stiffness , plays a crucial role in recruitment and function of cementum - forming cells . the mechanical signals , for example stiffness of the substrate , can have a significant influence on the adhesion , migration , proliferation and differentiation of numerous cell types such as fibroblasts and osteoblasts . it is now well documented that the elastic properties of the substrate plays a role in the differentiation of adult and embryonic stem cells . this can be extrapolated to the cementum , wherein an alteration of the nanomechanical properties during the progression of periodontal disease process may impede complete periodontal regeneration . the prime objective of this study was to determine the changes in the nanomechanical properties of the cervical third of the cementum in chronic periodontitis based on the values obtained from a total of 200 nanoindentations . because only 10 healthy cementum sections and 10 diseased cementum sections were taken for nanoindentation , a tooth - type statistical comparison was not possible however , further studies can focus on comparing the nanomechanical properties based on tooth type / upper arch and lower arch . the values obtained in the present study for the diseased cervical third cementum are found to be lesser than the healthy cementum sections , indicating a change in the nanostructure and mechanical integrity . this may have an effect on the recruitment on progenitor cells and formation of new attachment . thus , the understanding of the nanomechanical properties of the cervical third cementum may not only aid in determining the influence of mechanical signals of cementum in health and in chronic periodontitis on progenitor cells but also help in devising various nanomechanical design parameters required for engineering acellular extrinsic fiber cementum . in conclusion , there is a decrease in the nanomechanical properties of the diseased cervical third cementum . further analysis of the diseased root surface in wet conditions may help in understanding the nanostructural changes occurring in the cervical third of the root during periodontitis .

background : during the progression of periodontal disease , the cementum undergoes alterations in its structure and composition . understanding the nanostructure of cementum , in terms of its mechanical properties , will provide an insight into the milieu that periodontal ligament cells encounter in health and chronic periodontitis . this study aims to analyze the nanomechanical properties of the cervical third of the cementum ( transverse section ) in health and chronic periodontitis.materials and methods : twenty teeth ( 10 healthy and 10 periodontally diseased ) were collected and the nanomechanical properties of the transverse section of the cervical third cementum were evaluated with depth - sensing nanoindentation technique under dry conditions . a total of 100 nanoindentations were performed to analyze the modulus of elasticity and hardness of cervical third of the cementum.results:the nanomechanical properties of the healthy cervical third cementum sections were significantly higher ( p < 0.05 ) ( hardness : 0.720 0.305 gpa ; modulus : 15.420 3.902 gpa ) than the diseased cementum section ( hardness : 0.422 0.157 gpa ; modulus : 11.056 3.434 gpa).conclusion : the results of our study indicate that the hardness and modulus of elasticity of the cervical third cementum decreases significantly in chronic periodontitis .

Introduction MATERIALS AND METHODS Sample collection Scanning electron microscopy of the cervical third of the cementum Sample preparation for nanoindentation Nanoindentation of the tooth specimens Statistical analysis RESULTS Scanning electron microscopy of the cervical third cementum Assessment of the physical property of the cervical third of cementum Hardness Modulus of elasticity DISCUSSION CONCLUSION

in spite of allowing progenitor cells to selectively migrate and differentiate as in guided tissue regeneration , the possible reason for this could be attributed to the mechanical , physical and chemical changes that take place in the cementum during the progression of periodontitis . in addition to this , the regeneration of lost tissue can also be achieved by biomimicking the physical and mechanical properties of the tissue , which serves as a scaffold in nature . understanding the nanostructure of cementum may aid in designing a biomimetic scaffold that will match with the mechanical properties of the root surface . apart from the components of the cementum matrix , the local microenvironment of the extracellular matrix also plays a major role in periodontal regeneration . in addition to this , various studies have reported the importance of the mechanical properties of the matrix in directing stem cell differentiation . the cementum undergoes numerous physical , chemical , structural and cytotoxic alterations during the pathogenesis of periodontal disease . during the early stage of periodontitis , the acellular extrinsic fiber cementum gets irreversibly damaged as it is found in the cervical third of the root . although various studies have reported the cemental changes that take place during the progression of periodontal disease , the mechanical properties of the cementum are not completely understood . the mechanical properties of the cementum that are commonly analyzed are hardness , modulus of elasticity and surface roughness . although the mechanical properties of the cementum have been estimated on macro- and microscales , the nanostructure of the cementum has not yet been characterized in chronic periodontitis in detail . the aim of this study is to assess and compare the nanomechanical properties of acellular extrinsic fiber cementum at the cervical third of the root in health and in chronic periodontitis . a total of 20 teeth were collected from 12 subjects reporting to the outpatient department , department of periodontics . the healthy teeth ( n = 10 ) were collected from six individuals with age ranging from 30 to 40 years for whom orthodontic extractions were indicated . periodontally diseased teeth samples ( n = 10 ) were collected from six patients with age ranging from 30 to 40 years diagnosed with generalized severe chronic periodontitis . distribution of tooth types between the healthy and the diseased group the exclusion criteria were as follows : presence of gingival recession around the selected teeth , cigarette / tobacco smoking habit , patients who have undergone periodontal treatment in the last 5 years , patients who have taken any antibiotics for the past 3 months , presence of any systemic disease , root caries , fractured teeth and non - vital teeth and pregnant or lactating women . before nanoindentation , the topography of the sputter - coated outer surface of two healthy and two diseased transverse sections of 3 - 5-mm - thick cervical third cementum sections [ figures 1a , b and 2 ] were characterized using fei quanta feg 200 high - resolution scanning electron microscopy ( sem ) at various magnifications ranging from 6500 to 400 . the depth - sensing nanoindentation technique requires the sample 's surface to be flat and even . because the outer surface of the root is convex , the transverse sections of cementum were characterized for the nanomechanical properties . briefly , the freshly extracted teeth were cleaned and disinfected and the periodontal ligament fragments were removed by using milton 's solution ( 1% sodium hypochlorite ) for 10 min . the cervical third of the root was sectioned transversely to obtain 5-mm - thick sections [ figure 1a ] using a diamond wafering blade on a low - speed cutter under wet conditions . the cervical third sections were stored in deionized water at an ambient temperature of 23 2c in a polyethylene container until further analysis . the transverse sections of the cervical third of root [ figures 1b and 2 ] were embedded in epoxy resin and the resin blocks were trimmed and polished using a basic metallography polishing technique . ( a ) ( color online ) transverse section of the cervical third cementum after decoronation and ( b ) transverse section of the cervical third cementum before embedding in resin schematic of the sectioning of the cervical third of the root and the cementum surface characterized using nanoindentation and scanning electron microscopy the specimens were polished sequentially using sic grit papers ( 200 - 1000 sizes ) , then fine polishing using diamond suspension slurries ( 9 m , 6 m , 3 m and 1 m ) on a polishing cloth . the local elastic and plastic properties of the cementum were investigated by performing nanoindentation experiments with a three - sided berkovich - type diamond indenter . these tests were conducted under dry conditions with a csm nanoindenter ( peseux , switzerland ) [ figure 3 ] ; during indentation , a load - displacement curve was recorded , from which the contact area , hardness and elastic modulus were calculated using the oliver and pharr method . ten nanoindentations were performed per sample , adding up to 100 nanoindentations in each group . optical microscope image of the transverse sections of the cervical third cementum open arrow - dentin , white arrow - resin , black arrow showing the impression of the indent in the middle portion of the cervical cementum load / unload cycle parameters used during nanoindentation all the statistical analysis was performed using spss statistical software ( version 17.0 ) . the healthy teeth ( n = 10 ) were collected from six individuals with age ranging from 30 to 40 years for whom orthodontic extractions were indicated . periodontally diseased teeth samples ( n = 10 ) were collected from six patients with age ranging from 30 to 40 years diagnosed with generalized severe chronic periodontitis . distribution of tooth types between the healthy and the diseased group the exclusion criteria were as follows : presence of gingival recession around the selected teeth , cigarette / tobacco smoking habit , patients who have undergone periodontal treatment in the last 5 years , patients who have taken any antibiotics for the past 3 months , presence of any systemic disease , root caries , fractured teeth and non - vital teeth and pregnant or lactating women . before nanoindentation , the topography of the sputter - coated outer surface of two healthy and two diseased transverse sections of 3 - 5-mm - thick cervical third cementum sections [ figures 1a , b and 2 ] were characterized using fei quanta feg 200 high - resolution scanning electron microscopy ( sem ) at various magnifications ranging from 6500 to 400 . the depth - sensing nanoindentation technique requires the sample 's surface to be flat and even . because the outer surface of the root is convex , the transverse sections of cementum were characterized for the nanomechanical properties . the cervical third of the root was sectioned transversely to obtain 5-mm - thick sections [ figure 1a ] using a diamond wafering blade on a low - speed cutter under wet conditions . the transverse sections of the cervical third of root [ figures 1b and 2 ] were embedded in epoxy resin and the resin blocks were trimmed and polished using a basic metallography polishing technique . ( a ) ( color online ) transverse section of the cervical third cementum after decoronation and ( b ) transverse section of the cervical third cementum before embedding in resin schematic of the sectioning of the cervical third of the root and the cementum surface characterized using nanoindentation and scanning electron microscopy the specimens were polished sequentially using sic grit papers ( 200 - 1000 sizes ) , then fine polishing using diamond suspension slurries ( 9 m , 6 m , 3 m and 1 m ) on a polishing cloth . the local elastic and plastic properties of the cementum were investigated by performing nanoindentation experiments with a three - sided berkovich - type diamond indenter . these tests were conducted under dry conditions with a csm nanoindenter ( peseux , switzerland ) [ figure 3 ] ; during indentation , a load - displacement curve was recorded , from which the contact area , hardness and elastic modulus were calculated using the oliver and pharr method . optical microscope image of the transverse sections of the cervical third cementum open arrow - dentin , white arrow - resin , black arrow showing the impression of the indent in the middle portion of the cervical cementum load / unload cycle parameters used during nanoindentation the mean and standard deviation of the test parameters was estimated for the test and healthy control samples . the sem micrographs of the morphology of the healthy outer surface of the cervical third cementum sections are shown in figure 5 and those of the diseased sections are shown in figure 6 . sem characterization revealed the presence of mineralized collagen fibers in the healthy cementum , which were more predominant when compared with the diseased cementum . the sem micrographs of the diseased cementum showed areas of foreign bodies that could be deposits of calculus . scanning electron microscopy micrograph of the outer surface of the healthy cervical third cementum ( at 6011 magnification ) scanning electron microscopy micrograph of the outer surface of the diseased cervical third cementum section ( at 6011 magnification ) typical indentation profiles of the healthy and diseased cementum cervical sections are shown in figure 7 . ( color online ) typical nanoindentation profiles of healthy and diseased cervical third cementum the results showed that the hardness values varied between 0.546 and 1.124 gpa for the healthy cementum sections and , for the diseased cementum sections , the values ranged from 0.273 to 0.726 gpa . the mean hardness of the diseased cementum was significantly lower compared with the healthy cementum ( p < 0.05 ) [ table 3 ] . the mean hardness values for the cementum of the healthy samples and the diseased cementum sample are shown in figure 8 . nanomechanical properties of the healthy and the diseased cementum comparison of hardness values of healthy and diseased cervical third cementum ( n = 10 ) the modulus of elasticity of the healthy cementum was observed to be between 12.981 and 21.912 gpa and that of the diseased cementum varied between 6.781 and 13.443 gpa [ figure 5 ] . the difference between the mean modulus of elasticity of the healthy and diseased cementum [ table 3 ] was statistically significant ( p < 0.05 ) . the mean modulus of elasticity values for the cementum of the healthy samples and the diseased cementum samples are shown in figure 9 . comparison of modulus of elasticity values of healthy and diseased cervical third cementum ( n = 10 ) the sem micrographs of the morphology of the healthy outer surface of the cervical third cementum sections are shown in figure 5 and those of the diseased sections are shown in figure 6 . sem characterization revealed the presence of mineralized collagen fibers in the healthy cementum , which were more predominant when compared with the diseased cementum . scanning electron microscopy micrograph of the outer surface of the healthy cervical third cementum ( at 6011 magnification ) scanning electron microscopy micrograph of the outer surface of the diseased cervical third cementum section ( at 6011 magnification ) typical indentation profiles of the healthy and diseased cementum cervical sections are shown in figure 7 . ( color online ) typical nanoindentation profiles of healthy and diseased cervical third cementum the results showed that the hardness values varied between 0.546 and 1.124 gpa for the healthy cementum sections and , for the diseased cementum sections , the values ranged from 0.273 to 0.726 gpa . the mean hardness of the diseased cementum was significantly lower compared with the healthy cementum ( p < 0.05 ) [ table 3 ] . the mean hardness values for the cementum of the healthy samples and the diseased cementum sample are shown in figure 8 . nanomechanical properties of the healthy and the diseased cementum comparison of hardness values of healthy and diseased cervical third cementum ( n = 10 ) the modulus of elasticity of the healthy cementum was observed to be between 12.981 and 21.912 gpa and that of the diseased cementum varied between 6.781 and 13.443 gpa [ figure 5 ] . the difference between the mean modulus of elasticity of the healthy and diseased cementum [ table 3 ] was statistically significant ( p < 0.05 ) . the mean modulus of elasticity values for the cementum of the healthy samples and the diseased cementum samples are shown in figure 9 . comparison of modulus of elasticity values of healthy and diseased cervical third cementum ( n = 10 ) the results showed that the hardness values varied between 0.546 and 1.124 gpa for the healthy cementum sections and , for the diseased cementum sections , the values ranged from 0.273 to 0.726 gpa . the mean hardness of the diseased cementum was significantly lower compared with the healthy cementum ( p < 0.05 ) [ table 3 ] . the mean hardness values for the cementum of the healthy samples and the diseased cementum sample are shown in figure 8 . nanomechanical properties of the healthy and the diseased cementum comparison of hardness values of healthy and diseased cervical third cementum ( n = 10 ) the modulus of elasticity of the healthy cementum was observed to be between 12.981 and 21.912 gpa and that of the diseased cementum varied between 6.781 and 13.443 gpa [ figure 5 ] . the difference between the mean modulus of elasticity of the healthy and diseased cementum [ table 3 ] was statistically significant ( p < 0.05 ) . the mean modulus of elasticity values for the cementum of the healthy samples and the diseased cementum samples are shown in figure 9 . comparison of modulus of elasticity values of healthy and diseased cervical third cementum ( n = 10 ) even though regeneration of alveolar bone can be accomplished by the presently available therapeutic modalities , the regeneration of the acellular extrinsic fiber cementum still remains elusive . an important principle in functional tissue engineering is the determination of the biomechanical properties of the native tissue in health and in diseased conditions . in the case of periodontal tissue engineering , this information can be obtained by analyzing the mechanical properties of both the healthy and the diseased root surface . this study was undertaken to investigate the effect of chronic periodontitis on the nanomechanical properties of the cervical third of the cementum within a periodontal pocket environment . in order to evaluate the changes in the morphology of the diseased cementum the topography of the outer surface of the healthy and diseased transverse sections of the cervical third of the cementum was characterized using high - resolution sem . sem revealed morphological changes in the outer surface of the diseased cementum such as the absence of mineralized cemental collagen fibers that were a predominant feature in the healthy cementum sections . previous studies have estimated hardness and modulus of elasticity of healthy cementum . to the best our knowledge , this is the first study comparing the hardness and modulus of elasticity between healthy and diseased transverse sections of the cervical third cementum using depth - sensing nanoindentation techniques . the results of our study revealed a statistically significant difference in the hardness of the diseased cementum as compared with the healthy cementum ( p < 0.05 ) . this decrease in hardness of the diseased cementum could be due to the softening of the cementum induced by demineralization by organic acids of inflammatory exudates and resorption of collagen and protein polysaccharide matrix via enzyme activities within the confines of the periodontal pocket . in order to avoid the hypermineralized layer of cementum that forms when the root surface becomes exposed to the oral cavity due to gingival recession , periodontally compromised teeth with absence of gingival recession another important factor that must be taken into account is the total duration each of these diseased cementum sections had been exposed to the periodontal pocket environment since the onset of the periodontitis . there was a statistically significant ( p < 0.05 ) difference in the modulus of elasticity of the diseased cervical third cementum as compared with the healthy cementum . the results of our study are in agreement with various other studies that have characterized similar cemental sections from healthy teeth under similar conditions . , a cementum - like biomineralized microlayer was constructed using amelogenin - derived peptides and the authors compared the mechanical properties , namely modulus of elasticity and hardness using nanoindentation , with that of the native cementum . the hardness and modulus of elasticity values for the native healthy cementum obtained in their study are in accordance to the values observed in our study . it has been used in the field of endodontics to determine the nanomechanical properties of endodontically treated teeth and carious human teeth , in implantology to study the elasticity of the alveolar bone near the dental implant and in orthodontics . in the field of periodontics , nanoindentation has been used to determine the nanomechanical properties of the cement - dentinal junction , cementum in ank / ank mutant mouse bone - periodontal ligament and cementum complex . in addition , it allows the measurement of the mechanical properties of a very small selected region of the cementum . although there are few reports in the literature that have assessed the hardness of the cementum , most of these studies have used micromechanical testing techniques . also , healthy human dental cementum of premolar teeth analyzed using microindentation showed no significant differences in the hardness and elastic modulus of the cementum between the buccal and the lingual surfaces or between the upper and the lower teeth the importance of cervical third of the cementum is that it contains acellular extrinsic fiber cementum and its regeneration is considered to be the gold standard for periodontal regeneration . this can be extrapolated to the cementum , wherein an alteration of the nanomechanical properties during the progression of periodontal disease process may impede complete periodontal regeneration . the prime objective of this study was to determine the changes in the nanomechanical properties of the cervical third of the cementum in chronic periodontitis based on the values obtained from a total of 200 nanoindentations . because only 10 healthy cementum sections and 10 diseased cementum sections were taken for nanoindentation , a tooth - type statistical comparison was not possible however , further studies can focus on comparing the nanomechanical properties based on tooth type / upper arch and lower arch . the values obtained in the present study for the diseased cervical third cementum are found to be lesser than the healthy cementum sections , indicating a change in the nanostructure and mechanical integrity . thus , the understanding of the nanomechanical properties of the cervical third cementum may not only aid in determining the influence of mechanical signals of cementum in health and in chronic periodontitis on progenitor cells but also help in devising various nanomechanical design parameters required for engineering acellular extrinsic fiber cementum . in conclusion , there is a decrease in the nanomechanical properties of the diseased cervical third cementum . further analysis of the diseased root surface in wet conditions may help in understanding the nanostructural changes occurring in the cervical third of the root during periodontitis .

collection of high - resolution phenotypic data is useful in studies that aim to understand the interplay of genetics and environment in mediating organismal function . studies of this nature are also inherently large in scale , making it additionally necessary that methods employed for measuring phenotypes in this context be high in throughput . in establishing methods for phenomics - scale research , methods that are higher in throughput also tend to be lower in resolution , making it more difficult to detect small effects of genetics or environment . alternatively , methods that more carefully measure a desired phenotype also tend to be lower in throughput , making it difficult to survey genetic and environmental effects broadly . additionally , manual methods for quantifying phenotypes , including visual inspection , can be subject to variation due to differences in human perception . imaging technologies can provide a useful bridge between throughput and resolution in obtaining phenotypic observations . in general , an image is relatively easy to capture , facilitating throughput , and when taken at sufficient resolution , subtle phenotypes can be detected . imaging technologies tend to be modifiable to fit a system or process of interest and are generally scalable . because of this , imaging technologies are ideal for the development of large - scale studies of organismal function . the response of the primary root to a gravity stimulus is an intricate physiological process that occurs within a morphologically simple organ . the response involves activation of signaling pathways that propagate through the root organ and its progression is determined by environmental and genetic factors , including genetic factors influenced by the environment . the response of the primary root to a gravity stimulus has been studied at least since darwin , yet there is much to learn about how it works , particularly in the early signaling events and in the factors mediating response plasticity . gaining a detailed understanding of the dynamics of this response is important in finding ways to improve the ability of seedlings to successfully become established within a given environment . in addition , the shape of the root makes it amenable for image processing applications . taken together , the root gravitropic response is an ideal system for the development of high - throughput imaging technology for the purpose of conducting genomics - level studies of organismal function . in this report , a high - throughput , high - resolution method for image capture of the root gravitropic response using inexpensive , commercially - available flatbed scanners is presented . seedlings planted on agar plates were positioned on vertically - oriented flatbed scanners fitted with custom plexiglas plate holders . images were collected every few minutes at 4,800 dpi and saved on a local drive or data server . metadata associated with each image series is stored on a database and the stored images are processed . vuescan can be used to run over 2,100 different scanners on windows , mac , or linux operating systems ( see materials table ) . a scanner resolution of 4,800 dpi was used in this application to match the resolution achieved in previous studies using fixed ccd cameras . the flexibility of the vuescan software along with the common interface it uses for any scanner it runs allows users to readily adopt virtually any scanner hardware of sufficient resolution to the protocol presented in this paper . the technology is adaptable and scalable for use at institutions ranging from high schools to research universities . this protocol is most efficiently performed with two people , although it is possible for one to work alone . the arrangement working best in this laboratory was for one person to prepare plates for scanning while another works on scanner setup , then both work together to place plates in scanners and start the scanning process . it 's also important to note that the scanners in this project are vertically oriented with the scanner lids resting on the back of the scanner . a custom support was made to hold dishes in this vertical position and was affixed to the flatbed surface with 3 m command strips ( figure 2 ) . the removable document cover that comes with the scanner used in this protocol ( an epson v700 ) was lined on one side with black felt . the document cover was positioned against the flatbed with a bungee cord to hold the plates in place and to provide image contrast ( figure 3 ) . the epson perfection v700 was chosen because of its square profile ( making it easy to position vertically ) , its high resolution , and the additional options to scan from the both the bed and lid and to use the infrared channel . once the plates have been removed from the growth chamber , it is imperative that the protocol continue to the end . standard petri dishes containing 10 ml of transparent medium and 9 seeds planted across the middle of each plate were used . procedures for plate labeling , media preparation and planting can be found at : http://www.doane.edu/doane-phytomorph retrieve the first agar plate and absorb collected condensation on the lid and rim of the lid of the agar plate with a kimwipe . apply triton x-100 ( a detergent ) to lid with a kimwipe -- be generous . ( note that triton x-100 helps prevent the buildup of condensation on the lid as the plate is scanned . a generous application ( enough to create a film on the lid surface ) will help make sure that the lid stays transparent throughout the entire scanner run . ) wrap the plate with micropore tape to secure the lid , and to allow for ventilation . scanner setup and image collection this protocol assumes that more than 1 scanner is being used , and provides instructions to start multiple scanners from a single computer . each scanner will hold two plates , so keep this in mind when creating folders . one might choose to use metadata as components of the file name such as unique ids for each plate , seedling ages , seed size , and ids of stocks planted . an example of a folder name used in data collection containing these metadata is " 1652 - 2-sm-9 - 92 - 17 - 1653 - 2-lg-88 - 79 - 161 " . set outlet timers for designated collecting time ( 9 hr was used in this laboratory ) . ( note that scanners should be plugged into outlet timers in order to set the acquisition time . while the vuescan software allows a user to collect images repeatedly , it does not allow the user to indicate how many images to collect or how long to collect images for . ) turn on the first scanner and wait approximately 10 sec for the scanner to go through its initial warm - ups . vuescan version 9.0.20 was used in this protocol ( see materials table ) , though more recent versions can be used with little modification . make sure the ' more ' button has been pressed on the bottom panel of the user interface in order to display the menu options described below . set the auto repeat : drop - down box to none under the input tab and under the crop tab set preview area : to maximum ( figure 4 ) . create a crop box that would capture the region of interest by using the mouse to click and drag across the region of interest on the preview image . the typical settings used for the crop box were : x - offset 0.675 ; y - offset 1.924 in , though this was adjusted to capture the seedling area for each scanner . the crop box size used was 7.246 in wide by 1.1 in tall ( figure 5 ) . to move the crop box , hold the shift key while dragging with the mouse . make sure the crop box contains all the seedlings to be scanned plus any desired metadata that might be contained on a label ( figure 5 ) . under the crop tab , go to the output tab and select the correct file for the scanner ( figure 5 ) . repeat steps 1.7 - 1.12 on all scanners for one computer . choose the ' yes ' option when asked whether to open more than one instance of vuescan . ( note that all specifications can be altered to fit the needs of an individual laboratory including image color , resolution , etc . however , the settings used in this protocol can be directly applied to the particular scanning hardware of a given lab due to the common interface of the vuescan software . refer to the attached specifications list to view the parameters used in this project , using vuescan version 9.0.20 ) . under the input tab choose continuous in the auto repeat : field , or choose a longer time interval between images if desired . the time interval is the length of time the scanner pauses after saving the last image and beginning collection of the next image . in continuous mode , 3 - 4 min resolution repeat steps 1.14 - 1.15 for the rest of the scanners connected to a single computer . place prepared plates in the correct scanners with seedlings oriented horizontally ( do not gravistimulate ) . temporarily place a black , felt background against the plates so they do not fall from plexiglas template . ( note : in this project , black pieces of felt were attached to the document covers provided with the equipment to prevent glare and to provide contrast against root tissue . the specific background color used will depend on the color of tissue being imaged ) . have one person turn the plates 90 ( plates were turned counterclockwise in this protocol ) and immediately replace the felt background . the other person should be standing at the computer so that they can immediately press the ' scan ' button . ( note : immediately after gravistimulation ( rotation of the plates by 90 ) and placement of the felt background , ' scan ' should be pressed ) . repeat steps 1.17 - 1.21 for the rest of the scanners on a single computer . repeat steps 1.6 - 1.22 for the next set of scanners if applicable . do not leave the scanners until several images have been collected to make sure they are saving correctly . it is ideal to keep the scanners in an area that will be free of disturbances for the designated scan time . it is also prudent to consider the environmental conditions in the scanning area to ensure ideal phenotypic responses . when data collection is complete , press the green abort button on each vuescan window that coincides with each scanner . close out of all programs on the computer . restart the computer and shut off all the scanners before beginning another round of image collection . this protocol is most efficiently performed with two people , although it is possible for one to work alone . the arrangement working best in this laboratory was for one person to prepare plates for scanning while another works on scanner setup , then both work together to place plates in scanners and start the scanning process . it 's also important to note that the scanners in this project are vertically oriented with the scanner lids resting on the back of the scanner . a custom support was made to hold dishes in this vertical position and was affixed to the flatbed surface with 3 m command strips ( figure 2 ) . the removable document cover that comes with the scanner used in this protocol ( an epson v700 ) was lined on one side with black felt . the document cover was positioned against the flatbed with a bungee cord to hold the plates in place and to provide image contrast ( figure 3 ) . the epson perfection v700 was chosen because of its square profile ( making it easy to position vertically ) , its high resolution , and the additional options to scan from the both the bed and lid and to use the infrared channel . once the plates have been removed from the growth chamber , it is imperative that the protocol continue to the end . standard petri dishes containing 10 ml of transparent medium and 9 seeds planted across the middle of each plate were used . procedures for plate labeling , media preparation and planting can be found at : http://www.doane.edu/doane-phytomorph retrieve the first agar plate and absorb collected condensation on the lid and rim of the lid of the agar plate with a kimwipe . apply triton x-100 ( a detergent ) to lid with a kimwipe -- be generous . ( note that triton x-100 helps prevent the buildup of condensation on the lid as the plate is scanned . a generous application ( enough to create a film on the lid surface ) will help make sure that the lid stays transparent throughout the entire scanner run . ) wrap the plate with micropore tape to secure the lid , and to allow for ventilation . scanner setup and image collection this protocol assumes that more than 1 scanner is being used , and provides instructions to start multiple scanners from a single computer . each scanner will hold two plates , so keep this in mind when creating folders . one might choose to use metadata as components of the file name such as unique ids for each plate , seedling ages , seed size , and ids of stocks planted . an example of a folder name used in data collection containing these metadata is " 1652 - 2-sm-9 - 92 - 17 - 1653 - 2-lg-88 - 79 - 161 " . set outlet timers for designated collecting time ( 9 hr was used in this laboratory ) . ( note that scanners should be plugged into outlet timers in order to set the acquisition time . while the vuescan software allows a user to collect images repeatedly , it does not allow the user to indicate how many images to collect or how long to collect images for . ) turn on the first scanner and wait approximately 10 sec for the scanner to go through its initial warm - ups . vuescan version 9.0.20 was used in this protocol ( see materials table ) , though more recent versions can be used with little modification . make sure the ' more ' button has been pressed on the bottom panel of the user interface in order to display the menu options described below . set the auto repeat : drop - down box to none under the input tab and under the crop tab set preview area : to maximum ( figure 4 ) . press ' preview ' . create a crop box that would capture the region of interest by using the mouse to click and drag across the region of interest on the preview image . the typical settings used for the crop box were : x - offset 0.675 ; y - offset 1.924 in , though this was adjusted to capture the seedling area for each scanner . the crop box size used was 7.246 in wide by 1.1 in tall ( figure 5 ) . to move the crop box , hold the shift key while dragging with the mouse . make sure the crop box contains all the seedlings to be scanned plus any desired metadata that might be contained on a label ( figure 5 ) . under the crop tab , go to the output tab and select the correct file for the scanner ( figure 5 ) . repeat steps 1.7 - 1.12 on all scanners for one computer . choose the ' yes ' option when asked whether to open more than one instance of vuescan . ( note that all specifications can be altered to fit the needs of an individual laboratory including image color , resolution , etc . however , the settings used in this protocol can be directly applied to the particular scanning hardware of a given lab due to the common interface of the vuescan software . refer to the attached specifications list to view the parameters used in this project , using vuescan version 9.0.20 ) . under the input tab choose continuous in the auto repeat : field , or choose a longer time interval between images if desired . the time interval is the length of time the scanner pauses after saving the last image and beginning collection of the next image . in continuous mode , 3 - 4 min resolution repeat steps 1.14 - 1.15 for the rest of the scanners connected to a single computer . place prepared plates in the correct scanners with seedlings oriented horizontally ( do not gravistimulate ) . temporarily place a black , felt background against the plates so they do not fall from plexiglas template . ( note : in this project , black pieces of felt were attached to the document covers provided with the equipment to prevent glare and to provide contrast against root tissue . the specific background color used will depend on the color of tissue being imaged ) . have one person turn the plates 90 ( plates were turned counterclockwise in this protocol ) and immediately replace the felt background . the other person should be standing at the computer so that they can immediately press the ' scan ' button . ( note : immediately after gravistimulation ( rotation of the plates by 90 ) and placement of the felt background , ' scan ' should be pressed ) . repeat steps 1.17 - 1.21 for the rest of the scanners on a single computer . repeat steps 1.6 - 1.22 for the next set of scanners if applicable . do not leave the scanners until several images have been collected to make sure they are saving correctly . it is ideal to keep the scanners in an area that will be free of disturbances for the designated scan time . it is also prudent to consider the environmental conditions in the scanning area to ensure ideal phenotypic responses . when data collection is complete , press the green abort button on each vuescan window that coincides with each scanner . restart the computer and shut off all the scanners before beginning another round of image collection . representative images this approach enables rapid production of high - resolution time series of arabidopsis seedling growth . first and last images of a scanner run are shown in figures 7a and 7b . these issues include variation in germination , variation in seedling growth trajectory at the start of the run , and buildup of condensation during scanning . condensation can largely be resolved by increasing the amount of triton x-100 applied to the inside of the plate lid . other factors that could inhibit accurate image collection are incorrect configuration of the crop box with respect to the plate position and positioning plates such that they are skewed with respect to the crop box . image analysis application : image compression once a time sequence of scanner images has been obtained , it must be securely stored in a network accessible manner to facilitate image analysis . the image files associated with an individual scanner run occupy a significant amount of hard drive space . a single tiff file collected at 4,800 therefore , about 44 gb of hard drive space is required per run . to reduce storage and network transmission costs associated with image analysis it is desirable to reduce the amount of space needed to store image data while at the same time minimizing data loss . downstream analysis will involve identification of each seedling in subsequent image files associated with an experimental run . because segmentation of the seedling away from the rest of the image can also significantly reduce storage of unnecessary background pixels , this approach also leads to significant reduction in data size . furthermore , if downstream analysis is focused on root tissue it may not be necessary to retain color information since the root pixels are relatively narrow in their color space . a computer image processing protocol and code to reduce data size by both the workflow used to achieve this data compression is described in the following steps : start with a time series of scanner image files in a single folder . for each image , convert from an rgb to grey scale ( figure 8 , top ) . this is done by applying a threshold to convert pixels to black or white and then calculating the total pixel intensity of each image row . the row with the highest intensity is identified and each pixel is classified as ' plant ' or ' nonplant ' based on the intensity of its neighbors . the center of each ' plant ' within this row is found and from that point a crop box of a predetermined size is drawn ( figure 8 , bottom ) . create a separate folder for each side of the image ( left and right ) with separate subfolders for each seedling for storage of individual time series image files . the algorithm allows for an approximately 60% reduction in data size and is successful in in identifying all individual seedlings in 90% of the scanner image files analyzed thus far . the codes are freely available for download under the gnu general public license version 3 ( see materials table ) . the scanning procedure begins with seed planting ( up to nine arabidopsis seeds per plate ) and ends with data storage and image processing . plexiglas was cut such that the width fit the flatbed ( in this case 227 mm ) and the length was 128 mm . two circles with an 88 mm diameter were cut out of the remaining piece such that they were evenly distributed along the width and length of the support . this is the configuration of the scanner at step 1.21 of scanner setup and image collection . screen shot of vuescan software during steps 1.9 and 1.10 of scanner setup and image collection . the red box highlights the crop size while the blue box highlights specific settings for x- and y - offset used in order to capture seedlings and label information . the region of the flatbed to be scanned is shown as a dotted line in the preview area . pressing the @ button next to the default folder dialog box ( red arrow ) allows the user to select the appropriate destination folder . the above images are examples of those collected using the method described in this paper . panels a , b and c , d are the first and final images , respectively , from a single scan period . a , b show the full scanned area , while c , d are a cropped region of the scanned area , showing a single plate . panel b ( the same seedlings as image a ; 9 hr later ) shows that plates can accumulate condensation . panels c and d are considered to be good results due to robust growth of seedlings and image quality throughout the run . the image compression algorithm developed converts a scanner image to grey scale ( top ) . the image is divided into right and left halves and image borders are removed ( not shown ) . the positions of individual seedlings on each half are identified by finding the row with the largest total pixel intensity . those positions are used to define a new crop area , applied to all seedlings on the plate ( bottom ) . accurate phenotypic observation is crucial for understanding the manifestations of gene function within an organism . one way to acquire phenotypic information is through the capture of high - resolution image data . the scanner - based platform developed has enabled collection of many images ( 200 images / scan period ) at high - resolution ( 4,800 dpi ) over a number of hours . additionally , this platform is easily adapted to a variety of lab and classroom environments due to the flexibility of the vuescan software to run thousands of different scanners using a common interface . the method presented here fills a void in high - throughput image capture that extends from large scale phenotyping facilities and automated systems implementable in a single laboratory . the high - throughput platforms currently available tend to use specialized imaging hardware , including cameras mounted on robotic supports , to capture high - resolution images of primarily above ground plant tissues ( e.g. centre for plant integrative technology and the scanalyzer hts by lemnatec ) . specialized imaging systems using x - ray and mri technologies have also been developed to image below ground tissues with remarkable resolution as they grow in the soil environment ( e.g. centre for plant integrative technology ) . this development of more specialized technology is generally at the cost of throughput , making dynamic phenotypic studies more difficult . importantly , the cost and infrastructure needs for these high - end platforms make them mostly unfeasible for implementation in smaller laboratories . platforms have also been developed which use more standard image capture technology and are well suited to the measurement of dynamic responses such as the root response to a gravity stimulus . for example , ccd cameras have been used to capture individual seedling responses to light and gravity at high spatial and temporal resolution . other systems have been developed allowing measurement of root tip orientation of multiple roots from a single image ( e.g. roottipmulti by the iplant collaborative ) . in the former case , throughput is relatively low given that only one seedling is imaged by each camera at a time , while in the latter case throughput is higher , but generally at the cost of resolution . the procedure outlined in this paper presents a platform for capturing high - resolution images in high throughput with equipment and software that are readily available and relatively affordable . using this setup , 1,080 individual root responses can be collected each week in a single lab equipped with a bank of six scanners . in 15 months of collecting an average of 864 individual responses per week , about 15% of the individual collections failed due to setup error , network failure or equipment malfunction . another 22% responses failed due to lack of germination or insufficient root growth to elicit a growth response . the final data set consists of 27,475 individual seedling responses to a gravity stimulus from 163 recombinant inbred lines plus 99 near isogenic lines . the data were collected in a single laboratory , making this a very high - throughput approach . even given that the equipment used for acquisition is relatively inexpensive , it has functioned reliably for over two years even with heavy usage . while this protocol has been very useful for the research because of the throughput of about 50 gb of uncompressed image data per day , it was apparent that a large amount of space was needed to house images unless effective compression schemes could be developed . the storage problem was temporarily solved by purchasing external hard drives for each computer . in addition , later , compression algorithms were developed , as described above , which can help reduce the data size by up to 60% ( figure 8) . it is important to note that the speed at which data can be saved to a network associated storage device is dependent on the speed of the network connection . compression schemes have also been constrained due to the desire to prevent loss of image data . for example , in a scanner - based approach seedlings are exposed to high intensity light in the white and potentially infrared ranges during each scan . this likely affects seedling growth , though seedlings can still be observed to undergo robust responses to a gravity stimulus ( figure 7 ) . an area in active development is creation of analysis algorithms well matched to the resolution and throughput of these image data . the large data set generated using this scanner - based method has been ideal for development of robust tools for high - throughput phenotyping of seedling images . the compression algorithm employed on these images shown in figure 7 supports the claim that they are amenable to image analysis applications . additionally , the images generated can be analyzed by the previously published algorithm , roottrace , if they are collected at lower resolution ( less than 1,200 dpi ) , and individual seedlings are segmented from the image using the compression algorithm described above before analysis . root growth data could be extracted from images reduced to 1,200 dpi while tip angle data could be extracted from images reduced to 900 dpi ( unpublished observation ) . the procedure outlined in this paper fits into its own niche in the world of root imaging in that it is high throughput and high resolution while still being relatively affordable . an additional benefit of this approach is that it can easily be customized to accommodate the imaging needs of a particular research group .

research efforts in biology increasingly require use of methodologies that enable high - volume collection of high - resolution data . a challenge laboratories can face is the development and attainment of these methods . observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture . a particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector . imaging platforms used to measure the root gravitropic response can be expensive , relatively low in throughput , and/or labor intensive . these issues have been addressed by developing a high - throughput image capture method using inexpensive , yet high - resolution , flatbed scanners . using this method , images can be captured every few minutes at 4,800 dpi . the current setup enables collection of 216 individual responses per day . the image data collected is of ample quality for image analysis applications .

Introduction Protocol 1. Image Acquisition Protocol Plate Preparation Representative Results Discussion Disclosures

collection of high - resolution phenotypic data is useful in studies that aim to understand the interplay of genetics and environment in mediating organismal function . studies of this nature are also inherently large in scale , making it additionally necessary that methods employed for measuring phenotypes in this context be high in throughput . in establishing methods for phenomics - scale research , methods that are higher in throughput also tend to be lower in resolution , making it more difficult to detect small effects of genetics or environment . alternatively , methods that more carefully measure a desired phenotype also tend to be lower in throughput , making it difficult to survey genetic and environmental effects broadly . additionally , manual methods for quantifying phenotypes , including visual inspection , can be subject to variation due to differences in human perception . in general , an image is relatively easy to capture , facilitating throughput , and when taken at sufficient resolution , subtle phenotypes can be detected . imaging technologies tend to be modifiable to fit a system or process of interest and are generally scalable . because of this , imaging technologies are ideal for the development of large - scale studies of organismal function . the response of the primary root to a gravity stimulus is an intricate physiological process that occurs within a morphologically simple organ . the response involves activation of signaling pathways that propagate through the root organ and its progression is determined by environmental and genetic factors , including genetic factors influenced by the environment . the response of the primary root to a gravity stimulus has been studied at least since darwin , yet there is much to learn about how it works , particularly in the early signaling events and in the factors mediating response plasticity . gaining a detailed understanding of the dynamics of this response is important in finding ways to improve the ability of seedlings to successfully become established within a given environment . in addition , the shape of the root makes it amenable for image processing applications . taken together , the root gravitropic response is an ideal system for the development of high - throughput imaging technology for the purpose of conducting genomics - level studies of organismal function . in this report , a high - throughput , high - resolution method for image capture of the root gravitropic response using inexpensive , commercially - available flatbed scanners is presented . seedlings planted on agar plates were positioned on vertically - oriented flatbed scanners fitted with custom plexiglas plate holders . images were collected every few minutes at 4,800 dpi and saved on a local drive or data server . vuescan can be used to run over 2,100 different scanners on windows , mac , or linux operating systems ( see materials table ) . a scanner resolution of 4,800 dpi was used in this application to match the resolution achieved in previous studies using fixed ccd cameras . the flexibility of the vuescan software along with the common interface it uses for any scanner it runs allows users to readily adopt virtually any scanner hardware of sufficient resolution to the protocol presented in this paper . the technology is adaptable and scalable for use at institutions ranging from high schools to research universities . it 's also important to note that the scanners in this project are vertically oriented with the scanner lids resting on the back of the scanner . the removable document cover that comes with the scanner used in this protocol ( an epson v700 ) was lined on one side with black felt . the epson perfection v700 was chosen because of its square profile ( making it easy to position vertically ) , its high resolution , and the additional options to scan from the both the bed and lid and to use the infrared channel . once the plates have been removed from the growth chamber , it is imperative that the protocol continue to the end . procedures for plate labeling , media preparation and planting can be found at : http://www.doane.edu/doane-phytomorph retrieve the first agar plate and absorb collected condensation on the lid and rim of the lid of the agar plate with a kimwipe . a generous application ( enough to create a film on the lid surface ) will help make sure that the lid stays transparent throughout the entire scanner run . ) wrap the plate with micropore tape to secure the lid , and to allow for ventilation . scanner setup and image collection this protocol assumes that more than 1 scanner is being used , and provides instructions to start multiple scanners from a single computer . each scanner will hold two plates , so keep this in mind when creating folders . an example of a folder name used in data collection containing these metadata is " 1652 - 2-sm-9 - 92 - 17 - 1653 - 2-lg-88 - 79 - 161 " . ( note that scanners should be plugged into outlet timers in order to set the acquisition time . while the vuescan software allows a user to collect images repeatedly , it does not allow the user to indicate how many images to collect or how long to collect images for . ) turn on the first scanner and wait approximately 10 sec for the scanner to go through its initial warm - ups . vuescan version 9.0.20 was used in this protocol ( see materials table ) , though more recent versions can be used with little modification . make sure the ' more ' button has been pressed on the bottom panel of the user interface in order to display the menu options described below . create a crop box that would capture the region of interest by using the mouse to click and drag across the region of interest on the preview image . the typical settings used for the crop box were : x - offset 0.675 ; y - offset 1.924 in , though this was adjusted to capture the seedling area for each scanner . the crop box size used was 7.246 in wide by 1.1 in tall ( figure 5 ) . to move the crop box , hold the shift key while dragging with the mouse . ( note that all specifications can be altered to fit the needs of an individual laboratory including image color , resolution , etc . however , the settings used in this protocol can be directly applied to the particular scanning hardware of a given lab due to the common interface of the vuescan software . under the input tab choose continuous in the auto repeat : field , or choose a longer time interval between images if desired . the time interval is the length of time the scanner pauses after saving the last image and beginning collection of the next image . place prepared plates in the correct scanners with seedlings oriented horizontally ( do not gravistimulate ) . ( note : in this project , black pieces of felt were attached to the document covers provided with the equipment to prevent glare and to provide contrast against root tissue . the specific background color used will depend on the color of tissue being imaged ) . have one person turn the plates 90 ( plates were turned counterclockwise in this protocol ) and immediately replace the felt background . the other person should be standing at the computer so that they can immediately press the ' scan ' button . ( note : immediately after gravistimulation ( rotation of the plates by 90 ) and placement of the felt background , ' scan ' should be pressed ) . repeat steps 1.17 - 1.21 for the rest of the scanners on a single computer . repeat steps 1.6 - 1.22 for the next set of scanners if applicable . do not leave the scanners until several images have been collected to make sure they are saving correctly . it is ideal to keep the scanners in an area that will be free of disturbances for the designated scan time . close out of all programs on the computer . it 's also important to note that the scanners in this project are vertically oriented with the scanner lids resting on the back of the scanner . the removable document cover that comes with the scanner used in this protocol ( an epson v700 ) was lined on one side with black felt . the epson perfection v700 was chosen because of its square profile ( making it easy to position vertically ) , its high resolution , and the additional options to scan from the both the bed and lid and to use the infrared channel . once the plates have been removed from the growth chamber , it is imperative that the protocol continue to the end . procedures for plate labeling , media preparation and planting can be found at : http://www.doane.edu/doane-phytomorph retrieve the first agar plate and absorb collected condensation on the lid and rim of the lid of the agar plate with a kimwipe . apply triton x-100 ( a detergent ) to lid with a kimwipe -- be generous . ( note that triton x-100 helps prevent the buildup of condensation on the lid as the plate is scanned . a generous application ( enough to create a film on the lid surface ) will help make sure that the lid stays transparent throughout the entire scanner run . ) each scanner will hold two plates , so keep this in mind when creating folders . one might choose to use metadata as components of the file name such as unique ids for each plate , seedling ages , seed size , and ids of stocks planted . an example of a folder name used in data collection containing these metadata is " 1652 - 2-sm-9 - 92 - 17 - 1653 - 2-lg-88 - 79 - 161 " . set outlet timers for designated collecting time ( 9 hr was used in this laboratory ) . ( note that scanners should be plugged into outlet timers in order to set the acquisition time . turn on the first scanner and wait approximately 10 sec for the scanner to go through its initial warm - ups . vuescan version 9.0.20 was used in this protocol ( see materials table ) , though more recent versions can be used with little modification . make sure the ' more ' button has been pressed on the bottom panel of the user interface in order to display the menu options described below . set the auto repeat : drop - down box to none under the input tab and under the crop tab set preview area : to maximum ( figure 4 ) . press ' preview ' . create a crop box that would capture the region of interest by using the mouse to click and drag across the region of interest on the preview image . the typical settings used for the crop box were : x - offset 0.675 ; y - offset 1.924 in , though this was adjusted to capture the seedling area for each scanner . the crop box size used was 7.246 in wide by 1.1 in tall ( figure 5 ) . to move the crop box , hold the shift key while dragging with the mouse . make sure the crop box contains all the seedlings to be scanned plus any desired metadata that might be contained on a label ( figure 5 ) . ( note that all specifications can be altered to fit the needs of an individual laboratory including image color , resolution , etc . however , the settings used in this protocol can be directly applied to the particular scanning hardware of a given lab due to the common interface of the vuescan software . the time interval is the length of time the scanner pauses after saving the last image and beginning collection of the next image . ( note : in this project , black pieces of felt were attached to the document covers provided with the equipment to prevent glare and to provide contrast against root tissue . the other person should be standing at the computer so that they can immediately press the ' scan ' button . ( note : immediately after gravistimulation ( rotation of the plates by 90 ) and placement of the felt background , ' scan ' should be pressed ) . repeat steps 1.17 - 1.21 for the rest of the scanners on a single computer . repeat steps 1.6 - 1.22 for the next set of scanners if applicable . do not leave the scanners until several images have been collected to make sure they are saving correctly . it is ideal to keep the scanners in an area that will be free of disturbances for the designated scan time . when data collection is complete , press the green abort button on each vuescan window that coincides with each scanner . restart the computer and shut off all the scanners before beginning another round of image collection . representative images this approach enables rapid production of high - resolution time series of arabidopsis seedling growth . first and last images of a scanner run are shown in figures 7a and 7b . these issues include variation in germination , variation in seedling growth trajectory at the start of the run , and buildup of condensation during scanning . condensation can largely be resolved by increasing the amount of triton x-100 applied to the inside of the plate lid . other factors that could inhibit accurate image collection are incorrect configuration of the crop box with respect to the plate position and positioning plates such that they are skewed with respect to the crop box . image analysis application : image compression once a time sequence of scanner images has been obtained , it must be securely stored in a network accessible manner to facilitate image analysis . the image files associated with an individual scanner run occupy a significant amount of hard drive space . a single tiff file collected at 4,800 therefore , about 44 gb of hard drive space is required per run . to reduce storage and network transmission costs associated with image analysis it is desirable to reduce the amount of space needed to store image data while at the same time minimizing data loss . because segmentation of the seedling away from the rest of the image can also significantly reduce storage of unnecessary background pixels , this approach also leads to significant reduction in data size . furthermore , if downstream analysis is focused on root tissue it may not be necessary to retain color information since the root pixels are relatively narrow in their color space . a computer image processing protocol and code to reduce data size by both the workflow used to achieve this data compression is described in the following steps : start with a time series of scanner image files in a single folder . for each image , convert from an rgb to grey scale ( figure 8 , top ) . this is done by applying a threshold to convert pixels to black or white and then calculating the total pixel intensity of each image row . the row with the highest intensity is identified and each pixel is classified as ' plant ' or ' nonplant ' based on the intensity of its neighbors . the center of each ' plant ' within this row is found and from that point a crop box of a predetermined size is drawn ( figure 8 , bottom ) . create a separate folder for each side of the image ( left and right ) with separate subfolders for each seedling for storage of individual time series image files . the codes are freely available for download under the gnu general public license version 3 ( see materials table ) . this is the configuration of the scanner at step 1.21 of scanner setup and image collection . the region of the flatbed to be scanned is shown as a dotted line in the preview area . the above images are examples of those collected using the method described in this paper . panel b ( the same seedlings as image a ; 9 hr later ) shows that plates can accumulate condensation . panels c and d are considered to be good results due to robust growth of seedlings and image quality throughout the run . the image compression algorithm developed converts a scanner image to grey scale ( top ) . the image is divided into right and left halves and image borders are removed ( not shown ) . the positions of individual seedlings on each half are identified by finding the row with the largest total pixel intensity . those positions are used to define a new crop area , applied to all seedlings on the plate ( bottom ) . accurate phenotypic observation is crucial for understanding the manifestations of gene function within an organism . one way to acquire phenotypic information is through the capture of high - resolution image data . the scanner - based platform developed has enabled collection of many images ( 200 images / scan period ) at high - resolution ( 4,800 dpi ) over a number of hours . additionally , this platform is easily adapted to a variety of lab and classroom environments due to the flexibility of the vuescan software to run thousands of different scanners using a common interface . the method presented here fills a void in high - throughput image capture that extends from large scale phenotyping facilities and automated systems implementable in a single laboratory . the high - throughput platforms currently available tend to use specialized imaging hardware , including cameras mounted on robotic supports , to capture high - resolution images of primarily above ground plant tissues ( e.g. centre for plant integrative technology and the scanalyzer hts by lemnatec ) . specialized imaging systems using x - ray and mri technologies have also been developed to image below ground tissues with remarkable resolution as they grow in the soil environment ( e.g. this development of more specialized technology is generally at the cost of throughput , making dynamic phenotypic studies more difficult . importantly , the cost and infrastructure needs for these high - end platforms make them mostly unfeasible for implementation in smaller laboratories . platforms have also been developed which use more standard image capture technology and are well suited to the measurement of dynamic responses such as the root response to a gravity stimulus . for example , ccd cameras have been used to capture individual seedling responses to light and gravity at high spatial and temporal resolution . other systems have been developed allowing measurement of root tip orientation of multiple roots from a single image ( e.g. roottipmulti by the iplant collaborative ) . in the former case , throughput is relatively low given that only one seedling is imaged by each camera at a time , while in the latter case throughput is higher , but generally at the cost of resolution . the procedure outlined in this paper presents a platform for capturing high - resolution images in high throughput with equipment and software that are readily available and relatively affordable . using this setup , 1,080 individual root responses can be collected each week in a single lab equipped with a bank of six scanners . in 15 months of collecting an average of 864 individual responses per week , about 15% of the individual collections failed due to setup error , network failure or equipment malfunction . another 22% responses failed due to lack of germination or insufficient root growth to elicit a growth response . the data were collected in a single laboratory , making this a very high - throughput approach . even given that the equipment used for acquisition is relatively inexpensive , it has functioned reliably for over two years even with heavy usage . while this protocol has been very useful for the research because of the throughput of about 50 gb of uncompressed image data per day , it was apparent that a large amount of space was needed to house images unless effective compression schemes could be developed . the storage problem was temporarily solved by purchasing external hard drives for each computer . in addition , later , compression algorithms were developed , as described above , which can help reduce the data size by up to 60% ( figure 8) . it is important to note that the speed at which data can be saved to a network associated storage device is dependent on the speed of the network connection . compression schemes have also been constrained due to the desire to prevent loss of image data . for example , in a scanner - based approach seedlings are exposed to high intensity light in the white and potentially infrared ranges during each scan . an area in active development is creation of analysis algorithms well matched to the resolution and throughput of these image data . the large data set generated using this scanner - based method has been ideal for development of robust tools for high - throughput phenotyping of seedling images . the compression algorithm employed on these images shown in figure 7 supports the claim that they are amenable to image analysis applications . additionally , the images generated can be analyzed by the previously published algorithm , roottrace , if they are collected at lower resolution ( less than 1,200 dpi ) , and individual seedlings are segmented from the image using the compression algorithm described above before analysis . root growth data could be extracted from images reduced to 1,200 dpi while tip angle data could be extracted from images reduced to 900 dpi ( unpublished observation ) . an additional benefit of this approach is that it can easily be customized to accommodate the imaging needs of a particular research group .

cocoons of a. suraka and other saturniids , as well as b. mori , were used for the study . cocoons of north american saturniids , antheraea polyphemus ( cramer ) , hyalophora cecropia , and actias luna , were provided in february 2011 by breeders from new hampshire ( usa ) . cocoons of the malagasy saturniid argema mittrei ( gurin - mneville ) were collected in maroantsetra ( north - east madagascar ) in 2010 . cocoons of a. suraka were collected in maroantsetra ( may 2010 ) , kirindy ( west madagascar cocoons of b. mori were obtained from eggs ( carolina biological supply company , burlington , nc , in july 2009 ) raised with leaves of white mulberry ( morus alba l. ) in the laboratory ( department of entomology ) at the university of illinois at urbana - champaign ( uiuc ) . to examine the micro - structural properties of the silks , pieces of silk sheets were sputter - coated with gold and palladium and images were collected with the use of an environmental scanning electron microscope ( sem ) with a field - emission electron gun ( esem - feg ; fei co. , hillsboro , or ) in hivac mode at 5 kv and a spot size of 2.1 nm . the dimensions of the silk fibers were measured using imagej 1.49h ( national institutes of health ; bethesda , md ) . vertical and horizontal chord lengths of the fiber cross - section were used as parameters to evaluate the fiber size . the chord length is the length across the centre of the projection area of the cross section . any relationships between the fiber size and body size were studied by using adult wingspan and larval body length as reported in the literature . to test the mechanical properties of the silks , cocoons of a. suraka from kirindy and isalo were washed with a laboratory detergent to remove soil and other substances from the ground where the cocoons were collected , air - dried , and then cut longitudinally with a sharp scalpel . each silk sheet was placed under a cotton cloth and then ironed on cotton mode for 10 s with a domestic electrical cloth iron ( model 0005087553275 , stanley black & decker , new britain , ct ) to remove wrinkles . the ironing temperature was lowered ( due to the cotton cloth ) to an average of 132 c , which was measured from 10 samples using a non - contact infrared thermometer ( model lasergrip 774 , etekcity corporation , anaheim , ca ) . the same method is used by the farmers in maroantsetra , except the iron is not electrical but rather is powered by charcoal fuel . the cocoons of b. mori possess many layers that could be separated manually in different thicknesses depending on the objectives of the experiment . they were not ironed because the cocoons of b. mori naturally have no wrinkles ; ironing them would change the color of the fiber and other properties . a dog - bone - shaped stencil ( 30 mm in length , 15 mm in height ) was created with a shoulder at each end ( 10 mm width ) and a most reduced width in between ( rw : 4 mm ) ; the distance between shoulders ( bs ) reached 15 mm ( fig . 1 ) . the gauge length , ideally similar in size to bs , was measured for each sample , which was more or less larger than the stencil . the gauge width , ideally similar in size to rw , was measured at the shortest length of the dog - bone - shaped sample in the middle of the gl . the dog - bone - shaped stencil was designed to allow for uniform deformation and failure in the middle section of the sample due to maximum tensile loading ( roque et al . the stencil was placed on each ironed cocoon sheet of a. suraka and non - ironed cocoon sheet of b. mori . sample thickness was averaged by measuring in three locations on the gl with calipers . 1.stencil used to cut a piece of silk sheet for ts testing : isometric view ( a ) , front view ( b ) , and top view ( c ) . bs , distance between shoulders ( ideal sample gauge length ) ; rw , reduced width ( ideal sample gauge width ) . stencil used to cut a piece of silk sheet for ts testing : isometric view ( a ) , front view ( b ) , and top view ( c ) . bs , distance between shoulders ( ideal sample gauge length ) ; rw , reduced width ( ideal sample gauge width ) . once each dog - bone - shaped sample was created , its ts was tested using an electroforce biodynamic test instrument ( model 5100 , bose corporation , eden prairie , mn ; fig . 2 ) with a displacement speed of 0.02 mm / s . the deformation rate ( % /mn ) could be determined by taking into consideration the displacement speed ( in mm / mn ) by dividing it with the corresponding gl of the sample and multiplying all with 100% . the load cell limit was 225 n. two identical grips ( 40 mm wide and 38 mm tall ) were used to hold the sample . the stencil and the grips were custom - designed using solidworks 2013 computer - aided design ( cad ) software ( solidworks corporation , waltham , ma ) and then fabricated with two 3d printers ( eden 350 and abs - m30i , stratasys , ltd . , eden prairie , mn ) , respectively , using verowhite polymer ( stratasys , ltd . , eden prairie , mn ) . wintest 3.0 software ( bose corporation , eden prairie , mn ) was used to record load and displacement data . strain data in order to calculate the peak strength , i.e. , the ts , and the elastic modulus ( e ) , a measure of stiffness . stress is calculated by dividing the force by the cross - sectional area ( agnarsson et al . strain is determined by calculating the change in length divided by the initial length for each displacement . the elastic modulus was determined by finding the slope of the linear part of the stress elastic modulus were measured in megapascal ( mpa ) , equivalent to meganewton per square meter , or force per unit area . the failure mode of the samples of a. suraka ( inner and out layers ) and b. mori cocoons could be illustrated by representative stress 2.ts testing instrument where the dog - bone - shaped sample of antherina suraka cocoon is held at their two extremities by two squared grips . magnified view of intact samples ( from kirindy or isalo , madagascar ) at the beginning of the ts testing is shown . ts testing instrument where the dog - bone - shaped sample of antherina suraka cocoon is held at their two extremities by two squared grips . magnified view of intact samples ( from kirindy or isalo , madagascar ) at the beginning of the ts testing is shown . images of the samples were obtained using a canon eos-5d mark iii or mark ii camera with remote switch and canon 100 mm macro is lens ( canon u.s.a . , inc . , the image data were examined in the form of a binary image using labview 2013 ( national instruments , austin , tx ) to facilitate analyses of the fibers and the cells forming the cocoon sheets . a cell is defined as the empty closed space formed by at least three fibers crossing over at their ends . five properties of the cocoon were then measured : density and volume of cells , density and volume of fibers , and the cell shape factor . the densities of the cells or the fibers ( density of silk distribution ) were determined by counting the numbers of cells or fibers per square millimeter . the cell volume was calculated by multiplying the cell area with the thickness of the cocoon sheet . the thread volume was obtained by multiplying the component vector of the distance between nodes with the diameter of the thread and thickness . thus , the distance between nodes is defined as the length of the thread , which can be represented by its component vector ( d * sin ) , where is the angle of orientation of the fiber relative to the horizontal plane of the silk sheet . the use of a component vector ( d * sin ) instead of a simple distance ( d ) is necessary in order to obtain uniform data because the fibers are oriented in different angles . the cell shape factor , with a formula ( 4 * pi * area)/(perimeter2 ) , approaches a value of 1 for a circle , 0.78 for a square , and 0 for a line . all statistical analyses were performed using spss version 22 ( ibm corp . released 2013 , armonk , ny ) . for each cocoon , values of each parameter were tested for a normal distribution . values of parameters that were skewed were transformed to logarithm with base ten for normalization . levene 's test of equality of variances was performed followed by an independent t - test to compare parameters of inner and the outer layers of cocoons and of cocoons from two sites ( kirindy and isalo ) . in cases where the equality of variances was not assumed when performing the t - test , the welch satterthwaite method was used to adjust the degrees of freedom and the pooled estimate for the error term for the t - statistic was not used . evaluation of sem microscope images revealed that cocoon sheets consisted of nodes of multiple threads ( fig . the cross - section of the thread showed that it was composed of either two ( bombyx mori ) or multiple ( saturniid species ) strands that are themselves bundles of filaments . 4 ) is characterized by two strands of fibroin glued together by sericin ( sprague 1975 ) . 3.images collected through environmental scanning electron microscope : a piece of cocoon of antherina suraka ( saturniidae ) with multiple threads forming nodes ( a ) and cross - section of a thread ( c ) . 4.images collected through environmental scanning electron microscope : a piece of cocoon of bombyx mori ( bombycidae ) : degummed simple thread ( a ) and cross - section of a thread ( b ) . images collected through environmental scanning electron microscope : a piece of cocoon of antherina suraka ( saturniidae ) with multiple threads forming nodes ( a ) and cross - section of a thread ( c ) . images collected through environmental scanning electron microscope : a piece of cocoon of bombyx mori ( bombycidae ) : degummed simple thread ( a ) and cross - section of a thread ( b ) . the larger body size of saturniidae , associated with larger spinnerets , or silk - spinning organs , in the larval stage , was reflected in the size of threads produced . the chord lengths of a. suraka threads were about five times greater than the threads produced by b. mori ( table 1 ) . in species of saturniids , this body spinneret ratio was not obvious when comparing the size of the thread that we measured and the wingspan reported in the literature . antherina suraka ( tribe saturniini ) showed similar thread size to two other species from the same tribe , antheraea polyphemus and argema mittrei , although a. suraka wingspan is approximately two - thirds the size of the two latter species . the threads of these three saturniid species were in turn three times greater than the threads produced by actias luna , also belonging to the same tribe , saturniini , and hyalophora cecropia , belonging to another tribe , attacini , although ac . luna has a wingspan similar in size to that of a. suraka , and h. cecropia has a greater wingspan more similar in size to that of an . spinneret ratio was also not obvious when comparing the size of the thread that we measured and the larval body length of all the studied species reported in the literature . all the studied species in the tribe saturniini ( a. suraka , ac . luna , and an . mittrei , showed similar larval body length to b. mori , although the thread size and the wingspan of the latter were at least twice smaller ( table 1 ) . table 1.physical dimensions of silk threads of wild and domesticated silkworms measured from images obtained from a scanning electron microscope ( one thread is composed of two or multiple strands that are themselves bundles of multiple filaments)species ( number of individuals)vertical chord length mean ( sd ) ( in m)horizontal chord length mean ( sd ) ( in m)wing span ( in mm)last instar larval body length ( in mm)saturniidae ( tribe saturniini)antherina suraka ( 5)35.93 ( 13.81 ) , n = 1091.33 ( 35.76 ) , n = 418014075actias luna ( 1)13.07 ( 3.19 ) , n = 440.03 ( 9.73 ) , n = 99513575antheraea polyphemus ( 1)32.44 ( 0.84 ) , n = 287.54 ( 19.38 ) , n = 1111015075argema mittrei ( 1)44.10 ( 36.09 ) , n = 2106.89 ( 23.94 ) , n = 2130200150saturniidae ( tribe attacini)hyalophora cecropia ( 1)11.00 ( 5.15 ) , n = 534.69 ( 14.41 ) , n = 5110150100bombycidaebombyx mori ( 1)6.76 ( 0.01 ) , n = 214.66 ( 3.58 ) , n = 114075n , number of threads measured per sample . physical dimensions of silk threads of wild and domesticated silkworms measured from images obtained from a scanning electron microscope ( one thread is composed of two or multiple strands that are themselves bundles of multiple filaments ) n , number of threads measured per sample . the ranges of gl ( 814.56 mm ) and gw ( 3.065.24 mm ) for a. suraka samples ( n = 33 ) differed from those of b. mori ( gl : 9.512.63 mm ; gw : 3.345.09 mm ) samples ( n = 11 ) . the displacement rate varied considerably for a. suraka samples ( n = 33 ) , ranging from 8.24 to 15%/mn , in comparison with the more consistent rates for b. mori samples ( n = 11 ) , which ranged from 9.50 to 9.64%/mn . the failure mode of the samples of a. suraka ( inner and out layers ) and b. mori cocoons were illustrated by the representative stress - strain curves . stresses increased as strain increased , then peak stresses were followed by more or less abrupt failure depending on whether the sample was respectively b. mori or a. suraka ( fig . correlations between the mechanical and structural features of the silk sheet of a. suraka confirmed that the thickness was significantly negatively correlated with the elastic modulus and cell and thread density ( table 2 ) . the correlations showed that the thinner the silk sheets are , the stiffer they are . thus , thinner silk sheets present more fibers and cells ( more nodes or greater interfiber bonding ) , indicating higher cell and thread density , which form tighter meshes ( lower porosity ) as lower thread volume and smaller cell volume were observed . strain curves of bombyx mori ( bm ) cocoon layers and antherina suraka ( as ) inner and outer cocoon layers . table 2.pearson correlations between mechanical and structural features of antherina suraka silk sheetthicknesselastic modulustscell densitythread densitycell volumethread volumecell shape factorthickness1elastic modulus0.499**1ts0.3200.844**1cell density0.390 * 0.1120.1411thread density0.381 * 0.0910.1470.991**1cell volume0.657**0.3020.2740.873**0.879**1thread volume0.897**0.3060.1430.638**0.618**0.721**1cell shape factor0.1620.0780.0330.0260.0330.0650.1801values of elastic modulus , ts , cell volume , and thread volume were log - transformed to meet normality assumptions . an asterisk or a double asterisk indicates significant correlation ( p < 0.05 or p < 0.01 , respectively ) . representative stress strain curves of bombyx mori ( bm ) cocoon layers and antherina suraka ( as ) inner and outer cocoon layers . pearson correlations between mechanical and structural features of antherina suraka silk sheet values of elastic modulus , ts , cell volume , and thread volume were log - transformed to meet normality assumptions . an asterisk or a double asterisk indicates significant correlation ( p < 0.05 or p < 0.01 , respectively ) . results of thickness measurements and mechanical testing showed that the b. mori cocoon sheet , although fourfold thinner , has fourfold higher peak strength and is fivefold stiffer ( elastic modulus ) than that of a. suraka ( table 3 ) . mori , mean ( sd)ttestts ( mpa)5.43 ( 2.93)21.94 ( 6.07)t = 8.32 , df = 10.30 , p < 0.01na . suraka . mori= 10elastic modulus ( mpa)68.37 ( 44.50)350.44 ( 114.82)t = 7.95 , df = 11.02 , p < 0.01na . suraka = 33 , nb . mori= 11thickness ( mm)0.16 ( 0.06)0.04 ( 0.02)t = 9.99 , df = 44 , mori= 12n , number of cocoon sheets measured in that species.p < 0.01 indicates significant differences between means in the same row . features of cocoon sheets of antherina suraka and bombyx mori n , number of cocoon sheets measured in that species . a comparison of cocoons of a. suraka from two different localities , kirindy and isalo , did not reveal substantial differences in terms of peak stress , elastic modulus , thickness , mean thread volume , or cell and thread density ( fig . however , the mean cell volume was greater in cocoons from kirindy than in those from isalo ( table 4 ) . table 4.mechanical and structural features of cocoon sheets of antherina suraka collected in kirindy and isalofeature ( unit)kirindy mean ( sd)isalo mean ( sd)t - testts0.61 ( 0.26)0.72 ( 0.25)t = 1.195 , df = 31 , p = 0.241elastic modulus1.77 ( 0.28)1.73 ( 0.30)t = 0.414 , df = 31 , p = 0.682thickness ( mm)0.15 ( 0.06)0.17 ( 0.07)t = 0.703 , df = 31 , p = 0.488cell density ( cells / mm)4.0 ( 1.6)4.7 ( 1.4)t = 1.316 , df = 31 , p = 0.198thread density ( threads / mm)8.8 ( 4.3)11.1 ( 3.9)t = 1.654 , df = 31 , p = 0.108cell volume1.71 ( 0.35)1.96 ( 0.31)t = 2.172 , df = 31 , p = 0.038*thread volume2.11 ( 0.40)1.98 ( 0.30)t = 1.062 , df = 31 , p = 0.296cell shape factor0.68 ( 0.04)0.66 ( 0.03)t = 1.773 , df = 31 , p = 0.086values of ts , elastic modulus , cell volume , and thread volume were log - transformed to meet normality assumptions ; an asterisk ( * ) indicates significant differences ( p < 0.05 ) between means in the same row ; n= 15 for cocoons in kirindy and n= 18 for those in isalo . mechanical and structural features of cocoon sheets of antherina suraka collected in kirindy and isalo values of ts , elastic modulus , cell volume , and thread volume were log - transformed to meet normality assumptions ; an asterisk ( * ) indicates significant differences ( p < 0.05 ) between means in the same row ; n= 15 for cocoons in kirindy and n= 18 for those in isalo . a comparison of inner and outer cocoons , irrespective of source , however , did reveal many differences . with respect to mechanical properties , the inner cocoons of a. suraka had significantly greater stiffness than the outer cocoons ( table 5 ) . most of the structural properties were significantly different , except for the cell shape factor where inner and outer cocoons had similar shape ( fig . the inner cocoons had higher cell and thread density , and smaller mean cell and thread volume than the outer cocoons ( table 5 ) . in other words , the inner layers of cocoons of a. suraka showed denser fiber distribution and lower porosity than the outer ones . these results were consistent with the correlations between mechanical and structural parameters analyzed previously ( table 2 ) . 6.images of dog - bone - shaped samples of antherina suraka cocoons collected in kirindy ( madagascar ) : inner layer ( left ) ; outer layer ( right ) . table 5.mechanical and structural features of the internal and external cocoons of antherina surakafeature of cocoon layer ( unit)internal mean ( sd)external mean ( sd)t - testts0.75 ( 0.26)0.59 ( 0.22)t = 1.892 , df = 31 , p = 0.068elastic modulus1.87 ( 0.27)1.63 ( 0.25)t = 2.674 , df = 31 , p = 0.012*thickness ( mm)0.11 ( 0.04)0.21 ( 0.04)t = 6.610 , df = 31 , p < 0.001**cell density ( cells / mm)5.1 ( 1.2)3.7 ( 1.5)t = 2.930 , df = 31 , p = 0.006**thread density ( threads / mm)11.9 ( 3.3)8.3 ( 4.2)t = 2.670 , df = 31 , p = 0.012*cell volume2.07 ( 0.23)1.63 ( 0.32)t = 4.502 , df = 31 , p < 0.001**thread volume2.30 ( 0.27)1.79 ( 0.20)t = 6.244 , df = 31 , p < 0.001**cell shape factor0.68 ( 0.03)0.67 ( 0.04)t = 0.945 , df = 31 , p = 0.352values of ts , elastic modulus , cell volume , and thread volume were log - transformed to meet normality assumptions ; an asterisk or a double asterisk indicates significant differences ( p < 0.05 or p < 0.01 , respectively ) between means in the same row ; n= 16 for internal cocoons and n= 17 for external ones . images of dog - bone - shaped samples of antherina suraka cocoons collected in kirindy ( madagascar ) : inner layer ( left ) ; outer layer ( right ) . mechanical and structural features of the internal and external cocoons of antherina suraka values of ts , elastic modulus , cell volume , and thread volume were log - transformed to meet normality assumptions ; an asterisk or a double asterisk indicates significant differences ( p < 0.05 or p < 0.01 , respectively ) between means in the same row ; n= 16 for internal cocoons and n= 17 for external ones . differences in mechanical properties could be explained by taxonomic origins , fiber arrangement , protein composition , and structure of the silk fiber ( hayashi et al . cocoons of a. suraka and other saturniids differ in microstructural properties in comparison with cocoons of b. mori . environmental scanning electron microscope images showed that the cocoons of a. suraka resemble those of some caligula spp . ( saturniidae , lepidoptera ) in showing a looser scaffold structure characterized by large pores supported by bundles of fibers ; by contrast , the b. mori cocoon is characterized by high porosity and weak interlayer bonding ( chen et al . seem compact because the pores are microscopic but the pores in a. suraka are visible to the naked eye . spinneret ratio differs dramatically at the family level in comparisons of the thread size of b. mori with that of a. suraka . at the genus level , the thread size does not depend on body size ( adult wing span and larval body length ) : thread sizes in species of the same tribe are not necessarily similar . in view of the process by which lepidopteran larvae spin silk , examining potential relationships between spinneret structures and silk thread size the compact and thinner silk sheet of b. mori had greater ts and stiffness than the looser and thicker cocoon of a. suraka . these findings are consistent with those reported in a study on mechanical properties of b. mori cocoon , according to which thinner silk has proportionately higher elastic modulus and ts ( zhao et al . the inner layers were thinner and stiffer with lower porosity and denser silk distribution ( more interfiber bonding ) than the outer ones ; this finding , too , was confirmed for cocoon layers of b. mori ( zhao et al . 2012b ) and species belonging to the same family as a. suraka ( saturniidae ) and other wild silk moth families such as lasiocampidae ( chen et al . 2012c ) . chen et al . ( 2010 ) explained that the elastic modulus is controlled by the porosity of the silk composite according to the foam open cell model of zhu ( 1997 ) ; the decrease in the elastic modulus is due to the gradual loss of connectivity of sericin bonding between the fibers forming the nodes . the general curve shapes of the samples of a. suraka ( inner and out layers ) and b. mori were typical cocoon sheets , consisting of a peak stress followed by a failure ( zhao et al . 2005 ; chen et al . stress and strain values of the studied layers of cocoons of b. mori forming the curves were in the ranges indicated by zhao et al . 2005 . 2012 ) compared to the domesticated b. mori , which has been reared and subjected to artificial selection by humans for approximately 5,000 years ( kurin 2002 ) . bombyx mori produces high - quality silk that is woven worldwide to make fabrics primarily for clothing . as an entirely domesticated species , b. mori spins its cocoon in artificial frames , four - walled wooden structures provided by humans that permit construction of compact cocoons . thus , spacing available for spinning probably determines at least in part the form of cocoon . that physical space available during cocoon - spinning can influence compactness was demonstrated by waldbauer and sternburg ( 1967 ) , who found compact cocoons of the north american saturniid hyalophora cecropia only on twigs or branches of trees or on higher parts of shrubs , where the larvae could find a three - dimensional support on a fork of twigs or could create a closed space for spinning by attaching silk to leaves . by contrast , cocoons formed on twigs of shrubs near the ground where leaves were absent were looser in structure because there were no space constraints on spinning cocoons . among all the structural properties analyzed in this study , only thickness was correlated with elastic modulus : the inner cocoon was thinner with greater ts and stiffness than the outer cocoon . a study on b. mori cocoon confirmed that the silk layer became thinner but retained a superior protective function when larvae experienced external disturbances and/or were forced to spin another cocoon when the first one was removed ( huang et al . these mechanical properties of the a. suraka cocoon might be explained by its chemical composition , including the greater amount of polyalanine - sheet nanocrystals present in the silk fibers of its inner layer when compared to its outer one ( boulet - audet et al . 2015 ) ; these structures are indicative of the degree of crystallinity present ( porter et al . external environmental conditions such as temperature , humidity , and rain may perturb the larvae when spinning cocoons ( ramachandra et al . identifying environmental factors that influence both larval spinning behavior and silk attributes will be important in improving the a. suraka sericulture enterprise . in the lasiocampid wild silkworm gonomestica postica walker ( lepidoptera ) , physical properties of cocoons such as weight , size ( length and width ) , and breaking energy ( toughness ) were significantly lower when the larvae were reared indoors than outdoors ( teshome et al . 2014 ) , although no specific environmental factor was identified as being responsible for the differences . as well , in another species of saturniidae , antheraea pernyi ( gurin - mneville ) , the silk undergoes glass transition at 140 c and there might be annealing by dehydration of the material at 100 c ( guan reduce the risk of glass transition , we controlled the temperature of the iron at 130 c by limiting its duration and using a cloth to cover the cocoon to prevent too much deformation of the silk ; nonetheless , water might well have been driven out annealing the silk as the temperature was over 100 c. thus , the method of ironing used in this study and by the farmers , apart from removing wrinkles , possibly changes some mechanical properties of the cocoons such as toughness . like the saturniid fauna in other countries such as india , the saturniid species in madagascar have great potential for wild sericulture . the cocoons of a. suraka do not possess the strength of that of b. mori , but they do possess mechanical and physical properties that make them suitable for use as a raw material for jewelry and for use in sheet form as a patchwork fabric for curtains and lampshades . turning a. suraka silk into fabric is far less labor - intensive and time - consuming than silk fiber production for weaving from b. mori , rendering a. suraka silk more suitable for rural communities . the shiny brown color of a. suraka silk has considerable potential for artwork and decorative accessories . 2012 ; weber and craig 2014 ) and sericulture is currently expanding to other species of saturniidae , such as argema mittreii , with its silvery cocoon , and ceranchia appolina ( butler ) , with its lighter brown cocoon , which would diversify the field of wild sericulture in madagascar .

antherina suraka boisduval ( saturniidae , lepidoptera ) produces a silken cocoon that has been the focus of efforts to create a commercial wild silk industry in madagascar . in this study , structural and mechanical properties of the cocoon of a. suraka from two sites were measured and compared to the cocoon of bombyx mori l. ( bombycidae , lepidoptera ) the world 's most common source for silk . results of environmental scanning electron microscopy and mechanical testing showed that the silk sheet of a. suraka cocoon is less compact , with greater thickness and lower tensile strength and stiffness than that of b. mori . confirming these results , stiffness and cell and thread density were found to be negatively correlated with thickness , and the cell and thread volumes were positively correlated with thickness . antherina suraka showed no major differences between silk sheets from kirindy and isalo sites in either structural or mechanical properties , except for mean cell volume , which was greater in cocoons from kirindy . comparison between the two layers forming the cocoon showed that the inner layer has greater elastic modulus , denser silk distribution and lower porosity . cocoons from both kirindy and isalo are suitable for sericulture . although the inner layer of cocoon silk is of higher quality than the outer layer , the fact that both layers are of great but lower tensile strength than b. mori silk suggests that the current practice of sewing the two layers together for making one single layer fabric should be continued in efforts to produce a commercially viable product .

Materials and Methods Results Discussion

cocoons of a. suraka were collected in maroantsetra ( may 2010 ) , kirindy ( west madagascar cocoons of b. mori were obtained from eggs ( carolina biological supply company , burlington , nc , in july 2009 ) raised with leaves of white mulberry ( morus alba l. ) in the laboratory ( department of entomology ) at the university of illinois at urbana - champaign ( uiuc ) . to examine the micro - structural properties of the silks , pieces of silk sheets were sputter - coated with gold and palladium and images were collected with the use of an environmental scanning electron microscope ( sem ) with a field - emission electron gun ( esem - feg ; fei co. , hillsboro , or ) in hivac mode at 5 kv and a spot size of 2.1 nm . the dimensions of the silk fibers were measured using imagej 1.49h ( national institutes of health ; bethesda , md ) . to test the mechanical properties of the silks , cocoons of a. suraka from kirindy and isalo were washed with a laboratory detergent to remove soil and other substances from the ground where the cocoons were collected , air - dried , and then cut longitudinally with a sharp scalpel . the cocoons of b. mori possess many layers that could be separated manually in different thicknesses depending on the objectives of the experiment . the stencil was placed on each ironed cocoon sheet of a. suraka and non - ironed cocoon sheet of b. mori . , the ts , and the elastic modulus ( e ) , a measure of stiffness . the failure mode of the samples of a. suraka ( inner and out layers ) and b. mori cocoons could be illustrated by representative stress 2.ts testing instrument where the dog - bone - shaped sample of antherina suraka cocoon is held at their two extremities by two squared grips . ts testing instrument where the dog - bone - shaped sample of antherina suraka cocoon is held at their two extremities by two squared grips . , the image data were examined in the form of a binary image using labview 2013 ( national instruments , austin , tx ) to facilitate analyses of the fibers and the cells forming the cocoon sheets . five properties of the cocoon were then measured : density and volume of cells , density and volume of fibers , and the cell shape factor . the cell volume was calculated by multiplying the cell area with the thickness of the cocoon sheet . thus , the distance between nodes is defined as the length of the thread , which can be represented by its component vector ( d * sin ) , where is the angle of orientation of the fiber relative to the horizontal plane of the silk sheet . the cell shape factor , with a formula ( 4 * pi * area)/(perimeter2 ) , approaches a value of 1 for a circle , 0.78 for a square , and 0 for a line . levene 's test of equality of variances was performed followed by an independent t - test to compare parameters of inner and the outer layers of cocoons and of cocoons from two sites ( kirindy and isalo ) . the cross - section of the thread showed that it was composed of either two ( bombyx mori ) or multiple ( saturniid species ) strands that are themselves bundles of filaments . 3.images collected through environmental scanning electron microscope : a piece of cocoon of antherina suraka ( saturniidae ) with multiple threads forming nodes ( a ) and cross - section of a thread ( c ) . 4.images collected through environmental scanning electron microscope : a piece of cocoon of bombyx mori ( bombycidae ) : degummed simple thread ( a ) and cross - section of a thread ( b ) . images collected through environmental scanning electron microscope : a piece of cocoon of antherina suraka ( saturniidae ) with multiple threads forming nodes ( a ) and cross - section of a thread ( c ) . images collected through environmental scanning electron microscope : a piece of cocoon of bombyx mori ( bombycidae ) : degummed simple thread ( a ) and cross - section of a thread ( b ) . the chord lengths of a. suraka threads were about five times greater than the threads produced by b. mori ( table 1 ) . in species of saturniids , this body spinneret ratio was not obvious when comparing the size of the thread that we measured and the wingspan reported in the literature . antherina suraka ( tribe saturniini ) showed similar thread size to two other species from the same tribe , antheraea polyphemus and argema mittrei , although a. suraka wingspan is approximately two - thirds the size of the two latter species . the threads of these three saturniid species were in turn three times greater than the threads produced by actias luna , also belonging to the same tribe , saturniini , and hyalophora cecropia , belonging to another tribe , attacini , although ac . luna has a wingspan similar in size to that of a. suraka , and h. cecropia has a greater wingspan more similar in size to that of an . spinneret ratio was also not obvious when comparing the size of the thread that we measured and the larval body length of all the studied species reported in the literature . mittrei , showed similar larval body length to b. mori , although the thread size and the wingspan of the latter were at least twice smaller ( table 1 ) . the ranges of gl ( 814.56 mm ) and gw ( 3.065.24 mm ) for a. suraka samples ( n = 33 ) differed from those of b. mori ( gl : 9.512.63 mm ; gw : 3.345.09 mm ) samples ( n = 11 ) . the displacement rate varied considerably for a. suraka samples ( n = 33 ) , ranging from 8.24 to 15%/mn , in comparison with the more consistent rates for b. mori samples ( n = 11 ) , which ranged from 9.50 to 9.64%/mn . the failure mode of the samples of a. suraka ( inner and out layers ) and b. mori cocoons were illustrated by the representative stress - strain curves . correlations between the mechanical and structural features of the silk sheet of a. suraka confirmed that the thickness was significantly negatively correlated with the elastic modulus and cell and thread density ( table 2 ) . the correlations showed that the thinner the silk sheets are , the stiffer they are . thus , thinner silk sheets present more fibers and cells ( more nodes or greater interfiber bonding ) , indicating higher cell and thread density , which form tighter meshes ( lower porosity ) as lower thread volume and smaller cell volume were observed . strain curves of bombyx mori ( bm ) cocoon layers and antherina suraka ( as ) inner and outer cocoon layers . table 2.pearson correlations between mechanical and structural features of antherina suraka silk sheetthicknesselastic modulustscell densitythread densitycell volumethread volumecell shape factorthickness1elastic modulus0.499**1ts0.3200.844**1cell density0.390 * 0.1120.1411thread density0.381 * 0.0910.1470.991**1cell volume0.657**0.3020.2740.873**0.879**1thread volume0.897**0.3060.1430.638**0.618**0.721**1cell shape factor0.1620.0780.0330.0260.0330.0650.1801values of elastic modulus , ts , cell volume , and thread volume were log - transformed to meet normality assumptions . pearson correlations between mechanical and structural features of antherina suraka silk sheet values of elastic modulus , ts , cell volume , and thread volume were log - transformed to meet normality assumptions . results of thickness measurements and mechanical testing showed that the b. mori cocoon sheet , although fourfold thinner , has fourfold higher peak strength and is fivefold stiffer ( elastic modulus ) than that of a. suraka ( table 3 ) . features of cocoon sheets of antherina suraka and bombyx mori n , number of cocoon sheets measured in that species . a comparison of cocoons of a. suraka from two different localities , kirindy and isalo , did not reveal substantial differences in terms of peak stress , elastic modulus , thickness , mean thread volume , or cell and thread density ( fig . however , the mean cell volume was greater in cocoons from kirindy than in those from isalo ( table 4 ) . table 4.mechanical and structural features of cocoon sheets of antherina suraka collected in kirindy and isalofeature ( unit)kirindy mean ( sd)isalo mean ( sd)t - testts0.61 ( 0.26)0.72 ( 0.25)t = 1.195 , df = 31 , p = 0.241elastic modulus1.77 ( 0.28)1.73 ( 0.30)t = 0.414 , df = 31 , p = 0.682thickness ( mm)0.15 ( 0.06)0.17 ( 0.07)t = 0.703 , df = 31 , p = 0.488cell density ( cells / mm)4.0 ( 1.6)4.7 ( 1.4)t = 1.316 , df = 31 , p = 0.198thread density ( threads / mm)8.8 ( 4.3)11.1 ( 3.9)t = 1.654 , df = 31 , p = 0.108cell volume1.71 ( 0.35)1.96 ( 0.31)t = 2.172 , df = 31 , p = 0.038*thread volume2.11 ( 0.40)1.98 ( 0.30)t = 1.062 , df = 31 , p = 0.296cell shape factor0.68 ( 0.04)0.66 ( 0.03)t = 1.773 , df = 31 , p = 0.086values of ts , elastic modulus , cell volume , and thread volume were log - transformed to meet normality assumptions ; an asterisk ( * ) indicates significant differences ( p < 0.05 ) between means in the same row ; n= 15 for cocoons in kirindy and n= 18 for those in isalo . mechanical and structural features of cocoon sheets of antherina suraka collected in kirindy and isalo values of ts , elastic modulus , cell volume , and thread volume were log - transformed to meet normality assumptions ; an asterisk ( * ) indicates significant differences ( p < 0.05 ) between means in the same row ; n= 15 for cocoons in kirindy and n= 18 for those in isalo . with respect to mechanical properties , the inner cocoons of a. suraka had significantly greater stiffness than the outer cocoons ( table 5 ) . most of the structural properties were significantly different , except for the cell shape factor where inner and outer cocoons had similar shape ( fig . the inner cocoons had higher cell and thread density , and smaller mean cell and thread volume than the outer cocoons ( table 5 ) . in other words , the inner layers of cocoons of a. suraka showed denser fiber distribution and lower porosity than the outer ones . table 5.mechanical and structural features of the internal and external cocoons of antherina surakafeature of cocoon layer ( unit)internal mean ( sd)external mean ( sd)t - testts0.75 ( 0.26)0.59 ( 0.22)t = 1.892 , df = 31 , p = 0.068elastic modulus1.87 ( 0.27)1.63 ( 0.25)t = 2.674 , df = 31 , p = 0.012*thickness ( mm)0.11 ( 0.04)0.21 ( 0.04)t = 6.610 , df = 31 , p < 0.001**cell density ( cells / mm)5.1 ( 1.2)3.7 ( 1.5)t = 2.930 , df = 31 , p = 0.006**thread density ( threads / mm)11.9 ( 3.3)8.3 ( 4.2)t = 2.670 , df = 31 , p = 0.012*cell volume2.07 ( 0.23)1.63 ( 0.32)t = 4.502 , df = 31 , p < 0.001**thread volume2.30 ( 0.27)1.79 ( 0.20)t = 6.244 , df = 31 , p < 0.001**cell shape factor0.68 ( 0.03)0.67 ( 0.04)t = 0.945 , df = 31 , p = 0.352values of ts , elastic modulus , cell volume , and thread volume were log - transformed to meet normality assumptions ; an asterisk or a double asterisk indicates significant differences ( p < 0.05 or p < 0.01 , respectively ) between means in the same row ; n= 16 for internal cocoons and n= 17 for external ones . mechanical and structural features of the internal and external cocoons of antherina suraka values of ts , elastic modulus , cell volume , and thread volume were log - transformed to meet normality assumptions ; an asterisk or a double asterisk indicates significant differences ( p < 0.05 or p < 0.01 , respectively ) between means in the same row ; n= 16 for internal cocoons and n= 17 for external ones . differences in mechanical properties could be explained by taxonomic origins , fiber arrangement , protein composition , and structure of the silk fiber ( hayashi et al . cocoons of a. suraka and other saturniids differ in microstructural properties in comparison with cocoons of b. mori . environmental scanning electron microscope images showed that the cocoons of a. suraka resemble those of some caligula spp . ( saturniidae , lepidoptera ) in showing a looser scaffold structure characterized by large pores supported by bundles of fibers ; by contrast , the b. mori cocoon is characterized by high porosity and weak interlayer bonding ( chen et al . spinneret ratio differs dramatically at the family level in comparisons of the thread size of b. mori with that of a. suraka . in view of the process by which lepidopteran larvae spin silk , examining potential relationships between spinneret structures and silk thread size the compact and thinner silk sheet of b. mori had greater ts and stiffness than the looser and thicker cocoon of a. suraka . these findings are consistent with those reported in a study on mechanical properties of b. mori cocoon , according to which thinner silk has proportionately higher elastic modulus and ts ( zhao et al . the inner layers were thinner and stiffer with lower porosity and denser silk distribution ( more interfiber bonding ) than the outer ones ; this finding , too , was confirmed for cocoon layers of b. mori ( zhao et al . 2012b ) and species belonging to the same family as a. suraka ( saturniidae ) and other wild silk moth families such as lasiocampidae ( chen et al . ( 2010 ) explained that the elastic modulus is controlled by the porosity of the silk composite according to the foam open cell model of zhu ( 1997 ) ; the decrease in the elastic modulus is due to the gradual loss of connectivity of sericin bonding between the fibers forming the nodes . the general curve shapes of the samples of a. suraka ( inner and out layers ) and b. mori were typical cocoon sheets , consisting of a peak stress followed by a failure ( zhao et al . stress and strain values of the studied layers of cocoons of b. mori forming the curves were in the ranges indicated by zhao et al . 2012 ) compared to the domesticated b. mori , which has been reared and subjected to artificial selection by humans for approximately 5,000 years ( kurin 2002 ) . among all the structural properties analyzed in this study , only thickness was correlated with elastic modulus : the inner cocoon was thinner with greater ts and stiffness than the outer cocoon . a study on b. mori cocoon confirmed that the silk layer became thinner but retained a superior protective function when larvae experienced external disturbances and/or were forced to spin another cocoon when the first one was removed ( huang et al . these mechanical properties of the a. suraka cocoon might be explained by its chemical composition , including the greater amount of polyalanine - sheet nanocrystals present in the silk fibers of its inner layer when compared to its outer one ( boulet - audet et al . in the lasiocampid wild silkworm gonomestica postica walker ( lepidoptera ) , physical properties of cocoons such as weight , size ( length and width ) , and breaking energy ( toughness ) were significantly lower when the larvae were reared indoors than outdoors ( teshome et al . as well , in another species of saturniidae , antheraea pernyi ( gurin - mneville ) , the silk undergoes glass transition at 140 c and there might be annealing by dehydration of the material at 100 c ( guan reduce the risk of glass transition , we controlled the temperature of the iron at 130 c by limiting its duration and using a cloth to cover the cocoon to prevent too much deformation of the silk ; nonetheless , water might well have been driven out annealing the silk as the temperature was over 100 c. thus , the method of ironing used in this study and by the farmers , apart from removing wrinkles , possibly changes some mechanical properties of the cocoons such as toughness . the cocoons of a. suraka do not possess the strength of that of b. mori , but they do possess mechanical and physical properties that make them suitable for use as a raw material for jewelry and for use in sheet form as a patchwork fabric for curtains and lampshades . 2012 ; weber and craig 2014 ) and sericulture is currently expanding to other species of saturniidae , such as argema mittreii , with its silvery cocoon , and ceranchia appolina ( butler ) , with its lighter brown cocoon , which would diversify the field of wild sericulture in madagascar .

the benefits of physical exercise on cognitive function in the elderly have been demonstrated in many studies . several large - scale longitudinal studies showed that older people who have a high level of physical activity , have a significantly lower risk of developing alzheimer s disease and cognitive impairment.17 the results of a meta - analysis of 18 studies investigating the effectiveness of aerobic exercise concluded that fitness training could enhance the cognitive functioning of the elderly.8 this study also showed that a short duration , moderate - level training program could create an optimal effect on cognitive functions in the elderly . another meta - analysis investigating the change of duration and intensity of physical activity conducted by van gelder et al found that elderly people who participated in physical exercise for an average of 30 minutes per day or more could postpone their cognitive decline.9 however , studies on the benefits of physical training have focused closely on aerobic exercise such as walking , and strength exercise , such as weight lifting.4,6,7,1012 these aerobic and strength exercises require the participants to be highly mobile . the elderly with low mobility , or who are hospitalized , might have difficulty enjoying the full benefit of the exercise because of their limited locomotive ability . therefore exercise with reduced locomotion requirement , could provide the benefits of aerobic exercise to the elderly with restricted mobility . recently , there has been growing research interest in the therapeutic effects of mind body exercise.13,14 tai chi chuan , commonly known as tai chi , is a typical example of mind body exercise ; it is characterized by slow motion and emphasizes the conscious control of body movements , ie , it requires less locomotive mobility and is deemed appropriate for most elderly people.15 research has shown that the cognitive functions of the elderly could be well preserved with the aid of such mind body exercise , in a way similar to typical physical exercise.3 exercises with lower requirements of locomotive ability , such as coordination training ( ct ) and towel exercise ( te ) , are needed for the elderly with poor mobility . both ct and te require low locomotive ability , and thus are suitable for most elderly . the literature review showed that ct and te may also be beneficial for the cognitive functioning of the elderly . the purpose of this study was to compare the effectiveness of ct and te on the cognitive functioning and physical mobility of the elderly , with the aim of developing an exercise with a low mobility requirement , to benefit the cognitive functioning of the elderly . we hypothesized that the elderly in the ct group would show significant improvement in the cognitive measures compared with the elderly in the te group . forty elderly ( three male , 37 female ) with normal cognition were recruited from two elderly centers of the hong kong lutheran social service , aged 6690 ( mean = 79.0 , sd = 5.8 ) . targeted participants were asked to take the chinese version of mini - mental state examination ( cmmse ) as one of the screening criteria , and those who scored 18 were eligible for this study.16 other than that , there was no other inclusion or exclusion criterion in recruitment . the ethics approval of this study was obtained from the survey and behavioural research ethics committee of the chinese university of hong kong . participants confirmed their agreement to participate in this study by signing informed consent before the exercise began . a physiotherapist from the jockey club centre for positive ageing ( jccpa , see http://www.jccpa.org.hk ) developed an 8-week exercise program , called coordination training ( ct ) , which is a simplified version of tai chi . it was easy for the elderly to learn , and required a relatively low level of mobility to practice . the eleven movements included coordination of fingers , hands , eyes , and legs . a brief description of the eleven movements is set out in table 1 . the elements of movement 4 are tabulated in table 2 and graphically represented in figure 1 as an example . movement 4 helped to train participants coordination of upper limbs , and was intended to imitate the movements of tai chi . the training protocol of te was developed by the leisure and cultural services department , the government of the hong kong special administrative region in 2005.17 similar to movement 4 in ct , te was a type of stretching exercise mainly to train upper limb and bilateral arm movements , but utilize a towel as a tool . it was strongly promoted by the government because it was easy for elderly people with various locomotive abilities to master.18 te benefited the elderly by improving circulation and helping to control weight , and aimed to reduce the chance of falling.19 for the sake of convenience of participation and better monitoring of participants progress , those in one center were allocated to practice ct , and those in another center were allocated to practice te . te was chosen to compare with ct because these exercies were similar in a number of ways . both exercises required subjects to follow instructions , and to coordinate upper limb and bilateral arm movements . ct and te were conducted for 8 consecutive weeks , with one 40-minute session per week . both groups had a 10-minute warm - up period at the beginning and a 10-minute cool - down period at the end of the session to prevent injury . the remaining 20 minutes would be taken up with the actual ct or te exercise . both exercise groups were conducted by qualified instructors trained by the physiotherapist , mentioned above . for ct , there were three levels of difficulty : easy , medium , and difficult ( see table 2 ) . the level of difficulty was increased mainly by reducing the rest time ( demanding higher concentration as well as physical strength of participants ) , and by closing the eyes when performing the actions ( demanding higher psychomotor balance of participants ) . in this study , when the participants self - reported being able to handle the movement comfortably , which was confirmed by the trainer , they were required to practice the movement at an advanced difficulty level in order to avoid the ceiling effect.19 assessment tools including chinese mini - mental state examination ( cmmse ) , chinese dementia rating scale ( cdrs ) , and timed up - and - go test ( tug ) were administered to participants in both groups before and after the training sessions by trained occupational therapists and clinical psychologists . general cognitive status was assessed using the cmmse , which was translated and validated by chiu et al in the hong kong chinese population.20 the full mark was 30 . it examined five different aspects of cognitive ability , namely , attention , initiation - perseveration , construction , conceptualization , and memory . the maximum score of the unadjusted scale was 144 , with the cronbach s alpha of 0.89 . good psychometric properties were observed in both the original drs and the chinese version.21,22 tug was a good instrument to measure the general physical mobility of participants , and thus was administrated in this study to measure the effects of relevant exercises.23 the longer time spent to finish tug ( slower ) , the poorer the performance of participants , and vice versa . spss software v 15 ( ibm corp , somers , ny ) was used for data analyses . independent sample t - tests were conducted to compare the pre - test scores ( obtained in pre - test period ) between ct and te groups . paired sample t - tests were performed to compare the post - test scores ( obtained in the ninth week , after the 8-week exercise period ) with the pre - test scores in each group . analysis of covariance ( ancova ) was used to compare the scores of cmmse , cdrs , and tug of the two groups after the training program , using participants age and the pre - test scores as covariates . forty elderly ( three male , 37 female ) with normal cognition were recruited from two elderly centers of the hong kong lutheran social service , aged 6690 ( mean = 79.0 , sd = 5.8 ) . targeted participants were asked to take the chinese version of mini - mental state examination ( cmmse ) as one of the screening criteria , and those who scored 18 were eligible for this study.16 other than that , there was no other inclusion or exclusion criterion in recruitment . the ethics approval of this study was obtained from the survey and behavioural research ethics committee of the chinese university of hong kong . participants confirmed their agreement to participate in this study by signing informed consent before the exercise began . a physiotherapist from the jockey club centre for positive ageing ( jccpa , see http://www.jccpa.org.hk ) developed an 8-week exercise program , called coordination training ( ct ) , which is a simplified version of tai chi . it was easy for the elderly to learn , and required a relatively low level of mobility to practice . the elements of movement 4 are tabulated in table 2 and graphically represented in figure 1 as an example . movement 4 helped to train participants coordination of upper limbs , and was intended to imitate the movements of tai chi . the training protocol of te was developed by the leisure and cultural services department , the government of the hong kong special administrative region in 2005.17 similar to movement 4 in ct , te was a type of stretching exercise mainly to train upper limb and bilateral arm movements , but utilize a towel as a tool . it was strongly promoted by the government because it was easy for elderly people with various locomotive abilities to master.18 te benefited the elderly by improving circulation and helping to control weight , and aimed to reduce the chance of falling.19 for the sake of convenience of participation and better monitoring of participants progress , those in one center were allocated to practice ct , and those in another center were allocated to practice te . te was chosen to compare with ct because these exercies were similar in a number of ways . both exercises required subjects to follow instructions , and to coordinate upper limb and bilateral arm movements . ct and te were conducted for 8 consecutive weeks , with one 40-minute session per week . both groups had a 10-minute warm - up period at the beginning and a 10-minute cool - down period at the end of the session to prevent injury . the remaining 20 minutes would be taken up with the actual ct or te exercise . both exercise groups were conducted by qualified instructors trained by the physiotherapist , mentioned above . for ct , there were three levels of difficulty : easy , medium , and difficult ( see table 2 ) . the level of difficulty was increased mainly by reducing the rest time ( demanding higher concentration as well as physical strength of participants ) , and by closing the eyes when performing the actions ( demanding higher psychomotor balance of participants ) . in this study , when the participants self - reported being able to handle the movement comfortably , which was confirmed by the trainer , they were required to practice the movement at an advanced difficulty level in order to avoid the ceiling effect.19 assessment tools including chinese mini - mental state examination ( cmmse ) , chinese dementia rating scale ( cdrs ) , and timed up - and - go test ( tug ) were administered to participants in both groups before and after the training sessions by trained occupational therapists and clinical psychologists . general cognitive status was assessed using the cmmse , which was translated and validated by chiu et al in the hong kong chinese population.20 the full mark was 30 . it examined five different aspects of cognitive ability , namely , attention , initiation - perseveration , construction , conceptualization , and memory . the maximum score of the unadjusted scale was 144 , with the cronbach s alpha of 0.89 . good psychometric properties were observed in both the original drs and the chinese version.21,22 tug was a good instrument to measure the general physical mobility of participants , and thus was administrated in this study to measure the effects of relevant exercises.23 the longer time spent to finish tug ( slower ) , the poorer the performance of participants , and vice versa . spss software v 15 ( ibm corp , somers , ny ) was used for data analyses . independent sample t - tests were conducted to compare the pre - test scores ( obtained in pre - test period ) between ct and te groups . paired sample t - tests were performed to compare the post - test scores ( obtained in the ninth week , after the 8-week exercise period ) with the pre - test scores in each group . analysis of covariance ( ancova ) was used to compare the scores of cmmse , cdrs , and tug of the two groups after the training program , using participants age and the pre - test scores as covariates . forty people ( three males , 37 females ) aged 66 to 90 years ( mean = 79.0 , sd = 5.8 ) were recruited . the average ages of the elderly in the ct and te groups were 77.7 6.0 and 80.3 5.5 , respectively . no significant difference was found in demographic features or cognitive and physical functioning test scores between the two groups . comparisons of participants pre - test ( baseline ) and post - test cognitive functioning by cmmse amd cdrs scores and physical mobility by tug scores are shown in table 4 . paired t - tests showed that the cdrs scores of the ct group had improved significantly from 114.8 15.5 at pre - test to 119.3 18.0 at post - test ( cdrs t(17 ) = 2.25 , p = 0.045 ) . the cdrs scores of the te group improved slightly from 114.9 14.8 at pre - test to 116.9 12.5 at post - test . no significant change was found in cmmse ( t(18 ) = 0.931 , p = 0.368 ) , and tug ( t(17 ) = 0.334 , p = 0.747 ) in ct group , as well as cmmse ( t(19 ) = 0.665 , p = 0.516 ) , cdrs ( t(19 ) = 0.891 , p = 0.384 ) and tug ( t(19 ) = 1.908 , p = 0.086 ) in the te group . different ancova ( between - subject factor : group [ ct , te ] and covariates : age and the pre - test scores ) models show the following findings . for cmmse , the covariate age ( f(1,28 ) = 0.17 , p = 0.690 , p = 0.003 ) and the exercise groups ( f(1,28 ) = 3.41 , p = 0.570 , p = 0.139 ) were not significantly related to the cmmse post - test scores . only the covariate pre - test scores of cmmse were significantly related to the post - test scores ( f(1,28 ) = 16.32 , p < 0.001 , p = 0.428 ) . for cdrs , exercise groups were not significantly related to the cdrs post - test scores ( f(1,28 ) = 0.02 , p = 0.904 , p = 0.001 ) . only the covariate age ( f(1,28 ) = 9.14 , p = 0.005 , p = 0.462 ) and the covariate pre - test scores of cdrs ( f(1,28 ) = 59.12 , p < 0.001 , p = 0.738 ) were significantly related to the cdrs post - test scores . for tug , the covariate age ( f(1,26 ) = 0.01 , p = 0.940 , p = < 0.001 ) and the exercise groups ( f(1,26 ) = 0.11 , p = 0.740 , p = 0.005 ) were not significantly related to the tug post - test scores . only the covariate pre - test scores of tug were significantly related to the tug post test scores ( f(1,26 ) = 83.50 , the above findings served to compare the effectiveness of the two exercise programs , coordination training ( ct ) and towel exercise ( te ) , in improving cognitive functioning and physical mobility in the elderly . the results showed that ct group participants had significant improvements in global cognition after the 8-week exercise program . ct group gained significant improvement in cdrs scores after the exercise training , while the te group participants did not . the lack of significant group difference in the changes in cdrs might be caused by the small sample size . further investigation of the effectiveness of ct is recommended following this prospective study , through a large - scale clinical trial with appropriate numbers of samples in each group to detect the group differences . for the physical mobility measure , te tended to improve mobility while ct did not . this pattern was probably expected , because ct was designed to improve cognition , not mobility . the insignificant difference in physical mobility measure might suggest that ct , which required less in mobility , had a similar effect to te , a common physical exercise , on the cognitive and physical functioning of the elderly population . body exercise can improve cognitive functions and other health indicators , although the role of physical exercise in modulating cognitive decline is complex . the improvements can be described through ( 1 ) psychosocial indicators and ( 2 ) physiological responses . practicing regular physical exercise was found to be associated with better cognitive test performance and decreased arousal.3,24 a moderate exercise program followed twice a week significantly slowed , by one - third , the progressive deterioration in ability to perform activities of daily living in people with alzheimer s disease living in nursing homes.25 mind body exercises produce effects similar to those of regular cardiovascular exercises , suggesting an alternative model of exercise for the elderly , who are less able to exercise vigorously , to lower the risk of sport - related injuries and cardiac hazards.15 elderly people with the habit of regular physical exercise have been shown to be associated with socialization and environmental enrichment , which may also help attenuate the rate of cognitive decline.3 tai chi , a well - known mind body exercise , employs cognitive tools of both visualization and focused internal awareness to strengthen , relax , and integrate the body and mind.26 tai chi can also improve locomotion balance in seniors.27,28 a study evaluating a tai chi program called taiji ( tai chi ) buddies program found that the program encouraged social participation and supported partner involvement , which may have a positive influence on exercise persistence and the health and well - being of the support partner.28 a 12-week tai chi exercise program has been found adequate to reduce perceived stress and improve mood state , as well as increase perceived social support.29 the findings of this research showed that ct exercise , a simplified form of tai chi developed in this study specifically for the elderly with low activity , shares similar advantages , improving cognitive functions . body exercise enhances cardiovascular function , muscle strength , body balance , and physical function ; these improvements have a positive correlation with reduced stress , anxiety , and depression , resulting in an improved quality of life.24,30,31 a study utilizing electroencephalogram ( eeg ) recorded an increased cordance value at left hemisphere ( a sign of enhanced cerebral perfusion ) in a patient with chronic epilepsy after practicing dejian mind body intervention ( one of the components being mind body exercise).32 the changes in brain activities reflected by eeg underlie the observed improvements in cognitive functions.32 in addition , practicing mind - body exercise , which exerts similar effects to aerobic exercise , helps to increase volume in both gray and white matters primarily located in prefrontal and temporal cortices brain areas which are involved in age - related deterioration , as observed by mri images.33 as demonstrated by animal models , exercise - induced up - active pathways are associated with enhancement of several neurotransmitter systems afferent to the hippocampus , including the norepinephrine , serotonin , acetylcholine , and -aminobutyric acid systems , which are important to hippocampal function.34 these changes in brain activities and functioning demonstrate that regular , moderate physical exercise has beneficial effects on brain health . the findings of this study are consistent with previous reports that have shown that subjects practicing regular physical exercise are associated with better cognitive test performance , and there is a positive correlation between cardiovascular and mind body exercise and cognitive function among the chinese elderly.3,15 these exercises , however , might not be effective for the elderly suffering from moderate and severe dementia , who are likely to be immobile or even bed - bound . exercise applied in this study , which requires a lesser level of physical movement , sheds light on improving cognitive functions for dementia patients who may find difficulty undertaking regular physical exercise because they are physically less active or less mobile . additional , large - scale randomized control studies are recommended to elaborate on the efficacy of mind body exercise on cognitive functioning . the limitations of the study include the small sample size , and the absence of a control group ( without any exercise ) . participants in this study self - reported a habit of performing regular physical activities , and thus they are likely to be more health conscious with a lower cardiovascular burden.3 mind body exercise can improve cognitive functions and other health indicators , although the role of physical exercise in modulating cognitive decline is complex . the improvements can be described through ( 1 ) psychosocial indicators and ( 2 ) physiological responses . practicing regular physical exercise was found to be associated with better cognitive test performance and decreased arousal.3,24 a moderate exercise program followed twice a week significantly slowed , by one - third , the progressive deterioration in ability to perform activities of daily living in people with alzheimer s disease living in nursing homes.25 mind body exercises produce effects similar to those of regular cardiovascular exercises , suggesting an alternative model of exercise for the elderly , who are less able to exercise vigorously , to lower the risk of sport - related injuries and cardiac hazards.15 elderly people with the habit of regular physical exercise have been shown to be associated with socialization and environmental enrichment , which may also help attenuate the rate of cognitive decline.3 tai chi , a well - known mind body exercise , employs cognitive tools of both visualization and focused internal awareness to strengthen , relax , and integrate the body and mind.26 tai chi can also improve locomotion balance in seniors.27,28 a study evaluating a tai chi program called taiji ( tai chi ) buddies program found that the program encouraged social participation and supported partner involvement , which may have a positive influence on exercise persistence and the health and well - being of the support partner.28 a 12-week tai chi exercise program has been found adequate to reduce perceived stress and improve mood state , as well as increase perceived social support.29 the findings of this research showed that ct exercise , a simplified form of tai chi developed in this study specifically for the elderly with low activity , shares similar advantages , improving cognitive functions . body exercise enhances cardiovascular function , muscle strength , body balance , and physical function ; these improvements have a positive correlation with reduced stress , anxiety , and depression , resulting in an improved quality of life.24,30,31 a study utilizing electroencephalogram ( eeg ) recorded an increased cordance value at left hemisphere ( a sign of enhanced cerebral perfusion ) in a patient with chronic epilepsy after practicing dejian mind body intervention ( one of the components being mind body exercise).32 the changes in brain activities reflected by eeg underlie the observed improvements in cognitive functions.32 in addition , practicing mind - body exercise , which exerts similar effects to aerobic exercise , helps to increase volume in both gray and white matters primarily located in prefrontal and temporal cortices brain areas which are involved in age - related deterioration , as observed by mri images.33 as demonstrated by animal models , exercise - induced up - active pathways are associated with enhancement of several neurotransmitter systems afferent to the hippocampus , including the norepinephrine , serotonin , acetylcholine , and -aminobutyric acid systems , which are important to hippocampal function.34 these changes in brain activities and functioning demonstrate that regular , moderate physical exercise has beneficial effects on brain health . the findings of this study are consistent with previous reports that have shown that subjects practicing regular physical exercise are associated with better cognitive test performance , and there is a positive correlation between cardiovascular and mind body exercise and cognitive function among the chinese elderly.3,15 these exercises , however , might not be effective for the elderly suffering from moderate and severe dementia , who are likely to be immobile or even bed - bound . exercise applied in this study , which requires a lesser level of physical movement , sheds light on improving cognitive functions for dementia patients who may find difficulty undertaking regular physical exercise because they are physically less active or less mobile . additional , large - scale randomized control studies are recommended to elaborate on the efficacy of mind body exercise on cognitive functioning . the limitations of the study include the small sample size , and the absence of a control group ( without any exercise ) . participants in this study self - reported a habit of performing regular physical activities , and thus they are likely to be more health conscious with a lower cardiovascular burden.3 this prospective study attempted to provide evidence for the potential benefits of a customized coordination training exercise to improve the cognitive functioning of the elderly . the findings demonstrate that low physical level exercise similar to tai chi for example is beneficial for cognitive function and helps maintain the physical mobility of the elderly . the findings also give insight into developing further exercise regimes , which are more suitable for elderly people with a limited level of physical fitness or who are hospitalized .

background : studies on the effect of a low intensity coordination exercise on the elderly with limited mobility are sparse . this prospective study attempted to compare the effectiveness of a customized coordination exercise and a strength exercise in improving the cognitive functioning and physical mobility on the elderly.methods:participants from two centers for the elderly were allocated to practice either an 8-week coordination training ( ct ) program or an 8-week towel exercise ( te ) program . the chinese mini - mental state examination and chinese dementia rating scale ( cdrs ) were used to measure cognitive functioning of participants , and timed up - and - go test for physical mobility . these assessments were administered before and after the program.results:paired t - tests showed that the cdrs scores of the ct group improved significantly from 114.8 at pre - test to 119.3 after training ( p = 0.045 ) . the cdrs scores of the te group also improved from 114.9 at pre - test to 116.9 after training.conclusion:findings from this prospective study demonstrated that low - intensity level mind - body exercise could be beneficial to the cognitive functioning of older adults .

Introduction Method Subjects Coordination training (CT) Towel exercise (TE) Procedure Instruments Statistical analysis Results Discussion Mindbody exercise to improve cognitive function Psychosocial indicators Physiological responses to mindbody exercise Limitations of this study Conclusion

the benefits of physical exercise on cognitive function in the elderly have been demonstrated in many studies . several large - scale longitudinal studies showed that older people who have a high level of physical activity , have a significantly lower risk of developing alzheimer s disease and cognitive impairment.17 the results of a meta - analysis of 18 studies investigating the effectiveness of aerobic exercise concluded that fitness training could enhance the cognitive functioning of the elderly.8 this study also showed that a short duration , moderate - level training program could create an optimal effect on cognitive functions in the elderly . another meta - analysis investigating the change of duration and intensity of physical activity conducted by van gelder et al found that elderly people who participated in physical exercise for an average of 30 minutes per day or more could postpone their cognitive decline.9 however , studies on the benefits of physical training have focused closely on aerobic exercise such as walking , and strength exercise , such as weight lifting.4,6,7,1012 these aerobic and strength exercises require the participants to be highly mobile . the elderly with low mobility , or who are hospitalized , might have difficulty enjoying the full benefit of the exercise because of their limited locomotive ability . therefore exercise with reduced locomotion requirement , could provide the benefits of aerobic exercise to the elderly with restricted mobility . recently , there has been growing research interest in the therapeutic effects of mind body exercise.13,14 tai chi chuan , commonly known as tai chi , is a typical example of mind body exercise ; it is characterized by slow motion and emphasizes the conscious control of body movements , ie , it requires less locomotive mobility and is deemed appropriate for most elderly people.15 research has shown that the cognitive functions of the elderly could be well preserved with the aid of such mind body exercise , in a way similar to typical physical exercise.3 exercises with lower requirements of locomotive ability , such as coordination training ( ct ) and towel exercise ( te ) , are needed for the elderly with poor mobility . the literature review showed that ct and te may also be beneficial for the cognitive functioning of the elderly . the purpose of this study was to compare the effectiveness of ct and te on the cognitive functioning and physical mobility of the elderly , with the aim of developing an exercise with a low mobility requirement , to benefit the cognitive functioning of the elderly . we hypothesized that the elderly in the ct group would show significant improvement in the cognitive measures compared with the elderly in the te group . targeted participants were asked to take the chinese version of mini - mental state examination ( cmmse ) as one of the screening criteria , and those who scored 18 were eligible for this study.16 other than that , there was no other inclusion or exclusion criterion in recruitment . a physiotherapist from the jockey club centre for positive ageing ( jccpa , see http://www.jccpa.org.hk ) developed an 8-week exercise program , called coordination training ( ct ) , which is a simplified version of tai chi . it was easy for the elderly to learn , and required a relatively low level of mobility to practice . it was strongly promoted by the government because it was easy for elderly people with various locomotive abilities to master.18 te benefited the elderly by improving circulation and helping to control weight , and aimed to reduce the chance of falling.19 for the sake of convenience of participation and better monitoring of participants progress , those in one center were allocated to practice ct , and those in another center were allocated to practice te . both groups had a 10-minute warm - up period at the beginning and a 10-minute cool - down period at the end of the session to prevent injury . in this study , when the participants self - reported being able to handle the movement comfortably , which was confirmed by the trainer , they were required to practice the movement at an advanced difficulty level in order to avoid the ceiling effect.19 assessment tools including chinese mini - mental state examination ( cmmse ) , chinese dementia rating scale ( cdrs ) , and timed up - and - go test ( tug ) were administered to participants in both groups before and after the training sessions by trained occupational therapists and clinical psychologists . good psychometric properties were observed in both the original drs and the chinese version.21,22 tug was a good instrument to measure the general physical mobility of participants , and thus was administrated in this study to measure the effects of relevant exercises.23 the longer time spent to finish tug ( slower ) , the poorer the performance of participants , and vice versa . independent sample t - tests were conducted to compare the pre - test scores ( obtained in pre - test period ) between ct and te groups . paired sample t - tests were performed to compare the post - test scores ( obtained in the ninth week , after the 8-week exercise period ) with the pre - test scores in each group . analysis of covariance ( ancova ) was used to compare the scores of cmmse , cdrs , and tug of the two groups after the training program , using participants age and the pre - test scores as covariates . targeted participants were asked to take the chinese version of mini - mental state examination ( cmmse ) as one of the screening criteria , and those who scored 18 were eligible for this study.16 other than that , there was no other inclusion or exclusion criterion in recruitment . the ethics approval of this study was obtained from the survey and behavioural research ethics committee of the chinese university of hong kong . a physiotherapist from the jockey club centre for positive ageing ( jccpa , see http://www.jccpa.org.hk ) developed an 8-week exercise program , called coordination training ( ct ) , which is a simplified version of tai chi . it was easy for the elderly to learn , and required a relatively low level of mobility to practice . it was strongly promoted by the government because it was easy for elderly people with various locomotive abilities to master.18 te benefited the elderly by improving circulation and helping to control weight , and aimed to reduce the chance of falling.19 for the sake of convenience of participation and better monitoring of participants progress , those in one center were allocated to practice ct , and those in another center were allocated to practice te . both groups had a 10-minute warm - up period at the beginning and a 10-minute cool - down period at the end of the session to prevent injury . the level of difficulty was increased mainly by reducing the rest time ( demanding higher concentration as well as physical strength of participants ) , and by closing the eyes when performing the actions ( demanding higher psychomotor balance of participants ) . in this study , when the participants self - reported being able to handle the movement comfortably , which was confirmed by the trainer , they were required to practice the movement at an advanced difficulty level in order to avoid the ceiling effect.19 assessment tools including chinese mini - mental state examination ( cmmse ) , chinese dementia rating scale ( cdrs ) , and timed up - and - go test ( tug ) were administered to participants in both groups before and after the training sessions by trained occupational therapists and clinical psychologists . good psychometric properties were observed in both the original drs and the chinese version.21,22 tug was a good instrument to measure the general physical mobility of participants , and thus was administrated in this study to measure the effects of relevant exercises.23 the longer time spent to finish tug ( slower ) , the poorer the performance of participants , and vice versa . independent sample t - tests were conducted to compare the pre - test scores ( obtained in pre - test period ) between ct and te groups . paired sample t - tests were performed to compare the post - test scores ( obtained in the ninth week , after the 8-week exercise period ) with the pre - test scores in each group . analysis of covariance ( ancova ) was used to compare the scores of cmmse , cdrs , and tug of the two groups after the training program , using participants age and the pre - test scores as covariates . the average ages of the elderly in the ct and te groups were 77.7 6.0 and 80.3 5.5 , respectively . comparisons of participants pre - test ( baseline ) and post - test cognitive functioning by cmmse amd cdrs scores and physical mobility by tug scores are shown in table 4 . paired t - tests showed that the cdrs scores of the ct group had improved significantly from 114.8 15.5 at pre - test to 119.3 18.0 at post - test ( cdrs t(17 ) = 2.25 , p = 0.045 ) . the cdrs scores of the te group improved slightly from 114.9 14.8 at pre - test to 116.9 12.5 at post - test . no significant change was found in cmmse ( t(18 ) = 0.931 , p = 0.368 ) , and tug ( t(17 ) = 0.334 , p = 0.747 ) in ct group , as well as cmmse ( t(19 ) = 0.665 , p = 0.516 ) , cdrs ( t(19 ) = 0.891 , p = 0.384 ) and tug ( t(19 ) = 1.908 , p = 0.086 ) in the te group . different ancova ( between - subject factor : group [ ct , te ] and covariates : age and the pre - test scores ) models show the following findings . for cmmse , the covariate age ( f(1,28 ) = 0.17 , p = 0.690 , p = 0.003 ) and the exercise groups ( f(1,28 ) = 3.41 , p = 0.570 , p = 0.139 ) were not significantly related to the cmmse post - test scores . only the covariate pre - test scores of cmmse were significantly related to the post - test scores ( f(1,28 ) = 16.32 , p < 0.001 , p = 0.428 ) . for cdrs , exercise groups were not significantly related to the cdrs post - test scores ( f(1,28 ) = 0.02 , p = 0.904 , p = 0.001 ) . only the covariate age ( f(1,28 ) = 9.14 , p = 0.005 , p = 0.462 ) and the covariate pre - test scores of cdrs ( f(1,28 ) = 59.12 , p < 0.001 , p = 0.738 ) were significantly related to the cdrs post - test scores . for tug , the covariate age ( f(1,26 ) = 0.01 , p = 0.940 , p = < 0.001 ) and the exercise groups ( f(1,26 ) = 0.11 , p = 0.740 , p = 0.005 ) were not significantly related to the tug post - test scores . only the covariate pre - test scores of tug were significantly related to the tug post test scores ( f(1,26 ) = 83.50 , the above findings served to compare the effectiveness of the two exercise programs , coordination training ( ct ) and towel exercise ( te ) , in improving cognitive functioning and physical mobility in the elderly . the results showed that ct group participants had significant improvements in global cognition after the 8-week exercise program . ct group gained significant improvement in cdrs scores after the exercise training , while the te group participants did not . further investigation of the effectiveness of ct is recommended following this prospective study , through a large - scale clinical trial with appropriate numbers of samples in each group to detect the group differences . for the physical mobility measure , te tended to improve mobility while ct did not . the insignificant difference in physical mobility measure might suggest that ct , which required less in mobility , had a similar effect to te , a common physical exercise , on the cognitive and physical functioning of the elderly population . body exercise can improve cognitive functions and other health indicators , although the role of physical exercise in modulating cognitive decline is complex . practicing regular physical exercise was found to be associated with better cognitive test performance and decreased arousal.3,24 a moderate exercise program followed twice a week significantly slowed , by one - third , the progressive deterioration in ability to perform activities of daily living in people with alzheimer s disease living in nursing homes.25 mind body exercises produce effects similar to those of regular cardiovascular exercises , suggesting an alternative model of exercise for the elderly , who are less able to exercise vigorously , to lower the risk of sport - related injuries and cardiac hazards.15 elderly people with the habit of regular physical exercise have been shown to be associated with socialization and environmental enrichment , which may also help attenuate the rate of cognitive decline.3 tai chi , a well - known mind body exercise , employs cognitive tools of both visualization and focused internal awareness to strengthen , relax , and integrate the body and mind.26 tai chi can also improve locomotion balance in seniors.27,28 a study evaluating a tai chi program called taiji ( tai chi ) buddies program found that the program encouraged social participation and supported partner involvement , which may have a positive influence on exercise persistence and the health and well - being of the support partner.28 a 12-week tai chi exercise program has been found adequate to reduce perceived stress and improve mood state , as well as increase perceived social support.29 the findings of this research showed that ct exercise , a simplified form of tai chi developed in this study specifically for the elderly with low activity , shares similar advantages , improving cognitive functions . body exercise enhances cardiovascular function , muscle strength , body balance , and physical function ; these improvements have a positive correlation with reduced stress , anxiety , and depression , resulting in an improved quality of life.24,30,31 a study utilizing electroencephalogram ( eeg ) recorded an increased cordance value at left hemisphere ( a sign of enhanced cerebral perfusion ) in a patient with chronic epilepsy after practicing dejian mind body intervention ( one of the components being mind body exercise).32 the changes in brain activities reflected by eeg underlie the observed improvements in cognitive functions.32 in addition , practicing mind - body exercise , which exerts similar effects to aerobic exercise , helps to increase volume in both gray and white matters primarily located in prefrontal and temporal cortices brain areas which are involved in age - related deterioration , as observed by mri images.33 as demonstrated by animal models , exercise - induced up - active pathways are associated with enhancement of several neurotransmitter systems afferent to the hippocampus , including the norepinephrine , serotonin , acetylcholine , and -aminobutyric acid systems , which are important to hippocampal function.34 these changes in brain activities and functioning demonstrate that regular , moderate physical exercise has beneficial effects on brain health . the findings of this study are consistent with previous reports that have shown that subjects practicing regular physical exercise are associated with better cognitive test performance , and there is a positive correlation between cardiovascular and mind body exercise and cognitive function among the chinese elderly.3,15 these exercises , however , might not be effective for the elderly suffering from moderate and severe dementia , who are likely to be immobile or even bed - bound . additional , large - scale randomized control studies are recommended to elaborate on the efficacy of mind body exercise on cognitive functioning . the limitations of the study include the small sample size , and the absence of a control group ( without any exercise ) . participants in this study self - reported a habit of performing regular physical activities , and thus they are likely to be more health conscious with a lower cardiovascular burden.3 mind body exercise can improve cognitive functions and other health indicators , although the role of physical exercise in modulating cognitive decline is complex . practicing regular physical exercise was found to be associated with better cognitive test performance and decreased arousal.3,24 a moderate exercise program followed twice a week significantly slowed , by one - third , the progressive deterioration in ability to perform activities of daily living in people with alzheimer s disease living in nursing homes.25 mind body exercises produce effects similar to those of regular cardiovascular exercises , suggesting an alternative model of exercise for the elderly , who are less able to exercise vigorously , to lower the risk of sport - related injuries and cardiac hazards.15 elderly people with the habit of regular physical exercise have been shown to be associated with socialization and environmental enrichment , which may also help attenuate the rate of cognitive decline.3 tai chi , a well - known mind body exercise , employs cognitive tools of both visualization and focused internal awareness to strengthen , relax , and integrate the body and mind.26 tai chi can also improve locomotion balance in seniors.27,28 a study evaluating a tai chi program called taiji ( tai chi ) buddies program found that the program encouraged social participation and supported partner involvement , which may have a positive influence on exercise persistence and the health and well - being of the support partner.28 a 12-week tai chi exercise program has been found adequate to reduce perceived stress and improve mood state , as well as increase perceived social support.29 the findings of this research showed that ct exercise , a simplified form of tai chi developed in this study specifically for the elderly with low activity , shares similar advantages , improving cognitive functions . body exercise enhances cardiovascular function , muscle strength , body balance , and physical function ; these improvements have a positive correlation with reduced stress , anxiety , and depression , resulting in an improved quality of life.24,30,31 a study utilizing electroencephalogram ( eeg ) recorded an increased cordance value at left hemisphere ( a sign of enhanced cerebral perfusion ) in a patient with chronic epilepsy after practicing dejian mind body intervention ( one of the components being mind body exercise).32 the changes in brain activities reflected by eeg underlie the observed improvements in cognitive functions.32 in addition , practicing mind - body exercise , which exerts similar effects to aerobic exercise , helps to increase volume in both gray and white matters primarily located in prefrontal and temporal cortices brain areas which are involved in age - related deterioration , as observed by mri images.33 as demonstrated by animal models , exercise - induced up - active pathways are associated with enhancement of several neurotransmitter systems afferent to the hippocampus , including the norepinephrine , serotonin , acetylcholine , and -aminobutyric acid systems , which are important to hippocampal function.34 these changes in brain activities and functioning demonstrate that regular , moderate physical exercise has beneficial effects on brain health . the findings of this study are consistent with previous reports that have shown that subjects practicing regular physical exercise are associated with better cognitive test performance , and there is a positive correlation between cardiovascular and mind body exercise and cognitive function among the chinese elderly.3,15 these exercises , however , might not be effective for the elderly suffering from moderate and severe dementia , who are likely to be immobile or even bed - bound . additional , large - scale randomized control studies are recommended to elaborate on the efficacy of mind body exercise on cognitive functioning . the limitations of the study include the small sample size , and the absence of a control group ( without any exercise ) . participants in this study self - reported a habit of performing regular physical activities , and thus they are likely to be more health conscious with a lower cardiovascular burden.3 this prospective study attempted to provide evidence for the potential benefits of a customized coordination training exercise to improve the cognitive functioning of the elderly . the findings demonstrate that low physical level exercise similar to tai chi for example is beneficial for cognitive function and helps maintain the physical mobility of the elderly .

the world health organization ( who ) published the international classification of disability and health ( icf ) , which is a system to group and describe how a person is functioning in the environment based on a bio - psycho - social model . according to the icf an assistive device is described as an environmental factor which can either facilitate or inhibit a person s participation and activities . thus assistive devices are aimed at improving the functioning for disabled persons as stated in icf . the use of an assistive device can promote a person s quality of life ( qol ) by increasing his / her sense of competence , confidence and motivation to exploit the possibilities in their life . the use may provide opportunities by reducing difficulties in activities and decreasing dependence on others , and includes a broader psychosocial impact on a person s perceived qol [ 35 ] . qol is dynamic and changing over time and over a person s life and is experienced differently by different individuals , but the components are the same . renwick defines qol as the effect of the device on the degree to which a person enjoys the important possibilities of his / her life personal factors such as age , social habits and roles , and past and current experiences can be barriers to or facilitators of the use of assistive devices and personal factors can also influence the user s qol . psychosocial and cultural aspects , including the person s adaption to the disability in question , influence the meaning which the device holds for the person and whether the device will be used or not . use of a standing device can develop persons everyday activities in particular and according to nordstrm et al . the users of standing devices experienced that standing created freedom to perform activities and facilitated participation . the use of a standing device can also be part in treatment of bodily structures and prolonged standing may have beneficial effects on various bodily functions and structures , which in turn may affect the participation in activities in a positive way . the upright body position also allows communication on equal terms for persons with disabilities . standing devices in the present study follow the international classification and terminology iso 9999 comprising tilt tables , standing frames , standing frames with rear wheels , standing wheelchairs and standing shells . a previous study ( in press 2013 ) showed that the standing devices were frequently used and the users experienced an increased qol . however , the non - respond rate of 42% may indicate that the users were dissatisfied with the device or that the device was not used at all . the experiences of increased qol is consistent with arva et al . who concluded that standing enables participation in activities of daily living and that the upright position could promote the persons self - esteem and social interaction with other people . the usability of an assistive device is characterized by the relationship between the user , the assistive device , the activity and the context . non - use can be related to the feeling of being disabled and in turn affects the person s identity . people s reactions to their devices are complex and individual , because different persons have different needs , abilities , preferences and previous experiences . there is a knowledge gap in the significance of standing depending on that assistive devices hold different meanings for different users and there are several possible reasons for using or not using them . the psychosocial impact on the use of a powered wheelchair had a high value on qol , happiness and independence but also negative impact concerning self - esteem and a feeling of being stigmatized when using the device . the knowledge about the psychosocial impact of the use of standing devices is lacking , therefore it is important to get more knowledge in this area . based on this , the purpose of the present study was to investigate the psychosocial impact of standing devices as experienced by users . this study is the second part of a comprehensive survey conducted in the four northernmost counties and one county in central sweden and deals with the psychosocial impact of the standing device . the first part concerned the users characteristics , their degree of use of the standing device and their experiences of standing . the questionnaire consisted of background questions concerning the persons responding to the survey to determine whether they responded ( 1 ) without assistance , ( 2 ) receiving help or ( 3 ) through someone else answering on their behalf . the questionnaire had questions about perceived health , to be answered using a thermometer graded from 0 to 100 ( the eq5d thermometer ) , gender , age , diagnosis , movement skills , the type of standing device used , the time since the prescription and the standing frequency and duration . the impact of standing devices on functional independence , qol and wellbeing was assessed using the psychosocial impact of assistive devices scale ( piads ) . the scale of the questionnaire ranges from 3 ( the maximum negative impact ) to + 3 ( the maximum positive impact ) , and the results are presented with a total score and three sub - scores ( competence , adaptability and self - esteem ) . piads has proven to be a reliable , valid and responsive measure with good clinical utility . the scale seems to have the power to predict the abandonment and retention of an assistive device . a good example of previous use of the questionnaire is a study on the impact of the use of power wheelchairs on the activities and participation of people with stroke . the process was anchored by sending a request and information about the study to the manager for assistive devices in each county and statistics on the prescription of standing devices were obtained . the prescribers and/or the consultants for assistive devices who had knowledge about the potential participants received oral and written information about the study from the first author . the persons received information about the study and were informed that the participation was voluntary and that it was free to decline without declaration . those who accepted received the questionnaire and written information about the study by mail , together with a prepaid self - addressed envelope . the questionnaire was answered by the person himself / herself or a parent / related person . five hundred and forty - five ( 545 ) persons who had received a standing device were identified but 132 of those persons could not be reached or declined to participate . therefore only 413 questionnaires were sent out and 284 were returned , resulting in a response rate of 52% ( figure 1 ) . the participants were divided as belonging to all age groups , their age ranging from 2 to 86 years . only 22% of the respondents answered the questionnaire independently , while as many as 44% needed someone else to answer on their behalf . persons with acquired disabilities such as amyotrophic lateral sclerosis ( als ) and spinal cord injuries ( sci ) were most independent in this respect . three out of four persons with cerebral palsy ( cp ) had someone else answering the questionnaire on their behalf . the profiles of the participants are described in table 1 . as can be seen , the most common way to ambulate for the participants was to use a manual wheelchair , and a large proportion of those using a standing device were dependent on others for ambulation . figure 1.the number of questionnaires sent to users of standing devices and the number of eventual participants . n % who answered piads user6222 user with help of someone9734 someone else12544 missingage : 286 years median 37 ( sd 22.4)age groups 06 years217 712 years3713 1319 years269 2049 years10136 5064 years5419 65 years or older4416 missing1gender female10838 male17361 missing31diagnoses * congenital disease / injury12945 acquired disease / injury12745 undiagn./other diagn.186 missing104walking ability yes5118 no23382most common means of ambulation walking166 manual wheelchair20773 powered wheelchair6021 missing1need for help in ambulation independent7828 with some help5519 totally dependent15153type of standing device standing shell6623 standing frame6322 standing frame with rear wheels176 tilt table6623 wheelchair with stand - up function6925 other / missing31time since prescription 02 years6021 25 years7828 510 years6222 > 10 years8329 missing1*congenital disabilities : cp , syndromes , multi - disabilities , spina bifida . acquired disabilities : ms , als , tbi , stroke , virus , tumours . the number of questionnaires sent to users of standing devices and the number of eventual participants . congenital disabilities : cp , syndromes , multi - disabilities , spina bifida . acquired disabilities : ms , als , tbi , stroke , virus , tumours . the data for 164 of the 261 non - respondents were sufficient for a comparison with the respondents regarding age , sex gender and type of standing device . the mean age ( sd ) of the respondents was 37 22.4 years , while that of the non - respondents was 31 20.9 years . the proportion of men who responded to the survey was 61% , while the proportion of men in the group of non - respondents was 46% . the non - participants did not differ from the respondents with respect to the kind of prescribed device , except in the case of the standing wheelchair ; there were fewer users of standing wheelchairs amongst the non - respondents . twenty - five percent of the respondents had standing wheelchairs , while only 12% of those who refrained from responding to the survey had standing wheelchairs . the loss of participants was equally distributed in the northern region and the county in central sweden . the data were analyzed with descriptive statistics including percentages and medians . since the study was designed to be a survey of a sample population of people who used standing devices in sweden , no inferential statistics were calculated . the questionnaire consisted of background questions concerning the persons responding to the survey to determine whether they responded ( 1 ) without assistance , ( 2 ) receiving help or ( 3 ) through someone else answering on their behalf . the questionnaire had questions about perceived health , to be answered using a thermometer graded from 0 to 100 ( the eq5d thermometer ) , gender , age , diagnosis , movement skills , the type of standing device used , the time since the prescription and the standing frequency and duration . the impact of standing devices on functional independence , qol and wellbeing was assessed using the psychosocial impact of assistive devices scale ( piads ) . the scale of the questionnaire ranges from 3 ( the maximum negative impact ) to + 3 ( the maximum positive impact ) , and the results are presented with a total score and three sub - scores ( competence , adaptability and self - esteem ) . piads has proven to be a reliable , valid and responsive measure with good clinical utility . the scale seems to have the power to predict the abandonment and retention of an assistive device . a good example of previous use of the questionnaire is a study on the impact of the use of power wheelchairs on the activities and participation of people with stroke . the process was anchored by sending a request and information about the study to the manager for assistive devices in each county and statistics on the prescription of standing devices were obtained . the prescribers and/or the consultants for assistive devices who had knowledge about the potential participants received oral and written information about the study from the first author . the persons received information about the study and were informed that the participation was voluntary and that it was free to decline without declaration . those who accepted received the questionnaire and written information about the study by mail , together with a prepaid self - addressed envelope . the questionnaire was answered by the person himself / herself or a parent / related person . five hundred and forty - five ( 545 ) persons who had received a standing device were identified but 132 of those persons could not be reached or declined to participate . therefore only 413 questionnaires were sent out and 284 were returned , resulting in a response rate of 52% ( figure 1 ) . the participants were divided as belonging to all age groups , their age ranging from 2 to 86 years . only 22% of the respondents answered the questionnaire independently , while as many as 44% needed someone else to answer on their behalf . persons with acquired disabilities such as amyotrophic lateral sclerosis ( als ) and spinal cord injuries ( sci ) were most independent in this respect . three out of four persons with cerebral palsy ( cp ) had someone else answering the questionnaire on their behalf . the profiles of the participants are described in table 1 . as can be seen , the most common way to ambulate for the participants was to use a manual wheelchair , and a large proportion of those using a standing device were dependent on others for ambulation . figure 1.the number of questionnaires sent to users of standing devices and the number of eventual participants . n % who answered piads user6222 user with help of someone9734 someone else12544 missingage : 286 years median 37 ( sd 22.4)age groups 06 years217 712 years3713 1319 years269 2049 years10136 5064 years5419 65 years or older4416 missing1gender female10838 male17361 missing31diagnoses * congenital disease / injury12945 acquired disease / injury12745 undiagn./other diagn.186 missing104walking ability yes5118 no23382most common means of ambulation walking166 manual wheelchair20773 powered wheelchair6021 missing1need for help in ambulation independent7828 with some help5519 totally dependent15153type of standing device standing shell6623 standing frame6322 standing frame with rear wheels176 tilt table6623 wheelchair with stand - up function6925 other / missing31time since prescription 02 years6021 25 years7828 510 years6222 > 10 years8329 missing1*congenital disabilities : cp , syndromes , multi - disabilities , spina bifida . acquired disabilities : ms , als , tbi , stroke , virus , tumours . undiagnosed / other : persons with no diagnosis or an unusual diagnosis . the number of questionnaires sent to users of standing devices and the number of eventual participants . congenital disabilities : cp , syndromes , multi - disabilities , spina bifida . acquired disabilities : ms , als , tbi , stroke , virus , tumours . the data for 164 of the 261 non - respondents were sufficient for a comparison with the respondents regarding age , sex gender and type of standing device . the mean age ( sd ) of the respondents was 37 22.4 years , while that of the non - respondents was 31 20.9 years . the proportion of men who responded to the survey was 61% , while the proportion of men in the group of non - respondents was 46% . the non - participants did not differ from the respondents with respect to the kind of prescribed device , except in the case of the standing wheelchair ; there were fewer users of standing wheelchairs amongst the non - respondents . twenty - five percent of the respondents had standing wheelchairs , while only 12% of those who refrained from responding to the survey had standing wheelchairs . the loss of participants was equally distributed in the northern region and the county in central sweden . the data were analyzed with descriptive statistics including percentages and medians . since the study was designed to be a survey of a sample population of people who used standing devices in sweden , no inferential statistics were calculated . the psychosocial impact of the standing devices was perceived by the respondents as positive , deeming from their ratings ( table 2 ) . the medians for the total piads score and the piads sub - scores turned out to be positive , and even the first quartiles were on the positive side . the competence sub - score showed lower ratings than all the other sub - scores . table 2.piads scores.medianq1q3piads total ( n = 284)0.630.201.37adaptability ( n = 296)0.670.171.5competence ( n = 292)0.540.081.33self - esteem ( n = 296)0.620.121.37 the users answering the questionnaire without assistance awarded higher scores compared to those receiving help or having someone else answering on their behalf . this was the case for the piads total score and sub - scores ( figure 2 ) . figure 2.piads scores in relation to the level of assistance needed in responding to the questionnaire . piads scores in relation to the level of assistance needed in responding to the questionnaire . the highest value was found in the oldest group , aged 65 years or older ( median 0.77 ) , while the lowest value was found in the group aged 1319 years ( median 0.35 ) . persons with acquired diseases / injuries in general awarded higher piads scores compared to those with a congenital disease / injury . persons between 13 and 19 years of age differed as the group with the lowest piads total score and sub - scores , in contrast to children between 7 and 12 years of age , who gave higher scores , particularly concerning the dimension of self - esteem ( table 3 ) . table 3.piads scores in relation to different variables.piads totaladaptabilitycompetenceself - esteemsex female ( n = 108)0.630.670.500.62 male ( n = 173)0.650.830.580.62 missing ( n = 3)age groups 06 years ( n = 21)0.500.580.500.25 712 years ( n = 37)0.690.670.670.87 1319 years ( n = 26)0.350.330.250.37 2049 years ( n = 101)0.650.670.580.62 5064 years ( n = 54)0.650.830.500.75 65 years or older ( n = 44)0.771.000.580.62 missing ( n = 1)diagnoses * congenital disease / injury ( n = 129)0.540.670.500.56 acquired disease / injury ( n = 127)0.690.830.580.75 undiagn./other diagn . ( n = 28)0.560.670.580.62type of standing device standing shell ( n = 66)0.520.500.510.62 standing frame ( n = 63)0.690.750.500.62 standing frame with rear wheels ( n = 17)0.460.420.370.5 tilt table ( n = 66)0.630.830.580.75 wheelchair with stand - up function ( n = 69)0.650.670.620.62 other type / missing ( n = 3)time since prescription 02 years ( n = 60)0.711.000.580.69 25 years ( n = 78)0.420.670.420.37 510 years(n = 62)0.580.580.420.62 > 10 years ( n = 83)0.770.830.580.77 missing ( n = 1)walking ability with or without help yes ( n = 51)0.880.920.830.75 no ( n = 233)0.580.670.500.62most common means of ambulation walking ( n = 16)1.481.171.121.50 manual wheelchair ( n = 207)0.650.670.580.62 powered wheelchair ( n = 60)0.460.670.420.37 missing ( n = 1)need for help in ambulation independent ( n = 78)0.771.000.670.62 with some help ( n = 55)0.500.500.420.62 totally dependent ( n = 151)0.610.670.500.62frequency of standing * * often ( n = 167)0.690.670.580.75 quite often ( n = 86)0.540.920.500.50 rarely ( n = 31)0.310.330.210.37standing time < 15 min ( n = 20)0.420.330.500.37 1530 min ( n = 126)0.540.670.500.62 3060 min ( n = 111)0.690.830.580.75 > 60 min ( n = 14)0.380.420.330.31 short periods in different activities ( n = 13)1.071.50.920.87*congenital disease / injury : cp , syndromes , multi - disabled , spina bifida . acquired disease / injury : ms , als , sci , tbi , stroke , virus , tumours . undiagnosed / other diagnoses : persons with no diagnosis and unusual diagnoses.**often : several times a day , daily , almost daily . quite often : several times a week . rarely : once a week , almost never , never . congenital disease / injury : cp , syndromes , multi - disabled , spina bifida . acquired disease / injury : ms , als , sci , tbi , stroke , virus , tumours . several times a week . rarely : once a week , almost never , never . the piads total scores were quite similar for all the types of standing devices , except for standing shells and standing frames with rear wheels , which showed lower scores . when examining the piads scores in relation to the length of time the respondents had had their standing device , it was found that persons who had been using a standing device for 10 years or longer awarded the highest scores , while those who had received their device 25 years previously gave the lowest scores ( table 3 ) . respondents who possessed the ability to walk with or without help awarded higher piads scores compared to those who did not walk . it appeared that those who used walking as their most common means of ambulation assessed that standing had a greater psychosocial impact than was assessed by persons who had manual or powered wheelchairs as their most common means of ambulation . persons who were independent in ambulation awarded higher scores than persons who were totally dependent on help for ambulation , and also gave higher scores than those who needed some help for ambulation ( table 3 ) . an analysis of the persons who could walk showed that the majority of the 51 persons who had the ability to walk had congenital disabilities . nine persons with a congenital disability could walk independently , with or without a device , while no one with an acquired disability had an independent walking ability . forty - two persons could walk with help from someone and nine of them had an acquired disability . persons who had walking as the most common means of ambulation awarded a piads total score of 1.48 and persons who had the ability to walk with or without help scored higher than those who did not have the ability to walk . those who stood often ( several times / day , daily , almost daily ) awarded higher scores in the piads questionnaire compared to those who used their device less frequently . when standing was integrated in various activities , standing several times in different activities resulted in the highest scores , followed by standing for 3060 min each time . the ratings made according to the eq5d thermometer were spread across the whole range of the scale . the value was the same when the user rated without help and when someone else rated on behalf of the user ( 70 ) , but when the user rated with the help of someone else , the score was lower ( 61 ) . there was a trend towards a small positive correlation between the scoring according to the eq5d thermometer and the piads total score and the three sub - scores . age had an impact on the rating in that the values declined with age , from 73 on the scale for the youngest ( 16 years of age ) to 56 for the oldest ( 65 years of age or older ) . the psychosocial impact of the standing devices was perceived by the respondents as positive , deeming from their ratings ( table 2 ) . the medians for the total piads score and the piads sub - scores turned out to be positive , and even the first quartiles were on the positive side . the competence sub - score showed lower ratings than all the other sub - scores . table 2.piads scores.medianq1q3piads total ( n = 284)0.630.201.37adaptability ( n = 296)0.670.171.5competence ( n = 292)0.540.081.33self - esteem ( n = 296)0.620.121.37 the users answering the questionnaire without assistance awarded higher scores compared to those receiving help or having someone else answering on their behalf . this was the case for the piads total score and sub - scores ( figure 2 ) . figure 2.piads scores in relation to the level of assistance needed in responding to the questionnaire . piads scores in relation to the level of assistance needed in responding to the questionnaire . the highest value was found in the oldest group , aged 65 years or older ( median 0.77 ) , while the lowest value was found in the group aged 1319 years ( median 0.35 ) . persons with acquired diseases / injuries in general awarded higher piads scores compared to those with a congenital disease / injury . persons between 13 and 19 years of age differed as the group with the lowest piads total score and sub - scores , in contrast to children between 7 and 12 years of age , who gave higher scores , particularly concerning the dimension of self - esteem ( table 3 ) . table 3.piads scores in relation to different variables.piads totaladaptabilitycompetenceself - esteemsex female ( n = 108)0.630.670.500.62 male ( n = 173)0.650.830.580.62 missing ( n = 3)age groups 06 years ( n = 21)0.500.580.500.25 712 years ( n = 37)0.690.670.670.87 1319 years ( n = 26)0.350.330.250.37 2049 years ( n = 101)0.650.670.580.62 5064 years ( n = 54)0.650.830.500.75 65 years or older ( n = 44)0.771.000.580.62 missing ( n = 1)diagnoses * congenital disease / injury ( n = 129)0.540.670.500.56 acquired disease / injury ( n = 127)0.690.830.580.75 undiagn./other diagn . ( n = 28)0.560.670.580.62type of standing device standing shell ( n = 66)0.520.500.510.62 standing frame ( n = 63)0.690.750.500.62 standing frame with rear wheels ( n = 17)0.460.420.370.5 tilt table ( n = 66)0.630.830.580.75 wheelchair with stand - up function ( n = 69)0.650.670.620.62 other type / missing ( n = 3)time since prescription 02 years ( n = 60)0.711.000.580.69 25 years ( n = 78)0.420.670.420.37 510 years(n = 62)0.580.580.420.62 > 10 years ( n = 83)0.770.830.580.77 missing ( n = 1)walking ability with or without help yes ( n = 51)0.880.920.830.75 no ( n = 233)0.580.670.500.62most common means of ambulation walking ( n = 16)1.481.171.121.50 manual wheelchair ( n = 207)0.650.670.580.62 powered wheelchair ( n = 60)0.460.670.420.37 missing ( n = 1)need for help in ambulation independent ( n = 78)0.771.000.670.62 with some help ( n = 55)0.500.500.420.62 totally dependent ( n = 151)0.610.670.500.62frequency of standing * * often ( n = 167)0.690.670.580.75 quite often ( n = 86)0.540.920.500.50 rarely ( n = 31)0.310.330.210.37standing time < 15 min ( n = 20)0.420.330.500.37 1530 min ( n = 126)0.540.670.500.62 3060 min ( n = 111)0.690.830.580.75 > 60 min ( n = 14)0.380.420.330.31 short periods in different activities ( n = 13)1.071.50.920.87*congenital disease / injury : cp , syndromes , multi - disabled , spina bifida . acquired disease / injury : ms , als , sci , tbi , stroke , virus , tumours . undiagnosed / other diagnoses : persons with no diagnosis and unusual diagnoses.**often : several times a day , daily , almost daily . quite often : several times a week . rarely : once a week , almost never , never . piads scores in relation to different variables . congenital disease / injury : cp , syndromes , multi - disabled , spina bifida . acquired disease / injury : ms , als , sci , tbi , stroke , virus , tumours . the piads total scores were quite similar for all the types of standing devices , except for standing shells and standing frames with rear wheels , which showed lower scores . when examining the piads scores in relation to the length of time the respondents had had their standing device , it was found that persons who had been using a standing device for 10 years or longer awarded the highest scores , while those who had received their device 25 years previously gave the lowest scores ( table 3 ) . respondents who possessed the ability to walk with or without help awarded higher piads scores compared to those who did not walk . it appeared that those who used walking as their most common means of ambulation assessed that standing had a greater psychosocial impact than was assessed by persons who had manual or powered wheelchairs as their most common means of ambulation . persons who were independent in ambulation awarded higher scores than persons who were totally dependent on help for ambulation , and also gave higher scores than those who needed some help for ambulation ( table 3 ) . an analysis of the persons who could walk showed that the majority of the 51 persons who had the ability to walk had congenital disabilities . nine persons with a congenital disability could walk independently , with or without a device , while no one with an acquired disability had an independent walking ability . forty - two persons could walk with help from someone and nine of them had an acquired disability . persons who had walking as the most common means of ambulation awarded a piads total score of 1.48 and persons who had the ability to walk with or without help scored higher than those who did not have the ability to walk . those who stood often ( several times / day , daily , almost daily ) awarded higher scores in the piads questionnaire compared to those who used their device less frequently . when standing was integrated in various activities , standing several times in different activities resulted in the highest scores , followed by standing for 3060 min each time . the ratings made according to the eq5d thermometer were spread across the whole range of the scale . there were some differences depending on who answered the questionnaire . the value was the same when the user rated without help and when someone else rated on behalf of the user ( 70 ) , but when the user rated with the help of someone else , the score was lower ( 61 ) . there was a trend towards a small positive correlation between the scoring according to the eq5d thermometer and the piads total score and the three sub - scores . age had an impact on the rating in that the values declined with age , from 73 on the scale for the youngest ( 16 years of age ) to 56 for the oldest ( 65 years of age or older ) . based on our knowledge , this is one of the first studies that emphasize the meaning of using a standing device . the use of standing devices had a positive impact on psychosocial factors , which implies that the standing position holds a meaning for the users . the individually highest scores in the piads questionnaire were awarded by persons who had the ability to walk . the teenagers gave the lowest scores , which applied to both the total score and all the sub - scores , and persons with acquired disabilities gave higher scores compared to persons with congenital disabilities . the scores increased with a higher frequency of use and the persons who had had their device for 10 years or longer awarded higher scores than the persons who had had their device for a shorter time . analyzing the psychosocial impact in relation to the standing duration showed that standing for several short periods integrated in activities in daily life revealed higher scores compared to other selectable options . that finding coincides well with the icf s definition of assistive devices , which implies that an assistive device has the potential to facilitate activities . despite the fact that the physical effects of prolonged standing have been reported to be limited and inconclusive , this study provides a generally positive image of standing which reinforces the importance of standing from a psychosocial aspect . the teenagers ( 1319 years of age ) gave the lowest values for the piads total score and the three sub - scores ( concerning adaptability , competence and self - esteem ) , and the youngest group ( 06 years of age ) also awarded low scores concerning self - esteem . the low rating concerning self - esteem can be explained by the fact that it was parents / related persons who responded to the questionnaire on the children s behalf . this is in line with the findings of upton et al . who showed that the parents of children with health conditions tend to underestimate the child health - related qol . a previous study has shown that the parents psychological state should also be measured with respect to their physical and mental health , because the parents self - esteem is one factor that may affect proxy reports of qol . a previous study by nordstrm et al . showed that teenagers stood less frequently compared to the younger users and that the standing time decreased with increasing age . in the present study , frequent standing was shown to result in higher piads total scores and sub - scores . huang et al . showed that children with cp between 8 and 15 years of age had a negative impression of standing devices and experienced them as uncomfortable and limiting . muscle shortening in children with cp is common and that can contribute to the deterioration of standing skills . the shortening of muscles and chronic pain are common problems for youths with neuromuscular diseases . studies examining the period of transition from childhood to adult life have reported a decline in both health and functional ability . the low values for piads scores for teenagers could be a possible sign that they demonstrate a heightened level of self - consciousness and want to be like others . possibly , the standing device makes them feel different from other teenagers . according to larsson - lund and nygrd , an assistive device can not only facilitate an activity , but also be stigmatizing for the user . . showed that psychosocial aspects such as how the device influenced one s self - image and one s peers reactions to the assistive device were important from the teenager s perspective . people with acquired disabilities may value the psychosocial impact of their standing device higher because the standing position can contribute to the feeling of being like others , a sense of normality , being like before . louise - bender pape , kim and weiner concluded that the meaning of a device differs depending on whether the person using it has a congenital or an acquired disability . in contrast to the above - mentioned positive effect , a device can also clarify the consequences of a disability by highlighting the body s limitations for persons with progressive disabilities . persons with congenital disabilities do not have the time before and the time after the disease / injury to consider , and for them the use of standing devices may therefore have a natural meaning . however , deterioration in function is common for persons with congenital disabilities , e.g. persons with cp , for whom normal ageing may emerge earlier in life . this may contribute to the meaning of an assistive device being altered during a person s lifetime . this can be explained by the fact that the users of these devices were younger and had a congenital disability and that other persons awarded scores for them . all these factors affected the scores negatively . according to the process of prescribing assistive devices and to scherer and craddock , individual needs and goals should guide and govern the choice of device so that individual needs can be fulfilled . persons using their device for > 10 years awarded the highest scores and this could be an indication that the individual needs of this group have been fulfilled , in contrast to the lowest scoring group , who had had their device for 25 years . this may imply , in the latter case , that the assessment prior to the prescription of the device and the follow - ups had failed and that the psychosocial aspects of the device had not been taken into account . clinical experiences indicate that sometimes an adjustment or a replacement of the device has positive effects on the users experience in terms of the psychosocial impact of the device . as professionals we need to ensure that the person with a disability has been provided with the optimal standing device . surprisingly , persons who had the ability to walk awarded higher piads scores compared to those who did not . the majority of the persons who could walk had a congenital disability and it may be the case that standing was seen as a treatment which could improve their potential to walk . this is in line with a study by salem et al . , who showed that prolonged standing for children with cp improved their walking ability . furthermore , standing itself could also be the starting - point for independence and/or , according to mckeever et al . one can also speculate whether a user s ability to walk and to be independent in ambulation can mean that he / she has the ability to get in and out of the device independently , which in turn means that the device is used more and is perceived to have a greater psychosocial impact for the user . it is satisfying to note that those who stood most frequently rated the psychosocial impact of the standing device highest . being able to stand for several short periods in activities was highly valued . since the physical effects of standing are contradictory , we as professionals should focus on the user s perceived meaning of standing . if a standing device is used frequently and is perceived to have a positive psychosocial impact , this could mean that the person using it has the optimal standing device for their needs . according to alerby , a pen should be regarded as an integrated part of the body and not just an object that makes writing become a habit , and the assistive device should be considered in a similar way . the optimal standing device should therefore , adopting this view , be perceived as an extension of the body and not only an object whose purpose is to exercise the physical body . the highest piads scores in relation to the standing frequency and duration were found when standing was performed for several short periods in different activities . one explanation for this could be that , when the device is used in an activity , the standing position holds a meaning for the person using it . our mission as professionals is to broaden our view of the use of standing devices , i.e. to see the standing device as an aid that not only treats the body s structures or improves the user s abilities in activities , but also provides a psychosocial impact on the user s daily life , and to find meaningful goals for the user from a psychosocial aspect . we chose to use piads as an instrument for our purpose because it is an instrument that is especially designed to evaluate the psychosocial impact of the device . further , piads was developed with users involved and it also tested to be reliable in cases where someone else is answering the questionnaire on behalf of the user which is a common case among people who use standing devices . there are several limitations to take into account which affect the generalisation of this comprehensive survey . this fact raises the question of whether or not these non - respondents were users of standing devices with which they were dissatisfied . secondly , all the respondents were not autonomous and other persons were involved in answering the questionnaires . piads is supposed to work even if the survey is completed by another person , but that fact may have contributed to the high failure rate . many users of standing devices are not autonomous and it is important to obtain their knowledge with the help of those who know them best . however , we have to be aware that the outcome could have been different if all the users had been able to speak for themselves . it is known that the parents of children with health conditions tend to underestimate their children s health - related qol , and this kind of underestimation may have had an impact in this study . or could it be the case that those users who did not respond independently had a lower health status and consistently experienced a lower psychosocial impact from the device ? the fact that the requesting staff knew the participants could be a limitation from a confidentially perspective , therefore the participants were assured that their data would be presented in such a way that no single participant could be recognizable . the objective of this research study has been to measure the experience of standing of users of standing devices . for this purpose piads appears to serve a useful purpose . the main results of the study was that the psychosocial impact of standing devices was generally experienced positively , but there were some differences among the participants of the survey . it was shown that those respondents who possessed a higher physical capacity and an ability to respond independently considered it even more important to stand . being able to stand in activities and having the ability to walk seemed to be important . being a teenager was associated with lower scores , as was a standing time of > 60 min each time . the main results indicated that standing in a standing device had a value and we as professionals should ask the users about the intended purpose of their standing in order to prescribe the optimal device . the prescribers ought to try to influence the suppliers of standing devices to design a device that the users are asking for . future research should investigate the meaning which standing in the device holds for the person using it , and should focus on the psychosocial impact of using a standing device which the results of this study have confirmed .

purposethe aim of this study was to explore the psychosocial impact of standing devices as experienced by users.methodthis is the second part of a comprehensive survey in five counties in sweden where all the subjects with standing devices were invited to participate . the impact of standing devices on functional independence , quality of life and wellbeing was assessed using a questionnaire , psychosocial impact of assistive devices scale ( piads).resultsthe psychosocial impact of the standing devices was perceived as positive . the highest piads scores in relation to age were found in the oldest group , aged 65 years and older . the ability to walk and independence in ambulation resulted in higher scores than the use of a wheelchair and/or dependence on others . those who stood often awarded higher scores in the piads questionnaire compared to those who used the device less frequently . when standing was integrated in various activities , its psychosocial impact received high scores.conclusionthe psychosocial impact of standing devices was generally experienced positively . the main results indicated that standing in a standing device had a value and we as professionals should ask the users about the intended purpose of their standing in order to prescribe the optimal device . implications for rehabilitationstanding in standing devices has positive psychosocial impact for the user.as professionals we should broaden our view of the use of standing devices , i.e. to see the standing device as an aid that not only treats the body s structures or improves the user s abilities in activities , but also provides a psychosocial impact on the user s daily life , and to find meaningful goals for the user from a psychosocial perspective .

Introduction Methods Questionnaire Procedure Participants Analysis of the non-respondents Analysis of data Results Psychosocial impact of standing devices PIADS scores in relation to the participants sex, age and diagnosis PIADS scores in relation to the type of standing device used and the time since the device was prescribed PIADS scores in relation to ambulation and the need for help PIADS scores in relation to the standing frequency and duration Perceived health according to the EQ5D thermometer Discussion Conclusions

the use of an assistive device can promote a person s quality of life ( qol ) by increasing his / her sense of competence , confidence and motivation to exploit the possibilities in their life . the use may provide opportunities by reducing difficulties in activities and decreasing dependence on others , and includes a broader psychosocial impact on a person s perceived qol [ 35 ] . renwick defines qol as the effect of the device on the degree to which a person enjoys the important possibilities of his / her life personal factors such as age , social habits and roles , and past and current experiences can be barriers to or facilitators of the use of assistive devices and personal factors can also influence the user s qol . use of a standing device can develop persons everyday activities in particular and according to nordstrm et al . the use of a standing device can also be part in treatment of bodily structures and prolonged standing may have beneficial effects on various bodily functions and structures , which in turn may affect the participation in activities in a positive way . a previous study ( in press 2013 ) showed that the standing devices were frequently used and the users experienced an increased qol . the psychosocial impact on the use of a powered wheelchair had a high value on qol , happiness and independence but also negative impact concerning self - esteem and a feeling of being stigmatized when using the device . the knowledge about the psychosocial impact of the use of standing devices is lacking , therefore it is important to get more knowledge in this area . based on this , the purpose of the present study was to investigate the psychosocial impact of standing devices as experienced by users . this study is the second part of a comprehensive survey conducted in the four northernmost counties and one county in central sweden and deals with the psychosocial impact of the standing device . the first part concerned the users characteristics , their degree of use of the standing device and their experiences of standing . the questionnaire had questions about perceived health , to be answered using a thermometer graded from 0 to 100 ( the eq5d thermometer ) , gender , age , diagnosis , movement skills , the type of standing device used , the time since the prescription and the standing frequency and duration . the impact of standing devices on functional independence , qol and wellbeing was assessed using the psychosocial impact of assistive devices scale ( piads ) . a good example of previous use of the questionnaire is a study on the impact of the use of power wheelchairs on the activities and participation of people with stroke . the process was anchored by sending a request and information about the study to the manager for assistive devices in each county and statistics on the prescription of standing devices were obtained . as can be seen , the most common way to ambulate for the participants was to use a manual wheelchair , and a large proportion of those using a standing device were dependent on others for ambulation . n % who answered piads user6222 user with help of someone9734 someone else12544 missingage : 286 years median 37 ( sd 22.4)age groups 06 years217 712 years3713 1319 years269 2049 years10136 5064 years5419 65 years or older4416 missing1gender female10838 male17361 missing31diagnoses * congenital disease / injury12945 acquired disease / injury12745 undiagn./other diagn.186 missing104walking ability yes5118 no23382most common means of ambulation walking166 manual wheelchair20773 powered wheelchair6021 missing1need for help in ambulation independent7828 with some help5519 totally dependent15153type of standing device standing shell6623 standing frame6322 standing frame with rear wheels176 tilt table6623 wheelchair with stand - up function6925 other / missing31time since prescription 02 years6021 25 years7828 510 years6222 > 10 years8329 missing1*congenital disabilities : cp , syndromes , multi - disabilities , spina bifida . the non - participants did not differ from the respondents with respect to the kind of prescribed device , except in the case of the standing wheelchair ; there were fewer users of standing wheelchairs amongst the non - respondents . since the study was designed to be a survey of a sample population of people who used standing devices in sweden , no inferential statistics were calculated . the questionnaire had questions about perceived health , to be answered using a thermometer graded from 0 to 100 ( the eq5d thermometer ) , gender , age , diagnosis , movement skills , the type of standing device used , the time since the prescription and the standing frequency and duration . the impact of standing devices on functional independence , qol and wellbeing was assessed using the psychosocial impact of assistive devices scale ( piads ) . a good example of previous use of the questionnaire is a study on the impact of the use of power wheelchairs on the activities and participation of people with stroke . the process was anchored by sending a request and information about the study to the manager for assistive devices in each county and statistics on the prescription of standing devices were obtained . as can be seen , the most common way to ambulate for the participants was to use a manual wheelchair , and a large proportion of those using a standing device were dependent on others for ambulation . n % who answered piads user6222 user with help of someone9734 someone else12544 missingage : 286 years median 37 ( sd 22.4)age groups 06 years217 712 years3713 1319 years269 2049 years10136 5064 years5419 65 years or older4416 missing1gender female10838 male17361 missing31diagnoses * congenital disease / injury12945 acquired disease / injury12745 undiagn./other diagn.186 missing104walking ability yes5118 no23382most common means of ambulation walking166 manual wheelchair20773 powered wheelchair6021 missing1need for help in ambulation independent7828 with some help5519 totally dependent15153type of standing device standing shell6623 standing frame6322 standing frame with rear wheels176 tilt table6623 wheelchair with stand - up function6925 other / missing31time since prescription 02 years6021 25 years7828 510 years6222 > 10 years8329 missing1*congenital disabilities : cp , syndromes , multi - disabilities , spina bifida . the non - participants did not differ from the respondents with respect to the kind of prescribed device , except in the case of the standing wheelchair ; there were fewer users of standing wheelchairs amongst the non - respondents . since the study was designed to be a survey of a sample population of people who used standing devices in sweden , no inferential statistics were calculated . the psychosocial impact of the standing devices was perceived by the respondents as positive , deeming from their ratings ( table 2 ) . the medians for the total piads score and the piads sub - scores turned out to be positive , and even the first quartiles were on the positive side . table 2.piads scores.medianq1q3piads total ( n = 284)0.630.201.37adaptability ( n = 296)0.670.171.5competence ( n = 292)0.540.081.33self - esteem ( n = 296)0.620.121.37 the users answering the questionnaire without assistance awarded higher scores compared to those receiving help or having someone else answering on their behalf . figure 2.piads scores in relation to the level of assistance needed in responding to the questionnaire . piads scores in relation to the level of assistance needed in responding to the questionnaire . the highest value was found in the oldest group , aged 65 years or older ( median 0.77 ) , while the lowest value was found in the group aged 1319 years ( median 0.35 ) . persons with acquired diseases / injuries in general awarded higher piads scores compared to those with a congenital disease / injury . table 3.piads scores in relation to different variables.piads totaladaptabilitycompetenceself - esteemsex female ( n = 108)0.630.670.500.62 male ( n = 173)0.650.830.580.62 missing ( n = 3)age groups 06 years ( n = 21)0.500.580.500.25 712 years ( n = 37)0.690.670.670.87 1319 years ( n = 26)0.350.330.250.37 2049 years ( n = 101)0.650.670.580.62 5064 years ( n = 54)0.650.830.500.75 65 years or older ( n = 44)0.771.000.580.62 missing ( n = 1)diagnoses * congenital disease / injury ( n = 129)0.540.670.500.56 acquired disease / injury ( n = 127)0.690.830.580.75 undiagn./other diagn . the piads total scores were quite similar for all the types of standing devices , except for standing shells and standing frames with rear wheels , which showed lower scores . when examining the piads scores in relation to the length of time the respondents had had their standing device , it was found that persons who had been using a standing device for 10 years or longer awarded the highest scores , while those who had received their device 25 years previously gave the lowest scores ( table 3 ) . respondents who possessed the ability to walk with or without help awarded higher piads scores compared to those who did not walk . it appeared that those who used walking as their most common means of ambulation assessed that standing had a greater psychosocial impact than was assessed by persons who had manual or powered wheelchairs as their most common means of ambulation . persons who were independent in ambulation awarded higher scores than persons who were totally dependent on help for ambulation , and also gave higher scores than those who needed some help for ambulation ( table 3 ) . an analysis of the persons who could walk showed that the majority of the 51 persons who had the ability to walk had congenital disabilities . persons who had walking as the most common means of ambulation awarded a piads total score of 1.48 and persons who had the ability to walk with or without help scored higher than those who did not have the ability to walk . those who stood often ( several times / day , daily , almost daily ) awarded higher scores in the piads questionnaire compared to those who used their device less frequently . when standing was integrated in various activities , standing several times in different activities resulted in the highest scores , followed by standing for 3060 min each time . age had an impact on the rating in that the values declined with age , from 73 on the scale for the youngest ( 16 years of age ) to 56 for the oldest ( 65 years of age or older ) . the psychosocial impact of the standing devices was perceived by the respondents as positive , deeming from their ratings ( table 2 ) . the medians for the total piads score and the piads sub - scores turned out to be positive , and even the first quartiles were on the positive side . table 2.piads scores.medianq1q3piads total ( n = 284)0.630.201.37adaptability ( n = 296)0.670.171.5competence ( n = 292)0.540.081.33self - esteem ( n = 296)0.620.121.37 the users answering the questionnaire without assistance awarded higher scores compared to those receiving help or having someone else answering on their behalf . figure 2.piads scores in relation to the level of assistance needed in responding to the questionnaire . piads scores in relation to the level of assistance needed in responding to the questionnaire . the highest value was found in the oldest group , aged 65 years or older ( median 0.77 ) , while the lowest value was found in the group aged 1319 years ( median 0.35 ) . persons with acquired diseases / injuries in general awarded higher piads scores compared to those with a congenital disease / injury . table 3.piads scores in relation to different variables.piads totaladaptabilitycompetenceself - esteemsex female ( n = 108)0.630.670.500.62 male ( n = 173)0.650.830.580.62 missing ( n = 3)age groups 06 years ( n = 21)0.500.580.500.25 712 years ( n = 37)0.690.670.670.87 1319 years ( n = 26)0.350.330.250.37 2049 years ( n = 101)0.650.670.580.62 5064 years ( n = 54)0.650.830.500.75 65 years or older ( n = 44)0.771.000.580.62 missing ( n = 1)diagnoses * congenital disease / injury ( n = 129)0.540.670.500.56 acquired disease / injury ( n = 127)0.690.830.580.75 undiagn./other diagn . piads scores in relation to different variables . the piads total scores were quite similar for all the types of standing devices , except for standing shells and standing frames with rear wheels , which showed lower scores . when examining the piads scores in relation to the length of time the respondents had had their standing device , it was found that persons who had been using a standing device for 10 years or longer awarded the highest scores , while those who had received their device 25 years previously gave the lowest scores ( table 3 ) . respondents who possessed the ability to walk with or without help awarded higher piads scores compared to those who did not walk . it appeared that those who used walking as their most common means of ambulation assessed that standing had a greater psychosocial impact than was assessed by persons who had manual or powered wheelchairs as their most common means of ambulation . persons who were independent in ambulation awarded higher scores than persons who were totally dependent on help for ambulation , and also gave higher scores than those who needed some help for ambulation ( table 3 ) . an analysis of the persons who could walk showed that the majority of the 51 persons who had the ability to walk had congenital disabilities . persons who had walking as the most common means of ambulation awarded a piads total score of 1.48 and persons who had the ability to walk with or without help scored higher than those who did not have the ability to walk . those who stood often ( several times / day , daily , almost daily ) awarded higher scores in the piads questionnaire compared to those who used their device less frequently . when standing was integrated in various activities , standing several times in different activities resulted in the highest scores , followed by standing for 3060 min each time . age had an impact on the rating in that the values declined with age , from 73 on the scale for the youngest ( 16 years of age ) to 56 for the oldest ( 65 years of age or older ) . based on our knowledge , this is one of the first studies that emphasize the meaning of using a standing device . the use of standing devices had a positive impact on psychosocial factors , which implies that the standing position holds a meaning for the users . the individually highest scores in the piads questionnaire were awarded by persons who had the ability to walk . the teenagers gave the lowest scores , which applied to both the total score and all the sub - scores , and persons with acquired disabilities gave higher scores compared to persons with congenital disabilities . the scores increased with a higher frequency of use and the persons who had had their device for 10 years or longer awarded higher scores than the persons who had had their device for a shorter time . analyzing the psychosocial impact in relation to the standing duration showed that standing for several short periods integrated in activities in daily life revealed higher scores compared to other selectable options . despite the fact that the physical effects of prolonged standing have been reported to be limited and inconclusive , this study provides a generally positive image of standing which reinforces the importance of standing from a psychosocial aspect . according to larsson - lund and nygrd , an assistive device can not only facilitate an activity , but also be stigmatizing for the user . people with acquired disabilities may value the psychosocial impact of their standing device higher because the standing position can contribute to the feeling of being like others , a sense of normality , being like before . persons with congenital disabilities do not have the time before and the time after the disease / injury to consider , and for them the use of standing devices may therefore have a natural meaning . this may imply , in the latter case , that the assessment prior to the prescription of the device and the follow - ups had failed and that the psychosocial aspects of the device had not been taken into account . clinical experiences indicate that sometimes an adjustment or a replacement of the device has positive effects on the users experience in terms of the psychosocial impact of the device . as professionals we need to ensure that the person with a disability has been provided with the optimal standing device . surprisingly , persons who had the ability to walk awarded higher piads scores compared to those who did not . the majority of the persons who could walk had a congenital disability and it may be the case that standing was seen as a treatment which could improve their potential to walk . one can also speculate whether a user s ability to walk and to be independent in ambulation can mean that he / she has the ability to get in and out of the device independently , which in turn means that the device is used more and is perceived to have a greater psychosocial impact for the user . it is satisfying to note that those who stood most frequently rated the psychosocial impact of the standing device highest . since the physical effects of standing are contradictory , we as professionals should focus on the user s perceived meaning of standing . if a standing device is used frequently and is perceived to have a positive psychosocial impact , this could mean that the person using it has the optimal standing device for their needs . according to alerby , a pen should be regarded as an integrated part of the body and not just an object that makes writing become a habit , and the assistive device should be considered in a similar way . the optimal standing device should therefore , adopting this view , be perceived as an extension of the body and not only an object whose purpose is to exercise the physical body . the highest piads scores in relation to the standing frequency and duration were found when standing was performed for several short periods in different activities . our mission as professionals is to broaden our view of the use of standing devices , i.e. to see the standing device as an aid that not only treats the body s structures or improves the user s abilities in activities , but also provides a psychosocial impact on the user s daily life , and to find meaningful goals for the user from a psychosocial aspect . we chose to use piads as an instrument for our purpose because it is an instrument that is especially designed to evaluate the psychosocial impact of the device . the main results of the study was that the psychosocial impact of standing devices was generally experienced positively , but there were some differences among the participants of the survey . being able to stand in activities and having the ability to walk seemed to be important . the main results indicated that standing in a standing device had a value and we as professionals should ask the users about the intended purpose of their standing in order to prescribe the optimal device . future research should investigate the meaning which standing in the device holds for the person using it , and should focus on the psychosocial impact of using a standing device which the results of this study have confirmed .

the gastrointestinal ( gi ) tract contains a multitude of regulatory peptides that transmit information not only to the intestine and associated organs , but to other systems , such as the central nervous system ( cns ) and the cardiovascular system . at the beginning of the 20th century , experiments were carried out with mucosa extracts of the small intestine for treatment of diabetes mellitus , based on the idea that gastrointestinal hormones stimulated pancreatic endocrine function . historically , bayliss and starling in 1905 examined the effects of crude intestinal extracts on exocrine pancreatic secretions and reported the existence of a secretin , the first regulatory peptide to be identified , thus introducing the concept of hormones and describing their way of action . in 1906 , moore et al . discovered a chemical stimulant produced by the pancreas and , in 1930 , la barre studied the effects of intravenous administration of unclean the incretins identified in the 1930s were associated with intestinal synthesis of hormones similar to insulin and were , thus , responsible for the introduction of this term , which was originated from the junction of fragments of the words basically , an incretin describes a factor that reduces blood glucose levels without affecting exocrine pancreatic secretion . however , it would take more than 30 years before they showed perceptive implications on the regulation of blood glucose . incretin effect in 1964 , by observing that oral administration of glucose caused a greater increase in insulin secretion than the same amount of glucose administered intravenously , despite the higher blood glucose levels registered by the intravenous route . oral glucose administration resulted in increased insulin secretion , confirming the existence of a link between the intestine and the endocrine pancreas , leading to the assumption that gastrointestinal hormones could have an additional action on insulin secretion . therefore , for a given hormone to be included in a group of incretins , it must meet two essential criteria : be released in response to oral glucose intake and be able to achieve physiological concentrations resulting in insulin release . the revival of the term incretin was mostly due to creutzfeldt [ 2 , 5 ] , who emphasized the relationship glucose - insulin - intestine in association with the incretin effect , a feature that is of profound importance for its clinical application . in 1986 , studied the incretin effect ( insulin response after oral versus intravenous administration of either 25 , 50 , or 100 g of glucose ) by measuring the concentrations of c - peptide , a marker of endogenous insulin secretion . these investigators found that the level of incretin secretion was dependent on the amount of ingested glucose and that incretins were responsible for approximately 75% of the insulin response after the ingestion of 50 g of glucose . the study of incretin hormones was pursued by a number of researchers , but identification of the first incretin came from an unexpected source . brown of the university of british columbia , vancouver , canada , tried to isolate a hormone involved in the regulation of gastric acid secretion from pig intestinal extracts : enterogastrone . in collaboration with other researchers , he identified and isolated a hormone composed of 42 amino acids , to which he gave the name of gastric inhibitory polypeptide ( gip ) , now also known as glucose - dependent insulinotropic polypeptide , since it was shown to be able to stimulate insulin secretion in a glucose - dependent manner ; it is an incretin . later , a second incretin was isolated due to genetic studies on proglucagon coding sequences , and it was described as a this molecule was named glucagon - like peptide-1 ( glp-1 ) and as it met the criteria , it was classified as an incretin . both incretins are secreted in the intestinal mucosa ; gip is secreted from the k - cells ( enterochromaffin cells ) located mainly in the stomach , duodenal mucosa , and the proximal jejunum , whereas l - cells produce glp-1 and are located more distally in the ileum and colon . within minutes of nutrient ingestion , both incretins ( gip and glp-1 ) the metabolic , hormonal , and neuronal influences on the endocrine pancreas are collectively referred to as the enteroinsular axis . postprandial insulin secretion is directly stimulated by substrates of nerve stimulation , through enteropancreatic nerve activation by chyme and intestinal distension , and by a strong endocrine stimulus mediated by incretin hormones . besides gip and glp-1 , several other gastrointestinal hormones are released from endocrine cells and neurons in the digestive tract , which makes the gut the largest hormone producing organ in the body . the physiological functions of these peptides have been revealed during the last years , namely , those concerning the regulation of glucose homeostasis and their putative use as therapeutic target for obesity , and diabetes is an emerging and evolving challenge , as previously reviewed [ 1214 ] and briefly revisited in this section . ghrelin is secreted from the stomach in the fasting state and is an appetite - stimulating gi hormone , also known as the hunger hormone . cholecystokinin ( cck ) is mainly produced in the l - cells of the duodenum and small intestine in response to a meal , thus stimulating pancreatic hormone and bile secretion and inhibiting gastric emptying ; cck was the first gi hormone found to act as a hunger suppressant . the classical action of gastrin is the control of gallbladder contraction , satiety , and pancreatic and gastric acid secretion , but current knowledge points to participation in the control of glucose homeostasis , namely , by stimulation of glucagon release from human islets in vitro . peptide yy ( pyy ) is produced in the gi l - cells , mainly in the colon and rectum , and has been viewed as a meal termination signal and shows satiety peptide properties and its levels are low after overnight fasting and elevated after meal . pyy belongs to the pp fold family of peptides which includes pancreatic polypeptide ( pp ) and neuropeptide y ( npy ) . pp is secreted in the islets of langerhans and , in smaller amounts , by colon and rectum cells and has been associated with reduction of gastric emptying . its fasting levels are low , but their postprandial levels increase and are correlated with meal calorie content . oxyntomodulin ( oxm ) is secreted by the l - cells , in parallel with glp-1 production , and shows an incretin effect , reducing the appetite and the amount of ingested food , an effect that seems to be partly due to the inhibition of ghrelin secretion . amylin , also known as islet amyloid polypeptide ( iapp ) , is secreted together with insulin in pancreatic cells and has been suggested to play a role in glucose homeostasis by suppressing the release of glucagon from pancreatic cells , thus preventing the release of glucose from the liver , decreasing the gastric emptying , and stimulating the satiety center in the brain . although several gi hormones have been demonstrating impact on glucose metabolism , incretins hormones , mainly glp-1 , have been used as therapeutic target for diabetes treatment and will be the focus of this paper . approximately 30 to 60% of c - peptide and 80 to 90% of the insulin response after an oral glucose load are regulated by incretin hormones , depending on the amount of glucose . incretin action on pancreatic cells involves a series of events that potentiate the action of glucose , an important feature that is protective against the development of hypoglycaemia . one of the characteristics of these incretins , which makes them attractive as potential therapeutic agents , is that the associated insulin secretion ceases when euglycemia is achieved , thus minimizing the risk of hypoglycaemia . both gip and glp-1 exert their effects by binding to their specific receptors , the gip receptor ( gipr ) and the glp-1 receptor ( glp-1r ) , stimulating a cascade of events that culminate in stimulation of glucose - dependent insulin secretion in pancreatic cells . gip and glp-1 are rapidly metabolized ( t1/2 2 minutes ) by the ubiquitous enzyme dipeptidyl peptidase-4 ( dpp-4 ) to inactive metabolites and then eliminated by the urine . gip and glp-1 share common properties as incretins , but they also possess different biological characteristics . glp-1 acts in a positive way on the and cells , whereas gip acts preferentially on the and cells . the effects of glp-1 on pancreatic islet cells include increased insulin secretion by cells in a glucose - dependent manner , increased secretion of somatostatin by cells , and reduced secretion of glucagon by the cells . in addition to their insulinotropic effects , gip and glp-1 play critical roles in various biological processes in different tissues and organs that express gipr and glp-1r , including the pancreas , adipose tissues , bone , peripheral and central nervous systems ( cns ) , heart , kidney , liver , and gi tract [ 19 , 20 ] . here , we briefly revise the similarities and differences concerning the glp-1 and gip insulinotropic actions on pancreatic cells and their noninsulinotropic effects on pancreas and on extrapancreatic tissues , which have been revealed during the last years [ 21 , 22 ] . figure 1 schematically presents the major biological actions of glp-1 on pancreas and on tissue involved in their metabolic antidiabetic effects . one of the most important properties of gip and glp-1 is their ability to promote insulin secretion , maintaining glucose homeostasis without inducing hypoglycaemia . both gip and glp-1 are key mediators / regulators of pancreatic function and pancreatic cell mass . human beings spend most of their time in a postprandial state , and therefore it is important to emphasize that these peptides are almost undetectable during fasting and exist at high concentrations in the postprandial state . glp-1 and gip promote glucose - dependent insulin secretion and insulin biosynthesis , acting to regulate postprandial glucose disposal . binding of gip and glp-1 to their specific receptors ( gipr and glp-1r , resp . ) leads to the activation of adenylate cyclase and subsequent elevation of intracellular cyclic adenosine monophosphate ( camp ) levels , which then activates a signalling cascade that causes the increment of intracellular ca2 + concentrations triggering the fusion of insulin - containing granules with the plasma membrane and insulin secretion from the cells . increased ca2 + levels also promote transcription of the proinsulin gene , thereby increasing the insulin content of the cell . another important aspect of the insulinotropic effects of gip and glp-1 is their synergy with the sulfonylurea drugs , which is clinically relevant due to the risk of hypoglycemia when used in combined therapies [ 24 , 25 ] . ( b ) noninsulinotropic actions of glp-1 and gip ( i ) on pancreatic cells . although the major role of gip and glp-1 has generally been thought to stimulate insulin secretion by pancreatic cells , it is now known that gip and glp-1 exert noninsulinotropic actions , such as controlling pancreatic cell proliferation and survival . both hormones seem to be associated with antiapoptotic and proproliferative effects on pancreatic cell , but the signaling cascades involved display some differences that have been previously revealed with more details [ 2123 , 2628 ] . a critical difference in the antiapoptotic function of glp-1 is the requirement for pi3k , which is not required for the antiapoptotic action of gip , whose physiological impact remains to be fully clarified . another important aspect of gip and glp-1 action on cells is the stimulation of the proliferation of cells and/or progenitor cells [ 3032 ] . stimulation of cell proliferation and inhibition of apoptosis promote cell expansion , which was observed in diabetic mice and in cell cultures . glp-1 has a trophic action on cells in terms of amplification of insulin synthesis and in respect of cell hypertrophy . glp-1 increases cell mass by stimulating cell proliferation , inducing pancreatic islet neogenesis , and inhibiting cellular apoptosis . glp-1 also promotes cell differentiation , from exocrine ductal cells or immature islet stem cells , towards a greater degree of differentiation . an increase in the number and mass of cells has been demonstrated by direct action of gip [ 33 , 34 ] . further elucidation on the precise molecular mechanisms underlying the effects of gip and glp-1 on cell could reveal potential therapeutic targets to increase cell mass by inhibiting apoptosis and/or stimulating proliferation . ( ii ) effects on glucagon and somatostatin secretion from pancreatic and cells . the effects of glp-1 and gip on glucagon secretion from pancreatic cells are opposing . glp-1 suppresses glucagon secretion when plasma glucose levels are above fasting level , which is clinically important because glp-1 loses its inhibitory effect on glucagon secretion at hypoglycemic levels and does not attenuate the counterregulatory responses to hypoglycemia . furthermore , it has been recently reported that insulin stimulation and glucagon inhibition contribute equally to the effect of glp-1 on glucose turnover in t2 dm patients . although there are no glp-1 receptors on cells , insulin released by cells , in response to glp-1 , turns out to have an inhibitory action on the physiological secretion of glucagon . therefore , there is an improvement , at least partial , in the insulin / glucagon ratio , which improves glucose uptake by the liver and peripheral tissues , such as skeletal muscle . despite its clinical importance , the mechanism underlying the suppression of glucagon secretion by glp-1 remains to be clarified . in contrast with glp-1 effect , infusion of gip was shown to counteract suppression of glucagon secretion by glucose , which was observed in rats and further confirmed in healthy humans during euglycemic , but not during hyperglycemic , clamp studies , as well as in t2 dm patients [ 3841 ] . although its physiological importance remains unknown , enhancement of glucagon secretion by gip hinders clinical usage of gip as t2 dm treatment . gip has been proposed to have a physiological role in nutrient uptake into adipose tissues , thereby linking overnutrition to obesity . gip levels are high in obese t2 dm patients and fats strongly enhance gip secretion [ 4244 ] . the role played by gip in adipose tissue has been revealed during the last years . the first clue came from the evidence of fatty acid incorporation into rat epididymal fat pads induced by gip in the presence of insulin . these initial evidences were supported by gipr expressed in adipose tissues and then reinforced by studies of genetic ablation of gipr , which clarified some of the critical roles played by gip in fat accumulation . in fact , gipr - deficient mice fed high - fat diets showed higher energy expenditure indicating preferential use of fat as energy substrate , together with increased adiponectin secretion which promotes fat oxidation in muscle and increases the respiratory quotient [ 48 , 49 ] . in obese ob / ob mice , in which a defect in the leptin gene results in hyperphagia and subsequent obesity , genetic ablation of gipr improved not only obesity by increasing energy expenditure [ 47 , 51 ] , but also insulin insensitivity and glucose tolerance without seriously affecting insulin secretion . these findings were confirmed when a gipr antagonist was used in high - fat fed mice and in obese ob / ob mice treated [ 5356 ] . although gip was shown to increase the activity of lipoprotein lipase ( lpl ) , which hydrolyzes lipoprotein - associated triglycerides to produce free fatty acids available for local uptake , the molecular mechanism by which gip acts on adipocytes is largely unknown . further investigation might shed light on the molecular mechanisms underlying gip action in fat accumulation and might open up a possibility of gip - based antidiabetic therapy that does not promote obesity . importantly , glp-1 does not show any role in fat accumulation . while glp-1r is expressed in adipocytes , activation of glp-1r affects none of the aforementioned signaling molecules and does not increase lpl activity in adipocytes [ 58 , 59 ] . however , the insulin secretion evoked by glp-1 and the consequent suppression of the release of fatty acids is probably the most dominant effect observed on adipose tissue , with a simultaneous stimulation of glycogen synthesis . although glp-1 inhibits gastric emptying [ 60 , 61 ] , gip has been shown to have little effect on gastric emptying in humans and mice [ 41 , 62 ] . glp-1 slows gastric emptying and inhibits pentagastrin and meal - stimulated gastric acid secretion [ 63 , 64 ] . stimulation of glp-1 receptors in the pyloric sphincter causes a deceleration of gastric emptying and reduces postprandial blood glucose . ( 1996 ) examined the satiety effect of glp-1 on the central cns in fasted rats injected intracerebrally , in the ventricular area , with glp-1 versus saline ( control group ) , and measured food consumption at regular intervals . food intake decreased progressively with the increase of the concentration of injected glp-1 , whose receptors were detected within different areas of the brain , including densely innervate hypothalamic regions , such as the paraventricular , dorsomedial , and arcuate nuclei [ 34 , 67 ] . in the presence of food , glp-1 may mediate gut - brain signalling from the gastrointestinal tract to glp-1 receptors in the hypothalamus and brainstem . this constitutes a feeding control via neural and endocrine mechanisms [ 68 , 69 ] . several lines of evidence imply that not only glp-1 , but also gip , controls food intake and satiety . gipr deficiency seems to prevent ovariectomy - induced obesity , which might be linked to the reduced expression of npy in the hypothalamus and subsequent reduction of food intake . in fact , it was previously shown that cerebral infusion of npy stimulates neuronal secretion of gip , suggesting that gip acts as a negative regulator of npy , thus controlling food intake . thus , the antiobesity function of gip might result not only from the aforementioned effects on the adipose tissues but also from a direct effect on the brain . similar evidences have been obtained for glp-1 , such as the inhibition of food intake [ 66 , 72 ] by intracerebroventricular and peripheral infusion of glp-1r agonists , and further confirmed using the glp-1 and glp-1r antagonist exendin-(939 ) [ 73 , 74 ] . in addition to the pancreas , adipose tissue , stomach , and brain , receptors for gip and glp-1 are expressed in a wide variety of organs , including the bones , heart , and kidneys , where incretins seem to play important effects . gipr are expressed in bones and gip directly affects bone metabolism , having a role in bone formation , as suggested by the reduction of bone formation parameters and high turnover osteoporosis in gipr - deficient mice [ 75 , 76 ] . unlike gip , glp-1 has no direct effect on osteoblasts and osteoclasts , and glp-1 inhibits bone resorption indirectly through upregulation of calcitonin [ 77 , 78 ] . although exendin-4 has been shown to promote bone formation in rats , whether glp-1-based therapies show any effects on bone metabolism in human remains to be addressed in the future . recent studies have shown that incretins can regulate other vital functions , such as body temperature , blood pressure , heart rate , and fluid balance . the main cardiovascular and renal effects described will be reviewed in further sections of this paper . in addition , there is increasing interest in the potential role of incretin hormones in neurodegenerative disorders , including alzheimer 's , parkinson 's , and huntington 's diseases [ 8083 ] . glp-1 is responsible for most of the incretin effects , which in nondiabetic individuals is a normal physiological action . however , in t2 dm patients the incretin effect is blunted : the so - called incretin defect . the incretin defect , a metabolic deterioration associated with t2 dm , was demonstrated by nauck et al . ( 1986 ) . in their study , oral and intravenous glucose caused similar changes in plasma glucose concentration in subjects with t2 dm . in healthy individuals , the insulin secretory response after oral glucose ingestion exceeded the response elicited by intravenous administration of an equal amount of glucose . this incretin defect in t2 dm seems to have two main causes : reduced secretion of glp-1 and intense impairment of the insulinotropic effect of gip . in addition to the altered incretin effect , t2 dm is also associated with defective release of glp-1 . toft - nielsen et al . studied incretin secretion , including glp-1 , within 4 hours after a meal in individuals with t2 dm , and compared them with those who had a normal glucose tolerance . the results showed a significant reduction of the glp-1 response in patients with t2 dm . in addition , in a small study of identical twins , differing only in their t2 dm status , the glp-1 response was reduced only in the diabetic twin . several observations suggest that the abnormal glp-1 secretion is most likely a consequence rather than a cause of diabetes , including the study of knop et al . , which attempted to evaluate the reduced incretin effect as a cause or as a consequence , concluding that it is a characteristic consequence of the diabetic state rather than a primary event that leads to t2 dm . the pancreatic cell mass of a normal person can adapt to different insulin requirements when challenged with different glucose loads . however , the ability of pancreatic cells to release optimal and effective insulin may be compromised in diabetes . the inability of pancreatic cells to balance insulin resistance is a major problem in patients with impaired glucose tolerance or overt t2 dm . . it may also be due to the inability of the endocrine pancreas to maintain optimal cell mass capable of producing the required amount of effective insulin . the impact of a high workload and hyperglycaemia - induced oxidative stress can eventually lead to pancreatic cell death . some authors have shown that incretin pathways play important roles in the progression of t2 dm . the significant reduction in the incretin effect seen in patients with t2 dm has been attributed to several factors , including impaired secretion of glp-1 , accelerated metabolism of glp-1 and gip , and a defective responsiveness to both hormones . while the gip concentration is normal or modestly increased in patients with t2 dm , its insulinotropic actions are significantly diminished . this implies that a defect exists at the physiologic or even supraphysiologic levels in patients with t2 dm in response to gip . the impaired responsiveness to gip may suggest a possible link to gipr downregulation or desensitization . in contrast to gip , the secretion of glp-1 is reduced in obese subjects without diabetes , suggesting that incretin secretion is altered in the early stages of diabetes . in patients with t2 dm , the incretin effect is reduced or absent , which contributes to a defective first phase of insulin secretion . some authors report that the incretin effect is responsible for about 60% of the secretion of postprandial insulin , which is decreased in t2 dm . in these patients , gip secretion is normal , but its insulinotropic effect is markedly reduced , while the glp-1 secretion is reduced but preserves its insulinotropic action , meaning that it can still effectively stimulate insulin secretion . the cause for the differing properties of the gip and the glp-1 incretin effect in relation to changes in t2 dm is not fully understood . the finding that t2 dm patients have low concentrations of glp-1 , but their response of insulin secretion is preserved , supports the therapeutic potential of glp-1 treatments . thus , while gip has a low potential as a drug therapy , glp-1 , on the other hand , has a therapeutic potential as a promising pharmacological tool for the treatment of t2 dm , already proposed in the 1990s , when the incretin effect was reviewed . in contrast to other insulinotropic agents , such as sulphonylureas or glinides , the insulinotropic effect of glp-1 depends strictly on glucose , providing the ability to normalize glucose values without the risk of hypoglycaemia , which is a quite relevant therapeutic approach . furthermore , glp-1 possesses a variety of additional physiological effects that are attractive in the treatment of t2 dm , such as in the suppression of glucagon secretion from the pancreatic -cells , in a glucose - dependent manner . this can represent an important advantage for those patients with hyperglucagonemia refractory to glucose administration , but that are still responsive to glp-1 . global estimates of the prevalence of diabetes for 2030 indicate a growing burden of the disease , particularly in developing countries , where a 69% increase in numbers between 2010 and 2030 , ranging from 285 to 439 million adults ( aged 2079 years ) , was estimated . about 60% of the patients who are diagnosed with t2 dm do not achieve adequate glycaemic control and , therefore , have an increased risk for developing micro- and macrovascular complications . the spectrum of metabolic alterations includes insulin resistance in muscle and liver , as well as a progressive cell failure ( the classic triad ) . furthermore , from the triumvirate theory , defronzo ( 2009 ) suggested there is much more to the pathogenesis of t2 dm , suggesting five additional elements that make substantial contributions to the development and evolution of the disease : ( 1 ) alterations in the enteroendocrine physiology , ( 2 ) increased lipolysis in fat cells , ( 3 ) increased glucagon secretion , ( 4 ) increased renal reabsorption of glucose , and finally ( 5 ) cns insulin resistance with appetite dysregulation . because of the interrelation of these 8 factors to the pathophysiology of t2 dm and its associated morbidity and mortality , they have been referred to as the ominous octet . these eight interrelated factors have important implications in the optimization of the treatment for patients with t2 dm . first , it is because multiple abnormalities require the use of several drugs in order to correct the abnormal pathophysiology of t2 dm . second , treatment must address not only surrogate markers of the disease , such as elevated hba1c levels , but also known pathogenic mechanisms . finally , in order to prevent or slow progressive deterioration in cell function , the interval between the beginning of t2 dm and its diagnosis must be shortened so that treatment can be initiated as early as possible . in recognition of these important imperatives , treatment should include a combination of interventions . t2 dm treatment includes the physiological correction of insulin resistance and its defects in secretion . therefore , besides lifestyle changes , especially diet and exercise , drug therapy is the basis of the treatment , including medication that reduces insulin resistance [ such as biguanides or thiazolidinediones ( tzds ) ] , insulin secretagogue agents [ such as sulfonylureas ( su ) ] , and/or insulin therapy in more advanced stages of the disease . according to the main international institutions for diabetes care [ american diabetes association ( ada ) , european association for the study of diabetes ( easd ) , and international diabetes federation ( idf ) ] , drug treatment in t2 dm patients should be started when nutritional recommendations and physical activity are not effective to maintain hba1c levels below 7.0% , even in patients without complications , with relatively quality of life , and adhering to nutritional guidelines and physical activity [ 102 , 103 ] . in patients with t2 dm , the risk of complications is associated with the prior hyperglycaemic state , and any reduction in hba1c levels promotes a reduction in the risk for complications . treatment regimens that reduce the levels of hba1c to near or below 7% result in a significant reduction of risk of microvascular complications and diabetes - related death . current recommendations by the consensus of ada and easd justify the selection of appropriate treatment based on its ability to achieve and maintain glycaemic goals . table 1 features the main features of the antidiabetic armamentarium ( non - incretin - based therapies ) , focusing on mechanisms of action , major effects / advantages , adverse reactions , and the ability to decrease hba1c . the above discussion regarding the t2 dm pathophysiology reasonably suggests that ideal antidiabetic therapy should address the ominous octet . therefore , a drug ( or a combination of drugs ) that can ideally improve cell health ( tzds , incretin - based therapies , biguanides , and -glucosidase inhibitors ) , improve insulin resistance ( biguanides , tzds , and possibly incretin - based therapies ) , suppress glucagon secretion ( incretin - based therapies ) , suppress appetite ( glp-1 analogues , biguanides ) , improve lipid health ( tzd ) , and suppress renal glucose reabsorption causing the increase in urinary excretion ( sodium - glucose cotransporter 2 inhibitors ) would be the perfect therapy . incretin - based therapies ( addressing 4 out of the 8 pathophysiological defects ) with biguanide [ metformin ( met ) ] or tzd ( addressing insulin resistance in liver and skeletal muscle ) seem to be a very good option . efforts should be made for restoring the glp-1 physiologic function in t2 dm and , thus , correct the multiple metabolic abnormalities observed in patients with the disease . this enzyme , originally described as a lymphocyte cell surface protein cd26 , exists in two molecular forms with proteolytic activity : a membrane - spanning protein with a short intracellular tail and a soluble circulating protein , which lacks the intracellular tail and transmembrane regions . the soluble form ( sdpp-4 ) was first identified in serum and saliva and has been detected in cerebrospinal and seminal fluid and bile and accounts for a significant part of dpp-4 activity in human serum , as recently reviewed . the transmembrane protein is expressed on many different cell types and tissues , including the gut , liver , spleen , lungs , brain , heart , endothelial capillaries , acinar cells of mucous and salivary glands , pancreas , uterus , and immune organs such as thymus , spleen , and lymph node , with the highest levels found in the kidney [ 107109 ] . the dpp-4 gene family includes four enzymes dpp-4 , dpp-8 , dpp-9 , and fibroblast activation protein ( fap ) , in addition to the catalytically inactive proteins dpp-6 and dpp-10 . dpp-4 regulates the activity of the secretory hormones glp-1 and gip to maintain glucose homeostasis ( enhanced insulin secretion and glucagon suppression ) , thereby improving postprandial and fasting hyperglycaemia [ 110112 ] . glp-1 is degraded even before leaving the gut by dpp-4 molecules anchored to the luminal surface of endothelial cells of the mucosal capillaries . dpp-4 has several other physiological substrates [ including chemokines , neuropeptides , and regulatory peptides , such as neuropeptide y ( npy ) , substance p , or stromal cell - derived factor 1 ( sdf1 ) ] and its expression is highly regulated , as recently reviewed . several studies have shown that circulating dpp-4 activity is increased [ 113117 ] in diabetic patients and animals , but this finding is not consensual , as other studies reported decreased activity [ 118 , 119 ] . in humans , circulating levels of intact glp-1 decrease rapidly ( half - life of about 2 minutes ) due to inactivation by the dpp-4 , such that biologically active glp-1 represents only 10% to 20% of total plasma levels . therefore , strategies to increase glp-1 levels in plasma are based on ( a ) the use of glp-1 receptor agonists , such as exenatide , or glp-1 analogues , resistant to enzymatic inactivation , such as liraglutide , collectively known as incretin mimetics or glp-1 mimetics ; ( b ) the use of dpp-4 inhibitors , also known as gliptins . both agonists and analogues of glp-1 have demonstrated their efficacy in the treatment of t2 dm without causing hypoglycaemia but have the disadvantage of being injectable drugs . dpp-4 inhibitors , on the other hand , are orally active , but they may potentiate the action of other peptides that are also degraded by this enzyme . together , these therapeutic strategies are called incretin - based therapies or incretin modulators and represented , in themselves , a promising development for the treatment of t2 dm . this review will now focus on dpp-4 inhibitors , exploiting their antidiabetic properties in comparison ( and/or in association ) with the preexisting oral antidiabetic agents arsenal , as well as the potential for acting as a cytoprotective agent in extrapancreatic organs / tissue , including some of those targeted by diabetic complication ( heart , vessels , kidney , and retina ) . in sum , the place of gliptins in t2 dm therapy is revisited , questioning if these agents are essentially identical to the previous hypoglycemic drugs ( me too ) or if they have potential to notably improve the management of diabetes and prevent its serious complications , thus acting as the special one antidiabetic drugs . in t2 dm patients during hyperglycemic clamp studies , infusion of glp-1 , but not gip , stimulates insulin secretion , thus showing that the insulinotropic effect of glp-1 is reasonably well - preserved in t2 dm , despite possibly lower levels , when compared to nondiabetic individuals [ 120 , 121 ] . in contrast , despite relatively normal gip levels , the insulinotropic effect of gip is severely impaired , with the ability of gip to stimulate second - phase insulin secretion being absent , although a first - phase response is present . hence , the development of incretin - based therapies for t2 dm has thus far focused on glp-1 , rather than gip . an important feature of glp-1 is that it is rapidly secreted by the l - cells of the ileum , in just about 15 minutes after a meal , but it is also rapidly metabolized in the blood by dpp-4 , becoming an inactive fragment . the extremely rapid and widespread degradation of glp-1 by dpp-4 led to the proposal that enzyme inhibitors could be used therapeutically in t2 dm , protecting and strengthening the circulating levels of glp-1 [ 17 , 122 ] . so , gip is not an effective blood glucose - lowering agent in t2d subjects . as occurs with glp-1 , gip is rapidly in vivo inactivated by dpp-4 , converting full length gip(142 ) to inactive gip(342 ) within minutes of its secretion from the gut k - cell . thus , one of the objectives of dpp-4 inhibition is to stabilize gip , resulting therefore in greater insulinotropic activity , and to prolong the beneficial effects of endogenous glp-1 . the idea was promptly accepted by the pharmaceutical industry and many companies have embarked on the development of dpp-4 inhibitors for clinical use . the first dpp-4 inhibitor to reach the market was sitagliptin , followed by vildagliptin and more recently by saxagliptin , alogliptin , and linagliptin [ 9397 ] . long - term studies with dpp-4 inhibitors in clinical development have shown a good safety profile , tolerance , and no immune adverse effects . , when they confirmed a significant effect in reducing postprandial hyperglycaemia , by reducing fasting blood glucose levels and hba1c values after oral administration of sitagliptin daily for 4 weeks in patients with t2 dm . in another study with the long - acting dpp-4 inhibitor , vildagliptin , a sustained effect on hba1c this was a 52-week study where the existing treatment with metformin was accompanied by the dpp-4 inhibitor . these first two dpp-4 inhibitors showed good oral bioavailability and a relatively long action , so that one daily administration results in the inhibition of dpp-4 in 7090% , over a period of 24 hours , which is sufficient to fully protect the degradation of endogenous incretin hormones . clinically , both inhibitors were shown to have numerous advantages as they stimulate the synthesis of insulin , suppress glucagon secretion , lower levels of postprandial and fasting glucose , improve cell function , and lower hba1c values in t2 dm patients [ 91 , 128 ] . sitagliptin and vildagliptin have significant antidiabetic effects even when administered alone , resulting in improved glycaemic control , which is further improved when given in combination with other antidiabetic agents , including metformin , sulfonylureas , and thiazolidinediones . in contrast to incretin mimetics , the dpp-4 inhibitors do not cause a reduction in body weight . although various dpp-4 inhibitors have different pharmacokinetic and pharmodynamic profiles , they are remarkably similar with regard to their antihyperglycaemic properties with a very safe profile ( neutral concerning weight , without causing hypoglycaemia ) . these agents are all low - molecular - weight compounds , although they differ widely in terms of their chemical structure . chemical dpp-4 inhibitors have been mainly divided into two series / families : peptidomimetics and nonpeptidomimetics compounds . vildagliptin and saxagliptin are peptide - like compounds based on a dipeptide structure , whereas other dpp-4 inhibitors are nonpeptidomimetic substances with an ample chemical diversity , including b - amino acid - based compounds ( sitagliptin ) , modified pyrimidinediones ( alogliptin ) , and xanthines ( linagliptin ) . the nonpeptidomimetics compounds might assume special relevance because they show selectivity for dpp-4 versus other members of the dpp-4-like family of proteases , including dpp-2 , dpp-8 , and dpp-9 , thus avoiding interference with other putatively important functions ( although the in vivo functions remain largely unknown ) , as well as the possibility of undesirable side - effects . the pharmacokinetic profile and the clinical features of dpp-4 inhibitors have been reviewed in the last years [ 22 , 98 , 131133 ] , and the main features of the class , as well as of each of individual drugs , are summarized in the following subtitles and in tables 2 and 3 , respectively . sitagliptin , from merck sharp & dohme , was the first selective dpp-4 inhibitor in the market , approved in 2006 by fda and commercialized as januvia . sitagliptin promotes around 97% of dpp-4 inhibition and reduces blood glucose levels , either in the postprandial or the fasting state . it works differently from other drugs already available for diabetes and it is orally active . it presents a bioavailability of 87% and can be taken with or without food , with a recommended dose of 100 mg once daily in the eu and in the usa . the hepatic metabolism of sitagliptin is minimal ( mainly by cytochrome p450 3a4 ) and it is largely ( 7080% ) excreted by the urine in its unchanged form , with a half - life of around 12 hours . as a result of its metabolism and elimination , dose adjustment is required in patients with severe renal impairment , but not in those with mild or moderate renal or hepatic impairment [ 136 , 137 ] . no dosage adjustment is necessary on the basis of age , gender , race , or body mass index ; in addition , sitagliptin has a low propensity for pharmacokinetic drug interactions . randomized placebo- or active comparator - controlled trials have demonstrated the efficacy of sitagliptin in terms of improving glycaemic control in t2 dm patients , used as monotherapy , initial combination therapy ( usually with fixed - dose combinations of sitagliptin / metformin ) , or add - on therapy to metformin or to other antihyperglycaemic drugs , with or without metformin . sitagliptin showed efficacy in decreasing hba1c , fasting plasma glucose ( fpg ) , and 2 h postprandial glucose ( ppg ) levels , also increasing the proportion of patients achieving target hba1c levels . several randomized , double - blind , placebo - controlled trials with sitagliptin as monotherapy in adult patients with t2 dm and inadequate glycaemic control ( hba1c typically 710% ) showed statistically significant placebo - corrected reductions from baseline hba1c ( 0.61.1% ) , in fpg ( 1.01.8 mmol / l ) and in 2-h ppg ( 2.64.5 mmol / l ; p < 0.01 ) , among patients receiving sitagliptin . in addition , the proportion of patients achieving target hba1c levels ( < 7.0% ) at the end of the study period was significantly ( p < 0.001 ) superior among sitagliptin - treated patients ( 2158% ) compared to placebo recipients ( 517% ) [ 138142 ] . the results of randomized , double - blind , active comparator - controlled trials showed noninferiority of sitagliptin monotherapy versus metformin and versus voglibose in patients with normal renal function and noninferiority versus glipizide in patients with renal impairment [ 143145 ] . several randomized , double - blind trials in t2 dm adults inadequately controlled with diet and exercise showed improved glycaemic control from initial combination therapy with sitagliptin and other antihyperglycaemic agents , such as those with the biguanide metformin in fixed - dose combination formulations and those with the peroxisome proliferator - activated receptor- ( ppar ) agonist pioglitazone ( thiazolidinedione ) . initial combination sitagliptin ( 50 mg)/metformin ( 500 mg ) twice daily or 50 mg/1000 mg twice daily achieved significantly greater reductions in hba1c and fpg levels when compared to corresponding total daily dosages of sitagliptin or metformin monotherapy , and significantly more patients receiving combination therapy achieved target hba1c levels of < 7.0% [ 146151 ] . combination therapy with pioglitazone also had significantly greater effects on reductions from baseline hba1c levels than with pioglitazone monotherapy and improvement of other glycaemic parameters , including fgp reductions , together with a significantly greater proportion of patients achieving target hba1c levels [ 152154 ] , despite being less important than combination with metformin ( 57.3 versus 43.5% ) . a number of large randomized , double - blind , placebo - controlled [ 155158 ] , and active - comparator [ 159165 ] trials have evaluated the efficacy of sitagliptin as add - on therapy to metformin ( > 1500 mg / day ) in adults with inadequately controlled t2 dm . addition of sitagliptin ( 50 or 100 [ 155 , 157 , 158 ] mg / day ) to ongoing metformin for 1224 weeks was superior to placebo plus metformin in reducing placebo - corrected hba1c ( 0.651.0% ) , fpg ( 1.01.4 mmol / l ) , and 2 h ppg ( 1.93.0 mmol / l ) , with a greater proportion of patients achieving the target hba1c levels : 1447% for sitagliptin versus 318% placebo , across all four studies [ 155158 ] . various randomized studies of 1852 weeks ' duration compared the efficacy of sitagliptin ( 100 mg / day ) as add - on therapy to metformin with that of other orally administered antihyperglycaemic agents , including sulfonylureas ( glimepiride and glipizide ) and ppar agonists ( pioglitazone and rosiglitazone ) . two large studies comparing sitagliptin with sulfonylureas as add - on therapy to metformin demonstrated noninferiority between treatment groups [ 158 , 162 ] . the addition of sitagliptin to ongoing metformin achieved reductions in hba1c broadly similar to those observed when pioglitazone 45 mg / day or rosiglitazone 8 mg / day was added to metformin for 26 or 18 weeks , respectively [ 161 , 165 ] . other large randomized , placebo - controlled trials have evaluated the efficacy of sitagliptin as add - on therapy to ongoing treatment with a ppar agonist , a sulfonylurea , or insulin , with or without metformin , and the results , overall , showed that patients randomized to sitagliptin had statistically significant placebo - adjusted reductions of hba1c ( 0.60.9% ) , fpg ( 0.81.1 mmol / l ) , and 2-h ppg ( 2.02.7 mmol / l ) , as well as a great proportion of patients that achieved target hba1c ( 1345% versus 323% ) [ 166172 ] . sitagliptin has a neutral effect on body weight , as reported in almost all of the studies previously mentioned [ 138142 ] . concerning the impact of sitagliptin on lipid profile , the available data showed no consistency , but the majority of studies reported a beneficial effect on tgs , hdl - c , and ldl - c , as concluded in a systematic review and meta - analysis of 14 trials with incretin therapy in patients with t2 dm . in addition , some studies suggested a reduction of blood pressure in patients under sitagliptin treatment [ 174176 ] . the reduction of global cardiovascular risk factors by sitagliptin seems to be important for improving outcomes in patients with t2 dm . sitagliptin is well tolerated and the risk of adverse events , including hypoglycaemia , is very low [ 132 , 147 , 155 , 162 ] . the most common are gi disturbances , including abdominal pain , nausea , vomiting , and diarrhoea , which are rare when used in monotherapy . nasopharyngitis , upper respiratory tract infections , and headache occur in a low percentage of patients ( versus placebo ) . the prescribing information for sitagliptin includes information regarding postmarketing reports of acute pancreatitis , but the association between dpp-4 inhibitor use and pancreatitis remains controversial , as further discussed . vildagliptin , from novartis , commercialized firstly with the brand name of galvus , is a highly selective , reversible , inhibitor of the enzyme dpp-4 approved by the eu in 2007 for the treatment of t2 dm , with a recommended dose of 50 mg twice daily . vildagliptin treatment results in a rapid inhibition of dpp-4 ( around 95% of maximal inhibition ) , causing elevation of endogenous levels in fasting and postprandial incretin hormones , glp-1 and gip , thus improving cell sensitivity to glucose and resulting in the increased secretion of glucose - dependent insulin . furthermore , vildagliptin also enhances the sensitivity of cells to glucose , resulting in an improvement in the homeostasis of glucagon . oral vildagliptin had an absolute bioavailability of about 85% and can be administered with or without food , although food slightly delayed the tmax and decreased cmax by about 20% . vildagliptin is not metabolized by cytochrome p450 enzymes to a quantifiable extent and , thus , it is unlikely to affect the metabolism of other drugs or to be affected by them . the major metabolite ( carboxylic acid ) is obtained by hydrolysis and is pharmacologically inactive , the kidney being responsible for the hydrolysis and for excretion ( about 55% as unchanged parent and about 26% as metabolite ) . the efficacy of vildagliptin monotherapy with that of placebo in patients with t2 dm was analyzed in randomized , double - blind , multicentre trials of 12 , 24 , or 52 weeks ' duration . vildagliptin improved glycaemic control , viewed by reduction of hba1c , which was usually more effective for higher basal levels [ 178181 ] . in addition , fpg levels were also significantly reduced by vildagliptin versus placebo [ 178180 ] . the efficacy of vildagliptin monotherapy was also compared with that of other oral antihyperglycaemic agents , examining noninferiority of vildagliptin with the comparator . after 12 weeks ' therapy , a significantly greater reduction from baseline in hba1c was seen with vildagliptin than with voglibose . after 24 or 104 weeks ' therapy , the proportion of patients achieving the target hba1c of < 7.0% did not significantly differ between patients receiving vildagliptin and those receiving gliclazide or acarbose , while when compared with metformin the percentages were 35% with vildagliptin versus 45% with metformin . significantly more vildagliptin than voglibose recipients achieved an hba1c of < 6.5% . once again , the reduction of hba1c tended to be higher in patients with higher baseline levels [ 182186 ] . in addition , in vildagliptin recipients a significant reduction from baseline in fpg levels was seen ; however , the mean reduction was significantly higher with metformin , rosiglitazone , or gliclazide than with vildagliptin , and noninferiority between vildagliptin and acarbose was not established [ 182186 ] . on the contrary , the efficacy of vildagliptin administered in combination with metformin in the treatment of t2 dm patients was evaluated in randomized , double - blind , multicentre trials of 12 , 24 , and 52 weeks ' duration . combination therapy with vildagliptin 50 mg twice daily plus metformin , in 24-week trials , improved hba1c to a significantly greater extent than monotherapy with metformin or vildagliptin in patients with t2 dm poorly controlled by metformin monotherapy or who were treatment nave [ 187 , 188 ] ; patients receiving vildagliptin plus metformin showed reduced hba1c levels until week 12 thereafter and remained stable [ 187 , 188 ] . a greater proportion of patients receiving vildagliptin ( 50 mg twice daily ) plus metformin ( 500 or 1000 mg twice daily ) than vildagliptin or metformin monotherapy have achieved the target hba1c levels of < 7% ( 55.4% and 65.4% versus 40.0% and 43.5% ) , the reductions being higher for higher baseline levels . in addition , fpg levels were also reduced to a significantly greater extent with vildagliptin combined with metformin than monotherapies [ 187 , 188 ] . vildagliptin plus metformin demonstrated noninferiority to pioglitazone plus metformin concerning change in hba1c after 24 weeks in t2 dm patients inadequately controlled by metformin monotherapy . regarding change from baseline in fpg , noninferiority of vildagliptin plus metformin versus pioglitazone plus metformin was not established after 24 weeks ' therapy . following the 28-week single - blind phase , the mean change from baseline in hba1c at week 52 was identical ( 0.6% ) in both patients receiving vildagliptin plus metformin and those receiving pioglitazone plus metformin . the results of two 52-week trials showed noninferiority ( in terms of the change from baseline in hba1c ) of vildagliptin plus metformin combination therapy when compared with glimepiride or gliclazide plus metformin [ 191 , 192 ] ; the proportion of patients achieving an hba1c of < 7.0% was 29.6% and 54.1% with vildagliptin plus metformin , 31.9% with gliclazide plus metformin , and 55.5% with glimepiride plus metformin [ 191 , 192 ] . the reductions in hba1c in the vildagliptin plus metformin recipients tend to be higher in those with higher baseline hba1c levels [ 191 , 192 ] . noninferiority , in terms of the change from baseline in fpg , was demonstrated in the vildagliptin plus metformin combination versus gliclazide plus metformin and there were no differences versus glimepiride plus metformin [ 191 , 192 ] . the efficacy of vildagliptin administered in combination with pioglitazone or glimepiride was evaluated in randomized , double - blind , multicentre trials of 12 or 24 weeks ' duration , with t2 dm patients who had not received pharmacological therapy for at least 12 weeks or who were inadequately controlled with thiazolidinedione or sulfonylurea monotherapy . the combination of vildagliptin ( 50 mg twice daily ) with pioglitazone or with glimepiride significantly improved the glycaemic control , after 34 weeks , viewed by greater reductions in hba1c versus monotherapy with pioglitazone or with glimepiride [ 193 , 194 ] , and a significantly greater proportion of patients receiving combination with pioglitazone versus pioglitazone alone ( 36.4% versus 14.8% ) and with glimepiride versus glimepiride alone ( 21% versus 12% ) achieved an hba1c of < 7% [ 193 , 194 ] . once again , the reductions were higher for those patients with higher baseline hba1c levels . no differences were encountered for change from baseline in fpg between combined therapies versus monotherapies after 24 weeks ' therapy [ 193 , 194 ] . vildagliptin has been associated with low incidence of adverse reactions , including hypoglycaemia , and is neutral in terms of effects on body weight . no reactions were found to be associated with age , ethnicity , duration of exposure , or daily dose of the drug . the majority of the adverse reactions in the various studies were mild and transient , not requiring discontinuation of treatment . concerning vildagliptin monotherapy , at a dose of 100 mg per day , the adverse reactions reported , beyond those observed in patients receiving placebo , were dizziness , headache , peripheral oedema , constipation , nasopharyngitis , upper respiratory tract infection , and arthralgia [ 178181 ] . the use of vildagliptin in patients with moderate - to - severe renal or hepatic insufficiency is not recommended , and there is a requirement for liver enzyme monitoring to avoid possible hepatic adverse events . saxagliptin , from bristol - myers squibb , was approved by the fda in 2009 and marketed as onglyza and is another dpp-4 potent inhibitor with pharmacokinetic and pharmacodynamic properties suitable for once - daily dosing , with or without food , at any time of the day [ 195 , 196 ] . saxagliptin has a maximal inhibition of dpp-4 of around 95% and is metabolized hepatically by cytochrome p450 ( cyp ) 3a4/5 to an active metabolite , 5-hydroxy saxagliptin , which is also a selective , reversible , competitive dpp-4 inhibitor , but it is half as potent as the parent compound . the half - life after a single oral dose of 5 mg / day , the recommended dose , is 2.5 h for saxagliptin and 3.1 h for the active metabolite ; the elimination of saxagliptin is both hepatic and renal in the parental form or as metabolite . dose adjustments ( reduction to 2.5 mg / day ) are recommended in patients with moderate - to - severe renal impairment since systemic exposure to the drug increases in proportion to the degree of renal impairment ; renal function should be assessed prior to initiating saxagliptin therapy and monitored periodically thereafter ; its use is presently not recommended in patients with severe hepatic insufficiency . saxagliptin is approved as a combination therapy with metformin , sulfonylurea , thiazolidinedione , or insulin ( with or without metformin ) to improve glycaemic control in adult patients with t2 dm who do not achieve adequate glycaemia control with metformin , sulfonylurea , thiazolidinedione , or insulin in addition to diet and exercise , including patients with mild - to - severe renal impairment . the efficacy of saxagliptin as add - on therapy to various baseline antihyperglycaemic agents has been demonstrated in a number of clinical trials of 18 to 104 weeks ' duration . in combination with metformin , glibenclamide , thiazolidinedione , or insulin ( with or without metformin ) , saxagliptin was significantly more effective than placebo in lowering hba1c , fpg , and ppg levels , as previously reviewed . similar to other dpp-4 inhibitors , pooled data from saxagliptin monotherapy and combination therapy trials demonstrate that saxagliptin is generally well tolerated , with a very low risk of adverse events , including hypoglycaemia , and is generally weight neutral ; current prescribing information contains a warning regarding postmarketing reports of pancreatitis [ 199 , 200 ] . alogliptin was approved by fda in 2013 and is marketed by takeda pharmaceutical company as nesina . it is a highly selective dpp-4 inhibitor , with a maximal inhibition of > 90% ; the hepatic metabolism , mediated by cytochrome p450 ( cyp ) 2d6 , is minimal and it is largely ( 6070% ) excreted by the urine in its unchanged form ; its half - life varies between 11 and 22 h [ 96 , 98 ] . the pharmacokinetic properties of alogliptin did not alter to any clinically significant extent based on age , race , or sex . the recommended dose is 25 mg once a day . in several large trials of up to 26 weeks ' duration , alogliptin in monotherapy or in combination with other oral antihyperglycaemic agents ( metformin , glibenclamide , or pioglitazone ) or insulin therapy has improved glycaemic control in adult patients with inadequately controlled t2 dm [ 201 , 202 ] . as reported for the other drugs of this class , alogliptin is well tolerated , including elderly patients , and the incidence of hypoglycaemia is lower , with neutral effects on body weight and lipid parameters . considering the primarily renal elimination , alogliptin treatment should be accompanied by dose adjustment in patients with moderate - to - severe renal impairment . linagliptin was approved in 2011 by fda ( marketed as trajenta by eli lilly co. and boehringer ingelheim ) and is a xanthine derivative with singular pharmacokinetic properties when compared with previously commercialized dpp-4 inhibitors , which may offer some advantages in clinical practice [ 97 , 203 ] . therefore , at recommended therapeutic doses ( 5 mg once a day ) , linagliptin has a low oral bioavailability ( 30% ) , but a large apparent volume of distribution , demonstrating extensive distribution into tissues ; it has a long half - life ( > 100 h ) , due to its extensive binding to plasma proteins and its high - affinity binding to the dpp-4 enzyme , which produces a nonlinear pharmacokinetic profile . the nonlinear pharmacokinetics of linagliptin are best described by a two - compartmental model that incorporates target - mediated drug disposition resulting from high - affinity , saturable binding to dpp-4 . the strong link to dpp-4 ( which is inhibited in > 90% ) and the capacity to dissociate at a very low velocity prolong the in vivo action , allowing a once - a - day administration , thus improving compliance . a major pharmacokinetic property is the nonrenal elimination route , which allows its use in patients with renal impairment without dose adjustment or monitoring of renal function . in fact , linagliptin is poorly metabolized and mainly eliminated by biliary rout , with a very small renal elimination ( < 6% ) , which might explain the possibility of using linagliptin in renal insufficiency patients [ 98 , 206 , 207 ] , which is unique when compared to sitagliptin and saxagliptin , both requiring renal dose adjustment . despite the predominantly hepatic elimination , the main metabolite ( cd1790 ) is pharmacologically inactive , and no adjustments are currently recommended in patients with hepatic impairment . in addition , no meaningful impact of age , sex , or race on the pharmacokinetic properties of linagliptin has been observed . the efficacy of linagliptin is similar to that of the dpp-4 inhibitors previously discussed , when used as monotherapy , initial combination therapy ( with metformin or pioglitazone ) , or add - on therapy to other oral antihyperglycemic agents ( metformin and/or sulfonylurea ) or basal insulin ( with or without metformin and/or pioglitazone ) , improving the glycaemic control parameters , with a mean hba1c reduction between 0.5 and 0.7% [ 97 , 203 ] . data pooled from randomized , double - blind , placebo - controlled clinical trials lasting 24 weeks shows that linagliptin is well tolerated , with a low risk of hypoglycaemia , and is weight neutral . the efficacy of linagliptin may be limited in patients receiving concurrent inducers of cyp3a4 or p - gp ( e.g. , rifampin ) . a risk for hypoglycemia might exist when linagliptin , as well as another dpp-4 inhibitor , is used as a treatment adjunctive to an insulin secretagogue , and an initial dose decrease in background secretagogue medication should be considered to prevent hypoglycemic events . many other dpp-4 inhibitors have been developed and commercialized ( namely , in japan ) or are yet under clinical trials . anagliptin and teneligliptin were both already approved in japan in 2012 , while other agents ( such as gemigliptin and dutogliptin ) are yet in clinical trials or initiating approval procedures , mainly in asia countries . dpp-4 inhibitors undoubtedly constitute an innovative class of oral agents for the treatment of t2 dm which have enlarged the therapeutic possibilities . the main mechanism of action of dpp-4 inhibitors is essential to keep endogenous glp-1 from being degraded , by inhibiting dpp-4 . current indications for dpp-4 inhibitors recommend its use in combination with other antidiabetic agents , in particular with metformin , as second and third line therapy , and even over other antidiabetic therapies , especially if the patient is experiencing an increased incidence of hypoglycaemia . considering the above described characteristics of dpp-4 inhibitors , they could revolutionize the concept of diabetes management , either alone or in combination with other antidiabetic drugs . however , there are currently still some questions for which there is no complete answer : whether dpp-4 inhibitors can promote preservation of human cell function ; the most proper stage of disease to initiate therapy ; and the long - term safety of gliptins . t2 dm is characterized by a progressive loss of cells mass and function that is associated with insulin resistance . these defects are followed by a significant decrease in the incretin effect , which are mainly related to abnormalities in glp-1 secretion and action . since dpp-4 inhibitors are dependent on the endogenous secretion of incretins , that class of drugs will theoretically be useful in early stages of diabetes , when the patient still retains a cell population capable of responding to glp-1 stimulation . in fact , the possibility of using incretin modulators , including ( but not only ) dpp-4 inhibitors , in prediabetes is also under debate . on the other hand , and according to their benefit in reducing the levels of glucagon , dpp-4 inhibitors can also be used in the later stages of the disease , in combination with other oral antidiabetic agents , in poorly controlled patients , as is the current clinical indication . furthermore , the use of incretin modulators ( including dpp-4 inhibitors ) in conjugation with insulin in later stages of the disease has been extensively discussed during the last years . the safety and tolerability of dpp-4 inhibitors seem to be generally comparable to nongliptin treatments , although long - term studies and clinical practice followup are still needed , in particular to definitively evaluate if there is any reasonable association between the use of these agents and the risk of pancreatitis and pancreatic cancer , as suggested by some reports . table 2 provides a sum - up of dpp-4 inhibitors in terms of their mechanism of action , major biological effects / advantages , adverse reactions , and their ability to decrease hba1c , which can be compared with table 1 , which summarizes the same properties for the other ( non - incretin - based ) oral antidiabetic agents . in addition , the possibility of cytoprotective properties afforded by dpp-4 inhibitors on organs / tissues which are affected by diabetes ( such as the heart , vessels , kidneys , and retina ) and associated with serious diabetic complications might open new avenues for the use of these agents in the treatment of diabetic patients . the main challenges described above will be briefly revisited in the following subtitles . during the last years , several lines of evidences indicate that glp-1-based therapies could cause pancreatitis or pancreatic cancer [ 210 , 211 ] . these suggestions came from few preclinical studies [ 212214 ] , which are basically inconclusive because the histological changes are not reproduced in all studies and vary between different glp-1-based therapies and from very limited clinical data [ 215 , 216 ] , namely , observational studies [ 217221 ] , and from pancreases from organ donors with and without diabetes , which have limited value to conclude the issue , as commented by ryder ( 2013 ) . in addition , the increased reports of pancreatitis and pancreatic cancer by the fda adverse event reporting system for exenatide and sitagliptin are prone to bias , probably associated publicity surrounding these issues , and are thus not useful for establishing the incidence of adverse events . for that reason , it is decisively important to have data from well - controlled long - term studies , which are still lacking . the strongest evidences currently available come from two large cardiovascular safety studies with dpp-4 inhibitors and from meta - analysis recently published of nonrandomized and randomized clinical trials . the savor - timi 53 ( saxagliptin ) and examine ( alogliptin ) trials enrolled 16,492 and 5,380 patients over a median of 2.1 and 1.5 years , respectively , and concluded that there was no difference between the dpp-4 inhibitor treated and placebo groups with regard to pancreatitis or pancreatic cancer [ 225 , 226 ] . a meta - analysis of 53 randomized clinical trials ( including 20,312 patients treated with different dpp-4 inhibitors ) did not find an increased risk of pancreatitis in dpp-4-treated patients . identical conclusion was achieved by the analysis of 19 rcts comprising 10,246 patients treated for up to 2 years with sitagliptin . finally , li et al . ( 2014 ) recently reviewed the data concerning the risk of pancreatitis in patients with t2 dm under incretin - based therapies , by analyzing 60 studies ( n = 353,639 ) , consisting of 55 randomised controlled trials ( n = 33,350 ) and five observational studies ( three retrospective cohort studies and two case - control studies ; n = 320,289 ) , concluding that these drugs do not increase the risk of pancreatitis . in addition , fda and ema independently evaluated the safety data from postmarketing reports of pancreatitis and pancreatic cancer in patients using incretin - based drugs , analysing both animal and clinical information available , and concluded that a causal association between incretin - based drugs and pancreatitis or pancreatic cancer can not be established with the current data ; however , fda and the ema have not reached a final conclusion regarding such a causal relationship , and both agencies will continue to investigate the safety signal . so , at this stage , we should recognize that the link between these therapies and proven clinical pancreatitis and pancreatic cancer is not established . however , current evidence is not definitive and we should undoubtedly remain vigilant about the possibility of an association between glp-1-based therapies and pancreatic disease and more carefully designed and conducted studies are warranted to definitively conclude this issue . prediabetes has been defined as a state of impaired fasting glucose ( ifg ) concentration ranging between 110 and 126 mg / dl [ for the world health organization ( who ) ] or between 100 and 125 mg / dl ( for the american diabetes association ( ada ) ) and/or impaired glucose tolerance ( igt ) , characterized by a plasma glucose concentration 2 h after 75 g oral glucose load ranging between 140 and 199 mg / dl [ 231 , 232 ] . it is widely accepted that insulin resistance starts several years before the onset of diabetes and cell dysfunction is already present , even in the prediabetic stage . several pathophysiological mechanisms contribute to the evolution of t2 dm , including increased insulin resistance in the skeletal muscle and liver ; augmented hepatic glucose output ; impaired insulin secretion with progressive decline of pancreatic cell function . chronic hyperglycaemia and increased free fatty acids cause glucotoxicity and lipotoxicity , which accelerates cell failure by apoptosis and decreased proliferation . in addition , deficiency of incretin secretion by the gi tract and/or resistance to incretin action due to downregulation of their receptors have been associated with evolution of diabetes . since glp-1 is an insulin secretagogue and a suppressor of glucagon secretion , defects in glp-1 secretion could contribute to the pathogenesis of prediabetes . in fact , recent reports show that prediabetic patients with impaired glucose tolerance ( igt ) and insulin resistance have decreased glp-1 concentrations and early glucagon suppression [ 235 , 236 ] . considering the progressive decline of incretin effect in t2 dm patients and the beneficial effects of incretin modulators in the treatment of diabetes , their use has been extended to patients with prediabetes , in a few recent small studies , as reviewed by ahmadieh and azar ( 2014 ) . particularly , the putative preservation of cell function and mass afforded by these agents , in animal studies and in clinical trials , might help maintain good long - term metabolic control . however , the very small clinical experience on the use of dpp-4 inhibitors and glp-1 mimetics in individuals with impaired fasting glucose or impaired glucose tolerance and the unsolved aspects related to the possibility of pancreatic side - effects do not recommend incretin - based therapies as an option for treatment in patients with prediabetes . although incretin therapy has been mainly used in combination with oral antihyperglycemic agents , especially metformin , the potential use in association with insulin has been debated and increasingly tested during the last years . the complementary actions of the two approaches offer a promising strategy as a glucose - lowering treatment for t2 dm patients , as recently revised by vora et al . in fact , there are several benefits of combining incretin - based therapies with insulin therapy , including the lowering of hba1c due to combined reduction of fasting plasma glucose ( fpg ) by insulin and postprandial glycemia by incretins ; reduction of risk of hypoglycemia which is due to the protection against hypoglycemia with incretin therapy in association with the often observed reduction in insulin dose when using this combination ; the lower risk for weight gain given the protection afforded by incretin therapy , thus compensating the possible weight gain evoked by insulin therapy ; the potential for long - term disease modifying effects , namely , by cell function protection by insulin due to normalization of fasting glucose and prevention of glucotoxicity , combined with beta cell protection afforded by glp-1-based therapies . several clinical studies have been reinforcing the possibility of a beneficial combination between dpp-4 inhibitors and insulin therapy . several studies have analyzed the impact of adding dpp-4 inhibitor , during at least 24 weeks , on patients ongoing insulin therapy ( alone or with metformin ) with an insufficient glycemic control . despite some minor variations between dpp-4 inhibitors ( sitagliptin , vildagliptin , alogliptin , saxagliptin , and linagliptin ) , the combination treatment group ( versus the placebo arm ) showed a higher reduction of hba1c ( change between 0.5 and 0.8 ) and fpg ( change between 0.2 and 1.0 ) , as well as a reduced risk of hypoglycemia and weight gain [ 240248 ] . there was no evidence of additional concern for safety or tolerability by combining incretin therapy and insulin in these studies . further studies , comparing different dpp-4 inhibitors and distinct insulin therapies , with comparable protocols and cohorts , will be very important to clarify the long - term efficacy and safety of these combinations . the current knowledge indicates that the combination is a very promising glucose - lowering strategy for the treatment of t2 dm in patients who do need intensified therapy to control glycaemic levels . since incretin hormones response is typically blunted in patients with t2 dm , selective inhibition of dpp-4 can prolong their antihyperglycemic effects by increasing their circulating lifetime . furthermore , not only gip and glp-1 levels are affected by the modulation of dpp-4 activity , but also many other substrates , with a wide variety of physiological functions , can be modified , suggesting that dpp-4 inhibitors may participate in events other than the increase of incretin levels and glycemic control . although a number of recent experimental studies have demonstrated beneficial effects of incretin - based therapies in extrapancreatic organs or tissues , including the vasculature [ 116 , 250 , 251 ] , the kidney , the heart , and the brain , it remains unclear whether these effects are direct or mediated by the improvement of the glycemic control . it is also poorly understood whether those findings are observed in humans , indicating that further research is warranted to confirm the results of the preclinical studies . during the last years , our group has been studying the putative beneficial effects of dpp-4 inhibition with sitagliptin on several tissues in animal models of t2 dm and t1 dm . a therapeutic low dose of sitagliptin , during a 6-week treatment , in an animal model of t2 dm , the zucker diabetic fatty ( zdf ) rat , was able to promote a partial correction of glycaemia and hba1c levels when compared to controls , accompanied by a partial prevention of insulinopenia . furthermore , the zdf rats treated with sitagliptin showed reduced blood pressure , total cholesterol , and tgs levels , suggesting possible cardioprotective effects . in addition , this dpp-4 inhibitor also showed a positive impact on low - grade inflammation , with decreased serum hscrp levels , as well as an improvement in the redox status , which was accompanied by reduction of heart , pancreas , and kidney lipid peroxidation . moreover , sitagliptin treatment ameliorated both endocrine and exocrine pancreas lesions , as well as the glomerular , tubulointerstitial , and vascular lesions , together with a decrease in urea levels . besides the insulinotropic effects of glp-1r activation in pancreatic cells , the expression of this receptor in a wide range of tissues , including retina [ 256 , 257 ] and kidney , suggests the possibility of extrapancreatic effects . in fact , we observed that sitagliptin induced an increase in the levels of renal glp-1 and its colocalization with glp-1r in kidney tissue of diabetic zdf rats , suggesting that glp-1 may exert cytoprotective effects as we found an improvement of renal lesions , including glomerular , tubulointerstitial , and vascular lesions , as well as prevention of inflammation and apoptosis induced by diabetes . in another experimental study , abd el motteleb and elshazly ( 2013 ) described a protective effect of sitagliptin against l - name induced hypertensive nephropathy , related to increased levels of glp-1 , upregulation of glp-1r , and consequent overexpression of enos and increased serum no levels , together with improvement of redox status . although renoprotective properties of dpp-4 inhibitors have been suggested during the last years [ 109 , 260263 ] , namely , based on experimental data , few studies have been performed in humans to assess the effects of dpp-4 inhibitors on kidney function metrics . hattori ( 2011 ) evaluated the effect of sitagliptin ( 50 mg / day ) on albuminuria in t2 dm patients and found a significant decrease in hba1c , fpg , and ppg , as well as in glycated albumin after 3 and 6 months . significant reductions in hscrp and soluble vascular cell adhesion molecule 1 were also observed at 6 months . these authors also found that urinary albumin excretion ( measured as urinary albumin - to - creatinine ratio ) did not change in the 6 months before sitagliptin treatment and decreased in the 6 months after sitagliptin treatment , suggesting that sitagliptin lowered albuminuria without decreasing the estimated glomerular filtration rate . these effects seem to be related to blood glucose , inflammation reduction , or even increased levels of active glp-1 . recently , mori et al . ( 2014 ) , in an open - labelled , prospective randomized study , evaluated the effects of 50 mg / day of sitagliptin ( versus other oral glucose - lowering agents ) on urinary albumin excretion in t2 dm patients , during 6 weeks . though both of the treatments significantly reduced hba1c and fpg level , only sitagliptin significantly reduced urine albumin excretion , suggesting effects independent of the glucose - lowering effect of sitagliptin . a recent retrospective study performed for 2 years in t2 dm that aimed to determine the hypoglycemic effect of 2 years of sitagliptin administration revealed that the hba1c levels decreased and c - peptide immunoreactivity ( cpr ) index increased from baseline to 3 , 6 , 12 , 18 , and 24 months after sitagliptin initiation , suggesting that sitagliptin improves glycemic control via an improved intrinsic insulin response . our group has shown that sitagliptin induced a reduction in the nitrosative stress and inhibited inflammation and apoptosis of retinal cells in the zdf rat retinas . in a recent work , our group has also reported that sitagliptin prevented the diabetes - induced increase in dpp-4/cd26 activity and levels in serum and retina of streptozotocin- ( stz- ) induced t1 dm rats . furthermore , sitagliptin prevented the increase in blood - retinal barrier permeability and decreased the retinal inflammatory state and neuronal apoptosis , thus indicating that it has direct protective effects on the retina that are independent of its antihyperglycemic effects . there is growing evidence in the literature demonstrating the beneficial effects of sitagliptin on myocardial injury and cardiac function [ 267269 ] . ( 2013 ) have shown that treatment of t2 dm goto - kakizaki rats with sitagliptin ( 10 mg / kg / day ) for 10 weeks promoted glp-1-mediated cardioprotection primarily by limiting hyperglycaemia and hyperlipidemia . in another study , treatment with sitagliptin ( 300 mg / kg / day ) for 2 weeks in t1 dm fischer f344 rats with myocardial infarction ( mi ) attenuated several aspects of cardiac dysfunction and adverse remodeling in the post - mi setting , as revealed by the improvement in passive left ventricular compliance , increased endothelial cell density , reduced myocyte hypertrophy , and collagen 1 expression . since endothelial integrity and restitution of the lost cardiac microvasculature observed in mi are essentially mediated by stromal - derived factor ( sdf1 ) , a chemokine secreted by ischemic tissue but rapidly degraded by ddp-4 , it is possible that the benefit following mi in the diabetic animals is beyond its effect on glycemia . however , since dpp-4 activity determines the systemic and local concentrations of sdf-1 and the mobilization to the injured sites of stem cells involved in endothelial repair and angiogenesis , further studies are needed to clarify whether dpp-4 inhibition is able to reverse bone marrow dysfunction induced by diabetes and improve microvascular health in the ischemic tissue . regarding clinical data , mccormick et al . ( 2014 ) have recently shown that chronic dpp-4 inhibition with sitagliptin 100 mg / day for 4 weeks protected against ischemic left ventricular dysfunction during dobutamine stress in patients with t2 dm ( 19 patients ) and coronary artery disease , possibly by glp-1-mediated cardioprotection on ischemic regional wall segments . however , randomized studies involving large patient cohorts are required to ascertain whether these effects translate into an improvement in clinical outcomes . liu et al . have shown that vildagliptin treatment for 24 weeks led to an improvement in renal lesions , as revealed by inhibition of interstitial expansion , glomerulosclerosis , and thickening of the glomerular basement membrane in t1 dm rats . vildagliptin significantly reduced renal dpp-4 activity and increased plasma active glp-1 levels , which probably prevented oxidative dna damage mediated by suppression of tgf-1 and renal cell apoptosis by activating glp-1r and modulating the second messenger camp . in a t2 dm animal model , the zdf rat , vildagliptin treatment did not affect glucose levels or proteinuria , but it significantly decreased glomerulosclerosis and restores myogenic constriction of intrarenal arteries , suggesting that this dpp-4 inhibitor protects diabetic rats from loss of renal vascular reactivity and attenuates renal sclerosis independent of effects on blood glucose or proteinuria in t2 dm . few data exist concerning the effects of the dpp-4 inhibitor vildagliptin on the kidney of diabetic patients . ( 2013 ) have assessed the effect of vildagliptin ( 50 mg bid for 8 weeks ) on atherogenic low - density lipoprotein ( ldl ) heterogeneity and albuminuria in diabetics . treatment of t2 dm with vildagliptin for 8 weeks decreased significantly the serum small dense ldl levels by about 9% and the urinary albumin - to - creatinine ratio ( uacr ) by about 45% , suggesting that vildagliptin might prevent cardiovascular disease by improving ldl heterogeneity and improve renal function by decreasing albuminuria . accordingly , vildagliptin ( 3 mg / kg / day ) treatment for 12 weeks suppressed the expression of tgf- in the aorta of diabetic rats , by attenuating the deleterious effects of advanced glycation end products ( ages ) and their receptor rage axis , with suppression of oxidative stress generation and inflammation in aorta of diabetic and obese otsuka long tokushima fatty ( oleft ) rats . wang et al . investigated the impact of dpp-4 inhibition on cardiac microvascular injury in diabetes and the underlying mechanism involved . stz - induced diabetic rats treated with vildagliptin ( 1 mg / kg / day ) for 12 weeks improved cardiac function and glucose uptake , suggesting that glp-1 could protect the cardiac microvessels against oxidative stress , apoptosis , and the resultant microvascular barrier dysfunction in diabetes , en route to improved cardiac diastolic function and cardiac glucose metabolism . the protective effects of glp-1 were dependent on downstream inhibition of rho through a camp / pka - dependent manner , which may result in the cardioprotective effect on cardiovascular remodelling associated with oxidative stress . there is only one experimental study examining the effect of vildagliptin on retinal injury in diabetes . oleft rats at 22 weeks of age treated with vildagliptin ( 3 mg / kg ) for another 10 weeks presented a significant inhibition of the increase in body weight and decreased average fasting blood glucose . vildagliptin also inhibited inflammatory and thrombogenic reactions in the retinas of obese t2 dm rats , suggesting that it may play a protective role against diabetic retinopathy . ( 2009 ) performed a comparative study investigating the antidiabetic potency and duration of several dpp-4 inhibitors ( 0.13 mg / kg ) in rats with mild diabetes ( streptozotocin - nicotinamide - induced models ) . the potency order and duration of action for plasma dpp-4 inhibition and glucose tolerance improvement were as follows : saxagliptin > vildagliptin = sitagliptin . in this report , vildagliptin and sitagliptin improved glucose tolerance through increased insulin and glp-1 levels in plasma 8 h at a dose of 1 mg / kg . furthermore , saxagliptin potently improved glucose tolerance at a dose of 0.3 mg / kg , reflecting the long half - life of the enzyme complex formed by saxagliptin . these data suggest that the effects are mediated through glucose - dependent insulinotropic action via an increase in the glp-1 level . ( 2014 ) recently reported renoprotective effects of a dpp-4 inhibitor compound ( pkf275 - 055 ) in early stages of diabetic nephropathy in rats due to anti - inflammatory actions . regarding clinical data , the large , randomized , placebo - controlled savor - timi 53 ( saxagliptin assessment of vascular outcomes recorded in patients with diabetes mellitus ) trial showed that t2 dm patients with cardiovascular complications under saxagliptin treatment were significantly more likely , when compared to placebo - treated patients , to have improved albumin - to - creatinine ratio ( 10.7% in the saxagliptin group and 8.7% in the placebo group ) and less likely to have worsening ratio ( 13.3% in the saxagliptin group and 15.9% in the placebo group ) , suggesting a protection on albumin excretion rate . whether the effects observed are attributed , at least partially , to a better glucose control , or to a direct effect of saxagliptin , as suggested by preclinical data , remains to be fully clarified . a recent double - blind study using 50 patients with t2 dm ( mean duration of 4 years ) without signs of retinopathy has shown that saxagliptin administration ( 5 mg for 6 weeks ) significantly reduced retinal capillary blood flow , suggesting that it is able to reverse early hemodynamic and vascular remodeling processes in t2 dm . ( 2013 ) have demonstrated that linagliptin treatment on zdf rats for 8 weeks is beneficial in blunting obesity - associated cardiac diastolic dysfunction in the prediabetic state . furthermore , using the same approach , nistala et al . ( 2014 ) have reported that dpp-4 inhibition with linagliptin improved proteinuria along with filtration barrier remodelling , circulating , and kidney tissue dpp-4 activity , increased active glp-1 as well as sdf-1 , and improved oxidant markers and the podocyte - specific protein nephrin , suggesting that targeting dpp-4 may have a beneficial effect on the initial stages of obesity - related kidney disease . regarding human data , ( 2013 ) analyzed data from 4 similarly designed ( randomized , double - blind , and placebo - controlled ) phase iii trials , involving 217 individuals with t2 dm and prevalent albuminuria under treatment with renin - angiotensin - aldosterone system inhibitors . the authors showed that at 24 weeks linagliptin ( 5 mg / day ) was able to reduce ( 32% ) urinary albumin - to - creatinine ratio ( uacr ) when compared with patients ( 6% ) randomized to receive placebo . the lack of correlation between the degree of uacr reduction and the level of change in hba1c and sbp suggests that the improvement in urinary albumin excretion by linagliptin could be independent of glycemic and bp controls . the marlina - t2 dm ( efficacy , safety , and modification of albuminuria in type 2 diabetes subjects with renal disease with linagliptin ) trial is ongoing , in order to evaluate the albumin - lowering potential of linagliptin in t2 dm patients with renal impairment . sakata et al . ( 2013 ) reported an improvement in age - rage ( advanced glycation end products - advanced glycation end products ) axis and a reduction in albuminuria in japanese t2 dm patients treated with alogliptin ( 25 mg once daily ) during 12 weeks . ( 2014 ) performed a small , nonrandomized , crossover study with sitagliptin and alogliptin administration on top of angiotensin receptor blockers treatment in t2 dm patients with early nephropathy . four weeks of alogliptin ( 25 mg / day ) treatment after 4 weeks of sitagliptin ( 50 mg / day ) therapy was able to significantly reduce urinary albumin levels , whereas hba1c , blood pressure , serum lipids , and estimated glomerular filtration rate were found to be unchanged . the authors also observed a reduction in the urinary oxidative marker 8-hydroxy-20-deoxyguanosine and an increase in urinary camp level and serum sdf-1a level , suggesting a benefit of alogliptin treatment against early diabetic nephropathy related to antioxidative stress pathways . to conclude , clinical data addressing macro- and microvascular endpoints in t2 dm patients are warranted to provide information whether the promising preclinical findings can be translated into clinical benefit . ongoing clinical trial will probably shed light not only on the extrapancreatic benefits , but also on safety of dpp-4 inhibitors . figure 2 schematically represents the cytoprotective effects of dpp-4 inhibitors on extrapancreatic organs / tissues targeted by diabetes , including the heart , vessels , kidney , and retina , which could be important to control the severe micro- and macrovascular complications found in diabetic patients , including cardiovascular events , end - stage renal disease ( esrd ) , and blindness . our experimental data is suggestive of those putative protective effects and is in line with other previous studies already discussed above . if further confirmed in a near future , namely , in human organs / tissues , dpp-4 inhibitors might represent a key step forward in the management of t2 dm and its serious complications . dpp-4 inhibitors ( gliptins ) have unique benefits that complement and extend the current available therapeutic options for t2 dm . incretin - based therapies can modify various steps in the pathophysiology of t2 dm , including hypersecretion of glucagon , gastric emptying , postprandial hyperglycaemia , and possibly chronic dysfunction of pancreatic cells . overall , gliptins are efficient at reducing plasma glucose , similar to other therapeutic groups , and its use can be made in a combined form with other antidiabetic agents with distinct mechanism of action , in particular with metformin , the most widely used combination . the use of dpp-4 inhibitors has therapeutic benefits , such as improving the secretion of insulin and glucose - dependent suppression of glucagon synthesis . other benefits , including reduction of blood pressure and amelioration of lipid profile , have also been described . furthermore , dpp-4 inhibitors have the ability to improve metabolic control in t2 dm , with minimal risk of adverse effects , including hypoglycaemia , which is very important for the treatment of a large group of diabetic patients , including the elderly ones . the putative association of dpp-4 therapy with development of pancreatitis and pancreatic cancer remains to be confirmed . although several lines of evidences do not support such association , current evidence is not definitive and we should undoubtedly remain vigilant . in any case , currently the balance of evidence is strongly in support of benefits far outweighing potential risks . one of the most relevant and innovative aspects of these new therapies is that they seem to be able to protect the pancreas from progression of deterioration that inevitably seems to occur with the current treatments available , especially due to the ability of dpp-4 inhibitors to protect or even regenerate the pancreatic cell by mechanisms related to their antiapoptotic and proproliferative properties . t2 dm is characterized by a progressive loss of cells mass and function that is associated with insulin resistance , which starts early in the prediabetic states . these defects are followed by a significant decrease in the incretin effect , possibly due to abnormalities in secretion and action of glp-1 . in this sense , dpp-4 inhibitors will theoretically be useful in early stages of diabetes , when the patient still retains a cell population capable of responding to glp-1 stimulation . the possibility has been tested , but the current knowledge is scarce to fully recommend such use . on the other hand , given the complementary effects of dpp-4 and insulin , this association has been tested and in the near future additional data obtained from larger studies should better clarify the benefits and risks of this association in some subpopulations of diabetic patients . one of the most interesting and innovative aspects of incretin - based therapies , including dpp-4 inhibitors , is the putative cytoprotective effect on extrapancreatic organ or tissues target by diabetes , such as the heart , vessels , kidney , and retina . our group has shown beneficial effects of sitagliptin not only on the pancreas but also on the heart , kidney , and retina in animal models of t1 dm and t2 dm [ 116 , 252 , 255 , 256 , 258 ] , which are in line with other studies focused on cardiomyopathy , nephropathy , and retinopathy . if these potential extrapancreatic effects observed in experimental studies can be confirmed and reinforced in humans , then dpp-4 inhibitors could become a preferred treatment for t2 dm due to their ability to modify the natural history of disease by preventing its evolution to more serious stages , as well as due to the protection afforded against evolution of diabetes organ - target complications , thus preventing cardiovascular events , esrd , and progressive loss of vision . it remains to be seen , however , whether these benefits , mainly obtained from preclinical studies , will translate into clinical outcomes ( such as reduction in cardiovascular events and mortality , as well as amelioration of nephropathy and retinopathy ) in large - scale studies . however , to conclude , randomized studies involving large patient cohorts are required to ascertain whether these effects translate into an improvement in clinical outcomes .

incretin - based therapies , the most recent therapeutic options for type 2 diabetes mellitus ( t2 dm ) management , can modify various elements of the disease , including hypersecretion of glucagon , abnormal gastric emptying , postprandial hyperglycaemia , and , possibly , pancreatic cell dysfunction . dipeptidyl peptidase-4 ( dpp-4 ) inhibitors ( gliptins ) increase glucagon - like peptide-1 ( glp-1 ) availability and correct the incretin defect seen in t2 dm patients . clinical studies have shown good glycaemic control with minimal risk of hypoglycaemia or any other adverse effects , despite the reports of pancreatitis , whose association remains to be proved . recent studies have been focusing on the putative ability of dpp-4 inhibitors to preserve pancreas function , in particular due to the inhibition of apoptotic pathways and stimulation of cell proliferation . in addition , other cytoprotective effects on other organs / tissues that are involved in serious t2 dm complications , including the heart , kidney , and retina , have been increasingly reported . this review outlines the therapeutic potential of dpp-4 inhibitors for the treatment of t2 dm , focusing on their main features , clinical applications , and risks , and discusses the major challenges for the future , in particular the possibility of becoming the preferred therapy for t2 dm due to their ability to modify the natural history of the disease and ameliorate nephropathy , retinopathy , and cardiovascular complications .

1. Incretins in Healthy and Disease: Overview 2. Type 2 Diabetes Therapeutics with Incretin Modulators 3. Dipeptidyl Peptidase-4 Inhibitors 4. Major Challenges and Future Prospects 5. Concluding Remarks

later , a second incretin was isolated due to genetic studies on proglucagon coding sequences , and it was described as a this molecule was named glucagon - like peptide-1 ( glp-1 ) and as it met the criteria , it was classified as an incretin . oxyntomodulin ( oxm ) is secreted by the l - cells , in parallel with glp-1 production , and shows an incretin effect , reducing the appetite and the amount of ingested food , an effect that seems to be partly due to the inhibition of ghrelin secretion . in addition to their insulinotropic effects , gip and glp-1 play critical roles in various biological processes in different tissues and organs that express gipr and glp-1r , including the pancreas , adipose tissues , bone , peripheral and central nervous systems ( cns ) , heart , kidney , liver , and gi tract [ 19 , 20 ] . in addition to the pancreas , adipose tissue , stomach , and brain , receptors for gip and glp-1 are expressed in a wide variety of organs , including the bones , heart , and kidneys , where incretins seem to play important effects . however , in t2 dm patients the incretin effect is blunted : the so - called incretin defect . in addition , in a small study of identical twins , differing only in their t2 dm status , the glp-1 response was reduced only in the diabetic twin . the finding that t2 dm patients have low concentrations of glp-1 , but their response of insulin secretion is preserved , supports the therapeutic potential of glp-1 treatments . thus , while gip has a low potential as a drug therapy , glp-1 , on the other hand , has a therapeutic potential as a promising pharmacological tool for the treatment of t2 dm , already proposed in the 1990s , when the incretin effect was reviewed . furthermore , glp-1 possesses a variety of additional physiological effects that are attractive in the treatment of t2 dm , such as in the suppression of glucagon secretion from the pancreatic -cells , in a glucose - dependent manner . furthermore , from the triumvirate theory , defronzo ( 2009 ) suggested there is much more to the pathogenesis of t2 dm , suggesting five additional elements that make substantial contributions to the development and evolution of the disease : ( 1 ) alterations in the enteroendocrine physiology , ( 2 ) increased lipolysis in fat cells , ( 3 ) increased glucagon secretion , ( 4 ) increased renal reabsorption of glucose , and finally ( 5 ) cns insulin resistance with appetite dysregulation . because of the interrelation of these 8 factors to the pathophysiology of t2 dm and its associated morbidity and mortality , they have been referred to as the ominous octet . finally , in order to prevent or slow progressive deterioration in cell function , the interval between the beginning of t2 dm and its diagnosis must be shortened so that treatment can be initiated as early as possible . according to the main international institutions for diabetes care [ american diabetes association ( ada ) , european association for the study of diabetes ( easd ) , and international diabetes federation ( idf ) ] , drug treatment in t2 dm patients should be started when nutritional recommendations and physical activity are not effective to maintain hba1c levels below 7.0% , even in patients without complications , with relatively quality of life , and adhering to nutritional guidelines and physical activity [ 102 , 103 ] . in patients with t2 dm , the risk of complications is associated with the prior hyperglycaemic state , and any reduction in hba1c levels promotes a reduction in the risk for complications . table 1 features the main features of the antidiabetic armamentarium ( non - incretin - based therapies ) , focusing on mechanisms of action , major effects / advantages , adverse reactions , and the ability to decrease hba1c . therefore , a drug ( or a combination of drugs ) that can ideally improve cell health ( tzds , incretin - based therapies , biguanides , and -glucosidase inhibitors ) , improve insulin resistance ( biguanides , tzds , and possibly incretin - based therapies ) , suppress glucagon secretion ( incretin - based therapies ) , suppress appetite ( glp-1 analogues , biguanides ) , improve lipid health ( tzd ) , and suppress renal glucose reabsorption causing the increase in urinary excretion ( sodium - glucose cotransporter 2 inhibitors ) would be the perfect therapy . incretin - based therapies ( addressing 4 out of the 8 pathophysiological defects ) with biguanide [ metformin ( met ) ] or tzd ( addressing insulin resistance in liver and skeletal muscle ) seem to be a very good option . efforts should be made for restoring the glp-1 physiologic function in t2 dm and , thus , correct the multiple metabolic abnormalities observed in patients with the disease . together , these therapeutic strategies are called incretin - based therapies or incretin modulators and represented , in themselves , a promising development for the treatment of t2 dm . this review will now focus on dpp-4 inhibitors , exploiting their antidiabetic properties in comparison ( and/or in association ) with the preexisting oral antidiabetic agents arsenal , as well as the potential for acting as a cytoprotective agent in extrapancreatic organs / tissue , including some of those targeted by diabetic complication ( heart , vessels , kidney , and retina ) . in sum , the place of gliptins in t2 dm therapy is revisited , questioning if these agents are essentially identical to the previous hypoglycemic drugs ( me too ) or if they have potential to notably improve the management of diabetes and prevent its serious complications , thus acting as the special one antidiabetic drugs . in t2 dm patients during hyperglycemic clamp studies , infusion of glp-1 , but not gip , stimulates insulin secretion , thus showing that the insulinotropic effect of glp-1 is reasonably well - preserved in t2 dm , despite possibly lower levels , when compared to nondiabetic individuals [ 120 , 121 ] . hence , the development of incretin - based therapies for t2 dm has thus far focused on glp-1 , rather than gip . clinically , both inhibitors were shown to have numerous advantages as they stimulate the synthesis of insulin , suppress glucagon secretion , lower levels of postprandial and fasting glucose , improve cell function , and lower hba1c values in t2 dm patients [ 91 , 128 ] . the nonpeptidomimetics compounds might assume special relevance because they show selectivity for dpp-4 versus other members of the dpp-4-like family of proteases , including dpp-2 , dpp-8 , and dpp-9 , thus avoiding interference with other putatively important functions ( although the in vivo functions remain largely unknown ) , as well as the possibility of undesirable side - effects . the pharmacokinetic profile and the clinical features of dpp-4 inhibitors have been reviewed in the last years [ 22 , 98 , 131133 ] , and the main features of the class , as well as of each of individual drugs , are summarized in the following subtitles and in tables 2 and 3 , respectively . vildagliptin , from novartis , commercialized firstly with the brand name of galvus , is a highly selective , reversible , inhibitor of the enzyme dpp-4 approved by the eu in 2007 for the treatment of t2 dm , with a recommended dose of 50 mg twice daily . in addition , in vildagliptin recipients a significant reduction from baseline in fpg levels was seen ; however , the mean reduction was significantly higher with metformin , rosiglitazone , or gliclazide than with vildagliptin , and noninferiority between vildagliptin and acarbose was not established [ 182186 ] . on the contrary , the efficacy of vildagliptin administered in combination with metformin in the treatment of t2 dm patients was evaluated in randomized , double - blind , multicentre trials of 12 , 24 , and 52 weeks ' duration . saxagliptin is approved as a combination therapy with metformin , sulfonylurea , thiazolidinedione , or insulin ( with or without metformin ) to improve glycaemic control in adult patients with t2 dm who do not achieve adequate glycaemia control with metformin , sulfonylurea , thiazolidinedione , or insulin in addition to diet and exercise , including patients with mild - to - severe renal impairment . similar to other dpp-4 inhibitors , pooled data from saxagliptin monotherapy and combination therapy trials demonstrate that saxagliptin is generally well tolerated , with a very low risk of adverse events , including hypoglycaemia , and is generally weight neutral ; current prescribing information contains a warning regarding postmarketing reports of pancreatitis [ 199 , 200 ] . as reported for the other drugs of this class , alogliptin is well tolerated , including elderly patients , and the incidence of hypoglycaemia is lower , with neutral effects on body weight and lipid parameters . dpp-4 inhibitors undoubtedly constitute an innovative class of oral agents for the treatment of t2 dm which have enlarged the therapeutic possibilities . current indications for dpp-4 inhibitors recommend its use in combination with other antidiabetic agents , in particular with metformin , as second and third line therapy , and even over other antidiabetic therapies , especially if the patient is experiencing an increased incidence of hypoglycaemia . in fact , the possibility of using incretin modulators , including ( but not only ) dpp-4 inhibitors , in prediabetes is also under debate . on the other hand , and according to their benefit in reducing the levels of glucagon , dpp-4 inhibitors can also be used in the later stages of the disease , in combination with other oral antidiabetic agents , in poorly controlled patients , as is the current clinical indication . the safety and tolerability of dpp-4 inhibitors seem to be generally comparable to nongliptin treatments , although long - term studies and clinical practice followup are still needed , in particular to definitively evaluate if there is any reasonable association between the use of these agents and the risk of pancreatitis and pancreatic cancer , as suggested by some reports . table 2 provides a sum - up of dpp-4 inhibitors in terms of their mechanism of action , major biological effects / advantages , adverse reactions , and their ability to decrease hba1c , which can be compared with table 1 , which summarizes the same properties for the other ( non - incretin - based ) oral antidiabetic agents . in addition , the possibility of cytoprotective properties afforded by dpp-4 inhibitors on organs / tissues which are affected by diabetes ( such as the heart , vessels , kidneys , and retina ) and associated with serious diabetic complications might open new avenues for the use of these agents in the treatment of diabetic patients . in addition , the increased reports of pancreatitis and pancreatic cancer by the fda adverse event reporting system for exenatide and sitagliptin are prone to bias , probably associated publicity surrounding these issues , and are thus not useful for establishing the incidence of adverse events . ( 2014 ) recently reviewed the data concerning the risk of pancreatitis in patients with t2 dm under incretin - based therapies , by analyzing 60 studies ( n = 353,639 ) , consisting of 55 randomised controlled trials ( n = 33,350 ) and five observational studies ( three retrospective cohort studies and two case - control studies ; n = 320,289 ) , concluding that these drugs do not increase the risk of pancreatitis . in addition , fda and ema independently evaluated the safety data from postmarketing reports of pancreatitis and pancreatic cancer in patients using incretin - based drugs , analysing both animal and clinical information available , and concluded that a causal association between incretin - based drugs and pancreatitis or pancreatic cancer can not be established with the current data ; however , fda and the ema have not reached a final conclusion regarding such a causal relationship , and both agencies will continue to investigate the safety signal . several pathophysiological mechanisms contribute to the evolution of t2 dm , including increased insulin resistance in the skeletal muscle and liver ; augmented hepatic glucose output ; impaired insulin secretion with progressive decline of pancreatic cell function . considering the progressive decline of incretin effect in t2 dm patients and the beneficial effects of incretin modulators in the treatment of diabetes , their use has been extended to patients with prediabetes , in a few recent small studies , as reviewed by ahmadieh and azar ( 2014 ) . however , the very small clinical experience on the use of dpp-4 inhibitors and glp-1 mimetics in individuals with impaired fasting glucose or impaired glucose tolerance and the unsolved aspects related to the possibility of pancreatic side - effects do not recommend incretin - based therapies as an option for treatment in patients with prediabetes . in fact , there are several benefits of combining incretin - based therapies with insulin therapy , including the lowering of hba1c due to combined reduction of fasting plasma glucose ( fpg ) by insulin and postprandial glycemia by incretins ; reduction of risk of hypoglycemia which is due to the protection against hypoglycemia with incretin therapy in association with the often observed reduction in insulin dose when using this combination ; the lower risk for weight gain given the protection afforded by incretin therapy , thus compensating the possible weight gain evoked by insulin therapy ; the potential for long - term disease modifying effects , namely , by cell function protection by insulin due to normalization of fasting glucose and prevention of glucotoxicity , combined with beta cell protection afforded by glp-1-based therapies . several clinical studies have been reinforcing the possibility of a beneficial combination between dpp-4 inhibitors and insulin therapy . despite some minor variations between dpp-4 inhibitors ( sitagliptin , vildagliptin , alogliptin , saxagliptin , and linagliptin ) , the combination treatment group ( versus the placebo arm ) showed a higher reduction of hba1c ( change between 0.5 and 0.8 ) and fpg ( change between 0.2 and 1.0 ) , as well as a reduced risk of hypoglycemia and weight gain [ 240248 ] . the current knowledge indicates that the combination is a very promising glucose - lowering strategy for the treatment of t2 dm in patients who do need intensified therapy to control glycaemic levels . although a number of recent experimental studies have demonstrated beneficial effects of incretin - based therapies in extrapancreatic organs or tissues , including the vasculature [ 116 , 250 , 251 ] , the kidney , the heart , and the brain , it remains unclear whether these effects are direct or mediated by the improvement of the glycemic control . a therapeutic low dose of sitagliptin , during a 6-week treatment , in an animal model of t2 dm , the zucker diabetic fatty ( zdf ) rat , was able to promote a partial correction of glycaemia and hba1c levels when compared to controls , accompanied by a partial prevention of insulinopenia . besides the insulinotropic effects of glp-1r activation in pancreatic cells , the expression of this receptor in a wide range of tissues , including retina [ 256 , 257 ] and kidney , suggests the possibility of extrapancreatic effects . regarding clinical data , the large , randomized , placebo - controlled savor - timi 53 ( saxagliptin assessment of vascular outcomes recorded in patients with diabetes mellitus ) trial showed that t2 dm patients with cardiovascular complications under saxagliptin treatment were significantly more likely , when compared to placebo - treated patients , to have improved albumin - to - creatinine ratio ( 10.7% in the saxagliptin group and 8.7% in the placebo group ) and less likely to have worsening ratio ( 13.3% in the saxagliptin group and 15.9% in the placebo group ) , suggesting a protection on albumin excretion rate . the marlina - t2 dm ( efficacy , safety , and modification of albuminuria in type 2 diabetes subjects with renal disease with linagliptin ) trial is ongoing , in order to evaluate the albumin - lowering potential of linagliptin in t2 dm patients with renal impairment . figure 2 schematically represents the cytoprotective effects of dpp-4 inhibitors on extrapancreatic organs / tissues targeted by diabetes , including the heart , vessels , kidney , and retina , which could be important to control the severe micro- and macrovascular complications found in diabetic patients , including cardiovascular events , end - stage renal disease ( esrd ) , and blindness . if further confirmed in a near future , namely , in human organs / tissues , dpp-4 inhibitors might represent a key step forward in the management of t2 dm and its serious complications . dpp-4 inhibitors ( gliptins ) have unique benefits that complement and extend the current available therapeutic options for t2 dm . incretin - based therapies can modify various steps in the pathophysiology of t2 dm , including hypersecretion of glucagon , gastric emptying , postprandial hyperglycaemia , and possibly chronic dysfunction of pancreatic cells . overall , gliptins are efficient at reducing plasma glucose , similar to other therapeutic groups , and its use can be made in a combined form with other antidiabetic agents with distinct mechanism of action , in particular with metformin , the most widely used combination . furthermore , dpp-4 inhibitors have the ability to improve metabolic control in t2 dm , with minimal risk of adverse effects , including hypoglycaemia , which is very important for the treatment of a large group of diabetic patients , including the elderly ones . the putative association of dpp-4 therapy with development of pancreatitis and pancreatic cancer remains to be confirmed . one of the most relevant and innovative aspects of these new therapies is that they seem to be able to protect the pancreas from progression of deterioration that inevitably seems to occur with the current treatments available , especially due to the ability of dpp-4 inhibitors to protect or even regenerate the pancreatic cell by mechanisms related to their antiapoptotic and proproliferative properties . one of the most interesting and innovative aspects of incretin - based therapies , including dpp-4 inhibitors , is the putative cytoprotective effect on extrapancreatic organ or tissues target by diabetes , such as the heart , vessels , kidney , and retina . our group has shown beneficial effects of sitagliptin not only on the pancreas but also on the heart , kidney , and retina in animal models of t1 dm and t2 dm [ 116 , 252 , 255 , 256 , 258 ] , which are in line with other studies focused on cardiomyopathy , nephropathy , and retinopathy . if these potential extrapancreatic effects observed in experimental studies can be confirmed and reinforced in humans , then dpp-4 inhibitors could become a preferred treatment for t2 dm due to their ability to modify the natural history of disease by preventing its evolution to more serious stages , as well as due to the protection afforded against evolution of diabetes organ - target complications , thus preventing cardiovascular events , esrd , and progressive loss of vision .

central noradrenergic pathways are involved in regulating cardiovascular function , as well as in the control of food intake [ 3 , 4 ] . norepinephrine ( ne ) levels and sympathetic activity are responsive to stressors and glycemic fluctuations [ 57 ] . central - acting pharmacological compounds that have actions on norepinephrine and other biogenic amines are currently used ( e.g. , phentermine and related formulations ) or have the potential ( e.g. , tesofensine and bupropion / naltrexone ) to treat obesity [ 8 , 9 ] . however , elevations in heart rate and blood pressure are adverse effects that are often associated with the long - term use of these medications [ 10 , 11 ] . therefore , understanding how central - acting noradrenergic compounds impact feeding behavior and related neural structures can provide more targeted approaches for the treatment of obesity . nisoxetine ( 3-[2-methoxyphenoxy]-n - methyl-3-phenyl-1-propanamine ) is a highly selective central - acting norepinephrine reuptake inhibitor originally developed as an antidepressant [ 12 , 13 ] . in vitro studies with nisoxetine revealed that norepinephrine levels were 1,000-fold higher than serotonin ( 5-hydroxytryptamine ; 5ht ) levels and 300-fold higher than dopamine levels [ 13 , 14 ] . after studies suggested it did not have the desired antidepressant effect , nisoxetine has been used instead as a pharmacological tool to discriminate the involvement of the noradrenergic system . radiolabeled nisoxetine is also used to identify norepinephrine transporter ( net ) kinetics and density [ 14 , 16 , 17 ] . relevant to feeding behavior , nisoxetine has been used to distinguish the contribution of net inhibition on the feeding suppression of sibutramine , a potent ne and 5ht reuptake inhibitor , and bupropion , a potent ne and dopamine reuptake inhibitor [ 18 , 19 ] . even though ne reuptake inhibition has an additive effect when combined with other monoamine reuptake inhibition , examinations of the feeding effect of nisoxetine alone have had mixed findings [ 1821 ] . because sibutramine and its metabolites have relatively good bioavailability when orally administered [ 22 , 23 ] , an apparent discrepancy between studies is the route of administration of nisoxetine . that is , in experiments where the drug is compared with sibutramine and given by oral gavage , nisoxetine has little effect on food intake [ 19 , 21 ] . in contrast , nisoxetine administered by intraperitoneal ( ip ) injection produces a dose - dependent feeding suppression ( 0.163 mg / kg ) . this suggests that nisoxetine has poor oral bioavailability and has limited effectiveness on food intake when orally administered . since the susceptibility to diet - induced obesity has been associated with increased sympathetic and central norepinephrine activity [ 2426 ] , the feeding suppression produced by an ip injection of nisoxetine can be used to determine how dietary conditions alter the noradrenergic controls of feeding . one aspect that has not been investigated is whether doses of nisoxetine that suppress food intake alter cardiovascular and blood pressure parameters . the present studies used acute nisoxetine ( ip ) to identify its dose - dependent effects on feeding behavior and hemodynamic measures . from these experiments , a subthreshold dose of nisoxetine was used to determine the influence of weight gain following prolonged exposure to a high - fat diet on the noradrenergic controls of feeding . to further assess whether exposure to a high - fat diet alters neural responses to nisoxetine , adult male sprague dawley rats ( 8 week old ) acquired from harlan laboratories ( frederick , md ) were individually housed and placed on a 12/12 h light dark schedule ( lights off at 1700 h ) . rats were fed standard chow ( purina rat diet 5012 , 13% fat , 27% protein , 3.1 kcal / g ) , unless otherwise noted , and water was available at all times during the experiments . all procedures were approved by the institutional animal care and use committee of rutgers university and were in accordance with nih guidelines . animals ( n = 6 ) were food deprived 24 h before each injection . for this within - subject design , each animal received an ip injection of vehicle ( saline ) and nisoxetine hcl ( tocris biosciences ; 3 , 10 , and 30 mg / kg ) . each injection was randomized and separated by a one - week wash - out period . food intake measurements were recorded at 0.5 , 1 , 4 , and 24 h after injection . food intake was measured to the nearest 0.1 g. in an identical within - subject design as described above , animals ( n = 12 ) were 24 h food deprived and , upon re - feeding and injection , cardiovascular parameters were measured by using a noninvasive tail - cuff volume pressure recording system ( coda , kent scientific ) . animals were habituated to the coda system for three consecutive days one week before beginning the nisoxetine or saline dosing . on recording days , animals were acclimated to the coda system 5 min before the recordings . measurements for each time were determined by averaging the values for 3 successful recording trials at 1 , 4 , and 24 h after injection . these included measurements for mean blood pressure ( bp ) , diastolic bp , systolic bp , and heart rate . after 1-week acclimatization to standard chow , animals were placed on a high - fat diet ( hf ) , ( n = 9 ; research diets d12492 , 60% fat , 20% protein , 5.24 kcal / g ) or control diet ( cd ) , ( n = 8 ; research diets d12450b , 10% fat , 20% protein , 3.85 kcal / g ) for > 10 wks . body weights were measured twice a week . after a statistical separation of body weight between groups at 10 weeks , animals began testing with a subthreshold feeding dose of nisoxetine ( 3 mg / kg ) , as determined from section 2.2 . prior to each injection , animals were food deprived for 24 h. half of the animals in each group were injected with nisoxetine ( 3 mg / kg , ip ) , while the other half were injected with saline ( ip ) . in order to control for the palatability and caloric differences in diets , all animals were returned to their cages with standard chow and food intake measurements were recorded at 0.5 , 1 , 4 , and 24 h. immediately after the 24 h time point , all rats were returned to their respective diets ( i.e. , high fat or control ) . two weeks following the nisoxetine feeding suppression test , animals in both diet conditions were given an ip injection nisoxetine ( 3 mg / kg ) or saline . groups consisted of high fat ( nisoxetine ) ; n = 5 , high fat ( saline ) ; n = 4 , control diet ( nisoxetine ) ; n = 4 , control diet ( saline ) ; n = 4 . two hours later , rats were deeply anesthetized with sodium pentobarbital ( 100 mg / kg ) , exsanguinated with 0.9% saline , and perfused with 4% paraformaldehyde . brains were removed , stored overnight in 4% paraformaldehyde with 25% ( wt / vol ) sucrose at 28c , and then frozen and sectioned at 40 m on a cryostat through the forebrain and hindbrain regions . the immunohistochemistry procedure for free - floating sections with c - fos primary antibody ( 1 : 20,000 ; calbiochem , emd millipore , rabbit polyclonal , pc38 ) was similar to a previously published protocol . to control for staining variability , each immunohistochemistry run contained matched sections from all between treatment groups and saline controls . cell counts were performed using nih imagej software with value per animal for each region representing average counts from 4 anatomically matched sections . for the nisoxetine dose response , food intake ( kcal ) and individual hemodynamic parameters were analyzed using repeated measures anova . a two - way anova with repeated measures was used to determine whether exposure to the diet influenced the feeding suppression of nisoxetine . cell counts for each region were analyzed using a factorial anova to determine whether there was an interaction between nisoxetine and diet exposure . posthoc comparisons were made when appropriate with neuman - keuls test , unless otherwise noted . all statistical analyses were performed with statistica 7.1 software ( statsoft inc . ) and significance was set at = 0.05 . there was a dose - dependent effect of nisoxetine ( 030 mg / kg ) on the re - feeding response of standard chow . a repeated measures anova indicated a dose effect ( f(3 , 15 ) = 14.8 , p < 0.001 ) , time effect ( f(3,15 ) = 737.3 , p < 0.001 ) , and a dose x time effect that approached significance ( f(9 , 45 ) = 1.9 , p = 0.05 ) . post - hoc testing revealed the 10 mg / kg and 30 mg / kg were significantly different from all other doses ( p < 0.05 for both ) , see figure 1(a ) . when data were expressed as a ratio of individual saline intake to illustrate dose suppression , see figure 1(b ) , there was a significant suppression with 30 mg / kg and 10 mg / kg from 3 mg / kg at all time points ( p < 0.05 ) . data indicated that the 3 mg / kg dose was subthreshold for the nisoxetine - induced feeding suppression of standard chow in animals maintained on standard chow . there was an overall dose effect for mean bp ( f(3,30 ) = 6.1 , p < 0.005 ) , systolic bp ( f(3,30 ) = 5.8 , p < 0.005 ) , diastolic bp ( f(3,30 ) = 5.9 , p < 0.005 ) , and heart rate ( f(3,30 ) = 3.2 , p < 0.05 ) . post - hoc testing revealed that there was a significant decrease in mean bp at 1 h for 10 mg / kg and all time points for 30 mg / kg from saline ( p < 0.05 for all ) ; see figure 2(a ) . the 30 mg / kg dose reduced systolic bp at 4 h and 24 h and diastolic bp at all time points from saline ( p < 0.05 for all ) ; see figures 2(b ) and 2(c ) . there was an increase in heart rate at 24 h in 30 mg / kg dose from saline ( p < 0.05 ) ; see figure 2(d ) . as illustrated in figure 3(a ) , there was increased caloric intake with animals on the high - fat diet . the kcal intake showed a significant overall diet effect ( f(1,15 ) = 15.4 , p < 0.005 ) and time effect ( f(9,135 ) = 36.2 , p < 0.005 ) . this was accompanied by an increase in body weight over time ( f(9,135 ) = 668.4 , p < 0.005 ) , with a significant diet x time effect ( f(9,135 ) = 6.0 , p < 0.005 ) . post - hoc testing revealed that the high - fat diet group had a significantly higher body weight from the control diet group at week 10 ( p < 0.05 ) , see figure 3(b ) . rats with high - fat diet exposure showed a higher sensitivity to the feeding suppression of nisoxetine ( 3 mg / kg ) . there was an overall diet effect ( f(1,15 ) = 13.8 , p < 0.05 ) and nisoxetine effect ( f(1,15 ) = 32.1 , p < 0.005 ) . post - hoc testing indicated that the high - fat diet exposed group had decreased chow intake at the 24 h time point ( p < 0.05 ) . nisoxetine in the high - fat diet exposed group resulted in feeding suppression from the saline at the 1 h time point ( p < 0.05 for both ) . there was a greater feeding suppression from all groups with nisoxetine in the high - fat diet exposed group at the 4 and 24 h time points ( p < 0.05 for both ) , see figure 4 . several hindbrain and forebrain regions were examined for c - fos immunoreactivity , such as a2 cell group , locus coruleus , amygdala nuclei , and hypothalamic nuclei . the only structures to demonstrate immunopositive cells in response to nisoxetine ( 3 mg / kg ) at the 2 h time point in the arcuate nucleus , see figures 5(a ) and 5(b ) , there was an overall significant effect for nisoxetine ( f(1,13 ) = 27.1 , p < 0.005 ) and a significant diet x nisoxetine effect ( f(1,13 ) = 4.8 , p < 0.05 ) . post - hoc testing revealed animals injected with nisoxetine with high - fat diet exposure had more c - fos positive cells in the arcuate nucleus than animals injected with saline and exposed to either high - fat diet or control diet ( p < 0.05 for both ) . planned comparisons between animals receiving nisoxetine revealed the high - fat diet exposed animals had significantly more c - fos positive cells than animals exposed to the control diet ( p < 0.05 ) ; see figure 6(a ) . in the orbitofrontal cortex ( lateral and ventral regions ) , there was only a significant effect with nisoxetine increasing the number of immunopositive cells ( f(1,13 ) = 14.6 , p < 0.05 ) . there were no significant effects for diet or diet x nisoxetine , see figure 6(b ) . this study investigated the acute effects on feeding and hemodynamic measures of nisoxetine , a potent selective norepinephrine reuptake inhibitor , in adult male sprague dawley rats . it was determined that feeding a high - fat diet , which resulted in weight gain , increased sensitivity to the feeding suppression of low - dose nisoxetine . this increased sensitivity was accompanied by enhanced neural activation in the arcuate nucleus of the hypothalamus , a neural structure critical in the homeostatic control of feeding and body weight . the findings of this study indicated that nisoxetine , when given by intraperitoneal injection , produced a dose - dependent refeeding suppression of standard chow over 24 h. feeding suppression from saline was observed in the 10 mg / kg and 30 mg / kg doses , while the 3 mg / kg was subthreshold in normal - weight rats fed standard chow . in a study by billes and cowley comparing the additive effects of nisoxetine and a selective dopamine reuptake inhibitor ( gbr12783 ) on feeding measurements , intraperitoneal nisoxetine ( 0.163 mg / kg ) alone produced a similar dose - dependent refeeding ( i.e. , following a 16 h food deprivation ) suppression of 24 h intake of standard chow in mice . in a follow - up study , billies and cowley found the feeding suppression of nisoxetine ( 4 mg / kg ) did not affect locomotor activity over the 24 h feeding period , but did decrease average intrascapular temperature in mice . our investigation extended these findings over a narrower dose range in rats ( 330 mg / kg ) and measured the influence of these doses on hemodynamic measurements . our results indicated that the 30 mg / kg dose reduced mean blood pressure at 1 h , 4 h , and 24 h after injection . at the 24 h time point , the 30 mg / kg dose produced tachycardia , possibly compensatory to the depressor effect . this dose also still had an approximate 40% feeding suppression , 24 h after injection . it is interesting to note that the 10 mg / kg nisoxetine dose suppressed food intake by approximately 40% at 0.5 h , 1 h , and 4 h and only had an approximate 10% decrease of mean bp at the 1 h time point . one potential limitation to our findings is that the animals had a 24 h food deprivation prior to measuring the hemodynamic parameters . the 24 h food deprivation was done to facilitate comparisons between the feeding suppressive and hemodynamic effects of acute nisoxetine . although prolonged food deprivation ( 48 h ) does decrease heart rate and increase heart rate variability in lean and obese animals , the effects of the 24 h food deprivation in our study was controlled by randomizing the dosing scheme and having a vehicle control . altered hemodynamic measurements and hindquarter vasodilation have been demonstrated with acute administration of sibutramine ( a potent ne and 5-ht reuptake inhibitor ) , effects that are reported mediated by actions on the net [ 3032 ] . taken together , the decrease in intrascapular temperature observed by billes and cowley and the predominant decrease in blood pressure in our study suggests acute nisoxetine acts centrally to produce a short - term sympathoinhibitory effect . another novel finding of the present study was that animals fed a high - fat diet with increased body weight , demonstrated increased sensitivity to the feeding suppression of low - dose nisoxetine . that is , feeding suppression in animals with high - fat diet exposure was observed with the 3 mg / kg dose . this nisoxetine dose did not alter the food intake in standard chow - fed animals or animals fed a low - fat control diet ( 10% fat ) . however , the cardiovascular effects of nisoxetine were not assessed in the animals exposed to the high fat diet and it is possible the sensitivity to low - dose nisoxetine on hemodynamic measures was altered as well . in previous studies , the acute feeding effects of nisoxetine ( 3.5 mg / kg , ip ) were assessed in obese mice , but failed to demonstrate diet - induced alterations in feeding suppression . one difference between studies is the method used to measure the acute feeding response . in the study by billes and cowley , the feeding suppressive effects of nisoxetine were measured using a high - fat diet ( 45% fat , 20% protein ) . the high - fat diet was fed as a maintenance diet for the obese mice and the lean mice were exposed to the high - fat diet about 1 week prior to testing . because the fat content and the length of diet exposure can influence acute palatability and intake , in our study rats were maintained ( > 10 weeks ) on either a high - fat diet ( 60% fat , 20% protein ) or low - fat control diet ( 10% fat , 20% protein ) and were tested on standard chow . the standard chow was not novel per se , since all animals received the diet one week prior to beginning their exposure to a high - fat or control diet . animals with exposure to the high - fat diet demonstrated a reduced 24 h intake of standard chow , a suppressive effect that was enhanced with low dose of nisoxetine . it is possible that this alteration in the sensitivity to the feeding suppression of nisoxetine could have been masked if we had tested the animals with a more palatable high - fat diet , similar to the findings of billes and cowley . another possibility is that the control diet , compared with the high - fat diet , was more similar in relative macronutrient content to the standard chow ( 13% fat , 27% protein ) , so there was less of a contrast effect in the control diet - fed animals . in addition , we did not include a group fed the high - fat diet calorie matched to the control fed group , so we are unable to determine if the feeding suppression by low - dose nisoxetine was due to the high - fat diet feeding or resulting weight gain . future studies are needed to investigate whether nisoxetine or other noradrenergic - acting agents can reduce the intake of highly palatable foods and whether weight gain can alter the noradrenergic controls of palatable foods . the increased sensitivity in the feeding suppression of low - dose nisoxetine observed in the overweight animals suggests that the noradrenergic mechanisms involved in the neural and hormonal controls are altered in the development of diet - induced obesity . one limitation to our findings is that only a single dose of nisoxetine was used to determine the sensitivity to the feeing suppression . however , the increased sensitivity with high - fat feeding and weight gain was supported by our c - fos immunohistochemistry studies , which demonstrated that animals exposed to the high - fat diet had greater neural activation in the arcuate nucleus of the hypothalamus . nisoxetine also increased the number of c - fos positive cells in the orbitofrontal cortex , but this activation was not influenced by diet . an increase in the lateral orbitofrontal cortex has been demonstrated with another selective noradrenergic reuptake inhibitor , reboxetine . the differential dietary activation of nisoxetine in the arcuate nucleus , however , is of considerable importance because this region is critically involved in the central and peripheral integration of the expression of feeding behavior and body weight controls . although the exact role of ne in this region had not been delineated , it does appear ne plays a role in regulating neuronal activity of arcuate neurons . using an in vitro slice preparation , ne application altered the spontaneous discharge rate of arcuate neurons . most of the responses to ne were excitatory with 61.5% of these neurons increasing their firing rate , 22.9% decreasing their firing rate , and 15.6% were unresponsive . in addition , excitation of the arcuate neurons were mediated by both 1-adrenergic and -adrenergic receptors . hindbrain noradrenergic input also appears to modulate orexigenic arcuate neuronal population . using saporin - conjugated antidopamine hydroxylase ( dsap ) , a selective immunotoxin that destroys ne and epinephrine ( e ) containing neurons , loss of ne / e input in the arcuate nucleus resulted in an increase in baseline levels of neuropeptide y / agouti - related peptide ( npy / agrp ) mrna expression . norepinephrine / epinephrine in the arcuate nucleus appears to regulate glucoprivic feeding response , since animals with dsap immunotoxin lesions do not increase their food intake in response to 2-deoxy - d - glucose . studies from levin and colleagues demonstrated the ne turn - over rate in the arcuate nucleus / median eminence is increased in animals as a result of diet - induced obesity or in rats prone to diet - induced obesity [ 37 , 38 ] . in this sense , an increase to the feeding suppression to low - dose nisoxetine could have been a result of increased arcuate ne or increased net availability or both in this region . in addition , one aspect lacking in our study is that we did not determine the phenotype of the c - fos positive cells . future work is certainly needed to determine the interaction between diet - induced alterations in arcuate ne , net availability , and the feeding suppressive and cardiovascular effects of nisoxetine . obesity is associated with neural and metabolic alterations , which are involved in sustaining feeding behavior and increasing body weight . even though noradrenergic mechanisms involved in feeding and cardiovascular controls are altered with obesity , it remains unclear how the feeding alterations , brain norepinephrine , and cardiovascular alterations interact in the development of obesity . this study used a selective norepinephrine reuptake inhibitor , nisoxetine , to focus on the diet - induced alterations in feeding that emerge with onset of weight gain . data from these experiments demonstrate that the development of obesity resulted in an increased sensitivity to the feeding suppression effects of nisoxetine and increased nisoxetine - induced neural activation in feeding - related pathways . one thing to note is that the enhanced feeding suppressive effects of low - dose nisoxetine was demonstrated when tested on standard chow . changes in diet are often accompanied ( and are recommended by health care professionals ) with the prescribing pharmacotherapy for the treatment of obesity . therefore , our findings suggest that medications , which act on noradrenergic pathways , may facilitate the feeding suppression needed to reduce food intake and manage obesity , at least in the short - term . future studies will be conducted to determine the long - term impact of central noradrenergic modulation , food intake , weight management , and cardiovascular control .

central noradrenergic pathways are involved in feeding and cardiovascular control , physiological processes altered by obesity . the present studies determined how high - fat feeding and body weight gain alter the sensitivity to the feeding suppression and neural activation to a selective norepinephrine reuptake inhibitor , nisoxetine . acute administration of nisoxetine ( saline : 0 , 3 , 10 , and 30 mg / kg ; ip ) resulted in a dose - dependent reduction in the 24 h refeeding response in male sprague dawley rats maintained on standard chow . in a similar fashion , nisoxetine resulted in reductions in blood pressure and a compensatory increase in heart rate . from these studies , the 3 mg / kg dose was subthreshold . in a separate experiment , however , 10 wk exposure to a high - fat diet ( 60% fat ) resulted in weight gain and significant feeding suppression following administration of nisoxetine ( 3 mg / kg ) compared with animals fed a control diet ( 10% fat ) . nisoxetine ( 3 mg / kg ) also resulted in greater neural activation , as measured by c - fos immunohistochemistry , in the arcuate nucleus of the hypothalamus in animals exposed to the high - fat diet . such data indicate acute nisoxetine doses that suppress food intake can impact cardiovascular measures . it also suggests that the feeding suppression to a low - dose nisoxetine is enhanced as a result of high - fat diet and weight gain .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

central noradrenergic pathways are involved in regulating cardiovascular function , as well as in the control of food intake [ 3 , 4 ] . however , elevations in heart rate and blood pressure are adverse effects that are often associated with the long - term use of these medications [ 10 , 11 ] . nisoxetine ( 3-[2-methoxyphenoxy]-n - methyl-3-phenyl-1-propanamine ) is a highly selective central - acting norepinephrine reuptake inhibitor originally developed as an antidepressant [ 12 , 13 ] . after studies suggested it did not have the desired antidepressant effect , nisoxetine has been used instead as a pharmacological tool to discriminate the involvement of the noradrenergic system . relevant to feeding behavior , nisoxetine has been used to distinguish the contribution of net inhibition on the feeding suppression of sibutramine , a potent ne and 5ht reuptake inhibitor , and bupropion , a potent ne and dopamine reuptake inhibitor [ 18 , 19 ] . even though ne reuptake inhibition has an additive effect when combined with other monoamine reuptake inhibition , examinations of the feeding effect of nisoxetine alone have had mixed findings [ 1821 ] . that is , in experiments where the drug is compared with sibutramine and given by oral gavage , nisoxetine has little effect on food intake [ 19 , 21 ] . in contrast , nisoxetine administered by intraperitoneal ( ip ) injection produces a dose - dependent feeding suppression ( 0.163 mg / kg ) . since the susceptibility to diet - induced obesity has been associated with increased sympathetic and central norepinephrine activity [ 2426 ] , the feeding suppression produced by an ip injection of nisoxetine can be used to determine how dietary conditions alter the noradrenergic controls of feeding . one aspect that has not been investigated is whether doses of nisoxetine that suppress food intake alter cardiovascular and blood pressure parameters . the present studies used acute nisoxetine ( ip ) to identify its dose - dependent effects on feeding behavior and hemodynamic measures . from these experiments , a subthreshold dose of nisoxetine was used to determine the influence of weight gain following prolonged exposure to a high - fat diet on the noradrenergic controls of feeding . to further assess whether exposure to a high - fat diet alters neural responses to nisoxetine , adult male sprague dawley rats ( 8 week old ) acquired from harlan laboratories ( frederick , md ) were individually housed and placed on a 12/12 h light dark schedule ( lights off at 1700 h ) . rats were fed standard chow ( purina rat diet 5012 , 13% fat , 27% protein , 3.1 kcal / g ) , unless otherwise noted , and water was available at all times during the experiments . for this within - subject design , each animal received an ip injection of vehicle ( saline ) and nisoxetine hcl ( tocris biosciences ; 3 , 10 , and 30 mg / kg ) . food intake measurements were recorded at 0.5 , 1 , 4 , and 24 h after injection . food intake was measured to the nearest 0.1 g. in an identical within - subject design as described above , animals ( n = 12 ) were 24 h food deprived and , upon re - feeding and injection , cardiovascular parameters were measured by using a noninvasive tail - cuff volume pressure recording system ( coda , kent scientific ) . after 1-week acclimatization to standard chow , animals were placed on a high - fat diet ( hf ) , ( n = 9 ; research diets d12492 , 60% fat , 20% protein , 5.24 kcal / g ) or control diet ( cd ) , ( n = 8 ; research diets d12450b , 10% fat , 20% protein , 3.85 kcal / g ) for > 10 wks . after a statistical separation of body weight between groups at 10 weeks , animals began testing with a subthreshold feeding dose of nisoxetine ( 3 mg / kg ) , as determined from section 2.2 . prior to each injection , animals were food deprived for 24 h. half of the animals in each group were injected with nisoxetine ( 3 mg / kg , ip ) , while the other half were injected with saline ( ip ) . in order to control for the palatability and caloric differences in diets , all animals were returned to their cages with standard chow and food intake measurements were recorded at 0.5 , 1 , 4 , and 24 h. immediately after the 24 h time point , all rats were returned to their respective diets ( i.e. two weeks following the nisoxetine feeding suppression test , animals in both diet conditions were given an ip injection nisoxetine ( 3 mg / kg ) or saline . groups consisted of high fat ( nisoxetine ) ; n = 5 , high fat ( saline ) ; n = 4 , control diet ( nisoxetine ) ; n = 4 , control diet ( saline ) ; n = 4 . two hours later , rats were deeply anesthetized with sodium pentobarbital ( 100 mg / kg ) , exsanguinated with 0.9% saline , and perfused with 4% paraformaldehyde . the immunohistochemistry procedure for free - floating sections with c - fos primary antibody ( 1 : 20,000 ; calbiochem , emd millipore , rabbit polyclonal , pc38 ) was similar to a previously published protocol . a two - way anova with repeated measures was used to determine whether exposure to the diet influenced the feeding suppression of nisoxetine . there was a dose - dependent effect of nisoxetine ( 030 mg / kg ) on the re - feeding response of standard chow . a repeated measures anova indicated a dose effect ( f(3 , 15 ) = 14.8 , p < 0.001 ) , time effect ( f(3,15 ) = 737.3 , p < 0.001 ) , and a dose x time effect that approached significance ( f(9 , 45 ) = 1.9 , p = 0.05 ) . post - hoc testing revealed the 10 mg / kg and 30 mg / kg were significantly different from all other doses ( p < 0.05 for both ) , see figure 1(a ) . when data were expressed as a ratio of individual saline intake to illustrate dose suppression , see figure 1(b ) , there was a significant suppression with 30 mg / kg and 10 mg / kg from 3 mg / kg at all time points ( p < 0.05 ) . data indicated that the 3 mg / kg dose was subthreshold for the nisoxetine - induced feeding suppression of standard chow in animals maintained on standard chow . post - hoc testing revealed that there was a significant decrease in mean bp at 1 h for 10 mg / kg and all time points for 30 mg / kg from saline ( p < 0.05 for all ) ; see figure 2(a ) . the 30 mg / kg dose reduced systolic bp at 4 h and 24 h and diastolic bp at all time points from saline ( p < 0.05 for all ) ; see figures 2(b ) and 2(c ) . there was an increase in heart rate at 24 h in 30 mg / kg dose from saline ( p < 0.05 ) ; see figure 2(d ) . as illustrated in figure 3(a ) , there was increased caloric intake with animals on the high - fat diet . this was accompanied by an increase in body weight over time ( f(9,135 ) = 668.4 , p < 0.005 ) , with a significant diet x time effect ( f(9,135 ) = 6.0 , p < 0.005 ) . post - hoc testing revealed that the high - fat diet group had a significantly higher body weight from the control diet group at week 10 ( p < 0.05 ) , see figure 3(b ) . rats with high - fat diet exposure showed a higher sensitivity to the feeding suppression of nisoxetine ( 3 mg / kg ) . post - hoc testing indicated that the high - fat diet exposed group had decreased chow intake at the 24 h time point ( p < 0.05 ) . nisoxetine in the high - fat diet exposed group resulted in feeding suppression from the saline at the 1 h time point ( p < 0.05 for both ) . there was a greater feeding suppression from all groups with nisoxetine in the high - fat diet exposed group at the 4 and 24 h time points ( p < 0.05 for both ) , see figure 4 . several hindbrain and forebrain regions were examined for c - fos immunoreactivity , such as a2 cell group , locus coruleus , amygdala nuclei , and hypothalamic nuclei . the only structures to demonstrate immunopositive cells in response to nisoxetine ( 3 mg / kg ) at the 2 h time point in the arcuate nucleus , see figures 5(a ) and 5(b ) , there was an overall significant effect for nisoxetine ( f(1,13 ) = 27.1 , p < 0.005 ) and a significant diet x nisoxetine effect ( f(1,13 ) = 4.8 , p < 0.05 ) . post - hoc testing revealed animals injected with nisoxetine with high - fat diet exposure had more c - fos positive cells in the arcuate nucleus than animals injected with saline and exposed to either high - fat diet or control diet ( p < 0.05 for both ) . planned comparisons between animals receiving nisoxetine revealed the high - fat diet exposed animals had significantly more c - fos positive cells than animals exposed to the control diet ( p < 0.05 ) ; see figure 6(a ) . this study investigated the acute effects on feeding and hemodynamic measures of nisoxetine , a potent selective norepinephrine reuptake inhibitor , in adult male sprague dawley rats . it was determined that feeding a high - fat diet , which resulted in weight gain , increased sensitivity to the feeding suppression of low - dose nisoxetine . this increased sensitivity was accompanied by enhanced neural activation in the arcuate nucleus of the hypothalamus , a neural structure critical in the homeostatic control of feeding and body weight . the findings of this study indicated that nisoxetine , when given by intraperitoneal injection , produced a dose - dependent refeeding suppression of standard chow over 24 h. feeding suppression from saline was observed in the 10 mg / kg and 30 mg / kg doses , while the 3 mg / kg was subthreshold in normal - weight rats fed standard chow . in a study by billes and cowley comparing the additive effects of nisoxetine and a selective dopamine reuptake inhibitor ( gbr12783 ) on feeding measurements , intraperitoneal nisoxetine ( 0.163 mg / kg ) alone produced a similar dose - dependent refeeding ( i.e. , following a 16 h food deprivation ) suppression of 24 h intake of standard chow in mice . in a follow - up study , billies and cowley found the feeding suppression of nisoxetine ( 4 mg / kg ) did not affect locomotor activity over the 24 h feeding period , but did decrease average intrascapular temperature in mice . our investigation extended these findings over a narrower dose range in rats ( 330 mg / kg ) and measured the influence of these doses on hemodynamic measurements . our results indicated that the 30 mg / kg dose reduced mean blood pressure at 1 h , 4 h , and 24 h after injection . at the 24 h time point , the 30 mg / kg dose produced tachycardia , possibly compensatory to the depressor effect . it is interesting to note that the 10 mg / kg nisoxetine dose suppressed food intake by approximately 40% at 0.5 h , 1 h , and 4 h and only had an approximate 10% decrease of mean bp at the 1 h time point . the 24 h food deprivation was done to facilitate comparisons between the feeding suppressive and hemodynamic effects of acute nisoxetine . although prolonged food deprivation ( 48 h ) does decrease heart rate and increase heart rate variability in lean and obese animals , the effects of the 24 h food deprivation in our study was controlled by randomizing the dosing scheme and having a vehicle control . altered hemodynamic measurements and hindquarter vasodilation have been demonstrated with acute administration of sibutramine ( a potent ne and 5-ht reuptake inhibitor ) , effects that are reported mediated by actions on the net [ 3032 ] . taken together , the decrease in intrascapular temperature observed by billes and cowley and the predominant decrease in blood pressure in our study suggests acute nisoxetine acts centrally to produce a short - term sympathoinhibitory effect . another novel finding of the present study was that animals fed a high - fat diet with increased body weight , demonstrated increased sensitivity to the feeding suppression of low - dose nisoxetine . that is , feeding suppression in animals with high - fat diet exposure was observed with the 3 mg / kg dose . this nisoxetine dose did not alter the food intake in standard chow - fed animals or animals fed a low - fat control diet ( 10% fat ) . however , the cardiovascular effects of nisoxetine were not assessed in the animals exposed to the high fat diet and it is possible the sensitivity to low - dose nisoxetine on hemodynamic measures was altered as well . in previous studies , the acute feeding effects of nisoxetine ( 3.5 mg / kg , ip ) were assessed in obese mice , but failed to demonstrate diet - induced alterations in feeding suppression . in the study by billes and cowley , the feeding suppressive effects of nisoxetine were measured using a high - fat diet ( 45% fat , 20% protein ) . the high - fat diet was fed as a maintenance diet for the obese mice and the lean mice were exposed to the high - fat diet about 1 week prior to testing . because the fat content and the length of diet exposure can influence acute palatability and intake , in our study rats were maintained ( > 10 weeks ) on either a high - fat diet ( 60% fat , 20% protein ) or low - fat control diet ( 10% fat , 20% protein ) and were tested on standard chow . the standard chow was not novel per se , since all animals received the diet one week prior to beginning their exposure to a high - fat or control diet . animals with exposure to the high - fat diet demonstrated a reduced 24 h intake of standard chow , a suppressive effect that was enhanced with low dose of nisoxetine . it is possible that this alteration in the sensitivity to the feeding suppression of nisoxetine could have been masked if we had tested the animals with a more palatable high - fat diet , similar to the findings of billes and cowley . another possibility is that the control diet , compared with the high - fat diet , was more similar in relative macronutrient content to the standard chow ( 13% fat , 27% protein ) , so there was less of a contrast effect in the control diet - fed animals . in addition , we did not include a group fed the high - fat diet calorie matched to the control fed group , so we are unable to determine if the feeding suppression by low - dose nisoxetine was due to the high - fat diet feeding or resulting weight gain . the increased sensitivity in the feeding suppression of low - dose nisoxetine observed in the overweight animals suggests that the noradrenergic mechanisms involved in the neural and hormonal controls are altered in the development of diet - induced obesity . one limitation to our findings is that only a single dose of nisoxetine was used to determine the sensitivity to the feeing suppression . however , the increased sensitivity with high - fat feeding and weight gain was supported by our c - fos immunohistochemistry studies , which demonstrated that animals exposed to the high - fat diet had greater neural activation in the arcuate nucleus of the hypothalamus . nisoxetine also increased the number of c - fos positive cells in the orbitofrontal cortex , but this activation was not influenced by diet . an increase in the lateral orbitofrontal cortex has been demonstrated with another selective noradrenergic reuptake inhibitor , reboxetine . the differential dietary activation of nisoxetine in the arcuate nucleus , however , is of considerable importance because this region is critically involved in the central and peripheral integration of the expression of feeding behavior and body weight controls . using saporin - conjugated antidopamine hydroxylase ( dsap ) , a selective immunotoxin that destroys ne and epinephrine ( e ) containing neurons , loss of ne / e input in the arcuate nucleus resulted in an increase in baseline levels of neuropeptide y / agouti - related peptide ( npy / agrp ) mrna expression . norepinephrine / epinephrine in the arcuate nucleus appears to regulate glucoprivic feeding response , since animals with dsap immunotoxin lesions do not increase their food intake in response to 2-deoxy - d - glucose . studies from levin and colleagues demonstrated the ne turn - over rate in the arcuate nucleus / median eminence is increased in animals as a result of diet - induced obesity or in rats prone to diet - induced obesity [ 37 , 38 ] . in this sense , an increase to the feeding suppression to low - dose nisoxetine could have been a result of increased arcuate ne or increased net availability or both in this region . future work is certainly needed to determine the interaction between diet - induced alterations in arcuate ne , net availability , and the feeding suppressive and cardiovascular effects of nisoxetine . obesity is associated with neural and metabolic alterations , which are involved in sustaining feeding behavior and increasing body weight . even though noradrenergic mechanisms involved in feeding and cardiovascular controls are altered with obesity , it remains unclear how the feeding alterations , brain norepinephrine , and cardiovascular alterations interact in the development of obesity . this study used a selective norepinephrine reuptake inhibitor , nisoxetine , to focus on the diet - induced alterations in feeding that emerge with onset of weight gain . data from these experiments demonstrate that the development of obesity resulted in an increased sensitivity to the feeding suppression effects of nisoxetine and increased nisoxetine - induced neural activation in feeding - related pathways . one thing to note is that the enhanced feeding suppressive effects of low - dose nisoxetine was demonstrated when tested on standard chow . therefore , our findings suggest that medications , which act on noradrenergic pathways , may facilitate the feeding suppression needed to reduce food intake and manage obesity , at least in the short - term . future studies will be conducted to determine the long - term impact of central noradrenergic modulation , food intake , weight management , and cardiovascular control .

in keeping with the mission of a liberal arts education , denison s neuroscience concentration provides our students with a challenging interdisciplinary perspective on the complex relationships between brain and behavior . in this way , students who pursue the concentration are exposed to a number of perspectives within neuroscience , from cellular and molecular analysis to broader , more molar systems approaches to behavior . students pursuing our neuroscience concentration are most often biology or psychology majors , although there have been a few biochemistry majors completing the concentration . since 2000 , we have had close to 70 students earn the concentration , with an additional nine students completing self - designed majors in neuroscience , cognitive neuroscience , social neuroscience , or psychobiology . our curriculum is structured in such a way that all neuroscience students must complete introductory psychology and introduction to the science of biology prior to enrolling in our introduction to neuroscience ( neur 200 ) course . neur 200 emphasizes the basics of the field , including cellular physiology , ionic movements , refractory periods , receptor dynamics , post - synaptic potentials , neuropharmacology , and neuroanatomy . the two introductory courses are also required for the 200- and 300-level elective courses in psychology or biology . students must also complete four courses ( including neur 200 ) designed to provide breadth in the concentration . throughout the remainder of their tenure at denison , neuroscience students must complete biological psychology , biological psychology research , two upper - level electives depending upon the student s major and area(s ) of interest , and our capstone course , advanced neuroscience ( neur 400 ) . table 1 summarizes the neuroscience curriculum , identifying pre - requisite courses , courses required for breadth , and more advanced required and elective courses that offer depth in the concentration . over the last several years , capstone courses have become increasingly more visible on college and university campuses . indeed , the capstone experience is now fairly commonplace in smaller , private colleges , as well as in large public institutions ( badway and grubb , 1999 ) . capstone courses are believed to provide valuable experiences for students , including opportunities for synthesis and integration of information ( e.g. , henscheid et al . , 2000 ) , further development of critical thinking and writing and speaking skills ( cuseo , 1998 ) , and an ideal environment for assessing student learning ( moore , 2005 ) . in our program , the capstone experience for all neuroscience students is advanced neuroscience ( neur 400 ) . this course is designed for juniors and seniors who have completed the majority ( if not all ) of the courses required for the concentration . typically , the course enrolls 1525 students , and is taught in the spring semester . from the inception of our neuroscience program , we envisioned neur 400 to be a course that brings together critical concepts and ideas from the students previous coursework in a format emphasizing discussion of primary literature and incorporating multiple learning activities and projects throughout the semester . unlike the introductory course in neuroscience , neur 400 addresses contemporary molar issues in neuroscience . importantly , our advanced neuroscience course is team taught by faculty from biology , psychology , chemistry , philosophy and computer science . in this way , students are exposed to cutting edge issues within a number of sub - fields of neuroscience by faculty whose primary interests reflect those issues and problems . one faculty member serves as the instructor of record ( this person receives teaching credit for the course ) and is responsible for the course organization and administration ( syllabus creation , coordination among participating faculty , development of assignments , and grading ) . at denison , the instructor of record rotates every two years between the psychology and biology departments . in recent years , the course topics and readings have examined visual attention and computational neuroscience , autism , biochemistry of memory formation , explicit and implicit memory systems , amnesia , face recognition and cognitive neuroscience , artificial intelligence and face recognition , neuroscience of consciousness , neurophilosophy , nervous tissue differentiation and central nervous system development , glia and glioma , stress and neurodegeneration , affective neuroscience , educational neuroscience , and cultural neuroscience . each faculty participant assigns research articles and will typically lead either one , two , or three class meetings that can include lectures and research presentations , student - led discussions and presentations , or active learning exercises . students are also asked to post on blackboard , an on - line course management system , two or three discussion questions or talking points on the reading assignments prior to each class meeting . several different assignments have been designed to help students enhance their ability to read and critique neuroscience research , develop an integrative understanding of neuroscience core areas , examine the intersections that connect neuroscience with other disciplinary areas in sciences , arts , and humanities , and explore the relevance of neuroscience to contemporary issues and personal applications . we present three of the activities below . the dissemination of neuroscience research to scientists , educators , professionals , and the general public has increased through the availability of internet sites . in particular , the range and complexity of neuroscience research introduces unique challenges for the presentation of informative and useful research summaries to an informed lay audience . thus , the goal of this activity is to provide students with an opportunity to read a primary research article in a neuroscience journal and then to prepare and deliver an oral presentation about that research article to classmates in a session that is known as neuroscience in the news . each student selects a research article from any field of neuroscience published in the past three years based upon his or her personal interests . then they prepare a 5-minute presentation that will model the format of a news release and that will be similar in content and style to the neuroscience news reports on the webpage of the british neuroscience association ( bna ; http://www.bna.org.uk/news/ ) . in the presentation , the students objectives are to explain why they selected the particular research article ; describe the most significant theoretical issues , methods , and results ; summarize the most meaningful conclusions including the importance or relevance of the research and/or its applications ; and identify how the research contributed to an enhancement of personal knowledge and interests in neuroscience . it is stressed to the students that their neuroscience in the news presentation should be understandable to an informed lay audience as much as to neuroscientists . students can augment their oral presentation with a small number of powerpoint slides or video clips in order to show results , equipment , or other pertinent information . some of the recent neuroscience in the news presentations include does sleep deprivation put you in a better mood ? , exercise training increases the size of the hippocampus and improves your memory , videogames and cognitive training in the elderly , brain - machine - brain interfaces : a new way to connect to the world , brain imaging : visualizing the developing and maturing brain , and therapeutic potential of omega-3 pufas for peripheral nerve injuries . following each presentation many of our neuroscience students have educational and career goals involving the application of basic neuroscience research to medical , clinical , educational , or other professional settings . therefore , in this assignment , pairs of students work together to develop a brain briefing , a written document that explains how basic neuroscience research has relevance to a general audience of policy makers in clinical , health , educational , or other fields and professions ( e.g. , sports , law , economics , and robotics ) . the model for this assignment is the brain briefings newsletter published online by the society for neuroscience ( sfn ) ( lom , 2005 ) and more recently posted at the website , brainfacts.org ( http://www.brainfacts.org/ ) , a public information initiative of the kavli foundation , the gatsby charitable foundation , and the sfn . students first review several brain briefings on the website in order to get a grasp of the content , format , and style of these papers . then , each pair of students identify a particular area of application ( e.g. , medical treatments for alzheimer s disease ) that can be informed by neuroscience and conduct an online search to find four to six relevant research articles . the students brain briefing report is not meant to be a research abstract ; rather , their goal is to follow the format of the sfn brain briefings and provide information on recent neuroscience research that has exciting and valuable applications to neurological , psychological , and medical contexts . each briefing has a limit of 1000 words and includes references and one or two visual objects ( e.g. , research data , brain images , or other illustrations ) . the evaluation of the brain briefing is based upon how well students identify and explain the connections between the research outcomes with the potential applications . examples of brain briefings produced in the most recent class include an eye toward the future : sight restoration through neuroengineering and visual prosthesis , neuromarketing , transcranial direct current stimulation : shocking new therapeutic possibilities , and the neural truth : religion as an anesthetic . case studies are a type of non - experimental research methodology that typically investigate one individual in depth or over time . recently , case studies have also been used effectively in neuroscience pedagogy ( meil , 2007 ) and are frequently included in textbooks and in popular books written by neuroscientists or neurologists ( e.g. , oliver sacks ) . this assignment is a major and culminating project for the course and the overall learning goal is to develop a deep understanding of the neurological condition that is presented in the case study through the evaluation and synthesis of contemporary neuroscience research . students choose a case study after examining the strange brains and case studies internet resource compiled by william meil and jeremy skipper , http://lablab.hamilton.edu/lab-teaching/strange-brains-and-case-studies , or other resources noted by the instructor ( e.g. , ramachandran , 2004 ; bogousslavsky and boller , 2005 ; sacks , 2007 , 2010 ) . the 12-page written report for this project has three parts : 1 ) case description , 2 ) literature review , and 3 ) research proposal ( cf . meil , 2007 ) . in part one , students are asked to describe the most salient aspects of the case . for example , who was the person and what happened to them ? how did their condition influence their life ? why did you choose this case in order to deepen your study of neuroscience ? in part two , the literature review should include articles that help to answer questions such as what are the typical symptoms and what are the neurobiological or neuropsychological basis of the condition ? how is the condition treated and what is the treatment prognosis ? in this section the students are asked to evaluate and synthesize the results of at least eight empirical articles and to discuss how the literature review is relevant to course topics and readings . in the final section of the paper , students identify at least one unanswered question about the condition described in the case ( causes , symptoms , or treatment ) and then propose an experiment to address this question . the research proposal includes rationale , hypotheses , participants , materials and apparatus , procedures , and statistical analyses . finally , students are also asked to describe the potential significance of the proposed research for the field of neuroscience . the final three class meetings of the semester are devoted to oral presentations of the case studies . each presentation is 12 minutes in length and can include powerpoint slides . in the presentation , students describe the very most salient aspects of the case in terms of the underlying neurological issue . they also explain their decision to choose the case and how the case is important for the study of neuroscience . students briefly present the key research goals , methods , results , and conclusions from only two of the research articles selected from the literature review . the students also explain why or how these two articles have relevance to an understanding of the case study . finally , each student describes how the case study project has enhanced their study of neuroscience and the neuroscience topics that have been examined in the course . the topics of the case studies chosen by students have included creutzfeld - jakob disease , capgras syndrome , post - traumatic stress disorder , autism , tourette s syndrome , amnesia , epilepsy , dissociative identity disorder , charles bonnet syndrome , and auditory and visual hallucinations . the dissemination of neuroscience research to scientists , educators , professionals , and the general public has increased through the availability of internet sites . in particular , the range and complexity of neuroscience research introduces unique challenges for the presentation of informative and useful research summaries to an informed lay audience . thus , the goal of this activity is to provide students with an opportunity to read a primary research article in a neuroscience journal and then to prepare and deliver an oral presentation about that research article to classmates in a session that is known as neuroscience in the news . each student selects a research article from any field of neuroscience published in the past three years based upon his or her personal interests . then they prepare a 5-minute presentation that will model the format of a news release and that will be similar in content and style to the neuroscience news reports on the webpage of the british neuroscience association ( bna ; http://www.bna.org.uk/news/ ) . in the presentation , the students objectives are to explain why they selected the particular research article ; describe the most significant theoretical issues , methods , and results ; summarize the most meaningful conclusions including the importance or relevance of the research and/or its applications ; and identify how the research contributed to an enhancement of personal knowledge and interests in neuroscience . it is stressed to the students that their neuroscience in the news presentation should be understandable to an informed lay audience as much as to neuroscientists . students can augment their oral presentation with a small number of powerpoint slides or video clips in order to show results , equipment , or other pertinent information . some of the recent neuroscience in the news presentations include does sleep deprivation put you in a better mood ? , exercise training increases the size of the hippocampus and improves your memory , videogames and cognitive training in the elderly , brain - machine - brain interfaces : a new way to connect to the world , brain imaging : visualizing the developing and maturing brain , and therapeutic potential of omega-3 pufas for peripheral nerve injuries . following each presentation , there is a short period of questions and discussion among the students . many of our neuroscience students have educational and career goals involving the application of basic neuroscience research to medical , clinical , educational , or other professional settings . therefore , in this assignment , pairs of students work together to develop a brain briefing , a written document that explains how basic neuroscience research has relevance to a general audience of policy makers in clinical , health , educational , or other fields and professions ( e.g. , sports , law , economics , and robotics ) . the model for this assignment is the brain briefings newsletter published online by the society for neuroscience ( sfn ) ( lom , 2005 ) and more recently posted at the website , brainfacts.org ( http://www.brainfacts.org/ ) , a public information initiative of the kavli foundation , the gatsby charitable foundation , and the sfn . students first review several brain briefings on the website in order to get a grasp of the content , format , and style of these papers . then , each pair of students identify a particular area of application ( e.g. , medical treatments for alzheimer s disease ) that can be informed by neuroscience and conduct an online search to find four to six relevant research articles . the students brain briefing report is not meant to be a research abstract ; rather , their goal is to follow the format of the sfn brain briefings and provide information on recent neuroscience research that has exciting and valuable applications to neurological , psychological , and medical contexts . each briefing has a limit of 1000 words and includes references and one or two visual objects ( e.g. , research data , brain images , or other illustrations ) . the evaluation of the brain briefing is based upon how well students identify and explain the connections between the research outcomes with the potential applications . examples of brain briefings produced in the most recent class include an eye toward the future : sight restoration through neuroengineering and visual prosthesis , alzheimer s dementia , sleep and your emotions , neuromarketing , transcranial direct current stimulation : shocking new therapeutic possibilities , and the neural truth : religion as an anesthetic . case studies are a type of non - experimental research methodology that typically investigate one individual in depth or over time . recently , case studies have also been used effectively in neuroscience pedagogy ( meil , 2007 ) and are frequently included in textbooks and in popular books written by neuroscientists or neurologists ( e.g. , oliver sacks ) . this assignment is a major and culminating project for the course and the overall learning goal is to develop a deep understanding of the neurological condition that is presented in the case study through the evaluation and synthesis of contemporary neuroscience research . students choose a case study after examining the strange brains and case studies internet resource compiled by william meil and jeremy skipper , http://lablab.hamilton.edu/lab-teaching/strange-brains-and-case-studies , or other resources noted by the instructor ( e.g. , ramachandran , 2004 ; bogousslavsky and boller , 2005 ; sacks , 2007 , 2010 ) . the 12-page written report for this project has three parts : 1 ) case description , 2 ) literature review , and 3 ) research proposal ( cf . meil , 2007 ) . in part one , students are asked to describe the most salient aspects of the case . for example , who was the person and what happened to them ? how did their condition influence their life ? why did you choose this case in order to deepen your study of neuroscience ? in part two , the literature review should include articles that help to answer questions such as what are the typical symptoms and what are the neurobiological or neuropsychological basis of the condition ? how is the condition treated and what is the treatment prognosis ? in this section the students are asked to evaluate and synthesize the results of at least eight empirical articles and to discuss how the literature review is relevant to course topics and readings . in the final section of the paper , students identify at least one unanswered question about the condition described in the case ( causes , symptoms , or treatment ) and then propose an experiment to address this question . the research proposal includes rationale , hypotheses , participants , materials and apparatus , procedures , and statistical analyses . finally , students are also asked to describe the potential significance of the proposed research for the field of neuroscience . the final three class meetings of the semester are devoted to oral presentations of the case studies . each presentation is 12 minutes in length and can include powerpoint slides . in the presentation , students describe the very most salient aspects of the case in terms of the underlying neurological issue . they also explain their decision to choose the case and how the case is important for the study of neuroscience . students briefly present the key research goals , methods , results , and conclusions from only two of the research articles selected from the literature review . the students also explain why or how these two articles have relevance to an understanding of the case study . finally , each student describes how the case study project has enhanced their study of neuroscience and the neuroscience topics that have been examined in the course . the topics of the case studies chosen by students have included creutzfeld - jakob disease , capgras syndrome , post - traumatic stress disorder , autism , tourette s syndrome , amnesia , epilepsy , dissociative identity disorder , charles bonnet syndrome , and auditory and visual hallucinations . students are strongly encouraged to provide written feedback and rating scale responses for the course as part of the end - of - semester course evaluation program in place at denison . recent efforts on our campus have resulted in increased student participation in the course evaluation process by carving out class time during the last week of classes for this purpose . in neur 400 , participation rates for course evaluations are high , typically at 93% or better . students have evaluated the course quite favorably . in the past few offerings , the majority of students numeric ratings consisted of very good to excellent evaluations on items such as the course is challenging , their interest in neuroscience increased , their knowledge of the subject increased , and that faculty were clear , well - prepared , provided useful feedback , and were effective in their teaching . for example , one student commented that if i could i would take ( the class ) over again there has not been a single week that i did not learn something completely new . the depth of the material was something that i had not been exposed to previously ; this gave me an extreme increase in knowledge of the subject matter . we were particularly interested in student feedback regarding the team taught nature of the course , as this is something that the majority of undergraduate students have little or no experience with . generally , this was viewed as a positive aspect of the course . as one student noted , i like the variety of instructors and the variety of topics covered ; while another wrote the use of professors from different backgrounds presenting their primary focus of work and research to us was excellent , while still another student commented i really liked the idea of having different faculty come in to teach on a subject that was an area of expertise for them . we had a wonderful group of faculty who were really passionate about what they were teaching . finally , one student stated emphatically , the best part about this course has been the exposure to different professors through the rotation schedule . some students , however , found that having multiple instructors for the course was confusing and challenging , as noted by the following student comment , sometimes the switching of instructors can feel a little sporadic . the class felt scattered because we jumped from one topic to another so quickly . denison s required course evaluation form only contains a short list of course and instructor questions . in the future , we plan on administering an additional evaluation form in order to obtain information from specific open - ended questions about course content , organization , learning activities , and team - teaching . an integrative capstone course in neuroscience can provide a valuable culminating experience for students of the discipline ( wiertelak and ramirez , 2008 ) . we feel satisfied that our course challenges students to think about some of the larger contemporary issues and questions in neuroscience , to read and digest primary literature in the field , and to engage in multiple learning opportunities that enable integration of concepts and ideas acquired in previous courses . however , we do recognize that there are important challenges that our neuroscience faculty continue to discuss regarding ways to improve the course as our neuroscience program evolves . first and foremost , as a highly interdisciplinary field , neuroscience requires collaboration from individuals across departments . we feel that a successful team - taught capstone course in neuroscience , therefore , depends on faculty who are committed to an interdisciplinary neuroscience program , and who are willing to commit several hours out of their already busy schedules to the preparation that is necessary in order to meet with the class over two or three sessions with no monetary compensation or teaching credit provided . we have been quite fortunate over the years to have colleagues from across disciples eager to engage with our students in the capstone course . most recently , eight faculty participated and represented the departments of biology , chemistry , computer science , philosophy , and psychology . in addition , guest lecturers from denison s library and ohio state university also presented research . of course , when faculty are on leave or unable to participate for other reasons , the course content must be changed or modified in order to accommodate this change , or other faculty representing the same or some different area must be asked to participate in the course . also , it is our hope to have more faculty from other disciplines including the social sciences , humanities , and the arts participate in neur 400 in future semesters in order to highlight the importance of neuroscience in the interdisciplinary focus of a liberal arts college ( ramirez , 2007 ) . in addition to the importance of a core group of interested and willing faculty , the instructor of record also has a critical role in the success of such a course . s / he presents three to four lectures on neuroscience research and throughout the semester must keep the class on track , provide linkages and continuity between topics and speakers and bridge the topics coherently , particularly when faculty speakers change . the instructor also is responsible for developing all of the learning activities and assignments described in this paper . however , our neuroscience faculty have contributed ideas and support for these assignments which share a focus on integration and application of neuroscience research . these learning objectives address the goals of a capstone course within the structure of our neuroscience curriculum but we recognize that there are other learning goals and assignments that could be present in a capstone course . the course instructor also is solely responsible for grading all of the assignments , exams , and student participation . therefore , the instructor does have a challenge in ensuring that students will develop their integrative knowledge of neuroscience from the ongoing flow of topics , presenters , and learning activities that occur during the semester . student comments regarding course flow reinforce the importance of this role , and we continue to work on this in our discussions of our curriculum , particularly when the instructor of record changes between departments . another important consideration in planning for a neuroscience capstone course is that enrolled students should be near the end of their college career , preferably seniors or second - semester juniors . this is essential for the types of class discussions and thoughtful conversations that add to the success of such a course . the course as we design it assumes that students have mastered the basics of neuroscience and have completed adequate coursework that enables them to think critically , ask probing questions and offer meaningful comments in the classroom . for this to be the case , neuroscience curricula must be carefully structured around common core courses as well as prerequisite courses necessary for subsequent electives . finally , the success of an interdisciplinary and team - taught course depends , in part , upon multiple levels of support . there must be support from faculty across disciplines as we have already discussed , but also support from multiple departments and the administration . because the instructor of record earns one teaching credit for designing and coordinating neur 400 , this may very well put some strain on the home department of the instructor in terms of course offerings , enrollments , etc . having one of his / her courses a neuroscience course means that one fewer course in the home department ( psychology , biology , for example ) can be taught during that semester . clearly , then , it is imperative that there be departmental support for the program , transparency in what the commitment to neur courses will be , how frequently they will be taught ( every year ? every other year ? ) , and how many members of the department will be contributing regularly to the neuroscience courses and other activities associated with the concentration . on our campus , we had several departmental ( primarily within the psychology and biology departments ) discussions as the neuroscience program was in its early stages , and we continue to discuss particular aspects of the neur concentration and courses in departmental staff meetings as necessary ; in addition , neuroscience faculty meet regularly to plan future neur course offerings , discuss staffing issues , enrollments , and consider other important agenda items relevant to our neuroscience program . because our concentration and the capstone course require the involvement of multiple faculty representing multiple departments , it also is essential to have the support of a campus administration that encourages and rewards interdisciplinary efforts from faculty ( flint and dorr , 2010 ) . careful planning and communication with colleagues both within and across departments --- are key when creating an integrative and team - taught course for students of neuroscience .

capstone courses are becoming increasingly visible on college and university campuses . in this paper , we describe a capstone experience for undergraduate students pursuing our neuroscience concentration . the course is intended to provide an in - depth and interdisciplinary examination of contemporary topics in the field of neuroscience , and is designed for students who have completed the majority of requirements for the concentration . we describe the evolution of such a course , the goals and objectives of the course , and offer a workable model for similar courses in the context of a liberal arts institution . we summarize the positive aspects of such a course , describe the challenges involved in creating a course of this nature , and offer suggestions for successful similar capstone courses in neuroscience .

OVERVIEW OF THE NEUROSCIENCE CONCENTRATION THE CAPSTONE COURSE: ADVANCED NEUROSCIENCE LEARNING ACTIVITIES AND ASSIGNMENTS Neuroscience in the News Brain Briefings Neuroscience Case Studies STUDENT EVALUATIONS OF THE COURSE REFLECTIONS ON THE CAPSTONE: STRENGTHS, CHALLENGES AND SUGGESTIONS

in keeping with the mission of a liberal arts education , denison s neuroscience concentration provides our students with a challenging interdisciplinary perspective on the complex relationships between brain and behavior . in this way , students who pursue the concentration are exposed to a number of perspectives within neuroscience , from cellular and molecular analysis to broader , more molar systems approaches to behavior . students pursuing our neuroscience concentration are most often biology or psychology majors , although there have been a few biochemistry majors completing the concentration . since 2000 , we have had close to 70 students earn the concentration , with an additional nine students completing self - designed majors in neuroscience , cognitive neuroscience , social neuroscience , or psychobiology . neur 200 emphasizes the basics of the field , including cellular physiology , ionic movements , refractory periods , receptor dynamics , post - synaptic potentials , neuropharmacology , and neuroanatomy . the two introductory courses are also required for the 200- and 300-level elective courses in psychology or biology . students must also complete four courses ( including neur 200 ) designed to provide breadth in the concentration . throughout the remainder of their tenure at denison , neuroscience students must complete biological psychology , biological psychology research , two upper - level electives depending upon the student s major and area(s ) of interest , and our capstone course , advanced neuroscience ( neur 400 ) . table 1 summarizes the neuroscience curriculum , identifying pre - requisite courses , courses required for breadth , and more advanced required and elective courses that offer depth in the concentration . over the last several years , capstone courses have become increasingly more visible on college and university campuses . indeed , the capstone experience is now fairly commonplace in smaller , private colleges , as well as in large public institutions ( badway and grubb , 1999 ) . capstone courses are believed to provide valuable experiences for students , including opportunities for synthesis and integration of information ( e.g. in our program , the capstone experience for all neuroscience students is advanced neuroscience ( neur 400 ) . this course is designed for juniors and seniors who have completed the majority ( if not all ) of the courses required for the concentration . typically , the course enrolls 1525 students , and is taught in the spring semester . from the inception of our neuroscience program , we envisioned neur 400 to be a course that brings together critical concepts and ideas from the students previous coursework in a format emphasizing discussion of primary literature and incorporating multiple learning activities and projects throughout the semester . unlike the introductory course in neuroscience , neur 400 addresses contemporary molar issues in neuroscience . in this way , students are exposed to cutting edge issues within a number of sub - fields of neuroscience by faculty whose primary interests reflect those issues and problems . one faculty member serves as the instructor of record ( this person receives teaching credit for the course ) and is responsible for the course organization and administration ( syllabus creation , coordination among participating faculty , development of assignments , and grading ) . in recent years , the course topics and readings have examined visual attention and computational neuroscience , autism , biochemistry of memory formation , explicit and implicit memory systems , amnesia , face recognition and cognitive neuroscience , artificial intelligence and face recognition , neuroscience of consciousness , neurophilosophy , nervous tissue differentiation and central nervous system development , glia and glioma , stress and neurodegeneration , affective neuroscience , educational neuroscience , and cultural neuroscience . several different assignments have been designed to help students enhance their ability to read and critique neuroscience research , develop an integrative understanding of neuroscience core areas , examine the intersections that connect neuroscience with other disciplinary areas in sciences , arts , and humanities , and explore the relevance of neuroscience to contemporary issues and personal applications . we present three of the activities below . the dissemination of neuroscience research to scientists , educators , professionals , and the general public has increased through the availability of internet sites . in particular , the range and complexity of neuroscience research introduces unique challenges for the presentation of informative and useful research summaries to an informed lay audience . thus , the goal of this activity is to provide students with an opportunity to read a primary research article in a neuroscience journal and then to prepare and deliver an oral presentation about that research article to classmates in a session that is known as neuroscience in the news . each student selects a research article from any field of neuroscience published in the past three years based upon his or her personal interests . then they prepare a 5-minute presentation that will model the format of a news release and that will be similar in content and style to the neuroscience news reports on the webpage of the british neuroscience association ( bna ; http://www.bna.org.uk/news/ ) . in the presentation , the students objectives are to explain why they selected the particular research article ; describe the most significant theoretical issues , methods , and results ; summarize the most meaningful conclusions including the importance or relevance of the research and/or its applications ; and identify how the research contributed to an enhancement of personal knowledge and interests in neuroscience . some of the recent neuroscience in the news presentations include does sleep deprivation put you in a better mood ? , exercise training increases the size of the hippocampus and improves your memory , videogames and cognitive training in the elderly , brain - machine - brain interfaces : a new way to connect to the world , brain imaging : visualizing the developing and maturing brain , and therapeutic potential of omega-3 pufas for peripheral nerve injuries . following each presentation many of our neuroscience students have educational and career goals involving the application of basic neuroscience research to medical , clinical , educational , or other professional settings . therefore , in this assignment , pairs of students work together to develop a brain briefing , a written document that explains how basic neuroscience research has relevance to a general audience of policy makers in clinical , health , educational , or other fields and professions ( e.g. , sports , law , economics , and robotics ) . the model for this assignment is the brain briefings newsletter published online by the society for neuroscience ( sfn ) ( lom , 2005 ) and more recently posted at the website , brainfacts.org ( http://www.brainfacts.org/ ) , a public information initiative of the kavli foundation , the gatsby charitable foundation , and the sfn . students first review several brain briefings on the website in order to get a grasp of the content , format , and style of these papers . the students brain briefing report is not meant to be a research abstract ; rather , their goal is to follow the format of the sfn brain briefings and provide information on recent neuroscience research that has exciting and valuable applications to neurological , psychological , and medical contexts . the evaluation of the brain briefing is based upon how well students identify and explain the connections between the research outcomes with the potential applications . examples of brain briefings produced in the most recent class include an eye toward the future : sight restoration through neuroengineering and visual prosthesis , neuromarketing , transcranial direct current stimulation : shocking new therapeutic possibilities , and the neural truth : religion as an anesthetic . recently , case studies have also been used effectively in neuroscience pedagogy ( meil , 2007 ) and are frequently included in textbooks and in popular books written by neuroscientists or neurologists ( e.g. this assignment is a major and culminating project for the course and the overall learning goal is to develop a deep understanding of the neurological condition that is presented in the case study through the evaluation and synthesis of contemporary neuroscience research . the 12-page written report for this project has three parts : 1 ) case description , 2 ) literature review , and 3 ) research proposal ( cf . in part one , students are asked to describe the most salient aspects of the case . why did you choose this case in order to deepen your study of neuroscience ? in part two , the literature review should include articles that help to answer questions such as what are the typical symptoms and what are the neurobiological or neuropsychological basis of the condition ? in the final section of the paper , students identify at least one unanswered question about the condition described in the case ( causes , symptoms , or treatment ) and then propose an experiment to address this question . the research proposal includes rationale , hypotheses , participants , materials and apparatus , procedures , and statistical analyses . finally , students are also asked to describe the potential significance of the proposed research for the field of neuroscience . in the presentation , students describe the very most salient aspects of the case in terms of the underlying neurological issue . they also explain their decision to choose the case and how the case is important for the study of neuroscience . students briefly present the key research goals , methods , results , and conclusions from only two of the research articles selected from the literature review . the students also explain why or how these two articles have relevance to an understanding of the case study . finally , each student describes how the case study project has enhanced their study of neuroscience and the neuroscience topics that have been examined in the course . the topics of the case studies chosen by students have included creutzfeld - jakob disease , capgras syndrome , post - traumatic stress disorder , autism , tourette s syndrome , amnesia , epilepsy , dissociative identity disorder , charles bonnet syndrome , and auditory and visual hallucinations . the dissemination of neuroscience research to scientists , educators , professionals , and the general public has increased through the availability of internet sites . in particular , the range and complexity of neuroscience research introduces unique challenges for the presentation of informative and useful research summaries to an informed lay audience . thus , the goal of this activity is to provide students with an opportunity to read a primary research article in a neuroscience journal and then to prepare and deliver an oral presentation about that research article to classmates in a session that is known as neuroscience in the news . each student selects a research article from any field of neuroscience published in the past three years based upon his or her personal interests . then they prepare a 5-minute presentation that will model the format of a news release and that will be similar in content and style to the neuroscience news reports on the webpage of the british neuroscience association ( bna ; http://www.bna.org.uk/news/ ) . in the presentation , the students objectives are to explain why they selected the particular research article ; describe the most significant theoretical issues , methods , and results ; summarize the most meaningful conclusions including the importance or relevance of the research and/or its applications ; and identify how the research contributed to an enhancement of personal knowledge and interests in neuroscience . it is stressed to the students that their neuroscience in the news presentation should be understandable to an informed lay audience as much as to neuroscientists . some of the recent neuroscience in the news presentations include does sleep deprivation put you in a better mood ? , exercise training increases the size of the hippocampus and improves your memory , videogames and cognitive training in the elderly , brain - machine - brain interfaces : a new way to connect to the world , brain imaging : visualizing the developing and maturing brain , and therapeutic potential of omega-3 pufas for peripheral nerve injuries . , sports , law , economics , and robotics ) . the model for this assignment is the brain briefings newsletter published online by the society for neuroscience ( sfn ) ( lom , 2005 ) and more recently posted at the website , brainfacts.org ( http://www.brainfacts.org/ ) , a public information initiative of the kavli foundation , the gatsby charitable foundation , and the sfn . students first review several brain briefings on the website in order to get a grasp of the content , format , and style of these papers . the students brain briefing report is not meant to be a research abstract ; rather , their goal is to follow the format of the sfn brain briefings and provide information on recent neuroscience research that has exciting and valuable applications to neurological , psychological , and medical contexts . examples of brain briefings produced in the most recent class include an eye toward the future : sight restoration through neuroengineering and visual prosthesis , alzheimer s dementia , sleep and your emotions , neuromarketing , transcranial direct current stimulation : shocking new therapeutic possibilities , and the neural truth : religion as an anesthetic . recently , case studies have also been used effectively in neuroscience pedagogy ( meil , 2007 ) and are frequently included in textbooks and in popular books written by neuroscientists or neurologists ( e.g. this assignment is a major and culminating project for the course and the overall learning goal is to develop a deep understanding of the neurological condition that is presented in the case study through the evaluation and synthesis of contemporary neuroscience research . in part one , students are asked to describe the most salient aspects of the case . in part two , the literature review should include articles that help to answer questions such as what are the typical symptoms and what are the neurobiological or neuropsychological basis of the condition ? in this section the students are asked to evaluate and synthesize the results of at least eight empirical articles and to discuss how the literature review is relevant to course topics and readings . in the final section of the paper , students identify at least one unanswered question about the condition described in the case ( causes , symptoms , or treatment ) and then propose an experiment to address this question . finally , students are also asked to describe the potential significance of the proposed research for the field of neuroscience . in the presentation , students describe the very most salient aspects of the case in terms of the underlying neurological issue . they also explain their decision to choose the case and how the case is important for the study of neuroscience . students briefly present the key research goals , methods , results , and conclusions from only two of the research articles selected from the literature review . the students also explain why or how these two articles have relevance to an understanding of the case study . finally , each student describes how the case study project has enhanced their study of neuroscience and the neuroscience topics that have been examined in the course . the topics of the case studies chosen by students have included creutzfeld - jakob disease , capgras syndrome , post - traumatic stress disorder , autism , tourette s syndrome , amnesia , epilepsy , dissociative identity disorder , charles bonnet syndrome , and auditory and visual hallucinations . students are strongly encouraged to provide written feedback and rating scale responses for the course as part of the end - of - semester course evaluation program in place at denison . recent efforts on our campus have resulted in increased student participation in the course evaluation process by carving out class time during the last week of classes for this purpose . students have evaluated the course quite favorably . in the past few offerings , the majority of students numeric ratings consisted of very good to excellent evaluations on items such as the course is challenging , their interest in neuroscience increased , their knowledge of the subject increased , and that faculty were clear , well - prepared , provided useful feedback , and were effective in their teaching . the depth of the material was something that i had not been exposed to previously ; this gave me an extreme increase in knowledge of the subject matter . we were particularly interested in student feedback regarding the team taught nature of the course , as this is something that the majority of undergraduate students have little or no experience with . generally , this was viewed as a positive aspect of the course . some students , however , found that having multiple instructors for the course was confusing and challenging , as noted by the following student comment , sometimes the switching of instructors can feel a little sporadic . in the future , we plan on administering an additional evaluation form in order to obtain information from specific open - ended questions about course content , organization , learning activities , and team - teaching . an integrative capstone course in neuroscience can provide a valuable culminating experience for students of the discipline ( wiertelak and ramirez , 2008 ) . we feel satisfied that our course challenges students to think about some of the larger contemporary issues and questions in neuroscience , to read and digest primary literature in the field , and to engage in multiple learning opportunities that enable integration of concepts and ideas acquired in previous courses . however , we do recognize that there are important challenges that our neuroscience faculty continue to discuss regarding ways to improve the course as our neuroscience program evolves . we feel that a successful team - taught capstone course in neuroscience , therefore , depends on faculty who are committed to an interdisciplinary neuroscience program , and who are willing to commit several hours out of their already busy schedules to the preparation that is necessary in order to meet with the class over two or three sessions with no monetary compensation or teaching credit provided . of course , when faculty are on leave or unable to participate for other reasons , the course content must be changed or modified in order to accommodate this change , or other faculty representing the same or some different area must be asked to participate in the course . also , it is our hope to have more faculty from other disciplines including the social sciences , humanities , and the arts participate in neur 400 in future semesters in order to highlight the importance of neuroscience in the interdisciplinary focus of a liberal arts college ( ramirez , 2007 ) . in addition to the importance of a core group of interested and willing faculty , the instructor of record also has a critical role in the success of such a course . the instructor also is responsible for developing all of the learning activities and assignments described in this paper . however , our neuroscience faculty have contributed ideas and support for these assignments which share a focus on integration and application of neuroscience research . these learning objectives address the goals of a capstone course within the structure of our neuroscience curriculum but we recognize that there are other learning goals and assignments that could be present in a capstone course . the course instructor also is solely responsible for grading all of the assignments , exams , and student participation . therefore , the instructor does have a challenge in ensuring that students will develop their integrative knowledge of neuroscience from the ongoing flow of topics , presenters , and learning activities that occur during the semester . student comments regarding course flow reinforce the importance of this role , and we continue to work on this in our discussions of our curriculum , particularly when the instructor of record changes between departments . another important consideration in planning for a neuroscience capstone course is that enrolled students should be near the end of their college career , preferably seniors or second - semester juniors . this is essential for the types of class discussions and thoughtful conversations that add to the success of such a course . the course as we design it assumes that students have mastered the basics of neuroscience and have completed adequate coursework that enables them to think critically , ask probing questions and offer meaningful comments in the classroom . because the instructor of record earns one teaching credit for designing and coordinating neur 400 , this may very well put some strain on the home department of the instructor in terms of course offerings , enrollments , etc . having one of his / her courses a neuroscience course means that one fewer course in the home department ( psychology , biology , for example ) can be taught during that semester . clearly , then , it is imperative that there be departmental support for the program , transparency in what the commitment to neur courses will be , how frequently they will be taught ( every year ? , and how many members of the department will be contributing regularly to the neuroscience courses and other activities associated with the concentration . on our campus , we had several departmental ( primarily within the psychology and biology departments ) discussions as the neuroscience program was in its early stages , and we continue to discuss particular aspects of the neur concentration and courses in departmental staff meetings as necessary ; in addition , neuroscience faculty meet regularly to plan future neur course offerings , discuss staffing issues , enrollments , and consider other important agenda items relevant to our neuroscience program . careful planning and communication with colleagues both within and across departments --- are key when creating an integrative and team - taught course for students of neuroscience .

although the efficacy and effectiveness , as well as the cost - effectiveness , of screening and brief alcohol interventions ( sbi ) have been shown to be strong , the implementation of sbi into routine care practice has not been satisfactory ; it seems to be difficult to motivate health care professionals to deliver sbi [ 16 ] . many implementation projects have tried to overcome known barriers such as lack of time , resources , training , and negative attitudes to working with sbi with limited success [ 4 , 5 , 79 ] . in a survey in the united kingdom comparing role security and therapeutic commitment between 1999 and 2009 among general practitioners ( gps ) , it was seen that the main issue was lack of therapeutic commitment . the study also highlighted that lack of time and resources rather than negative attitudes was related to the lack of therapeutic commitment to sbi . implementation of new methods has repeatedly been shown to need more than simple training sessions to be effective [ 4 , 10 ] . implementation research suggests that a multifaceted strategy addressing several barriers may be more effective than simple training sessions . in addition , implementation efforts involving more professionals than gps can improve how professionals work together towards increased sbi activities [ 4 , 10 , 11 ] . in a recent study from five european countries involving 120 primary care units , no evidence that sbi rates were influenced by role security or therapeutic commitment was found . other factors , not specifically studied , such as clinical priorities and management support might be more important for implementation . this study underlines a review by nilsen et al . , which concluded that motivation to engage in sbi should be viewed as a dynamic process encompassing the characteristics of the individual health professional , the patients , the clinical setting , and the wider context . thus , there is a knowledge gap on how to engage primary care staff in brief alcohol advice . how do we overcome the perceived lack of knowledge and reluctance to ask patients about their alcohol habits ? tentative answers might be found by giving office - based support material and management support rather than trying to change already positive attitudes among staff [ 4 , 10 ] . how staff 's performance changes over time if these preconditions are offered has not been sufficiently studied . most previous researches have measured role security and therapeutic commitment at one point in time or just after a training session using standardized questionnaires [ 5 , 10 , 12 ] . the few studies that have measured role security and therapeutic commitment before and after an educational session do not provide information about how a change in engagement is accomplished or mediated [ 8 , 14 ] . thus , there is a lack of qualitative studies following staff practices over time in implementation projects aiming to establish new routines . the aim of this study was to explore how the perceptions and experiences of working with risky drinkers change over time among primary health care staff during a systematic implementation project . the study was part of the swedish implementation study spira ( secondary prevention implementation research on alcohol ) that aimed to study the implementation process of different sbi methods at primary health care centres ( phcs ) in sweden . this study was approved by the regional ethical review board in gothenburg , sweden ( 405 - 10/2010 ) . the spira study was conducted during 20102012 in 16 phcs from three different regions in sweden . in brief , the spira study started with a baseline measurement of sbi rates over 2 days . after the baseline period , a 3-hour training session on how to perform sbi was given to all staff . it was then followed by the first implementation period of 4 weeks during which staff were asked to routinely offer sbi to their patients . six months after the first implementation period , a booster education session of 1 - 2 hours was given followed by a second 4-week sbi implementation period . a significant increase in sbi rates was seen during the implementation periods , results reported elsewhere . as part of the study , qualitative data were collected during a series of focus group interviews with staff at baseline and 6 months after the second implementation period . for some participating units , a total of 30 focus group interviews were included in this study ; 16 were conducted at baseline and 14 at follow - up . two of the phcs did not participate in the follow - up because they were recruited too late , which explains why there are fewer interviews for the follow - up . the phcs included in the spira study were located in three counties , selected to ensure representation from various parts of sweden and to include both rural and urban areas . all phcs in these counties were invited to participate in the study . because we did not reach the intended number of phcs with this procedure , we proceeded with snowball sampling using our research networks . we aimed to explore how the perceptions and experiences of working with risky drinkers change over time among primary health care staff during a systematic implementation project ; therefore some of the staff were allowed to refrain from participating in the implementation . all staff who had actively participated in the implementation process were invited to participate in the focus group interviews . the individuals who volunteered to participate were then given more information about the aim and content of the study . reasons for not participating included sick leave , vacation , terminated employment , lack of time , or other commitments at the phc . our aim was to have the exact same participating staff at all different measurement points but that was not possible because all staff were not available at the time of the interviews due to the reasons stated above . the focus groups consisted of a mix of professionals working at the phc and included physicians , registered nurses , nurse assistants , social workers , psychologists , physiotherapists , and others ( social manager , health educator , rehab coordinator , midwife , secretary , behaviourist , head of unit , dietician , secretary , and unknown ) ; the details are presented in tables 1 and 2 . two of the interviews were conducted as individual interviews because the work situation did not allow more than one person to participate at a time . the guide focused on themes including the staff 's experiences with working with risky drinkers and sbi . at the follow - up , themes regarding how their experiences with working with risky drinkers and sbi had changed during the implementation period were added . the interviews took place in a separate room at each of the phcs where the participants worked except for the two individual interviews , which were performed by phone . participation was voluntary and the participants were informed that they could abandon participation at any time . the participants were informed about the purpose of the interview and they were encouraged to discuss freely around the themes and to bring their perspectives into the open . the interviews lasted for 2330 minutes at baseline and 619 minutes at follow - up . the data were inductively analysed using content analysis , meaning that coding and categorization of data were done in a structured way , gradually deriving the categories from the data in an explorative and descriptive way [ 17 , 18 ] . the analysis was conducted in several steps with the aim of identifying the experiences of the staff regarding working with risky drinkers and especially how these changed during the implementation process . initially , the first author listened to all recordings and ensured that the transcripts were accurate . then all texts were read through several times by the first author to provide a sense of the whole . a qualitative analysis software program , nvivo 10 , was used to facilitate the analysis . the first step was performed using open coding by reading the texts line by line to identify meaning units , which were labelled with preliminary codes . the coded meaning units were then combined into preliminary categories based on similarity of content . this first analysis was mainly performed by the first author but continuously discussed with coauthor fredrik spak to prevent researcher bias and strengthen the internal validity . disagreements were discussed until consensus was reached . in the second analysis step , the purpose and the specific aim of this study were taken into deeper consideration and the analysis process continued with identification of meaning units responding to the aim . the meaning units were then labelled with codes and the codes were compared regarding similarities and differences and then categorized based on similarity of content to build categories . in the third analysis step , the categories were discussed and then sorted and abstracted into main categories and subcategories that captured the main content of the data with regard to the aim of the study . this deeper analysis of the content was mainly performed by the first author but continuously supervised and discussed with coauthor ulrika mssener . the coding and interpretative levels of the categories quotations were identified to illustrate the categorization and translated from swedish . in the results , / / in a quotation shows that text has been omitted or means a pause or a short silence . the analysis of the interviews before and after the implementation phases revealed two main categories : ( 1 ) awareness of shortcomings , reflecting thoughts before the implementation phases , and ( 2 ) change in practice as expressed by the participants after participating in the study ( table 3 ) . thus , the analysis displayed a pattern of change during the implementation period with regard to awareness , knowledge , and confidence that led to a change in practice . before the implementation , the participants reported a lack of resources and engagement in working with alcohol and sbi . before the implementation period , the participants reported a lack of engagement regarding working with alcohol - related questions . a majority of the participants expressed that they did not work as much with screening and brief interventions as they thought was needed and that they were motivated to do more sbi work but lacked the tools to do so . they also expressed that they lacked knowledge regarding both alcohol and risky drinking and how to advise risky drinkers . this was creating a lack of confidence and insecurity in asking about alcohol , not least because this was regarded as a sensitive issue . most of the participants seemed to have an awareness of their lack of appropriate engagement despite their positive attitudes.i am too bad at asking questions about alcohol actually . but i do n't do it.i believe health care has an obligation to ask questions about how life styles interfere with health . it should not be strange that health care brings it up ; it has to be natural . to not do it is almost misconduct.despite awareness of lack of engagement staff expressed a positive attitude towards implementing sbi . this seemed to be grounded in the belief that the staff actually could facilitate change in patients ' alcohol habits . it was also evident that the staff believed that alcohol problems could be the underlying cause of many of the symptoms that patients present at the phcs and that , through active work on alcohol , they might identify that cause and be able to help patients to a greater extent.but it is good in order to identify what is the actual problem with the patient , because it can be alcohol that lies behind a lot of what the patients seek care for . i believe health care has an obligation to ask questions about how life styles interfere with health . it should not be strange that health care brings it up ; it has to be natural . but it is good in order to identify what is the actual problem with the patient , because it can be alcohol that lies behind a lot of what the patients seek care for . there was a wish for alcohol work to be more visible for both patients and staff in order to facilitate the work . the participants thought that if there was advertising material around the phcs , both staff and patients would remember and embrace the alcohol work to a greater extent . the participants could identify several situations where more systematic alcohol work could be performed and which they regarded as underutilized at the moment . some participants also expressed motivation to learn more and work more with alcohol prevention and expressed that the implementation efforts that were planned were much needed and perceived that they would be very useful.i feel that this is very useful . lack of knowledge regarding several important issues for alcohol preventive work was expressed among the interviewees . almost all participants described that they were aware of their shortcomings in responding to risky drinkers and requested more training as planned in the implementation project.for some of us it would facilitate with more education in order to understand the limits for risky drinking.it would facilitate us a lot if we knew where to refer patients for additional help when we are insufficient / / and at the same time know what to do by yourself . how far should you go in trying to talk to people before you try to get help elsewhere?lack of knowledge on how to encounter risky drinkers was emphasized and it was revealed that this shortcoming had led to avoidance to ask about alcohol.the times i might have avoided asking is because i do n't know how to deal with the answer . i simply do n't have the knowledge.it can be quite hard if you ask and you get an answer . i think that is hard.the interviewees believed that the alcohol preventive work would be more efficient if they had more knowledge because patients tend to listen more if they perceive that the person is knowledgeable and confident in discussing the issue.i think that the patient understands if you have the right knowledge ; they listen more to you . you can explain and talk about it in such a way , with such a tone , that they will take it in a better way and understand . for some of us it would facilitate with more education in order to understand the limits for risky drinking . it would facilitate us a lot if we knew where to refer patients for additional help when we are insufficient / how far should you go in trying to talk to people before you try to get help elsewhere ? the times i might have avoided asking is because i do n't know how to deal with the answer . i think that the patient understands if you have the right knowledge ; they listen more to you . you can explain and talk about it in such a way , with such a tone , that they will take it in a better way and understand . before the implementation period , most of the participants felt insecure asking about alcohol and intervening with risky drinkers , and this insecurity meant that patients were not being asked about their alcohol habits . one of the factors that contributed to the feelings of insecurity seemed to be the fact that alcohol was regarded as a sensitive issue and that the staff were afraid of offending patients . some of the interviewees described how they sometimes came up with excuses to not ask about alcohol and that the alcohol questions were easy to forget.if you ask the question in the wrong manner , or in a way that the patients experience as offending // the relationship can be affected.but some staff had a different view and expressed that they were confident in asking about alcohol and had never felt that the patients were offended.that it is a sign of us caring . i rather think that more patients are dissatisfied because they are not asked.among those who expressed concern about offending patients , some solutions were proposed . they highlighted the importance of patients not feeling singled out and questions about alcohol should be brought up in a natural way or in a natural context . it was suggested that if more patients were asked about alcohol , the issue would become more natural and less sensitive to both the patient and the staff , and the patients would feel less singled out.it might be better accepted among the patients if they know that everyone is asked the question , so you they do n't feel so singled out and that it is something that we always do . if you ask the question in the wrong manner , or in a way that the patients experience as offending // the relationship can be affected . that it is a sign of us caring . it might be better accepted among the patients if they know that everyone is asked the question , so you they do n't feel so singled out and that it is something that we always do . after the implementation period , a majority of the participants expressed that their level of knowledge and their confidence in working with sbi had changed . the participants perceived that they asked more patients about their alcohol habits after the implementation of the project . it was also seen that the phcs had integrated sbi into routine practice and that the staff asked special patients group more systematically about alcohol . the participants appeared to have been given some of the tools to perform sbi work that they lacked at the baseline . they were also more aware about alcohol both at an individual level and at a phc level and discussed alcohol more among their colleagues . in general , the participants were positive about continuing to work with alcohol prevention . at the follow - up , many of the participants perceived that the sbi activities at their phcs had increased . they were convinced that , in general , they asked more patients about alcohol habits after the implementation and had integrated alcohol preventive measures into the daily routines to a greater extent . for example , at some phcs , the staff have started to systematically ask patients with certain diagnoses ; others screened and intervened for alcohol problems systematically at certain visits such as annual health check-ups.yes , of course you ask more frequently , more often , i do that.in fact , i never asked my patients this question before we entered this project , and now it is at every annually check-up.some participants stated that they had started with new tools and approaches , such as health questionnaires or health consultations that included questions about alcohol . increased systematic work was also evident from more notes about alcohol habits in the medical records.you can see a lot more notes in the medical records about the patients ' alcohol habits.not only did sbi activities increase during the implementation , but also the participants expressed that they experienced greater general awareness about alcohol and risky drinking at the phcs and discussed the issue to a greater extent among colleagues at the phcs after the implementation.we talk about alcohol more in general now . before nobody talked about it . yes , of course you ask more frequently , more often , i do that . in fact , i never asked my patients this question before we entered this project , and now it is at every annually check - up . you can see a lot more notes in the medical records about the patients ' alcohol habits . the greater awareness of alcohol and establishment of new routines seem to have been mediated by increased knowledge and skills about alcohol prevention . also , the participants knew more about their own limitations and when and where to refer patients if needed.one thing that is important to know is what to do with patients who have a risky dinking behaviour or misuse . i think we have talked a lot about that here at the phc so it feels like you know what to with the patient.they also experienced increased knowledge about alcohol and risky drinking and felt that they knew how to respond to risky drinkers to a greater extent.there has been the issue of not knowing what to answer when you get a question back . now you know a little bit more about where the patient can turn for help and what you can do . maybe you ca n't do so much more than say cut down your intake to half but you know what advice you can give . one thing that is important to know is what to do with patients who have a risky dinking behaviour or misuse . i think we have talked a lot about that here at the phc so it feels like you know what to with the patient . there has been the issue of not knowing what to answer when you get a question back . now you know a little bit more about where the patient can turn for help and what you can do . maybe you ca n't do so much more than say cut down your intake to half but you know what advice you can give . at the follow - up interviews , it was highlighted by most of the participants that they experienced more confidence in screening and intervening for risky drinking . they expressed that it had become easier to ask patients about their alcohol habits ; they dared to ask to a greater extent after the implementation and were more confident about intervening for risky drinking.yes , i believe it feels more secure to ask patients , to bring it up with the patients . sometimes you can feel that it is sensitive , snooping into their life when it comes to alcohol . yes , i believe it feels more secure to ask patients , to bring it up with the patients . sometimes you can feel that it is sensitive , snooping into their life when it comes to alcohol . after the implementation , the participants also expressed that they were more comfortable in persisting with the alcohol issue and that they did not drop the subject if they met resistance from the patient , which implies greater confidence in their own ability to intervene with risky drinkers.but i am not as afraid anymore to continue . before i could stop when i felt , oh , now i am in deep water . i do n't anymore , i feel that i can continue , can coax so to speak . go around and continue . because if i back off , i confirm for that patient that this is a sensitive issue ; if i continue , i can sometimes also explain that we always ask like this when it comes to alcohol because it can lead to pain in the body or bad sleeping habits or.the factors that seemed to contribute to this were that they did not regard alcohol as a sensitive issue as they did before and that they had more experience and knowledge as well as more tools . before i could stop when i felt , oh , now i am in deep water . i do n't anymore , i feel that i can continue , can coax so to speak . go around and continue . because if i back off , i confirm for that patient that this is a sensitive issue ; if i continue , i can sometimes also explain that we always ask like this when it comes to alcohol because it can lead to pain in the body or bad sleeping habits or . the fear of offending patients that was expressed at the baseline was not highlighted at all at the follow - up . many of the participants said that they had never experienced that patients were offended by being asked about alcohol . one reason that the participants felt contributed to alcohol becoming a less sensitive issue was that the patients felt less singled out when more patients were asked about their alcohol habits.and when someone looks a little hesitant or wandering , we say we ask everyone , both men and women , old and young so there is nothing odd about that . and we ask everyone , both men and women , old and young so there is nothing odd about that . another factor that seemed to contribute to the decreased insecurity in screening and intervening for risky drinking was increased experience . the participants agreed that the more they worked with these issues , the more confidence they got . they expressed that with more experience it was easier , more natural , and less inconvenient to ask about alcohol.it is easier . it gave me experience , practice makes perfect , and if you have done it a couple of times , it becomes more natural to do it . it gave me experience , practice makes perfect , and if you have done it a couple of times , it becomes more natural to do it . the aim of this study was to explore how the primary health care staff 's experiences of working with risky drinking changed during an implementation process that included two educational sessions and office - based material support . the project focused on inspiring staff to start offering sbi to patients and consequently getting more and more confident in applying sbi . at the baseline , the staff perceived that they did not work enough with sbi although they were motivated to do more . it appears that by participating in the focus group the staff were also given an additional chance to reflect upon their own attitudes and engagement , which might also have contributed to the results . at the follow - up 12 months later , a number of positive changes could be noted . now , most staff perceived that sbi activities had clearly increased and a number of barriers such as lack of knowledge and confidence in asking about alcohol had been overcome . from the interviews , it became clear that the project had inspired staff to try using the material provided and they had learned by practicing to a great extent . the lack of knowledge and confidence in bringing up the issue of alcohol was strongly emphasized at the baseline and mentioned as an important reason for not bringing up the issue . the staff also expressed insecurity in how to introduce the issue and how to intervene , as seen previously in most studies [ 5 , 6 , 12 , 14 ] . however , this is somewhat surprising since sweden has made a strong national effort over 5 years to educate large sections of primary health care in sbi from 2004 to 2010 at a total cost of 25 million euros . the project was a government initiative addressing primary , child , maternity , and occupational health care . a multifaceted approach with educational courses , workshops , and seminars was applied in order to encourage learning - by - doing . one important lesson learned from this project was the benefit of involving nurses in sbi in contrast to many other countries . it has been repeatedly suggested that nurses are an underutilized resource in alcohol prevention work [ 4 , 11 ] . since the phcs in this study were representative of phcs in sweden , in both rural and urban areas , the lack of knowledge and confidence in working with sbi could probably be generalized to large sections of swedish primary health care . so the effect of training postgraduate phc professionals appears to have had limited reach in sweden despite the great national effort . a better way forward might call for more systematic training in alcohol prevention work in medical schools and nursing schools . based on what happened over time in the group of health care professionals participating in the present study , we identified a number of important changes after training and active use of the office - based material during the implementation phases . the health providers in the study felt that both knowledge and confidence had increased at the follow - up and they stated that they had more confidence in bringing up the issue and giving a response and were able to go into the issue in greater depth . this indicates that the implementation was successful in increasing knowledge and confidence and that that effect lasted longer than the implementation period itself . involving staff other than gps may explain some of the positive outcomes in the present study . it was also obvious that some of the staff still regard alcohol as a sensitive issue and that this affected their lack of confidence in talking about alcohol and their reluctance to bring up the issue with patients . the fact that staff consider alcohol a sensitive and difficult issue has been shown in previous studies [ 2022 ] . this is a challenge that must be faced but as some of the caregivers stated , it might be easier for both staff and patients if more patients were asked routinely because it might decrease the risk of causing offense or feelings of being singled out . the fact that the alcohol issue is sensitive was not emphasized as much at the follow - up as at the baseline , indicating that more knowledge , experience , and confidence in talking about alcohol also reduces the sensitiveness of the issue . staff had started to establish new routines when to ask patients about their alcohol habits and this also decreased the sensitivity of the subject . although not measured , most staff stated that more patients were asked about alcohol at the follow - up , indicating success with the frequency of patients being asked , although this was not measured in more exact terms . however , staff expressed decreasing engagement over time ; sbi rates increased initially during the implementation phase but they decreased somewhat over time . we studied engagement about 6 months after the last educational sessions and do not know whether the sbi activity will continue to fade out . however , there will probably be a need for repeated booster educational sessions and continuous managerial support . variability in engagement among staff was noted in the interviews with early adopters and laggers who were more reluctant to adopt a new routine . some phc centres decided to systematically screen certain groups of patients and were thus starting to integrate sbi into the daily routines , creating a precondition for continuous engagement over time . another promising observation that might help establish new routines was increased awareness and discussion among the staff about the negative health consequences of alcohol . among the lessons learned from this study , it is obvious that the previous educational efforts extended to the primary care sector in sweden were not evident . this calls for more effort to integrate alcohol prevention training in medical and nursing schools . being part of an implementation project with education , new office - based material , and a commitment to try using the new knowledge and tools made them more confident and facilitated a change in practice . the effects of the study seemed to last for at least 6 months after the termination of the study . the positive changes in attitudes and engagement during the study could potentially secure continuous positive development . also , the decision to specify when to ask patients about their alcohol consumption decreased the sensitivity of asking the question and increased confidence . in summary , the study shows that staff gain knowledge and confidence in working with alcohol screening and brief intervention when participating in an implementation study with an educational approach . in this study , the effects of participation lasted up to 6 months after the termination of the study . this adds new knowledge to the science of implementation studies concerning alcohol prevention measures , which have otherwise shown disappointing results , emphasizing the importance of practicing and learning in daily meetings with patients / clients [ 4 , 5 , 79 ] . in this study a semistructured interview guide was developed and used in order to explore how the perceptions and experiences of working with risky drinkers change over time . we used mainly focus group interviews to gather data because they are an effective method to explore attitudes and needs of professionals and may encourage the participants to share and discuss views , attitudes , and experiences [ 1618 ] . in focus group interviews , there is a risk that participants do not feel free to discuss sensitive or personal experiences and perceptions , especially if they know the other participants , as was the case in this study . the focus group interviews facilitated a relaxed discussion and provided a satisfactory framework in that it helped direct the participants toward the issues focusing on them but still allowed the participates to express their answers in their own way . the focus groups included participants from different categories of staff , which may add to the results with more in - depth perceptions and experiences from professionals with different educational and professional backgrounds sharing their different perspectives [ 16 , 17 ] . we think it is was a benefit that different professionals were included but one must be aware that it may have affected the results because there is a hierarchy among these professionals , even though this was not perceived when listening to and analysing the interviews . the results predominantly showed that the implementation was successful and staff seem to have been learning - by - doing , gaining more confidence and security in offering sbi . but considering the fact that the phcs volunteered to participate , as did the staff at the phcs , this might have led to participants being more positive towards sbi work than occurs in general , thus making successful implementation more likely . as always when conducting qualitative research , voluntary participation may lead to the study sample differing from the broader population . one strength in this study was that the interviews revealed both challenges and shortcomings among the participants as well as positive standpoints , indicating that the staff felt they could speak freely . our data are based on a relatively small sample and a specific group of professionals , and the results can not be generalized to other groups . it is a limitation that the authors did not conduct the interviews themselves , that we did not include an observer , and that the moderator of the interviews was not included in the analysis process . this has been addressed by the first author listening to the recorded interviews several times . several steps have been taken to ensure the validity of the results . in the present analysis two of the authors the meaning units , codes , and categories were discussed by the authors in different combinations and at several points during the process . the coauthors were all senior researchers and well experienced in either qualitative research or the alcohol research field with focus on early identification and intervention with risky drinkers . some of the results are supported by previous results indicating an acceptable trustworthiness . staff at phcs in sweden are aware of the negative health consequences of alcohol and perceive that they seldom engage in alcohol screening and brief intervention . however , they appear highly motivated to work more actively , which implies that , with the right tools and incentives , a positive change can be achieved as shown in this study . staff perceived that lack of knowledge and the sensitivity of the topic contribute to low confidence in working with alcohol issues . participating in an implementation study where staff agree to perform sbi after a training session appears to have continued the learning - by - doing process . thus , 6 months after the termination of the project , positive attitudes and perceived engagement were prevailing . the more the patients or patient groups who were routinely offered sbi were , the less the staff perceived the alcohol issue to be sensitive , which increased their confidence in bringing up alcohol , leading to a sense of more knowledge , which also facilitated a change in practice .

objective . to explore how the perceptions and experiences of working with risky drinkers change over time among primary health care staff during a systematic implementation project . methods . qualitative focus group interviews took place before and after the implementation of the project . results . the staff displayed a positive change during the implementation period with regard to awareness , knowledge , and confidence that led to a change in routine practice . throughout the project , staff were committed to engaging with risky drinkers and appeared to have been learning - by - doing . conclusions . the results indicated a positive attitude to alcohol prevention work but staff lack knowledge and confidence in the area . the more practical experience during the study is , the more confidence seems to have been gained . this adds new knowledge to the science of implementation studies concerning alcohol prevention measures , which have otherwise shown disappointing results , emphasizing the importance of learning in practice .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions

although the efficacy and effectiveness , as well as the cost - effectiveness , of screening and brief alcohol interventions ( sbi ) have been shown to be strong , the implementation of sbi into routine care practice has not been satisfactory ; it seems to be difficult to motivate health care professionals to deliver sbi [ 16 ] . many implementation projects have tried to overcome known barriers such as lack of time , resources , training , and negative attitudes to working with sbi with limited success [ 4 , 5 , 79 ] . the study also highlighted that lack of time and resources rather than negative attitudes was related to the lack of therapeutic commitment to sbi . implementation of new methods has repeatedly been shown to need more than simple training sessions to be effective [ 4 , 10 ] . , which concluded that motivation to engage in sbi should be viewed as a dynamic process encompassing the characteristics of the individual health professional , the patients , the clinical setting , and the wider context . how do we overcome the perceived lack of knowledge and reluctance to ask patients about their alcohol habits ? the few studies that have measured role security and therapeutic commitment before and after an educational session do not provide information about how a change in engagement is accomplished or mediated [ 8 , 14 ] . the aim of this study was to explore how the perceptions and experiences of working with risky drinkers change over time among primary health care staff during a systematic implementation project . the study was part of the swedish implementation study spira ( secondary prevention implementation research on alcohol ) that aimed to study the implementation process of different sbi methods at primary health care centres ( phcs ) in sweden . in brief , the spira study started with a baseline measurement of sbi rates over 2 days . after the baseline period , a 3-hour training session on how to perform sbi was given to all staff . it was then followed by the first implementation period of 4 weeks during which staff were asked to routinely offer sbi to their patients . six months after the first implementation period , a booster education session of 1 - 2 hours was given followed by a second 4-week sbi implementation period . a significant increase in sbi rates was seen during the implementation periods , results reported elsewhere . as part of the study , qualitative data were collected during a series of focus group interviews with staff at baseline and 6 months after the second implementation period . for some participating units , a total of 30 focus group interviews were included in this study ; 16 were conducted at baseline and 14 at follow - up . two of the phcs did not participate in the follow - up because they were recruited too late , which explains why there are fewer interviews for the follow - up . all phcs in these counties were invited to participate in the study . we aimed to explore how the perceptions and experiences of working with risky drinkers change over time among primary health care staff during a systematic implementation project ; therefore some of the staff were allowed to refrain from participating in the implementation . all staff who had actively participated in the implementation process were invited to participate in the focus group interviews . the individuals who volunteered to participate were then given more information about the aim and content of the study . our aim was to have the exact same participating staff at all different measurement points but that was not possible because all staff were not available at the time of the interviews due to the reasons stated above . the guide focused on themes including the staff 's experiences with working with risky drinkers and sbi . at the follow - up , themes regarding how their experiences with working with risky drinkers and sbi had changed during the implementation period were added . the interviews took place in a separate room at each of the phcs where the participants worked except for the two individual interviews , which were performed by phone . the analysis was conducted in several steps with the aim of identifying the experiences of the staff regarding working with risky drinkers and especially how these changed during the implementation process . in the second analysis step , the purpose and the specific aim of this study were taken into deeper consideration and the analysis process continued with identification of meaning units responding to the aim . in the third analysis step , the categories were discussed and then sorted and abstracted into main categories and subcategories that captured the main content of the data with regard to the aim of the study . in the results , / / in a quotation shows that text has been omitted or means a pause or a short silence . the analysis of the interviews before and after the implementation phases revealed two main categories : ( 1 ) awareness of shortcomings , reflecting thoughts before the implementation phases , and ( 2 ) change in practice as expressed by the participants after participating in the study ( table 3 ) . thus , the analysis displayed a pattern of change during the implementation period with regard to awareness , knowledge , and confidence that led to a change in practice . before the implementation , the participants reported a lack of resources and engagement in working with alcohol and sbi . before the implementation period , the participants reported a lack of engagement regarding working with alcohol - related questions . a majority of the participants expressed that they did not work as much with screening and brief interventions as they thought was needed and that they were motivated to do more sbi work but lacked the tools to do so . most of the participants seemed to have an awareness of their lack of appropriate engagement despite their positive attitudes.i am too bad at asking questions about alcohol actually . it should not be strange that health care brings it up ; it has to be natural . to not do it is almost misconduct.despite awareness of lack of engagement staff expressed a positive attitude towards implementing sbi . this seemed to be grounded in the belief that the staff actually could facilitate change in patients ' alcohol habits . it was also evident that the staff believed that alcohol problems could be the underlying cause of many of the symptoms that patients present at the phcs and that , through active work on alcohol , they might identify that cause and be able to help patients to a greater extent.but it is good in order to identify what is the actual problem with the patient , because it can be alcohol that lies behind a lot of what the patients seek care for . some participants also expressed motivation to learn more and work more with alcohol prevention and expressed that the implementation efforts that were planned were much needed and perceived that they would be very useful.i feel that this is very useful . almost all participants described that they were aware of their shortcomings in responding to risky drinkers and requested more training as planned in the implementation project.for some of us it would facilitate with more education in order to understand the limits for risky drinking.it would facilitate us a lot if we knew where to refer patients for additional help when we are insufficient / / and at the same time know what to do by yourself . how far should you go in trying to talk to people before you try to get help elsewhere?lack of knowledge on how to encounter risky drinkers was emphasized and it was revealed that this shortcoming had led to avoidance to ask about alcohol.the times i might have avoided asking is because i do n't know how to deal with the answer . before the implementation period , most of the participants felt insecure asking about alcohol and intervening with risky drinkers , and this insecurity meant that patients were not being asked about their alcohol habits . one of the factors that contributed to the feelings of insecurity seemed to be the fact that alcohol was regarded as a sensitive issue and that the staff were afraid of offending patients . some of the interviewees described how they sometimes came up with excuses to not ask about alcohol and that the alcohol questions were easy to forget.if you ask the question in the wrong manner , or in a way that the patients experience as offending // the relationship can be affected.but some staff had a different view and expressed that they were confident in asking about alcohol and had never felt that the patients were offended.that it is a sign of us caring . they highlighted the importance of patients not feeling singled out and questions about alcohol should be brought up in a natural way or in a natural context . it was suggested that if more patients were asked about alcohol , the issue would become more natural and less sensitive to both the patient and the staff , and the patients would feel less singled out.it might be better accepted among the patients if they know that everyone is asked the question , so you they do n't feel so singled out and that it is something that we always do . after the implementation period , a majority of the participants expressed that their level of knowledge and their confidence in working with sbi had changed . the participants perceived that they asked more patients about their alcohol habits after the implementation of the project . it was also seen that the phcs had integrated sbi into routine practice and that the staff asked special patients group more systematically about alcohol . the participants appeared to have been given some of the tools to perform sbi work that they lacked at the baseline . in general , the participants were positive about continuing to work with alcohol prevention . they were convinced that , in general , they asked more patients about alcohol habits after the implementation and had integrated alcohol preventive measures into the daily routines to a greater extent . for example , at some phcs , the staff have started to systematically ask patients with certain diagnoses ; others screened and intervened for alcohol problems systematically at certain visits such as annual health check-ups.yes , of course you ask more frequently , more often , i do that.in fact , i never asked my patients this question before we entered this project , and now it is at every annually check-up.some participants stated that they had started with new tools and approaches , such as health questionnaires or health consultations that included questions about alcohol . increased systematic work was also evident from more notes about alcohol habits in the medical records.you can see a lot more notes in the medical records about the patients ' alcohol habits.not only did sbi activities increase during the implementation , but also the participants expressed that they experienced greater general awareness about alcohol and risky drinking at the phcs and discussed the issue to a greater extent among colleagues at the phcs after the implementation.we talk about alcohol more in general now . in fact , i never asked my patients this question before we entered this project , and now it is at every annually check - up . the greater awareness of alcohol and establishment of new routines seem to have been mediated by increased knowledge and skills about alcohol prevention . also , the participants knew more about their own limitations and when and where to refer patients if needed.one thing that is important to know is what to do with patients who have a risky dinking behaviour or misuse . i think we have talked a lot about that here at the phc so it feels like you know what to with the patient.they also experienced increased knowledge about alcohol and risky drinking and felt that they knew how to respond to risky drinkers to a greater extent.there has been the issue of not knowing what to answer when you get a question back . at the follow - up interviews , it was highlighted by most of the participants that they experienced more confidence in screening and intervening for risky drinking . they expressed that it had become easier to ask patients about their alcohol habits ; they dared to ask to a greater extent after the implementation and were more confident about intervening for risky drinking.yes , i believe it feels more secure to ask patients , to bring it up with the patients . after the implementation , the participants also expressed that they were more comfortable in persisting with the alcohol issue and that they did not drop the subject if they met resistance from the patient , which implies greater confidence in their own ability to intervene with risky drinkers.but i am not as afraid anymore to continue . because if i back off , i confirm for that patient that this is a sensitive issue ; if i continue , i can sometimes also explain that we always ask like this when it comes to alcohol because it can lead to pain in the body or bad sleeping habits or.the factors that seemed to contribute to this were that they did not regard alcohol as a sensitive issue as they did before and that they had more experience and knowledge as well as more tools . because if i back off , i confirm for that patient that this is a sensitive issue ; if i continue , i can sometimes also explain that we always ask like this when it comes to alcohol because it can lead to pain in the body or bad sleeping habits or . one reason that the participants felt contributed to alcohol becoming a less sensitive issue was that the patients felt less singled out when more patients were asked about their alcohol habits.and when someone looks a little hesitant or wandering , we say we ask everyone , both men and women , old and young so there is nothing odd about that . the participants agreed that the more they worked with these issues , the more confidence they got . the aim of this study was to explore how the primary health care staff 's experiences of working with risky drinking changed during an implementation process that included two educational sessions and office - based material support . the project focused on inspiring staff to start offering sbi to patients and consequently getting more and more confident in applying sbi . at the baseline , the staff perceived that they did not work enough with sbi although they were motivated to do more . it appears that by participating in the focus group the staff were also given an additional chance to reflect upon their own attitudes and engagement , which might also have contributed to the results . now , most staff perceived that sbi activities had clearly increased and a number of barriers such as lack of knowledge and confidence in asking about alcohol had been overcome . from the interviews , it became clear that the project had inspired staff to try using the material provided and they had learned by practicing to a great extent . the lack of knowledge and confidence in bringing up the issue of alcohol was strongly emphasized at the baseline and mentioned as an important reason for not bringing up the issue . however , this is somewhat surprising since sweden has made a strong national effort over 5 years to educate large sections of primary health care in sbi from 2004 to 2010 at a total cost of 25 million euros . the project was a government initiative addressing primary , child , maternity , and occupational health care . a multifaceted approach with educational courses , workshops , and seminars was applied in order to encourage learning - by - doing . it has been repeatedly suggested that nurses are an underutilized resource in alcohol prevention work [ 4 , 11 ] . since the phcs in this study were representative of phcs in sweden , in both rural and urban areas , the lack of knowledge and confidence in working with sbi could probably be generalized to large sections of swedish primary health care . a better way forward might call for more systematic training in alcohol prevention work in medical schools and nursing schools . based on what happened over time in the group of health care professionals participating in the present study , we identified a number of important changes after training and active use of the office - based material during the implementation phases . the health providers in the study felt that both knowledge and confidence had increased at the follow - up and they stated that they had more confidence in bringing up the issue and giving a response and were able to go into the issue in greater depth . this indicates that the implementation was successful in increasing knowledge and confidence and that that effect lasted longer than the implementation period itself . involving staff other than gps may explain some of the positive outcomes in the present study . it was also obvious that some of the staff still regard alcohol as a sensitive issue and that this affected their lack of confidence in talking about alcohol and their reluctance to bring up the issue with patients . this is a challenge that must be faced but as some of the caregivers stated , it might be easier for both staff and patients if more patients were asked routinely because it might decrease the risk of causing offense or feelings of being singled out . the fact that the alcohol issue is sensitive was not emphasized as much at the follow - up as at the baseline , indicating that more knowledge , experience , and confidence in talking about alcohol also reduces the sensitiveness of the issue . however , staff expressed decreasing engagement over time ; sbi rates increased initially during the implementation phase but they decreased somewhat over time . we studied engagement about 6 months after the last educational sessions and do not know whether the sbi activity will continue to fade out . being part of an implementation project with education , new office - based material , and a commitment to try using the new knowledge and tools made them more confident and facilitated a change in practice . the effects of the study seemed to last for at least 6 months after the termination of the study . the positive changes in attitudes and engagement during the study could potentially secure continuous positive development . in summary , the study shows that staff gain knowledge and confidence in working with alcohol screening and brief intervention when participating in an implementation study with an educational approach . in this study , the effects of participation lasted up to 6 months after the termination of the study . this adds new knowledge to the science of implementation studies concerning alcohol prevention measures , which have otherwise shown disappointing results , emphasizing the importance of practicing and learning in daily meetings with patients / clients [ 4 , 5 , 79 ] . in this study a semistructured interview guide was developed and used in order to explore how the perceptions and experiences of working with risky drinkers change over time . we used mainly focus group interviews to gather data because they are an effective method to explore attitudes and needs of professionals and may encourage the participants to share and discuss views , attitudes , and experiences [ 1618 ] . in focus group interviews , there is a risk that participants do not feel free to discuss sensitive or personal experiences and perceptions , especially if they know the other participants , as was the case in this study . the focus group interviews facilitated a relaxed discussion and provided a satisfactory framework in that it helped direct the participants toward the issues focusing on them but still allowed the participates to express their answers in their own way . the focus groups included participants from different categories of staff , which may add to the results with more in - depth perceptions and experiences from professionals with different educational and professional backgrounds sharing their different perspectives [ 16 , 17 ] . the results predominantly showed that the implementation was successful and staff seem to have been learning - by - doing , gaining more confidence and security in offering sbi . but considering the fact that the phcs volunteered to participate , as did the staff at the phcs , this might have led to participants being more positive towards sbi work than occurs in general , thus making successful implementation more likely . as always when conducting qualitative research , voluntary participation may lead to the study sample differing from the broader population . our data are based on a relatively small sample and a specific group of professionals , and the results can not be generalized to other groups . it is a limitation that the authors did not conduct the interviews themselves , that we did not include an observer , and that the moderator of the interviews was not included in the analysis process . several steps have been taken to ensure the validity of the results . in the present analysis two of the authors the meaning units , codes , and categories were discussed by the authors in different combinations and at several points during the process . the coauthors were all senior researchers and well experienced in either qualitative research or the alcohol research field with focus on early identification and intervention with risky drinkers . some of the results are supported by previous results indicating an acceptable trustworthiness . staff at phcs in sweden are aware of the negative health consequences of alcohol and perceive that they seldom engage in alcohol screening and brief intervention . however , they appear highly motivated to work more actively , which implies that , with the right tools and incentives , a positive change can be achieved as shown in this study . staff perceived that lack of knowledge and the sensitivity of the topic contribute to low confidence in working with alcohol issues . participating in an implementation study where staff agree to perform sbi after a training session appears to have continued the learning - by - doing process . thus , 6 months after the termination of the project , positive attitudes and perceived engagement were prevailing . the more the patients or patient groups who were routinely offered sbi were , the less the staff perceived the alcohol issue to be sensitive , which increased their confidence in bringing up alcohol , leading to a sense of more knowledge , which also facilitated a change in practice .

under various experimental conditions the onset of atrial fibrillation ( af ) has been attributed to aberrant electrical impulses originating in the area of the left atrium ( la ) providing a left to right atrial ( ra ) gradient for the initiation and perpetuation of reentry circuits . in turn these multiple circuits constitute the electrophysiological substrate for the disorganized and chaotic rhythm in the atrium known as af . animal models have been developed to study this phenomenon . a seminal report in goats generated a new understanding of an underlying mechanism for af and coined the phrase atrial fibrillation begets atrial fibrillation . after several weeks of pacing induced af it was noted that longer and longer episodes were occurring without pacing until af became continuous . electrophysiological remodeling , in the form of a consistent shortening of atrial refractoriness provided the substrate for triggers to initiate af . perpetuation of af has been attributed to the establishment of multiple reentry circuits which became the hallmark of the of the multiple wavelet theory of af . surgical procedures to treat patients with af by physically interrupting the reentrant circuits are now performed in patients worldwide based on the multiple wavelet concept for af . it is important to note that the investigators of these initial experimental studies also found structural remodeling four weeks after the onset of af in the form of changes such as increased myocyte size , myolysis and marked glycogen accumulation within the atrial myocytes . it is not easy to understand how certain changes in cellular structure , such as glycogen accumulation can play a role in the perpetuation of af . the present study describes the differences in glycogen content and distribution between the right atrial appendage ( raa ) and left atrial appendage ( laa ) of five normal goats . we hypothesized that glycogen content and distribution found in the laa would induce an favorable substrate for impulse conduction and thus explain the more frequent af origin in this chamber . ethical statement : goat tissue samples were obtained at a local federally inspected slaughter house . permission to harvest these tissues was granted by a public health veterinarian , on site , representing the united states department of agriculture ( usda ) . the tissue samples were separately preserved in 10% buffered formalin ( n = 3 ) and in 95% ethanol n = 2 ) . fixed tissue samples were routinely processed , paraffin - embedded and sectioned at 4 m . following sectioning , the tissues were stained separately with either hematoxylin and eosin ( h and e ) or periodic acid - schiff ( pas ) stain performed with or without diastase digestion for demonstration of glycogen . digital images of the pas - stained sections were acquired by olympus dp25 digital photomicroscopy . five random , color photomicrographs were obtained from all regions of the myocardium ( subendocardial , subepicardial and intervening myocardium ) . the digital images were transferred to nih image j and the total area of the image calculated ( m ) by setting the scale to scale bar acquired with the image . the color threshold was set to identify only glycogen ( magenta stain from pas ) , which was converted to a black and white image and then set to binary for measurement . the binary image was regularly compared to the original to confirm scope and intensity of glycogen staining . glycogen was reported as area covered ( m ) by staining in the binary image , which could then be compared to the total area of the image . a standard two tailed t - test was used to compare equal numbers of the 20 sections , taken from four goats , of the laa and raa samples . fixed tissue samples were routinely processed , paraffin - embedded and sectioned at 4 m . following sectioning , the tissues were stained separately with either hematoxylin and eosin ( h and e ) or periodic acid - schiff ( pas ) stain performed with or without diastase digestion for demonstration of glycogen . digital images of the pas - stained sections were acquired by olympus dp25 digital photomicroscopy . five random , color photomicrographs were obtained from all regions of the myocardium ( subendocardial , subepicardial and intervening myocardium ) . the digital images were transferred to nih image j and the total area of the image calculated ( m ) by setting the scale to scale bar acquired with the image . the color threshold was set to identify only glycogen ( magenta stain from pas ) , which was converted to a black and white image and then set to binary for measurement . the binary image was regularly compared to the original to confirm scope and intensity of glycogen staining . glycogen was reported as area covered ( m ) by staining in the binary image , which could then be compared to the total area of the image . a standard two tailed t - test was used to compare equal numbers of the 20 sections , taken from four goats , of the laa and raa samples . glycogen could be demonstrated in all laas and raas ; however , there was marked variability in the intensity and distribution of glycogen deposition from animal to animal . glycogen distribution was similar in both formalin - fixed and ethanol - fixed tissue samples . the formalin fixed tissue samples provided for more crisp images for photography and morphometric analysis ( because of superiority of formalin for fixation ) . glycogen deposition in both raas and laas was most consistently located in the subepicardial and subendocardial myocardium [ figures 1 and 2 respectively ] . glycogen was also present in the mid - myocardium ; however , this was most conspicuous in laas ( only ) and in goats with the highest levels of myocardial glycogen . although the glycogen content was markedly variable from animal to animal , the laas consistently had higher glycogen levels based upon pas staining . heart , goat . in the raas glycogen deposition delicately highlighted the intercalated discs ( arrows ) . also glycogen deposition was seen along and just beneath the sarcolemmal membrane ( solid arrowhead ) . some cells showed little periodic - acid schiff ( pas ) stain without diastase treatment ( open arrowhead ) . in contrast to the raas , in the laas glycogen often concentrated against intercalated discs ( arrows ) with dense tails of glycogen extending into the cell along the lateral wall at the myocyte - myocyte junction ( solid arrowhead ) . bar = 50 m at the cellular level , glycogen in the raas occasionally highlighted the intercalated discs [ arrows , figures 1 and 2 ] and was distributed along and just beneath the sarcolemmal membrane [ solid arrowhead , figures 1 and 2 ] . this was not observed at other myocyte to myocyte connections [ figure 1 , open arrowhead ] . in the laas , the glycogen often concentrated against the intercalated disc [ arrows , figure 2 ] , particularly in regions of heavy glycogen accumulation . furthermore , not only was the glycogen found just beneath the sarcoplasmic membrane , but extended into and occupied the sarcoplasm [ figure 2 , arrowhead ] . in heavily stained regions , morphometric analysis [ figure 3 ] shows that pas stained regions of the laas averaged 2.6 3 m compared to raas , 0.8 1.3 m , p = 0.02 . laas glycogen levels ( per m ) always exceeded raas levels in each individual animal . it should be noted that besides the three fold greater prevalence of the mean glycogen concentration in the laa compared to the raa , the larger standard deviation in the laa indicates the greater heterogeneity of glycogen in the laa . glycogen was quantitated based upon area covered by pas - stained glycogen as a percent of the area of the total image . the pas - stained area of laas ( 2.6 3.0 m ) was significantly greater than raas ( 0.8 1.3m ) , p = 0.02 histological sections made from tissues in normal goats showed a differential distribution and concentration of glycogen in left versus right atrial tissues . the concentration of glycogen in the laa was not only greater than in the raa but the density and location of the glycogen was critically distinct [ figures 1 and 2 ] . we suggest that the potential arrhythmogenic aspect of these glycogen differences are a potential contributory mechanism for the initiation and maintenance for af and in particular the greater propensity for the development of an af substrate in the left than in the right atrium . the present study demonstrates that in the normal caprine heart there are intrinsic quantitative differences in glycogen concentrations between the laa and raa . both sides showed subepicardial presence of glycogen [ figures 4 and 5 ] that is notably displayed in a greater concentration in the laa . the significantly greater glycogen in the laa is heterogeneously found as high - density depositions against the intercalated discs and extending into the myocytes . also condensed glycogen was observed at the sided to side junction of adjacent myocytes . in the raa granules of glycogen although there were individual myocytes in which the glycogen granules outlined the intercalated disc and side to side myocyte junctions , many other cells did not show this pattern [ figure 1 , open arrow head ] . raas myocardium showing pas staining ( never as conspicuous as the laa ) bar 1 mm . please note that there is subepicardial glycogen concentration laas myocardium also exhibited consistent regions of subepicardial and subendocardial pas - positive glycogen staining ( arrowheads ) however , the intensity of raas staining was never as conspicuous or as widely distributed as laas glycogen particularly within the mid - myocardium ( arrows ) . bar = 1 mm of interest , one of the striking characteristic structural changes noted in the studies of the allessie group in the goat and also observed by others clinically was the accumulation of glycogen in the atrial myocytes associated with myolysis and a loss of contractile elements . it has been suggested that as a results of reduced contractility during af , lowered oxygen supply - demand ratio switches the energy metabolism of atrial cardiomyocytes from the use of fatty acids to the use of glucose leading to the accumulation of glycogen . however , this would not explain the presence and differential concentrations of glycogen in normal atria as shown in the present study . early on , studies showed that anisotropic conduction , that is , differential longitudinal versus lateral conduction velocity of atrial myocytes predisposed to microreentry in old compared to young atrial bundles . more recently the contribution of the intrinsic autonomic nervous system innervating the atria appears to play a critical role in the initiation and persistence of af . we propose that the differential qualitative and quantitative distribution of glycogen in the normal goat heart demonstrated in the present study provides another intrinsic factor in the susceptibility of the atria for af . in the present study , there was a dense accumulation of glycogen concentrating at the intercalated discs and extending into the myocytes along the side to side connections between myocytes . as noted in [ figures 1 and 2 ] , this effect was notably observed in the laa albeit in a heterogeneous manner . we propose that the large glycogen molecules provide an impediment to electrical conduction both longitudinally and laterally . these heterogeneously distributed islands of impaired conduction create a non - uniform wavefront of activation with areas of slow conduction predisposing for reentry circuit development . for example , muir subjected stands of purkinje cells to centrifugation and found that the centrifugal movement of the pas - positive material , glycogen ] appears to be arrested by transverse partitions , which correspond to the intercellular junctions . in uncentrifuged strands there is considerable variation in the concentration of stainable glycogen on the two sides of the intercellular junctions more recently , embi et al . , confirmed , in vivo , in atrial myocytes , this impermeability to be the result of the selectivity of the gap junction pore to allow particles of a given molecular size to pass from cell to cell . the observations of muir can then be explained as follows : glycogen particles have been reported to be 24.8 1.8 nm in diameter , whereas the atrial myocyte gap junction pore has a reported radius of 0.6 - 1.5 nm . as the intracellular glycogen level increases during af , it could cause fractionated intercellular communication , thus disrupting the stability of the atrial syncytium . dhillon et al . , studied gap junction resistivity and measured conduction velocity in guinea pig atria and found significant differences between the left and right atria . they found that the left atrial gap junction resistivity was significantly higher and conduction velocity significantly lower compared to the right atrium . moreover , addition of a gap junction uncoupling agent slowed atrial conduction more in the left than in the right atrial appendage . in another report from tai et al . , in a model of af caused by ventricular pacing induced heart failure , they compared activation of the left and right atria in control and heart failure dogs . they designated 3 different forms of activation : type 1 , a single broad wavefront ; type 2 , a non - uniform wavefront associated with conduction block and/or slow conduction or the presence of two wavelets ; type 3 , the presence of 3 or more wavelets associated with areas of slow conduction and multiple arcs of conduction block . in the right atrium only 2 of the 5 control dogs had type 2 activation whereas in the left atrium , all five control dogs had type 2 activation . furthermore , the left atrium had a greater dispersion of refractoriness ( a biomarker for af susceptibility ) than the right atrium . other evidence supporting glycogen as a contributing factor promoting af relates to the finding that angiotensin ii receptor blockers ( arbs ) have had significant improvement in reducing the metabolic parameters such as glucose levels in large human clinical trials . the drug irbesartan is such a blocker and has been known to reduce glucose levels amongst other effects such as attenuating atrial stunning after cardioversion , and most important for our hypothesis it has been used as a stand alone or adjunctive therapy in controlling af . arbs have also been used as intensive therapy to treat patients with glycogen storage disease . it is interesting to note that other areas of the normal heart have appreciable amount of glycogen . indeed , the purkinje system is known to contain a high glycogen content yet conduction velocity in purkinje fibers is on the order of 2 - 4 times greater than in the atrium . here again , it is not the concentration but the localization of the glycogen in relation to the myocytes that appears to determine conduction velocity and the activation wavefront . in an electron microscopic study of the purkinje system , legato clearly showed that the strands of purkinje cell were separated by pools of glycogen . this arrangement would favor rapid longitudinal conduction through unimpeded intercalated discs without loss by side to side cellular connections . in fact , this arrangement is associated with low internal resistance in these purkinje strands . please note that the evidence obtained in our study and supported findings were restricted to normal atrial tissues taken from goat hearts . the translation of our hypothesis , developed in the normal goat heart to conditions of established af , is moot since we do not know the role of glycogen in perpetuating af . however , many studies in different species of normal animals have shown the inducibility for atrial fibrillation and the greater propensity of the left rather than the right atrium to initiate and maintain af . indeed in humans ( with valvular disease and af ) , the presence of myocytes with the total intercellular space filled with glycogen has been documented . histological sections made from tissues in normal goats showed a differential distribution and concentration of glycogen in left versus right atrial tissues . the concentration of glycogen in the laa was not only greater than in the raa but the density and location of the glycogen was critically distinct [ figures 1 and 2 ] . we suggest that the potential arrhythmogenic aspect of these glycogen differences are a potential contributory mechanism for the initiation and maintenance for af and in particular the greater propensity for the development of an af substrate in the left than in the right atrium . the present study demonstrates that in the normal caprine heart there are intrinsic quantitative differences in glycogen concentrations between the laa and raa . both sides showed subepicardial presence of glycogen [ figures 4 and 5 ] that is notably displayed in a greater concentration in the laa . the significantly greater glycogen in the laa is heterogeneously found as high - density depositions against the intercalated discs and extending into the myocytes . also condensed glycogen was observed at the sided to side junction of adjacent myocytes . in the raa granules of glycogen although there were individual myocytes in which the glycogen granules outlined the intercalated disc and side to side myocyte junctions , many other cells did not show this pattern [ figure 1 , open arrow head ] . raas myocardium showing pas staining ( never as conspicuous as the laa ) bar 1 mm . please note that there is subepicardial glycogen concentration laas myocardium also exhibited consistent regions of subepicardial and subendocardial pas - positive glycogen staining ( arrowheads ) however , the intensity of raas staining was never as conspicuous or as widely distributed as laas glycogen particularly within the mid - myocardium ( arrows ) . bar = 1 mm of interest , one of the striking characteristic structural changes noted in the studies of the allessie group in the goat and also observed by others clinically was the accumulation of glycogen in the atrial myocytes associated with myolysis and a loss of contractile elements . it has been suggested that as a results of reduced contractility during af , lowered oxygen supply - demand ratio switches the energy metabolism of atrial cardiomyocytes from the use of fatty acids to the use of glucose leading to the accumulation of glycogen . however , this would not explain the presence and differential concentrations of glycogen in normal atria as shown in the present study . early on , studies showed that anisotropic conduction , that is , differential longitudinal versus lateral conduction velocity of atrial myocytes predisposed to microreentry in old compared to young atrial bundles . more recently the contribution of the intrinsic autonomic nervous system innervating the atria appears to play a critical role in the initiation and persistence of af . we propose that the differential qualitative and quantitative distribution of glycogen in the normal goat heart demonstrated in the present study provides another intrinsic factor in the susceptibility of the atria for af . in the present study , there was a dense accumulation of glycogen concentrating at the intercalated discs and extending into the myocytes along the side to side connections between myocytes . as noted in [ figures 1 and 2 ] , this effect was notably observed in the laa albeit in a heterogeneous manner . we propose that the large glycogen molecules provide an impediment to electrical conduction both longitudinally and laterally . these heterogeneously distributed islands of impaired conduction create a non - uniform wavefront of activation with areas of slow conduction predisposing for reentry circuit development . the centrifugal movement of the pas - positive material , glycogen ] appears to be arrested by transverse partitions , which correspond to the intercellular junctions . in uncentrifuged strands there is considerable variation in the concentration of stainable glycogen on the two sides of the intercellular junctions more recently , embi et al . , confirmed , in vivo , in atrial myocytes , this impermeability to be the result of the selectivity of the gap junction pore to allow particles of a given molecular size to pass from cell to cell . the observations of muir can then be explained as follows : glycogen particles have been reported to be 24.8 1.8 nm in diameter , whereas the atrial myocyte gap junction pore has a reported radius of 0.6 - 1.5 nm . as the intracellular glycogen level increases during af , it could cause fractionated intercellular communication , thus disrupting the stability of the atrial syncytium . dhillon et al . , studied gap junction resistivity and measured conduction velocity in guinea pig atria and found significant differences between the left and right atria . they found that the left atrial gap junction resistivity was significantly higher and conduction velocity significantly lower compared to the right atrium . moreover , addition of a gap junction uncoupling agent slowed atrial conduction more in the left than in the right atrial appendage . in another report from tai et al . , in a model of af caused by ventricular pacing induced heart failure , they compared activation of the left and right atria in control and heart failure dogs . they designated 3 different forms of activation : type 1 , a single broad wavefront ; type 2 , a non - uniform wavefront associated with conduction block and/or slow conduction or the presence of two wavelets ; type 3 , the presence of 3 or more wavelets associated with areas of slow conduction and multiple arcs of conduction block . in the right atrium only 2 of the 5 control dogs had type 2 activation whereas in the left atrium , all five control dogs had type 2 activation . furthermore , the left atrium had a greater dispersion of refractoriness ( a biomarker for af susceptibility ) than the right atrium . other evidence supporting glycogen as a contributing factor promoting af relates to the finding that angiotensin ii receptor blockers ( arbs ) have had significant improvement in reducing the metabolic parameters such as glucose levels in large human clinical trials . the drug irbesartan is such a blocker and has been known to reduce glucose levels amongst other effects such as attenuating atrial stunning after cardioversion , and most important for our hypothesis it has been used as a stand alone or adjunctive therapy in controlling af . arbs have also been used as intensive therapy to treat patients with glycogen storage disease . it is interesting to note that other areas of the normal heart have appreciable amount of glycogen . indeed , the purkinje system is known to contain a high glycogen content yet conduction velocity in purkinje fibers is on the order of 2 - 4 times greater than in the atrium . here again , it is not the concentration but the localization of the glycogen in relation to the myocytes that appears to determine conduction velocity and the activation wavefront . in an electron microscopic study of the purkinje system , legato clearly showed that the strands of purkinje cell were separated by pools of glycogen . this arrangement would favor rapid longitudinal conduction through unimpeded intercalated discs without loss by side to side cellular connections . in fact , this arrangement is associated with low internal resistance in these purkinje strands . please note that the evidence obtained in our study and supported findings were restricted to normal atrial tissues taken from goat hearts . the translation of our hypothesis , developed in the normal goat heart to conditions of established af , is moot since we do not know the role of glycogen in perpetuating af . however , many studies in different species of normal animals have shown the inducibility for atrial fibrillation and the greater propensity of the left rather than the right atrium to initiate and maintain af . indeed in humans ( with valvular disease and af ) , the presence of myocytes with the total intercellular space filled with glycogen has been documented . the present study demonstrates that glycogen is heterogeneously distributed in both atria in the normal goat heart . glycogen granules were observed scattered throughout the raa myocytes and at the intercalated discs as well as the side to side connections between some but not all myocytes . in contrast , in the laa dense glycogen deposits were coalesced against the intercalated discs and side to side connections between myocytes with tailing into the cell interior . for the first time , evidence is presented suggesting that this results in an impediment of cell to cell conduction leading to a non - uniform wavefront of activation . in conjunction with areas of slowed conduction , these conditions predispose the la , when vulnerable , that is , in response to premature beats , for reentrant based af .

background : previous experimental studies have demonstrated electrophysiological and structural remodeling in pacing induced atrial fibrillation . the latter has been characterized by glycogen accumulation but no connection to atrial fibrillation induction and maintenance has as yet been proposed.aims:we determined the presence of glycogen in the right and left atrial appendages in the goat heart , in order to find any intrinsic disparity in distribution and concentration between these sites.materials and methods : atrial appendages from 5 goats were stained by the periodic acid schiffmethod to determine the presence of glycogen and the concentration of glycogen by morphometric analysis.results:we are reporting for the first time that the right atrial appendage showed scattered glycogen granules throughout the atrial myocytes which delineated the intercalated discs ; whereas glycogen in the left atrial appendage was more dense within cells and coalesced against the intercalated discs and side to side junctions between myocytes . also , morphometric analysis determined that the stained regions of the right atrial appendages averaged , 0.8 1.3 m2 compared to the left atrial appendage sections , 2.6 3 m2 , p = 0.02 . we show that glycogen is heterogeneously distributed in both atria in the normal goat heart ; however , the density of glycogen deposits concentrating against the intercalated discs and side to side connections in the left atrial appendage is a critically distinct difference . impediment of cell to cell conduction could result in a non - uniform wavefront of activation , with areas of slowed conduction , predisposing the left atrium to reentrant based atrial fibrillation.conclusion:these findings provide a basis for the well - known greater propensity for atrial fibrillation in the left versus the right atrium .

Introduction Materials and Methods Histochemical procedures Morphometric analysis Statistical analysis Results Discussion Major findings of the present study Intrinsic factors predisposing the normal atria for atrial fibrillation Glycogen/atrial fibrillation hypothesis Evidence supporting glycogen as a contributing factor promoting AF Myocardial sites with intrinsic glycogen concentrations Limitations Conclusions

under various experimental conditions the onset of atrial fibrillation ( af ) has been attributed to aberrant electrical impulses originating in the area of the left atrium ( la ) providing a left to right atrial ( ra ) gradient for the initiation and perpetuation of reentry circuits . in turn these multiple circuits constitute the electrophysiological substrate for the disorganized and chaotic rhythm in the atrium known as af . electrophysiological remodeling , in the form of a consistent shortening of atrial refractoriness provided the substrate for triggers to initiate af . perpetuation of af has been attributed to the establishment of multiple reentry circuits which became the hallmark of the of the multiple wavelet theory of af . it is important to note that the investigators of these initial experimental studies also found structural remodeling four weeks after the onset of af in the form of changes such as increased myocyte size , myolysis and marked glycogen accumulation within the atrial myocytes . it is not easy to understand how certain changes in cellular structure , such as glycogen accumulation can play a role in the perpetuation of af . the present study describes the differences in glycogen content and distribution between the right atrial appendage ( raa ) and left atrial appendage ( laa ) of five normal goats . we hypothesized that glycogen content and distribution found in the laa would induce an favorable substrate for impulse conduction and thus explain the more frequent af origin in this chamber . following sectioning , the tissues were stained separately with either hematoxylin and eosin ( h and e ) or periodic acid - schiff ( pas ) stain performed with or without diastase digestion for demonstration of glycogen . five random , color photomicrographs were obtained from all regions of the myocardium ( subendocardial , subepicardial and intervening myocardium ) . the digital images were transferred to nih image j and the total area of the image calculated ( m ) by setting the scale to scale bar acquired with the image . the binary image was regularly compared to the original to confirm scope and intensity of glycogen staining . glycogen was reported as area covered ( m ) by staining in the binary image , which could then be compared to the total area of the image . a standard two tailed t - test was used to compare equal numbers of the 20 sections , taken from four goats , of the laa and raa samples . following sectioning , the tissues were stained separately with either hematoxylin and eosin ( h and e ) or periodic acid - schiff ( pas ) stain performed with or without diastase digestion for demonstration of glycogen . five random , color photomicrographs were obtained from all regions of the myocardium ( subendocardial , subepicardial and intervening myocardium ) . the digital images were transferred to nih image j and the total area of the image calculated ( m ) by setting the scale to scale bar acquired with the image . the binary image was regularly compared to the original to confirm scope and intensity of glycogen staining . glycogen was reported as area covered ( m ) by staining in the binary image , which could then be compared to the total area of the image . a standard two tailed t - test was used to compare equal numbers of the 20 sections , taken from four goats , of the laa and raa samples . glycogen could be demonstrated in all laas and raas ; however , there was marked variability in the intensity and distribution of glycogen deposition from animal to animal . glycogen deposition in both raas and laas was most consistently located in the subepicardial and subendocardial myocardium [ figures 1 and 2 respectively ] . glycogen was also present in the mid - myocardium ; however , this was most conspicuous in laas ( only ) and in goats with the highest levels of myocardial glycogen . in the raas glycogen deposition delicately highlighted the intercalated discs ( arrows ) . in contrast to the raas , in the laas glycogen often concentrated against intercalated discs ( arrows ) with dense tails of glycogen extending into the cell along the lateral wall at the myocyte - myocyte junction ( solid arrowhead ) . bar = 50 m at the cellular level , glycogen in the raas occasionally highlighted the intercalated discs [ arrows , figures 1 and 2 ] and was distributed along and just beneath the sarcolemmal membrane [ solid arrowhead , figures 1 and 2 ] . in the laas , the glycogen often concentrated against the intercalated disc [ arrows , figure 2 ] , particularly in regions of heavy glycogen accumulation . in heavily stained regions , morphometric analysis [ figure 3 ] shows that pas stained regions of the laas averaged 2.6 3 m compared to raas , 0.8 1.3 m , p = 0.02 . it should be noted that besides the three fold greater prevalence of the mean glycogen concentration in the laa compared to the raa , the larger standard deviation in the laa indicates the greater heterogeneity of glycogen in the laa . the pas - stained area of laas ( 2.6 3.0 m ) was significantly greater than raas ( 0.8 1.3m ) , p = 0.02 histological sections made from tissues in normal goats showed a differential distribution and concentration of glycogen in left versus right atrial tissues . the concentration of glycogen in the laa was not only greater than in the raa but the density and location of the glycogen was critically distinct [ figures 1 and 2 ] . we suggest that the potential arrhythmogenic aspect of these glycogen differences are a potential contributory mechanism for the initiation and maintenance for af and in particular the greater propensity for the development of an af substrate in the left than in the right atrium . the present study demonstrates that in the normal caprine heart there are intrinsic quantitative differences in glycogen concentrations between the laa and raa . both sides showed subepicardial presence of glycogen [ figures 4 and 5 ] that is notably displayed in a greater concentration in the laa . the significantly greater glycogen in the laa is heterogeneously found as high - density depositions against the intercalated discs and extending into the myocytes . in the raa granules of glycogen although there were individual myocytes in which the glycogen granules outlined the intercalated disc and side to side myocyte junctions , many other cells did not show this pattern [ figure 1 , open arrow head ] . please note that there is subepicardial glycogen concentration laas myocardium also exhibited consistent regions of subepicardial and subendocardial pas - positive glycogen staining ( arrowheads ) however , the intensity of raas staining was never as conspicuous or as widely distributed as laas glycogen particularly within the mid - myocardium ( arrows ) . bar = 1 mm of interest , one of the striking characteristic structural changes noted in the studies of the allessie group in the goat and also observed by others clinically was the accumulation of glycogen in the atrial myocytes associated with myolysis and a loss of contractile elements . it has been suggested that as a results of reduced contractility during af , lowered oxygen supply - demand ratio switches the energy metabolism of atrial cardiomyocytes from the use of fatty acids to the use of glucose leading to the accumulation of glycogen . however , this would not explain the presence and differential concentrations of glycogen in normal atria as shown in the present study . early on , studies showed that anisotropic conduction , that is , differential longitudinal versus lateral conduction velocity of atrial myocytes predisposed to microreentry in old compared to young atrial bundles . more recently the contribution of the intrinsic autonomic nervous system innervating the atria appears to play a critical role in the initiation and persistence of af . we propose that the differential qualitative and quantitative distribution of glycogen in the normal goat heart demonstrated in the present study provides another intrinsic factor in the susceptibility of the atria for af . in the present study , there was a dense accumulation of glycogen concentrating at the intercalated discs and extending into the myocytes along the side to side connections between myocytes . as noted in [ figures 1 and 2 ] , this effect was notably observed in the laa albeit in a heterogeneous manner . these heterogeneously distributed islands of impaired conduction create a non - uniform wavefront of activation with areas of slow conduction predisposing for reentry circuit development . for example , muir subjected stands of purkinje cells to centrifugation and found that the centrifugal movement of the pas - positive material , glycogen ] appears to be arrested by transverse partitions , which correspond to the intercellular junctions . in uncentrifuged strands there is considerable variation in the concentration of stainable glycogen on the two sides of the intercellular junctions more recently , embi et al . , confirmed , in vivo , in atrial myocytes , this impermeability to be the result of the selectivity of the gap junction pore to allow particles of a given molecular size to pass from cell to cell . the observations of muir can then be explained as follows : glycogen particles have been reported to be 24.8 1.8 nm in diameter , whereas the atrial myocyte gap junction pore has a reported radius of 0.6 - 1.5 nm . as the intracellular glycogen level increases during af , it could cause fractionated intercellular communication , thus disrupting the stability of the atrial syncytium . they found that the left atrial gap junction resistivity was significantly higher and conduction velocity significantly lower compared to the right atrium . moreover , addition of a gap junction uncoupling agent slowed atrial conduction more in the left than in the right atrial appendage . , in a model of af caused by ventricular pacing induced heart failure , they compared activation of the left and right atria in control and heart failure dogs . they designated 3 different forms of activation : type 1 , a single broad wavefront ; type 2 , a non - uniform wavefront associated with conduction block and/or slow conduction or the presence of two wavelets ; type 3 , the presence of 3 or more wavelets associated with areas of slow conduction and multiple arcs of conduction block . in the right atrium only 2 of the 5 control dogs had type 2 activation whereas in the left atrium , all five control dogs had type 2 activation . furthermore , the left atrium had a greater dispersion of refractoriness ( a biomarker for af susceptibility ) than the right atrium . it is interesting to note that other areas of the normal heart have appreciable amount of glycogen . indeed , the purkinje system is known to contain a high glycogen content yet conduction velocity in purkinje fibers is on the order of 2 - 4 times greater than in the atrium . here again , it is not the concentration but the localization of the glycogen in relation to the myocytes that appears to determine conduction velocity and the activation wavefront . in an electron microscopic study of the purkinje system , legato clearly showed that the strands of purkinje cell were separated by pools of glycogen . this arrangement would favor rapid longitudinal conduction through unimpeded intercalated discs without loss by side to side cellular connections . the translation of our hypothesis , developed in the normal goat heart to conditions of established af , is moot since we do not know the role of glycogen in perpetuating af . however , many studies in different species of normal animals have shown the inducibility for atrial fibrillation and the greater propensity of the left rather than the right atrium to initiate and maintain af . indeed in humans ( with valvular disease and af ) , the presence of myocytes with the total intercellular space filled with glycogen has been documented . histological sections made from tissues in normal goats showed a differential distribution and concentration of glycogen in left versus right atrial tissues . the concentration of glycogen in the laa was not only greater than in the raa but the density and location of the glycogen was critically distinct [ figures 1 and 2 ] . we suggest that the potential arrhythmogenic aspect of these glycogen differences are a potential contributory mechanism for the initiation and maintenance for af and in particular the greater propensity for the development of an af substrate in the left than in the right atrium . the present study demonstrates that in the normal caprine heart there are intrinsic quantitative differences in glycogen concentrations between the laa and raa . both sides showed subepicardial presence of glycogen [ figures 4 and 5 ] that is notably displayed in a greater concentration in the laa . the significantly greater glycogen in the laa is heterogeneously found as high - density depositions against the intercalated discs and extending into the myocytes . in the raa granules of glycogen although there were individual myocytes in which the glycogen granules outlined the intercalated disc and side to side myocyte junctions , many other cells did not show this pattern [ figure 1 , open arrow head ] . please note that there is subepicardial glycogen concentration laas myocardium also exhibited consistent regions of subepicardial and subendocardial pas - positive glycogen staining ( arrowheads ) however , the intensity of raas staining was never as conspicuous or as widely distributed as laas glycogen particularly within the mid - myocardium ( arrows ) . bar = 1 mm of interest , one of the striking characteristic structural changes noted in the studies of the allessie group in the goat and also observed by others clinically was the accumulation of glycogen in the atrial myocytes associated with myolysis and a loss of contractile elements . it has been suggested that as a results of reduced contractility during af , lowered oxygen supply - demand ratio switches the energy metabolism of atrial cardiomyocytes from the use of fatty acids to the use of glucose leading to the accumulation of glycogen . however , this would not explain the presence and differential concentrations of glycogen in normal atria as shown in the present study . early on , studies showed that anisotropic conduction , that is , differential longitudinal versus lateral conduction velocity of atrial myocytes predisposed to microreentry in old compared to young atrial bundles . more recently the contribution of the intrinsic autonomic nervous system innervating the atria appears to play a critical role in the initiation and persistence of af . we propose that the differential qualitative and quantitative distribution of glycogen in the normal goat heart demonstrated in the present study provides another intrinsic factor in the susceptibility of the atria for af . in the present study , there was a dense accumulation of glycogen concentrating at the intercalated discs and extending into the myocytes along the side to side connections between myocytes . as noted in [ figures 1 and 2 ] , this effect was notably observed in the laa albeit in a heterogeneous manner . these heterogeneously distributed islands of impaired conduction create a non - uniform wavefront of activation with areas of slow conduction predisposing for reentry circuit development . the centrifugal movement of the pas - positive material , glycogen ] appears to be arrested by transverse partitions , which correspond to the intercellular junctions . in uncentrifuged strands there is considerable variation in the concentration of stainable glycogen on the two sides of the intercellular junctions more recently , embi et al . , confirmed , in vivo , in atrial myocytes , this impermeability to be the result of the selectivity of the gap junction pore to allow particles of a given molecular size to pass from cell to cell . the observations of muir can then be explained as follows : glycogen particles have been reported to be 24.8 1.8 nm in diameter , whereas the atrial myocyte gap junction pore has a reported radius of 0.6 - 1.5 nm . as the intracellular glycogen level increases during af , it could cause fractionated intercellular communication , thus disrupting the stability of the atrial syncytium . , studied gap junction resistivity and measured conduction velocity in guinea pig atria and found significant differences between the left and right atria . they found that the left atrial gap junction resistivity was significantly higher and conduction velocity significantly lower compared to the right atrium . moreover , addition of a gap junction uncoupling agent slowed atrial conduction more in the left than in the right atrial appendage . , in a model of af caused by ventricular pacing induced heart failure , they compared activation of the left and right atria in control and heart failure dogs . they designated 3 different forms of activation : type 1 , a single broad wavefront ; type 2 , a non - uniform wavefront associated with conduction block and/or slow conduction or the presence of two wavelets ; type 3 , the presence of 3 or more wavelets associated with areas of slow conduction and multiple arcs of conduction block . in the right atrium only 2 of the 5 control dogs had type 2 activation whereas in the left atrium , all five control dogs had type 2 activation . furthermore , the left atrium had a greater dispersion of refractoriness ( a biomarker for af susceptibility ) than the right atrium . it is interesting to note that other areas of the normal heart have appreciable amount of glycogen . indeed , the purkinje system is known to contain a high glycogen content yet conduction velocity in purkinje fibers is on the order of 2 - 4 times greater than in the atrium . here again , it is not the concentration but the localization of the glycogen in relation to the myocytes that appears to determine conduction velocity and the activation wavefront . in an electron microscopic study of the purkinje system , legato clearly showed that the strands of purkinje cell were separated by pools of glycogen . this arrangement would favor rapid longitudinal conduction through unimpeded intercalated discs without loss by side to side cellular connections . the translation of our hypothesis , developed in the normal goat heart to conditions of established af , is moot since we do not know the role of glycogen in perpetuating af . however , many studies in different species of normal animals have shown the inducibility for atrial fibrillation and the greater propensity of the left rather than the right atrium to initiate and maintain af . indeed in humans ( with valvular disease and af ) , the presence of myocytes with the total intercellular space filled with glycogen has been documented . the present study demonstrates that glycogen is heterogeneously distributed in both atria in the normal goat heart . glycogen granules were observed scattered throughout the raa myocytes and at the intercalated discs as well as the side to side connections between some but not all myocytes . in contrast , in the laa dense glycogen deposits were coalesced against the intercalated discs and side to side connections between myocytes with tailing into the cell interior . for the first time , evidence is presented suggesting that this results in an impediment of cell to cell conduction leading to a non - uniform wavefront of activation . in conjunction with areas of slowed conduction , these conditions predispose the la , when vulnerable , that is , in response to premature beats , for reentrant based af .

, stroke patients can usually recover to some extent , which may be related to structural and functional modifications in surviving brain tissue . studies on stroke rats and patients have revealed that spontaneous recovery of the motor function after stroke is associated with brain plasticity [ 15 ] . neuroimaging techniques , especially the multimodality mris , have significantly contributed to our understanding of the mechanisms of stroke recovery by characterizing brain structural and functional changes after stroke [ 610 ] . electroencephalogram ( eeg ) has high temporal resolution ; however , it exhibits low spatial resolution and can not record signals from deep brain tissues . positron emission tomography ( pet ) is a radioactive technique with a relatively low spatial resolution . however , mri is a noninvasive technique with high spatial resolution and is especially suitable for longitudinal connectivity studies . moreover , mri is a multimodality imaging technique that can be used to investigate both anatomical and functional connectivities . structural mri studies have revealed extensive atrophy in brain regions that connect with stroke lesions . more importantly , increased grey matter volume and cortical thickness were also found in the motor - related areas and hippocampus either during spontaneous recovery or after treatments [ 1113 ] . task - based functional mri ( fmri ) or pet has been extensively used to investigate brain activation changes during stroke recovery [ 1416 ] and has provided important information on the patterns of functional reorganization after stroke [ 1419 ] . movements of the stroke - affected hand are initially activated extensive brain regions in stroke patients [ 16 , 2022 ] and rats [ 6 , 2330 ] . however , this initial widespread activation does not predict good functional recovery [ 3133 ] , whereas normalization of the activation pattern to prestroke level is associated with good outcome . knowing brain structural or functional alteration in a particular region does not tell us how this region interacts with other regions , which modulates behavior in concert . recently , connectivity - based methods have been used in stroke patients and rats to demonstrate anatomical and functional connectivity changes after stroke ; these changes may be related to clinical deficits and functional recovery . these methods may provide great insight into network dysfunction [ 21 , 3639 ] and functional reorganization [ 40 , 41 ] from a system perspective ; they will also contribute to predict outcomes and to develop new therapeutic interventions [ 40 , 41 ] . in this review , we focused on recent neuroimaging studies employing connectivity - based analyses to investigate connectivity changes in the motor network after ischemic stroke . connectivity models are based on the concept that the brain is organized by segregation of specialized and anatomically distinct brain regions which are functionally integrated in a network mediating motor , sensory , or cognitive processing . there are four common connectivity models : the anatomical connectivity , functional connectivity , effective connectivity , and network models . diffusion tensor imaging ( dti ) is the most common method that can detect changes in anatomical connectivity in vivo . in normal white matter , water molecules move relatively freely in a direction parallel to fiber tracts in contrast to the restricted movement across the tracts ; this phenomenon is referred to as diffusion anisotropy . based on diffusion anisotropy , the white matter fiber tracts can be reconstructed using diffusion tensor tractography ( dtt ) [ 44 , 45 ] . fractional anisotropy ( fa ) and mean diffusivity ( md ) are the most commonly used dti measures ; md reflects the average diffusion amplitude , and fa represents the degree of directionality of microstructures , such as axons , myelin , and microtubules [ 44 , 46 ] . dti measures can be analyzed by either data - driven methods ( voxel - based analysis and tract - based spatial statistics ) or hypothesis - driven methods ( region of interest ( roi ) analysis and tractography - based analysis ) . dti and dtt have been used in the evaluation of the white matter damage and reorganization in stroke patients [ 4751 ] . in different poststroke stages , alterations of dti measures represent different pathological processes : md decreases at acute stage represent cell swelling ; md increases and fa decreases at subacute stage denote cell lysis and demyelination ; and fa increases at chronic stage suggests axonal regeneration or remyelination [ 5255 ] . it is well recognized that diffusion - weighted imaging ( dwi ) is capable of yielding considerably more information than that contained in the diffusion metrics derived from dti due to the fact that dti is based upon a gaussian approximation of the diffusion displacement probability function . however , non - gaussian diffusion in the brain really exists [ 56 , 57 ] and is believed to arise from diffusion restricted by barriers , such as cell membranes and organelles , as well as the presence of distinct water compartments [ 58 , 59 ] . diffusional kurtosis imaging ( dki ) has been proposed to quantify non - gaussian diffusion through the estimation of the diffusional kurtosis [ 6062 ] . the mean kurtosis ( mk ) , a principal metric of the diffusional non - gaussianity , is of potential interest to the study of white and gray matter integrity . dki can provide additional information ( non - gaussian diffusion ) that can not be obtained from dti , and it has been suggested that dki may provide enhanced contrast between ischaemic and normal tissues . besides , mk is sensitive to hyperacute and acute stroke changes and exhibits more significant differences between ischaemic and normal tissues than measures ( md and fa ) derived from dti . thus , diffusional kurtosis is sensitive to diffusional heterogeneity and dki may be a promising method in the assessment of ischemic stroke [ 61 , 63 , 64 ] . the resting - state functional connectivity ( rsfc ) measures temporal coherence of low - frequency fluctuations ( < 0.1 hz ) of the blood oxygenation level - dependent ( bold ) signals between spatially remote brain regions . correlation of these signals with underlying neural activity indicates that these fluctuations are of functional significance [ 6567 ] . strong rsfc is first reported in motor network and then is confirmed in other functional systems [ 69 , 70 ] . the rsfc is present between brain regions with both monosynaptic and polysynaptic connections and depends on intact connections within a specific polysynaptic pathway . it is a promising means of assessing intrinsic information transfer within a functional network while avoiding task - induced confounds . a hypothesis - driven method has been extensively used to analyze rsfc changes . in this method , an roi should be predefined based on hypotheses , and then the rsfc pattern of this roi is obtained by computing correlations in the fmri time courses between the roi and every voxel of the whole brain . the independent component analysis ( ica ) is the most popular data - driven method to assess rsfc [ 74 , 75 ] . it has been used to extract brain functional networks and to identify intranetwork rsfc changes . the functional connectivity density ( fcd ) mapping is another data - driven method and measures the number of functional connections per voxel [ 77 , 78 ] . these rsfc analytic methods are suited for the investigation of how multiple distributed networks are disrupted by stroke and are reorganized after stroke and what patterns of connectivity are most likely to be behaviorally relevant [ 40 , 41 ] . in contrast to the nondirectional , correlative nature of functional connectivity , effective connectivity measures the causal influence that one brain area exerts over another under the assumption of a given mechanistic model . this approach can provide crucial knowledge about the direction of information flow and ultimately what kinds of computations are being performed in the system by showing which nodes in a network are driven by which other nodes [ 35 , 40 , 41 , 79 ] . effective connectivity is based on mathematical models that are usually applied to task - based neuroimaging data . the psychophysiological interactions ( ppi ) model is a relatively simple method to estimate effective connectivity from neuroimaging data . this exploratory method explains activity of a cortical area by means of an interaction term between the influence of another area and some experimental or psychological parameter [ 80 , 81 ] . granger causality analysis of effective connectivity is another exploratory method that identifies those voxels that are sources or targets of directed influence for any seed region . in contrast to these exploratory methods , structural equation modelling ( sem ) and dynamic causal modeling ( dcm ) are hypothesis - driven approaches that require a priori defined network of brain regions to estimate effective connectivity from neuroimaging data . in addition , the effective connectivity analysis can also be used to resting - state fmri data . in the framework of graph theory , the brain is described as a graph comprising a certain number of nodes ( corresponding to brain regions ) that are connected by edges ( corresponding to anatomical connectivity , functional connectivity , effective connectivity , or other measures of interregional interactions ) [ 8588 ] . the network 's structure can be assessed by measuring its clustering coefficient , a measure of segregation , that reflects the degree to which nodes are clustered , and the shortest path length , a measure of integration , that reflects the minimal number of edges between any pair of nodes . a high clustering coefficient and a low average shortest path length indicate a small - world network topology , which is proposed to be an optimal network configuration for global information transfer and local processing [ 85 , 86 , 88 ] . in addition to measures that assess the efficiency of the whole network , the node degree ( i.e. , the number of edges connected to a given node ) and the betweenness centrality ( i.e. , the fraction of the shortest paths that pass through a given node ) are used to assess the importance of a given node . brain regions featuring a high node degree and a high centrality are assumed to serve as brain reorganization after stroke is a dynamic process , which considerably differs across patients , depending on lesion location , time since stroke , severity of motor impairment , premorbid state , and even genetics . these contributing factors make it unlikely that one universal measure exists which is suitable for all patients . consequently , a variety of connectivity analyses have been performed to investigate motor recovery mechanisms after ischemic stroke . dti and dtt are powerful methods to detect not only impairments but also modifications in anatomical connectivity after stroke [ 89 , 90 ] . the motor recovery mechanisms in ischemic stroke patients have been summarized as ( 1 ) recovery of a damaged lateral corticospinal tract ( cst ) ; ( 2 ) subcortical perilesional reorganization ; ( 3 ) ipsilateral motor pathway from the unaffected motor cortex to the affected extremities ; and ( 4 ) collateral pathway of the pyramidal tract . . investigated longitudinal cst white matter connectivity changes during stroke recovery using probabilistic dtt in 10 patients with cerebral infarcts . they found that the anatomical connectivity of the cst at the cortical regions within the affected hemisphere was enhanced over time and that the enhanced connectivity was correlated with stroke recovery . examined the relationship between microstructural status of brain white matter tracts and motor skill of the stroke - affected hand in patients with chronic stroke . they found that motor skill significantly and positively correlated with fa values of the ipsilesional and contralesional cst in these patients . they also found that patients with better motor skill had elevated fa of the bilateral csts compared to controls . however , our previous study of dynamic evolution of the diffusion indices in the degenerated cst did not show any significant plastic changes after ischemic stroke although a slightly elevated fa was found 1 year after stroke . the discrepancy between these studies may be ascribed to methodological differences because we only studied the cst section at the level of pons , which may miss the portion of the cst that exhibits plastic changes . at subacute stage of stroke , decreased fa in ipsilesional white matter has been commonly reported , which reflects demyelination or axonal loss [ 8 , 47 , 94 ] . this initial decrease of fa may be chronically followed by normalization or elevation in the borderzone of the ischemic lesion , which could be further enhanced by treatments with neural progenitor cells , sildenafil , or erythropoietin . pathological examination confirmed a high density of axons and myelin in this region [ 9 , 95 ] . the subcortical perilesional reorganization is also reported at the levels of the corona radiata and pons in stroke patients [ 97101 ] . these findings suggest that rearrangement of white matter in the ischemic perilesional areas is accompanied by preservation or restoration of neuronal connectivity and may be used to predict motor outcome after stroke . the ipsilateral motor pathway from the unaffected motor cortex to the affected extremities has been regarded as one of the recovery mechanisms of stroke [ 102106 ] . however , this mechanism is not supported by other studies [ 107 , 108 ] . the location and size of the lesions , the different recovery rates , and the analytic methods used in these studies may partly account for the discrepancy . as reviewed by jang , understanding the ipsilateral motor pathway mechanism is important because it is related to poor motor outcome and can be changed with time or manipulated by various rehabilitative interventions [ 102 , 108113 ] . the pyramidal tract is known to possess collateral pathways in the human brain [ 114116 ] . the aberrant pyramidal tract refers to the collateral pathway of the pyramidal tract through the medial lemniscus in the brainstem , which separates from the original pyramidal tract at the level of the midbrain and the pons and descends through the medial lemniscus . recently , several studies have suggested that the aberrant pyramidal tract may contribute to motor recovery in stroke [ 117120 ] . other motor - related pathways , including the corticorubrospinal and corticoreticulospinal tracts and the transcallosal motor pathways , may also contribute to the potential for functional recovery [ 121 , 122 ] . for example , rber et al . reported increased white matter integrity in the corticorubrospinal tract within the vicinity of the red nuclei . they also found strong correlations between microstructural properties of this tract and the level of motor function in chronic stroke patients , which highlight a compensatory function of the corticorubrospinal system . in a longitudinal rsfc study of rats after unilateral stroke , the authors found considerable loss of rsfc between the ipsilesional and contralesional primary sensorimotor cortex regions , alongside significant sensorimotor function deficits in the first days after stroke . the interhemispheric rsfc restored in the following weeks but remained significantly reduced up to 10 weeks after stroke in rats with lesions that comprised both the subcortical and cortical tissues . intrahemispheric rsfc between the primary somatosensory and motor cortex areas was preserved in the lesion border zone and moderately enhanced contralesionally . the temporal pattern of changes in rsfc between the bilateral primary motor and somatosensory cortices correlated significantly with the evolution of sensorimotor function scores . subsequently , these authors confirmed that the decreased interhemispheric rsfc between the ipsilesional and contralesional primary sensorimotor cortex regions is associated with a decrease in transcallosal manganese transfer between these regions . using the same stroke rat model , these authors recently found that the degree of functional recovery after stroke was associated with the extent of preservation or restoration of the ipsilesional corticospinal tracts in combination with reinstatement of the interhemispheric neuronal signal synchronization and normalization of small - world cortical network organization . similar rsfc changes were also found in stroke patients . immediately after stroke , significantly decreased rsfc of the ipsilesional primary sensorimotor cortex , especially the interhemispheric rsfc , was consistently reported [ 36 , 124126 ] . these decreased rsfcs were then gradually increased during recovery process and finally restored to the levels of near normal , normal , or above normal [ 36 , 124126 ] . in acute stroke patients , carter and colleagues reported that disruption of the interhemispheric rsfc within the attention network was significantly correlated with abnormal detection of visual stimuli . in the somatomotor network , in contrast , intrahemispheric rsfcs within the normal or damaged hemispheres were not correlated with performance in either of the two networks . recently , a longitudinal study revealed that the rsfcs of the ipsilesional primary motor cortex ( m1 ) with the contralesional thalamus , supplementary motor area ( sma ) , and middle frontal gyrus at onset were positively correlated with motor recovery at 6 months after stroke . in subacute stroke patients , the interhemispheric rsfc was negatively correlated with the extent of corticospinal damage . although corticospinal damage accounted for much of the variance in motor performance , the behavioral impact of rsfc was greater in subjects with mild or moderate corticospinal damage and less in those with severe corticospinal damage . in chronic stroke patients , different outcomes are found to be associated with different change patterns of the rsfcs . moreover , the longitudinal changes of the rsfcs after stroke were correlated with modification of motor function . besides the altered rsfcs within the motor network after stroke , reduced interhemispheric connectivity was also found between the attention - related areas in stroke patients with neglect [ 36 , 128 ] and the language areas in stroke patients with aphasia . however , the effect of anatomical damage may extend beyond the lesioned area but remain within the bounds of the existing network connections . this concept is well elucidated by the finding of double dissociation of two cognitive control networks in patients with focal brain lesions . the degree of network damage correlates with a decrease in rsfc within that network while sparing the nonlesioned network . applied dynamic causal modeling to investigate changes of effective connectivity among the m1 , lateral premotor cortex ( pmc ) , and sma in subacute stroke patients when they performed visually paced hand movements with their left , right , or both hands . independently from hand movements , the intrahemispheric effective connectivity between the ipsilesional sma and m1 and the interhemispheric effective connectivity of both the smas were significantly reduced . furthermore , movements of the stroke - affected hand showed additional inhibitory influences from the contralesional to the ipsilesional m1 that correlated with the degree of motor impairment . for bimanual movements , interhemispheric communication between the ipsilesional sma and contralesional m1 the study suggested that the motor deficit of patients with a single subcortical lesion was associated with pathological interhemispheric interactions among the key motor areas . a dysfunction of effective connectivity between the ipsilesional and contralesional m1 , and between the ipsilesional sma and contralesional m1 underlied hand motor disability after stroke . changes in effective connectivity among the m1 , sma , and cerebellum ( ce ) were also assessed in chronic stroke patients using dynamic causal modeling . relative to healthy controls , stroke patients exhibited decreased intrinsic neural coupling between the m1 and ce , but showed increased sma to m1 and sma to cerebellum couplings . the results demonstrate that connectivity alterations between motor areas may help to counterbalance a functionally abnormal m1 in chronic stroke patients . the temporal evolution of intra- and interhemispheric effective connectivity was also investigated during motor recovery from the acute to the early chronic phase after stroke [ 131 , 132 ] . results showed reduced positive coupling of the ipsilesional sma and pmc with the ipsilesional m1 in the acute stage . coupling parameters among these areas increased with recovery and predicted a better outcome . likewise , negative influences from the ipsilesional areas to the contralesional m1 were attenuated in the acute stage . in the subacute stage , the contralesional m1 exerted a positive influence on the ipsilesional m1 . negative influences from the ipsilesional areas on the contralesional m1 subsequently normalized , but patients with poorer outcome in the chronic stage now showed enhanced negative coupling from the contralesional upon the ipsilesional m1 . these findings show that the reinstatement of effective connectivity in the ipsilesional hemisphere is an important feature of motor recovery after stroke . the shift of an early , supportive role of the contralesional m1 to enhanced inhibitory coupling might indicate maladaptive processes which could be a target of noninvasive brain stimulation techniques . sharma et al . investigated well - recovered stroke patients performing a motor imagery task , and found that the regional activations had returned to normal in the patients . in addition to reduced effective connectivity among the motor - related brain areas , they found significantly enhanced positive influences from the prefrontal cortex to both the sma and pmc in stroke patients using an sem analysis of effective connectivity . the authors suggest that enhanced coupling of the prefrontal areas might reflect the enhanced role of cognitive - related areas that facilitate movement planning to overcome the functional deficits caused by the damage to the motor pathways . the effective connectivity can also be investigated during resting state . using an sem analysis , inman and coauthors focused on the intrinsic effective connectivity of top - down motor control in stroke patients exhibiting significant motor deficit . they found alterations in resting - state effective connectivity from the frontoparietal guidance systems to the motor network in stroke survivors . more specifically , diminished connectivity was found in connections from the superior parietal cortex to the m1 and sma . these findings suggest that characterizing the deficits in resting - state connectivity of top - down processes in stroke survivors may help optimize cognitive and physical rehabilitation therapies by individually targeting specific neural pathway . the first preliminary study on the functional network efficiency changes after stroke was performed using graph theoretical measures on electroencephalography ( eeg ) data of 1 stroke patient and 8 healthy subjects when they performed a finger tapping task . the authors found significant decrease in global and local efficiency in the patient 's networks , reflecting a lower capacity to integrate communication between distant brain regions and a lower tendency to be modular . they also showed that these changes were associated with significant increases in the disconnected nodes and in the links of some other crucial vertices . the authors concluded that overall connectivity after stroke was governed by a lower number of brain regions in which increased connectivity could not compensate for the drastic reduction in information propagation . the poststroke longitudinal changes in motor network efficiency have been reported in a resting - state fmri study in which the authors used graph theory to assess changes in the topological configuration of the motor network from the acute phase to the chronic phase after subcortical stroke . they found that over a year of recovery , motor execution network showed lower local efficiency within the network suggesting a shift towards a nonoptimal network configuration with less functional segregation . the overall decrease in network efficiency was paralleled by a stronger betweenness centrality of the ipsilesional m1 and cerebellum , the latter being a measure of the functional importance of a node for information processing . the increased importance of the ipsilesional m1 within the motor network after recovery was also indicated by stronger functional connectivity of this area with the contralesional motor areas . recently , the functional network characteristics of the whole brain were also investigated in stroke patients from the perspective of graph theory . a longitudinal design was adopted in the study and the network measures were calculated based on the fmri data when subjects were performing a finger tapping task . the authors showed that the brain networks shifted towards a nonoptimal topological configuration with low small worldness during the process of recovery . however , in an experimental stroke model , a rather different pattern of changes of network features were reported . they found that the clustering coefficient , shortest path length , and small worldness of the motor network of stroke rats were significantly increased in the first poststroke days , and then these network measures declined towards a normal level . the authors speculate that the initial increase of network measures may be associated with initial excessive neuronal clustering and wiring , whereas the later decline toward a baseline small - world topology may be related to the refinement of the reorganized network . they also explain the discrepancy between their study and the findings of wang et al . by the differences between the ( re)organization of rat and human brain and the differences in the analytical methods . using dtt data , crofts and colleagues investigated the anatomical network efficiency changes after stroke . they proposed a measure of communicability that estimates the ease through which information can travel across a network . they found reduced communicability in stroke patients in both regions surrounding the lesions in the affected hemisphere and homologous locations in the contralesional hemisphere . they also identified regions with increased communicability in patients that could represent adaptive , plastic changes after stroke . much evidence has suggested that stroke patients will benefit significantly from rehabilitative therapies beyond spontaneous recovery of function [ 138 , 139 ] . recently , connectivity analyses have been used to investigate the intervention effects on brain connectivity . the preliminary experimental studies have suggested that treatments with neural progenitor cells , sildenafil , or erythropoietin induced increased fa in the ischemic lesion borderzone , which reflects a high density of axons and myelin in this region [ 9 , 97 ] . in chronic stroke patients participating in a regimen of electrical stimulation targeting the paretic arm , after an 8-week therapy , these patients exhibited decreased mean diffusivity ( md ) in the middle cerebellar peduncle and posterior limb of the internal capsule following treatment , which suggests that active rehabilitative therapies augmented by electrical stimulation may induce positive behavioral changes by increasing white matter tract integrity in regions specific to sensory - motor function . in stroke patients with aphasia , both intense intonation - based speech therapy and constraint induced language therapy could induce increased fibers or white matter integrity in fiber tracts involved in language function . wu and colleagues observed the longitudinal changes of dti measures after acupuncture treatment in rats with transient focal cerebral ischaemia . they found significantly increased fa at the edge of the ischemic lesions in stroke rats with acupuncture treatment . the effect of acupuncture therapy for postponing wallerian degeneration secondary to cerebral infarction has also been observed by dti in stroke patients . the authors found that acupuncture treatment was effective for protecting white matter integrity in stroke patients . the rsfc was assessed before and after a 12-week robot - aided motor rehabilitation program . the authors found that the rsfc between the ipsilesional and contralesional m1 reduced after a bout of motor rehabilitation . greater reduction in the interhemispheric rsfc was associated with greater gains in motor function induced by the 12-week robotic therapy program . these findings suggest that greater reduction in interhemispheric rsfc in response to a bout of motor rehabilitation may predict greater efficacy of the full rehabilitation program . a recent study examined the effects of upper - extremity robot - assisted rehabilitation ( manus ) versus an electroencephalography - based brain computer interface setup with motor imagery ( mi eeg - bci ) and compared pretreatment and posttreatment rsfcs . the authors found that the individual gain in motor function over 12 weeks could be predicted by the rsfc changes before and after treatment . both the motor function gain and increases in rsfcs of the sma , the contralesional and ipsilesional motor cortex , and parts of the visuospatial system with mostly association cortex regions and the cerebellum were correlated with individual upper - extremity function improvement . a placebo - controlled , double - blind , and crossover design study was performed to investigate the effects of noradrenergic stimulation on the cortical motor system in hemiparetic stroke patients . effective connectivity analyses with dcm revealed that in stroke patients neural coupling with the sma or vpmc was significantly reduced compared with healthy controls . this hypoconnectivity was partially normalized when patients received reboxetine ( rbx ) , especially for the coupling between the ipsilesional sma and m1 . the data suggest that noradrenergic stimulation may help to modulate the pathologically altered motor network architecture in stroke patients , resulting in increased coupling of the ipsilesional motor areas and thereby improved motor function . resting - state fmri data were analyzed using the sem to evaluate therapy - related changes in motor network effective connectivity in stroke patients after 3 weeks of upper - extremity rehabilitation in the accelerated skill acquisition program ( asap ) . the authors found that the behavioral improvement after training of the impaired upper extremity is accompanied by increased influence of the affected hemispheric pmc upon the unaffected hemispheric pmc and on the affected hemispheric m1 . the relationship between behavioral recovery and interhemispheric and intrahemispheric communication has been investigated by inhibiting the contralesional m1 using1-hz repetitive transcranial magnetic stimulation ( rtms ) . after inhibiting the contralesional m1 using 1-hz repetitive transcranial magnetic stimulation ( rtms ) the connectivity analysis revealed that the behavioral improvements were significantly correlated with a reduction of the negative influences originating from the contralesional m1 during paretic hand movements . concurrently , endogenous coupling between the ipsilesional sma and m1 was significantly enhanced after rtms was applied over the contralesional m1 . the connectivity analyses suggest that both a reduction of pathological transcallosal influences and a restitution of ipsilesional effective connectivity between the sma and m1 underlie improved motor performance . a growing body of evidence from connectivity - based analyses of functional imaging data has told us that a focal stroke lesion may affect not only the lesion site but also the network to which it belongs . thus the connectivity - based analytic methods may be more appropriate for elucidating stroke - induced impairments from a network perspective and for clarifying the mechanisms of motor recovery after stroke . moreover , connectivity analyses are likely to be better suited to investigate the mechanisms through which therapeutic interventions may facilitate the recovery of motor function and help us to develop new intervention therapies targeting the restoration of the function of the motor network . finally , connectivity measures may serve to monitor the process of stroke recovery and to predict the outcomes of stroke patients at an early stage . however , conflicting findings exist and the exact mechanisms leading to changes in brain connectivity after stroke remain elucidated . therefore , longitudinal studies with large sample size employing different neuroimaging modalities covering the whole period from the superacute stage to the late chronic stage are needed to further our understanding of how different types of brain connectivity evolve after stroke and how they relate to motor deficits and clinical outcome .

the motor function is controlled by the motor system that comprises a series of cortical and subcortical areas interacting via anatomical connections . the motor function will be disturbed when the stroke lesion impairs either any of these areas or their connections . more and more evidence indicates that the reorganization of the motor network including both areas and their anatomical and functional connectivity might contribute to the motor recovery after stroke . here , we review recent studies employing models of anatomical , functional , and effective connectivity on neuroimaging data to investigate how ischemic stroke influences the connectivity of motor areas and how changes in connectivity relate to impaired function and functional recovery . we suggest that connectivity changes constitute an important pathophysiological aspect of motor impairment after stroke and important mechanisms of motor recovery . we also demonstrate that therapeutic interventions may facilitate motor recovery after stroke by modulating the connectivity among the motor areas . in conclusion , connectivity analyses improved our understanding of the mechanisms of motor recovery after stroke and may help to design hypothesis - driven treatment strategies and sensitive measures for outcome prediction in stroke patients .

1. Introduction 2. Neuroimaging Methods for Brain Connectivity 3. Brain Connectivity Changes After Stroke 4. Intervention Effects on Connectivity in Stroke Patients 5. Conclusions

, stroke patients can usually recover to some extent , which may be related to structural and functional modifications in surviving brain tissue . studies on stroke rats and patients have revealed that spontaneous recovery of the motor function after stroke is associated with brain plasticity [ 15 ] . neuroimaging techniques , especially the multimodality mris , have significantly contributed to our understanding of the mechanisms of stroke recovery by characterizing brain structural and functional changes after stroke [ 610 ] . moreover , mri is a multimodality imaging technique that can be used to investigate both anatomical and functional connectivities . more importantly , increased grey matter volume and cortical thickness were also found in the motor - related areas and hippocampus either during spontaneous recovery or after treatments [ 1113 ] . task - based functional mri ( fmri ) or pet has been extensively used to investigate brain activation changes during stroke recovery [ 1416 ] and has provided important information on the patterns of functional reorganization after stroke [ 1419 ] . movements of the stroke - affected hand are initially activated extensive brain regions in stroke patients [ 16 , 2022 ] and rats [ 6 , 2330 ] . however , this initial widespread activation does not predict good functional recovery [ 3133 ] , whereas normalization of the activation pattern to prestroke level is associated with good outcome . recently , connectivity - based methods have been used in stroke patients and rats to demonstrate anatomical and functional connectivity changes after stroke ; these changes may be related to clinical deficits and functional recovery . these methods may provide great insight into network dysfunction [ 21 , 3639 ] and functional reorganization [ 40 , 41 ] from a system perspective ; they will also contribute to predict outcomes and to develop new therapeutic interventions [ 40 , 41 ] . in this review , we focused on recent neuroimaging studies employing connectivity - based analyses to investigate connectivity changes in the motor network after ischemic stroke . connectivity models are based on the concept that the brain is organized by segregation of specialized and anatomically distinct brain regions which are functionally integrated in a network mediating motor , sensory , or cognitive processing . there are four common connectivity models : the anatomical connectivity , functional connectivity , effective connectivity , and network models . dti measures can be analyzed by either data - driven methods ( voxel - based analysis and tract - based spatial statistics ) or hypothesis - driven methods ( region of interest ( roi ) analysis and tractography - based analysis ) . dti and dtt have been used in the evaluation of the white matter damage and reorganization in stroke patients [ 4751 ] . it is well recognized that diffusion - weighted imaging ( dwi ) is capable of yielding considerably more information than that contained in the diffusion metrics derived from dti due to the fact that dti is based upon a gaussian approximation of the diffusion displacement probability function . the mean kurtosis ( mk ) , a principal metric of the diffusional non - gaussianity , is of potential interest to the study of white and gray matter integrity . dki can provide additional information ( non - gaussian diffusion ) that can not be obtained from dti , and it has been suggested that dki may provide enhanced contrast between ischaemic and normal tissues . the resting - state functional connectivity ( rsfc ) measures temporal coherence of low - frequency fluctuations ( < 0.1 hz ) of the blood oxygenation level - dependent ( bold ) signals between spatially remote brain regions . correlation of these signals with underlying neural activity indicates that these fluctuations are of functional significance [ 6567 ] . a hypothesis - driven method has been extensively used to analyze rsfc changes . in this method , an roi should be predefined based on hypotheses , and then the rsfc pattern of this roi is obtained by computing correlations in the fmri time courses between the roi and every voxel of the whole brain . the functional connectivity density ( fcd ) mapping is another data - driven method and measures the number of functional connections per voxel [ 77 , 78 ] . these rsfc analytic methods are suited for the investigation of how multiple distributed networks are disrupted by stroke and are reorganized after stroke and what patterns of connectivity are most likely to be behaviorally relevant [ 40 , 41 ] . in contrast to the nondirectional , correlative nature of functional connectivity , effective connectivity measures the causal influence that one brain area exerts over another under the assumption of a given mechanistic model . effective connectivity is based on mathematical models that are usually applied to task - based neuroimaging data . the psychophysiological interactions ( ppi ) model is a relatively simple method to estimate effective connectivity from neuroimaging data . in contrast to these exploratory methods , structural equation modelling ( sem ) and dynamic causal modeling ( dcm ) are hypothesis - driven approaches that require a priori defined network of brain regions to estimate effective connectivity from neuroimaging data . in the framework of graph theory , the brain is described as a graph comprising a certain number of nodes ( corresponding to brain regions ) that are connected by edges ( corresponding to anatomical connectivity , functional connectivity , effective connectivity , or other measures of interregional interactions ) [ 8588 ] . the network 's structure can be assessed by measuring its clustering coefficient , a measure of segregation , that reflects the degree to which nodes are clustered , and the shortest path length , a measure of integration , that reflects the minimal number of edges between any pair of nodes . , the fraction of the shortest paths that pass through a given node ) are used to assess the importance of a given node . brain regions featuring a high node degree and a high centrality are assumed to serve as brain reorganization after stroke is a dynamic process , which considerably differs across patients , depending on lesion location , time since stroke , severity of motor impairment , premorbid state , and even genetics . consequently , a variety of connectivity analyses have been performed to investigate motor recovery mechanisms after ischemic stroke . dti and dtt are powerful methods to detect not only impairments but also modifications in anatomical connectivity after stroke [ 89 , 90 ] . the motor recovery mechanisms in ischemic stroke patients have been summarized as ( 1 ) recovery of a damaged lateral corticospinal tract ( cst ) ; ( 2 ) subcortical perilesional reorganization ; ( 3 ) ipsilateral motor pathway from the unaffected motor cortex to the affected extremities ; and ( 4 ) collateral pathway of the pyramidal tract . they found that the anatomical connectivity of the cst at the cortical regions within the affected hemisphere was enhanced over time and that the enhanced connectivity was correlated with stroke recovery . examined the relationship between microstructural status of brain white matter tracts and motor skill of the stroke - affected hand in patients with chronic stroke . however , our previous study of dynamic evolution of the diffusion indices in the degenerated cst did not show any significant plastic changes after ischemic stroke although a slightly elevated fa was found 1 year after stroke . the discrepancy between these studies may be ascribed to methodological differences because we only studied the cst section at the level of pons , which may miss the portion of the cst that exhibits plastic changes . this initial decrease of fa may be chronically followed by normalization or elevation in the borderzone of the ischemic lesion , which could be further enhanced by treatments with neural progenitor cells , sildenafil , or erythropoietin . the subcortical perilesional reorganization is also reported at the levels of the corona radiata and pons in stroke patients [ 97101 ] . these findings suggest that rearrangement of white matter in the ischemic perilesional areas is accompanied by preservation or restoration of neuronal connectivity and may be used to predict motor outcome after stroke . the ipsilateral motor pathway from the unaffected motor cortex to the affected extremities has been regarded as one of the recovery mechanisms of stroke [ 102106 ] . the location and size of the lesions , the different recovery rates , and the analytic methods used in these studies may partly account for the discrepancy . the aberrant pyramidal tract refers to the collateral pathway of the pyramidal tract through the medial lemniscus in the brainstem , which separates from the original pyramidal tract at the level of the midbrain and the pons and descends through the medial lemniscus . recently , several studies have suggested that the aberrant pyramidal tract may contribute to motor recovery in stroke [ 117120 ] . other motor - related pathways , including the corticorubrospinal and corticoreticulospinal tracts and the transcallosal motor pathways , may also contribute to the potential for functional recovery [ 121 , 122 ] . they also found strong correlations between microstructural properties of this tract and the level of motor function in chronic stroke patients , which highlight a compensatory function of the corticorubrospinal system . subsequently , these authors confirmed that the decreased interhemispheric rsfc between the ipsilesional and contralesional primary sensorimotor cortex regions is associated with a decrease in transcallosal manganese transfer between these regions . using the same stroke rat model , these authors recently found that the degree of functional recovery after stroke was associated with the extent of preservation or restoration of the ipsilesional corticospinal tracts in combination with reinstatement of the interhemispheric neuronal signal synchronization and normalization of small - world cortical network organization . similar rsfc changes were also found in stroke patients . immediately after stroke , significantly decreased rsfc of the ipsilesional primary sensorimotor cortex , especially the interhemispheric rsfc , was consistently reported [ 36 , 124126 ] . in acute stroke patients , carter and colleagues reported that disruption of the interhemispheric rsfc within the attention network was significantly correlated with abnormal detection of visual stimuli . recently , a longitudinal study revealed that the rsfcs of the ipsilesional primary motor cortex ( m1 ) with the contralesional thalamus , supplementary motor area ( sma ) , and middle frontal gyrus at onset were positively correlated with motor recovery at 6 months after stroke . in chronic stroke patients , different outcomes are found to be associated with different change patterns of the rsfcs . moreover , the longitudinal changes of the rsfcs after stroke were correlated with modification of motor function . besides the altered rsfcs within the motor network after stroke , reduced interhemispheric connectivity was also found between the attention - related areas in stroke patients with neglect [ 36 , 128 ] and the language areas in stroke patients with aphasia . however , the effect of anatomical damage may extend beyond the lesioned area but remain within the bounds of the existing network connections . this concept is well elucidated by the finding of double dissociation of two cognitive control networks in patients with focal brain lesions . applied dynamic causal modeling to investigate changes of effective connectivity among the m1 , lateral premotor cortex ( pmc ) , and sma in subacute stroke patients when they performed visually paced hand movements with their left , right , or both hands . independently from hand movements , the intrahemispheric effective connectivity between the ipsilesional sma and m1 and the interhemispheric effective connectivity of both the smas were significantly reduced . furthermore , movements of the stroke - affected hand showed additional inhibitory influences from the contralesional to the ipsilesional m1 that correlated with the degree of motor impairment . for bimanual movements , interhemispheric communication between the ipsilesional sma and contralesional m1 the study suggested that the motor deficit of patients with a single subcortical lesion was associated with pathological interhemispheric interactions among the key motor areas . a dysfunction of effective connectivity between the ipsilesional and contralesional m1 , and between the ipsilesional sma and contralesional m1 underlied hand motor disability after stroke . changes in effective connectivity among the m1 , sma , and cerebellum ( ce ) were also assessed in chronic stroke patients using dynamic causal modeling . relative to healthy controls , stroke patients exhibited decreased intrinsic neural coupling between the m1 and ce , but showed increased sma to m1 and sma to cerebellum couplings . the results demonstrate that connectivity alterations between motor areas may help to counterbalance a functionally abnormal m1 in chronic stroke patients . the temporal evolution of intra- and interhemispheric effective connectivity was also investigated during motor recovery from the acute to the early chronic phase after stroke [ 131 , 132 ] . these findings show that the reinstatement of effective connectivity in the ipsilesional hemisphere is an important feature of motor recovery after stroke . the shift of an early , supportive role of the contralesional m1 to enhanced inhibitory coupling might indicate maladaptive processes which could be a target of noninvasive brain stimulation techniques . investigated well - recovered stroke patients performing a motor imagery task , and found that the regional activations had returned to normal in the patients . in addition to reduced effective connectivity among the motor - related brain areas , they found significantly enhanced positive influences from the prefrontal cortex to both the sma and pmc in stroke patients using an sem analysis of effective connectivity . the authors suggest that enhanced coupling of the prefrontal areas might reflect the enhanced role of cognitive - related areas that facilitate movement planning to overcome the functional deficits caused by the damage to the motor pathways . the effective connectivity can also be investigated during resting state . using an sem analysis , inman and coauthors focused on the intrinsic effective connectivity of top - down motor control in stroke patients exhibiting significant motor deficit . they found alterations in resting - state effective connectivity from the frontoparietal guidance systems to the motor network in stroke survivors . these findings suggest that characterizing the deficits in resting - state connectivity of top - down processes in stroke survivors may help optimize cognitive and physical rehabilitation therapies by individually targeting specific neural pathway . the authors concluded that overall connectivity after stroke was governed by a lower number of brain regions in which increased connectivity could not compensate for the drastic reduction in information propagation . the poststroke longitudinal changes in motor network efficiency have been reported in a resting - state fmri study in which the authors used graph theory to assess changes in the topological configuration of the motor network from the acute phase to the chronic phase after subcortical stroke . the increased importance of the ipsilesional m1 within the motor network after recovery was also indicated by stronger functional connectivity of this area with the contralesional motor areas . recently , the functional network characteristics of the whole brain were also investigated in stroke patients from the perspective of graph theory . they found that the clustering coefficient , shortest path length , and small worldness of the motor network of stroke rats were significantly increased in the first poststroke days , and then these network measures declined towards a normal level . the authors speculate that the initial increase of network measures may be associated with initial excessive neuronal clustering and wiring , whereas the later decline toward a baseline small - world topology may be related to the refinement of the reorganized network . by the differences between the ( re)organization of rat and human brain and the differences in the analytical methods . they found reduced communicability in stroke patients in both regions surrounding the lesions in the affected hemisphere and homologous locations in the contralesional hemisphere . they also identified regions with increased communicability in patients that could represent adaptive , plastic changes after stroke . recently , connectivity analyses have been used to investigate the intervention effects on brain connectivity . in chronic stroke patients participating in a regimen of electrical stimulation targeting the paretic arm , after an 8-week therapy , these patients exhibited decreased mean diffusivity ( md ) in the middle cerebellar peduncle and posterior limb of the internal capsule following treatment , which suggests that active rehabilitative therapies augmented by electrical stimulation may induce positive behavioral changes by increasing white matter tract integrity in regions specific to sensory - motor function . in stroke patients with aphasia , both intense intonation - based speech therapy and constraint induced language therapy could induce increased fibers or white matter integrity in fiber tracts involved in language function . they found significantly increased fa at the edge of the ischemic lesions in stroke rats with acupuncture treatment . the effect of acupuncture therapy for postponing wallerian degeneration secondary to cerebral infarction has also been observed by dti in stroke patients . the authors found that acupuncture treatment was effective for protecting white matter integrity in stroke patients . the authors found that the rsfc between the ipsilesional and contralesional m1 reduced after a bout of motor rehabilitation . greater reduction in the interhemispheric rsfc was associated with greater gains in motor function induced by the 12-week robotic therapy program . these findings suggest that greater reduction in interhemispheric rsfc in response to a bout of motor rehabilitation may predict greater efficacy of the full rehabilitation program . the authors found that the individual gain in motor function over 12 weeks could be predicted by the rsfc changes before and after treatment . both the motor function gain and increases in rsfcs of the sma , the contralesional and ipsilesional motor cortex , and parts of the visuospatial system with mostly association cortex regions and the cerebellum were correlated with individual upper - extremity function improvement . a placebo - controlled , double - blind , and crossover design study was performed to investigate the effects of noradrenergic stimulation on the cortical motor system in hemiparetic stroke patients . effective connectivity analyses with dcm revealed that in stroke patients neural coupling with the sma or vpmc was significantly reduced compared with healthy controls . the data suggest that noradrenergic stimulation may help to modulate the pathologically altered motor network architecture in stroke patients , resulting in increased coupling of the ipsilesional motor areas and thereby improved motor function . resting - state fmri data were analyzed using the sem to evaluate therapy - related changes in motor network effective connectivity in stroke patients after 3 weeks of upper - extremity rehabilitation in the accelerated skill acquisition program ( asap ) . the authors found that the behavioral improvement after training of the impaired upper extremity is accompanied by increased influence of the affected hemispheric pmc upon the unaffected hemispheric pmc and on the affected hemispheric m1 . after inhibiting the contralesional m1 using 1-hz repetitive transcranial magnetic stimulation ( rtms ) the connectivity analysis revealed that the behavioral improvements were significantly correlated with a reduction of the negative influences originating from the contralesional m1 during paretic hand movements . the connectivity analyses suggest that both a reduction of pathological transcallosal influences and a restitution of ipsilesional effective connectivity between the sma and m1 underlie improved motor performance . a growing body of evidence from connectivity - based analyses of functional imaging data has told us that a focal stroke lesion may affect not only the lesion site but also the network to which it belongs . thus the connectivity - based analytic methods may be more appropriate for elucidating stroke - induced impairments from a network perspective and for clarifying the mechanisms of motor recovery after stroke . moreover , connectivity analyses are likely to be better suited to investigate the mechanisms through which therapeutic interventions may facilitate the recovery of motor function and help us to develop new intervention therapies targeting the restoration of the function of the motor network . finally , connectivity measures may serve to monitor the process of stroke recovery and to predict the outcomes of stroke patients at an early stage . however , conflicting findings exist and the exact mechanisms leading to changes in brain connectivity after stroke remain elucidated . therefore , longitudinal studies with large sample size employing different neuroimaging modalities covering the whole period from the superacute stage to the late chronic stage are needed to further our understanding of how different types of brain connectivity evolve after stroke and how they relate to motor deficits and clinical outcome .

accurately computing the free energy of binding a ligand to a protein is a task of essential importance in biochemical and biophysical studies that still presents us considerable difficulty to overcome . an effective approach in the current literature is to use the relationship between the potential of mean force ( pmf ) and the binding affinity . the equilibrium approaches , based on pmf or not , are not brute force in nature but require delicate choices of biasing / constraining potentials during the simulation processes . the nonequilibrium steered molecular dynamics ( smd ) approach , seemingly brute force , can be very efficient in sampling forced transition paths from the bound state to the dissociated state of the ligand but has not been used reliably for free - energy calculations with quantitative accuracy . in this paper , we present a hybrid steered molecular dynamics ( hsmd ) approach that produces binding affinities in quantitative agreement with experimental measurements ( within a factor of 1.5 in terms of the dissociation constant kd defined as the ligand concentration at which the holoprotein concentration equals the apoprotein concentration ) . the hsmd approach is based on the relationship between the pmf and the binding affinity in the established literature . the widely used smd involves pulling one center of mass of one selection of the ligand atoms using a spring of finite , carefully chosen , stiffness . in contrast , the hsmd approach involves pulling n ( n = 1 , 2 , 3 , ... ) centers of mass of n selected segments of the ligand ( using n springs of infinite stiffness to disallow any fluctuation of the pulling centers along the way ) to produce a 3n - dimensional ( 3n - d ) pmf curve leading from the binding site on or inside the protein to the dissociated state in the bulk region far from the protein . this pmf difference between the bound state and the dissociated state gives a large ( but not dominant ) part of the absolute binding free energy . another part of the hsmd approach is the equilibrium molecular dynamics ( md ) sampling of the ligand s fluctuations at the binding site that also contribute to the binding free energy . the third , final , part of the hsmd approach is smd stretching and equilibrium md sampling of the ligand in the dissociated state when one of the n pulling centers is fixed at a point in the bulk far from the protein . protein complexes whose binding affinities were experimentally measured and whose crystal structures are available : oca glb , caprylic acid ( 25 atoms , neutral ) bound to bovine -lactoglobulin ; and gsh sjgst(y7f ) , glutathione ( 36 atoms , singly negatively charged ) bound to schistosoma japonicum glutathione s - transferase tyrosine 7 to phenylalanine mutant . the computing times required were approximately 62 wall - clock hours for oca glb ( all - atom model of 95 296 atoms ) and 88 wall - clock hours for gsh sjgst(y7f ) ( all - atom model of 114 538 atoms ) , respectively , using 32 xeon e5 - 2628 processors ( 512 cores ) in parallel . in each of the two cases , following the standard literature , the binding affinity at one binding site is1where c0 is the standard concentration . for clarity and for convenience of unit conversion , we use two different but equivalent forms : c0 = 1 m on the left - hand side and c0 = 6.02 10/ on the right - hand side of eq 1 . the three - dimensional ( 3d ) integrations are over the x- , y- , and z - coordinates of the ligand s position r1 that can be chosen as the center of mass of one segment of or the whole ligand . the integral has the units of that renders the right - hand side dimensionless , as it should be . the subscripts site and bulk indicate that r1 is near the pmf minimum and r1 = r1 in the bulk region far from the protein , respectively . for a ligand whose size is not small and whose shape is not simple , the position of one segment center r1 will not be sufficient / efficient to represent its location and situation . instead , the ligand can be better described with the positions ( r1 , r2 , ... , rn ) of n centers of mass of its n chosen segments . figure 1 shows an example of n = 3 , where the positions ( r1 , r2 , r3 ) of the three -carbons of glutathione are chosen to quantify the location and situation of the ligand . these n positions fluctuate without being in any way biased / constrained during the equilibrium md simulation of the bound state . they are steered during the smd runs from the bound state to the dissociated state for constructing the 3n - d pmf w[r1 , r2 , ... , rn ] as a function of these positions . in the dissociated state , one of them will be fixed at r1 = r1 while all others ( r2 , ... , rn ) rotate and fluctuate according to the stochastic dynamics of the system without any other bias / constraint . the three -carbons of gsh are shown as yellow balls marked with their position vectors . the protein surface is shown as wire frames and the ligand is shown as licorice , all colored according to atom names . note that in the relationship between the 3d and 3n - d pmfs2the 3(n 1)-d integration over the ( n 1 ) positions ( r2 , ... , rn ) is effectively in a defined neighborhood of r1 because the ligand as one whole molecule dictates that the n centers can not be stretched much farther from one another than its molecular size . when r1 is near the binding site , when the ligand is in the dissociated state , r1 needs to be so far from the protein that integration over ( r2 , ... , rn ) will be all in the region far from the protein . c is the normalization constant that will be canceled out in the following expressions . making use of eq 2 twice in eq 1 ( for the binding site and for the bulk ) , one has the following expression for the binding affinity.3 now inserting the boltzmann factor at a single state ( r10 , r20 , ... , rn ) chosen from the bound state ensemble and the boltzmann factor at the corresponding dissociated state ( r1 , r2 , ... , rn ) , the binding affinity can be expressed as three contributing factors : the partial partition function at the binding site zn0 , the pmf difference between two chosen states ( r10 , r20 , ... , rn0 ) and ( r1 , r2 , ... , rn ) , and the partial partition function in the dissociated state zn. mathematically4here ( r1 , r2 , ... , rn ) can be connected to ( r10 , r20 , ... , rn0 ) via many curves in the 3n - d space , but the pmf is a function of state . computation of the pmf difference between the two states can be achieved along a single curve passing through them both . the partial partition function zn0 of the bound state has the integration over all n centers and thus has the units of .5 the partial partition function zn of the dissociated state has the integration over n 1 centers and thus has the units of .6 again , the use of c0 = 6.02 10/ on the right - hand side of eq 3 renders it a pure number as desired . the dissociation constant will conveniently be in the unit of m = moles per liter . the 3n - d pmf difference7is between one chosen bound state and its corresponding dissociated state . this pmf difference can be computed by means of the smd simulations described in the latter part of this section . note that the one chosen position of the ligand in the bound state8is the starting point for smd runs . it does not have to be the minimum of the pmf but any one state in its close neighborhood . note that we take the collection of coordinate vectors , e.g. , eq 8 , as a single - row 1 3n matrix . its transpose in the following eq 13 is a single - column 3n 1 matrix . the one state chosen from the dissociated state ensemble9is related to the smd starting point by a large enough displacement in the 3n - d space.10here vd is the constant velocity of the smd pulling and t is the time it takes to steer / pull the ligand from the binding site to the bulk . the reference point for the pmf , w[r1 , r2 , ... , rn ] = 0 , is chosen as when the ligand is far from the protein . the absolute free energy of binding is11 with all this , one needs to compute three factors to determine the absolute free energy of binding . ( 1 ) the first factor is the pmf difference between one chosen bound state and its corresponding dissociated state that can be computed by running smd simulations of pulling the ligand forward and backward along a 3n - d line connecting the two states . note that the pmf is a function of state ( a point in the 3n - d space ) and the pmf difference is independent of the paths connecting the two end points . ( 2 ) the second factor is the partial partition function in the bound state that can be approximated as gaussian in cases of strong binding or can be numerically evaluated by running equilibrium md simulation . ( 3 ) the third factor is the partial partition function in the dissociated state that needs to be computed case by case for n = 2 , 3 , .... when the binding is tight , one can approximate the integral of eq 5 as gaussian in the neighborhood of the pmf minimum . the coordinates of the minimum of a gaussian distribution are equal to the average coordinates , of course , ( r1 , r2 , ... , rn ) . carrying out the gaussian integral , one has12here the dimensionless quantity n / kbt gives a measure of how far ( r10 , r20 , ... , rn0 ) , the initial state chosen for smd , is from the pmf minimum ( r1 , r2 , ... n is the 3n 3n matrix of the fluctuations / deviations of the pulling center coordinates x1 , in the bound state ensemble:14 n1 is the inverse matrix of n which can be accurately evaluated by running equilibrium md in the bound state of the ligand this approximation is generally valid if the ligand does not deviate much from the binding site . however , it is invalid if the binding is extremely weak or the binding site is not well localized . then one would have to evaluate the 3n - d integral in eq 5 directly by numerical means . to decide whether the gaussian approximation is suitable or not , one can evaluate how far from the gaussian distribution is the distribution of the fluctuations at the binding site . note that this evaluation does not require additional equilibrium md runs in the bound state of the ligand for example , by computing the first to the fourth moments of the fluctuations and checking their relationships , one can readily determine the non - gaussian - ness of the fluctuations . unlike the partial partition function zn0 of the bound state , the computation of the partial partition function of the dissociated state , zn in eq 6 , needs to be done individually for each case of n = 1 , 2 , .... in the following , we detail three cases : n = 1 , 2 , and 3 . when one center of mass is steered , n = 1 , z1 = 1 , all we have to do is to evaluate the fluctuations around the binding site ( the 3 3 matrix 1 ) along with the pmf difference . in this case , we have the dissociate constant , within the gaussian approximation of the fluctuations at the binding site15and the absolute free energy of binding the ligand to the protein16 it needs to be pointed out that the method of pulling one center of the ligand is only practical for small ligands of simple shapes . furthermore , the gaussian approximation of the ligand fluctuations at the binding site in eq 12 is inapplicable to cases of large fluctuations . for example , glycerol binds to glpf inside the conducting channel and its fluctuations inside the channel are large and non - gaussian . in the computation of its binding affinity , some special attention needs to be paid to the integral of eq 5 instead of using the gaussian approximation in eq 12 . when two centers of mass are steered , n = 2 , one has17which is an integration over the second steering center when the first steering center is fixed in a position r1 far from the protein . all one needs to do now is to evaluate the integral of eq 17 around the position ( r1 , r2 ) . over there , far from the protein , the ligand s environment is spherically symmetrical around the position of the first pulling center r1 that is fixed while the second pulling center r2 is free to sample all space available . therefore , the 3d integral becomes the following one - dimensional integral:18where r = |r2 w[r ] here , as a function of r , is the pmf ( reversible work ) for stretching the ligand between the two pulling centers . it can be evaluated by conducting smd runs of steering the second pulling center r2 to and from the first pulling center that is fixed at r1 along the axis passing through ( r1 , r2 ) . in this , we have the absolute free energy of binding the ligand to the binding site19and the dissociation constant20here the partial partition function of the dissociated state z2 is given in eq 18 and the partial partition function of the bound state is approximated below as gaussian.21where 2 is a 6 6 matrix of coordinate deviations defined in eq 14 and 2 is defined in eq 13 with n = 2 . it is worth noting that , for protein ligand complexes whose binding is strong , the computation of z20 and z2 can be carried out efficiently with sufficient accuracy . particularly , if the orientational entropy plays a large role , one needs to pull three or more centers of the ligand segments . when three centers of mass are steered , n = 3 , we need to evaluate the integral of eq 6 around the position ( r1 , r2 , r3 ) when the ligand is far from the protein . over there , the ligand s environment is spherically symmetrical around the position of the first pulling center r1 that is fixed while the second and the third pulling centers r2 and r3 are free to sample all space available . the pmf w[r1 , r2 , r3 ] is only dependent upon three of the six degrees of freedom contained in ( r2 , r3 ) . namely22where r21 = |r2 r1| and r31 = |r3 r1| are the distances between the second / third pulling center and the first pulling center that is fixed at r1. is the angle between ( r2 r1 ) and ( r3 r1 ) . therefore , the six - dimensional integral of eq 6 becomes the following 3d integral:23 in terms of the 3d probability distribution (r21 , r31 , ) , the integral becomes24which can be evaluated by equilibrium sampling in the 3d space ( r21 , r31 , ) from md runs with r1 being fixed at r1. is the angle between ( r2 r1 ) and ( r3 r1 ) , of course . if the equilibrium sampling in 3d can not be achieved with sufficient accuracy , we can approximate the pmf as three separable terms:25correspondingly , we obtain26here is a factor negating the possible error introduced in eq 25 . the pmf w[r21 ] , as a function of the distance between the two pulling centers r21 , can be evaluated by conducting smd runs of steering the second pulling center r2 to and from the first pulling center that is fixed at r1 along the axis passing through ( r1 , r2 ) . the pmf w[r31 ] , as a function of the distance between the two pulling centers r31 , can be evaluated by conducting smd runs of steering the third pulling center r3 to and from the first pulling center that is fixed at r1 along the axis passing through ( r1 , r3 ) . the -integral can be approximated by conducting equilibrium md runs with the first pulling center fixed at r1 to sample angular distribution . note that the direct sampling method in eq 24 is exact but it requires sufficient sampling in the 3d phase space ( r21 , r31 , ) , which is not an easy task . the approximation eq 26 can be evaluated with much less sampling effort in the dissociated state , but it involves the approximation in eq 25 that is somewhat arbitrary . when the two methods produce similar results ( as is the case in this work ) , one can be confident of the computation , of course . after all this , we have the free energy of binding the ligand to the binding site:27 the corresponding binding affinity is 28 note that , by pulling three centers of the ligand , it is easier to compute the pmf difference w[r10 , r20 , r30 ] w[r1 , r2 , r3 ] as the overall orientation of the ligand is fixed along the pulling paths . also , the computation of z30 is not difficult for complexes of strong binding whose equilibrium fluctuations can be well approximated as gaussian:29where 3 is a 9 9 matrix of coordinate deviations defined in eq 14 and 3 is defined in eq 13 with n = 3 . however , the computation of z3 will be considerably more elaborate than z2. nevertheless , for long ligands of irregular shapes , pulling three or more centers should be the optimal method for accurately computing the binding free energy or binding affinity . in an smd simulation of the current literature , one steers / pulls one center of mass of one selection of atoms , using a spring with a carefully chosen stiffness ( spring constant ) . the use of a spring of finite stiffness introduces fluctuations and dissipation in the added degrees of freedom . in this paper , we choose n segments ( mutually exclusive n selections of atoms ) of the ligand molecule for steering / pulling with n infinitely stiff springs ( n = 1 , 2 , 3 , ... ) . namely , the n centers of mass of the chosen n segments will be controlled as functions of time t30while all the other degrees of freedom of the system are freely subject to stochastic dynamics . here ri = ( xi , yi , zi ) is the center of mass coordinates of the ith segments . the + and signs are for the forward and reverse pulling paths , respectively . { ri } denotes ( r1 , r2 , ... , rn ) , etc . the path from the bound state to the dissociated state is divided into a number of sections . within a given section whose end states are marked as a and b , respectively , multiple forward and reverse pulling paths are sampled along which the work done to the system is recorded . the gibbs free energy difference ( namely , the pmf or the reversible work ) is computed via the brownian dynamics fluctuation dissipation theorem ( bd - fdt ) as follows:31here the brackets with subscript f and r represent the statistical average over the forward / reverse paths . w{ria } { ri } is the work done to the system along a forward path when the ligand is steered from a to r. w{ri}{ria } = w{rib}{ria } w{rib}{ri } is the work for the part of a reverse path when the ligand is pulled from r to a. { ria } , { ri } , and { rib } are the coordinates of the centers of mass of the steered segments of the ligand at the end state a , the general state r , and the end state b of the system , respectively . at each end of a section , a / b , the system is equilibrated for a time long enough to reach conditioned equilibrium while the steered centers are fixed at { ria}/{rib}. in this way , running smd section by section , we map the pmf w[{ri } ] as a function of the steered centers along a chosen path from the ligand s bound state to its dissociated state . in all the equilibrium md and nonequilibrium smd runs we implemented langevin stochastic dynamics with namd to simulate the systems at a constant temperature of 298 k and a constant pressure of 1 bar . full electrostatics was implemented by means of particle mesh ewald ( pme ) at 128 128 128 . the time step was 1 fs for short - range interactions and 2 fs for long - range interactions . selected -carbons on -helices and -sheets far from the binding site are fixed to their crystal structure coordinates , fully respecting the experimentally determined ligand protein structures . the pulling was along the z - axis at a speed of 2.5 / ns in all smd runs . namely , vd = ( 0 , 0 , 2.5 / ns ) . the simulation system of the oca lgb complex is illustrated in figure 2 , which was set up by taking the crystal structure of the protein ligand complex from the pdb ( code 3nq9 ) , putting it in the center of a box of water , neutralizing the system , and then salinating it with na and cl ions to the concentration of 150 mm . running equilibrium md for 25 ns leads to the equilibrated system shown in figure 2 . in particular , the equilibrium structure of oca bound to the protein is shown in figure 2d , where the c8 ( position vector r1 ) and c1 atoms ( position vector r2 ) were highlighted with the green and red balloons , respectively . these two atoms were chosen as the two steering / pulling centers for the nonequilibrium smd runs . at the binding site , the two pulling centers ( r1 , r2 ) fluctuate inside the binding pocket with small amplitudes ( shown in the supporting information , figure s1 ) . these fluctuations give us the following statistics at the binding site : the determinant of the deviation matrix det(2 ) = 2.42 10 and the deviation of the smd initial state from the pmf minimum 2 = 1.68 kcal / mol . lgb complex ( 95 296 atoms , 100 100 92 in dimension ) . na and cl ions are represented by vdw ( spheres ) colored in yellow and cyan , respectively ; water is represented by red and white dots ; protein is represented by ribbons colored by residue types ; and the ligand ( not easily visible ) is represented by licorices colored according to atom names . caprylic acid , visible on top of the figure , is represented by licorice while protein is represented by ribbons and by cpk ( ball - and - stick ) , both colored according to residue types . ( c ) caprylic acid ( in licorice colored in green ) resides in the binding pocket of the protein ( in surface representation colored according to atom names ) . the two groups ( highlighted in red and green respectively ) are selected for steering / pulling . conducting multiple smd runs starting from the initial state ( r10 , r20 ) to the final state ( r1 , r2 ) along the z - axis , we sampled four forward and four reverse pulling paths connecting the two states . the curves of work done to the system along the pulling paths are shown in the supporting information , figure s2 . from those work curves , we computed the pmf as a function of the z - displacement z shown in figure 3a . this pmf curve gives us the pmf difference between the one chosen bound state and its corresponding dissociated state:32 note that the pmf rises gradually all the way until z = 8 as the ligand is steered out of the binding pocket . this behavior clearly reflects the hydrophobic nature of the deep binding pocket of lgb and that of oca s long hydrocarbon chain . the van der waals attraction between oca and lgb is gradually reduced along the dissociation path , and meanwhile , the hydrophobic surfaces of lgb and oca are increasingly exposed to water as they are steered apart from one another . these two together are responsible for binding oca s hydrocarbon chain inside lgb s -barrel , giving rise to a large part of the binding free energy . there are two other parts of the binding free energy : first , fluctuations of oca inside the barrel as represented by z20 . second , the rotation and fluctuations of oca in the dissociated state represented by z2. in the dissociated state , we fixed the c8 atom at r1 and steered the c1 atom ( position vector r2 ) toward and away from c8 , sampling four forward paths and four reverse paths ( shown in the supporting information , figure s2 ) . from the work curves along those pulling paths , we obtained the pmf w[r ] in the dissociated state as a function of the c1c8 distance r shown in figure 3b . carrying out the integral of eq 18 using this pmf curve , we obtained the partial partition function in the dissociated state z2 = 2.01 10 . ( a ) pmf w[r1 , r2 ] as a function of the ligand displacement from its binding site along the pulling path when two centers are steered away from the protein . ( b ) pmf w[r ] in the dissociated state as a function of the distance r between the two steered centers ( the c1 and c8 atoms ) . w[r0 ] = 0 where r0 = 8.40 is the distance between c1 and c8 atoms of oca at the binding site . it is not surprising to note that the ligand conformation in the bound state represented by the distance between the two pulling centers ( c1 and c8 atoms)33is not optimal in the dissociated state . for a range of r values , the pmf has lower values:34 in the bulk region , away from the protein , the linear oca hydrocarbon carbon chain will fluctuate more freely than in the binding pocket of lgb . these effects constitute a significant contribution to the binding free energy represented in the partial partition function z2. putting all the afore - presented results together into eq 19 , we obtain the absolute binding energy of 5.7 0.6 kcal / mol ( corresponding to a dissociation constant of kd = 71 m ) that is in comparison with the in vitro result of 5.5 kcal / mol ( kd = 92.6 m ) . the simulation system box of the gsh sjgst(y7f ) complex was set up by taking the crystal structure of the protein ligand complex from the pdb ( code 1u87 ) , putting it in the center of a box of water , neutralizing the system , and then salinating it with na and cl ions to the concentration of 150 mm . running equilibrium md for 30 ns leads to the equilibrated system shown in figure 4 . in particular , the equilibrium structure of glutathione is shown in figure 4d , where the three -carbons ca1 ( position vector r2 ) , ca2 ( position vector r1 ) , and ca3 ( position vector r3 ) , were highlighted with the red , green , and blue balloons , respectively . these three atoms were chosen as the three steering / pulling centers for the nonequilibrium smd runs . at the binding site , the three pulling centers ( r1 , r2 , r3 ) fluctuate inside the binding pocket with small amplitudes ( shown in the supporting information , figure s3 ) . these fluctuations give us the following statistics at the binding site : the determinant of the deviation matrix det(3 ) = 2.53 10 and the deviation of the smd initial state from the pmf minimum 3 = 5.78 kcal / mol . conducting multiple smd runs starting from the initial state ( r10 , r20 , r30 ) to the final state ( r1 , r2 , r3 ) along the z - axis , we sampled four forward and four reverse pulling paths connecting the two states . the curves of work done to the system along the pulling paths are shown in the supporting information , figure s4 . from those work curves , we computed the pmf as a function of the z - displacement ( shown in figure 5 ) . this pmf curve gives us the pmf difference between the one chosen bound state and its corresponding dissociated state.35 ( a ) the in silico system box ( 114 538 atoms , 100 100 113 in dimension ) . na and cl ions are represented by vdw ( spheres ) colored in yellow and cyan respectively ; water is represented by red ( oxygen ) and white ( hydrogen ) balls and sticks ( cpk ) ; protein is represented by ribbons colored according to residue types ; and the ligand gsh ( not easily visible ) is represented by licorices colored according to atom names . ( b ) gsh ( licorice colored according to atom names ) in complex with the protein ( ribbons colored according to residue types and cpk colored according to atom names ) . ( c ) gsh ( licorice colored according to atom names ) in the binding pocket . here the protein surface ( colored according to atom names ) near or around the binding site is shown . ( d ) gsh in licorice with its three -carbons bubbled in red ( ca1 ) , green ( ca2 ) , and blue ( ca3 ) balloons . pmf w[r1 , r2 , r3 ] along the path of pulling gsh out of the binding pocket . pulling its center of mass ( n = 1 ) would not be efficient for sampling the relevant phase space . pulling three centers ( n = 3 ) turned out to be effective as indicated in the pmf curve of figure 5 . the pmf rises most rapidly during the first 1.5 of displacement , showing effectiveness of pulling the three -carbons of gsh simultaneously to separate its backbone from the protein . from 1.5 to 7 the oscillatory rise in pmf indicates separation of gsh side chains from the protein . after that , the pmf gradually rises some more and then levels off after 15 , indicating the ligand is in the bulk region ( dissociated from the protein ) . pulling three centers here is advantageous over pulling one center because the separation of the ligand from the protein , if pulled by one center , involves tight entanglement of the ligand s fluctuations in conformation and in orientation with the fluctuations of the many residues at and near the binding site . by pulling three centers , this disentanglement of two sets of fluctuations turned out to be very effective . in the dissociated state , we computed the partial partition function z3 in two ways . first , we used eq 24 on the long time equilibrium fluctuations of ca1 ( r2 ) and ca3 ( r3 ) around ca2 ( fixed at r1 ) shown in the supporting information , figure s5 . second , we computed the pmfs in eq 26 . fixing ca2 at r1 , we conducted smd runs steering ca1 ( r2 ) toward and away from ca2 , sampling four forward and four reverse pulling paths ( shown in the supporting information , figure s6a ) . likewise , steering ca3 ( r3 ) toward and away from ca2 , we sampled four forward and four reverse pulling paths ( shown in the supporting information , figure s6b ) . from those work curves , we extracted the pmf w[r21 ] as a function of the ca1ca2 distance in figure 6a and the pmf w[r31 ] as a function of the ca3ca2 distance in figure 6b . all these put together into eq 26 , we obtained the partition function z3 in the dissociated state . in two different ways described above , we obtained identical results for the partial partition function of the dissociated state , z3 = 1.2 10 . ( a ) pmf w[r21 ] as a function of the ca1ca2 distance in the dissociated state . ( b ) pmf w[r31 ] as a function of the ca3ca2 distance in the dissociated state . ca2 is fixed at r1. ca1 ( position vector r2 ) and ca3 ( position vector r3 ) are steered to and from ca2 in ( a ) and ( b ) , respectively . it is interesting to note that the pmf w[r31 ] ( figure 6b ) has a deep , nearly harmonic well centered at a ca3ca2 distance r31 |r3 this indicates that binding gsh to the protein does not significantly stretch ca3 from ca2 . in contrast , the pmf w[r21 ] as a function of the ca1ca2 distance ( figure 6a ) behaves very differently . the pmf well is anharmonic and its minimum is lower than the pmf value at the ca1ca2 distance |r2 binding gsh to the protein actually causes ca1 to stretch away from ca2 and reduces the fluctuations of ca1 . putting together the bound state fluctuations , the pmf difference , and the dissociated state fluctuations into eq 27 , we obtain the absolute binding energy of 7.0 0.9 kcal / mol ( corresponding to a dissociation constant of kd = 8.2 m ) that is in comparison with the itc result of 6.75 kcal / mol ( kd = 13 m ) . in terms of a computational approach , we have developed a hybrid steered molecular dynamics approach for computing absolute binding energy from the pmf along a dissociation path . applying this hsmd approach with high - performance parallel processing , one can achieve , within a few wall - clock days , the computation of the binding affinity of one ligand the hsmd approach is brute force in the sense that one does not have to delicately devise biasing and constraining potentials during the course of simulations . also , it does not involve sophisticated ways of removing the artifacts introduced by biasing / constraining the ligand in other pmf - based and non - pmf - based approaches . hsmd can be implemented straightforwardly by steering / pulling the n centers of mass of n chosen segments of a ligand using n infinitely stiff springs along one predetermined dissociate path , disallowing any deviations from the path . this use of a single path is correct because the pmf is a function of state . therefore , the pmf difference between two states is independent of the paths connecting them . all other contributions , in addition to the pmf difference between the two end states of this one dissociation path , are rigorously accounted for in the partial partition functions of the bound and the dissociated states . the segments chosen to be steered , however , need to be the most stable parts of the ligand in the bound state because the coordinate integrations of these centers then can be approximated as gaussian . the gaussian approximation is expected to be valid for a wide range of ligand protein complexes because there should always be at least one center of a ligand that does not deviate greatly from its bound - state coordinates but is tightly bound to the protein except for the cases of very weak binding . another possible difficulty for the hsmd approach lies in the coordinate integration in the dissociated state when three segments centers of mass are steered . the approximation in eq 26 is based on the assumptions that two of the three pulling centers do not have significant contribution from the stereo collision between them and that the angle between the lines they form with the other center is approximately free to bend . when these two assumptions are not valid , the coordinate integration in eq 23 will necessitate further new schemes of approximation . in terms of biophysics , we have provided atomistic details in support of the binding mechanisms of two ligand protein complexes elucidated in the experimental investigations . for both oca glb and gsh sjgst(y7f ) complexes , our equilibrium md simulations confirm the experimentally determined binding conformations . during the long time dynamics , the ligands were found to fluctuate with small deviations at the binding sites determined in the crystal structures of the ligand protein complexes ( see figures 2b , c and 4b , c and the supporting information , figures s1 and s3 ) . also , our computed dissociation constants agree with the experimentally measured values within a factor of 1.5 . therefore , it is expected that hsmd can be used to reliably predict binding affinities of ligand protein complexes whose structures are available in the pdb without data for binding affinities yet and that the hsmd predictions would be validated by future experimental measurements . finally , the agreement between the computed results in this work and the experimental data was based on the charmm force field , indicating its accuracy . the hsmd approach , however , is independent of which force field to use . it can be implemented with other force fields , of course , which may or may not produce similar results .

computing the free energy of binding a ligand to a protein is a difficult task of essential importance for which purpose various theoretical / computational approaches have been pursued . in this paper , we develop a hybrid steered molecular dynamics ( hsmd ) method capable of resolving one ligand protein complex within a few wall - clock days with high enough accuracy to compare with the experimental data . this hsmd approach is based on the relationship between the binding affinity and the potential of mean force ( pmf ) in the established literature . it involves simultaneously steering n ( n = 1 , 2 , 3 , ... ) centers of mass of n selected segments of the ligand using n springs of infinite stiffness . steering the ligand from a single initial state chosen from the bound state ensemble to the corresponding dissociated state , disallowing any fluctuations of the pulling centers along the way , one can determine a 3n - dimensional pmf curve connecting the two states by sampling a small number of forward and reverse pulling paths . this pmf constitutes a large but not the sole contribution to the binding free energy . two other contributors are ( 1 ) the partial partition function containing the equilibrium fluctuations of the ligand at the binding site and the deviation of the initial state from the pmf minimum and ( 2 ) the partial partition function containing rotation and fluctuations of the ligand around one of the pulling centers that is fixed at a position far from the protein . we implement this hsmd approach for two ligand protein complexes whose structures were determined and whose binding affinities were measured experimentally : caprylic acid binding to bovine -lactoglobulin and glutathione binding to schistosoma japonicum glutathione s - transferase tyrosine 7 to phenylalanine mutant . our computed binding affinities agree with the experimental data within a factor of 1.5 . the total time of computation for these two all - atom model systems ( consisting of 96k and 114k atoms , respectively ) was less than one wall - clock week using 512 cores ( 32 xeon e5 - 2680 processors ) .

Introduction Methods Results Discussion

accurately computing the free energy of binding a ligand to a protein is a task of essential importance in biochemical and biophysical studies that still presents us considerable difficulty to overcome . an effective approach in the current literature is to use the relationship between the potential of mean force ( pmf ) and the binding affinity . the nonequilibrium steered molecular dynamics ( smd ) approach , seemingly brute force , can be very efficient in sampling forced transition paths from the bound state to the dissociated state of the ligand but has not been used reliably for free - energy calculations with quantitative accuracy . in this paper , we present a hybrid steered molecular dynamics ( hsmd ) approach that produces binding affinities in quantitative agreement with experimental measurements ( within a factor of 1.5 in terms of the dissociation constant kd defined as the ligand concentration at which the holoprotein concentration equals the apoprotein concentration ) . the hsmd approach is based on the relationship between the pmf and the binding affinity in the established literature . in contrast , the hsmd approach involves pulling n ( n = 1 , 2 , 3 , ... ) centers of mass of n selected segments of the ligand ( using n springs of infinite stiffness to disallow any fluctuation of the pulling centers along the way ) to produce a 3n - dimensional ( 3n - d ) pmf curve leading from the binding site on or inside the protein to the dissociated state in the bulk region far from the protein . this pmf difference between the bound state and the dissociated state gives a large ( but not dominant ) part of the absolute binding free energy . another part of the hsmd approach is the equilibrium molecular dynamics ( md ) sampling of the ligand s fluctuations at the binding site that also contribute to the binding free energy . the third , final , part of the hsmd approach is smd stretching and equilibrium md sampling of the ligand in the dissociated state when one of the n pulling centers is fixed at a point in the bulk far from the protein . protein complexes whose binding affinities were experimentally measured and whose crystal structures are available : oca glb , caprylic acid ( 25 atoms , neutral ) bound to bovine -lactoglobulin ; and gsh sjgst(y7f ) , glutathione ( 36 atoms , singly negatively charged ) bound to schistosoma japonicum glutathione s - transferase tyrosine 7 to phenylalanine mutant . the computing times required were approximately 62 wall - clock hours for oca glb ( all - atom model of 95 296 atoms ) and 88 wall - clock hours for gsh sjgst(y7f ) ( all - atom model of 114 538 atoms ) , respectively , using 32 xeon e5 - 2628 processors ( 512 cores ) in parallel . the subscripts site and bulk indicate that r1 is near the pmf minimum and r1 = r1 in the bulk region far from the protein , respectively . instead , the ligand can be better described with the positions ( r1 , r2 , ... , rn ) of n centers of mass of its n chosen segments . in the dissociated state , one of them will be fixed at r1 = r1 while all others ( r2 , ... , rn ) rotate and fluctuate according to the stochastic dynamics of the system without any other bias / constraint . note that in the relationship between the 3d and 3n - d pmfs2the 3(n 1)-d integration over the ( n 1 ) positions ( r2 , ... , rn ) is effectively in a defined neighborhood of r1 because the ligand as one whole molecule dictates that the n centers can not be stretched much farther from one another than its molecular size . when r1 is near the binding site , when the ligand is in the dissociated state , r1 needs to be so far from the protein that integration over ( r2 , ... , rn ) will be all in the region far from the protein . making use of eq 2 twice in eq 1 ( for the binding site and for the bulk ) , one has the following expression for the binding affinity.3 now inserting the boltzmann factor at a single state ( r10 , r20 , ... , rn ) chosen from the bound state ensemble and the boltzmann factor at the corresponding dissociated state ( r1 , r2 , ... , rn ) , the binding affinity can be expressed as three contributing factors : the partial partition function at the binding site zn0 , the pmf difference between two chosen states ( r10 , r20 , ... , rn0 ) and ( r1 , r2 , ... , rn ) , and the partial partition function in the dissociated state zn. computation of the pmf difference between the two states can be achieved along a single curve passing through them both . the partial partition function zn0 of the bound state has the integration over all n centers and thus has the units of .5 the partial partition function zn of the dissociated state has the integration over n 1 centers and thus has the units of .6 again , the use of c0 = 6.02 10/ on the right - hand side of eq 3 renders it a pure number as desired . the one state chosen from the dissociated state ensemble9is related to the smd starting point by a large enough displacement in the 3n - d space.10here vd is the constant velocity of the smd pulling and t is the time it takes to steer / pull the ligand from the binding site to the bulk . ( 1 ) the first factor is the pmf difference between one chosen bound state and its corresponding dissociated state that can be computed by running smd simulations of pulling the ligand forward and backward along a 3n - d line connecting the two states . note that the pmf is a function of state ( a point in the 3n - d space ) and the pmf difference is independent of the paths connecting the two end points . ( 2 ) the second factor is the partial partition function in the bound state that can be approximated as gaussian in cases of strong binding or can be numerically evaluated by running equilibrium md simulation . ( 3 ) the third factor is the partial partition function in the dissociated state that needs to be computed case by case for n = 2 , 3 , .... when the binding is tight , one can approximate the integral of eq 5 as gaussian in the neighborhood of the pmf minimum . carrying out the gaussian integral , one has12here the dimensionless quantity n / kbt gives a measure of how far ( r10 , r20 , ... , rn0 ) , the initial state chosen for smd , is from the pmf minimum ( r1 , r2 , ... n is the 3n 3n matrix of the fluctuations / deviations of the pulling center coordinates x1 , in the bound state ensemble:14 n1 is the inverse matrix of n which can be accurately evaluated by running equilibrium md in the bound state of the ligand this approximation is generally valid if the ligand does not deviate much from the binding site . to decide whether the gaussian approximation is suitable or not , one can evaluate how far from the gaussian distribution is the distribution of the fluctuations at the binding site . note that this evaluation does not require additional equilibrium md runs in the bound state of the ligand for example , by computing the first to the fourth moments of the fluctuations and checking their relationships , one can readily determine the non - gaussian - ness of the fluctuations . unlike the partial partition function zn0 of the bound state , the computation of the partial partition function of the dissociated state , zn in eq 6 , needs to be done individually for each case of n = 1 , 2 , .... in the following , we detail three cases : n = 1 , 2 , and 3 . when one center of mass is steered , n = 1 , z1 = 1 , all we have to do is to evaluate the fluctuations around the binding site ( the 3 3 matrix 1 ) along with the pmf difference . in this case , we have the dissociate constant , within the gaussian approximation of the fluctuations at the binding site15and the absolute free energy of binding the ligand to the protein16 it needs to be pointed out that the method of pulling one center of the ligand is only practical for small ligands of simple shapes . when two centers of mass are steered , n = 2 , one has17which is an integration over the second steering center when the first steering center is fixed in a position r1 far from the protein . over there , far from the protein , the ligand s environment is spherically symmetrical around the position of the first pulling center r1 that is fixed while the second pulling center r2 is free to sample all space available . in this , we have the absolute free energy of binding the ligand to the binding site19and the dissociation constant20here the partial partition function of the dissociated state z2 is given in eq 18 and the partial partition function of the bound state is approximated below as gaussian.21where 2 is a 6 6 matrix of coordinate deviations defined in eq 14 and 2 is defined in eq 13 with n = 2 . when three centers of mass are steered , n = 3 , we need to evaluate the integral of eq 6 around the position ( r1 , r2 , r3 ) when the ligand is far from the protein . the pmf w[r21 ] , as a function of the distance between the two pulling centers r21 , can be evaluated by conducting smd runs of steering the second pulling center r2 to and from the first pulling center that is fixed at r1 along the axis passing through ( r1 , r2 ) . the pmf w[r31 ] , as a function of the distance between the two pulling centers r31 , can be evaluated by conducting smd runs of steering the third pulling center r3 to and from the first pulling center that is fixed at r1 along the axis passing through ( r1 , r3 ) . after all this , we have the free energy of binding the ligand to the binding site:27 the corresponding binding affinity is 28 note that , by pulling three centers of the ligand , it is easier to compute the pmf difference w[r10 , r20 , r30 ] w[r1 , r2 , r3 ] as the overall orientation of the ligand is fixed along the pulling paths . in this paper , we choose n segments ( mutually exclusive n selections of atoms ) of the ligand molecule for steering / pulling with n infinitely stiff springs ( n = 1 , 2 , 3 , ... ) . the path from the bound state to the dissociated state is divided into a number of sections . within a given section whose end states are marked as a and b , respectively , multiple forward and reverse pulling paths are sampled along which the work done to the system is recorded . { ria } , { ri } , and { rib } are the coordinates of the centers of mass of the steered segments of the ligand at the end state a , the general state r , and the end state b of the system , respectively . in this way , running smd section by section , we map the pmf w[{ri } ] as a function of the steered centers along a chosen path from the ligand s bound state to its dissociated state . the simulation system of the oca lgb complex is illustrated in figure 2 , which was set up by taking the crystal structure of the protein ligand complex from the pdb ( code 3nq9 ) , putting it in the center of a box of water , neutralizing the system , and then salinating it with na and cl ions to the concentration of 150 mm . these fluctuations give us the following statistics at the binding site : the determinant of the deviation matrix det(2 ) = 2.42 10 and the deviation of the smd initial state from the pmf minimum 2 = 1.68 kcal / mol . conducting multiple smd runs starting from the initial state ( r10 , r20 ) to the final state ( r1 , r2 ) along the z - axis , we sampled four forward and four reverse pulling paths connecting the two states . this pmf curve gives us the pmf difference between the one chosen bound state and its corresponding dissociated state:32 note that the pmf rises gradually all the way until z = 8 as the ligand is steered out of the binding pocket . these two together are responsible for binding oca s hydrocarbon chain inside lgb s -barrel , giving rise to a large part of the binding free energy . from the work curves along those pulling paths , we obtained the pmf w[r ] in the dissociated state as a function of the c1c8 distance r shown in figure 3b . carrying out the integral of eq 18 using this pmf curve , we obtained the partial partition function in the dissociated state z2 = 2.01 10 . ( a ) pmf w[r1 , r2 ] as a function of the ligand displacement from its binding site along the pulling path when two centers are steered away from the protein . it is not surprising to note that the ligand conformation in the bound state represented by the distance between the two pulling centers ( c1 and c8 atoms)33is not optimal in the dissociated state . these effects constitute a significant contribution to the binding free energy represented in the partial partition function z2. these fluctuations give us the following statistics at the binding site : the determinant of the deviation matrix det(3 ) = 2.53 10 and the deviation of the smd initial state from the pmf minimum 3 = 5.78 kcal / mol . conducting multiple smd runs starting from the initial state ( r10 , r20 , r30 ) to the final state ( r1 , r2 , r3 ) along the z - axis , we sampled four forward and four reverse pulling paths connecting the two states . this pmf curve gives us the pmf difference between the one chosen bound state and its corresponding dissociated state.35 ( a ) the in silico system box ( 114 538 atoms , 100 100 113 in dimension ) . pulling three centers here is advantageous over pulling one center because the separation of the ligand from the protein , if pulled by one center , involves tight entanglement of the ligand s fluctuations in conformation and in orientation with the fluctuations of the many residues at and near the binding site . in the dissociated state , we computed the partial partition function z3 in two ways . first , we used eq 24 on the long time equilibrium fluctuations of ca1 ( r2 ) and ca3 ( r3 ) around ca2 ( fixed at r1 ) shown in the supporting information , figure s5 . in two different ways described above , we obtained identical results for the partial partition function of the dissociated state , z3 = 1.2 10 . putting together the bound state fluctuations , the pmf difference , and the dissociated state fluctuations into eq 27 , we obtain the absolute binding energy of 7.0 0.9 kcal / mol ( corresponding to a dissociation constant of kd = 8.2 m ) that is in comparison with the itc result of 6.75 kcal / mol ( kd = 13 m ) . in terms of a computational approach , we have developed a hybrid steered molecular dynamics approach for computing absolute binding energy from the pmf along a dissociation path . applying this hsmd approach with high - performance parallel processing , one can achieve , within a few wall - clock days , the computation of the binding affinity of one ligand the hsmd approach is brute force in the sense that one does not have to delicately devise biasing and constraining potentials during the course of simulations . hsmd can be implemented straightforwardly by steering / pulling the n centers of mass of n chosen segments of a ligand using n infinitely stiff springs along one predetermined dissociate path , disallowing any deviations from the path . all other contributions , in addition to the pmf difference between the two end states of this one dissociation path , are rigorously accounted for in the partial partition functions of the bound and the dissociated states . the approximation in eq 26 is based on the assumptions that two of the three pulling centers do not have significant contribution from the stereo collision between them and that the angle between the lines they form with the other center is approximately free to bend . in terms of biophysics , we have provided atomistic details in support of the binding mechanisms of two ligand protein complexes elucidated in the experimental investigations . during the long time dynamics , the ligands were found to fluctuate with small deviations at the binding sites determined in the crystal structures of the ligand protein complexes ( see figures 2b , c and 4b , c and the supporting information , figures s1 and s3 ) . also , our computed dissociation constants agree with the experimentally measured values within a factor of 1.5 . therefore , it is expected that hsmd can be used to reliably predict binding affinities of ligand protein complexes whose structures are available in the pdb without data for binding affinities yet and that the hsmd predictions would be validated by future experimental measurements . finally , the agreement between the computed results in this work and the experimental data was based on the charmm force field , indicating its accuracy .

ovarian cancer is the most lethal and second most common gynecologic malignancy in the united states , with an estimated 21,980 new cases and 14,270 deaths expected in 2014 . first progression typically occurs within 18 months , and overall survival ( os ) is typically < 4 years [ 25 ] . most patients are present with advanced disease , and the current standard of care in the primary setting is surgical debulking followed by platinum - based chemotherapy . ovarian cancer is a heterogeneous disease with respect to histopathology , molecular biology , and clinical outcome , suggesting that a single standard treatment is unlikely to benefit all patients . histologically , most ovarian cancer arises from the distal fallopian tube or ovarian surface , and the majority of these epithelial ovarian cancers ( eoc ) are serous / papillary pathological subtype , followed by endometrioid , mucinous , clear cell , and undifferentiated . these different subtypes together with other clinical factors including age , performance status , figo stage , differentiation , ascites presence , and surgical debulking status the cancer genome atlas project found that more than 30 growth - stimulating genes were altered across different ovarian cancer subtypes . these alterations included : pi3k pathway activation , brca1 or brca2 mutations , other dna repair defects and varied expression status of er , cyclin e2 , and kit . this molecular heterogeneity may be linked to clinical heterogeneity , such as histological subtype presentation , disease prognosis , and chemotherapy efficacy . carboplatin / paclitaxel has been widely accepted as the standard of care in treating primary eoc for nearly two decades [ 25 ] . multiple alternate regimens have been investigated , most of them based on the platinum / taxane standard , but augmented with additional chemotherapies and/or altered sequencing ( table 1 ) [ 25 ] . many studies have randomized patients across various regimens in an effort to identify regimens superior to the carboplatin / paclitaxel standard . these studies have consistently demonstrated remarkably similar progression - free survival ( pfs ) and os between the standard of care and the various alternates , highlighting the therapeutic equivalence of the various regimens and the associated empiric treatment ambiguity.table 1patient characteristics of control arm and assay - informed arm cohortspatient characteristicscontrol armassay - informed armdu bois jnci 2003pfisterer jnci 2006du bois jco 2006bookman jco 2009herzog ajog 2010number of patients7831,3081,2824,312192median age5760595959pathological subtypeserous / papillary70 % 71 % 73 % 80 % 71 % other30 % 29 % 27 % 20 % 29 % figo stage distributionstage i<1 % < 1 % stage ii8 % 9 % 9 % stage iii75 % 74 % 74 % 85 % 84 % stage iv17 % 17 % 17 % 15 % 16 % debulking statusoptimal63 % 67 % 68 % 68 % 52 % sub - optimal37 % 33 % 32 % 32 % 48 % treatment regimenscpcispcpcptcpcpecpcpgcpdct / cpcg / cpcpcpgcispctother c carboplatin , p paclitaxel , g gemcitabine , d doxorubicin , e epirubicin , t topotecan , cis cisplatin patient characteristics of control arm and assay - informed arm cohorts c carboplatin , p paclitaxel , g gemcitabine , d doxorubicin , e epirubicin , t topotecan , cis cisplatin for patients with recurrent , persistent , or progressive disease , chemotherapy choice is currently based , in part , on the duration and type of response to initial therapy . for platinum - sensitive disease [ progression - free interval ( pfi ) 6 months from the end of platinum / taxane therapy ] , a platinum - based combination regimen is usually empirically selected . for platinum - resistant disease ( pfi < 6 months ) , physicians empirically select from an array of non - platinum regimens , including pegylated liposomal doxorubicin ( pld ) , topotecan , gemcitabine , etoposide , taxanes , and targeted therapies , all of which have been evaluated and demonstrated to be clinically equivalent and acceptable for use in this patient population . while marker identification and development in ovarian cancer is generally limited to early detection , monitoring progression , or detecting recurrence , there are some encouraging preliminary studies linking markers with drug response , thereby demonstrating early potential for informing effective individualized chemotherapy selection . for example , expression of copper importers / exporters , ercc1 , tau , gst - pi , mlh1 , and xiap , and mutations of mlh1 , brca1/brca2 , and p53 have been linked to platinum response , and expression of tgfbi , survivin , and mutation of tubulin are associated with response to paclitaxel [ 821 ] . however , none of these biomarkers have demonstrated sufficient clinical validation required to inform clinical treatment decisions . the national institutes of health ( nih ) biomarkers definitions working group , which includes leaders in the field from the food and drug administration ( fda ) , nih , academia and industry , defines a marker as a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes , pathogenic processes , or pharmacologic responses to a therapeutic intervention . a marker was similarly described by hayes et al . as a molecular , cellular , tissue , or process - based alteration that provides indication of current , or more importantly , future behavior of a cancer . a chemoresponse assay reports a panel of markers characterizing a tumor s response to multiple chemotherapy agents . each of the multiple chemotherapy assay results reported is a singular marker associated with a distinct treatment . such assays provide tumor response information aimed at aiding in the selection of effective , individualized treatment regimens . chemoresponse assays provide the same utility as other treatment markers that are associated with patient outcome when the given marker s associated treatment is clinically administered ( e.g. , kras and cetuximab / panitumumab , egfr and erlotinib , her2 and trastuzumab ) . chemoresponse assays are generally based on phenotypic rather than molecular characterization , thus enabling assays to simultaneously report multiple treatment markers , each associated with a distinct treatment , for a given patient . the concept of a chemoresponse assay or chemotherapy sensitivity and resistance assay ( csra ) , originated in the 1950s . there are different types of csras such as the adenosine triphosphate ( atp ) assay , human tumor cloning assay ( htca ) , methylthiazolyl - diphenyl - tetrazolium bromide ( mtt ) assay , extreme drug resistance ( edr ) assay , as well as assays utilizing drug - induced apoptosis as the end point [ 24 , 26 , 27 ] . a high - impact htca study published in 1978 was followed by decades of research from various academic groups and a few commercial entities with mixed results [ 2831 ] . while other csra reviews have been published previously [ 24 , 26 ] , this review will focus on progress made during the most recent decade . the value of csras to inform effective treatment selection for individual patients remains a compelling clinical question and a highly debated topic among oncologists . while the advantages and disadvantages of various csra methods have been published , clinical validations demonstrating association of assay results with patient outcomes through prospective studies have the most value . a prospective histoculture drug response assay ( hdra ) study in advanced eoc patients ( n = 104 ) treated with carboplatin and paclitaxel after cytoreductive surgery demonstrated a lower recurrence rate and extended pfs , both of which were statistically significant , in the hdra - sensitive group as compared to the hdra - resistant group . another prospective study , utilizing an atp - based chemoresponse assay , evaluated response rate and pfs in platinum - resistant recurrent eoc patients ( n = 180 ) randomized to assay - directed or physician s empiric therapy choice , demonstrating trends for improved response rate and pfs for assay - informed treatment . and , finally , a prospective study of 113 recurrent eoc patients showed that patients whose treatment was determined by an atp - based chemoresponse assay had statistically longer pfs and higher overall response rates compared with patients receiving physicians - choice therapy . numerous retrospective data have also been published in the past decade , with the majority reporting statistically significant associations between assay results and clinical outcomes [ 3537 ] . the results from these various studies reasonably demonstrate the clinical potential of chemoresponse assays in both primary and recurrent eoc . although several chemoresponse assays clinical validity and clinical utility have been evaluated in clinical trials , there are currently only two assays commercially available in the united states : the microculture - kinetic ( mick ) assay ( diatech oncology , franklin , tn ) and the chemofx assay ( precision therapeutics , inc . , pittsburgh , pa ) . the mick assay is based on drug - induced apoptosis and was originally developed in hematologic malignancies where it was noted that chemotherapeutic drugs have the ability to rapidly induce apoptosis in tumor cells in short - term culture . the assay was later applied to solid tumors , including breast , lung , and gynecologic malignancies [ 27 , 3841 ] . clinical validation of the mick assay in 73 ovarian cancer patients demonstrated that clinical treatment with the assay - indicated best chemotherapy is an independent predictor of os in multivariate analysis of chemotherapy - nave stage iii or iv primary ovarian cancer patients . a clinical utility study of 44 cancer patients showed that oncologists used the mick assay to determine chemotherapy selection in 28 patients ( 64 % ) and did not use the assay in treatment selection for the other 16 patients ( 36 % ) . the median os was 10.1 months for the assay - informed patients vs. 4.1 months for the assay - uninformed patients ( p = 0.02 ) . however , the 44 tumors in the study included a variety of tissue types , such as breast and non - small lung cancers ; only two ovarian cancer tumors were included in the study . therefore , the clinical validation and clinical utility of this assay in ovarian cancer requires further investigation . the chemofx chemoresponse assay has been extensively evaluated in patients with ovarian cancer and will be the focus of the remainder of this review . chemofx is a chemoresponse assay that characterizes both the sensitivity and resistance of a patient s tumor to various physician - selected , clinically applicable chemotherapy treatments . it quantifies chemotherapy effect by direct visualization and enumeration of live cells following exposure to these treatments . the assay is performed in a clinical laboratory improvement amendments ( clia ) and new york state department of health ( nysdoh ) approved facility . icc immunocytochemistry , auc area under curve , s sensitive , is intermediate sensitive , r resistant chemofx assay process . icc immunocytochemistry , auc area under curve , s sensitive , is intermediate sensitive , r resistant in contrast to other csras , this chemoresponse assay is characterized by several features that make it more reproducible and clinically accessible.the assay uniquely insures that tumor cells are proliferating prior to chemotherapy exposure , thereby measuring treatment efficacy at halting proliferation and/or killing tumor cells . this approach accommodates the cell cycle - specific , cytostatic , and cytotoxic natures of various chemotherapies.the assay s primary culture process is optimized to generate sufficient proliferating tumor cells for testing . as a result , 9 out of 10 ovarian cancer samples meeting the incoming sample criteria , such as sufficient sample size and absence of microorganism contamination , are successfully reported.the culture process favors epithelial tumor cell proliferation and incorporates an immunocytochemistry ( icc ) step to insure that the majority of cells tested are epithelial.the assay process is highly automated . cell seeding into microtiter plates , serial treatment dilution and application , cell fixation , fluorescence staining , as well as cell enumeration are performed using automated liquid handling robotics , computer - assisted microscopy , and automated cell - counting algorithms and software . the automated process strongly contributes to the high throughput and reproducibility of the assay .lastly , this assay requires significantly less tissue ( a minimum of 35 mm ) , as compared to historical assays . tumor tissue from surgical excision , biopsy , or paracentesis is compatible , making the assay highly clinically accessible . the assay uniquely insures that tumor cells are proliferating prior to chemotherapy exposure , thereby measuring treatment efficacy at halting proliferation and/or killing tumor cells . this approach accommodates the cell cycle - specific , cytostatic , and cytotoxic natures of various chemotherapies . the assay s primary culture process is optimized to generate sufficient proliferating tumor cells for testing . as a result , 9 out of 10 ovarian cancer samples meeting the incoming sample criteria , such as sufficient sample size and absence of microorganism contamination , are successfully reported . the culture process favors epithelial tumor cell proliferation and incorporates an immunocytochemistry ( icc ) step to insure that the majority of cells tested are epithelial . cell seeding into microtiter plates , serial treatment dilution and application , cell fixation , fluorescence staining , as well as cell enumeration are performed using automated liquid handling robotics , computer - assisted microscopy , and automated cell - counting algorithms and software . lastly , this assay requires significantly less tissue ( a minimum of 35 mm ) , as compared to historical assays . tumor tissue from surgical excision , biopsy , or paracentesis is compatible , making the assay highly clinically accessible . the analytical performance of this assay has been previously reported [ 42 , 43 , 46 ] . heinzman et al . demonstrated a coefficient of variation ( cov ) of 3.64.6 % for sk - ov-3 cells treated with doxorubicin , across three operators and 9 days . in addition to variability across operators and days , process variability due to inter- and intraday stability of the chemotherapeutic treatments has also been reported . the assay has demonstrated the necessary analytical performance characteristics required by both clia and nysdoh . demonstrated the association of assay response with pfs in 256 eoc patients . in patients with either an exact or partial match between treatments assayed and those that were clinically administered , the hazard ratio ( hr ) for progression in patients clinically treated with an assay - resistant ( r ) vs. assay - sensitive ( s ) treatment was 2.1 ( 95 % ci 1.23.6 , p = 0.01 ) . in the subset of 135 patients with an exact match , the hr for progression in patients clinically administered an assay - r vs. assay - s treatment was 2.9 ( 95 % ci 1.46.3 , p < 0.01 ) . the median pfs for patients treated with r therapies was 9 and 14 months for those treated with intermediate sensitive ( is ) therapies . furthermore , at the time of study completion with a median follow - up time of 14.6 months , 60 % of patients treated with s therapies remained relapse - free . herzog et al . subsequently reported an association between assay response and os in 192 patients with advanced eoc following first - line platinum - based chemotherapy . median os was 72.5 , 48.6 , and 28.2 months for patients who were treated with agents reported as s , is , and r , respectively ( hr = 0.7 , 95 % ci 0.500.97 , p = 0.03 ) . multivariate cox regression analysis demonstrated that the assay prediction of response to platinum agents was a predictor of os independent of other prognostic factors of stage , age , and optimal debulking ( hr = 0.68 , 95 % ci 0.490.95 , p = 0.023 ) . in another more recent observational study of 276 women with figo stage iii - iv eoc cancer uniformly treated with first - line carboplatin-/paclitaxel - based therapy , patients with assay - r results for carboplatin were at increased risk of disease progression ( as defined by pfs ) compared with patients with s or is assay results ( hr = 1.87 , 95 % ci 1.292.70 , p = 0.0009 ) ; these results were consistent after controlling for clinical covariates ( hr = 1.71 , 95 % ci 1.122.62 , p = 0.013 ) . median pfs for patients who were assay - r to carboplatin was 11.8 vs. 16.6 months for assay - is and assay - s patients . this study demonstrates that assay resistance to carboplatin is associated with reduced pfs in eoc patients treated with standard of care carboplatin / paclitaxel , supporting the assay s ability to identify platinum - resistant patients . furthermore , of those patients who were resistant to carboplatin in vitro , 59 % of them displayed assay sensitivity ( s or is ) to at least one other agent . finally , a prospective study of 262 women with recurrent or persistent eoc reported that patients treated with an assay - s regimen experienced significantly improved pfs ( hr = 0.67 , 95 % ci 0.500.91 , p = 0.009 ) and os ( hr = 0.61 , 95 % ci 0.410.89 , p = 0.010 ) compared with those treated with assay - is or assay - r regimens , resulting in a 14-month improvement in median os . assay - pfs association was consistent in both platinum - sensitive and platinum - resistant tumors ( hr : 0.71 and 0.66 , respectively ) and was independent of other covariates ( hr = 0.66 , 95 % ci 0.470.94 , p = 0.020 ) . moreover , the results indicated that more than 50 % of the patients had at least one s therapy identified by the assay , whereas only 25 % of them were empirically treated with an s drug , suggesting that the number of patients potentially experiencing improved os may more than double when physicians reference the assay . a further analysis of the 262 recurrent or persistent eoc patients reported by rutherford et al . was presented at the 2013 european cancer organization ( ecco ) biennial meeting and addressed the assay s ability to function as a predictive marker . a prognostic assay identifies patients likely to respond / not respond to ( any ) therapy , while a predictive assay identifies a patient 's likely response to specific therapies , which is particularly important for individualized chemotherapy selection . four different analytical methods were used in the study to assess the predictive value of the assay . these analyses provide the evidence that this chemoresponse assay is a predictive marker , demonstrating its ability to discern specific therapies that are likely to be more effective among multiple alternatives . as briefly outlined earlier , a chemoresponse assay , such as chemofx , is a panel of treatment markers , with the assay result for each treatment evaluated functioning as a distinct marker . when ordering the assay , a physician selects each of the multiple treatments under consideration for a given patient for inclusion in the assay . clinically validated chemotherapy regimens , consistent with guidelines such as nccn , comprise the available treatment choices . clinical trials designed to evaluate marker or assay efficacy are fundamentally different than trials designed to evaluate drug efficacy . various different marker trial designs have been extensively studied and reported in the recent literature . however , discussion of effective marker validation trial designs that are appropriate for multiple markers / therapies to be assessed simultaneously ( e.g. , chemoresponse assays ) remains very limited in the current trial design literature . marker validation trial designs for this type of multiple markers / therapies assay may vary , to some extent , from marker trial designs appropriate for a single molecular biomarker associated with a single therapy ( e.g. , kras / panitumumab , egfr / erlotinib ) . primarily , three marker study designs have been outlined in the literature for marker validation : enrichment , strategy , and stratified [ 5153 ] . marker negative patients are excluded in the enrichment design , and thus , it is not applicable to chemoresponse assay evaluation . historically , the strategy design , in many ways similar to a standard drug trial design , has been considered the gold standard for marker validation as it attempts to emulate what might occur in clinical practice . a variant of the strategy design has been recommended by the american society of clinical oncology ( asco ) and blue cross blue shield ( bcbs ) technical evaluation center ( tec ) assessments of validations of chemoresponse assays [ 54 , 55 ] date back to the mid 1990s . however , multiple recent and updated marker trial design publications , including an evaluation by the center for medical technology policy ( cmtp ) in 2013 , indicate that the strategy design is less than ideal for marker validation , in that it requires a larger sample size and can not distinguish between a more effective treatment and marker efficacy , when compared to alternate marker trial designs . show that the required sample size for a strategy design can exceed several thousand patients depending on the prevalence of the marker in the study population , presenting a large challenge in a small incidence / prevalence disease like eoc . further and specific to chemoresponse assays where multiple markers are evaluated simultaneously , the pan - resistant and pan - sensitive patients dilute the ability to assess assay impact on patient outcome . additionally , overlapping treatments between study arms still further increase the required sample size , rendering the strategy approach essentially pragmatically infeasible . finally , a potential physician treatment bias or learning effect may be associated with the strategy design . the strategy trial design attempted by cree et al . showed a trend toward improved response rate and pfs in assay - informed patients as compared to those treated with the physician s empiric choice , but did not achieve statistical significance . asserted that physicians learned from the assay - informed arm and began administering treatments similar to those recommended for assay - informed patients to patients in the physician - directed arm as the study progressed . analysis confirmed this effect ; in early physician - choice arm patients , pfs was significantly shorter than that in subsequent year patients . the stratified design has been reported as more efficient , capable of answering the relevant clinical questions , and able to assess both prognostic and predictive marker properties , which is often at issue with evaluations of markers [ 52 , 56 , 57 ] . have concluded that trial designs , such as the stratified design that use the marker to guide analysis , but not treatment assignment ( i.e. , blind or non - interventional designs ) , are recommended for marker validation . the stratified design has been successfully implemented in multiple clinical validations of clinical guideline recommended markers , including kras , egfr , oncotype dx ( genomic health , inc . , redwood city , ca , usa ) , and veristrat ( biodesix , boulder , co , usa ) . the prospective clinical validation trial for the chemofx assay required that both the prognostic and predictive properties of the assay be evaluated using an analytical method very similar to the stratified approach [ 49 , 50 ] . an important aspect of clinical utility considers how use of an assay or marker affects patient outcome in terms of treatment selection , survival , and morbidity . other considerations include the impact of the assay or marker usage on physician treatment plans as well as immediate and downstream healthcare costs [ 5759 ] . to further demonstrate the clinical utility of this chemoresponse assay , we conducted a comparative analysis based on a two - arm marker strategy approach . a 192-patient cohort , serving as the assay - informed arm , was compared to a non - assay - informed ( historical control ) arm , comprised of patients treated by non - assay - informed physicians from four large cooperative group drug studies in primary eoc , totaling more than 7,000 patients [ 246 ] . patient characteristics in both the assay - informed and control arms were similar with the exception that between 11 and 16 % more optimally debulked patients were included in the multiple literature cohorts that comprise the control arm ( table 1 ) . additionally , while the assay - informed arm and the largest control arm cohort consisted of only advanced stage patients [ 5 , 44 ] , the other three control arm cohorts also included a small portion of earlier stage patients . based on traditionally accepted adverse clinical variables , a worse prognosis was projected for the assay - informed cohort given the greater proportion of late - stage and sub - optimally debulked patients . despite worse prognostic clinical factors , patients in the assay - informed arm experienced a 10 % improvement in median os compared with the literature - derived control arm ( 48 vs. 44 months , respectively ) . furthermore , at study completion ( 6 years follow - up ) , 39 % of the assay - informed patients were alive compared to 29 % of control arm patients ( table 2).table 2comparison of median os in control , assay - informed , and assay - informed sensitive cohortssurvivaldu bois jnci 2003pfisterer jnci 2006du bois jco 2006bookman jco 2009control arm averageassay - informed herzog ajog 2010assay - informed herzog ajog 2010 sensitive groupmedian os44 month44 month44 month44 month44 month48 month72.5 month1-year92 % 91 % 90 % 90 % 91 % 85 % 90 % 2-year74 % 72 % 73 % 75 % 74 % 72 % 86 % 3-year59 % 57 % 58 % 60 % 59 % 59 % 75 % 4-year47 % 45 % 46 % 45 % 46 % 51 % 70 % 5-year38 % 35 % 38 % 35 % 37 % 44 % 60 % 6-year29 % na28 % 30 % 29 % 39 % 55 % annual os rates in the four cohorts comprising the control arm are based on extrapolation of the published kaplan meier survival curves [ 25 , 45 ] comparison of median os in control , assay - informed , and assay - informed sensitive cohorts annual os rates in the four cohorts comprising the control arm are based on extrapolation of the published kaplan meier survival curves [ 25 , 45 ] median os for the assay - informed arm , stratified by assay response category , was s = 72.5 ( n = 20 ) , is = 48.6 ( n = 133 ) , and r = 28.2 months ( n = 39 ) ( table 2 ; fig . 2 ) , representing a 28.5 month ( 72.5 vs. 44 , 65 % ) increased os for patients treated with assay - s regimens and a 15.8 month ( 28.2 vs. 44 , 36 % ) decreased os for patients treated with assay - r regimens , as compared to the control arm . when comparing annual os for assay - informed patients treated with an s regimen to control arm patients , 1025 % more assay - informed patients were living in years 2 thru 6 . notably , 55 % of assay - informed patients treated with an s regimen were alive at year 6 compared to 29 % in the control arm , despite the disparity in adverse clinical factors favoring the control arm ( table 2).fig . 2kaplan meier survival curves comparing the control arm cohort ( black ) and the assay - informed arm cohort . survival curves for the assay - informed cohort were stratified according to assay response category of clinically administered therapy ( s sensitive , green ; is intermediate sensitive , light green ; r resistant , red ) kaplan meier survival curves comparing the control arm cohort ( black ) and the assay - informed arm cohort . survival curves for the assay - informed cohort were stratified according to assay response category of clinically administered therapy ( s sensitive , green ; is intermediate sensitive , light green ; r resistant , red ) an analysis of survival from patients in the control arm cohorts [ 246 ] indicates that the various treatment regimens had similar efficacy when therapies were randomly assigned in phase iii clinical trials [ 25 , 44 ] . therefore , even though therapies from the same pool of approved and recommended treatment options were administered to patients in the comparative analysis , patients whose treatment was assay - informed had improved survival when compared to patients whose treatment was randomly assigned . furthermore , in the assay - informed arm , final treatment decisions for patients were made by their physicians , and assays were used to assist treatment selections in some cases and not in others . it is therefore rational to hypothesize that if physicians routinely had chemoresponse information available when choosing chemotherapeutic regimens for patients with ovarian cancer , os might be further improved . average chemotherapy costs for patients with recurrent ovarian cancer treated with or without use of the assay have also been evaluated . results based on ubs warburg market share data demonstrated mean costs for chemotherapy treatment were $ 48,758 for patients treated empirically ( no assay ) , $ 33,187 for patients with assay results available ( 65 % adhered to assay results ) , and $ 23,986 for patients modeled to have 100 % adherence to assay results . spanning the median os of 44 months , the majority of eoc patients experience multiple episodes of disease recurrence . therefore , treatment costs typically include both surgery and multiple chemotherapy interventions . considering that assay - informed treatment selection may result in delayed cancer progression and increased os , if one or more of the multiple chemotherapy interventions were delayed or avoided in assay - informed patients , treatment costs may be reduced by the costs associated with less effective chemotherapy regimens . use of chemoresponse assays during primary therapy may help to identify patients with platinum - resistant disease , potentially allowing for consideration of alternate clinically validated or similarly appropriate [ 6567 ] treatments , as well as prognostic stratification of patients in prospective clinical trials and/or modification of primary therapies off trial such as the addition of bevacizumab or other targeted therapies to standard carboplatin / paclitaxel treatment [ 6264 ] . likewise , in the recurrent disease setting where there is no single standard of care , crsas may assist oncologists with prioritization of the various single - agent therapies used with or without platinum therapies [ 5456 ] . additionally , in both primary and recurrent eoc , in the event of a severe drug reaction , physicians may employ this assay to identify an effective ( s or is ) therapy with which to replace the toxic agent . chemofx is a chemoresponse assay that characterizes both the sensitivity and resistance of a patient s tumor to various physician - selected , clinically applicable chemotherapy treatments . it quantifies chemotherapy effect by direct visualization and enumeration of live cells following exposure to these treatments . the assay is performed in a clinical laboratory improvement amendments ( clia ) and new york state department of health ( nysdoh ) approved facility . icc immunocytochemistry , auc area under curve , s sensitive , is intermediate sensitive , r resistant chemofx assay process . icc immunocytochemistry , auc area under curve , s sensitive , is intermediate sensitive , r resistant in contrast to other csras , this chemoresponse assay is characterized by several features that make it more reproducible and clinically accessible.the assay uniquely insures that tumor cells are proliferating prior to chemotherapy exposure , thereby measuring treatment efficacy at halting proliferation and/or killing tumor cells . this approach accommodates the cell cycle - specific , cytostatic , and cytotoxic natures of various chemotherapies.the assay s primary culture process is optimized to generate sufficient proliferating tumor cells for testing . as a result , 9 out of 10 ovarian cancer samples meeting the incoming sample criteria , such as sufficient sample size and absence of microorganism contamination , are successfully reported.the culture process favors epithelial tumor cell proliferation and incorporates an immunocytochemistry ( icc ) step to insure that the majority of cells tested are epithelial.the assay process is highly automated . cell seeding into microtiter plates , serial treatment dilution and application , cell fixation , fluorescence staining , as well as cell enumeration are performed using automated liquid handling robotics , computer - assisted microscopy , and automated cell - counting algorithms and software . the automated process strongly contributes to the high throughput and reproducibility of the assay .lastly , this assay requires significantly less tissue ( a minimum of 35 mm ) , as compared to historical assays . tumor tissue from surgical excision , biopsy , or paracentesis is compatible , making the assay highly clinically accessible . the assay uniquely insures that tumor cells are proliferating prior to chemotherapy exposure , thereby measuring treatment efficacy at halting proliferation and/or killing tumor cells . this approach accommodates the cell cycle - specific , cytostatic , and cytotoxic natures of various chemotherapies . the assay s primary culture process is optimized to generate sufficient proliferating tumor cells for testing . as a result , 9 out of 10 ovarian cancer samples meeting the incoming sample criteria , such as sufficient sample size and absence of microorganism contamination , are successfully reported . the culture process favors epithelial tumor cell proliferation and incorporates an immunocytochemistry ( icc ) step to insure that the majority of cells tested are epithelial . cell seeding into microtiter plates , serial treatment dilution and application , cell fixation , fluorescence staining , as well as cell enumeration are performed using automated liquid handling robotics , computer - assisted microscopy , and automated cell - counting algorithms and software . lastly , this assay requires significantly less tissue ( a minimum of 35 mm ) , as compared to historical assays . tumor tissue from surgical excision , biopsy , or paracentesis is compatible , making the assay highly clinically accessible . the analytical performance of this assay has been previously reported [ 42 , 43 , 46 ] . demonstrated a coefficient of variation ( cov ) of 3.64.6 % for sk - ov-3 cells treated with doxorubicin , across three operators and 9 days . in addition to variability across operators and days , process variability due to inter- and intraday stability of the chemotherapeutic treatments has also been reported . the assay has demonstrated the necessary analytical performance characteristics required by both clia and nysdoh . demonstrated the association of assay response with pfs in 256 eoc patients . in patients with either an exact or partial match between treatments assayed and those that were clinically administered , the hazard ratio ( hr ) for progression in patients clinically treated with an assay - resistant ( r ) vs. assay - sensitive ( s ) treatment was 2.1 ( 95 % ci 1.23.6 , p = 0.01 ) . in the subset of 135 patients with an exact match , the hr for progression in patients clinically administered an assay - r vs. assay - s treatment was 2.9 ( 95 % ci 1.46.3 , p < 0.01 ) . the median pfs for patients treated with r therapies was 9 and 14 months for those treated with intermediate sensitive ( is ) therapies . furthermore , at the time of study completion with a median follow - up time of 14.6 months , 60 % of patients treated with s therapies remained relapse - free . herzog et al . subsequently reported an association between assay response and os in 192 patients with advanced eoc following first - line platinum - based chemotherapy . median os was 72.5 , 48.6 , and 28.2 months for patients who were treated with agents reported as s , is , and r , respectively ( hr = 0.7 , 95 % ci 0.500.97 , p = 0.03 ) . multivariate cox regression analysis demonstrated that the assay prediction of response to platinum agents was a predictor of os independent of other prognostic factors of stage , age , and optimal debulking ( hr = 0.68 , 95 % ci 0.490.95 , p = 0.023 ) . in another more recent observational study of 276 women with figo stage iii - iv eoc cancer uniformly treated with first - line carboplatin-/paclitaxel - based therapy , patients with assay - r results for carboplatin were at increased risk of disease progression ( as defined by pfs ) compared with patients with s or is assay results ( hr = 1.87 , 95 % ci 1.292.70 , p = 0.0009 ) ; these results were consistent after controlling for clinical covariates ( hr = 1.71 , 95 % ci 1.122.62 , p = 0.013 ) . median pfs for patients who were assay - r to carboplatin was 11.8 vs. 16.6 months for assay - is and assay - s patients . this study demonstrates that assay resistance to carboplatin is associated with reduced pfs in eoc patients treated with standard of care carboplatin / paclitaxel , supporting the assay s ability to identify platinum - resistant patients . furthermore , of those patients who were resistant to carboplatin in vitro , 59 % of them displayed assay sensitivity ( s or is ) to at least one other agent . finally , a prospective study of 262 women with recurrent or persistent eoc reported that patients treated with an assay - s regimen experienced significantly improved pfs ( hr = 0.67 , 95 % ci 0.500.91 , p = 0.009 ) and os ( hr = 0.61 , 95 % ci 0.410.89 , p = 0.010 ) compared with those treated with assay - is or assay - r regimens , resulting in a 14-month improvement in median os . assay - pfs association was consistent in both platinum - sensitive and platinum - resistant tumors ( hr : 0.71 and 0.66 , respectively ) and was independent of other covariates ( hr = 0.66 , 95 % ci 0.470.94 , p = 0.020 ) . moreover , the results indicated that more than 50 % of the patients had at least one s therapy identified by the assay , whereas only 25 % of them were empirically treated with an s drug , suggesting that the number of patients potentially experiencing improved os may more than double when physicians reference the assay . a further analysis of the 262 recurrent or persistent eoc patients reported by rutherford et al . was presented at the 2013 european cancer organization ( ecco ) biennial meeting and addressed the assay s ability to function as a predictive marker . a prognostic assay identifies patients likely to respond / not respond to ( any ) therapy , while a predictive assay identifies a patient 's likely response to specific therapies , which is particularly important for individualized chemotherapy selection . four different analytical methods were used in the study to assess the predictive value of the assay . these analyses provide the evidence that this chemoresponse assay is a predictive marker , demonstrating its ability to discern specific therapies that are likely to be more effective among multiple alternatives . as briefly outlined earlier , a chemoresponse assay , such as chemofx , is a panel of treatment markers , with the assay result for each treatment evaluated functioning as a distinct marker . when ordering the assay , a physician selects each of the multiple treatments under consideration for a given patient for inclusion in the assay . clinically validated chemotherapy regimens , consistent with guidelines such as nccn , comprise the available treatment choices . clinical trials designed to evaluate marker or assay efficacy are fundamentally different than trials designed to evaluate drug efficacy . various different marker trial designs have been extensively studied and reported in the recent literature . however , discussion of effective marker validation trial designs that are appropriate for multiple markers / therapies to be assessed simultaneously ( e.g. , chemoresponse assays ) remains very limited in the current trial design literature . marker validation trial designs for this type of multiple markers / therapies assay may vary , to some extent , from marker trial designs appropriate for a single molecular biomarker associated with a single therapy ( e.g. , kras / panitumumab , egfr / erlotinib ) . primarily , three marker study designs have been outlined in the literature for marker validation : enrichment , strategy , and stratified [ 5153 ] . marker negative patients are excluded in the enrichment design , and thus , it is not applicable to chemoresponse assay evaluation . historically , the strategy design , in many ways similar to a standard drug trial design , has been considered the gold standard for marker validation as it attempts to emulate what might occur in clinical practice . a variant of the strategy design has been recommended by the american society of clinical oncology ( asco ) and blue cross blue shield ( bcbs ) technical evaluation center ( tec ) assessments of validations of chemoresponse assays [ 54 , 55 ] date back to the mid 1990s . however , multiple recent and updated marker trial design publications , including an evaluation by the center for medical technology policy ( cmtp ) in 2013 , indicate that the strategy design is less than ideal for marker validation , in that it requires a larger sample size and can not distinguish between a more effective treatment and marker efficacy , when compared to alternate marker trial designs . show that the required sample size for a strategy design can exceed several thousand patients depending on the prevalence of the marker in the study population , presenting a large challenge in a small incidence / prevalence disease like eoc . further and specific to chemoresponse assays where multiple markers are evaluated simultaneously , the pan - resistant and pan - sensitive patients dilute the ability to assess assay impact on patient outcome . additionally , overlapping treatments between study arms still further increase the required sample size , rendering the strategy approach essentially pragmatically infeasible . finally , a potential physician treatment bias or learning effect may be associated with the strategy design . the strategy trial design attempted by cree et al . showed a trend toward improved response rate and pfs in assay - informed patients as compared to those treated with the physician s empiric choice , but did not achieve statistical significance . asserted that physicians learned from the assay - informed arm and began administering treatments similar to those recommended for assay - informed patients to patients in the physician - directed arm as the study progressed . analysis confirmed this effect ; in early physician - choice arm patients , pfs was significantly shorter than that in subsequent year patients . the stratified design has been reported as more efficient , capable of answering the relevant clinical questions , and able to assess both prognostic and predictive marker properties , which is often at issue with evaluations of markers [ 52 , 56 , 57 ] . friedlin et al . have concluded that trial designs , such as the stratified design that use the marker to guide analysis , but not treatment assignment ( i.e. , blind or non - interventional designs ) , are recommended for marker validation . the stratified design has been successfully implemented in multiple clinical validations of clinical guideline recommended markers , including kras , egfr , oncotype dx ( genomic health , inc . , redwood city , ca , usa ) , and veristrat ( biodesix , boulder , co , usa ) . the prospective clinical validation trial for the chemofx assay required that both the prognostic and predictive properties of the assay be evaluated using an analytical method very similar to the stratified approach [ 49 , 50 ] . an important aspect of clinical utility considers how use of an assay or marker affects patient outcome in terms of treatment selection , survival , and morbidity . other considerations include the impact of the assay or marker usage on physician treatment plans as well as immediate and downstream healthcare costs [ 5759 ] . to further demonstrate the clinical utility of this chemoresponse assay , we conducted a comparative analysis based on a two - arm marker strategy approach . a 192-patient cohort , serving as the assay - informed arm , was compared to a non - assay - informed ( historical control ) arm , comprised of patients treated by non - assay - informed physicians from four large cooperative group drug studies in primary eoc , totaling more than 7,000 patients [ 246 ] . patient characteristics in both the assay - informed and control arms were similar with the exception that between 11 and 16 % more optimally debulked patients were included in the multiple literature cohorts that comprise the control arm ( table 1 ) . additionally , while the assay - informed arm and the largest control arm cohort consisted of only advanced stage patients [ 5 , 44 ] , the other three control arm cohorts also included a small portion of earlier stage patients . based on traditionally accepted adverse clinical variables , a worse prognosis was projected for the assay - informed cohort given the greater proportion of late - stage and sub - optimally debulked patients . despite worse prognostic clinical factors , patients in the assay - informed arm experienced a 10 % improvement in median os compared with the literature - derived control arm ( 48 vs. 44 months , respectively ) . furthermore , at study completion ( 6 years follow - up ) , 39 % of the assay - informed patients were alive compared to 29 % of control arm patients ( table 2).table 2comparison of median os in control , assay - informed , and assay - informed sensitive cohortssurvivaldu bois jnci 2003pfisterer jnci 2006du bois jco 2006bookman jco 2009control arm averageassay - informed herzog ajog 2010assay - informed herzog ajog 2010 sensitive groupmedian os44 month44 month44 month44 month44 month48 month72.5 month1-year92 % 91 % 90 % 90 % 91 % 85 % 90 % 2-year74 % 72 % 73 % 75 % 74 % 72 % 86 % 3-year59 % 57 % 58 % 60 % 59 % 59 % 75 % 4-year47 % 45 % 46 % 45 % 46 % 51 % 70 % 5-year38 % 35 % 38 % 35 % 37 % 44 % 60 % 6-year29 % na28 % 30 % 29 % 39 % 55 % annual os rates in the four cohorts comprising the control arm are based on extrapolation of the published kaplan meier survival curves [ 25 , 45 ] comparison of median os in control , assay - informed , and assay - informed sensitive cohorts annual os rates in the four cohorts comprising the control arm are based on extrapolation of the published kaplan meier survival curves [ 25 , 45 ] median os for the assay - informed arm , stratified by assay response category , was s = 72.5 ( n = 20 ) , is = 48.6 ( n = 133 ) , and r = 28.2 months ( n = 39 ) ( table 2 ; fig . 2 ) , representing a 28.5 month ( 72.5 vs. 44 , 65 % ) increased os for patients treated with assay - s regimens and a 15.8 month ( 28.2 vs. 44 , 36 % ) decreased os for patients treated with assay - r regimens , as compared to the control arm . when comparing annual os for assay - informed patients treated with an s regimen to control arm patients , 1025 % more assay - informed patients were living in years 2 thru 6 . notably , 55 % of assay - informed patients treated with an s regimen were alive at year 6 compared to 29 % in the control arm , despite the disparity in adverse clinical factors favoring the control arm ( table 2).fig . 2kaplan meier survival curves comparing the control arm cohort ( black ) and the assay - informed arm cohort . survival curves for the assay - informed cohort were stratified according to assay response category of clinically administered therapy ( s sensitive , green ; is intermediate sensitive , light green ; r resistant , red ) kaplan meier survival curves comparing the control arm cohort ( black ) and the assay - informed arm cohort . survival curves for the assay - informed cohort were stratified according to assay response category of clinically administered therapy ( s sensitive , green ; is intermediate sensitive , light green ; r resistant , red ) an analysis of survival from patients in the control arm cohorts [ 246 ] indicates that the various treatment regimens had similar efficacy when therapies were randomly assigned in phase iii clinical trials [ 25 , 44 ] . therefore , even though therapies from the same pool of approved and recommended treatment options were administered to patients in the comparative analysis , patients whose treatment was assay - informed had improved survival when compared to patients whose treatment was randomly assigned . furthermore , in the assay - informed arm , final treatment decisions for patients were made by their physicians , and assays were used to assist treatment selections in some cases and not in others . it is therefore rational to hypothesize that if physicians routinely had chemoresponse information available when choosing chemotherapeutic regimens for patients with ovarian cancer , os might be further improved . average chemotherapy costs for patients with recurrent ovarian cancer treated with or without use of the assay have also been evaluated . results based on ubs warburg market share data demonstrated mean costs for chemotherapy treatment were $ 48,758 for patients treated empirically ( no assay ) , $ 33,187 for patients with assay results available ( 65 % adhered to assay results ) , and $ 23,986 for patients modeled to have 100 % adherence to assay results . spanning the median os of 44 months , the majority of eoc patients experience multiple episodes of disease recurrence . therefore , treatment costs typically include both surgery and multiple chemotherapy interventions . considering that assay - informed treatment selection may result in delayed cancer progression and increased os , if one or more of the multiple chemotherapy interventions were delayed or avoided in assay - informed patients , treatment costs may be reduced by the costs associated with less effective chemotherapy regimens . use of chemoresponse assays during primary therapy may help to identify patients with platinum - resistant disease , potentially allowing for consideration of alternate clinically validated or similarly appropriate [ 6567 ] treatments , as well as prognostic stratification of patients in prospective clinical trials and/or modification of primary therapies off trial such as the addition of bevacizumab or other targeted therapies to standard carboplatin / paclitaxel treatment [ 6264 ] . likewise , in the recurrent disease setting where there is no single standard of care , crsas may assist oncologists with prioritization of the various single - agent therapies used with or without platinum therapies [ 5456 ] . additionally , in both primary and recurrent eoc , in the event of a severe drug reaction , physicians may employ this assay to identify an effective ( s or is ) therapy with which to replace the toxic agent . despite several years of chemoresponse assay development and clinical experience with these assays , studies have largely been confined to single - institutional , retrospective evaluations . recent large , prospective , multi - site clinical studies that correlate chemofx assay results with overall and progression - free survival in both primary and recurrent ovarian cancers indicate that the assay may offer significant clinical benefit for patients , is predictive of treatment outcomes , and is potentially economically beneficial by reducing the chance that ineffective chemotherapy is administered . this overview supports the inclusion of chemoresponse assay results , along with other clinical factors and biomarkers , to support the individualized selection of effective chemotherapy agents for treatment of patients with ovarian cancer .

the objective of this review is to summarize recent scientific and medical literature regarding chemoresponse assays or chemotherapy sensitivity and resistance assays ( csras ) , specifically as applied to epithelial ovarian cancer . a total of sixty - seven articles , identified through pubmed using the key words in vitro chemoresponse assay , chemo sensitivity resistance assay , atp , hdra , edr , mick , and chemofx , were reviewed . recent publications on marker validation , including relevant clinical trial designs , were also included . recent csra research and clinical studies are outlined in this review . published findings demonstrate benefits regarding patient outcome with respect to recent csras . specifically , analytical and clinical validations , as well as clinical utility and economic benefit , of the most common clinically used csra in the united states support its use to aid in making effective , individualized clinical treatment selections for patients with ovarian cancer .

Ovarian cancer Chemoresponse assays: a panel of treatment response markers ChemoFx Assay process Analytical and clinical validation Clinical utility and economic analysis Conclusions

ovarian cancer is the most lethal and second most common gynecologic malignancy in the united states , with an estimated 21,980 new cases and 14,270 deaths expected in 2014 . most patients are present with advanced disease , and the current standard of care in the primary setting is surgical debulking followed by platinum - based chemotherapy . ovarian cancer is a heterogeneous disease with respect to histopathology , molecular biology , and clinical outcome , suggesting that a single standard treatment is unlikely to benefit all patients . histologically , most ovarian cancer arises from the distal fallopian tube or ovarian surface , and the majority of these epithelial ovarian cancers ( eoc ) are serous / papillary pathological subtype , followed by endometrioid , mucinous , clear cell , and undifferentiated . these studies have consistently demonstrated remarkably similar progression - free survival ( pfs ) and os between the standard of care and the various alternates , highlighting the therapeutic equivalence of the various regimens and the associated empiric treatment ambiguity.table 1patient characteristics of control arm and assay - informed arm cohortspatient characteristicscontrol armassay - informed armdu bois jnci 2003pfisterer jnci 2006du bois jco 2006bookman jco 2009herzog ajog 2010number of patients7831,3081,2824,312192median age5760595959pathological subtypeserous / papillary70 % 71 % 73 % 80 % 71 % other30 % 29 % 27 % 20 % 29 % figo stage distributionstage i<1 % < 1 % stage ii8 % 9 % 9 % stage iii75 % 74 % 74 % 85 % 84 % stage iv17 % 17 % 17 % 15 % 16 % debulking statusoptimal63 % 67 % 68 % 68 % 52 % sub - optimal37 % 33 % 32 % 32 % 48 % treatment regimenscpcispcpcptcpcpecpcpgcpdct / cpcg / cpcpcpgcispctother c carboplatin , p paclitaxel , g gemcitabine , d doxorubicin , e epirubicin , t topotecan , cis cisplatin patient characteristics of control arm and assay - informed arm cohorts c carboplatin , p paclitaxel , g gemcitabine , d doxorubicin , e epirubicin , t topotecan , cis cisplatin for patients with recurrent , persistent , or progressive disease , chemotherapy choice is currently based , in part , on the duration and type of response to initial therapy . for platinum - resistant disease ( pfi < 6 months ) , physicians empirically select from an array of non - platinum regimens , including pegylated liposomal doxorubicin ( pld ) , topotecan , gemcitabine , etoposide , taxanes , and targeted therapies , all of which have been evaluated and demonstrated to be clinically equivalent and acceptable for use in this patient population . such assays provide tumor response information aimed at aiding in the selection of effective , individualized treatment regimens . chemoresponse assays provide the same utility as other treatment markers that are associated with patient outcome when the given marker s associated treatment is clinically administered ( e.g. the concept of a chemoresponse assay or chemotherapy sensitivity and resistance assay ( csra ) , originated in the 1950s . there are different types of csras such as the adenosine triphosphate ( atp ) assay , human tumor cloning assay ( htca ) , methylthiazolyl - diphenyl - tetrazolium bromide ( mtt ) assay , extreme drug resistance ( edr ) assay , as well as assays utilizing drug - induced apoptosis as the end point [ 24 , 26 , 27 ] . while other csra reviews have been published previously [ 24 , 26 ] , this review will focus on progress made during the most recent decade . another prospective study , utilizing an atp - based chemoresponse assay , evaluated response rate and pfs in platinum - resistant recurrent eoc patients ( n = 180 ) randomized to assay - directed or physician s empiric therapy choice , demonstrating trends for improved response rate and pfs for assay - informed treatment . numerous retrospective data have also been published in the past decade , with the majority reporting statistically significant associations between assay results and clinical outcomes [ 3537 ] . although several chemoresponse assays clinical validity and clinical utility have been evaluated in clinical trials , there are currently only two assays commercially available in the united states : the microculture - kinetic ( mick ) assay ( diatech oncology , franklin , tn ) and the chemofx assay ( precision therapeutics , inc . the assay was later applied to solid tumors , including breast , lung , and gynecologic malignancies [ 27 , 3841 ] . clinical validation of the mick assay in 73 ovarian cancer patients demonstrated that clinical treatment with the assay - indicated best chemotherapy is an independent predictor of os in multivariate analysis of chemotherapy - nave stage iii or iv primary ovarian cancer patients . however , the 44 tumors in the study included a variety of tissue types , such as breast and non - small lung cancers ; only two ovarian cancer tumors were included in the study . therefore , the clinical validation and clinical utility of this assay in ovarian cancer requires further investigation . the chemofx chemoresponse assay has been extensively evaluated in patients with ovarian cancer and will be the focus of the remainder of this review . chemofx is a chemoresponse assay that characterizes both the sensitivity and resistance of a patient s tumor to various physician - selected , clinically applicable chemotherapy treatments . cell seeding into microtiter plates , serial treatment dilution and application , cell fixation , fluorescence staining , as well as cell enumeration are performed using automated liquid handling robotics , computer - assisted microscopy , and automated cell - counting algorithms and software . the automated process strongly contributes to the high throughput and reproducibility of the assay .lastly , this assay requires significantly less tissue ( a minimum of 35 mm ) , as compared to historical assays . cell seeding into microtiter plates , serial treatment dilution and application , cell fixation , fluorescence staining , as well as cell enumeration are performed using automated liquid handling robotics , computer - assisted microscopy , and automated cell - counting algorithms and software . as briefly outlined earlier , a chemoresponse assay , such as chemofx , is a panel of treatment markers , with the assay result for each treatment evaluated functioning as a distinct marker . when ordering the assay , a physician selects each of the multiple treatments under consideration for a given patient for inclusion in the assay . various different marker trial designs have been extensively studied and reported in the recent literature . however , discussion of effective marker validation trial designs that are appropriate for multiple markers / therapies to be assessed simultaneously ( e.g. primarily , three marker study designs have been outlined in the literature for marker validation : enrichment , strategy , and stratified [ 5153 ] . marker negative patients are excluded in the enrichment design , and thus , it is not applicable to chemoresponse assay evaluation . however , multiple recent and updated marker trial design publications , including an evaluation by the center for medical technology policy ( cmtp ) in 2013 , indicate that the strategy design is less than ideal for marker validation , in that it requires a larger sample size and can not distinguish between a more effective treatment and marker efficacy , when compared to alternate marker trial designs . an important aspect of clinical utility considers how use of an assay or marker affects patient outcome in terms of treatment selection , survival , and morbidity . other considerations include the impact of the assay or marker usage on physician treatment plans as well as immediate and downstream healthcare costs [ 5759 ] . to further demonstrate the clinical utility of this chemoresponse assay , we conducted a comparative analysis based on a two - arm marker strategy approach . furthermore , at study completion ( 6 years follow - up ) , 39 % of the assay - informed patients were alive compared to 29 % of control arm patients ( table 2).table 2comparison of median os in control , assay - informed , and assay - informed sensitive cohortssurvivaldu bois jnci 2003pfisterer jnci 2006du bois jco 2006bookman jco 2009control arm averageassay - informed herzog ajog 2010assay - informed herzog ajog 2010 sensitive groupmedian os44 month44 month44 month44 month44 month48 month72.5 month1-year92 % 91 % 90 % 90 % 91 % 85 % 90 % 2-year74 % 72 % 73 % 75 % 74 % 72 % 86 % 3-year59 % 57 % 58 % 60 % 59 % 59 % 75 % 4-year47 % 45 % 46 % 45 % 46 % 51 % 70 % 5-year38 % 35 % 38 % 35 % 37 % 44 % 60 % 6-year29 % na28 % 30 % 29 % 39 % 55 % annual os rates in the four cohorts comprising the control arm are based on extrapolation of the published kaplan meier survival curves [ 25 , 45 ] comparison of median os in control , assay - informed , and assay - informed sensitive cohorts annual os rates in the four cohorts comprising the control arm are based on extrapolation of the published kaplan meier survival curves [ 25 , 45 ] median os for the assay - informed arm , stratified by assay response category , was s = 72.5 ( n = 20 ) , is = 48.6 ( n = 133 ) , and r = 28.2 months ( n = 39 ) ( table 2 ; fig . 2 ) , representing a 28.5 month ( 72.5 vs. 44 , 65 % ) increased os for patients treated with assay - s regimens and a 15.8 month ( 28.2 vs. 44 , 36 % ) decreased os for patients treated with assay - r regimens , as compared to the control arm . furthermore , in the assay - informed arm , final treatment decisions for patients were made by their physicians , and assays were used to assist treatment selections in some cases and not in others . it is therefore rational to hypothesize that if physicians routinely had chemoresponse information available when choosing chemotherapeutic regimens for patients with ovarian cancer , os might be further improved . average chemotherapy costs for patients with recurrent ovarian cancer treated with or without use of the assay have also been evaluated . results based on ubs warburg market share data demonstrated mean costs for chemotherapy treatment were $ 48,758 for patients treated empirically ( no assay ) , $ 33,187 for patients with assay results available ( 65 % adhered to assay results ) , and $ 23,986 for patients modeled to have 100 % adherence to assay results . use of chemoresponse assays during primary therapy may help to identify patients with platinum - resistant disease , potentially allowing for consideration of alternate clinically validated or similarly appropriate [ 6567 ] treatments , as well as prognostic stratification of patients in prospective clinical trials and/or modification of primary therapies off trial such as the addition of bevacizumab or other targeted therapies to standard carboplatin / paclitaxel treatment [ 6264 ] . likewise , in the recurrent disease setting where there is no single standard of care , crsas may assist oncologists with prioritization of the various single - agent therapies used with or without platinum therapies [ 5456 ] . chemofx is a chemoresponse assay that characterizes both the sensitivity and resistance of a patient s tumor to various physician - selected , clinically applicable chemotherapy treatments . cell seeding into microtiter plates , serial treatment dilution and application , cell fixation , fluorescence staining , as well as cell enumeration are performed using automated liquid handling robotics , computer - assisted microscopy , and automated cell - counting algorithms and software . the automated process strongly contributes to the high throughput and reproducibility of the assay .lastly , this assay requires significantly less tissue ( a minimum of 35 mm ) , as compared to historical assays . cell seeding into microtiter plates , serial treatment dilution and application , cell fixation , fluorescence staining , as well as cell enumeration are performed using automated liquid handling robotics , computer - assisted microscopy , and automated cell - counting algorithms and software . median os was 72.5 , 48.6 , and 28.2 months for patients who were treated with agents reported as s , is , and r , respectively ( hr = 0.7 , 95 % ci 0.500.97 , p = 0.03 ) . four different analytical methods were used in the study to assess the predictive value of the assay . as briefly outlined earlier , a chemoresponse assay , such as chemofx , is a panel of treatment markers , with the assay result for each treatment evaluated functioning as a distinct marker . when ordering the assay , a physician selects each of the multiple treatments under consideration for a given patient for inclusion in the assay . , chemoresponse assays ) remains very limited in the current trial design literature . primarily , three marker study designs have been outlined in the literature for marker validation : enrichment , strategy , and stratified [ 5153 ] . marker negative patients are excluded in the enrichment design , and thus , it is not applicable to chemoresponse assay evaluation . however , multiple recent and updated marker trial design publications , including an evaluation by the center for medical technology policy ( cmtp ) in 2013 , indicate that the strategy design is less than ideal for marker validation , in that it requires a larger sample size and can not distinguish between a more effective treatment and marker efficacy , when compared to alternate marker trial designs . show that the required sample size for a strategy design can exceed several thousand patients depending on the prevalence of the marker in the study population , presenting a large challenge in a small incidence / prevalence disease like eoc . further and specific to chemoresponse assays where multiple markers are evaluated simultaneously , the pan - resistant and pan - sensitive patients dilute the ability to assess assay impact on patient outcome . the stratified design has been reported as more efficient , capable of answering the relevant clinical questions , and able to assess both prognostic and predictive marker properties , which is often at issue with evaluations of markers [ 52 , 56 , 57 ] . the stratified design has been successfully implemented in multiple clinical validations of clinical guideline recommended markers , including kras , egfr , oncotype dx ( genomic health , inc . an important aspect of clinical utility considers how use of an assay or marker affects patient outcome in terms of treatment selection , survival , and morbidity . other considerations include the impact of the assay or marker usage on physician treatment plans as well as immediate and downstream healthcare costs [ 5759 ] . to further demonstrate the clinical utility of this chemoresponse assay , we conducted a comparative analysis based on a two - arm marker strategy approach . furthermore , at study completion ( 6 years follow - up ) , 39 % of the assay - informed patients were alive compared to 29 % of control arm patients ( table 2).table 2comparison of median os in control , assay - informed , and assay - informed sensitive cohortssurvivaldu bois jnci 2003pfisterer jnci 2006du bois jco 2006bookman jco 2009control arm averageassay - informed herzog ajog 2010assay - informed herzog ajog 2010 sensitive groupmedian os44 month44 month44 month44 month44 month48 month72.5 month1-year92 % 91 % 90 % 90 % 91 % 85 % 90 % 2-year74 % 72 % 73 % 75 % 74 % 72 % 86 % 3-year59 % 57 % 58 % 60 % 59 % 59 % 75 % 4-year47 % 45 % 46 % 45 % 46 % 51 % 70 % 5-year38 % 35 % 38 % 35 % 37 % 44 % 60 % 6-year29 % na28 % 30 % 29 % 39 % 55 % annual os rates in the four cohorts comprising the control arm are based on extrapolation of the published kaplan meier survival curves [ 25 , 45 ] comparison of median os in control , assay - informed , and assay - informed sensitive cohorts annual os rates in the four cohorts comprising the control arm are based on extrapolation of the published kaplan meier survival curves [ 25 , 45 ] median os for the assay - informed arm , stratified by assay response category , was s = 72.5 ( n = 20 ) , is = 48.6 ( n = 133 ) , and r = 28.2 months ( n = 39 ) ( table 2 ; fig . 2 ) , representing a 28.5 month ( 72.5 vs. 44 , 65 % ) increased os for patients treated with assay - s regimens and a 15.8 month ( 28.2 vs. 44 , 36 % ) decreased os for patients treated with assay - r regimens , as compared to the control arm . furthermore , in the assay - informed arm , final treatment decisions for patients were made by their physicians , and assays were used to assist treatment selections in some cases and not in others . it is therefore rational to hypothesize that if physicians routinely had chemoresponse information available when choosing chemotherapeutic regimens for patients with ovarian cancer , os might be further improved . average chemotherapy costs for patients with recurrent ovarian cancer treated with or without use of the assay have also been evaluated . results based on ubs warburg market share data demonstrated mean costs for chemotherapy treatment were $ 48,758 for patients treated empirically ( no assay ) , $ 33,187 for patients with assay results available ( 65 % adhered to assay results ) , and $ 23,986 for patients modeled to have 100 % adherence to assay results . use of chemoresponse assays during primary therapy may help to identify patients with platinum - resistant disease , potentially allowing for consideration of alternate clinically validated or similarly appropriate [ 6567 ] treatments , as well as prognostic stratification of patients in prospective clinical trials and/or modification of primary therapies off trial such as the addition of bevacizumab or other targeted therapies to standard carboplatin / paclitaxel treatment [ 6264 ] . recent large , prospective , multi - site clinical studies that correlate chemofx assay results with overall and progression - free survival in both primary and recurrent ovarian cancers indicate that the assay may offer significant clinical benefit for patients , is predictive of treatment outcomes , and is potentially economically beneficial by reducing the chance that ineffective chemotherapy is administered . this overview supports the inclusion of chemoresponse assay results , along with other clinical factors and biomarkers , to support the individualized selection of effective chemotherapy agents for treatment of patients with ovarian cancer .

many japanese women experience health issues associated with menstruation ; these can include menstrual pain also referred to as dysmenorrhea , heavy menstrual bleeding ( hmb ) also referred to as menorrhagia , and premenstrual syndrome ( pms).13 dysmenorrhea is the most common gynecological complaint associated with menstruation with a prevalence of 25% among all women , and reaching up to 90% among adolescents.4 it has also been reported that one - third of japanese women require analgesic therapy for dysmenorrhea.5 in some women , dysmenorrhea can not be attributed to any specific cause and is referred to as primary or idiopathic . it was reported that 47% of patients who consulted a physician for menstrual cramping had primary dysmenorrhea , based on a survey in 2000.6 in other cases , it is associated with a preexisting gynecological disorder , and the disease is referred to as secondary or organic dysmenorrhea . regardless of the cause , dysmenorrhea can have a substantial impact on patient quality of life,7,8 yet many patients do not seek treatment.9 in a patient survey , some untreated women have expressed feelings of resistance or aversion toward seeking therapy , and many suggested that gynecologist consultations were unnecessary for their disorder.1 however , a substantial proportion of women who did seek medical treatment agreed that their daily lives were significantly improved after therapy , and it was also estimated that gynecologist visits saved over 7,000 jpy ( 70 usd ) monthly costs per - patient , occurring due to time off work.1 according to the guidelines for gynecological practice in japan , by the japan society of obstetrics and gynecology ( jsog ) and japan association of obstetricians and gynecologists ( jaog ) ( 2011 edition ) , low - dose estrogen progestins ( leps ) and nonsteroidal anti - inflammatory drugs ( nsaids ) are primarily recommended for primary dysmenorrhea , and traditional chinese medicines ( tcms ) could be used for primary dysmenorrhea.10 other current clinical practice guidelines for the treatment of dysmenorrhea include the use of over - the - counter analgesics , nsaids , and oral contraceptives such as leps , progestin - only therapies , and the levonorgestrel - releasing intrauterine system.11,12 two kinds of combined oral contraceptives ( cocs ) are available on the japanese market : leps , ( norethisterone / estrogen and drospirenone / estrogen ) , which are reimbursed for dysmenorrhea treatment , and the other cocs for contraceptive purposes , which are not reimbursed . for example , leps have been reported to be effective in alleviating symptoms in up to 80% of women.13 additional studies have shown efficacy of both gonadotropin - releasing hormone ( gnrh ) analogs and testosterone derivatives for treating underlying causes of secondary dysmenorrhea , such as endometriosis.1417 existing evidence on treatment patterns , health care resource use , and associated costs in japanese patients with dysmenorrhea is limited . information on treatment patterns and the economic burden of disease would be useful to guide the allocation of health care resources for the treatment of dysmenorrhea . furthermore , the analysis of treatment patterns and resource utilization may shed light on any potential challenges related to the current diagnosis and management of dysmenorrhea in japan . the objectives of this study were to describe treatment patterns and estimate health care resource use and costs among japanese women with newly diagnosed dysmenorrhea in a real - world setting . this included a detailed description of the baseline characteristics and comorbidities of these patients , their initial and subsequent therapies for dysmenorrhea , and the probability of surgical procedure related to dysmenorrhea . to assess health care resource utilization and costs among patients , a comparison was made between patients with dysmenorrhea ( cases ) and those without dysmenorrhea ( matched control group ) . furthermore , all results were differentiated between women with primary and secondary dysmenorrhea and treatment patterns were evaluated by prescriber s specialty . the reason why we took prescriber s specialty into consideration is that the japanese health care system allows patients free access to any clinic irrespective of specialty . this was a retrospective claims analysis of the japan medical data center ( jmdc ) database , which includes deidentified medical ( inpatient and outpatient ) and pharmacy claims from up to 3.0 million beneficiaries ( both employees and their dependents ) from 2005 onward . the jmdc database covers nearly 10% of the 30 million individuals in the japanese social insurance system , equating to approximately 2.5% of the total japanese population . data were extracted for patients with records between july 1 , 2009 , and june 30 , 2014 , and included information on patient demographics , diagnoses , drug prescriptions , medical procedures , medical facility characteristics , and reimbursement costs . included patients were women between ages 18 and 49 years who had two separate diagnoses of dysmenorrhea within 3 months . in japan , patients with dysmenorrhea are generally asked to come back to the clinic within the 3 months following a first medical consultation , as it was considered that only one visit would not be enough for selecting patients requiring medical care for dysmenorrhea . diagnoses were identified by icd-10 ( international classification of diseases 10th revision ) codes n944 ( primary dysmenorrhea ) or n946 ( dysmenorrhea , unspecified ) or one of the following medical information system development center ( medis - dc ) standard disease names : functional dysmenorrhea , menstrual pain , or dysmenorrhea . medis - dc consists of standardized names of diseases and injuries for reimbursement by the national health insurance.18 the index date was defined as the date of the first diagnosis of dysmenorrhea . primary dysmenorrhea was defined as patients without underlying causes ( endometriosis , adenomyosis , and fibroids ) during the preindex period and from 60 days after the index date , whereas secondary dysmenorrhea was defined as patients presenting at least one diagnosis of one of these conditions during this period . this criterion was defined based on the publications by copher et al19,20 and an assumption that imaging procedures for detecting underlying causes would be performed within 2 months following the first visit . patients were also required to have insurance records for at least 6 months with a diagnosis of dysmenorrhea prior to their initial diagnosis ( preindex period ) and for a continuous postindex period of at least 1 year , to be included in the study . patients were excluded if they had been diagnosed with gynecological cancers or bleeding disorders during the study period ( including both pre- and postindex ) , if they underwent gynecological surgery ( hysterectomy , endometrial ablation , or myomectomy ) during the preindex period , or if they were treated with hormonal agents during the preindex period ( figure 1 ) . for the resource utilization analysis , matched controls were identified among females in the jmdc database who did not suffer from dysmenorrhea ( ie , no diagnosis between july 1 , 2009 , and june 30 , 2014 ) . controls and cases were matched 2:1 by year of birth ( within 5 years ) , charlson comorbidity index ( cci ) at baseline ( 15 days from index date ) , and at least the same 18 months of follow - up period . the study assessed therapies that fit into one of the following categories : hormonal treatments ( leps , progestin , testosterone derivatives , and gnrh analogs ) , nsaids , hemostatic agents , and tcm therapies . relevant gynecological surgeries , including hysterectomies , endometrial ablations , and myomectomies , were also considered . the treatment patterns were analyzed based on prescription records , with a treatment line defined as a group of consecutive prescriptions of the same agent or combination of agents ( combination therapy ) , with no interruption exceeding 90 days . for each treatment line , the start date , end date , duration , and treatment category were determined . when a period exceeding 90 days was observed between the end of one treatment line and the beginning of a new one , therapy switches were defined when an overlap shorter than 21 days between the end of one therapy and the beginning of a new one was observed and the interval did not exceed 90 days . if an overlap occurred for 21 days or more , however , this was considered a combination therapy . because surgery was considered as a permanent treatment , a separate combination therapy type ( surgery combination ) was assigned . treatment patterns were analyzed among the entire cohort and according to the type of dysmenorrhea ( primary or secondary ) . a subgroup analysis was also conducted according to the prescriber s specialty for the first treatment line . the resources used and their associated costs were calculated based on the 1-year follow - up period after the index date for each patient . data were collected on inpatient costs , outpatient costs , and costs associated with both prescriptions and imaging procedures . from this information , resource utilization data collected in this analysis included the number of inpatient admissions , cumulative length of stay , number of outpatient visits , and number of imaging procedures . imaging procedures , which included echography , computed tomography scan , and magnetic resonance imaging , were determined to be related to a diagnosis of dysmenorrhea when they occurred within a 1-month period around the date of the diagnosis ( 15 days ) . the use of imaging procedures in controls was evaluated during the same 1-month period as it was for their corresponding matched cases . nonadjusted comparisons were conducted for outcomes with respect to primary vs secondary dysmenorrhea and cases vs controls . for continuous variables , a fisher s f mean values were then compared using a student s t - test ( for variables showing homoscedasticity ) or satterthwaite test ( heteroscedasticity ) . for categorical variables , a or fisher s exact test ( in cases of insufficient theoretical size of subgroups ) time - to - event variables were assessed using kaplan meier curves and log - rank tests for between - group comparisons . ( ie , end - of - treatment events such as treatment switch , discontinuation of treatment , surgery , add - on therapy , and step - down therapy ) were analyzed using the competing risks survival method , estimating the cumulative incidence . the analyses of costs and resources were conducted through generalized linear models ( glms ) , testing normal , poisson , negative binomial , zero - inflated poisson , and zero - inflated negative binomial distributions . the goodness of fit of the models was assessed with the deviance , and the dispersion with the pearson value divided by the number of degrees of freedom . the models minimizing the deviance or with the best compromise deviance / dispersion ( in case of high dispersion ) were retained . models were adjusted for age , the category ( based on quartiles ) of total health care costs over the preindex period , the quantity corresponding to the modeled variable computed over the preindex period ( eg , the inpatient costs over the preindex period while modeling the inpatient costs over the 1-year postindex period ) , hospitalization during the preindex period , and the type of health insurance membership ( employee or dependent ) . all analyses were performed using sas analytics pro release 9.3 and r ( version 3.2.1 , sas institute , cary , nc , usa ) software ( for time - to - event data ) . in general , a p - value of 0.05 was determined to represent statistical significance . this study was approved by the ethics committee of the nonprofit organization , clinical research promotion network japan . the informed consent of patients was not applicable based on the ethical guidelines for epidemiological research issued by the ministry of health , welfare and labor . this was a retrospective claims analysis of the japan medical data center ( jmdc ) database , which includes deidentified medical ( inpatient and outpatient ) and pharmacy claims from up to 3.0 million beneficiaries ( both employees and their dependents ) from 2005 onward . the jmdc database covers nearly 10% of the 30 million individuals in the japanese social insurance system , equating to approximately 2.5% of the total japanese population . data were extracted for patients with records between july 1 , 2009 , and june 30 , 2014 , and included information on patient demographics , diagnoses , drug prescriptions , medical procedures , medical facility characteristics , and reimbursement costs . included patients were women between ages 18 and 49 years who had two separate diagnoses of dysmenorrhea within 3 months . in japan , patients with dysmenorrhea are generally asked to come back to the clinic within the 3 months following a first medical consultation , as it was considered that only one visit would not be enough for selecting patients requiring medical care for dysmenorrhea . diagnoses were identified by icd-10 ( international classification of diseases 10th revision ) codes n944 ( primary dysmenorrhea ) or n946 ( dysmenorrhea , unspecified ) or one of the following medical information system development center ( medis - dc ) standard disease names : functional dysmenorrhea , menstrual pain , or dysmenorrhea . medis - dc consists of standardized names of diseases and injuries for reimbursement by the national health insurance.18 the index date was defined as the date of the first diagnosis of dysmenorrhea . primary dysmenorrhea was defined as patients without underlying causes ( endometriosis , adenomyosis , and fibroids ) during the preindex period and from 60 days after the index date , whereas secondary dysmenorrhea was defined as patients presenting at least one diagnosis of one of these conditions during this period . this criterion was defined based on the publications by copher et al19,20 and an assumption that imaging procedures for detecting underlying causes would be performed within 2 months following the first visit . patients were also required to have insurance records for at least 6 months with a diagnosis of dysmenorrhea prior to their initial diagnosis ( preindex period ) and for a continuous postindex period of at least 1 year , to be included in the study . patients were excluded if they had been diagnosed with gynecological cancers or bleeding disorders during the study period ( including both pre- and postindex ) , if they underwent gynecological surgery ( hysterectomy , endometrial ablation , or myomectomy ) during the preindex period , or if they were treated with hormonal agents during the preindex period ( figure 1 ) . for the resource utilization analysis , matched controls were identified among females in the jmdc database who did not suffer from dysmenorrhea ( ie , no diagnosis between july 1 , 2009 , and june 30 , 2014 ) . controls and cases were matched 2:1 by year of birth ( within 5 years ) , charlson comorbidity index ( cci ) at baseline ( 15 days from index date ) , and at least the same 18 months of follow - up period . the study assessed therapies that fit into one of the following categories : hormonal treatments ( leps , progestin , testosterone derivatives , and gnrh analogs ) , nsaids , hemostatic agents , and tcm therapies . relevant gynecological surgeries , including hysterectomies , endometrial ablations , and myomectomies , were also considered . the treatment patterns were analyzed based on prescription records , with a treatment line defined as a group of consecutive prescriptions of the same agent or combination of agents ( combination therapy ) , with no interruption exceeding 90 days . for each treatment line , the start date , end date , duration , and treatment category were determined . when a period exceeding 90 days was observed between the end of one treatment line and the beginning of a new one , therapy switches were defined when an overlap shorter than 21 days between the end of one therapy and the beginning of a new one was observed and the interval did not exceed 90 days . if an overlap occurred for 21 days or more , however , this was considered a combination therapy . because surgery was considered as a permanent treatment , a separate combination therapy type ( surgery combination ) was assigned . treatment patterns were analyzed among the entire cohort and according to the type of dysmenorrhea ( primary or secondary ) . a subgroup analysis was also conducted according to the prescriber s specialty for the first treatment line . the resources used and their associated costs were calculated based on the 1-year follow - up period after the index date for each patient . data were collected on inpatient costs , outpatient costs , and costs associated with both prescriptions and imaging procedures . from this information , resource utilization data collected in this analysis included the number of inpatient admissions , cumulative length of stay , number of outpatient visits , and number of imaging procedures . imaging procedures , which included echography , computed tomography scan , and magnetic resonance imaging , were determined to be related to a diagnosis of dysmenorrhea when they occurred within a 1-month period around the date of the diagnosis ( 15 days ) . the use of imaging procedures in controls was evaluated during the same 1-month period as it was for their corresponding matched cases . nonadjusted comparisons were conducted for outcomes with respect to primary vs secondary dysmenorrhea and cases vs controls . for continuous variables , a fisher s f mean values were then compared using a student s t - test ( for variables showing homoscedasticity ) or satterthwaite test ( heteroscedasticity ) . for categorical variables , a or fisher s exact test ( in cases of insufficient theoretical size of subgroups ) was performed to compare the distributions . time - to - event variables were assessed using kaplan meier curves and log - rank tests for between - group comparisons . ( ie , end - of - treatment events such as treatment switch , discontinuation of treatment , surgery , add - on therapy , and step - down therapy ) were analyzed using the competing risks survival method , estimating the cumulative incidence . the analyses of costs and resources were conducted through generalized linear models ( glms ) , testing normal , poisson , negative binomial , zero - inflated poisson , and zero - inflated negative binomial distributions . the goodness of fit of the models was assessed with the deviance , and the dispersion with the pearson value divided by the number of degrees of freedom . the models minimizing the deviance or with the best compromise deviance / dispersion ( in case of high dispersion ) were retained . models were adjusted for age , the category ( based on quartiles ) of total health care costs over the preindex period , the quantity corresponding to the modeled variable computed over the preindex period ( eg , the inpatient costs over the preindex period while modeling the inpatient costs over the 1-year postindex period ) , hospitalization during the preindex period , and the type of health insurance membership ( employee or dependent ) . all analyses were performed using sas analytics pro release 9.3 and r ( version 3.2.1 , sas institute , cary , nc , usa ) software ( for time - to - event data ) . in general , a p - value of 0.05 was determined to represent statistical significance . this study was approved by the ethics committee of the nonprofit organization , clinical research promotion network japan . the informed consent of patients was not applicable based on the ethical guidelines for epidemiological research issued by the ministry of health , welfare and labor . a total of 6,315 patients with dysmenorrhea , 3,441 ( 54.5% ) primary and 2,874 ( 45.5% ) secondary , were identified , with an average follow - up duration of 4.00.9 years . patients with secondary dysmenorrhea were significantly older compared with patients with primary dysmenorrhea ( 35.78.0 vs 31.67.9 years ; p<0.0001 ; table 2 ) , and over half of the secondary dysmenorrhea cohort were older than 35 years ( 59.3% vs 36.8% in the primary dysmenorrhea cohort ) . primary ovarian dysfunction was the most common comorbidity ( 22.1% ) at baseline and was more frequent in patients with primary dysmenorrhea than secondary dysmenorrhea ( 25.0% vs 18.6% ; p<0.0001 ; table 2 ) . the other comorbidities were more frequently reported in patients with secondary dysmenorrhea than primary dysmenorrhea , including erosion and ectropion of cervix uteri ( 21.8% vs 15.6% ; p<0.0001 ) , anemia ( 19.7% vs 6.2% ; p<0.0001 ) , acute vaginitis ( 14.5% vs 10.2% ; p<0.0001 ) , and low back pain ( 11.2% vs 8.1% ; p<0.0001 ) , respectively . over three - quarters of patients had a cci score of 0 , and less than 4% had a score greater than 1 ( table 2 ) . for both cohorts , first diagnoses of dysmenorrhea were reported in clinics ( 019-bed ) ( 76.4% ) and in hospitals ( 20-bed ) ( 23.6% ) . in more detail , 81.3% of primary and 70.6% of secondary dysmenorrhea cases were reported in clinics and 18.7% of primary and 29.4% of secondary dysmenorrhea cases were reported in hospitals . patients who were diagnosed in large hospitals ( 500-bed ) represented 5.7% of the primary dysmenorrhea cohort and 11.7% of the secondary dysmenor - rhea cohort . thus , patients in the secondary dysmenorrhea cohort were significantly more likely to be diagnosed in hospitals ( p<0.0001 ) and very large ( 500-bed ) facilities ( p<0.0001 ) compared to the primary dysmenorrhea cohort . the majority of patients were diagnosed in obstetrics and gynecology facilities ( 53.0% and 64.6% for primary and secondary dysmenorrhea cohorts , respectively ) , whereas the remainder were typically diagnosed in general internal medicine facilities ( 32.1% and 27.6% for primary and secondary dysmenorrhea cohorts , respectively ) . at least one diagnostic imaging procedure within 2 weeks before or after diagnosis was reported for 38.7% of patients with primary dysmenorrhea and 69.2% of those with secondary dysmenorrhea ; in most cases , this was echography . among women with secondary dysmenorrhea , a total of 83.4% of patients in the primary dysmenorrhea cohort and 89.2% in the secondary dysmenorrhea cohort were treated with at least one pharmacological agent or surgical procedure . median times to any treatment initiation after diagnosis of dysmenorrhea were 9 and 3 days , in primary and secondary dysmenorrhea cohorts , respectively . in the primary dysmenorrhea cohort , tcm therapies were the most frequently prescribed first - line treatment and were administered in 38.8% of all patients with primary dysmenorrhea ( including treated and untreated patients ) , leps were prescribed in 27.4% of patients , and the use of hemostatic agents was reported in 12.8% ( figure 2 ) . in the secondary dysmenorrhea cohort , the proportion of patients treated with leps was higher , with 50.2% receiving these agents as a first - line treatment , whereas 19.5% were treated with tcm therapies ( figure 3 ) . substantial differences in the treatment of dysmenorrhea were observed according to the specialty of the prescriber : leps were prescribed in first line by obstetricians and gynecologists in 57.2% and 67.7% of treated patients from the primary and secondary dysmenorrhea cohorts , respectively , while internal medicine physicians prescribed leps to 11.6% and 35.7% of treated patients , respectively . hemostatic agents were prescribed in first line in 3.8% and 3.4% by obstetricians and gynecologists , and in 25.4% and 21.8% by internal medicine physicians , of treated patients in the primary and secondary dysmenorrhea cohorts , respectively . tcms were prescribed in first line in 34.0% and 20.6% by obstetricians and gynecologists , and in 56.1% and 25.3% by internal medicine physicians , of treated patients in the primary and secondary dysmenorrhea cohorts , respectively . the time to treatment discontinuation was longer for leps compared with other therapies . in patients with primary dysmenorrhea , 35.1% were still receiving lep treatment at 1 year , compared to 10.4% for tcm therapies . similarly , 33.9% of patients with secondary dysmenorrhea continued to receive leps at 1 year , while only 11.5% continued taking tcm therapies for this duration . hemostatic agents and nsaids were administered only briefly , with a median treatment duration of 5 days for both agents , regardless of the cohort . of those patients who did not continue with their first - line treatment for the first 12 months postindex date , only a minority switched to a new therapy ( 17.1% and 21.3% for the primary and secondary dysmenorrhea cohorts , respectively ) and most did not receive any second - line treatment ( 73.7% and 63.3% , respectively ) . the most widely used treatments in both second and third lines were also leps and tcm therapies . the probability of surgery at 1 year was 4% in the secondary dysmenorrhea cohort and 0.2% in the primary dysmenorrhea cohort . patients with dysmenorrhea and the matched control cohort of patients without dysmenorrhea had similar baseline characteristics and resource utilization levels prior to the index period , with all groups reporting a mean number of inpatient admissions of 1.1 for the pre - index period . the analysis of inpatient care demonstrated a significant increase in the number of hospital admissions and length of stay due to dysmenorrhea in the secondary dysmenorrhea cohort vs the controls ( 5.7 additional admissions per 100 persons over 12 months ; 2.2 times longer cumulative length of stay ; both p<0.0001 ) , whereas there was no change in admissions compared to controls in the primary dysmenorrhea cohort . outpatient care visits occurred substantially more often in patients with dysmenorrhea vs controls , with primary and secondary dysmenorrhea cohorts reporting 8.0 and 8.5 additional outpatient visits over 12 months , respectively , after adjusting for baseline characteristics ( both p<0.0001 ; table 3 ) . primary and secondary dysmenorrhea cohorts had significantly higher mean total health care costs than controls ( mean sd : 191,680 jpy ( 1,917 usd ) 261,226 jpy ( 2,612 usd ) vs 83,615 jpy ( 836 usd ) 246,093 jpy ( 2,461usd ) for primary dysmenorrhea cohort and 246,488 jpy ( 2,465 usd ) 295,936 jpy ( 2,959 usd ) vs 90,711 jpy ( 907 usd ) 297,513 jpy ( 2,975 usd ) for secondary dysmenorrhea cohort ; p<0.001 ; table 3 ) . after adjusting for baseline characteristics , costs were 2.2 times and 2.9 times higher in patients in the primary and secondary dysmenorrhea cohorts , respectively , than controls ( both p<0.0001 ) . furthermore , total costs in secondary cases were higher by 33.5% compared to primary cases ( p<0.001 ; table 3 ) . a total of 6,315 patients with dysmenorrhea , 3,441 ( 54.5% ) primary and 2,874 ( 45.5% ) secondary , were identified , with an average follow - up duration of 4.00.9 years . patients with secondary dysmenorrhea were significantly older compared with patients with primary dysmenorrhea ( 35.78.0 vs 31.67.9 years ; p<0.0001 ; table 2 ) , and over half of the secondary dysmenorrhea cohort were older than 35 years ( 59.3% vs 36.8% in the primary dysmenorrhea cohort ) . primary ovarian dysfunction was the most common comorbidity ( 22.1% ) at baseline and was more frequent in patients with primary dysmenorrhea than secondary dysmenorrhea ( 25.0% vs 18.6% ; p<0.0001 ; table 2 ) . the other comorbidities were more frequently reported in patients with secondary dysmenorrhea than primary dysmenorrhea , including erosion and ectropion of cervix uteri ( 21.8% vs 15.6% ; p<0.0001 ) , anemia ( 19.7% vs 6.2% ; p<0.0001 ) , acute vaginitis ( 14.5% vs 10.2% ; p<0.0001 ) , and low back pain ( 11.2% vs 8.1% ; p<0.0001 ) , respectively . over three - quarters of patients had a cci score of 0 , and less than 4% had a score greater than 1 ( table 2 ) . for both cohorts , first diagnoses of dysmenorrhea were reported in clinics ( 019-bed ) ( 76.4% ) and in hospitals ( 20-bed ) ( 23.6% ) . in more detail , 81.3% of primary and 70.6% of secondary dysmenorrhea cases were reported in clinics and 18.7% of primary and 29.4% of secondary dysmenorrhea cases were reported in hospitals . patients who were diagnosed in large hospitals ( 500-bed ) represented 5.7% of the primary dysmenorrhea cohort and 11.7% of the secondary dysmenor - rhea cohort . thus , patients in the secondary dysmenorrhea cohort were significantly more likely to be diagnosed in hospitals ( p<0.0001 ) and very large ( 500-bed ) facilities ( p<0.0001 ) compared to the primary dysmenorrhea cohort . the majority of patients were diagnosed in obstetrics and gynecology facilities ( 53.0% and 64.6% for primary and secondary dysmenorrhea cohorts , respectively ) , whereas the remainder were typically diagnosed in general internal medicine facilities ( 32.1% and 27.6% for primary and secondary dysmenorrhea cohorts , respectively ) . at least one diagnostic imaging procedure within 2 weeks before or after diagnosis was reported for 38.7% of patients with primary dysmenorrhea and 69.2% of those with secondary dysmenorrhea ; in most cases , this was echography . among women with secondary dysmenorrhea , a total of 83.4% of patients in the primary dysmenorrhea cohort and 89.2% in the secondary dysmenorrhea cohort were treated with at least one pharmacological agent or surgical procedure . median times to any treatment initiation after diagnosis of dysmenorrhea were 9 and 3 days , in primary and secondary dysmenorrhea cohorts , respectively . in the primary dysmenorrhea cohort , tcm therapies were the most frequently prescribed first - line treatment and were administered in 38.8% of all patients with primary dysmenorrhea ( including treated and untreated patients ) , leps were prescribed in 27.4% of patients , and the use of hemostatic agents was reported in 12.8% ( figure 2 ) . in the secondary dysmenorrhea cohort , the proportion of patients treated with leps was higher , with 50.2% receiving these agents as a first - line treatment , whereas 19.5% were treated with tcm therapies ( figure 3 ) . substantial differences in the treatment of dysmenorrhea were observed according to the specialty of the prescriber : leps were prescribed in first line by obstetricians and gynecologists in 57.2% and 67.7% of treated patients from the primary and secondary dysmenorrhea cohorts , respectively , while internal medicine physicians prescribed leps to 11.6% and 35.7% of treated patients , respectively . hemostatic agents were prescribed in first line in 3.8% and 3.4% by obstetricians and gynecologists , and in 25.4% and 21.8% by internal medicine physicians , of treated patients in the primary and secondary dysmenorrhea cohorts , respectively . tcms were prescribed in first line in 34.0% and 20.6% by obstetricians and gynecologists , and in 56.1% and 25.3% by internal medicine physicians , of treated patients in the primary and secondary dysmenorrhea cohorts , respectively . in patients with primary dysmenorrhea , 35.1% were still receiving lep treatment at 1 year , compared to 10.4% for tcm therapies . similarly , 33.9% of patients with secondary dysmenorrhea continued to receive leps at 1 year , while only 11.5% continued taking tcm therapies for this duration . hemostatic agents and nsaids were administered only briefly , with a median treatment duration of 5 days for both agents , regardless of the cohort . of those patients who did not continue with their first - line treatment for the first 12 months postindex date , only a minority switched to a new therapy ( 17.1% and 21.3% for the primary and secondary dysmenorrhea cohorts , respectively ) and most did not receive any second - line treatment ( 73.7% and 63.3% , respectively ) . the most widely used treatments in both second and third lines were also leps and tcm therapies . the probability of surgery at 1 year was 4% in the secondary dysmenorrhea cohort and 0.2% in the primary dysmenorrhea cohort . patients with dysmenorrhea and the matched control cohort of patients without dysmenorrhea had similar baseline characteristics and resource utilization levels prior to the index period , with all groups reporting a mean number of inpatient admissions of 1.1 for the pre - index period . the analysis of inpatient care demonstrated a significant increase in the number of hospital admissions and length of stay due to dysmenorrhea in the secondary dysmenorrhea cohort vs the controls ( 5.7 additional admissions per 100 persons over 12 months ; 2.2 times longer cumulative length of stay ; both p<0.0001 ) , whereas there was no change in admissions compared to controls in the primary dysmenorrhea cohort . outpatient care visits occurred substantially more often in patients with dysmenorrhea vs controls , with primary and secondary dysmenorrhea cohorts reporting 8.0 and 8.5 additional outpatient visits over 12 months , respectively , after adjusting for baseline characteristics ( both p<0.0001 ; table 3 ) . primary and secondary dysmenorrhea cohorts had significantly higher mean total health care costs than controls ( mean sd : 191,680 jpy ( 1,917 usd ) 261,226 jpy ( 2,612 usd ) vs 83,615 jpy ( 836 usd ) 246,093 jpy ( 2,461usd ) for primary dysmenorrhea cohort and 246,488 jpy ( 2,465 usd ) 295,936 jpy ( 2,959 usd ) vs 90,711 jpy ( 907 usd ) 297,513 jpy ( 2,975 usd ) for secondary dysmenorrhea cohort ; p<0.001 ; table 3 ) . after adjusting for baseline characteristics , costs were 2.2 times and 2.9 times higher in patients in the primary and secondary dysmenorrhea cohorts , respectively , than controls ( both p<0.0001 ) . furthermore , total costs in secondary cases were higher by 33.5% compared to primary cases ( p<0.001 ; table 3 ) . this analysis demonstrated that the most commonly prescribed treatments for dysmenorrhea in japan excluding non - reimbursed agents were leps and tcm therapies , and patients with secondary dysmenorrhea had the highest utilization rates of leps . important differences in treatment patterns were observed according to the specialty of the prescriber of the first treatment line : obstetricians and gynecologists mainly prescribed leps , whereas internal medicine physicians prescribed tcm therapies most frequently . this might be due to differences in severity of dysmenorrhea among women who seek medical care from obstetricians and gynecologists vs internal medicine physicians and because women suffering from more severe dysmenorrhea may be more likely to consult an obstetrician or gynecologist and to receive leps . our findings were generally compatible with the guidelines for gynecological practice by the jsog and jaog 2011 edition.10 although such guidance suggests nsaid treatment for initial pain relief , our findings show relatively low levels of nsaid use . one of the reasons may be that many patients may have used over - the - counter therapies including nsaids , which are not captured by the database.1 persistence on leps was higher in this analysis than for any of the other therapies assessed , including tcm therapies . it was found by adding up the proportions of patients without defined treatment ( 13.0% of both primary and secondary dysmenorrhea cases ) and those with hemostatic agents ( 11.2% ) and nsaids ( 1.6% ) that around 25% of patients diagnosed with dysmenorrhea received no reimbursed treatment or short - term treatments only . this study also demonstrated that affected patients have more frequent physician visits , equating to roughly one additional visit every 45 days that can be attributed to dysmenorrhea . this high frequency suggests that the disease has a strong impact on the quality of life of these patients . furthermore , dysmenorrhea does require inpatient care in some cases , with approximately six additional inpatient admissions per 100 patients over 12 months attributed to dysmenorrhea in patients with secondary dysmenorrhea . as a result , health care costs in women with dysmenorrhea are two to three times higher than costs in women who do not suffer from this disorder . estimates of the prevalence of dysmenorrhea in japan ranged from 15.8% to 25%.4,21 a survey conducted in 2004 by the josei rodo kyokai ( the japan association for the advancement of working women ) reported that 80% of working women has menstrual pain and 3% of women had severe symptoms resulting in absence from work.22 the prevalence of dysmenor - rhea in this jmdc database was only 1.6% , and it is possible that the restriction to patients with at least two recorded diagnoses within 3 months led to the exclusion of less severe cases . compared to a study by tanaka et al,1 which was a population - based survey of japanese women aged 1549 years , patients in this analysis had a higher rate of lep utilization . one potential explanation is an increase in the general utilization of leps over time that has been captured by our more recently collected jmdc data . comparatively , our analysis also has a more restrictive patient sample than that of tanaka et al,2 who surveyed women with menstrual symptoms in general , as our study only included patients who consulted for dysmenorrhea at least two times within 3 months , and not for any other menstrual symptoms . in addition to an estimate of prevalence and resource utilization , tanaka et al1 estimated the willingness to pay for a drug that would eliminate all menstrual symptoms . the average willingness - to - pay amount for a drug that would eliminate all menstrual symptoms in outpatients was 4,834 jpy ( 48 usd ) per month . furthermore , willingness - to - pay to eliminate interference of their menstrual symptoms with the activities of daily life was estimated at 3,304 jpy ( 33 usd ) per month . interestingly , the annual pharmacy costs attributable to dysmenorrhea , which were derived from differences of annual pharmacy costs between cases and controls in the current analysis , were estimated to be 31,840 jpy ( 318 usd ) , or 2,653 jpy ( 26.5 usd ) per month , which was below those of the average monthly costs that outpatients in the aforementioned survey were willing to pay for a drug that would eliminate all menstrual symptoms , and costs that would eliminate interference with daily life due to menstrual symptoms . however , the jmdc pharmacy costs might be less than the pharmacy costs that patients actually paid , as the analysis excluded nonreimbursed therapies , such as nsaids and cocs . therefore , it is uncertain that the total pharmacy costs paid by patients and health insurance are below the willingness - to - pay amount for dysmenorrhea treatments . study limitations include those related to any retrospective claims data analysis , including the lack of data supporting the specific reason for each treatment choice . furthermore , dysmenorrhea is certainly underreported in claims databases and women included in this study may represent severe cases consulting a practitioner . it was previously estimated that 64.6% of women with menstrual symptoms do not seek medical care.1 in addition , treatment patterns observed in this analysis may not fully reflect all medications taken to treat dysmenorrhea if they are not reimbursed by insurance , such as cocs or over - the - counter pain medications such as nsaids . as a result , women who were identified as having no treatment may have received nonreimbursed or over - the - counter therapies during the study period ; this could have artificially increased the observed no treatment rate . because the jmdc is an administrative database , its diagnostic reporting may be incomplete as physicians may report codes for which reimbursement is provided instead of the full or actual diagnoses . for example , nsaids prescribed for diagnosis with other pain symptoms not associated with menstruation were not included in this study . patients who require surgery may not receive a diagnosis of dysmenorrhea as they will instead receive a diagnostic code for the underlying condition requiring surgery ( eg , fibroids ) . the burden of the disease was estimated over the first year of follow - up and not over the whole available period . building on this research , future analyses may look at costs beyond 12 months , which are likely lower than in the year following diagnosis . finally , assumptions used to define treatment patterns were somewhat arbitrary , including the use of 90 days as the cut - off point for treatment discontinuation . these assumptions were , however , supported by expert opinion , owing to the fact that physicians typically prescribe such therapies for a maximum of 3 months . considerable heterogeneity in treatment patterns was observed among patients with dysmenorrhea , with relatively low utilization of leps in patients with primary dysmenorrhea compared to secondary dysmenorrhea , and those treated by internal medicine physicians compared to obstetricians and gynecologists . total health care costs were significantly higher among women with dysmenor - rhea compared with similar women who do not suffer from this condition , and excess costs are primarily driven by outpatient care . further research is recommended to evaluate whether a different allocation of resources , for example with higher utilization of leps , may yield better health outcomes and reduce the economic burden of dysmenorrhea .

purposethis study aimed to describe treatment patterns and estimate health care resource utilization and associated costs among japanese women with dysmenorrhea , using a claims database.methodsthis was a retrospective analysis using health insurance data from the japan medical data center , assessing female patients aged 1849 years with newly diagnosed primary or secondary dysmenorrhea . treatment pattern analyses focused on hormonal medications , analgesics , hemostatic agents , traditional chinese medicine ( tcm ) , and gynecological surgeries . data were collected on health care resource utilization and costs associated with medications , imaging procedures , and inpatient and outpatient care in both patients and matched controls.resultsthe analysis included 6,315 women with dysmenorrhea ( 3,441 primary ; 2,874 secondary ) . the most commonly prescribed initial therapies were low - dose estrogen progestins ( leps , 37.7% ) and tcm ( 30.0% ) , with substantial differences between primary ( leps : 27.4% , tcm : 38.8% ) and secondary ( leps : 50.2% , tcm : 19.5% ) dysmenorrhea cohorts . surgery was conducted in < 5% of all patients . both primary and secondary cohorts of dysmenorrhea had significantly higher mean total health care costs compared to controls within the 1-year period following diagnosis ( case - primary : 191,680 jpy [ 1,916 usd ] ; secondary : 246,488 jpy [ 2,465 usd ] , control - primary : 83,615 jpy [ 836 usd ] ; secondary : 90,711 jpy [ 907 usd ] ) ( p<0.0001 ) . after adjusting for baseline characteristics , these costs were 2.2 and 2.9 times higher for primary and secondary dysmenorrhea cohorts , respectively , compared with matched controls , ( both p<0.0001 ) . the main driver of these excess costs was outpatient care , with eight additional physician visits per year among dysmenorrhea patients compared to controls ( p<0.0001).conclusionconsiderable heterogeneity in treatment patterns was observed , with relatively low utilization of leps in patients with primary dysmenorrhea and those treated by internal medicine physicians . total annual health care costs were approximately 23 times higher in patients with dysmenorrhea compared to women without the condition .

Introduction Methods Study design and population Treatment patterns Resource utilization and cost calculations Statistical analysis Results Patient characteristics Treatment patterns Resource utilization and costs associated with dysmenorrhea Discussion Conclusion

many japanese women experience health issues associated with menstruation ; these can include menstrual pain also referred to as dysmenorrhea , heavy menstrual bleeding ( hmb ) also referred to as menorrhagia , and premenstrual syndrome ( pms).13 dysmenorrhea is the most common gynecological complaint associated with menstruation with a prevalence of 25% among all women , and reaching up to 90% among adolescents.4 it has also been reported that one - third of japanese women require analgesic therapy for dysmenorrhea.5 in some women , dysmenorrhea can not be attributed to any specific cause and is referred to as primary or idiopathic . regardless of the cause , dysmenorrhea can have a substantial impact on patient quality of life,7,8 yet many patients do not seek treatment.9 in a patient survey , some untreated women have expressed feelings of resistance or aversion toward seeking therapy , and many suggested that gynecologist consultations were unnecessary for their disorder.1 however , a substantial proportion of women who did seek medical treatment agreed that their daily lives were significantly improved after therapy , and it was also estimated that gynecologist visits saved over 7,000 jpy ( 70 usd ) monthly costs per - patient , occurring due to time off work.1 according to the guidelines for gynecological practice in japan , by the japan society of obstetrics and gynecology ( jsog ) and japan association of obstetricians and gynecologists ( jaog ) ( 2011 edition ) , low - dose estrogen progestins ( leps ) and nonsteroidal anti - inflammatory drugs ( nsaids ) are primarily recommended for primary dysmenorrhea , and traditional chinese medicines ( tcms ) could be used for primary dysmenorrhea.10 other current clinical practice guidelines for the treatment of dysmenorrhea include the use of over - the - counter analgesics , nsaids , and oral contraceptives such as leps , progestin - only therapies , and the levonorgestrel - releasing intrauterine system.11,12 two kinds of combined oral contraceptives ( cocs ) are available on the japanese market : leps , ( norethisterone / estrogen and drospirenone / estrogen ) , which are reimbursed for dysmenorrhea treatment , and the other cocs for contraceptive purposes , which are not reimbursed . for example , leps have been reported to be effective in alleviating symptoms in up to 80% of women.13 additional studies have shown efficacy of both gonadotropin - releasing hormone ( gnrh ) analogs and testosterone derivatives for treating underlying causes of secondary dysmenorrhea , such as endometriosis.1417 existing evidence on treatment patterns , health care resource use , and associated costs in japanese patients with dysmenorrhea is limited . the objectives of this study were to describe treatment patterns and estimate health care resource use and costs among japanese women with newly diagnosed dysmenorrhea in a real - world setting . to assess health care resource utilization and costs among patients , a comparison was made between patients with dysmenorrhea ( cases ) and those without dysmenorrhea ( matched control group ) . furthermore , all results were differentiated between women with primary and secondary dysmenorrhea and treatment patterns were evaluated by prescriber s specialty . this was a retrospective claims analysis of the japan medical data center ( jmdc ) database , which includes deidentified medical ( inpatient and outpatient ) and pharmacy claims from up to 3.0 million beneficiaries ( both employees and their dependents ) from 2005 onward . the study assessed therapies that fit into one of the following categories : hormonal treatments ( leps , progestin , testosterone derivatives , and gnrh analogs ) , nsaids , hemostatic agents , and tcm therapies . treatment patterns were analyzed among the entire cohort and according to the type of dysmenorrhea ( primary or secondary ) . data were collected on inpatient costs , outpatient costs , and costs associated with both prescriptions and imaging procedures . imaging procedures , which included echography , computed tomography scan , and magnetic resonance imaging , were determined to be related to a diagnosis of dysmenorrhea when they occurred within a 1-month period around the date of the diagnosis ( 15 days ) . models were adjusted for age , the category ( based on quartiles ) of total health care costs over the preindex period , the quantity corresponding to the modeled variable computed over the preindex period ( eg , the inpatient costs over the preindex period while modeling the inpatient costs over the 1-year postindex period ) , hospitalization during the preindex period , and the type of health insurance membership ( employee or dependent ) . this was a retrospective claims analysis of the japan medical data center ( jmdc ) database , which includes deidentified medical ( inpatient and outpatient ) and pharmacy claims from up to 3.0 million beneficiaries ( both employees and their dependents ) from 2005 onward . data were extracted for patients with records between july 1 , 2009 , and june 30 , 2014 , and included information on patient demographics , diagnoses , drug prescriptions , medical procedures , medical facility characteristics , and reimbursement costs . the study assessed therapies that fit into one of the following categories : hormonal treatments ( leps , progestin , testosterone derivatives , and gnrh analogs ) , nsaids , hemostatic agents , and tcm therapies . treatment patterns were analyzed among the entire cohort and according to the type of dysmenorrhea ( primary or secondary ) . data were collected on inpatient costs , outpatient costs , and costs associated with both prescriptions and imaging procedures . models were adjusted for age , the category ( based on quartiles ) of total health care costs over the preindex period , the quantity corresponding to the modeled variable computed over the preindex period ( eg , the inpatient costs over the preindex period while modeling the inpatient costs over the 1-year postindex period ) , hospitalization during the preindex period , and the type of health insurance membership ( employee or dependent ) . a total of 6,315 patients with dysmenorrhea , 3,441 ( 54.5% ) primary and 2,874 ( 45.5% ) secondary , were identified , with an average follow - up duration of 4.00.9 years . patients with secondary dysmenorrhea were significantly older compared with patients with primary dysmenorrhea ( 35.78.0 vs 31.67.9 years ; p<0.0001 ; table 2 ) , and over half of the secondary dysmenorrhea cohort were older than 35 years ( 59.3% vs 36.8% in the primary dysmenorrhea cohort ) . primary ovarian dysfunction was the most common comorbidity ( 22.1% ) at baseline and was more frequent in patients with primary dysmenorrhea than secondary dysmenorrhea ( 25.0% vs 18.6% ; p<0.0001 ; table 2 ) . the other comorbidities were more frequently reported in patients with secondary dysmenorrhea than primary dysmenorrhea , including erosion and ectropion of cervix uteri ( 21.8% vs 15.6% ; p<0.0001 ) , anemia ( 19.7% vs 6.2% ; p<0.0001 ) , acute vaginitis ( 14.5% vs 10.2% ; p<0.0001 ) , and low back pain ( 11.2% vs 8.1% ; p<0.0001 ) , respectively . thus , patients in the secondary dysmenorrhea cohort were significantly more likely to be diagnosed in hospitals ( p<0.0001 ) and very large ( 500-bed ) facilities ( p<0.0001 ) compared to the primary dysmenorrhea cohort . the majority of patients were diagnosed in obstetrics and gynecology facilities ( 53.0% and 64.6% for primary and secondary dysmenorrhea cohorts , respectively ) , whereas the remainder were typically diagnosed in general internal medicine facilities ( 32.1% and 27.6% for primary and secondary dysmenorrhea cohorts , respectively ) . at least one diagnostic imaging procedure within 2 weeks before or after diagnosis was reported for 38.7% of patients with primary dysmenorrhea and 69.2% of those with secondary dysmenorrhea ; in most cases , this was echography . median times to any treatment initiation after diagnosis of dysmenorrhea were 9 and 3 days , in primary and secondary dysmenorrhea cohorts , respectively . in the primary dysmenorrhea cohort , tcm therapies were the most frequently prescribed first - line treatment and were administered in 38.8% of all patients with primary dysmenorrhea ( including treated and untreated patients ) , leps were prescribed in 27.4% of patients , and the use of hemostatic agents was reported in 12.8% ( figure 2 ) . substantial differences in the treatment of dysmenorrhea were observed according to the specialty of the prescriber : leps were prescribed in first line by obstetricians and gynecologists in 57.2% and 67.7% of treated patients from the primary and secondary dysmenorrhea cohorts , respectively , while internal medicine physicians prescribed leps to 11.6% and 35.7% of treated patients , respectively . hemostatic agents were prescribed in first line in 3.8% and 3.4% by obstetricians and gynecologists , and in 25.4% and 21.8% by internal medicine physicians , of treated patients in the primary and secondary dysmenorrhea cohorts , respectively . tcms were prescribed in first line in 34.0% and 20.6% by obstetricians and gynecologists , and in 56.1% and 25.3% by internal medicine physicians , of treated patients in the primary and secondary dysmenorrhea cohorts , respectively . in patients with primary dysmenorrhea , 35.1% were still receiving lep treatment at 1 year , compared to 10.4% for tcm therapies . of those patients who did not continue with their first - line treatment for the first 12 months postindex date , only a minority switched to a new therapy ( 17.1% and 21.3% for the primary and secondary dysmenorrhea cohorts , respectively ) and most did not receive any second - line treatment ( 73.7% and 63.3% , respectively ) . patients with dysmenorrhea and the matched control cohort of patients without dysmenorrhea had similar baseline characteristics and resource utilization levels prior to the index period , with all groups reporting a mean number of inpatient admissions of 1.1 for the pre - index period . the analysis of inpatient care demonstrated a significant increase in the number of hospital admissions and length of stay due to dysmenorrhea in the secondary dysmenorrhea cohort vs the controls ( 5.7 additional admissions per 100 persons over 12 months ; 2.2 times longer cumulative length of stay ; both p<0.0001 ) , whereas there was no change in admissions compared to controls in the primary dysmenorrhea cohort . outpatient care visits occurred substantially more often in patients with dysmenorrhea vs controls , with primary and secondary dysmenorrhea cohorts reporting 8.0 and 8.5 additional outpatient visits over 12 months , respectively , after adjusting for baseline characteristics ( both p<0.0001 ; table 3 ) . primary and secondary dysmenorrhea cohorts had significantly higher mean total health care costs than controls ( mean sd : 191,680 jpy ( 1,917 usd ) 261,226 jpy ( 2,612 usd ) vs 83,615 jpy ( 836 usd ) 246,093 jpy ( 2,461usd ) for primary dysmenorrhea cohort and 246,488 jpy ( 2,465 usd ) 295,936 jpy ( 2,959 usd ) vs 90,711 jpy ( 907 usd ) 297,513 jpy ( 2,975 usd ) for secondary dysmenorrhea cohort ; p<0.001 ; table 3 ) . after adjusting for baseline characteristics , costs were 2.2 times and 2.9 times higher in patients in the primary and secondary dysmenorrhea cohorts , respectively , than controls ( both p<0.0001 ) . a total of 6,315 patients with dysmenorrhea , 3,441 ( 54.5% ) primary and 2,874 ( 45.5% ) secondary , were identified , with an average follow - up duration of 4.00.9 years . patients with secondary dysmenorrhea were significantly older compared with patients with primary dysmenorrhea ( 35.78.0 vs 31.67.9 years ; p<0.0001 ; table 2 ) , and over half of the secondary dysmenorrhea cohort were older than 35 years ( 59.3% vs 36.8% in the primary dysmenorrhea cohort ) . primary ovarian dysfunction was the most common comorbidity ( 22.1% ) at baseline and was more frequent in patients with primary dysmenorrhea than secondary dysmenorrhea ( 25.0% vs 18.6% ; p<0.0001 ; table 2 ) . the other comorbidities were more frequently reported in patients with secondary dysmenorrhea than primary dysmenorrhea , including erosion and ectropion of cervix uteri ( 21.8% vs 15.6% ; p<0.0001 ) , anemia ( 19.7% vs 6.2% ; p<0.0001 ) , acute vaginitis ( 14.5% vs 10.2% ; p<0.0001 ) , and low back pain ( 11.2% vs 8.1% ; p<0.0001 ) , respectively . thus , patients in the secondary dysmenorrhea cohort were significantly more likely to be diagnosed in hospitals ( p<0.0001 ) and very large ( 500-bed ) facilities ( p<0.0001 ) compared to the primary dysmenorrhea cohort . the majority of patients were diagnosed in obstetrics and gynecology facilities ( 53.0% and 64.6% for primary and secondary dysmenorrhea cohorts , respectively ) , whereas the remainder were typically diagnosed in general internal medicine facilities ( 32.1% and 27.6% for primary and secondary dysmenorrhea cohorts , respectively ) . median times to any treatment initiation after diagnosis of dysmenorrhea were 9 and 3 days , in primary and secondary dysmenorrhea cohorts , respectively . in the primary dysmenorrhea cohort , tcm therapies were the most frequently prescribed first - line treatment and were administered in 38.8% of all patients with primary dysmenorrhea ( including treated and untreated patients ) , leps were prescribed in 27.4% of patients , and the use of hemostatic agents was reported in 12.8% ( figure 2 ) . substantial differences in the treatment of dysmenorrhea were observed according to the specialty of the prescriber : leps were prescribed in first line by obstetricians and gynecologists in 57.2% and 67.7% of treated patients from the primary and secondary dysmenorrhea cohorts , respectively , while internal medicine physicians prescribed leps to 11.6% and 35.7% of treated patients , respectively . hemostatic agents were prescribed in first line in 3.8% and 3.4% by obstetricians and gynecologists , and in 25.4% and 21.8% by internal medicine physicians , of treated patients in the primary and secondary dysmenorrhea cohorts , respectively . tcms were prescribed in first line in 34.0% and 20.6% by obstetricians and gynecologists , and in 56.1% and 25.3% by internal medicine physicians , of treated patients in the primary and secondary dysmenorrhea cohorts , respectively . in patients with primary dysmenorrhea , 35.1% were still receiving lep treatment at 1 year , compared to 10.4% for tcm therapies . of those patients who did not continue with their first - line treatment for the first 12 months postindex date , only a minority switched to a new therapy ( 17.1% and 21.3% for the primary and secondary dysmenorrhea cohorts , respectively ) and most did not receive any second - line treatment ( 73.7% and 63.3% , respectively ) . patients with dysmenorrhea and the matched control cohort of patients without dysmenorrhea had similar baseline characteristics and resource utilization levels prior to the index period , with all groups reporting a mean number of inpatient admissions of 1.1 for the pre - index period . the analysis of inpatient care demonstrated a significant increase in the number of hospital admissions and length of stay due to dysmenorrhea in the secondary dysmenorrhea cohort vs the controls ( 5.7 additional admissions per 100 persons over 12 months ; 2.2 times longer cumulative length of stay ; both p<0.0001 ) , whereas there was no change in admissions compared to controls in the primary dysmenorrhea cohort . outpatient care visits occurred substantially more often in patients with dysmenorrhea vs controls , with primary and secondary dysmenorrhea cohorts reporting 8.0 and 8.5 additional outpatient visits over 12 months , respectively , after adjusting for baseline characteristics ( both p<0.0001 ; table 3 ) . primary and secondary dysmenorrhea cohorts had significantly higher mean total health care costs than controls ( mean sd : 191,680 jpy ( 1,917 usd ) 261,226 jpy ( 2,612 usd ) vs 83,615 jpy ( 836 usd ) 246,093 jpy ( 2,461usd ) for primary dysmenorrhea cohort and 246,488 jpy ( 2,465 usd ) 295,936 jpy ( 2,959 usd ) vs 90,711 jpy ( 907 usd ) 297,513 jpy ( 2,975 usd ) for secondary dysmenorrhea cohort ; p<0.001 ; table 3 ) . after adjusting for baseline characteristics , costs were 2.2 times and 2.9 times higher in patients in the primary and secondary dysmenorrhea cohorts , respectively , than controls ( both p<0.0001 ) . this analysis demonstrated that the most commonly prescribed treatments for dysmenorrhea in japan excluding non - reimbursed agents were leps and tcm therapies , and patients with secondary dysmenorrhea had the highest utilization rates of leps . it was found by adding up the proportions of patients without defined treatment ( 13.0% of both primary and secondary dysmenorrhea cases ) and those with hemostatic agents ( 11.2% ) and nsaids ( 1.6% ) that around 25% of patients diagnosed with dysmenorrhea received no reimbursed treatment or short - term treatments only . furthermore , dysmenorrhea does require inpatient care in some cases , with approximately six additional inpatient admissions per 100 patients over 12 months attributed to dysmenorrhea in patients with secondary dysmenorrhea . considerable heterogeneity in treatment patterns was observed among patients with dysmenorrhea , with relatively low utilization of leps in patients with primary dysmenorrhea compared to secondary dysmenorrhea , and those treated by internal medicine physicians compared to obstetricians and gynecologists . total health care costs were significantly higher among women with dysmenor - rhea compared with similar women who do not suffer from this condition , and excess costs are primarily driven by outpatient care .

causality assessment of adverse drug reactions ( adrs ) is formally undertaken by the pharmaceutical industry , regulators and researchers in clinical trials but rarely by clinicians . for example , regulatory authorities use causality assessment to assess spontaneous adr reports to help with signal detection and inform risk benefit decisions regarding medicines . anecdotally , clinicians ' assessment of adr causality is generally done informally and sometimes subconsciously , which leads to variability in decision making , specifically in terms of when to alter drug therapy and reporting of adrs . the naranjo tool is probably the most widely used worldwide . in two recent large paediatric adr studies , the naranjo tool was found to be inadequate for adr causality assessment and have poor reproducibility.[2 , 3 ] for instance , in the assessment of adrs detected in children acutely admitted to hospital over a 12 month period , interrater reliability using the naranjo tool was poor . the investigators concluded that some of the questions in the tool were not appropriate , leading to a lack of sensitivity , with the overall score obtained being artificially lowered . in addition , the weighting for each question within the naranjo tool was not justified in the original publication . subsequently , a new causality assessment tool , the liverpool causality assessment tool ( lcat , see appendix s1 ) , was developed , formally tested and internally validated . this tool aimed to overcome the issues identified with the naranjo tool , while ( 1 ) making it as easy , or easier , to use than the naranjo tool and ( 2 ) maintaining the basic principles of causality assessment . given that there is variability in clinical decision making around adrs , we aimed to develop a means to disseminate this new approach of adr causality assessment to practitioners . we developed an interactive , webbased elearning package designed to improve assessment by individual practitioners : the liverpool adr causality assessment elearning package ( lacaep ) ( see box 1 for the attributes of this package ) . the use of elearning packages for medical training has been shown to have good uptake and to be effective . content and navigation the elearning package takes approximately 1 hour to completethe package contains interactive bespoke learning activities that require the user to interact with the software in order to continue , and will offer instructive feedback . the package includes : an interactive diagram ( based on the causality flowchart ) that allows the use to zoom / pan / rollover to navigate the tool and to gain more information about items in the diagramlogical question arrangement that underlines the sequential nature of the assessment toolreallife case studies : use reallife case studies to build a causality assessment by answering each of the questions on the lcat in turnexpert opinion : the package includes the availability of an expert or panel of expert characters who can provide feedback and hints on decisions made by the user during the case studies exercise the interface contains a straightforward navigation system , with a short tutorial available explaining the functionability of all buttons in the interfacea content menu and glossary are includedthe package has no formal assessment but will require users to complete interactive activities in order to progress the elearning package takes approximately 1 hour to complete the package contains interactive bespoke learning activities that require the user to interact with the software in order to continue , and will offer instructive feedback . the package includes : an interactive diagram ( based on the causality flowchart ) that allows the use to zoom / pan / rollover to navigate the tool and to gain more information about items in the diagramlogical question arrangement that underlines the sequential nature of the assessment toolreallife case studies : use reallife case studies to build a causality assessment by answering each of the questions on the lcat in turnexpert opinion : the package includes the availability of an expert or panel of expert characters who can provide feedback and hints on decisions made by the user during the case studies exercise an interactive diagram ( based on the causality flowchart ) that allows the use to zoom / pan / rollover to navigate the tool and to gain more information about items in the diagram logical question arrangement that underlines the sequential nature of the assessment tool reallife case studies : use reallife case studies to build a causality assessment by answering each of the questions on the lcat in turn expert opinion : the package includes the availability of an expert or panel of expert characters who can provide feedback and hints on decisions made by the user during the case studies exercise the interface contains a straightforward navigation system , with a short tutorial available explaining the functionability of all buttons in the interface a content menu and glossary are included the package has no formal assessment but will require users to complete interactive activities in order to progress reporting and user tracking the package bookmarks user progress between sessions and retain options chosen in completed activitiesadministrators of the package will be able to access the following information ( via the learning management system where the package will be hosted ) : the participant demographic details ; the current progress of the participant ; which activities they have undertaken ; the outcome of each assessment made , i.e. what was the classification , what path did they take on the lcat to get there and was the classification correct . at the end of the package , the user is asked to complete a feedback survey which assesses the usability and usefulness of the package and of the lcat the package bookmarks user progress between sessions and retain options chosen in completed activities administrators of the package will be able to access the following information ( via the learning management system where the package will be hosted ) : the participant demographic details ; the current progress of the participant ; which activities they have undertaken ; the outcome of each assessment made , i.e. what was the classification , what path did they take on the lcat to get there and was the classification correct . the participant demographic details ; the current progress of the participant ; which activities they have undertaken ; the outcome of each assessment made , i.e. what was the classification , what path did they take on the lcat to get there and was the classification correct . at the end of the package , the user is asked to complete a feedback survey which assesses the usability and usefulness of the package and of the lcat accessibility an ebook provides alternative testbased content for the packagethe package has been tested for sharable content object reference model ( scorm ) compliance with the adl test suitethe package complied with world wide web consortium ( w3c ) web standards wherever possibleflash or javascriptbased content is accompanied by alternative html content an ebook provides alternative testbased content for the package the package has been tested for sharable content object reference model ( scorm ) compliance with the adl test suite the package complied with world wide web consortium ( w3c ) web standards wherever possible flash or javascriptbased content is accompanied by alternative html content the purpose of this pilot randomised controlled trial was to gain feedback on the usability and usefulness of the lcat and the lacaep . feedback obtained for the latter will be used to identify areas for improvement and development . this trial also aimed to generate data on effect size enabling a larger hypothesis testing study to be conducted . this was a pilot , singleblind , parallel group study conducted by the university of liverpool . this pilot aimed to inform a larger scale study that would formally compare the effect that the lacaep has on improving the consistency of assigning causality using the lcat . the study obtained organisational approval from nhs north of england , and permission was obtained from the head of schools at the north west and mersey deaneries to include trainees from that region . eligibility criteria were defined to ensure that improvements in classifications were attributed to the intervention and so that prior knowledge or experience of the participants could be managed as appropriate . in the uk , trainees in paediatrics progress through specialty training ( st ) levels , st 1 being the first year of training . eligible participants were specialist trainees in paediatrics ( st level 1 and above ) within the mersey ( n = 165 ) and north west deaneries ( n = 214 ) . trainees who had previously received formal training in causality assessment or had obtained a professional qualification in clinical pharmacology or pharmacy were excluded . eligible trainees were recruited through email and by advertising the trial on the alder hey children 's hospital intranet and in workplaces . invitations to participate all participants completed a consent form when they registered to participate and returned an electric or hard copy . random allocation sequence was generated by computer by an independent statistician and was stratified by speciality training level . both the control and intervention arm received access to the lcat to assist causality assessment . participants in the intervention arm accessed an interactive training module with selfdirected elearning components that guided users when making causality assessments using the lcat ( lacaep ) . appendix s2 and s3 show illustrative screenshots of the introduction page and a worked example , respectively . although trainees were aware of the allocated arm , data analysts were kept blinded to the allocation until after the analyses were finalised . each trainee was issued a username , password and web link which would allow access to the trial platform according to the randomisation schedule . following any training , trainees in both arms were required to assess the same 20 adr cases using the lcat tool . the elearning package was tested rigorously by the study team for functionality and content before the trial was opened . several iterations of testing were undertaken until all aspects of the trial and package were suitable for a full pilot study to commence . participants were able to access the trial platform from 29 february to 15 march 2013 . email reminders were sent to those who had not completed the assessments during this period to improve completion rates . trainees who completed the trial were given a training certificate for their training records and entered into a cash prize draw . adr case studies used both for post intervention assessment and within the training phase of the elearning tool were taken from two previous adr studies.[6 , 7 ] within these studies , causality classification ( unlikely , possible , probable or definite ) was reached by consensus by a multidisciplinary panel of experts . for the purpose of this trial , gold standard. case studies were selected using quota sampling methods from this cohort of cases to mirror the distribution of possible , probable , definite and unlikely adrs observed in these studies . the number of correct classifications when compared against the gold standard was defined as the primary efficacy outcome . as a second efficacy outcome , the route taken on the lcat flowchart was recorded to ensure that classifications were obtained following a route defined by a multidisciplinary panel of experts . upon completing the intervention , trainees were encouraged to provide feedback on both lcat and lacaep ( appendix s4 ) by completing an optional survey , built in to the package , made up of a series of open and closed questions . as this was a pilot study , intended to generate data on effect size to enable a larger hypothesis study to be conducted , no formal power analysis was completed prior to the trial . a pragmatic sample of 80 participants of the total 379 trainees at that time was considered a minimum requirement . closed items on the feedback questionnaire were analysed quantitatively and reported as count data and percentages . no formal qualitative analysis was conducted on open items which are presented verbatim . to ensure participants remained anonymous , each was given a unique participant number made up of a letter to indicate the intervention arm ( a for the intervention arm and b for the control arm ) and a sequential number within arm . overall series agreement postintervention was summarised for each treatment group , both overall and split by speciality training level ( groups : 3 and below , 4 and above ) , using descriptive statistics , means with 95% confidence intervals ( ci ) or medians with an interquartile range if the scores was nonnormally distributed . the effect of the intervention was summarised using descriptive statistics , means with 95% confidence intervals ( or medians with an interquartile range if the scores was nonnormally distributed ) . all statistical analysis was carried out using the statistical software package r ( version 2.13.2 , r core team ( 2013 ) . as this was a pilot study , intended to generate data on effect size to enable a larger hypothesis study to be conducted , no formal power analysis was completed prior to the trial . a pragmatic sample of 80 participants of the total 379 trainees at that time was considered a minimum requirement . closed items on the feedback questionnaire no formal qualitative analysis was conducted on open items which are presented verbatim . to ensure participants remained anonymous , each was given a unique participant number made up of a letter to indicate the intervention arm ( a for the intervention arm and b for the control arm ) and a sequential number within arm . overall series agreement postintervention was summarised for each treatment group , both overall and split by speciality training level ( groups : 3 and below , 4 and above ) , using descriptive statistics , means with 95% confidence intervals ( ci ) or medians with an interquartile range if the scores was nonnormally distributed . the effect of the intervention was summarised using descriptive statistics , means with 95% confidence intervals ( or medians with an interquartile range if the scores was nonnormally distributed ) . all statistical analysis was carried out using the statistical software package r ( version 2.13.2 , r core team ( 2013 ) . all paediatric trainees within the mersey ( n = 165 ) and north west ( n = 214 ) deanery were approached to participate . sixty participants provided consent during the recruitment phase ; three were found to be ineligible upon screening ; one had a pharmacology phd , one had pharmaceutical industry experience , and the st level was unknown for the third . the 57 remaining participants were randomised 1 : 1 to the two intervention arms . twentynine participants were randomised to the intervention ( training ) arm , 13 were st level 13 , and 16 were st level 48 . twentyone ( 72% ) of those randomised to the intervention arm started the training package and assessment , and of those , 18 ( 62% ) completed the assessment and the feedback questionnaire . twentyeight participants were randomised to receive no training , 13 were st level 13 and 15 st level 48 . twentythree ( 82% ) of those randomised to the control arm started the assessment , 17 ( 61% ) completed the assessment , and 16 ( 57% ) completed the feedback questionnaire . thirtyfour participants provided feedback on the lcat , and 18 provided feedback on the lacaep . results of the feedback questionnaire is given in tables 1 , 2 , 3 , 4 . summary statistics of categorical answers to feedback questionnaire free text responses to q4 of the feedback survey : please write any comments you might have about this tool free text responses to q7 of the feedback survey : give an example of what you have learnt free text responses to q11 of the feedback survey : please write any comments about the elearning package feedback about the lcat was generally positive . threequarters ( n = 26 , 76% , table 1 ) of participants found the lcat easy to use , approximately the same proportion ( n = 25 , 74% , table 1 ) said that they would or would probably use the tool in their role , and twothirds ( n = 23 , 68% , table 1 ) stated that they would be likely or very likely to recommend the tool to others . the majority of participants ( n = 14 , 78% , table 1 ) felt that the elearning package was useful , and many learned something from the package ( n = 13 , 72% , table 1 ) . almost all of those that said that they had learnt something from the package felt that they could use what they have learnt in practice ( n = 12 , 92% , table 1 ) . the majority of participants in the intervention arm ( 11 , 61% , table 1 ) said that they would be unlikely to recommend the training to others . four participants felt that the feedback was inadequate and more explanation was needed ( a1 , a5 , a7 and a17 , table 4 ) . a7 felt that the language was unhelpful and that the package needed work ( table 4 ) , while a10 also thought that the package needed work due to its technical problems ( table 4 ) . the average score by correct classification was 9.22 ( 95% ci , 7.96 to 10.48 ) in the intervention arm and 7.88 in the control arm ( 95% ci , 6.76 to 9.00 ) . the effect of the intervention was to increase the score by 1.34 on average ( 95% ci , 0.3 to 3.0 ) . participants in the intervention arm of st level 13 and st level 48 had scores on average of 9.14 ( 95% ci , 6.45 to 11.84 ) and 9.27 ( 95% ci , 7.65 to 10.89 ) , respectively . similarly , participants in the control arm that were st level 13 and st level 48 had an average score of 7.86 ( 95% ci , 5.39 to 10.33 ) and 7.90 ( 95% ci , 6.53 to 9.27 ) . outcome data for 35 participants split by group at trial closure the maximum score is 20 . the secondary outcome , score based on correct route , ranged from a minimum of 2 to a maximum of 8 out of 20 across both arms . the effect of the training package increased the score by correct route by 1.01 correct classifications ( 95% ci , 0.3 to 2.3 ) . all paediatric trainees within the mersey ( n = 165 ) and north west ( n = 214 ) deanery were approached to participate . sixty participants provided consent during the recruitment phase ; three were found to be ineligible upon screening ; one had a pharmacology phd , one had pharmaceutical industry experience , and the st level was unknown for the third . the 57 remaining participants were randomised 1 : 1 to the two intervention arms . twentynine participants were randomised to the intervention ( training ) arm , 13 were st level 13 , and 16 were st level 48 . twentyone ( 72% ) of those randomised to the intervention arm started the training package and assessment , and of those , 18 ( 62% ) completed the assessment and the feedback questionnaire . twentyeight participants were randomised to receive no training , 13 were st level 13 and 15 st level 48 . twentythree ( 82% ) of those randomised to the control arm started the assessment , 17 ( 61% ) completed the assessment , and 16 ( 57% ) completed the feedback questionnaire . . results of the feedback questionnaire is given in tables 1 , 2 , 3 , 4 . summary statistics of categorical answers to feedback questionnaire free text responses to q4 of the feedback survey : please write any comments you might have about this tool free text responses to q7 of the feedback survey : give an example of what you have learnt free text responses to q11 of the feedback survey : please write any comments about the elearning package feedback about the lcat was generally positive . threequarters ( n = 26 , 76% , table 1 ) of participants found the lcat easy to use , approximately the same proportion ( n = 25 , 74% , table 1 ) said that they would or would probably use the tool in their role , and twothirds ( n = 23 , 68% , table 1 ) stated that they would be likely or very likely to recommend the tool to others the majority of participants ( n = 14 , 78% , table 1 ) felt that the elearning package was useful , and many learned something from the package ( n = 13 , 72% , table 1 ) . almost all of those that said that they had learnt something from the package felt that they could use what they have learnt in practice ( n = 12 , 92% , table 1 ) . the majority of participants in the intervention arm ( 11 , 61% , table 1 ) said that they would be unlikely to recommend the training to others . four participants felt that the feedback was inadequate and more explanation was needed ( a1 , a5 , a7 and a17 , table 4 ) . a7 felt that the language was unhelpful and that the package needed work ( table 4 ) , while a10 also thought that the package needed work due to its technical problems ( table 4 ) . the average score by correct classification was 9.22 ( 95% ci , 7.96 to 10.48 ) in the intervention arm and 7.88 in the control arm ( 95% ci , 6.76 to 9.00 ) . the effect of the intervention was to increase the score by 1.34 on average ( 95% ci , 0.3 to 3.0 ) . participants in the intervention arm of st level 13 and st level 48 had scores on average of 9.14 ( 95% ci , 6.45 to 11.84 ) and 9.27 ( 95% ci , 7.65 to 10.89 ) , respectively . similarly , participants in the control arm that were st level 13 and st level 48 had an average score of 7.86 ( 95% ci , 5.39 to 10.33 ) and 7.90 ( 95% ci , 6.53 to 9.27 ) . outcome data for 35 participants split by group at trial closure the maximum score is 20 . the secondary outcome , score based on correct route , ranged from a minimum of 2 to a maximum of 8 out of 20 across both arms . the effect of the training package increased the score by correct route by 1.01 correct classifications ( 95% ci , 0.3 to 2.3 ) . in our programme of research into adrs in children , we have developed the lcat , which has been internally validated . in order to progress this further , we went on to develop an elearning package ( lacaep ) , the utility of which was tested in this pilot trial . before embarking on a larger trial , it is also important to assess the feedback received from the participants on the tools used . feedback on the lcat was mostly positive , with trainees indicating they had learnt something about adr assessment from the tool and would use it in their clinical practice . the user feedback on both the lcat and lacaep has highlighted a number of areas that need to be addressed in the educational package such as giving more explanation of some of the terms used and the routes taken to determine causality . our data indicate that the lacaep did not improve causality assessment in trainees , but participants who were given training by the lacaep in causality assessment obtained a higher score by approximately 1.5 ( out of 20 ) on average for correct classification ( mean = 1.34 , 95% ci , 0.3 to 3.0 ) . this was a pilot study to inform a main trial , and data were not available to inform a sample size calculation prior to recruitment . we selected a pragmatic sample size ( n = 80 ) based on the eligible population , but we did not reach this recruitment target . however , the inclusion of 35 participants was adequate to fulfil the aims of this pilot study . we consider that a difference of 2 correctly classified adrs out of 20 between the groups would be the minimum worthwhile clinical difference . based on the results obtained and this consideration , a trial with 90 participants ( 45 per group ) is required to have adequate power to detect a true clinically relevant difference of 2.0 at the 5% significance level and 80% power . first , it shows that a novel trial design where participants can take part remotely is feasible and results in a reasonable proportion of participants completing the trial ; overall , 61.4% of participants who were randomised completed the trial . second , this pilot offers a template for easy expansion to a larger trial that will represent the package utility in a larger cohort , not only in size but also in geographical expansion to represent a wider proportion of trainees across the uk . a similar elearning approach has been used by gordon et al . who conducted an rct to investigate the effectiveness of an elearning course on paediatric prescribing in north west england . this research attained a sample size of 206 from a pool of 1150 ( 17.9% ) of which 113 completed the trial ( 54.9% ) . this is comparable with our sample size of 57 from a pool of 379 ( 15.0% ) of which 35 completed the assessment phase of the trial ( 59.5% ) . third , the use of trainees as participants means that differences observed are indeed down to the intervention and not prior knowledge or experience . the inclusion of a preintervention assessment of all participants would have been the optimum approach to determining the impact of the intervention . however , the study relied on the availability of trainees to participate , so the design was adapted to minimise the time commitment required from participants , with the aim of enhancing participation rates . second , and for the same reason , the study was held remotely hosted on a server such that trainees could participate in their own time , and so , the possibility of participants discussing their responses can not be eradicated . however , the study management team did not consider this likely as participants consented to not discussing aspects of the trial with their peers . third , as this trial relied on the participation of volunteers , the generalisability of the results may be questionable . we have recently shown the importance of adrs in paediatric medicine,[6 , 7 , 8 ] and previously in adult medicine.[2 , 3 ] the burden overall is very large leading to a great deal of morbidity in patients , occasional mortality , unnecessary investigations , increased length of stay in hospital and a huge cost burden . it is incumbent on all healthcare professionals to recognise adrs and act accordingly ( stop the drug and/or reduce the dose , and report the adr to their own hospitals and regulatory authorities ) . however , because adrs can affect any bodily system , and can present in a multitude of ways , they are sometimes difficult to recognise , and even when recognised , there may be difficulties in assigning causality . many causality assessment tools have been developed ; more complicated tools may potentially be more accurate but extremely difficult to use in clinical practice . our aim with the lcat was to develop a userfriendly and easytocomplete tool which would improve assessment of adrs and their reporting in daily clinical practice . the feedback from participants that they would use such a tool in their clinical practice is thus encouraging . although this trial included medical trainees , the lcat was developed by a multidisciplinary team and has been used by nurses and pharmacists for the evaluation of causality in a research setting.[6 , 7 ] therefore , we anticipate it being used by both medical and nonmedical professionals in a clinical setting . nevertheless , the appropriate use of the tool needs an educational package such as the one developed as part of this study . feedback on the lcat and lacaep was mainly positive although we have identified areas of the lacaep that need improving before conducting a trial to formally assess the effectiveness of the tool . this study was not powered to detect a difference between allocation arms though preliminary findings show a nonsignificant improvement of 1.5 ( out of 20 ) on average in the lacaep arm . the data collected were sufficient to enable a formal sample size calculation for a main study , and thus , our next step will be to improve the educational tool and then test it again in an appropriately powered trial of 90 participants ( n = 45 per group ) . all authors are members of the research team on the adverse drug reactions in children project . outputs of this project included the liverpool causality assessment tool and the liverpool adr causality assessment elearning package . the authors thank the nihr for providing funding for the adric ( adverse drug reactions in children programme grant ) and the merseyside and cheshire health innovation and education cluster ( hiec ) for funding the development of the educational tool . all authors state that they had complete access to the study data that support the publication . all authors are members of the research team on the adverse drug reactions in children project . outputs of this project included the liverpool causality assessment tool and the liverpool adr causality assessment elearning package . the authors thank the nihr for providing funding for the adric ( adverse drug reactions in children programme grant ) and the merseyside and cheshire health innovation and education cluster ( hiec ) for funding the development of the educational tool . all authors state that they had complete access to the study data that support the publication .

abstractobjectivescausality assessment of adverse drug reactions ( adrs ) by healthcare professionals is often informal which can lead to inconsistencies in practice . the liverpool causality assessment tool ( lcat ) offers a systematic approach . an interactive , webbased , elearning package , the liverpool adr causality assessment elearning package ( lacaep ) , was designed to improve causality assessment using the lcat . this study aimed to ( 1 ) get feedback on usability and usefulness on the lacaep , identify areas for improvement and development , and generate data on effect size to inform a larger scale study ; and ( 2 ) test the usability and usefulness of the lcat.methodsa pilot , singleblind , parallelgroup , randomised controlled trial hosted by the university of liverpool was undertaken . participants were paediatric medical trainees at specialty training level 1 + within the mersey and northwest england deaneries . participants were randomised ( 1 : 1 ) access to the lacaep or no training . the primary efficacy outcome was score by correct classification , predefined by a multidisciplinary panel of experts . following participation , feedback on both the lcat and the lacaep was obtained , via a built in survey , from participants.key findingsof 57 randomised , 35 completed the study . feedback was mainly positive although areas for improvement were identified . seventyfour per cent of participants found the lcat easy to use and 78% found the lacaep training useful . sixtyone per cent would be unlikely to recommend the training . scores ranged from 4 to 13 out of 20 . the lacaep increased scores by 1.3 , but this was not significant.conclusionsimproving the lacaep before testing it in an appropriately powered trial , informed by the differences observed , is required . rigorous evaluation will enable a quality resource that will be of value in healthcare professional training .

Introduction Methods Statistical considerations Results Study population Feedback on the Postintervention scores: by correct classification and correct route Discussion Conclusions Declarations Conflict of interest Funding Authors' contributions Supporting information

causality assessment of adverse drug reactions ( adrs ) is formally undertaken by the pharmaceutical industry , regulators and researchers in clinical trials but rarely by clinicians . anecdotally , clinicians ' assessment of adr causality is generally done informally and sometimes subconsciously , which leads to variability in decision making , specifically in terms of when to alter drug therapy and reporting of adrs . in two recent large paediatric adr studies , the naranjo tool was found to be inadequate for adr causality assessment and have poor reproducibility. [2 , 3 ] for instance , in the assessment of adrs detected in children acutely admitted to hospital over a 12 month period , interrater reliability using the naranjo tool was poor . in addition , the weighting for each question within the naranjo tool was not justified in the original publication . subsequently , a new causality assessment tool , the liverpool causality assessment tool ( lcat , see appendix s1 ) , was developed , formally tested and internally validated . this tool aimed to overcome the issues identified with the naranjo tool , while ( 1 ) making it as easy , or easier , to use than the naranjo tool and ( 2 ) maintaining the basic principles of causality assessment . given that there is variability in clinical decision making around adrs , we aimed to develop a means to disseminate this new approach of adr causality assessment to practitioners . we developed an interactive , webbased elearning package designed to improve assessment by individual practitioners : the liverpool adr causality assessment elearning package ( lacaep ) ( see box 1 for the attributes of this package ) . the package includes : an interactive diagram ( based on the causality flowchart ) that allows the use to zoom / pan / rollover to navigate the tool and to gain more information about items in the diagramlogical question arrangement that underlines the sequential nature of the assessment toolreallife case studies : use reallife case studies to build a causality assessment by answering each of the questions on the lcat in turnexpert opinion : the package includes the availability of an expert or panel of expert characters who can provide feedback and hints on decisions made by the user during the case studies exercise the interface contains a straightforward navigation system , with a short tutorial available explaining the functionability of all buttons in the interfacea content menu and glossary are includedthe package has no formal assessment but will require users to complete interactive activities in order to progress the elearning package takes approximately 1 hour to complete the package contains interactive bespoke learning activities that require the user to interact with the software in order to continue , and will offer instructive feedback . the package includes : an interactive diagram ( based on the causality flowchart ) that allows the use to zoom / pan / rollover to navigate the tool and to gain more information about items in the diagramlogical question arrangement that underlines the sequential nature of the assessment toolreallife case studies : use reallife case studies to build a causality assessment by answering each of the questions on the lcat in turnexpert opinion : the package includes the availability of an expert or panel of expert characters who can provide feedback and hints on decisions made by the user during the case studies exercise an interactive diagram ( based on the causality flowchart ) that allows the use to zoom / pan / rollover to navigate the tool and to gain more information about items in the diagram logical question arrangement that underlines the sequential nature of the assessment tool reallife case studies : use reallife case studies to build a causality assessment by answering each of the questions on the lcat in turn expert opinion : the package includes the availability of an expert or panel of expert characters who can provide feedback and hints on decisions made by the user during the case studies exercise the interface contains a straightforward navigation system , with a short tutorial available explaining the functionability of all buttons in the interface a content menu and glossary are included the package has no formal assessment but will require users to complete interactive activities in order to progress reporting and user tracking the package bookmarks user progress between sessions and retain options chosen in completed activitiesadministrators of the package will be able to access the following information ( via the learning management system where the package will be hosted ) : the participant demographic details ; the current progress of the participant ; which activities they have undertaken ; the outcome of each assessment made , i.e. what was the classification , what path did they take on the lcat to get there and was the classification correct . at the end of the package , the user is asked to complete a feedback survey which assesses the usability and usefulness of the package and of the lcat the package bookmarks user progress between sessions and retain options chosen in completed activities administrators of the package will be able to access the following information ( via the learning management system where the package will be hosted ) : the participant demographic details ; the current progress of the participant ; which activities they have undertaken ; the outcome of each assessment made , i.e. what was the classification , what path did they take on the lcat to get there and was the classification correct . what was the classification , what path did they take on the lcat to get there and was the classification correct . at the end of the package , the user is asked to complete a feedback survey which assesses the usability and usefulness of the package and of the lcat accessibility an ebook provides alternative testbased content for the packagethe package has been tested for sharable content object reference model ( scorm ) compliance with the adl test suitethe package complied with world wide web consortium ( w3c ) web standards wherever possibleflash or javascriptbased content is accompanied by alternative html content an ebook provides alternative testbased content for the package the package has been tested for sharable content object reference model ( scorm ) compliance with the adl test suite the package complied with world wide web consortium ( w3c ) web standards wherever possible flash or javascriptbased content is accompanied by alternative html content the purpose of this pilot randomised controlled trial was to gain feedback on the usability and usefulness of the lcat and the lacaep . feedback obtained for the latter will be used to identify areas for improvement and development . this trial also aimed to generate data on effect size enabling a larger hypothesis testing study to be conducted . this was a pilot , singleblind , parallel group study conducted by the university of liverpool . this pilot aimed to inform a larger scale study that would formally compare the effect that the lacaep has on improving the consistency of assigning causality using the lcat . the study obtained organisational approval from nhs north of england , and permission was obtained from the head of schools at the north west and mersey deaneries to include trainees from that region . eligible participants were specialist trainees in paediatrics ( st level 1 and above ) within the mersey ( n = 165 ) and north west deaneries ( n = 214 ) . both the control and intervention arm received access to the lcat to assist causality assessment . participants in the intervention arm accessed an interactive training module with selfdirected elearning components that guided users when making causality assessments using the lcat ( lacaep ) . the elearning package was tested rigorously by the study team for functionality and content before the trial was opened . adr case studies used both for post intervention assessment and within the training phase of the elearning tool were taken from two previous adr studies. [6 , 7 ] within these studies , causality classification ( unlikely , possible , probable or definite ) was reached by consensus by a multidisciplinary panel of experts . the number of correct classifications when compared against the gold standard was defined as the primary efficacy outcome . as a second efficacy outcome , the route taken on the lcat flowchart was recorded to ensure that classifications were obtained following a route defined by a multidisciplinary panel of experts . upon completing the intervention , trainees were encouraged to provide feedback on both lcat and lacaep ( appendix s4 ) by completing an optional survey , built in to the package , made up of a series of open and closed questions . as this was a pilot study , intended to generate data on effect size to enable a larger hypothesis study to be conducted , no formal power analysis was completed prior to the trial . overall series agreement postintervention was summarised for each treatment group , both overall and split by speciality training level ( groups : 3 and below , 4 and above ) , using descriptive statistics , means with 95% confidence intervals ( ci ) or medians with an interquartile range if the scores was nonnormally distributed . as this was a pilot study , intended to generate data on effect size to enable a larger hypothesis study to be conducted , no formal power analysis was completed prior to the trial . a pragmatic sample of 80 participants of the total 379 trainees at that time was considered a minimum requirement . sixty participants provided consent during the recruitment phase ; three were found to be ineligible upon screening ; one had a pharmacology phd , one had pharmaceutical industry experience , and the st level was unknown for the third . the 57 remaining participants were randomised 1 : 1 to the two intervention arms . twentynine participants were randomised to the intervention ( training ) arm , 13 were st level 13 , and 16 were st level 48 . twentyone ( 72% ) of those randomised to the intervention arm started the training package and assessment , and of those , 18 ( 62% ) completed the assessment and the feedback questionnaire . twentyeight participants were randomised to receive no training , 13 were st level 13 and 15 st level 48 . twentythree ( 82% ) of those randomised to the control arm started the assessment , 17 ( 61% ) completed the assessment , and 16 ( 57% ) completed the feedback questionnaire . thirtyfour participants provided feedback on the lcat , and 18 provided feedback on the lacaep . summary statistics of categorical answers to feedback questionnaire free text responses to q4 of the feedback survey : please write any comments you might have about this tool free text responses to q7 of the feedback survey : give an example of what you have learnt free text responses to q11 of the feedback survey : please write any comments about the elearning package feedback about the lcat was generally positive . threequarters ( n = 26 , 76% , table 1 ) of participants found the lcat easy to use , approximately the same proportion ( n = 25 , 74% , table 1 ) said that they would or would probably use the tool in their role , and twothirds ( n = 23 , 68% , table 1 ) stated that they would be likely or very likely to recommend the tool to others . the majority of participants ( n = 14 , 78% , table 1 ) felt that the elearning package was useful , and many learned something from the package ( n = 13 , 72% , table 1 ) . the majority of participants in the intervention arm ( 11 , 61% , table 1 ) said that they would be unlikely to recommend the training to others . the average score by correct classification was 9.22 ( 95% ci , 7.96 to 10.48 ) in the intervention arm and 7.88 in the control arm ( 95% ci , 6.76 to 9.00 ) . the effect of the intervention was to increase the score by 1.34 on average ( 95% ci , 0.3 to 3.0 ) . the secondary outcome , score based on correct route , ranged from a minimum of 2 to a maximum of 8 out of 20 across both arms . the effect of the training package increased the score by correct route by 1.01 correct classifications ( 95% ci , 0.3 to 2.3 ) . the 57 remaining participants were randomised 1 : 1 to the two intervention arms . twentynine participants were randomised to the intervention ( training ) arm , 13 were st level 13 , and 16 were st level 48 . twentyone ( 72% ) of those randomised to the intervention arm started the training package and assessment , and of those , 18 ( 62% ) completed the assessment and the feedback questionnaire . twentyeight participants were randomised to receive no training , 13 were st level 13 and 15 st level 48 . twentythree ( 82% ) of those randomised to the control arm started the assessment , 17 ( 61% ) completed the assessment , and 16 ( 57% ) completed the feedback questionnaire . results of the feedback questionnaire is given in tables 1 , 2 , 3 , 4 . summary statistics of categorical answers to feedback questionnaire free text responses to q4 of the feedback survey : please write any comments you might have about this tool free text responses to q7 of the feedback survey : give an example of what you have learnt free text responses to q11 of the feedback survey : please write any comments about the elearning package feedback about the lcat was generally positive . threequarters ( n = 26 , 76% , table 1 ) of participants found the lcat easy to use , approximately the same proportion ( n = 25 , 74% , table 1 ) said that they would or would probably use the tool in their role , and twothirds ( n = 23 , 68% , table 1 ) stated that they would be likely or very likely to recommend the tool to others the majority of participants ( n = 14 , 78% , table 1 ) felt that the elearning package was useful , and many learned something from the package ( n = 13 , 72% , table 1 ) . almost all of those that said that they had learnt something from the package felt that they could use what they have learnt in practice ( n = 12 , 92% , table 1 ) . the majority of participants in the intervention arm ( 11 , 61% , table 1 ) said that they would be unlikely to recommend the training to others . the average score by correct classification was 9.22 ( 95% ci , 7.96 to 10.48 ) in the intervention arm and 7.88 in the control arm ( 95% ci , 6.76 to 9.00 ) . the effect of the intervention was to increase the score by 1.34 on average ( 95% ci , 0.3 to 3.0 ) . the secondary outcome , score based on correct route , ranged from a minimum of 2 to a maximum of 8 out of 20 across both arms . the effect of the training package increased the score by correct route by 1.01 correct classifications ( 95% ci , 0.3 to 2.3 ) . in order to progress this further , we went on to develop an elearning package ( lacaep ) , the utility of which was tested in this pilot trial . before embarking on a larger trial , it is also important to assess the feedback received from the participants on the tools used . feedback on the lcat was mostly positive , with trainees indicating they had learnt something about adr assessment from the tool and would use it in their clinical practice . the user feedback on both the lcat and lacaep has highlighted a number of areas that need to be addressed in the educational package such as giving more explanation of some of the terms used and the routes taken to determine causality . our data indicate that the lacaep did not improve causality assessment in trainees , but participants who were given training by the lacaep in causality assessment obtained a higher score by approximately 1.5 ( out of 20 ) on average for correct classification ( mean = 1.34 , 95% ci , 0.3 to 3.0 ) . this was a pilot study to inform a main trial , and data were not available to inform a sample size calculation prior to recruitment . we selected a pragmatic sample size ( n = 80 ) based on the eligible population , but we did not reach this recruitment target . we consider that a difference of 2 correctly classified adrs out of 20 between the groups would be the minimum worthwhile clinical difference . first , it shows that a novel trial design where participants can take part remotely is feasible and results in a reasonable proportion of participants completing the trial ; overall , 61.4% of participants who were randomised completed the trial . second , this pilot offers a template for easy expansion to a larger trial that will represent the package utility in a larger cohort , not only in size but also in geographical expansion to represent a wider proportion of trainees across the uk . this is comparable with our sample size of 57 from a pool of 379 ( 15.0% ) of which 35 completed the assessment phase of the trial ( 59.5% ) . third , the use of trainees as participants means that differences observed are indeed down to the intervention and not prior knowledge or experience . however , the study relied on the availability of trainees to participate , so the design was adapted to minimise the time commitment required from participants , with the aim of enhancing participation rates . second , and for the same reason , the study was held remotely hosted on a server such that trainees could participate in their own time , and so , the possibility of participants discussing their responses can not be eradicated . however , the study management team did not consider this likely as participants consented to not discussing aspects of the trial with their peers . third , as this trial relied on the participation of volunteers , the generalisability of the results may be questionable . our aim with the lcat was to develop a userfriendly and easytocomplete tool which would improve assessment of adrs and their reporting in daily clinical practice . although this trial included medical trainees , the lcat was developed by a multidisciplinary team and has been used by nurses and pharmacists for the evaluation of causality in a research setting. nevertheless , the appropriate use of the tool needs an educational package such as the one developed as part of this study . feedback on the lcat and lacaep was mainly positive although we have identified areas of the lacaep that need improving before conducting a trial to formally assess the effectiveness of the tool . this study was not powered to detect a difference between allocation arms though preliminary findings show a nonsignificant improvement of 1.5 ( out of 20 ) on average in the lacaep arm . the data collected were sufficient to enable a formal sample size calculation for a main study , and thus , our next step will be to improve the educational tool and then test it again in an appropriately powered trial of 90 participants ( n = 45 per group ) . all authors are members of the research team on the adverse drug reactions in children project . outputs of this project included the liverpool causality assessment tool and the liverpool adr causality assessment elearning package . the authors thank the nihr for providing funding for the adric ( adverse drug reactions in children programme grant ) and the merseyside and cheshire health innovation and education cluster ( hiec ) for funding the development of the educational tool . all authors state that they had complete access to the study data that support the publication . all authors are members of the research team on the adverse drug reactions in children project . outputs of this project included the liverpool causality assessment tool and the liverpool adr causality assessment elearning package . the authors thank the nihr for providing funding for the adric ( adverse drug reactions in children programme grant ) and the merseyside and cheshire health innovation and education cluster ( hiec ) for funding the development of the educational tool . all authors state that they had complete access to the study data that support the publication .

participants were selected from the framingham heart study multidetector ct substudy of the community - based framingham heart study offspring cohort ; inclusion criteria have been described previously ( 1 ) . beginning in 1971 , the offspring study enrolled the offspring and spouses of the offspring whose parents were in the original cohort of the framingham heart study . a subset of the offspring cohort had measurements of adipokine concentrations at their seventh examination cycle between 1998 and 2001 . of 1,418 participants in the offspring cohort of the multidetector ct study , 1,342 participants had interpretable ct measurements of visceral , subcutaneous , pericardial , and intrathoracic fat volume between 2002 and 2005 . after excluding participants with clinically prevalent cardiovascular disease ( cvd ) , prior coronary artery bypass graft , and an incomplete covariate profile , the final sample size was 916 ( 501 women and 415 men ) . the institutional review boards of boston university medical center and massachusetts general hospital approved this study . written informed consent was obtained from all participants . circulating concentrations of adiponectin and resistin were measured by enzyme - linked immunosorbent assay ( r&d systems , minneapolis , mn ) in plasma collected after a > 8-h fast and frozen at 80c . single , random plasma adiponectin and resistin concentrations have been shown to be stable over a 1-year period ( 9 ) . an 8-slice multidetector ct ( lightspeed ultra ; general electric , milwaukee , wi ) was used to image the abdomen and thorax . while lying in a supine position , participants had an average of 25 contiguous 5-mm - thick cross - sectional images ( 120 kvp ; 40 ma ; gantry rotation time , 500 ms ; table feed , 3:1 ) of the abdomen and 48 contiguous 2.5-mm - thick cross - sectional images of the heart ( 120 kvp ; 400 ma ; temporal resolution , 330 ms ) . the specific protocols used to obtain images of the abdomen and thorax have been described elsewhere ( 5,10 ) . visceral , subcutaneous , pericardial , and thoracic fat volumes were measured ( aquarius 3d workstation ; terarecon , san mateo , ca ) using an image display window width of 195 to 45 hounsfield units ( hu ) and window center of 120 hu . the readers manually traced the abdominal muscular wall separating the visceral and subcutaneous depots and outlined the pericardium to distinguish pericardial from thoracic fat . total thoracic fat volume consisted of adipose tissue from the right pulmonary artery to the diaphragm and from the chest wall to the descending aorta , including fat within the pericardial sac . interreader reproducibility was excellent for visceral , subcutaneous , pericardial , and thoracic fat volume measurements ( 1,10 ) . intrathoracic fat was derived after subtracting pericardial fat from total thoracic fat to identify two mutually exclusive fat compartments . bmi was measured as weight ( in kilograms ) divided by the square of height ( in meters ) . participants were classified as current smokers if they smoked on average at least one cigarette per day in the previous year . women who drank 7 alcoholic drinks per week and men who consumed 14 alcoholic drinks per week were considered alcohol users . the physical activity index was based on the average number of hours per day spent sleeping and doing sedentary , slight , moderate , and heavy activity . women were considered postmenopausal if they had not had any menstrual periods for at least 1 year . hypertension was diagnosed as a systolic blood pressure 140 mmhg , a diastolic blood pressure 90 mmhg , or treatment with antihypertensive agents . fasting morning plasma samples were collected to measure fasting plasma glucose , total cholesterol , hdl cholesterol , and triglycerides . diabetes was defined as a fasting plasma glucose 126 mg / dl ( 7 mmol / l ) or treatment with insulin or a hypoglycemic medication . adiponectin , vat , sat , pericardial fat , and intrathoracic fat volumes were approximately normally distributed . all analyses were a priori performed specific to sex due to strong sex interactions we had previously observed with sat and vat ( 1 ) . sex - specific and age - adjusted pearson correlation coefficients were calculated to determine the correlation between each fat compartment , adiponectin , and log resistin . sex - specific multivariable linear regression was used to evaluate the associations of adiponectin and log resistin ( dependent variables ; separate models for each ) with fat depots ( independent variables ) , after adjustment for age , smoking , alcohol use , menopausal status ( women only ) , and hormone replacement therapy ( women only ) . vat , sat , pericardial fat , and intrathoracic fat were standardized to a means sd of 0 1 ; the covariate - adjusted average change in adiponectin and log resistin per sd of adipose tissue volume was estimated . using this approach , the multivariable models for vat , pericardial fat , and intrathoracic fat were then additionally adjusted for bmi and waist circumference in order to assess whether relations were maintained after adjusting for measures of generalized adiposity . as secondary analyses , multivariable models for vat , sat , pericardial fat , and intrathoracic fat were also additionally adjusted for physical activity and education status . multivariable linear and logistic regressions were used to relate vat and sat ( independent variables ) to cardiometabolic risk factors ( dependent variables ) . models for systolic blood pressure , fasting plasma glucose , log triglycerides , hdl cholesterol , and metabolic syndrome were evaluated ( separate model for each risk factor ) . age , smoking , alcohol use , menopausal status ( women only ) , hormone replacement therapy ( women only ) , and treatment of hypertension , dyslipidemia , and diabetes were entered as covariates in each of the models . each multivariable model was also adjusted for either adiponectin or log resistin ( separate models for each adjustment ) to assess the extent of attenuation of the association of vat and sat with each cardiometabolic risk factor upon adjustment for these adipokines . all analyses were performed using sas version 9.1 . a two - tailed value of p < 0.05 was considered statistically significant . circulating concentrations of adiponectin and resistin were measured by enzyme - linked immunosorbent assay ( r&d systems , minneapolis , mn ) in plasma collected after a > 8-h fast and frozen at 80c . single , random plasma adiponectin and resistin concentrations have been shown to be stable over a 1-year period ( 9 ) . an 8-slice multidetector ct ( lightspeed ultra ; general electric , milwaukee , wi ) was used to image the abdomen and thorax . while lying in a supine position , participants had an average of 25 contiguous 5-mm - thick cross - sectional images ( 120 kvp ; 40 ma ; gantry rotation time , 500 ms ; table feed , 3:1 ) of the abdomen and 48 contiguous 2.5-mm - thick cross - sectional images of the heart ( 120 kvp ; 400 ma ; temporal resolution , 330 ms ) . the specific protocols used to obtain images of the abdomen and thorax have been described elsewhere ( 5,10 ) . visceral , subcutaneous , pericardial , and thoracic fat volumes were measured ( aquarius 3d workstation ; terarecon , san mateo , ca ) using an image display window width of 195 to 45 hounsfield units ( hu ) and window center of 120 hu . the readers manually traced the abdominal muscular wall separating the visceral and subcutaneous depots and outlined the pericardium to distinguish pericardial from thoracic fat . total thoracic fat volume consisted of adipose tissue from the right pulmonary artery to the diaphragm and from the chest wall to the descending aorta , including fat within the pericardial sac . interreader reproducibility was excellent for visceral , subcutaneous , pericardial , and thoracic fat volume measurements ( 1,10 ) . intrathoracic fat was derived after subtracting pericardial fat from total thoracic fat to identify two mutually exclusive fat compartments . bmi was measured as weight ( in kilograms ) divided by the square of height ( in meters ) . participants were classified as current smokers if they smoked on average at least one cigarette per day in the previous year . women who drank 7 alcoholic drinks per week and men who consumed 14 alcoholic drinks per week were considered alcohol users . the physical activity index was based on the average number of hours per day spent sleeping and doing sedentary , slight , moderate , and heavy activity . women were considered postmenopausal if they had not had any menstrual periods for at least 1 year . hypertension was diagnosed as a systolic blood pressure 140 mmhg , a diastolic blood pressure 90 mmhg , or treatment with antihypertensive agents . fasting morning plasma samples were collected to measure fasting plasma glucose , total cholesterol , hdl cholesterol , and triglycerides . diabetes was defined as a fasting plasma glucose 126 mg / dl ( 7 mmol / l ) or treatment with insulin or a hypoglycemic medication . adiponectin , vat , sat , pericardial fat , and intrathoracic fat volumes were approximately normally distributed . all analyses were a priori performed specific to sex due to strong sex interactions we had previously observed with sat and vat ( 1 ) . sex - specific and age - adjusted pearson correlation coefficients were calculated to determine the correlation between each fat compartment , adiponectin , and log resistin . sex - specific multivariable linear regression was used to evaluate the associations of adiponectin and log resistin ( dependent variables ; separate models for each ) with fat depots ( independent variables ) , after adjustment for age , smoking , alcohol use , menopausal status ( women only ) , and hormone replacement therapy ( women only ) . vat , sat , pericardial fat , and intrathoracic fat were standardized to a means sd of 0 1 ; the covariate - adjusted average change in adiponectin and log resistin per sd of adipose tissue volume was estimated . using this approach , the multivariable models for vat , pericardial fat , and intrathoracic fat were then additionally adjusted for bmi and waist circumference in order to assess whether relations were maintained after adjusting for measures of generalized adiposity . as secondary analyses , multivariable models for vat , sat , pericardial fat , and intrathoracic fat were multivariable linear and logistic regressions were used to relate vat and sat ( independent variables ) to cardiometabolic risk factors ( dependent variables ) . models for systolic blood pressure , fasting plasma glucose , log triglycerides , hdl cholesterol , and metabolic syndrome were evaluated ( separate model for each risk factor ) . age , smoking , alcohol use , menopausal status ( women only ) , hormone replacement therapy ( women only ) , and treatment of hypertension , dyslipidemia , and diabetes were entered as covariates in each of the models . each multivariable model was also adjusted for either adiponectin or log resistin ( separate models for each adjustment ) to assess the extent of attenuation of the association of vat and sat with each cardiometabolic risk factor upon adjustment for these adipokines . all analyses were performed using sas version 9.1 . a two - tailed value of p < 0.05 was considered statistically significant . the mean age was 59 years ( table 1 ) , and slightly more than one - quarter were obese . clinical characteristics of 916 study participants data are means sd or median ( 25th75th percentile ) . * defined as 7 drinks / week ( for women ) or 14 drinks / week ( for men ) . conversion to si units : multiply waist circumference by 2.54 for cm , triglycerides by 0.01129 for mmol / l , hdl and total cholesterol by 0.02586 for mmol / l , and fasting plasma glucose by 0.05551 for mmol / l . vat , sat , pericardial fat , and intrathoracic fat were negatively correlated with adiponectin ( r = 0.19 to 0.34 , p < 0.001 [ women ] ; r = 0.15 to 0.26 , p < 0.01 [ men ] except sat ) and positively correlated with resistin ( r = 0.160.21 , p < 0.001 [ women ] ; r = 0.110.14 , p < 0.05 [ men ] except vat ) ( table 2 ) . age - adjusted pearson correlation coefficients between fat depot volumes and adipokines in women , adiponectin concentration was 2.1 0.3 g / ml lower per sd increase of vat , 1.2 0.3 g / ml lower per sd increase of sat , 1.2 0.3 g / ml lower per sd increase of pericardial fat , and 1.2 0.3 g / ml lower per sd increase of intrathoracic fat ( p < 0.001 ) in multivariable - adjusted models ( table 3 ) . the regression coefficients for adiponectin concentration were larger per sd of vat than per sd of sat ( p < 0.001 ) . in men , the multivariable - adjusted effect sizes of vat , sat , pericardial fat , and intrathoracic fat on adiponectin concentration were weaker but still significant in nearly all cases ( table 3 ) . a sex - by - vat interaction was observed for adiponectin ( p < 0.001 ) , as well as a sex - by - sat ( p = 0.001 ) and sex by pericardial fat interaction ( p = 0.03 ) . sex - specific multivariable - adjusted * regressions for fat depot volumes with adipokines * adjusted for age , smoking , alcohol use , menopausal status ( women only ) , and hormone replacement therapy ( women only ) . effect size refers to the average change in adiponectin or resistin concentration per sd of visceral , subcutaneous , pericardial , or intrathoracic fat volume . p value for sex interaction obtained from multivariable regression analysis on entire sample , adjusted for sex , age , smoking , alcohol use , menopausal status ( women only ) , hormone replacement therapy ( women only ) , and a sex - by - fat interaction term . r of model with covariates only is 2% in women and 4% in men . r of model with covariates only is 4% in women and 3% in men . in women , vat remained associated with adiponectin after additional adjustment for bmi and waist circumference ( p = < 0.001 ) ; pericardial fat and intrathoracic fat did not remain associated with adiponectin ( p = 0.15 [ pericardial fat ] , p = 0.5 [ intrathoracic fat ] ) . in contrast , among men , vat , pericardial fat , and intrathoracic fat were associated with adiponectin after further adjustment for bmi and waist circumference ( all p < 0.02 ) . covariates ( age , sex , smoking , alcohol use , menopausal status , and hormone replacement therapy ) explained 2% of the circulating variability in adiponectin concentration in women and 4% in men ; adding vat to this model increased the model r to 13% in women and 10% in men ( table 3 ) . multivariable - adjusted models for sat , pericardial fat , and intrathoracic fat accounted for a smaller portion of the variation in adiponectin concentration ( r range 48% ) . additional adjustment for bmi and waist circumference attenuated associations between vat , pericardial fat , intrathoracic fat , and resistin ( all p > 0.2 ) . results were similar for men , except intrathoracic fat remained associated with resistin after additional adjustment for bmi and waist circumference ( p = 0.02 ) . none of the multivariable - adjusted models explained a substantial portion of the variability in resistin concentration ( r range 36% ) . when physical activity and education status were included as additional covariates , relations between adipose tissue volumes , adiponectin , and resistin were not materially different ( data not shown ) . vat was associated with all cardiometabolic risk factors in both sexes as previously reported ( 1 ) , except for systolic blood pressure in men ( online appendix tables 1a and 1b [ available at http://care.diabetesjournals.org/cgi/content/full/dc08-1733/dc1 ] ) . in women , we observed some attenuation of the effect size of vat on log triglycerides and hdl cholesterol after additional adjustment for adiponectin , but strong associations were still observed . after multivariable adjustment , hdl cholesterol was 5.1 0.7 mg / dl ( 0.13 0.02 mmol / l ) lower per sd increase of vat . after further adjustment for adiponectin , mmol / l ) lower per sd increase of vat . fasting plasma glucose and diabetes did not appreciably change after adjustment for adiponectin . for sat , similar results were observed . in men , there was more attenuation of the effect size of vat on fasting plasma glucose , but not for diabetes , after additional adjustment for adiponectin . the effect size of vat on hdl cholesterol was minimally weakened after additional adjustment for adiponectin . relations between vat , log triglycerides , and metabolic syndrome , as well as between sat and cardiometabolic risk factors , did not appreciably change after adjustment for adiponectin in men . none of the associations between vat , sat , and cardiometabolic risk factors appreciably changed upon adjustment for resistin ( online appendix tables 1a and 1b ) . the mean age was 59 years ( table 1 ) , and slightly more than one - quarter were obese . clinical characteristics of 916 study participants data are means sd or median ( 25th75th percentile ) . * defined as 7 drinks / week ( for women ) or 14 drinks / week ( for men ) . conversion to si units : multiply waist circumference by 2.54 for cm , triglycerides by 0.01129 for mmol / l , hdl and total cholesterol by 0.02586 for mmol / l , and fasting plasma glucose by 0.05551 for mmol / l . vat , sat , pericardial fat , and intrathoracic fat were negatively correlated with adiponectin ( r = 0.19 to 0.34 , p < 0.001 [ women ] ; r = 0.15 to 0.26 , p < 0.01 [ men ] except sat ) and positively correlated with resistin ( r = 0.160.21 , p < 0.001 [ women ] ; r = 0.110.14 , p < 0.05 [ men ] except vat ) ( table 2 ) . in women , adiponectin concentration was 2.1 0.3 g / ml lower per sd increase of vat , 1.2 0.3 g / ml lower per sd increase of sat , 1.2 0.3 g / ml lower per sd increase of pericardial fat , and 1.2 0.3 g / ml lower per sd increase of intrathoracic fat ( p < 0.001 ) in multivariable - adjusted models ( table 3 ) . the regression coefficients for adiponectin concentration were larger per sd of vat than per sd of sat ( p < 0.001 ) . in men , the multivariable - adjusted effect sizes of vat , sat , pericardial fat , and intrathoracic fat on adiponectin concentration were weaker but still significant in nearly all cases ( table 3 ) . a sex - by - vat interaction was observed for adiponectin ( p < 0.001 ) , as well as a sex - by - sat ( p = 0.001 ) and sex by pericardial fat interaction ( p = 0.03 ) . sex - specific multivariable - adjusted * regressions for fat depot volumes with adipokines * adjusted for age , smoking , alcohol use , menopausal status ( women only ) , and hormone replacement therapy ( women only ) . effect size refers to the average change in adiponectin or resistin concentration per sd of visceral , subcutaneous , pericardial , or intrathoracic fat volume . p value for sex interaction obtained from multivariable regression analysis on entire sample , adjusted for sex , age , smoking , alcohol use , menopausal status ( women only ) , hormone replacement therapy ( women only ) , and a sex - by - fat interaction term . r of model with covariates only is 2% in women and 4% in men . r of model with covariates only is 4% in women and 3% in men . in women , vat remained associated with adiponectin after additional adjustment for bmi and waist circumference ( p = < 0.001 ) ; pericardial fat and intrathoracic fat did not remain associated with adiponectin ( p = 0.15 [ pericardial fat ] , p = 0.5 [ intrathoracic fat ] ) . in contrast , among men , vat , pericardial fat , and intrathoracic fat were associated with adiponectin after further adjustment for bmi and waist circumference ( all p < 0.02 ) . covariates ( age , sex , smoking , alcohol use , menopausal status , and hormone replacement therapy ) explained 2% of the circulating variability in adiponectin concentration in women and 4% in men ; adding vat to this model increased the model r to 13% in women and 10% in men ( table 3 ) . multivariable - adjusted models for sat , pericardial fat , and intrathoracic fat accounted for a smaller portion of the variation in adiponectin concentration ( r range 48% ) . additional adjustment for bmi and waist circumference attenuated associations between vat , pericardial fat , intrathoracic fat , and resistin ( all p > 0.2 ) . results were similar for men , except intrathoracic fat remained associated with resistin after additional adjustment for bmi and waist circumference ( p = 0.02 ) . none of the multivariable - adjusted models explained a substantial portion of the variability in resistin concentration ( r range 36% ) . when physical activity and education status were included as additional covariates , relations between adipose tissue volumes , adiponectin , and resistin were not materially different ( data not shown ) . vat was associated with all cardiometabolic risk factors in both sexes as previously reported ( 1 ) , except for systolic blood pressure in men ( online appendix tables 1a and 1b [ available at http://care.diabetesjournals.org/cgi/content/full/dc08-1733/dc1 ] ) . in women , we observed some attenuation of the effect size of vat on log triglycerides and hdl cholesterol after additional adjustment for adiponectin , but strong associations were still observed . after multivariable adjustment , hdl cholesterol was 5.1 0.7 mg / dl ( 0.13 0.02 mmol / l ) lower per sd increase of vat . after further adjustment for adiponectin , fasting plasma glucose and diabetes did not appreciably change after adjustment for adiponectin . for sat , , there was more attenuation of the effect size of vat on fasting plasma glucose , but not for diabetes , after additional adjustment for adiponectin . the effect size of vat on hdl cholesterol was minimally weakened after additional adjustment for adiponectin . relations between vat , log triglycerides , and metabolic syndrome , as well as between sat and cardiometabolic risk factors , did not appreciably change after adjustment for adiponectin in men . none of the associations between vat , sat , and cardiometabolic risk factors appreciably changed upon adjustment for resistin ( online appendix tables 1a and 1b ) . in the community - based framingham offspring cohort , vat , sat , pericardial fat , and intrathoracic fat were negatively correlated with adiponectin but positively correlated with resistin . for both sexes , the strongest correlation was between vat and adiponectin , but the correlation coefficients were still weak . furthermore , the addition of vat only increased the model r for adiponectin from 24% to 1013% . vat is not a primary determinant of systemic total adiponectin concentration , and circulating levels of adiponectin can not be used as a surrogate for vat . adjustment for adiponectin attenuated relations between vat , sat , and cardiometabolic risk factors , but adiponectin did not fully account for the strong relations between vat and cardiometabolic risk factors . easily obtainable anthropometric measures ( bmi and waist circumference ) did not explain the associations between vat and adiponectin in women or men and between pericardial fat , intrathoracic fat , and adiponectin in men . therefore , ectopic fat depots may provide some additional insight about systemic adiponectin concentrations not apparent with measures of general adiposity . the modest inverse association between vat and adiponectin has been observed previously ( 6,8,11 ) . we observed strong sex interactions in the effect size of vat on systemic adiponectin concentration . other studies have demonstrated significant sex interactions in the associations between vat and cardiometabolic risk factors ( 1,7 ) . sex differences in free fatty acid delivery to the liver may account for the observed sex interactions , but the mechanisms are still not clear . the role of resistin in human obesity is controversial , but a recent candidate gene study ( 13 ) suggests an association between resistin and regional fat distribution and metabolic complications in hiv lipodystrophy . the strong relations between vat , diabetes , dyslipidemia , hypertension , and metabolic syndrome have been observed previously . the results of the present study reveal that adiponectin does not fully explain the relations between vat , systolic blood pressure , diabetes , and metabolic syndrome . the associations between vat , log triglycerides , hdl cholesterol , and fasting plasma glucose were attenuated , but not fully accounted for , by adiponectin . these findings contrast with prior work in the health , aging , and body composition cohort , where the association between vat and diabetes in a multivariable model was minimally attenuated by adjustment for combination of adiponectin , leptin , and plasminogen activator inhibitor-1 ( 11 ) . however , pai-1 was the only adipokine found to be independently associated with incident diabetes . given the modest correlations between vat , sat , and adiponectin , it is not surprising that we did not observe more attenuation in these models . adjustment for resistin did not weaken any of the associations between vat and cardiometabolic risk factors . the differential associations between vat , sat , and adiponectin may be explained by differential adipocyte adipokine production and regulatory feedback of local inflammatory signals . several small studies in lean and obese nondiabetic participants have shown that adiponectin mrna levels are lower in vat compared with sat ( 14,15 ) , although these findings are not universal ( 16 ) . vat is associated with several inflammatory markers , even after adjustment for bmi and waist circumference ( 17 ) . tumor necrosis factor- , interleukin-6 , and c - reactive protein can suppress adiponectin gene expression . in addition , adiponectin binds to complement factors and injured vessel walls ( 18 ) ; therefore , adiponectin concentrations may be further reduced in the presence of a high vat volume and systemic inflammation . in humans , resistin is primarily derived from macrophages that have infiltrated adipose tissue ( 19 ) . a rodent study ( 20 ) revealed that macrophage content is higher in vat compared with sat in db / db mice but had similar concentrations in control mice . given the modest correlations between vat and adiponectin , adiponectin does not appear to be a good serum proxy for vat . adjustment for adiponectin affected relations between vat and fasting plasma glucose in men , hdl cholesterol , and log triglycerides , albeit minimally . these findings suggest that additional circulating biomarkers and hormones may mediate the relations between vat and cardiometabolic risk factors . it is also possible that high molecular weight adiponectin , rather than total adiponectin , may be a more potent mediator of the associations between vat and cardiometabolic risk factors . the strengths of this study include a well - characterized study sample and precise volumetric quantification of adipose tissue . the limitations include the cross - sectional design of the study , lack of ethnic diversity , noncontemporaneous ct and adipokine concentrations , and adiponectin assay . given the cross - sectional , observational nature of our study design , causality can not be inferred . because the sample is comprised of primarily white participants , generalizability to other ethnic groups is uncertain . adipokine concentrations , as well as glucose , cholesterol , and anthropometric measurements , were obtained several years before the cts were performed . serum adiponectin levels and cardiometabolic risk factors may have changed at the time of the cts , and regional fat depot measurements may have been different at the time of adipokine concentrations . however , adipokine measurements have been shown to be stable over a 1-year period ( 9 ) . nonetheless , we can not rule out misclassification based on the 4-year time lag between the adipokine and ct measurements that may have affected our results . any minor variation in adipokine measurements should not impact the relative association among vat , sat , pericardial fat , intrathoracic fat , and adipokines , which is the primary focus of this study . we measured systemic levels and not local adipokine concentrations , and systemic levels may not reflect local production and release by all regional fat depots . adipokines produced by vat are released into portal circulation , where hormones can be metabolized . therefore , systemic adiponectin concentration may underestimate adiponectin concentration that is actually secreted by vat . however , a recent study did not demonstrate a difference between portal and systemic adiponectin concentrations ( 21 ) ; therefore , this is unlikely to explain our results . lastly , high molecular weight adiponectin is more strongly associated with regional adiposity and metabolic diseases than total adiponectin ( 22 ) . thus , our results may have been stronger if we had measured high molecular weight adiponectin instead of total adiponectin . adiponectin and resistin are correlated with fat depots cross - sectionally , but none of these adipokines can serve as surrogates for these fat depots . relations among vat , sat , and cardiometabolic risk factors were not fully explained by adiponectin or resistin concentrations .

objectiveto test the association of regional fat depots with circulating adiponectin and resistin concentrations and to assess the potential mediating effect of adipokines on associations between abdominal fat depots and cardiometabolic risk factors.research design and methodsparticipants from the framingham heart study offspring cohort ( n = 916 , 55% women ; mean age 59 years ) free of cardiovascular disease underwent computed tomography measurement of visceral adipose tissue ( vat ) , subcutaneous adipose tissue ( sat ) , pericardial fat , and intrathoracic fat volumes and assays of circulating adiponectin and resistin.resultsvat , sat , pericardial fat , and intrathoracic fat were negatively correlated with adiponectin ( r = 0.19 to 0.34 , p < 0.001 [ women ] ; r = 0.15 to 0.26 , p < 0.01 [ men ] except sat ) and positively correlated with resistin ( r = 0.160.21 , p < 0.001 [ women ] ; r = 0.110.14 , p < 0.05 [ men ] except vat ) . vat increased the multivariable model r2 for adiponectin from 24% to 1013% and for resistin from 34% to 36% . adjustment for adipokines did not fully attenuate associations between vat , sat , and cardiometabolic risk factors.conclusionsadiponectin and resistin are correlated with fat depots cross - sectionally , but none of the adipokines can serve as surrogates for the fat depots . relations between vat , sat , and cardiometabolic risk factors were not fully explained by adiponectin or resistin concentrations .

RESEARCH DESIGN AND METHODS Adipokine measurements Volumetric adipose tissue imaging Abdominal and thoracic adipose tissue measurements Risk factor and covariate assessment Statistical analysis RESULTS Study sample characteristics Age-adjusted correlations between fat depots and adipokines Multivariable-adjusted regressions with fat depots and adiponectin Multivariable-adjusted regressions with fat depots and resistin Secondary analyses Multivariable-adjusted regressions with VAT, SAT, and cardiometabolic risk factors additionally adjusted for adipokines CONCLUSIONS Supplementary Material

participants were selected from the framingham heart study multidetector ct substudy of the community - based framingham heart study offspring cohort ; inclusion criteria have been described previously ( 1 ) . beginning in 1971 , the offspring study enrolled the offspring and spouses of the offspring whose parents were in the original cohort of the framingham heart study . of 1,418 participants in the offspring cohort of the multidetector ct study , 1,342 participants had interpretable ct measurements of visceral , subcutaneous , pericardial , and intrathoracic fat volume between 2002 and 2005 . after excluding participants with clinically prevalent cardiovascular disease ( cvd ) , prior coronary artery bypass graft , and an incomplete covariate profile , the final sample size was 916 ( 501 women and 415 men ) . single , random plasma adiponectin and resistin concentrations have been shown to be stable over a 1-year period ( 9 ) . visceral , subcutaneous , pericardial , and thoracic fat volumes were measured ( aquarius 3d workstation ; terarecon , san mateo , ca ) using an image display window width of 195 to 45 hounsfield units ( hu ) and window center of 120 hu . interreader reproducibility was excellent for visceral , subcutaneous , pericardial , and thoracic fat volume measurements ( 1,10 ) . adiponectin , vat , sat , pericardial fat , and intrathoracic fat volumes were approximately normally distributed . sex - specific multivariable linear regression was used to evaluate the associations of adiponectin and log resistin ( dependent variables ; separate models for each ) with fat depots ( independent variables ) , after adjustment for age , smoking , alcohol use , menopausal status ( women only ) , and hormone replacement therapy ( women only ) . vat , sat , pericardial fat , and intrathoracic fat were standardized to a means sd of 0 1 ; the covariate - adjusted average change in adiponectin and log resistin per sd of adipose tissue volume was estimated . using this approach , the multivariable models for vat , pericardial fat , and intrathoracic fat were then additionally adjusted for bmi and waist circumference in order to assess whether relations were maintained after adjusting for measures of generalized adiposity . as secondary analyses , multivariable models for vat , sat , pericardial fat , and intrathoracic fat were also additionally adjusted for physical activity and education status . age , smoking , alcohol use , menopausal status ( women only ) , hormone replacement therapy ( women only ) , and treatment of hypertension , dyslipidemia , and diabetes were entered as covariates in each of the models . each multivariable model was also adjusted for either adiponectin or log resistin ( separate models for each adjustment ) to assess the extent of attenuation of the association of vat and sat with each cardiometabolic risk factor upon adjustment for these adipokines . a two - tailed value of p < 0.05 was considered statistically significant . circulating concentrations of adiponectin and resistin were measured by enzyme - linked immunosorbent assay ( r&d systems , minneapolis , mn ) in plasma collected after a > 8-h fast and frozen at 80c . single , random plasma adiponectin and resistin concentrations have been shown to be stable over a 1-year period ( 9 ) . visceral , subcutaneous , pericardial , and thoracic fat volumes were measured ( aquarius 3d workstation ; terarecon , san mateo , ca ) using an image display window width of 195 to 45 hounsfield units ( hu ) and window center of 120 hu . interreader reproducibility was excellent for visceral , subcutaneous , pericardial , and thoracic fat volume measurements ( 1,10 ) . adiponectin , vat , sat , pericardial fat , and intrathoracic fat volumes were approximately normally distributed . sex - specific multivariable linear regression was used to evaluate the associations of adiponectin and log resistin ( dependent variables ; separate models for each ) with fat depots ( independent variables ) , after adjustment for age , smoking , alcohol use , menopausal status ( women only ) , and hormone replacement therapy ( women only ) . vat , sat , pericardial fat , and intrathoracic fat were standardized to a means sd of 0 1 ; the covariate - adjusted average change in adiponectin and log resistin per sd of adipose tissue volume was estimated . using this approach , the multivariable models for vat , pericardial fat , and intrathoracic fat were then additionally adjusted for bmi and waist circumference in order to assess whether relations were maintained after adjusting for measures of generalized adiposity . as secondary analyses , multivariable models for vat , sat , pericardial fat , and intrathoracic fat were multivariable linear and logistic regressions were used to relate vat and sat ( independent variables ) to cardiometabolic risk factors ( dependent variables ) . age , smoking , alcohol use , menopausal status ( women only ) , hormone replacement therapy ( women only ) , and treatment of hypertension , dyslipidemia , and diabetes were entered as covariates in each of the models . each multivariable model was also adjusted for either adiponectin or log resistin ( separate models for each adjustment ) to assess the extent of attenuation of the association of vat and sat with each cardiometabolic risk factor upon adjustment for these adipokines . a two - tailed value of p < 0.05 was considered statistically significant . the mean age was 59 years ( table 1 ) , and slightly more than one - quarter were obese . vat , sat , pericardial fat , and intrathoracic fat were negatively correlated with adiponectin ( r = 0.19 to 0.34 , p < 0.001 [ women ] ; r = 0.15 to 0.26 , p < 0.01 [ men ] except sat ) and positively correlated with resistin ( r = 0.160.21 , p < 0.001 [ women ] ; r = 0.110.14 , p < 0.05 [ men ] except vat ) ( table 2 ) . age - adjusted pearson correlation coefficients between fat depot volumes and adipokines in women , adiponectin concentration was 2.1 0.3 g / ml lower per sd increase of vat , 1.2 0.3 g / ml lower per sd increase of sat , 1.2 0.3 g / ml lower per sd increase of pericardial fat , and 1.2 0.3 g / ml lower per sd increase of intrathoracic fat ( p < 0.001 ) in multivariable - adjusted models ( table 3 ) . the regression coefficients for adiponectin concentration were larger per sd of vat than per sd of sat ( p < 0.001 ) . in men , the multivariable - adjusted effect sizes of vat , sat , pericardial fat , and intrathoracic fat on adiponectin concentration were weaker but still significant in nearly all cases ( table 3 ) . a sex - by - vat interaction was observed for adiponectin ( p < 0.001 ) , as well as a sex - by - sat ( p = 0.001 ) and sex by pericardial fat interaction ( p = 0.03 ) . effect size refers to the average change in adiponectin or resistin concentration per sd of visceral , subcutaneous , pericardial , or intrathoracic fat volume . in women , vat remained associated with adiponectin after additional adjustment for bmi and waist circumference ( p = < 0.001 ) ; pericardial fat and intrathoracic fat did not remain associated with adiponectin ( p = 0.15 [ pericardial fat ] , p = 0.5 [ intrathoracic fat ] ) . in contrast , among men , vat , pericardial fat , and intrathoracic fat were associated with adiponectin after further adjustment for bmi and waist circumference ( all p < 0.02 ) . covariates ( age , sex , smoking , alcohol use , menopausal status , and hormone replacement therapy ) explained 2% of the circulating variability in adiponectin concentration in women and 4% in men ; adding vat to this model increased the model r to 13% in women and 10% in men ( table 3 ) . multivariable - adjusted models for sat , pericardial fat , and intrathoracic fat accounted for a smaller portion of the variation in adiponectin concentration ( r range 48% ) . additional adjustment for bmi and waist circumference attenuated associations between vat , pericardial fat , intrathoracic fat , and resistin ( all p > 0.2 ) . results were similar for men , except intrathoracic fat remained associated with resistin after additional adjustment for bmi and waist circumference ( p = 0.02 ) . none of the multivariable - adjusted models explained a substantial portion of the variability in resistin concentration ( r range 36% ) . when physical activity and education status were included as additional covariates , relations between adipose tissue volumes , adiponectin , and resistin were not materially different ( data not shown ) . vat was associated with all cardiometabolic risk factors in both sexes as previously reported ( 1 ) , except for systolic blood pressure in men ( online appendix tables 1a and 1b [ available at http://care.diabetesjournals.org/cgi/content/full/dc08-1733/dc1 ] ) . in women , we observed some attenuation of the effect size of vat on log triglycerides and hdl cholesterol after additional adjustment for adiponectin , but strong associations were still observed . fasting plasma glucose and diabetes did not appreciably change after adjustment for adiponectin . in men , there was more attenuation of the effect size of vat on fasting plasma glucose , but not for diabetes , after additional adjustment for adiponectin . the effect size of vat on hdl cholesterol was minimally weakened after additional adjustment for adiponectin . relations between vat , log triglycerides , and metabolic syndrome , as well as between sat and cardiometabolic risk factors , did not appreciably change after adjustment for adiponectin in men . none of the associations between vat , sat , and cardiometabolic risk factors appreciably changed upon adjustment for resistin ( online appendix tables 1a and 1b ) . the mean age was 59 years ( table 1 ) , and slightly more than one - quarter were obese . vat , sat , pericardial fat , and intrathoracic fat were negatively correlated with adiponectin ( r = 0.19 to 0.34 , p < 0.001 [ women ] ; r = 0.15 to 0.26 , p < 0.01 [ men ] except sat ) and positively correlated with resistin ( r = 0.160.21 , p < 0.001 [ women ] ; r = 0.110.14 , p < 0.05 [ men ] except vat ) ( table 2 ) . in women , adiponectin concentration was 2.1 0.3 g / ml lower per sd increase of vat , 1.2 0.3 g / ml lower per sd increase of sat , 1.2 0.3 g / ml lower per sd increase of pericardial fat , and 1.2 0.3 g / ml lower per sd increase of intrathoracic fat ( p < 0.001 ) in multivariable - adjusted models ( table 3 ) . the regression coefficients for adiponectin concentration were larger per sd of vat than per sd of sat ( p < 0.001 ) . in men , the multivariable - adjusted effect sizes of vat , sat , pericardial fat , and intrathoracic fat on adiponectin concentration were weaker but still significant in nearly all cases ( table 3 ) . a sex - by - vat interaction was observed for adiponectin ( p < 0.001 ) , as well as a sex - by - sat ( p = 0.001 ) and sex by pericardial fat interaction ( p = 0.03 ) . effect size refers to the average change in adiponectin or resistin concentration per sd of visceral , subcutaneous , pericardial , or intrathoracic fat volume . in women , vat remained associated with adiponectin after additional adjustment for bmi and waist circumference ( p = < 0.001 ) ; pericardial fat and intrathoracic fat did not remain associated with adiponectin ( p = 0.15 [ pericardial fat ] , p = 0.5 [ intrathoracic fat ] ) . in contrast , among men , vat , pericardial fat , and intrathoracic fat were associated with adiponectin after further adjustment for bmi and waist circumference ( all p < 0.02 ) . covariates ( age , sex , smoking , alcohol use , menopausal status , and hormone replacement therapy ) explained 2% of the circulating variability in adiponectin concentration in women and 4% in men ; adding vat to this model increased the model r to 13% in women and 10% in men ( table 3 ) . multivariable - adjusted models for sat , pericardial fat , and intrathoracic fat accounted for a smaller portion of the variation in adiponectin concentration ( r range 48% ) . additional adjustment for bmi and waist circumference attenuated associations between vat , pericardial fat , intrathoracic fat , and resistin ( all p > 0.2 ) . results were similar for men , except intrathoracic fat remained associated with resistin after additional adjustment for bmi and waist circumference ( p = 0.02 ) . none of the multivariable - adjusted models explained a substantial portion of the variability in resistin concentration ( r range 36% ) . when physical activity and education status were included as additional covariates , relations between adipose tissue volumes , adiponectin , and resistin were not materially different ( data not shown ) . vat was associated with all cardiometabolic risk factors in both sexes as previously reported ( 1 ) , except for systolic blood pressure in men ( online appendix tables 1a and 1b [ available at http://care.diabetesjournals.org/cgi/content/full/dc08-1733/dc1 ] ) . in women , we observed some attenuation of the effect size of vat on log triglycerides and hdl cholesterol after additional adjustment for adiponectin , but strong associations were still observed . after further adjustment for adiponectin , fasting plasma glucose and diabetes did not appreciably change after adjustment for adiponectin . for sat , , there was more attenuation of the effect size of vat on fasting plasma glucose , but not for diabetes , after additional adjustment for adiponectin . relations between vat , log triglycerides , and metabolic syndrome , as well as between sat and cardiometabolic risk factors , did not appreciably change after adjustment for adiponectin in men . none of the associations between vat , sat , and cardiometabolic risk factors appreciably changed upon adjustment for resistin ( online appendix tables 1a and 1b ) . in the community - based framingham offspring cohort , vat , sat , pericardial fat , and intrathoracic fat were negatively correlated with adiponectin but positively correlated with resistin . furthermore , the addition of vat only increased the model r for adiponectin from 24% to 1013% . adjustment for adiponectin attenuated relations between vat , sat , and cardiometabolic risk factors , but adiponectin did not fully account for the strong relations between vat and cardiometabolic risk factors . easily obtainable anthropometric measures ( bmi and waist circumference ) did not explain the associations between vat and adiponectin in women or men and between pericardial fat , intrathoracic fat , and adiponectin in men . other studies have demonstrated significant sex interactions in the associations between vat and cardiometabolic risk factors ( 1,7 ) . the strong relations between vat , diabetes , dyslipidemia , hypertension , and metabolic syndrome have been observed previously . the results of the present study reveal that adiponectin does not fully explain the relations between vat , systolic blood pressure , diabetes , and metabolic syndrome . the associations between vat , log triglycerides , hdl cholesterol , and fasting plasma glucose were attenuated , but not fully accounted for , by adiponectin . these findings contrast with prior work in the health , aging , and body composition cohort , where the association between vat and diabetes in a multivariable model was minimally attenuated by adjustment for combination of adiponectin , leptin , and plasminogen activator inhibitor-1 ( 11 ) . given the modest correlations between vat , sat , and adiponectin , it is not surprising that we did not observe more attenuation in these models . adjustment for resistin did not weaken any of the associations between vat and cardiometabolic risk factors . the differential associations between vat , sat , and adiponectin may be explained by differential adipocyte adipokine production and regulatory feedback of local inflammatory signals . adjustment for adiponectin affected relations between vat and fasting plasma glucose in men , hdl cholesterol , and log triglycerides , albeit minimally . these findings suggest that additional circulating biomarkers and hormones may mediate the relations between vat and cardiometabolic risk factors . it is also possible that high molecular weight adiponectin , rather than total adiponectin , may be a more potent mediator of the associations between vat and cardiometabolic risk factors . the limitations include the cross - sectional design of the study , lack of ethnic diversity , noncontemporaneous ct and adipokine concentrations , and adiponectin assay . serum adiponectin levels and cardiometabolic risk factors may have changed at the time of the cts , and regional fat depot measurements may have been different at the time of adipokine concentrations . any minor variation in adipokine measurements should not impact the relative association among vat , sat , pericardial fat , intrathoracic fat , and adipokines , which is the primary focus of this study . we measured systemic levels and not local adipokine concentrations , and systemic levels may not reflect local production and release by all regional fat depots . adiponectin and resistin are correlated with fat depots cross - sectionally , but none of these adipokines can serve as surrogates for these fat depots . relations among vat , sat , and cardiometabolic risk factors were not fully explained by adiponectin or resistin concentrations .

obesity , declared a global epidemic by the world health organization , is associated with a number of illnesses such as diabetes , cardiovascular disease and cancer ( http://www.who.int ) . equally , obesity is a significant risk factor for obstructive sleep apnea ( osa ) , the most prevalent sleep disordered breathing problem . osa affects 226% of the general population , depending on gender , age and definition of the used criteria [ 2 , 3 ] . to make matters worse , obesity is dramatically on the rise . in 2005 , 400 million adults worldwide were obese . the who projects that in 2015 , 700 million adults will be obese ( http://www.who.int ) . the exact pathophysiology of osa in obese patients remains poorly understood , but it is thought that in obese patients the local fatty tissue deposition in the neck results in reduction of the lumen of the upper airway , thereby reducing airflow and inducing airway collapse . in patients with morbid obesity , who have failed conservative treatment , bariatric surgery ( bs ) can be considered . bs is not only the most effective , long - term treatment modality in these patients for losing weight , but is also known to have a positive effect on comorbidities . the benefits of bariatric surgery are increasingly reported , but concern about the safety is also heightened . patients with osa are particularly vulnerable during anesthesia and sedation and at an increased risk of developing respiratory and cardiopulmonary complications postoperatively . furthermore , a recent study by the longitudinal assessment of bariatric surgery group ( labs ) shows that a history of osa is significantly associated with an increased risk of major perisurgical adverse outcomes in patients undergoing bs . additionally , osa was found to triple the risk of perioperative death in a recent single - surgeon report of 1000 roux - en - y gastric bypass procedures . anesthetist and surgeons should be aware that undiagnosed osa is common ; osa remains undiagnosed in an estimated 93% of women and 82% of men [ 6 , 8 ] . bearing this in mind , and with the aim of preventing osa - related complications of bs , we were interested to see which percentage of patients undergoing bariatric surgery in our clinic had osa . we were interested in measuring the predictive value of various clinical parameters : body mass index ( bmi ) , neck circumference ( nc ) and epworth sleepiness scale ( ess ) . we performed a prospective , multidisciplinary , single - center observational study involving a consecutive series of patients being evaluated for bs in our clinic from june 2009 until june 2010 . patients meeting the international federation for the surgery of obesity ( ifso ) ( http://www.ifso.com ) criteria were eligible for bs , specifically patients aged 1865 years , with a bmi > 40 kg / m or bmi > 35 kg / m with associated comorbidity ( e.g. , hypertension , diabetes , osa or joint problems ) . secondly , patients were required to have made sufficient attempts at weight loss using conservative measures and must be motivated for dietary and behavior modification . various bs types are performed in our clinic : laparoscopic gastric banding , swedish type of adjustable gastric banding ( sagb ) , laparoscopic gastric bypass and gastric sleeve resection . all patients eligible for bs underwent a mandatory preoperative screening for osa in addition to our routine preoperative workup . apart from patients with osa previously diagnosed elsewhere , preoperatively all patients on the waiting list for bs visited the ent outpatient department . information was gained using patient history , ent and general examination and a full overnight polysomnography ( psg ) . weight , length ( bmi ) and nc at the level of the cricothyroid membrane were measured . the following bmi grading system was implemented : obese ( bmi 3034.9 kg / m ) , severely obese ( bmi 3539.9 kg / m ) , morbidly obese ( bmi 4049.9 kg / m ) and super obese ( bmi > 50 the patients completed a questionnaire containing various questions concerning possible daily or nocturnal symptoms , intoxications , medication and medical history . patients scored themselves on a scale of 03 on how easily they would fall asleep in eight different situations , giving an overall score between 0 and 24 ; the higher the score , the sleepier was the individual . besides patients with osa previously diagnosed elsewhere , all patients underwent a full night comprehensive sleep study using a digital embla recorder ( flaga medical devices , reykjavik , iceland ) . this records the sleep architecture ( derived from electroencephalogram , electrooculogram and submental electromyogram ) , respiration ( thoracic and abdominal measurement ) , movements of limbs , nasal airflow and the intensity of the snoring ( the last two measured by pressure sensor ) . transcutaneous pulse oximetry was used to monitor oxygen saturation ( sao2 ) and heart rate . hypopneas were defined as periods of reduction of > 30% oronasal airflow for at least 10s and a 4% decrease in oxygen saturation . was calculated as the sum of total events ( apneas and hypopneas ) per hour of sleep . an ahi of 515/h is mild osas , an ahi of 1530/h is moderate and ahi > 30/h is severe osas , as assessed by polysomnography [ 2 , 10 ] . statistical analysis was performed using microsoft excel and spss statistical software ( version 18 , spss inc . , the distribution of recorded variables was characterized by calculating the mean and standard deviation . since some parameters ( especially the ahi ) were expected to be non - normally distributed , also the median and range were calculated . data are given for both the total study population and subdivided for women and men . the results of women and men were compared using an unpaired t test , with additional non - parametric mann differences were considered significant when p < 0.05 . the prevalence of osa and osa severity was subdivided for obesity severity subgroups . the relation between the ahi and patient characteristics was further evaluated employing stepwise linear regression and binomial logistic regression . the sensitivity and specificity of the clinical predictor variables for the presence versus absence of osa ( ahi > 5/h ) and moderate to severe osa ( ahi > 15/h ) were used to plot receiver operating characteristic ( roc ) curves . we performed a prospective , multidisciplinary , single - center observational study involving a consecutive series of patients being evaluated for bs in our clinic from june 2009 until june 2010 . patients meeting the international federation for the surgery of obesity ( ifso ) ( http://www.ifso.com ) criteria were eligible for bs , specifically patients aged 1865 years , with a bmi > 40 kg / m or bmi > 35 kg / m with associated comorbidity ( e.g. , hypertension , diabetes , osa or joint problems ) . secondly , patients were required to have made sufficient attempts at weight loss using conservative measures and must be motivated for dietary and behavior modification . various bs types are performed in our clinic : laparoscopic gastric banding , swedish type of adjustable gastric banding ( sagb ) , laparoscopic gastric bypass and gastric sleeve resection . all patients eligible for bs underwent a mandatory preoperative screening for osa in addition to our routine preoperative workup . apart from patients with osa previously diagnosed elsewhere , preoperatively all patients on the waiting list for bs visited the ent outpatient department . information was gained using patient history , ent and general examination and a full overnight polysomnography ( psg ) . weight , length ( bmi ) and nc at the level of the cricothyroid membrane were measured . the following bmi grading system was implemented : obese ( bmi 3034.9 kg / m ) , severely obese ( bmi 3539.9 kg / m ) , morbidly obese ( bmi 4049.9 kg / m ) and super obese ( bmi > 50 the patients completed a questionnaire containing various questions concerning possible daily or nocturnal symptoms , intoxications , medication and medical history . patients scored themselves on a scale of 03 on how easily they would fall asleep in eight different situations , giving an overall score between 0 and 24 ; the higher the score , the sleepier was the individual . besides patients with osa previously diagnosed elsewhere , all patients underwent a full night comprehensive sleep study using a digital embla recorder ( flaga medical devices , reykjavik , iceland ) . this records the sleep architecture ( derived from electroencephalogram , electrooculogram and submental electromyogram ) , respiration ( thoracic and abdominal measurement ) , movements of limbs , nasal airflow and the intensity of the snoring ( the last two measured by pressure sensor ) . transcutaneous pulse oximetry was used to monitor oxygen saturation ( sao2 ) and heart rate . hypopneas were defined as periods of reduction of > 30% oronasal airflow for at least 10s and a 4% decrease in oxygen saturation . was calculated as the sum of total events ( apneas and hypopneas ) per hour of sleep . an ahi of 515/h is mild osas , an ahi of 1530/h is moderate and ahi > 30/h is severe osas , as assessed by polysomnography [ 2 , 10 ] . statistical analysis was performed using microsoft excel and spss statistical software ( version 18 , spss inc . , the distribution of recorded variables was characterized by calculating the mean and standard deviation . since some parameters ( especially the ahi ) were expected to be non - normally distributed , also the median and range were calculated . data are given for both the total study population and subdivided for women and men . the results of women and men were compared using an unpaired t test , with additional non - parametric mann differences were considered significant when p < 0.05 . the prevalence of osa and osa severity was subdivided for obesity severity subgroups . the relation between the ahi and patient characteristics was further evaluated employing stepwise linear regression and binomial logistic regression the sensitivity and specificity of the clinical predictor variables for the presence versus absence of osa ( ahi > 5/h ) and moderate to severe osa ( ahi > 15/h ) were used to plot receiver operating characteristic ( roc ) curves . ten patients did not show up for their ent outpatient clinic and psg appointment . of the remaining 279 patients , 214 ( 76.7% ) patient baseline characteristics are shown in table 1.table 1patient characteristics : clinical and polysomnographic parameterspatient characteristicsmeanmedianrangewomen214 ( 76.7%)men65 ( 23.3%)age ( years)45.1 10.646.0(1767)bmi ( kg / m)44.2 6.442.8(3366)neck circumference ( cm)42.6 4.842.0(3459.8)ess4.3 3.83.0(017)ahi ( per h)23.9 27.712.4(0142)ai11 21.41.6(0127)arousal index ( per h)7.5 8.45(054.7)mean sao2 ( % ) 93.8 3.394.7(7499)minimum sao2 ( % ) 80.8 10.783.0(5095)desaturation index ( di)16.3 23.45.3(0106) indicates standard deviationai apnea index ; ahi apnea hypopnea index ; bmi body mass index , ess epworth sleepiness scale ; osa obstructive sleep apnea ; sao2 oxygen saturation patient characteristics : clinical and polysomnographic parameters indicates standard deviation ai apnea index ; ahi apnea hypopnea index ; bmi body mass index , ess epworth sleepiness scale ; osa obstructive sleep apnea ; sao2 oxygen saturation an unpaired t test was conducted to compare baseline characteristics in men and women . there was a significant difference in the ahi ( p < 0.0003 ) , ai ( p < 0.0004 ) , arousal index ( p < 0.006 ) , di ( p < 0.0003 ) , mean o2 saturation ( p = 0.001 ) , minimum o2 saturation ( p < 0.0007 ) and nc ( p < 0.0003 ) between men and women . in our study population , men were found to have a higher ahi , ai , arousal index and di , and lower mean and minimum o2 saturation ( table 2 ) . application of a non - parametric test provided no new insights.table 2patient characteristics subdivided for women and menpatient characteristicsmenwomenage ( years)48.5 9.344.0 10.8bmi ( kg / m)44.3 7.144.2 6.2neck circumference ( cm)48.0 3.941.2 4.0ess5.0 4.24.2 3.6ahi ( h)45.9 29.917.3 23.3ai ( h)25.7 27.17.0 17.5arousal index ( h)10.6 8.96.7 8.1mean sao2 ( % ) 92.3 3.094.1 3.4minimum sao2 ( % ) 74.6 11.382.5 9.9desaturation index ( di)32.2 26.811.8 20.3 indicates standard deviationai apnea index ; ahi apnea hypopnea index ; bmi body mass index , ess epworth sleepiness scale ; osa obstructive sleep apnea ; sao2 oxygen saturation patient characteristics subdivided for women and men indicates standard deviation ai apnea index ; ahi apnea hypopnea index ; bmi body mass index , ess epworth sleepiness scale ; osa obstructive sleep apnea ; sao2 oxygen saturation three ( 1.1% ) of the patients were obese , 75 ( 26.9% ) severely obese , 149 ( 53.6% ) morbidly obese , 51 ( 18.3% ) super obese . osa stratified by bmi and the severity of osa by bmi are depicted in table 3 and fig . 1 , respectively . a total of 112 ( 40.1% ) patients underwent or are on the waiting list for sagb ( average bmi 41 sd 4 kg / m ) , 155 ( 55.6% ) laparoscopic gastric bypass ( average bmi 46.1 sd 6.7 kg / m ) and 12 ( 4.3% ) gastric sleeve surgery ( average bmi 49.4 sd 8.5 patients had been previously diagnosed with osas ( ahi : 42.5 sd 27.2 per h ) in our hospital ( 12 pts ) or elsewhere ( 25 pts ) before being placed on the waiting list for bs . based on the psg results , 195 ( 69.9% ) patients were diagnosed with osa , specifically 67 ( 34.7% ) with mild osa , 48 ( 24.9% ) with moderate osa and 78 ( 40.4% ) with severe osa . 69.2% of the patients diagnosed with osa were female and 30.7% male.table 3number of patients with osa stratified by bmiosa stratified by bmiosano osatotal no.osa ( % ) obese ( 3034.9 kg / m)12333.3severely obese ( 3539.9 kg / m)49267565.3morbidly obese ( 4049.9 kg / m)1034614969.1super obese ( > 50 kg / m)41105180.4bmi body mass index , osa obstructive sleep apneafig . 1severity osa stratified by bmi number of patients with osa stratified by bmi bmi body mass index , osa obstructive sleep apnea severity osa stratified by bmi stepwise linear regression was performed with ahi as the dependent variable . independent variables evaluated were the bmi , nc and ess [ adjusted r = 0.236 ; f = 22.9 , p < 0.0001 ( using the stepwise method ) ] . in men , only the bmi was associated with the ahi ( adjusted r = 0.167 ; f = 10.2 , p = 0.003 ) . addition of nc made significant improvement to the model , but ess did not ( adjusted r = 0.113 ; f = 11.5 , p < 0.0001 ) . the ahi data are not strictly normally distributed . however , also after normalizing the data with a square root transformation , there was no improvement in the association between nc , ess and the dependent variable ( ahi ) . binomial logistic regression was used to identify independent variables associated with the presence or absence of ( 1 ) an ahi greater than 5/h or ( 2 ) 15/h . results showed that in women , the bmi was the only significant predictor of an ahi greater than 5/h ( odds ratio [ or ] = 1.072 , p = 0.018 , 95% ci 1.0121.135 ) and of an ahi greater than 15/h ( [ or ] = 1.102 , p = 0.001 , 95% ci 1.0421.165 ) . in men , nc was a significant predictor of an ahi greater than 15/h ( [ or ] = 1.278 , p = 0.026 , 95% ci 1.0301.586 ) . the results of the roc curves were disappointing ; of all three clinical parameters , no cutoff values were found to have both a sensible sensitivity ( > 0.8 ) and a useful specificity ( > 0.9 ) . the neck circumference was found to be the most accurate predictor of the presence of osa when the ahi as greater than 5/h ( fig . 2 ) . 2roc curve comparing sensitivity and specificity of neck circumference ( nc ) , body mass index ( bmi ) and epworth sleepiness scale ( ess ) of an ahi > 5/h . the mean area under the curve ( auc ) for nc , bmi and ess were 0.69 0.4 ( 95% ci 0.620.77 ) , 0.61 0.4 ( 95% ci 0.530.69 ) , 0.54 0.4 ( 95% ci 0.450.62 ) , respectively . no cutoff values were found to have both a sensible sensitivity ( > 0.8 ) and a useful specificity ( > 0.9)fig . 3roc curve comparing sensitivity and specificity of neck circumference ( nc ) , body mass index ( bmi ) and epworth sleepiness scale ( ess ) of an ahi > 15/h . the mean area under the curve ( auc ) for nc , bmi and ess were 0.76 0.4 ( 95% ci 0.690.82 ) , 0.62 0.4 ( 95% ci 0.540.69 ) and 0.59 0.4 ( 95% ci 0.510.67 ) , respectively . no cutoff values were found to have both a sensible sensitivity ( > 0.8 ) and a useful specificity ( > 0.9 ) roc curve comparing sensitivity and specificity of neck circumference ( nc ) , body mass index ( bmi ) and epworth sleepiness scale ( ess ) of an ahi > 5/h . the mean area under the curve ( auc ) for nc , bmi and ess were 0.69 0.4 ( 95% ci 0.620.77 ) , 0.61 0.4 ( 95% ci 0.530.69 ) , 0.54 0.4 ( 95% ci 0.450.62 ) , respectively . no cutoff values were found to have both a sensible sensitivity ( > 0.8 ) and a useful specificity ( > 0.9 ) roc curve comparing sensitivity and specificity of neck circumference ( nc ) , body mass index ( bmi ) and epworth sleepiness scale ( ess ) of an ahi > 15/h . the mean area under the curve ( auc ) for nc , bmi and ess were 0.76 0.4 ( 95% ci 0.690.82 ) , 0.62 0.4 ( 95% ci 0.540.69 ) and 0.59 0.4 ( 95% ci 0.510.67 ) , respectively . no cutoff values were found to have both a sensible sensitivity ( > 0.8 ) and a useful specificity ( > 0.9 ) in this series of consecutive patients undergoing bs , we found a 69.9% prevalence of osa . more than 40% of these patients were diagnosed with severe osa . of the 195 patients diagnosed with osa , 69.2% were female : a 1:2.3 ratio ( : ) , which is opposite to the typical osa male female ratio of 2:1 ( : ) . the raised percentage of women with osa is caused by a skewed gender distribution within our study population . more than three - quarters of our study population was female ; comparative to earlier reports that women seek surgical weight loss treatment nearly four times more often than men . unlike many other studies , we used no selective inclusion criteria such as the ess as a screening tool . irrespective of history or clinical findings , all patients being evaluated for bs underwent a polysomnography , unless performed previously . our results are consistent with similar studies ( see table 3 ) . using synonyms for : bariatric surgery , obstructive sleep apnea and polysomnography , an online systematic search was performed of the medline and embase database on the 4th april 2011 . ten relevant articles were found . in each study , all patients being evaluated for bariatric surgery underwent a polysomnography as part of routine screening for osa regardless of symptoms and without use of screening tools such as the ess . four articles were omitted from our overview ( see table 4 ) , owing to various applied inclusion criteria ( a bmi 40 kg / m or asian race ) and articles , which did not apply the aasm osa guidelines [ 1215].table 4outcomes of similar studies , in which all patients being evaluated for bariatric surgery underwent a polysomnography , irrespective of history or clinical findingsreferencetotal nosa nprevalence osa ( % ) severe osa ( % ) mean ahi ( h)mean bmi ( kg / m)frey et al . 17612671.648.028.042.0all1268100479.233.125.748.8ahi apnea hypopnea index , bmi body mass index , osa obstructive sleep apneaonly studies presenting percentage severe osa dataonly studies presenting mean ahi dataonly studies presenting mean bmi data outcomes of similar studies , in which all patients being evaluated for bariatric surgery underwent a polysomnography , irrespective of history or clinical findings ahi apnea hypopnea index , bmi body mass index , osa obstructive sleep apnea only studies presenting percentage severe osa data only studies presenting mean ahi data only studies presenting mean bmi data to our knowledge , following sareli et al . and lopez et al . [ 16 , 17 ] , our results are more in line with the results of frey et al . and lee et al . , but it should be noted that lee et al . studied a predominantly asian population several studies reported that the prevalence of osa increased as the bmi increased , which may explain why our prevalence was lowest of all [ 17 , 18 ] . we have two main study limitations , the first being that osa was an inclusion criterion for bariatric surgery , in accordance with the ifso guidelines : a potential bias , which could result in an overestimation of the prevalence of osa in our bariatric surgery population . a mere 13.3% ( 37 ) patients were diagnosed with osa before being placed on the waiting list for bs ; 62.2% of these patients had a bmi greater than 40 kg / m and were therefore eligible for bs regardless of the presence of osa . as this group was minimal , we chose to include these patients in the series so as to avoid underestimating the prevalence of osa in our bariatric surgery population . the second limitation of the study is absent data , in particular from patients who had previously been diagnosed with osa elsewhere . these patients did not visit our outpatient department , consequently patient information such as ess or nc was unavailable . ess data were available for 78.5% of the patients , and nc measurements for 82.4% . ess is considered a useful screening tool for osa in the adult population ; but as reported by sareli et al . , in the bariatric population , ess can not independently reliably predict the presence of osa . our data support this observation ; we found ess not to be significantly related to the presence of osa in patients undergoing bs . hence , ess is not a reliable predictor of osa in this patient population , despite often being used in bs centers as a screening tool . we used various statistical techniques to analyze the data . the various regression models and the roc curves give discrepant results , mainly due to the non - normal distribution of the data and data values of zero or close to zero . the poor statistical power and discrepancy of the results we contend that all three clinical parameters are inadequate predictors of osa and that psg is an essential component of the preoperative workup of patients undergoing bs . despite the high test probability of moderate to severe osa in obese patients , the high costs and patient burden of psgs as well as the increasing prevalence of obesity and bs , the task force of the aasm does not advise the use of unattended portable monitoring ( pm ) for general screening , as there is yet insufficient evidence to guide the use of pm . a recent , unique study by hallowell et al . compared a series of consecutive patients who underwent mandatory osa evaluation with a second group who were selected for a preoperative sleep study based on clinical suspicion and a raised ess in preparation for the bariatric surgery program . the authors suggest that osa is grossly underdiagnosed in the bariatric population and concludes that clinical evaluation including the ess is inadequate to identify the true prevalence of osa . we found that a substantial number of patients , even patients with extremely high ahis , were completely unaware of their osa . a mere 13.3% of the patients were diagnosed with osa before being placed on the waiting list for bs ; 37 ( 13.3% ) patients had an ahi > 60/h and only 11 patients were aware of their extreme osa . patients are often asymptomatic or relate complaints of fatigue and hypersomnolence to their obesity and/or other comorbidities ( e.g. , diabetes ) and do not realize that these are actually osa related . more often than in the general population , these patients sleep / live alone and a history of a bed partner is often lacking , which collaborates with patients being often unaware of their breathing abnormalities during sleep . the finding of osa may in fact influence the indication for bs being a bmi > 40 kg / m in patients without comorbidity , or bmi > 35 kg / m with comorbidity . therefore , in otherwise healthy patients with a bmi between 35 kg / m and 40 kg / m , the finding of osa widens the indication for bs . patients with osa have been shown to have increased preoperative risk and specific perioperative measures have to been taken [ 4 , 23 ] . to what extent perioperative cpap therapy should also be applied in mild osa remains to be elucidated . intubation might be difficult , and specific methods of intubation can be indicated . in case intubation is impossible and a tracheostomy must be performed , longer than usual tracheal cannulas might be necessary . postoperatively , the use of morphinomimetic painkillers and other muscle tone reducing medication is contraindicated in patients with osa , or can only be used with postoperative monitoring . increased neck circumference , bmi or ess can not reliably predict the presence of osa . we are currently following these patients and aim to publish a paper showing the results of postoperative psg results shortly .

the aim of this study was to evaluate prevalence of obstructive sleep apnea among patients undergoing bariatric surgery and the predictive value of various clinical parameters : body mass index ( bmi ) , neck circumference ( nc ) and the epworth sleepiness scale ( ess ) . we performed a prospective , multidisciplinary , single - center observational study including all patients on the waiting list for bariatric surgery between june 2009 and june 2010 , irrespective of history or clinical findings . patients visited our ent outpatient clinic for patient history , ent and general examination and underwent a full night polysomnography , unless performed previously . as much as 69.9% of the patients fulfilled the criteria for osa ( mean bmi 44.2 sd 6.4 kg / m2 ) ; 40.4% of the patients met the criteria for severe osa . the regression models found bmi to be the best clinical predictor , while the roc curve found the nc to be the most accurate predictor of the presence of osa . the discrepancy of the results and the poor statistical power suggest that all three clinical parameters are inadequate predictors of osa . in conclusion , in this large patient series , 69.9% of patients undergoing bs meet the criteria for osa . more than 40% of these patients have severe osa . a mere 13.3% of the patients were diagnosed with osa before being placed on the waiting list for bs . on statistical analysis , increased neck circumference , bmi and the ess were found to be insufficient predictors of the presence of osa . polysomnography is an essential component of the preoperative workup of patients undergoing bs . when osa is found , specific perioperative measures are indicated .

Introduction Methods Patients Polysomnography Statistical analysis Results Discussion Conclusion

equally , obesity is a significant risk factor for obstructive sleep apnea ( osa ) , the most prevalent sleep disordered breathing problem . the exact pathophysiology of osa in obese patients remains poorly understood , but it is thought that in obese patients the local fatty tissue deposition in the neck results in reduction of the lumen of the upper airway , thereby reducing airflow and inducing airway collapse . furthermore , a recent study by the longitudinal assessment of bariatric surgery group ( labs ) shows that a history of osa is significantly associated with an increased risk of major perisurgical adverse outcomes in patients undergoing bs . bearing this in mind , and with the aim of preventing osa - related complications of bs , we were interested to see which percentage of patients undergoing bariatric surgery in our clinic had osa . we were interested in measuring the predictive value of various clinical parameters : body mass index ( bmi ) , neck circumference ( nc ) and epworth sleepiness scale ( ess ) . we performed a prospective , multidisciplinary , single - center observational study involving a consecutive series of patients being evaluated for bs in our clinic from june 2009 until june 2010 . all patients eligible for bs underwent a mandatory preoperative screening for osa in addition to our routine preoperative workup . apart from patients with osa previously diagnosed elsewhere , preoperatively all patients on the waiting list for bs visited the ent outpatient department . information was gained using patient history , ent and general examination and a full overnight polysomnography ( psg ) . weight , length ( bmi ) and nc at the level of the cricothyroid membrane were measured . the following bmi grading system was implemented : obese ( bmi 3034.9 kg / m ) , severely obese ( bmi 3539.9 kg / m ) , morbidly obese ( bmi 4049.9 kg / m ) and super obese ( bmi > 50 the patients completed a questionnaire containing various questions concerning possible daily or nocturnal symptoms , intoxications , medication and medical history . besides patients with osa previously diagnosed elsewhere , all patients underwent a full night comprehensive sleep study using a digital embla recorder ( flaga medical devices , reykjavik , iceland ) . this records the sleep architecture ( derived from electroencephalogram , electrooculogram and submental electromyogram ) , respiration ( thoracic and abdominal measurement ) , movements of limbs , nasal airflow and the intensity of the snoring ( the last two measured by pressure sensor ) . the sensitivity and specificity of the clinical predictor variables for the presence versus absence of osa ( ahi > 5/h ) and moderate to severe osa ( ahi > 15/h ) were used to plot receiver operating characteristic ( roc ) curves . we performed a prospective , multidisciplinary , single - center observational study involving a consecutive series of patients being evaluated for bs in our clinic from june 2009 until june 2010 . all patients eligible for bs underwent a mandatory preoperative screening for osa in addition to our routine preoperative workup . apart from patients with osa previously diagnosed elsewhere , preoperatively all patients on the waiting list for bs visited the ent outpatient department . information was gained using patient history , ent and general examination and a full overnight polysomnography ( psg ) . weight , length ( bmi ) and nc at the level of the cricothyroid membrane were measured . the following bmi grading system was implemented : obese ( bmi 3034.9 kg / m ) , severely obese ( bmi 3539.9 kg / m ) , morbidly obese ( bmi 4049.9 kg / m ) and super obese ( bmi > 50 the patients completed a questionnaire containing various questions concerning possible daily or nocturnal symptoms , intoxications , medication and medical history . besides patients with osa previously diagnosed elsewhere , all patients underwent a full night comprehensive sleep study using a digital embla recorder ( flaga medical devices , reykjavik , iceland ) . this records the sleep architecture ( derived from electroencephalogram , electrooculogram and submental electromyogram ) , respiration ( thoracic and abdominal measurement ) , movements of limbs , nasal airflow and the intensity of the snoring ( the last two measured by pressure sensor ) . the relation between the ahi and patient characteristics was further evaluated employing stepwise linear regression and binomial logistic regression the sensitivity and specificity of the clinical predictor variables for the presence versus absence of osa ( ahi > 5/h ) and moderate to severe osa ( ahi > 15/h ) were used to plot receiver operating characteristic ( roc ) curves . of the remaining 279 patients , 214 ( 76.7% ) patient baseline characteristics are shown in table 1.table 1patient characteristics : clinical and polysomnographic parameterspatient characteristicsmeanmedianrangewomen214 ( 76.7%)men65 ( 23.3%)age ( years)45.1 10.646.0(1767)bmi ( kg / m)44.2 6.442.8(3366)neck circumference ( cm)42.6 4.842.0(3459.8)ess4.3 3.83.0(017)ahi ( per h)23.9 27.712.4(0142)ai11 21.41.6(0127)arousal index ( per h)7.5 8.45(054.7)mean sao2 ( % ) 93.8 3.394.7(7499)minimum sao2 ( % ) 80.8 10.783.0(5095)desaturation index ( di)16.3 23.45.3(0106) indicates standard deviationai apnea index ; ahi apnea hypopnea index ; bmi body mass index , ess epworth sleepiness scale ; osa obstructive sleep apnea ; sao2 oxygen saturation patient characteristics : clinical and polysomnographic parameters indicates standard deviation ai apnea index ; ahi apnea hypopnea index ; bmi body mass index , ess epworth sleepiness scale ; osa obstructive sleep apnea ; sao2 oxygen saturation an unpaired t test was conducted to compare baseline characteristics in men and women . there was a significant difference in the ahi ( p < 0.0003 ) , ai ( p < 0.0004 ) , arousal index ( p < 0.006 ) , di ( p < 0.0003 ) , mean o2 saturation ( p = 0.001 ) , minimum o2 saturation ( p < 0.0007 ) and nc ( p < 0.0003 ) between men and women . application of a non - parametric test provided no new insights.table 2patient characteristics subdivided for women and menpatient characteristicsmenwomenage ( years)48.5 9.344.0 10.8bmi ( kg / m)44.3 7.144.2 6.2neck circumference ( cm)48.0 3.941.2 4.0ess5.0 4.24.2 3.6ahi ( h)45.9 29.917.3 23.3ai ( h)25.7 27.17.0 17.5arousal index ( h)10.6 8.96.7 8.1mean sao2 ( % ) 92.3 3.094.1 3.4minimum sao2 ( % ) 74.6 11.382.5 9.9desaturation index ( di)32.2 26.811.8 20.3 indicates standard deviationai apnea index ; ahi apnea hypopnea index ; bmi body mass index , ess epworth sleepiness scale ; osa obstructive sleep apnea ; sao2 oxygen saturation patient characteristics subdivided for women and men indicates standard deviation ai apnea index ; ahi apnea hypopnea index ; bmi body mass index , ess epworth sleepiness scale ; osa obstructive sleep apnea ; sao2 oxygen saturation three ( 1.1% ) of the patients were obese , 75 ( 26.9% ) severely obese , 149 ( 53.6% ) morbidly obese , 51 ( 18.3% ) super obese . osa stratified by bmi and the severity of osa by bmi are depicted in table 3 and fig . a total of 112 ( 40.1% ) patients underwent or are on the waiting list for sagb ( average bmi 41 sd 4 kg / m ) , 155 ( 55.6% ) laparoscopic gastric bypass ( average bmi 46.1 sd 6.7 kg / m ) and 12 ( 4.3% ) gastric sleeve surgery ( average bmi 49.4 sd 8.5 patients had been previously diagnosed with osas ( ahi : 42.5 sd 27.2 per h ) in our hospital ( 12 pts ) or elsewhere ( 25 pts ) before being placed on the waiting list for bs . based on the psg results , 195 ( 69.9% ) patients were diagnosed with osa , specifically 67 ( 34.7% ) with mild osa , 48 ( 24.9% ) with moderate osa and 78 ( 40.4% ) with severe osa . 69.2% of the patients diagnosed with osa were female and 30.7% male.table 3number of patients with osa stratified by bmiosa stratified by bmiosano osatotal no.osa ( % ) obese ( 3034.9 kg / m)12333.3severely obese ( 3539.9 kg / m)49267565.3morbidly obese ( 4049.9 kg / m)1034614969.1super obese ( > 50 kg / m)41105180.4bmi body mass index , osa obstructive sleep apneafig . 1severity osa stratified by bmi number of patients with osa stratified by bmi bmi body mass index , osa obstructive sleep apnea severity osa stratified by bmi stepwise linear regression was performed with ahi as the dependent variable . the results of the roc curves were disappointing ; of all three clinical parameters , no cutoff values were found to have both a sensible sensitivity ( > 0.8 ) and a useful specificity ( > 0.9 ) . the neck circumference was found to be the most accurate predictor of the presence of osa when the ahi as greater than 5/h ( fig . 2roc curve comparing sensitivity and specificity of neck circumference ( nc ) , body mass index ( bmi ) and epworth sleepiness scale ( ess ) of an ahi > 5/h . the mean area under the curve ( auc ) for nc , bmi and ess were 0.69 0.4 ( 95% ci 0.620.77 ) , 0.61 0.4 ( 95% ci 0.530.69 ) , 0.54 0.4 ( 95% ci 0.450.62 ) , respectively . no cutoff values were found to have both a sensible sensitivity ( > 0.8 ) and a useful specificity ( > 0.9)fig . 3roc curve comparing sensitivity and specificity of neck circumference ( nc ) , body mass index ( bmi ) and epworth sleepiness scale ( ess ) of an ahi > 15/h . the mean area under the curve ( auc ) for nc , bmi and ess were 0.76 0.4 ( 95% ci 0.690.82 ) , 0.62 0.4 ( 95% ci 0.540.69 ) and 0.59 0.4 ( 95% ci 0.510.67 ) , respectively . no cutoff values were found to have both a sensible sensitivity ( > 0.8 ) and a useful specificity ( > 0.9 ) roc curve comparing sensitivity and specificity of neck circumference ( nc ) , body mass index ( bmi ) and epworth sleepiness scale ( ess ) of an ahi > 5/h . the mean area under the curve ( auc ) for nc , bmi and ess were 0.69 0.4 ( 95% ci 0.620.77 ) , 0.61 0.4 ( 95% ci 0.530.69 ) , 0.54 0.4 ( 95% ci 0.450.62 ) , respectively . no cutoff values were found to have both a sensible sensitivity ( > 0.8 ) and a useful specificity ( > 0.9 ) roc curve comparing sensitivity and specificity of neck circumference ( nc ) , body mass index ( bmi ) and epworth sleepiness scale ( ess ) of an ahi > 15/h . the mean area under the curve ( auc ) for nc , bmi and ess were 0.76 0.4 ( 95% ci 0.690.82 ) , 0.62 0.4 ( 95% ci 0.540.69 ) and 0.59 0.4 ( 95% ci 0.510.67 ) , respectively . no cutoff values were found to have both a sensible sensitivity ( > 0.8 ) and a useful specificity ( > 0.9 ) in this series of consecutive patients undergoing bs , we found a 69.9% prevalence of osa . more than 40% of these patients were diagnosed with severe osa . of the 195 patients diagnosed with osa , 69.2% were female : a 1:2.3 ratio ( : ) , which is opposite to the typical osa male female ratio of 2:1 ( : ) . irrespective of history or clinical findings , all patients being evaluated for bs underwent a polysomnography , unless performed previously . using synonyms for : bariatric surgery , obstructive sleep apnea and polysomnography , an online systematic search was performed of the medline and embase database on the 4th april 2011 . in each study , all patients being evaluated for bariatric surgery underwent a polysomnography as part of routine screening for osa regardless of symptoms and without use of screening tools such as the ess . four articles were omitted from our overview ( see table 4 ) , owing to various applied inclusion criteria ( a bmi 40 kg / m or asian race ) and articles , which did not apply the aasm osa guidelines [ 1215].table 4outcomes of similar studies , in which all patients being evaluated for bariatric surgery underwent a polysomnography , irrespective of history or clinical findingsreferencetotal nosa nprevalence osa ( % ) severe osa ( % ) mean ahi ( h)mean bmi ( kg / m)frey et al . 17612671.648.028.042.0all1268100479.233.125.748.8ahi apnea hypopnea index , bmi body mass index , osa obstructive sleep apneaonly studies presenting percentage severe osa dataonly studies presenting mean ahi dataonly studies presenting mean bmi data outcomes of similar studies , in which all patients being evaluated for bariatric surgery underwent a polysomnography , irrespective of history or clinical findings ahi apnea hypopnea index , bmi body mass index , osa obstructive sleep apnea only studies presenting percentage severe osa data only studies presenting mean ahi data only studies presenting mean bmi data to our knowledge , following sareli et al . we have two main study limitations , the first being that osa was an inclusion criterion for bariatric surgery , in accordance with the ifso guidelines : a potential bias , which could result in an overestimation of the prevalence of osa in our bariatric surgery population . a mere 13.3% ( 37 ) patients were diagnosed with osa before being placed on the waiting list for bs ; 62.2% of these patients had a bmi greater than 40 kg / m and were therefore eligible for bs regardless of the presence of osa . as this group was minimal , we chose to include these patients in the series so as to avoid underestimating the prevalence of osa in our bariatric surgery population . the second limitation of the study is absent data , in particular from patients who had previously been diagnosed with osa elsewhere . ess data were available for 78.5% of the patients , and nc measurements for 82.4% . , in the bariatric population , ess can not independently reliably predict the presence of osa . our data support this observation ; we found ess not to be significantly related to the presence of osa in patients undergoing bs . hence , ess is not a reliable predictor of osa in this patient population , despite often being used in bs centers as a screening tool . the various regression models and the roc curves give discrepant results , mainly due to the non - normal distribution of the data and data values of zero or close to zero . the poor statistical power and discrepancy of the results we contend that all three clinical parameters are inadequate predictors of osa and that psg is an essential component of the preoperative workup of patients undergoing bs . despite the high test probability of moderate to severe osa in obese patients , the high costs and patient burden of psgs as well as the increasing prevalence of obesity and bs , the task force of the aasm does not advise the use of unattended portable monitoring ( pm ) for general screening , as there is yet insufficient evidence to guide the use of pm . the authors suggest that osa is grossly underdiagnosed in the bariatric population and concludes that clinical evaluation including the ess is inadequate to identify the true prevalence of osa . a mere 13.3% of the patients were diagnosed with osa before being placed on the waiting list for bs ; 37 ( 13.3% ) patients had an ahi > 60/h and only 11 patients were aware of their extreme osa . the finding of osa may in fact influence the indication for bs being a bmi > 40 kg / m in patients without comorbidity , or bmi > 35 kg / m with comorbidity . therefore , in otherwise healthy patients with a bmi between 35 kg / m and 40 kg / m , the finding of osa widens the indication for bs . patients with osa have been shown to have increased preoperative risk and specific perioperative measures have to been taken [ 4 , 23 ] . increased neck circumference , bmi or ess can not reliably predict the presence of osa .

much of the extant research on weight loss and weight management in adults has focused on the factors associated with adherence during , or after , programs which are typically staged in academic or clinical settings , using randomized controlled designs [ 1 , 2 ] . despite the controlled settings and the high level of staff training in these programs , mean dropout from such interventions remains at 2040% [ 3 , 4 ] despite incentives to complete regular assessments and frequent follow - up contact from staff members . therefore , the accuracy of dropout rates reported by such interventions may be misleading , and these studies may not help researchers understand the process of dropout in community - based settings . to evaluate and understand attrition in community - based settings , grave et al . examined the predictors of dropout from the quovadis observational study of community - based weight loss programs in italy . grave and colleagues addressed an important research question : are all drop - outs treatment failures ? findings showed that , despite significantly lower mean percentage weight loss than program completers at 36-month follow - up , dropouts achieved encouraging outcomes . for example , dropouts reported a higher mean percentage weight loss of 9.6% and 6.5% , respectively , than continuers ( 5.2% ) . other studies have shown that program participants who disengage may still join other behaviors or programs . tracked elderly participants in a community center for three years and noticed that 21% of participants tried out or transferred between programs within the center , while others concurrently participated in exercise programs outside of the center . in addition , stiggelbout and colleagues found that 31% of older adults who dropped out of a diverse range of organized exercise programs simply switched to another type of exercise . thus , people who drop out from a program at one stage of their life may return to the same program after more than a year away , or after addressing personal or health problems , or they may express intentions to return in the future . research examining other health behaviors , such as smoking cessation , has shown that it can take a sequence of repeated , unsuccessful attempts before long - term healthy behaviors are maintained . for example , related research has found that , following short - term commercial weight management interventions ( of six months or less ) , some participants report strong intentions in maintaining newly learned behaviors , increased self - efficacy in achieving a future weight target , and positive changes in habitual physical activity at one - year follow - up . it is possible , therefore , that unsuccessful attempts can be part of the path to a healthy lifestyle because they contain some useful lessons in health education ( i.e. , exercise technique and eating preferences ) , stress management , or self - regulation . as weight management programs increasingly become translated into community settings , a certain degree of dropout can be expected as participants manage multiple time commitments and as program staff members manage multiple job responsibilities . translation into community settings will bring along with it a greater need for accountability and compliance . private fitness centers , health departments , and public and private insurers will want to know if these programs are sustainable or profitable . for example , programs established by health insurance agencies tend to have compliance guidelines to which participants must adhere to remain eligible to receive subsidized services . fitness centers and community clinics benefit from these subsidized programs because they earn money by providing professional services to the policy holder . the main reason for stringent compliance guidelines is that providing such programs can be costly , particularly for insurance - funded programs , which aim to address various health risks that impact chronic disease and curb insurance expenses associated with chronic disease . thus , a dropout from an insurance - sponsored program may either voluntarily quit the program or be administratively removed due to noncompliance . little is known about the reasons for , and consequences of , dropout from insurance - sponsored health promotion programs . understanding more about these patterns of behavior may help insurance agencies maximize the benefit of the programs they deliver in communities while keeping costs at a reasonable level . finally , if positive consequences of dropout are found , these data will help support the continued spending of public and private insurance dollars on health promotion programs . thus , the purpose of this study was to examine reasons for and consequences associated with dropout ( or removal ) from a state - wide , insurance - funded weight management program . the research questions included the following : ( 1 ) what are the reasons people drop out from an insurance - funded weight management program ? ( 2 ) what are the positive and negative consequences associated with dropout ? and ( 3 ) what are the differences in consequences and future health behavior intentions for those who drop out early , middle , or late into the program ? west virginia is a small , appalachian state ranked among the least healthy states in the us based on adult prevalence of obesity ( 33% versus 27.5% us median ) , diabetes ( 12% versus 8.7% us median ) , and heart disease ( 6% versus 4% us median ) , all of which rank in the top 10 in the united states . the estimated direct medical costs associated with obesity in west virginia ( wv ) increased by $ 51 million usd between 2001 and 2009 , which is a meaningful increase for a state with a population of just under two million . these negative health trends , and the economic consequences of them , have driven the need for programs which can help curtail the growth of obesity and promote healthy lifestyles . to combat these trends , the state 's largest public insurer , the west virginia public employees insurance agency ( peia ) , has established a comprehensive weight management program to provide policy holders with opportunities to learn how to be physically active and eat a healthful diet . the program is a two - year benefit offered by peia for people with a bmi of 25 kg / m or higher or an eligible waist circumference ( i.e. , 35 inches or greater for women and 40 inches or greater for men ) . the program , which requires a $ 20 monthly copay , includes access to a fitness facility in the local community and services provided by a personal trainer , dietitian , and exercise physiologist . previous single - site and large - scale evaluations of this program 's effectiveness have shown low reach , moderate to high effectiveness in short - term and long - term weight loss , and strong potential for sustainability [ 10 , 1315 ] . the requirements for ongoing participation ( for up to two years ) are maintaining at least eight monthly visits to the fitness facility , attending appointments with professionals , and showing improvements in weight or other measured health parameters . for example , in the first six months , participants receive 120 minutes of personal training per month , three 3060-minute consultations with a dietician , and two 60-minute fitness assessments by an exercise physiologist . however , there is no standard model of service delivery as each professional is allowed the freedom to deliver fitness and dietary services within their own scope of practice . facility staff are responsible for entering objective data on participant attendance and body measurements ( i.e. , waist , weight , body fat , and blood pressure ) on a monthly basis into a secure , web - based platform used for evaluation and billing . those participants that do not meet the eight visits per month minimum attendance criteria for two or more consecutive months are removed from the program . therefore , participants may be administratively removed for noncompliance with these requirements or other issues may arise that cause the termination of their program ( e.g. , medical issues and change of insurance ) . in addition , participants may voluntarily choose to drop out from the program for personal reasons . when they are removed or choose to drop out from the program , they receive a letter informing them of their change in status . the study used a mixed methods design including quantitative data on objective outcomes ( i.e. , length in the program , % weight loss ) as well as self - reported reasons and consequences in both quantitative and qualitative forms collected from the program evaluation survey . all program participants over the age of 18 who exited the program during 2014 ( n = 973 ) received the program evaluation survey containing questions related to their participation in the program . the survey was sent first via email through a unique link to the participants who provided their email addresses at the start of the program . the electronic version of the survey was sent twice via survey monkey with a one - week interval between the first attempt and the reminder . if they did not respond to the email survey , a hard copy of the survey was mailed , which is the first step for participants without an email address . after a week without response from the paper - based survey , a reminder post card was sent to the participant . using this dillman modified recruitment method , 400 of 973 participants responded to the survey resulting in a 41% response rate . the program evaluation survey sent to the participants included questions regarding their reasons for dropping out of the program , their satisfaction with different services of the program , and their future intentions . first , all participants were asked to choose between the reasons for leaving the program from why have you chosen to leave the weight management program at this time ? next , two sets of questions assessed participants ' satisfaction with different services of the program . these questions asked participants to rate their exercise facility , personal training , and dietary services using a scaled response from 1 ( not at all satisfied ) to 4 ( completely satisfied ) . the final question analyzed in this study inquired about the participants ' intentions of carrying out the following health - related behaviors in the future , in a categorical format ( yes , no , or i do n't know ) : ( a ) continue as a private fitness member at the facility ; ( b ) exercise for 30 minutes per day , five days per week ; ( c ) exercise for 60 minutes per day , five days per week ; ( d ) seek out a registered dietician ; ( e ) enroll in a group exercise program ; or ( f ) enroll in a group nutrition program . after inquiring about their future intentions , two demographic questions ( i.e. , gender and date of birth ) preceded a final question where they could express other thoughts about the program . because of the wording of this last question , many of its responses were related to satisfaction or dissatisfaction with the program ( i.e. , do you have anything else you would like to add about your level of satisfaction so far with the weight management program and the different services you have received ? ) . in addition to satisfaction / dissatisfaction , participants also tended to express the positive and negative consequences related to their participation in the program and thus these data were included in the subsequent qualitative analysis . then , a frequency analysis of their overall intentions for future health - related behaviors was performed . finally , the two open - ended questions were analyzed through content analysis to address the research questions . for the analysis of the open - ended data , two researchers independently analyzed and open - coded different subsamples of the data using descriptive coding . subsequently , they met , contrasted their codes , and agreed on pattern codes ( i.e. , categories ) that provided more meaningful and parsimonious units of analysis . these categories and their codes were organized into a coding book that served as a source for the later provisional coding of the entire sample , allowing a combination of inductive and deductive coding . the software program nvivo 10 was utilized for this step , which allowed for a check of the interrater reliability . a near identical process was used to analyze the consequences and dropout open - ended items . however , data from the items were analyzed independently , that is , by separate sets of researchers and using separate coding books . among all codes across both analyses , these values are considered moderate to strong agreement values . in any instance where the two raters disagreed , a third rater was engaged so that consensus could be reached in each coding moment before finalizing the code . for the final research question , respondents were classified into three groups depending on the timing of their program exit : ( 1 ) early drops ( participation of 6 months or less ) ; ( 2 ) mid drops ( between 7 and 12 months of participation ) ; and ( 3 ) late drops ( between 13 and 24 months of participation ) . two - way chi - square analysis was used to explore the future intention data as well as the patterns of consequences experienced across the three groups . these bivariate analyses were used to determine if there was any relationship between the length of time in the program prior to exit and their coded consequences and stated intentions . effect size estimates for all analyses are reported as the contingency coefficient , and values > .3 can be considered moderate . the respondents were aged between 24 and 70 years , with a mean age of 48.6 years ( sd = 11.1 ) . the mean length of participation in the program was 9.8 months ( sd = 5.6 ) , with 33.7% exiting the program within the first six months , 35.7% exiting between 7 and 12 months , and 30.6% completing 1324 months . among the male participants , mean weight at the beginning of the program was 262.2 lbs ( sd = 57.3 ) , while women had a mean weight of 210 lbs ( sd = 49.6 ) . men 's mean waist circumference at the beginning of the program was 46.9 inches ( sd = 6.9 ) and women 's was 42.4 inches ( sd = 9.8 ) . on a 6-point likert scale , with one meaning not at all satisfied and six meaning completely satisfied , participants had a mean satisfaction of 4.4 ( sd = 1.5 ) . at the time of survey completion , the average percent body weight loss for the respondents was 2.27% ( sd = 4.9 ) . additionally , upon program exit , 21% of respondents lost at least 5% of their baseline body weight during the course of the program , while 26.7% had gained weight . to check for demographic differences between the survey respondents and nonrespondents , a series of independent samples t - tests analyses indicated that mean bmi , waist circumference , body fat percentage , and length of participation in the program of participants who dropped out from the program and responded the survey were not significantly different from the participants who dropped out and did not respond the survey ( p > .05 ) . however , the nonrespondents ' age ( m = 44.6 , sd = 11.6 ) was significantly lower than the respondents ' ( m = 48.6 , sd = 11.1 ) , t(971 ) = 5.33 , p < .001 . despite this small difference in age , survey respondents and nonrespondents appear to report similar profiles related to their weight and program participation , thus reducing the concern for selection bias in the subsample of respondents . among the 400 survey respondents , 375 participants responded to the question about their reasons to drop out from the program , while 272 responded to the final open - ended question . reasons for dropout were allowed to emerge from either of these two questions . in total , reasons to drop out from the program were coded 420 times , with some participants indicating more than one reason and some leaving the question blank . seven major themes regarding the participants ' reasons to drop out from the program emerged from the survey responses : competing priority ( 36.9% ) , medical ( 22.8% ) , negative experience ( 13.1% ) , programmatic issues ( 12.4% ) , administrative drop ( 7.4% ) , insurance coverage change ( 6.4% ) , and completed attempt ( 1.0% ) . the competing priority theme was the largest emergent theme with 155 occurrences ( 36.9% ) and included the subthemes of time ( 18.3% ) , caregiving ( 5.7% ) , distance ( 5.2% ) , lack of motivation ( 4.1% ) , personal reasons ( 2.1% ) , and grief ( 1.4% ) . time was a frequently coded subtheme , including statements of limited or lack of time to comply with the program requirements ( e.g. , not enough time at the moment ) . caregiving meant having to take care of older family members ( e.g. , family commitments due to sick family member ) or children ( e.g. , my child playing sports kept me from coming at the scheduled times of the classes that i enjoyed taking ) . distance included the gym being far from home or work and moving to another place . lack of motivation included need for external motivation , not being able to meet requirements , and just not wanting to participate anymore ( e.g. , i was no longer invested in the program and wanted to do something else ) . personal reasons contained personal or family reasons that were not specified ( e.g. , personal family difficulties were drawing me away from working out regularly ) . finally , grief regarded difficulties in adherence due to grieving the death of a close person ( e.g. , death of my spouse , so i haven't adhered as well as i should have ) . the medical reasons theme included other physical limitation ( 20.9% ) , injury at facility ( 1.7% ) , and weight loss surgery ( 0.2% ) . other physical limitation was the most frequently coded subtheme and included a variety of health issues other than injury at facility and weight loss surgery ( e.g. , i have been diagnosed with blood clots in the lungs and can not work out for several months until i heal ) . getting injured at the facility was reported by seven respondents , which prevented further exercise participation ( e.g. , weight loss surgery ( e.g. , bariatric surgery ) was indicated by one participant . the theme negative experience ( 13.1% ) included the subthemes of dissatisfaction ( 7.4% ) , negative feedback ( 2.4% ) , price ( 2.1% ) , and lack of support ( 1.2% ) . dissatisfaction included disappointment with results , training , and services in general ( e.g. , negative feedback included negative feedback from measurements ( 1.0% ) and pain ( 1.4% ) . the negative feedback made me feel like a failure instead of acknowledging my improvement , so i quit going , while the second involved any kind of pain related to the participation of the program ( e.g. , price comprised unwillingness or impossibility of paying for direct or indirect costs of the program ( e.g. , cost of daycare while i participate in the program ) . finally , lack of support included family and social support . lack of family support ( 1.0% ) was coded when family members were unsupportive of the participant 's compliance with the program ( e.g. , conflict with my spouse , he felt i was spending too much time at the gym ) , while lack of social support ( 0.2% ) contained only one respondent who wished to have a buddy exerciser : programmatic issues was divided into the subthemes facility problem ( 11.7% ) and gym culture ( 0.7% ) . the subtheme facility problem was further divided into professionals ( 6.2% ) , facility closure ( 4.5% ) , and facility hours ( 1.0% ) . the professionals subtheme regarded problems with the staff delivering the services offered by the program , such as trainer 's or dietitian 's ineffectiveness , limited availability , and lack of support ( e.g. , did n't feel i had the support needed from staff ) . facility closure represented when the gym closed or stopped offering the program and this situation triggered the participants ' dropout ( e.g. , the facility closed and i chose not to select an alternate facility ) . finally , facility hours included dissatisfaction with the facility hours ( e.g. , hours at the facility did not coincide with my work hours ) . gym culture included expressions of uncomfortable feelings due to the gym environment ( e.g. , [ i ] did not like the gym environment ) . the themes completed attempt , insurance coverage , and administrative drop ( 14.8% total response among the three categories ) were less meaningful since they involved administrative issues . insurance coverage contained participants who left for another job or retired , thus losing the insurance benefits , and administrative drop encompassed respondents who were removed by the program administration due to noncompliance . completed attempt included people who believed that they had already completed the program and ceased going to the facility . among the 400 survey respondents , 272 responded to the final open - ended question about their experiences in the program . two major themes emerged from these 272 responses : positive and negative consequences from participation in the program . positive consequences were coded 150 times ( 88.2% ) and negative consequences 20 times ( 11.8% ) . positive consequences included three subthemes : psychological ( 52.7% ) , behavioral ( 22.0% ) , and physical ( 25.3% ) . the negative consequences were composed of two subthemes : psychological ( 80% ) and physical ( 20% ) consequences . the first was attitude change ( 41.3% ) , which included the codes positive intentions ( 32% ) , perception of success ( 6.7% ) , and appreciate exercise ( 2.7% ) . the most common subtheme within the attitude change was positive intentions , which comprised intentions such as continuing to exercise , joining another weight management or exercise program , and seeking nutrition guidance ( e.g. , i will continue to work at home on my own equipment ) . the second most common was perception of success , including subjective comments regarding achieving success ( e.g. , i have worked very hard and have had great success ) . the last subtheme within the attitude change was appreciate exercise , which included liking exercise and realizing positives of exercising ( e.g. , the program made me realize how good i feel when i do work out ) . another subtheme included in the positive psychological consequences was learning ( 8% ) , which contained several learning experiences , such as learning about exercise , eating , self - motivation , and weight loss ( e.g. , i learned a great deal from both the trainer and dietitian i have continued to utilize info from both ) . finally , the last subtheme within the positive psychological consequences theme was feelings ( 3.3% ) , which contained general positive feelings such as being proud ( e.g. , i 'm very proud of myself for what i have accomplished with some help from the program ) . the second major theme within the positive consequences was behavioral ( 22% ) , which comprised maintenance ( 14.7% ) and changes ( 7.3% ) . behavioral maintenance included continuing to exercise and eating healthy ( e.g. , as of right now i am once again working on my fitness and diet on my own ) . behavioral changes referred to changes in exercise and eating habits without referring to the maintenance of these changes ( e.g. , i have changed my eating and exercise habits since i began [ the ] program ) . the final major subtheme within the positive consequences was physical ( 25.3% ) , which included the subthemes weight loss ( 15.3% ) , health improvement ( 5.3% ) , and fitness improvement ( 4.7% ) . weight loss subtheme was subcoded into lost weight ( 13.3% ) and continue to lose weight ( 2% ) , which differed regarding when the weight loss happened ( during the program or after dropping out of it ) . i lost 45 pounds in the program and one of continue to lose weight is since leaving the program i have lost about 15 pounds . health improvement regarded improvements in health - related markers ( e.g. , hemoglobin a1c ) , stress , energy , and pain ( e.g. , while working through this program my a1c level dropped from 7 to 5.7 ) . fitness improvement was related to enhancement in physical capability such as strength , flexibility , stamina , and resistance ( e.g. , 6 months ago it took me 30 minutes for a mile . now i can do 3 miles in 31 minutes ) . among the negative consequences ( n = 20 ) , the psychological consequences was the most frequent coded subtheme and was further subcategorized into attitudes ( 40% ) , feelings ( 35% ) , and lack of learning ( 5% ) . attitudes included dissatisfaction with weight loss ( 15% ) and lower motivation for being dropped ( 25% ) . the first comprised expressions that weight loss was not enough to satisfy the participant ( e.g. , i only lost 1015 lbs and after i felt that was n't enough ) . the latter regarded the participant 's decrease in motivation as a consequence of being removed from the program ( e.g. , i was kicked out of the program twice , it has been very discouraging ) . still within the negative psychological consequences , the code feelings included negative feelings such as guilt , disappointment , and shame ( e.g. , i feel guilty for not continuing exercising as faithfully on my on ) . finally , the last negative psychological consequence was lack of learning and it was coded once : the physical negative consequences were comprised of only one subtheme : weight gain ( 20% ) . it included statements of weight gain during the program or after leaving it ( e.g. , i actually gained more weight after enrolling this program ) . regarding the respondents ' ( n = 400 ) intentions for future health - related behaviors , 64% intended to exercise 30 minutes per day , five days per week , 16% intended to exercise 60 minutes per day , five days per week , 11% intended to join a group exercise program , and 21% intended to continue as a private member of the fitness facility that they were attending during the program . additionally , 7% intended to seek the help of a registered dietitian to make nutrition changes and 13% intended to enroll in a group nutrition program . when comparing these intentions among respondents who dropped out of the program early ( 6 months or less of participation ) , in the middle ( between 7 and 12 months ) , or later ( between months 13 and 24 ) chi - square analysis showed that respondents who stayed for seven or more months in the program were more likely to intend to continue exercising five days a week for at least 30 minutes ( 68.6% combined for the mid and later groups ) compared to early dropouts ( 55% ) ; however this effect was not significant , (4 , n = 382 ) = 7.09 , p = .131 , cc = 0.14 . this trend was not found for intentions to exercise five days a week for 60 minutes , which overall remained low in the overall sample ( 16.1% ) . however , early dropouts ( 8.2% ) were significantly more likely to intend to seek a dietitian help to make weight changes than middle dropouts ( 5.8% ) , (4 , n = 371 ) = 12.97 , p = .011 , cc = 0.18 . the pattern of intentions to seek out group exercise classes or group nutrition services did not differ in a meaningful way across the three groups . in general , the final set of chi - square analyses targeted the relationship between stage of dropout ( early , middle , and late ) and consequences expressed . first , two analyses were conducted to see if the percentage of respondents reporting positive or negative consequences differed across the three groups . positive consequences experienced in the program did show an increasing trend across the three groups , from 30.4% to 31.3% to 38.8% , but this relationship was not significant (2 , n = 268 ) = 1.59 , p = .451 , cc = 0.07 . overall , one - third of those who answered this open - ended question reported a positive consequence of program participation . those participants who dropped out after 12 months ( 32.5% ) were nearly twice as likely to report a positive psychological outcome compared to those who dropped out early ( 17.4% ) ; however , this small effect failed to reach significance , (2 , n = 268 ) = 5.30 , p = .071 , cc = 0.14 . a different trend was revealed for the negative consequences , with those who dropped out in the middle group reporting a negative outcome 35 times as often ( 12.5% ) as the early ( 2.2% ) or late ( 3.8% ) dropouts , (2 , n = 268 ) = 9.723 , p = .008 , cc = 0.19 . this pattern held true for all of the subthemes of negative consequences , with the exception of dissatisfaction with weight loss . the results of this mixed methods study support the idea that there are both positive and negative consequences experienced in a sample of weight management program dropouts . overall , 1 in 5 respondents lost a clinically significant amount of weight during the program ( > 5% of baseline body weight ) , regardless of the time of program exit , and 1 in 3 experienced a positive consequence , while only 6% expressed a negative outcome of participation . additionally , nearly 90% of all of the consequences that emerged from the data were positive . thus , some of the dropouts appear to have experienced substantial success in terms of outcomes or lessons learned through participation , and five times as many respondents reported a positive consequence compared to those who reported a negative consequence of participation . skill acquisition were major themes , including positive health intentions , perceived success , learning skills , and new appreciation of exercise . these findings support previous theories of health behavior change including the theory of planned behavior 's connection between intentions , self - efficacy , and past behavior as well as similar work on barriers to the self - management of diabetes . specifically , based on these models , if programs can help individuals increase their health intentions , self - efficacy , and specific behavioral skills , regardless if they dropout or not , then they will increase the probability of future health behavior change among participants . approximately two - thirds of the respondents indicated they intend to continue exercising five days a week for 30 minutes , an amount of physical activity that would meet guidelines for general health . some respondents may have received as much out of the program as they needed , and they felt ready to move onto a self - managed program . others simply learned some new skills and experienced a change in attitude toward eating or physical activity . since intentions are widely regarded as being the closest psychological predictor of future behavior , it is possible that if these intentions are sustained , a subsequent attempt at behavior change will stick [ 6 , 23 ] . thus , the failure of dropout may have resulted in a valuable learning experience where participants identified activities they enjoyed and learned new meals to prepare for themselves and their families . these findings support previous research that also found positive intentions among former program participants [ 9 , 24 ] and research that has identified a subset of dropouts who may have experienced substantial success . additionally , it is possible that a shift in identity occurred moving some former participants towards a newer , active identity . using self - determination theory as another frame of reference , if participants of health behavior interventions can develop more intrinsic forms of motivation , by identifying activities they enjoy and that confirm their sense of self , their future efforts may be sustained over a longer period of time . not all participants reported positive outcomes , however , and approximately 1 in 4 of the dropouts gained weight during the program . it is important to note that most of the negative consequences reported were also psychological in nature ( including lower motivation and dissatisfaction with weight loss ) , and there were no strong themes for things such as injury or worsened disability . integrating the objective and subjective data on consequences allows an observation that approximately 20% ( percent of sample with clinically significant weight loss ) to 33% ( percent of respondents who self - reported a positive consequence ) of dropouts of this community - based weight management program experienced a meaningful positive consequence resulting from program participation . future research may consider capturing some of these possible benefits along with reporting the outcomes of those who complete intervention programs . the average respondent dropped out approximately 10 months into this two - year - long program , and negative consequences were reported at a higher rate among middle dropouts compared to early or late dropouts . this particular phase of the program ( between 7 and 12 months of participation ) appears to be the highest risk of dropout and may warrant additional attention . competing priorities ( i.e. , lack of time ) was the most widely cited reason for program exit ( noted by 37% of respondents ) , suggesting that the working adults in the program often struggle to manage the multiple commitments of work , family , and health . this evidence supports the idea that some people need more support than others based of the timing of their attempt and the strength of their motivation at program entry . these data also support the finding from grave et al . that some participants may need help managing logistics to increase long - term adherence . in this particular program , there is also a significant change to the services provided to participants during this time frame . in particular , their access to personal training drops from 120 minutes per month to 60 minutes per month , and they are not scheduled for another fitness or dietary reassessment until month 13 . this reduction in professional services and the lack of accountability that comes along with those reductions may cause some participants to lose motivation and commitment . it is also possible that adhering to the facility - based program that requires at least eight in - person visits per month is not sustainable for everyone over two years . anecdotally , many participants report traveling substantial distances to reach the facility , particularly in rural parts of the state . adding further support in the form of behavioral services may be needed considering that many program enrollees enter with various comorbidities or physical limitations . this issue highlights a key difference in populations who are enrolled in community - based programs compared to clinical trials ; prior to group assignment , many participants with comorbidities are often excluded from trials . adding behavioral services ( in various forms and at various stages of the program ) could help participants learn new skills and stay engaged long enough to maintain the multiple health behaviors they have initiated . the final discussion point worth noting relates to the differences observed when working with high risk adults and translating programs into communities . though this program is a fitness facility based program , the weight management program participants are often dissimilar from typical gym members . they are typically obese , possess low fitness at baseline , and present with additional health risk factors including hypertension , hyperlipidemia , and/or diabetes . as insurance companies try to build connections with the fitness industry , it is imperative that fitness staff are trained to safely and accurately assess , prescribe modified exercise to , and train high risk clients . a mismatch between the needs of these participants and the staff training may be contributing to weak outcomes , injuries , and program noncompliance . these issues were noted by some participants who dropped out , thus highlighting the difficulty of ensuring high treatment fidelity in a large , community - based program . the results of this study should be interpreted given the following limitations . despite the analyses indicating the responders are not that different from the nonresponders , there is still the potential for self - selection bias in the data . with a 40% response rate , it is possible that survey responders were more likely to report positive consequences than those who chose not to respond . next , in our analyses of those who were dropped from the program , we did not differentiate between those who left the program voluntarily and those who were dropped due to noncompliance . all participants self - enroll in the program and decide on their own to attend the facility or to stop coming . some participants are conscientious enough to call program staff , and the others simply stop going to the facility and then are dropped within 2 - 3 months for noncompliance . finally , the study is limited because there is no follow - up data indicating if the positive intentions expressed at program exit led to future observable behaviors . future studies will explore these patterns over time and should address the relative importance of the psychosocial and environmental factors and barriers that emerged among participants . there is strength in the study 's design and data given the unique mix of quantitative , objectively observed changes in body weight and attendance along with the qualitative reasons , and consequences expressed by several hundred respondents . the loss of internal validity when programs are disseminated into multiple community settings is unavoidable . however , we can not expect to treat or reverse the obesity epidemic in the us with the limited reach of randomized controlled trials in clinical settings . we must embrace the messiness of community - based programs , especially those funded by insurance agencies , because they are sustainable and possess strong external validity . findings of the current study can be used to inform and improve future community - based research and multisite intervention programs with similar rural populations in the united states that experience the greatest health disparities in chronic disease .

the majority of weight management research is based on data from randomized controlled studies conducted in clinical settings . as these findings are translated into community - based settings , additional research is needed to understand patterns of lifestyle change and dropout . the purpose of this study was to examine reasons for and consequences associated with dropout ( or removal ) from an insurance - funded weight management program . using a mixed methods approach with objectively measured changes in body weight and attendance along with quantitative and qualitative survey data , patterns of intention and behavior change were explored . the results from a sample of 400 respondents support the idea that there are both positive and negative consequences of program participation . overall , 1 in 5 respondents lost a clinically significant amount of weight during the program ( > 5% of baseline body weight ) and 1 in 3 experienced a positive consequence , while only 6% expressed a negative outcome of participation . additionally , nearly 90% of all of the consequences that emerged from the data were positive . attitude change was a major theme , including positive health intentions , perceived success , learning skills , and new appreciation of exercise .

1. Introduction 2. Method 3. Results 4. Discussion

much of the extant research on weight loss and weight management in adults has focused on the factors associated with adherence during , or after , programs which are typically staged in academic or clinical settings , using randomized controlled designs [ 1 , 2 ] . therefore , the accuracy of dropout rates reported by such interventions may be misleading , and these studies may not help researchers understand the process of dropout in community - based settings . to evaluate and understand attrition in community - based settings , grave et al . examined the predictors of dropout from the quovadis observational study of community - based weight loss programs in italy . tracked elderly participants in a community center for three years and noticed that 21% of participants tried out or transferred between programs within the center , while others concurrently participated in exercise programs outside of the center . for example , related research has found that , following short - term commercial weight management interventions ( of six months or less ) , some participants report strong intentions in maintaining newly learned behaviors , increased self - efficacy in achieving a future weight target , and positive changes in habitual physical activity at one - year follow - up . as weight management programs increasingly become translated into community settings , a certain degree of dropout can be expected as participants manage multiple time commitments and as program staff members manage multiple job responsibilities . translation into community settings will bring along with it a greater need for accountability and compliance . the main reason for stringent compliance guidelines is that providing such programs can be costly , particularly for insurance - funded programs , which aim to address various health risks that impact chronic disease and curb insurance expenses associated with chronic disease . thus , a dropout from an insurance - sponsored program may either voluntarily quit the program or be administratively removed due to noncompliance . little is known about the reasons for , and consequences of , dropout from insurance - sponsored health promotion programs . understanding more about these patterns of behavior may help insurance agencies maximize the benefit of the programs they deliver in communities while keeping costs at a reasonable level . finally , if positive consequences of dropout are found , these data will help support the continued spending of public and private insurance dollars on health promotion programs . thus , the purpose of this study was to examine reasons for and consequences associated with dropout ( or removal ) from a state - wide , insurance - funded weight management program . the research questions included the following : ( 1 ) what are the reasons people drop out from an insurance - funded weight management program ? ( 2 ) what are the positive and negative consequences associated with dropout ? west virginia is a small , appalachian state ranked among the least healthy states in the us based on adult prevalence of obesity ( 33% versus 27.5% us median ) , diabetes ( 12% versus 8.7% us median ) , and heart disease ( 6% versus 4% us median ) , all of which rank in the top 10 in the united states . the estimated direct medical costs associated with obesity in west virginia ( wv ) increased by $ 51 million usd between 2001 and 2009 , which is a meaningful increase for a state with a population of just under two million . these negative health trends , and the economic consequences of them , have driven the need for programs which can help curtail the growth of obesity and promote healthy lifestyles . to combat these trends , the state 's largest public insurer , the west virginia public employees insurance agency ( peia ) , has established a comprehensive weight management program to provide policy holders with opportunities to learn how to be physically active and eat a healthful diet . the program , which requires a $ 20 monthly copay , includes access to a fitness facility in the local community and services provided by a personal trainer , dietitian , and exercise physiologist . previous single - site and large - scale evaluations of this program 's effectiveness have shown low reach , moderate to high effectiveness in short - term and long - term weight loss , and strong potential for sustainability [ 10 , 1315 ] . , waist , weight , body fat , and blood pressure ) on a monthly basis into a secure , web - based platform used for evaluation and billing . those participants that do not meet the eight visits per month minimum attendance criteria for two or more consecutive months are removed from the program . therefore , participants may be administratively removed for noncompliance with these requirements or other issues may arise that cause the termination of their program ( e.g. in addition , participants may voluntarily choose to drop out from the program for personal reasons . when they are removed or choose to drop out from the program , they receive a letter informing them of their change in status . the study used a mixed methods design including quantitative data on objective outcomes ( i.e. , length in the program , % weight loss ) as well as self - reported reasons and consequences in both quantitative and qualitative forms collected from the program evaluation survey . the survey was sent first via email through a unique link to the participants who provided their email addresses at the start of the program . after a week without response from the paper - based survey , a reminder post card was sent to the participant . the program evaluation survey sent to the participants included questions regarding their reasons for dropping out of the program , their satisfaction with different services of the program , and their future intentions . first , all participants were asked to choose between the reasons for leaving the program from why have you chosen to leave the weight management program at this time ? next , two sets of questions assessed participants ' satisfaction with different services of the program . these questions asked participants to rate their exercise facility , personal training , and dietary services using a scaled response from 1 ( not at all satisfied ) to 4 ( completely satisfied ) . because of the wording of this last question , many of its responses were related to satisfaction or dissatisfaction with the program ( i.e. , do you have anything else you would like to add about your level of satisfaction so far with the weight management program and the different services you have received ? ) in addition to satisfaction / dissatisfaction , participants also tended to express the positive and negative consequences related to their participation in the program and thus these data were included in the subsequent qualitative analysis . for the analysis of the open - ended data , two researchers independently analyzed and open - coded different subsamples of the data using descriptive coding . a near identical process was used to analyze the consequences and dropout open - ended items . however , data from the items were analyzed independently , that is , by separate sets of researchers and using separate coding books . these bivariate analyses were used to determine if there was any relationship between the length of time in the program prior to exit and their coded consequences and stated intentions . the mean length of participation in the program was 9.8 months ( sd = 5.6 ) , with 33.7% exiting the program within the first six months , 35.7% exiting between 7 and 12 months , and 30.6% completing 1324 months . among the male participants , mean weight at the beginning of the program was 262.2 lbs ( sd = 57.3 ) , while women had a mean weight of 210 lbs ( sd = 49.6 ) . men 's mean waist circumference at the beginning of the program was 46.9 inches ( sd = 6.9 ) and women 's was 42.4 inches ( sd = 9.8 ) . additionally , upon program exit , 21% of respondents lost at least 5% of their baseline body weight during the course of the program , while 26.7% had gained weight . to check for demographic differences between the survey respondents and nonrespondents , a series of independent samples t - tests analyses indicated that mean bmi , waist circumference , body fat percentage , and length of participation in the program of participants who dropped out from the program and responded the survey were not significantly different from the participants who dropped out and did not respond the survey ( p > .05 ) . despite this small difference in age , survey respondents and nonrespondents appear to report similar profiles related to their weight and program participation , thus reducing the concern for selection bias in the subsample of respondents . among the 400 survey respondents , 375 participants responded to the question about their reasons to drop out from the program , while 272 responded to the final open - ended question . in total , reasons to drop out from the program were coded 420 times , with some participants indicating more than one reason and some leaving the question blank . seven major themes regarding the participants ' reasons to drop out from the program emerged from the survey responses : competing priority ( 36.9% ) , medical ( 22.8% ) , negative experience ( 13.1% ) , programmatic issues ( 12.4% ) , administrative drop ( 7.4% ) , insurance coverage change ( 6.4% ) , and completed attempt ( 1.0% ) . the competing priority theme was the largest emergent theme with 155 occurrences ( 36.9% ) and included the subthemes of time ( 18.3% ) , caregiving ( 5.7% ) , distance ( 5.2% ) , lack of motivation ( 4.1% ) , personal reasons ( 2.1% ) , and grief ( 1.4% ) . time was a frequently coded subtheme , including statements of limited or lack of time to comply with the program requirements ( e.g. lack of motivation included need for external motivation , not being able to meet requirements , and just not wanting to participate anymore ( e.g. the negative feedback made me feel like a failure instead of acknowledging my improvement , so i quit going , while the second involved any kind of pain related to the participation of the program ( e.g. , price comprised unwillingness or impossibility of paying for direct or indirect costs of the program ( e.g. lack of family support ( 1.0% ) was coded when family members were unsupportive of the participant 's compliance with the program ( e.g. , conflict with my spouse , he felt i was spending too much time at the gym ) , while lack of social support ( 0.2% ) contained only one respondent who wished to have a buddy exerciser : programmatic issues was divided into the subthemes facility problem ( 11.7% ) and gym culture ( 0.7% ) . the professionals subtheme regarded problems with the staff delivering the services offered by the program , such as trainer 's or dietitian 's ineffectiveness , limited availability , and lack of support ( e.g. facility closure represented when the gym closed or stopped offering the program and this situation triggered the participants ' dropout ( e.g. insurance coverage contained participants who left for another job or retired , thus losing the insurance benefits , and administrative drop encompassed respondents who were removed by the program administration due to noncompliance . among the 400 survey respondents , 272 responded to the final open - ended question about their experiences in the program . two major themes emerged from these 272 responses : positive and negative consequences from participation in the program . positive consequences were coded 150 times ( 88.2% ) and negative consequences 20 times ( 11.8% ) . the negative consequences were composed of two subthemes : psychological ( 80% ) and physical ( 20% ) consequences . the first was attitude change ( 41.3% ) , which included the codes positive intentions ( 32% ) , perception of success ( 6.7% ) , and appreciate exercise ( 2.7% ) . the most common subtheme within the attitude change was positive intentions , which comprised intentions such as continuing to exercise , joining another weight management or exercise program , and seeking nutrition guidance ( e.g. the second most common was perception of success , including subjective comments regarding achieving success ( e.g. the last subtheme within the attitude change was appreciate exercise , which included liking exercise and realizing positives of exercising ( e.g. , i 'm very proud of myself for what i have accomplished with some help from the program ) . the second major theme within the positive consequences was behavioral ( 22% ) , which comprised maintenance ( 14.7% ) and changes ( 7.3% ) . weight loss subtheme was subcoded into lost weight ( 13.3% ) and continue to lose weight ( 2% ) , which differed regarding when the weight loss happened ( during the program or after dropping out of it ) . fitness improvement was related to enhancement in physical capability such as strength , flexibility , stamina , and resistance ( e.g. among the negative consequences ( n = 20 ) , the psychological consequences was the most frequent coded subtheme and was further subcategorized into attitudes ( 40% ) , feelings ( 35% ) , and lack of learning ( 5% ) . the latter regarded the participant 's decrease in motivation as a consequence of being removed from the program ( e.g. , i was kicked out of the program twice , it has been very discouraging ) . still within the negative psychological consequences , the code feelings included negative feelings such as guilt , disappointment , and shame ( e.g. it included statements of weight gain during the program or after leaving it ( e.g. regarding the respondents ' ( n = 400 ) intentions for future health - related behaviors , 64% intended to exercise 30 minutes per day , five days per week , 16% intended to exercise 60 minutes per day , five days per week , 11% intended to join a group exercise program , and 21% intended to continue as a private member of the fitness facility that they were attending during the program . when comparing these intentions among respondents who dropped out of the program early ( 6 months or less of participation ) , in the middle ( between 7 and 12 months ) , or later ( between months 13 and 24 ) chi - square analysis showed that respondents who stayed for seven or more months in the program were more likely to intend to continue exercising five days a week for at least 30 minutes ( 68.6% combined for the mid and later groups ) compared to early dropouts ( 55% ) ; however this effect was not significant , (4 , n = 382 ) = 7.09 , p = .131 , cc = 0.14 . in general , the final set of chi - square analyses targeted the relationship between stage of dropout ( early , middle , and late ) and consequences expressed . first , two analyses were conducted to see if the percentage of respondents reporting positive or negative consequences differed across the three groups . overall , one - third of those who answered this open - ended question reported a positive consequence of program participation . a different trend was revealed for the negative consequences , with those who dropped out in the middle group reporting a negative outcome 35 times as often ( 12.5% ) as the early ( 2.2% ) or late ( 3.8% ) dropouts , (2 , n = 268 ) = 9.723 , p = .008 , cc = 0.19 . this pattern held true for all of the subthemes of negative consequences , with the exception of dissatisfaction with weight loss . the results of this mixed methods study support the idea that there are both positive and negative consequences experienced in a sample of weight management program dropouts . overall , 1 in 5 respondents lost a clinically significant amount of weight during the program ( > 5% of baseline body weight ) , regardless of the time of program exit , and 1 in 3 experienced a positive consequence , while only 6% expressed a negative outcome of participation . additionally , nearly 90% of all of the consequences that emerged from the data were positive . thus , some of the dropouts appear to have experienced substantial success in terms of outcomes or lessons learned through participation , and five times as many respondents reported a positive consequence compared to those who reported a negative consequence of participation . skill acquisition were major themes , including positive health intentions , perceived success , learning skills , and new appreciation of exercise . these findings support previous theories of health behavior change including the theory of planned behavior 's connection between intentions , self - efficacy , and past behavior as well as similar work on barriers to the self - management of diabetes . specifically , based on these models , if programs can help individuals increase their health intentions , self - efficacy , and specific behavioral skills , regardless if they dropout or not , then they will increase the probability of future health behavior change among participants . approximately two - thirds of the respondents indicated they intend to continue exercising five days a week for 30 minutes , an amount of physical activity that would meet guidelines for general health . some respondents may have received as much out of the program as they needed , and they felt ready to move onto a self - managed program . others simply learned some new skills and experienced a change in attitude toward eating or physical activity . not all participants reported positive outcomes , however , and approximately 1 in 4 of the dropouts gained weight during the program . it is important to note that most of the negative consequences reported were also psychological in nature ( including lower motivation and dissatisfaction with weight loss ) , and there were no strong themes for things such as injury or worsened disability . integrating the objective and subjective data on consequences allows an observation that approximately 20% ( percent of sample with clinically significant weight loss ) to 33% ( percent of respondents who self - reported a positive consequence ) of dropouts of this community - based weight management program experienced a meaningful positive consequence resulting from program participation . the average respondent dropped out approximately 10 months into this two - year - long program , and negative consequences were reported at a higher rate among middle dropouts compared to early or late dropouts . this particular phase of the program ( between 7 and 12 months of participation ) appears to be the highest risk of dropout and may warrant additional attention . , lack of time ) was the most widely cited reason for program exit ( noted by 37% of respondents ) , suggesting that the working adults in the program often struggle to manage the multiple commitments of work , family , and health . this evidence supports the idea that some people need more support than others based of the timing of their attempt and the strength of their motivation at program entry . these data also support the finding from grave et al . this issue highlights a key difference in populations who are enrolled in community - based programs compared to clinical trials ; prior to group assignment , many participants with comorbidities are often excluded from trials . adding behavioral services ( in various forms and at various stages of the program ) could help participants learn new skills and stay engaged long enough to maintain the multiple health behaviors they have initiated . though this program is a fitness facility based program , the weight management program participants are often dissimilar from typical gym members . these issues were noted by some participants who dropped out , thus highlighting the difficulty of ensuring high treatment fidelity in a large , community - based program . the results of this study should be interpreted given the following limitations . despite the analyses indicating the responders are not that different from the nonresponders , there is still the potential for self - selection bias in the data . next , in our analyses of those who were dropped from the program , we did not differentiate between those who left the program voluntarily and those who were dropped due to noncompliance . future studies will explore these patterns over time and should address the relative importance of the psychosocial and environmental factors and barriers that emerged among participants . there is strength in the study 's design and data given the unique mix of quantitative , objectively observed changes in body weight and attendance along with the qualitative reasons , and consequences expressed by several hundred respondents . however , we can not expect to treat or reverse the obesity epidemic in the us with the limited reach of randomized controlled trials in clinical settings . we must embrace the messiness of community - based programs , especially those funded by insurance agencies , because they are sustainable and possess strong external validity . findings of the current study can be used to inform and improve future community - based research and multisite intervention programs with similar rural populations in the united states that experience the greatest health disparities in chronic disease .

the restorative treatment of deep carious lesions in young permanent molars presents a challenge for dentists because complete caries removal can expose the pulp tissue , leading to more complex treatments such as an endodontic procedure . to avoid this , the complete removal of the carious tissue is no longer recommended because partial caries removal or the sealing of the infected dentin tissue , allows dentin repair , prevents unnecessary tissue loss , and permits the conservative treatment of deep carious lesions . unlike the step - wise excavation technique , a single clinical session ( without tooth reopening ) is the current trend in carious lesion management . after cavity sealing / dental restoration , tissue remineralization , reduction in bacterial counts , and histological dentin reorganization with intertubular dentin thickening and formation of a dense collagen network were reported . if the glass ionomer cement ( gic ) is used as a liner , an ion exchange of calcium , phosphate , and fluorine between demineralized dentin occurs . this fact generated a discussion about the role of the lining material on the reorganization of the remaining carious tissue . some authors reported that gic can supply bioactive molecules promoting dentin regeneration , whereas others argued that caries arrestment is not dependent on the cavity liner materials . therefore , this study aimed to evaluate the impact of the liner material on the morphological and mineral changes of carious dentin of permanent teeth after tooth restoration . this study was approved by the ethics committee of the state university of ponta grossa ( ponta grossa , paran , brazil ) under protocol # 78/2011 and evaluated infected permanent dentin at baseline and 60 days after cavity sealing ( 60-day ) , using laser fluorescence ( lf ) readings and morphological and mineral analysis under scanning electron microscopy ( sem ) . after an initial screening of 772 children from rural area schools , 35 children of both genders with ages ranging from 7 to 15 years ( average = 11.0 2.7 ) were included in the study . the carious lesions should reach the inner portion of dentin ( 2/3 or more of the dentin thickness ) . teeth should not exhibit signs or symptoms of pulp pathology ( i.e. fistula , tooth mobility , periapical alterations , and spontaneous pain ) . if any of these signs were observed , the tooth was excluded . the teeth were randomly designed to group 1 ( n = 23 - gic liner + composite resin restoration ) or group 2 ( n = 22 - inert material wax + composite restoration ) . after local anesthesia and rubber dam isolation , teeth were cleaned with a new toothbrush and water , washed thoroughly with air / water spray , and air dried without desiccation . at baseline , we examined the characteristics of the mesial portion of the tooth , whereas the distal portion was examined 60 days after restoration at the reopening of the tooth . the research protocol consisted of lf readings ( as described later in this session ) followed by the removal of the carious dentin ; this process was repeated at baseline and at the 60-day follow - up period . to facilitate the removal of the mesial and distal portions of the dentin , the carious lesion was divided at baseline into two equal portions using a hollenback carver in a buccal - lingual direction . the dentin samples were removed using a sterile dentin excavator , and immediately after removal , the dentin fragments were processed for morphological and mineral analysis . the cavity preparation involved only the removal of the carious tissue from the cavosurface margin to promote appropriate cavity sealing . in the gic group ( g1 ) , the pulpal floor of the cavity was covered with high - viscosity gic ( ketac molar easymix , 3 m espe , st . polyacrylic acid was not used to aid cavity reopening 60 days after the procedure . in the wax group ( g2 ) , a piece of utility wax ( lysanda , so paulo , sp , brazil ) approximately 1 mm thick was placed on the pulpal floor . all cavities were etched with 37% phosphoric acid gel ( villevie , joinville , sc , brazil ) , rinsed with water and slightly air dried . a two - step etch - and - rinse adhesive ( adper single bond 2 , 3 m espe ) was applied according to the manufacturer 's instructions , and the composite resin ( llis , fgm , joinville , sc , brazil ) was placed in slim increments . after the composite resin placement , a sealant ( fluroshield , dentsply , petrpolis , rj , brazil ) was applied on the restoration margins for extra protection . the light - curing process was performed with a quartz - tungsten - halogen - light under ramped curing ( dx turbo led 1200 , d - x , ribeiro preto , so paulo , brazil ) . the initial light intensity was 450 mw / cm , with an automatic increase to 1200 mw / cm after 10 s. sixty days after the procedure , the marginal integrity of the restorations was evaluated using the united states public health service criteria : ( 1 ) cavosurface marginal discoloration , ( 2 ) recurrent caries , ( 3 ) contour or loss of substance ( wear ) , ( 4 ) marginal integrity , and ( 5 ) surface texture . these variables were ranked with the following scores : alpha ( no defect clinically detectable , needing only a polish ) , bravo ( clinically acceptable , but repair is needed ) , and charlie ( clinically unacceptable , needs restoration replacement ) . if there were signs or symptoms of pulp pathology or restorations defects that compromised cavity sealing , the tooth was excluded . then , the restorative material was removed with a sterile diamond bur ( # 1092 - kg sorensen , so paulo , brazil ) under water cooling until the gic and wax were reached . dentin samples from the distal portion of the tooth were removed for morphological and mineral analysis in the same manner as described for the mesial portion . after the collection of the dentin samples in both periods , the teeth were restored with composite resin restorations . all of the clinical procedures were performed by a single operator ( eunice kuhn ) , a trained and experienced pediatric dentist . dentin lf was measured with diagnodent 2095 ( kavo , biberach , germany ) following the manufacturer 's instructions . the device was calibrated against a porcelain reference object and recalibrated on a sound surface of each tooth before the examination . optimal cutoff limits of the lf device to detect in vivo occlusal caries lesion depth are as follows : 014 - sound teeth ; 1521 - enamel lesions ; 2237 - caries lesion in the outer half of the dentin ; and > 38 - caries lesion in the inner half of the dentin . three measurements on carious dentin in the mesial ( baseline measurement ) or distal portion ( 60 days after tooth restoration ) of the cavity were taken , and the mean value from each period was calculated to represent the individual tooth . dentin fragments from the mesial ( baseline ) and distal ( 60-day sample ) portions were stored into a 2% glutaraldehyde solution with a sodium phosphate buffer of 0.1 m ( ph 7.4 ) for 7 days , rinsed thoroughly with 50% sodium phosphate buffer ( 0.3 m ) and distilled water solution ( three 30-min rinses ) , and dehydrated in acetone at 30% , 50% , and 70% for 10 min , 90% for 20 min , 100% for 10 min , and 100% for 20 min . samples were kept at 37c for at least 3 days to remove any residual water . they were analyzed with sem using secondary electron mode with a voltage of 12 kv . the same sample was analyzed using dispersive x - ray spectrometry energy to measure the relative percentage of calcium , phosphorus , and fluorine among the 12 most common chemical elements in the dentin substrate . the weight percentages of calcium , phosphorus , and fluorine before and after tooth restoration were assessed at a magnification of 200 for 100 s. all of the microphotographs were analyzed by the same examiner ( ana claudia rodrigues chibinski ) , who was blinded to the group to which the specimen belonged . the characteristics evaluated were the presence of bacteria , the collagen network , and the mineralization of inter- and peri - tubular dentin . the software statistical package for social sciences ( ibm corporation , spss 17.0 , chicago , illinois , usa ) was used for statistical analysis with the significance level set at 0.05 . before submitting the data to statistical analysis , kolmogorov smirnov test was performed to assess the normality of the data , and bartlett 's test was used to verify if the assumption of equal variances was valid . as the data from the lf reading failed in both analysis of variance ( anova ) assumptions ( p < 0.05 ) , we used nonparametric statistics for data analysis . in each group , the lf readings before and after cavity sealing were compared by wilcoxon signed - rank test . in each period , the lf readings of the gic and wax groups were statistically analyzed with mann for the mineral data , the anova assumptions were valid ( p > 0.05 ) , and therefore , parametric statistics were employed . for each tooth , the percentage value of each mineral obtained at 60 days was subtracted from the baseline value . a paired t - test was used to compare the differences in wt% of calcium , phosphorus , and fluorine between the two study groups . after an initial screening of 772 children from rural area schools , 35 children of both genders with ages ranging from 7 to 15 years ( average = 11.0 2.7 ) were included in the study . children with systemic pathologies the carious lesions should reach the inner portion of dentin ( 2/3 or more of the dentin thickness ) . teeth should not exhibit signs or symptoms of pulp pathology ( i.e. fistula , tooth mobility , periapical alterations , and spontaneous pain ) . if any of these signs were observed , the tooth was excluded . the teeth were randomly designed to group 1 ( n = 23 - gic liner + composite resin restoration ) or group 2 ( n = 22 - inert material wax + composite restoration ) . after local anesthesia and rubber dam isolation , teeth were cleaned with a new toothbrush and water , washed thoroughly with air / water spray , and air dried without desiccation . at baseline , we examined the characteristics of the mesial portion of the tooth , whereas the distal portion was examined 60 days after restoration at the reopening of the tooth . the research protocol consisted of lf readings ( as described later in this session ) followed by the removal of the carious dentin ; this process was repeated at baseline and at the 60-day follow - up period . to facilitate the removal of the mesial and distal portions of the dentin , the carious lesion was divided at baseline into two equal portions using a hollenback carver in a buccal - lingual direction . the dentin samples were removed using a sterile dentin excavator , and immediately after removal , the dentin fragments were processed for morphological and mineral analysis . the cavity preparation involved only the removal of the carious tissue from the cavosurface margin to promote appropriate cavity sealing . in the gic group ( g1 ) , the pulpal floor of the cavity was covered with high - viscosity gic ( ketac molar easymix , 3 m espe , st . polyacrylic acid was not used to aid cavity reopening 60 days after the procedure . in the wax group ( g2 ) , a piece of utility wax ( lysanda , so paulo , sp , brazil ) approximately 1 mm thick was placed on the pulpal floor . all cavities were etched with 37% phosphoric acid gel ( villevie , joinville , sc , brazil ) , rinsed with water and slightly air dried . a two - step etch - and - rinse adhesive ( adper single bond 2 , 3 m espe ) was applied according to the manufacturer 's instructions , and the composite resin ( llis , fgm , joinville , sc , brazil ) was placed in slim increments . after the composite resin placement , a sealant ( fluroshield , dentsply , petrpolis , rj , brazil ) the light - curing process was performed with a quartz - tungsten - halogen - light under ramped curing ( dx turbo led 1200 , d - x , ribeiro preto , so paulo , brazil ) . the initial light intensity was 450 mw / cm , with an automatic increase to 1200 mw / cm after 10 s. sixty days after the procedure , the marginal integrity of the restorations was evaluated using the united states public health service criteria : ( 1 ) cavosurface marginal discoloration , ( 2 ) recurrent caries , ( 3 ) contour or loss of substance ( wear ) , ( 4 ) marginal integrity , and ( 5 ) surface texture . these variables were ranked with the following scores : alpha ( no defect clinically detectable , needing only a polish ) , bravo ( clinically acceptable , but repair is needed ) , and charlie ( clinically unacceptable , needs restoration replacement ) . if there were signs or symptoms of pulp pathology or restorations defects that compromised cavity sealing , the tooth was excluded . then , the restorative material was removed with a sterile diamond bur ( # 1092 - kg sorensen , so paulo , brazil ) under water cooling until the gic and wax were reached . dentin samples from the distal portion of the tooth were removed for morphological and mineral analysis in the same manner as described for the mesial portion . after the collection of the dentin samples in both periods , the teeth were restored with composite resin restorations . all of the clinical procedures were performed by a single operator ( eunice kuhn ) , a trained and experienced pediatric dentist . dentin lf was measured with diagnodent 2095 ( kavo , biberach , germany ) following the manufacturer 's instructions . the device was calibrated against a porcelain reference object and recalibrated on a sound surface of each tooth before the examination . optimal cutoff limits of the lf device to detect in vivo occlusal caries lesion depth are as follows : 014 - sound teeth ; 1521 - enamel lesions ; 2237 - caries lesion in the outer half of the dentin ; and > 38 - caries lesion in the inner half of the dentin . three measurements on carious dentin in the mesial ( baseline measurement ) or distal portion ( 60 days after tooth restoration ) of the cavity were taken , and the mean value from each period was calculated to represent the individual tooth . dentin fragments from the mesial ( baseline ) and distal ( 60-day sample ) portions were stored into a 2% glutaraldehyde solution with a sodium phosphate buffer of 0.1 m ( ph 7.4 ) for 7 days , rinsed thoroughly with 50% sodium phosphate buffer ( 0.3 m ) and distilled water solution ( three 30-min rinses ) , and dehydrated in acetone at 30% , 50% , and 70% for 10 min , 90% for 20 min , 100% for 10 min , and 100% for 20 min . samples were kept at 37c for at least 3 days to remove any residual water . they were analyzed with sem using secondary electron mode with a voltage of 12 kv . the same sample was analyzed using dispersive x - ray spectrometry energy to measure the relative percentage of calcium , phosphorus , and fluorine among the 12 most common chemical elements in the dentin substrate . the weight percentages of calcium , phosphorus , and fluorine before and after tooth restoration were assessed at a magnification of 200 for 100 s. all of the microphotographs were analyzed by the same examiner ( ana claudia rodrigues chibinski ) , who was blinded to the group to which the specimen belonged . the characteristics evaluated were the presence of bacteria , the collagen network , and the mineralization of inter- and peri - tubular dentin . the tooth was employed as an experimental unit for data analysis . the software statistical package for social sciences ( ibm corporation , spss 17.0 , chicago , illinois , usa ) was used for statistical analysis with the significance level set at 0.05 . before submitting the data to statistical analysis , kolmogorov smirnov test was performed to assess the normality of the data , and bartlett 's test was used to verify if the assumption of equal variances was valid . as the data from the lf reading failed in both analysis of variance ( anova ) assumptions ( p < 0.05 ) , we used nonparametric statistics for data analysis . in each group , the lf readings before and after cavity sealing were compared by wilcoxon signed - rank test . in each period , the lf readings of the gic and wax groups were statistically analyzed with mann for the mineral data , the anova assumptions were valid ( p > 0.05 ) , and therefore , parametric statistics were employed . for each tooth , the percentage value of each mineral obtained at 60 days was subtracted from the baseline value . a paired t - test was used to compare the differences in wt% of calcium , phosphorus , and fluorine between the two study groups . of the 45 permanent molars at baseline , four teeth were not evaluated in the 60-day assessment . in g1 ( the gic group ) , the teeth were not evaluated due to pulp necrosis . in g2 ( the wax group ) , three students did not attend the 60-day recall , so we could not evaluate their teeth . in addition , after 60 days , one restoration was classified as charlie , so it was not included in the sample . no significant difference in lf readings was detected for the gic and wax groups in either period ( mann whitney u - test , p > 0.05 ) . significant reductions in lf means were detected between periods for both groups ( wilcoxon signed - rank test , p < 0.05 ) [ table 1 ] . means and standard deviations of laser fluorescence for both groups at baseline and in the 60 days follow - up baseline samples from both groups showed highly infected tissue and a clearly exposed collagen matrix [ figures 1 and 2 ] . after cavity sealing , fewer bacteria with a more compact arrangement of collagen fibers were seen in both groups [ figures 1 and 2 ] . a similar gain in calcium and phosphorus was detected for both groups ( t - test , p > 0.05 ) [ table 2 ] . an uptake of fluorine was significantly detected only for the gic group ( t - test , p = 0.032 ) [ table 2 ] . after restoration , we observed some dentinal tubules without microorganisms ( arrows ) for g1 and g2 , and a decrease in the bacterial contamination is evident although some microorganisms can still be seen in the intertubular dentin ( pointer ) the demineralized dentin tissue with exposed collagen fibers was observed at baseline for both groups . however , the remineralization process also occurred on g2 in peri- and inter - tubular dentin ( arrows ) means and standard deviations of the 60-day vs. baseline difference in the weight percentage of calcium , fluorine and phosphorus for both groups no significant difference in lf readings was detected for the gic and wax groups in either period ( mann whitney u - test , p > 0.05 ) . significant reductions in lf means were detected between periods for both groups ( wilcoxon signed - rank test , p < 0.05 ) [ table 1 ] . means and standard deviations of laser fluorescence for both groups at baseline and in the 60 days follow - up baseline samples from both groups showed highly infected tissue and a clearly exposed collagen matrix [ figures 1 and 2 ] . after cavity sealing , fewer bacteria with a more compact arrangement of collagen fibers were seen in both groups [ figures 1 and 2 ] . a similar gain in calcium and phosphorus was detected for both groups ( t - test , p > 0.05 ) [ table 2 ] . an uptake of fluorine was significantly detected only for the gic group ( t - test , p = 0.032 ) [ table 2 ] . after restoration , we observed some dentinal tubules without microorganisms ( arrows ) for g1 and g2 , and a decrease in the bacterial contamination is evident although some microorganisms can still be seen in the intertubular dentin ( pointer ) the demineralized dentin tissue with exposed collagen fibers was observed at baseline for both groups . however , the remineralization process also occurred on g2 in peri- and inter - tubular dentin ( arrows ) means and standard deviations of the 60-day vs. baseline difference in the weight percentage of calcium , fluorine and phosphorus for both groups the clinical success of conservative procedures ( step - wise excavation / indirect pulp capping ) is achieved when pulp vitality is maintained , and no adverse symptoms are reported after treatment . in the present study , therefore , the overall success rate is 98% ( 95% confidence interval 88%100% ) , which is in agreement with other published papers . although this study was not pioneering research in demonstrating caries arrestment after dentin carious lesions sealing , few papers have compared a bioactive versus inert material as a liner . this article provided evidence that dentin reorganization and mineral changes were not dependent on the material placed in contact with the carious tissue , suggesting that the carious arrestment is a host - driven process rather than a material - induced process . the concept that dentin remineralization is a material - driven process was based on the benefits of the fluoride in the enamel remineralization . some authors believe that fluoride release from gic could favor the remineralization of carious dentin . however , if fluoride was necessary for dentin remineralization , we should have detected remineralization ( by increased percentage of calcium and phosphorus ) only in the gic group , and this was not observed . the cessation of the caries process allows the biological response of the teeth . sealing the cavity isolates bacteria from the oral environment and active biofilm , arresting the carious process , and providing time for the defense mechanism from the pulp - dentin complex . in general , repair and regeneration in the dentin - pulp complex are similar to natural wound - healing responses seen in many of the body 's systems . to respond to the inflammatory process induced by the caries lesion , odontoblasts produce a reactionary tertiary dentin matrix , along with high levels of growth factor secretion in an effort to remodel and repair the extracellular matrix damaged by the disease process . thus , the dense collagen matrix seen 60 days after sealing is the result of tissue remodeling . considering that we used a destructive method , the images obtained were not from the same area , but the dentin sample collected was from the same tooth ( which was reopened after 60 days ) and depth . this was possible because the carious tissue on the cavity floor was divided into two portions ( mesial and distal ) . the decreased means of lf after restoration are consistent with the bacterial reduction detected in the sem images . the lower lf readings are associated with the reduction of the bacterial aggregates and their metabolic by - products such as protoporphyrin ix , mesoporphyrin , and coproporphyrin , but not the mineral content of the dentin . however , the means of lf in the 60-day sample are still far from those reported for sound dentin ( which ranges between 3 and 6 ) and dentin caries ( which ranges between 35 and 40 ) . a possible explanation is that the residual metabolic products are still readable by an lf device even after cavity sealing . by no means has the present study suggested the placement of an inert material ( such as wax ) inside the cavity , which was used only as a negative control for research purposes . the selection of the liner / provisional or definitive restorative material should consider its biocompatibility , longevity and ability to bond to the dental structures . caries arrestment with dentin reorganization occurs regardless of the lining material placed in contact with the infected dentin . 525/10 ) from the brazilian research agency , the araucaria foundation for supporting scientific and technological development of paran , brazil . 525/10 ) from the brazilian research agency , the araucaria foundation for supporting scientific and technological development of paran , brazil .

aim : this study evaluated the impact of liner material on the fluorescence , morphological and mineral characteristics of permanent carious dentin after cavity sealing.methods:thirty children ( 11.0 2.7 years old ) presenting at least one active deep carious lesion in permanent molars were selected . fragments of carious dentin were removed from teeth before lining the cavity ( baseline samples ) with high - viscosity glass ionomer cement ( g1 ) or an inert material ( wax - g2 ) . cavities were restored with composite resin and reopened 60 days later , and other fragments were removed ( 60-day sample ) . the laser fluorescence ( lf ) readings and morphological and mineral changes of both groups were compared.results:after 60 days , forty teeth were available for evaluation . lower lf means were obtained ( wilcoxon signed - rank test ; p < 0.05 ) , and enhanced calcium and phosphorus levels were detected for both groups ( t - test , p < 0.05 ) . an uptake of fluorine was observed only in g1 ( t - test ; p < 0.05 ) . regardless of the group , baseline samples exhibited clear signs of bacterial invasion , and the collagen fibers were exposed ; the 60-day samples showed a better - organized tissue with a more compact intertubular dentin.conclusion:caries arrestment with dentin reorganization occurs regardless of the lining material placed in contact with the infected dentin .

INTRODUCTION METHODS Sample selection and inclusion criteria Clinical procedures Laser fluorescence readings Morphological and mineral analysis Statistical analysis RESULTS Laser fluorescence readings Morphological and mineral analysis DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

the restorative treatment of deep carious lesions in young permanent molars presents a challenge for dentists because complete caries removal can expose the pulp tissue , leading to more complex treatments such as an endodontic procedure . to avoid this , the complete removal of the carious tissue is no longer recommended because partial caries removal or the sealing of the infected dentin tissue , allows dentin repair , prevents unnecessary tissue loss , and permits the conservative treatment of deep carious lesions . after cavity sealing / dental restoration , tissue remineralization , reduction in bacterial counts , and histological dentin reorganization with intertubular dentin thickening and formation of a dense collagen network were reported . if the glass ionomer cement ( gic ) is used as a liner , an ion exchange of calcium , phosphate , and fluorine between demineralized dentin occurs . this fact generated a discussion about the role of the lining material on the reorganization of the remaining carious tissue . some authors reported that gic can supply bioactive molecules promoting dentin regeneration , whereas others argued that caries arrestment is not dependent on the cavity liner materials . therefore , this study aimed to evaluate the impact of the liner material on the morphological and mineral changes of carious dentin of permanent teeth after tooth restoration . this study was approved by the ethics committee of the state university of ponta grossa ( ponta grossa , paran , brazil ) under protocol # 78/2011 and evaluated infected permanent dentin at baseline and 60 days after cavity sealing ( 60-day ) , using laser fluorescence ( lf ) readings and morphological and mineral analysis under scanning electron microscopy ( sem ) . after an initial screening of 772 children from rural area schools , 35 children of both genders with ages ranging from 7 to 15 years ( average = 11.0 2.7 ) were included in the study . the teeth were randomly designed to group 1 ( n = 23 - gic liner + composite resin restoration ) or group 2 ( n = 22 - inert material wax + composite restoration ) . after local anesthesia and rubber dam isolation , teeth were cleaned with a new toothbrush and water , washed thoroughly with air / water spray , and air dried without desiccation . at baseline , we examined the characteristics of the mesial portion of the tooth , whereas the distal portion was examined 60 days after restoration at the reopening of the tooth . the research protocol consisted of lf readings ( as described later in this session ) followed by the removal of the carious dentin ; this process was repeated at baseline and at the 60-day follow - up period . the dentin samples were removed using a sterile dentin excavator , and immediately after removal , the dentin fragments were processed for morphological and mineral analysis . the cavity preparation involved only the removal of the carious tissue from the cavosurface margin to promote appropriate cavity sealing . in the gic group ( g1 ) , the pulpal floor of the cavity was covered with high - viscosity gic ( ketac molar easymix , 3 m espe , st . in the wax group ( g2 ) , a piece of utility wax ( lysanda , so paulo , sp , brazil ) approximately 1 mm thick was placed on the pulpal floor . all cavities were etched with 37% phosphoric acid gel ( villevie , joinville , sc , brazil ) , rinsed with water and slightly air dried . a two - step etch - and - rinse adhesive ( adper single bond 2 , 3 m espe ) was applied according to the manufacturer 's instructions , and the composite resin ( llis , fgm , joinville , sc , brazil ) was placed in slim increments . after the composite resin placement , a sealant ( fluroshield , dentsply , petrpolis , rj , brazil ) was applied on the restoration margins for extra protection . the initial light intensity was 450 mw / cm , with an automatic increase to 1200 mw / cm after 10 s. sixty days after the procedure , the marginal integrity of the restorations was evaluated using the united states public health service criteria : ( 1 ) cavosurface marginal discoloration , ( 2 ) recurrent caries , ( 3 ) contour or loss of substance ( wear ) , ( 4 ) marginal integrity , and ( 5 ) surface texture . these variables were ranked with the following scores : alpha ( no defect clinically detectable , needing only a polish ) , bravo ( clinically acceptable , but repair is needed ) , and charlie ( clinically unacceptable , needs restoration replacement ) . dentin samples from the distal portion of the tooth were removed for morphological and mineral analysis in the same manner as described for the mesial portion . after the collection of the dentin samples in both periods , the teeth were restored with composite resin restorations . all of the clinical procedures were performed by a single operator ( eunice kuhn ) , a trained and experienced pediatric dentist . optimal cutoff limits of the lf device to detect in vivo occlusal caries lesion depth are as follows : 014 - sound teeth ; 1521 - enamel lesions ; 2237 - caries lesion in the outer half of the dentin ; and > 38 - caries lesion in the inner half of the dentin . three measurements on carious dentin in the mesial ( baseline measurement ) or distal portion ( 60 days after tooth restoration ) of the cavity were taken , and the mean value from each period was calculated to represent the individual tooth . dentin fragments from the mesial ( baseline ) and distal ( 60-day sample ) portions were stored into a 2% glutaraldehyde solution with a sodium phosphate buffer of 0.1 m ( ph 7.4 ) for 7 days , rinsed thoroughly with 50% sodium phosphate buffer ( 0.3 m ) and distilled water solution ( three 30-min rinses ) , and dehydrated in acetone at 30% , 50% , and 70% for 10 min , 90% for 20 min , 100% for 10 min , and 100% for 20 min . the weight percentages of calcium , phosphorus , and fluorine before and after tooth restoration were assessed at a magnification of 200 for 100 s. all of the microphotographs were analyzed by the same examiner ( ana claudia rodrigues chibinski ) , who was blinded to the group to which the specimen belonged . the characteristics evaluated were the presence of bacteria , the collagen network , and the mineralization of inter- and peri - tubular dentin . the software statistical package for social sciences ( ibm corporation , spss 17.0 , chicago , illinois , usa ) was used for statistical analysis with the significance level set at 0.05 . before submitting the data to statistical analysis , kolmogorov smirnov test was performed to assess the normality of the data , and bartlett 's test was used to verify if the assumption of equal variances was valid . as the data from the lf reading failed in both analysis of variance ( anova ) assumptions ( p < 0.05 ) , we used nonparametric statistics for data analysis . in each group , the lf readings before and after cavity sealing were compared by wilcoxon signed - rank test . in each period , the lf readings of the gic and wax groups were statistically analyzed with mann for the mineral data , the anova assumptions were valid ( p > 0.05 ) , and therefore , parametric statistics were employed . a paired t - test was used to compare the differences in wt% of calcium , phosphorus , and fluorine between the two study groups . after an initial screening of 772 children from rural area schools , 35 children of both genders with ages ranging from 7 to 15 years ( average = 11.0 2.7 ) were included in the study . the teeth were randomly designed to group 1 ( n = 23 - gic liner + composite resin restoration ) or group 2 ( n = 22 - inert material wax + composite restoration ) . after local anesthesia and rubber dam isolation , teeth were cleaned with a new toothbrush and water , washed thoroughly with air / water spray , and air dried without desiccation . at baseline , we examined the characteristics of the mesial portion of the tooth , whereas the distal portion was examined 60 days after restoration at the reopening of the tooth . the research protocol consisted of lf readings ( as described later in this session ) followed by the removal of the carious dentin ; this process was repeated at baseline and at the 60-day follow - up period . to facilitate the removal of the mesial and distal portions of the dentin , the carious lesion was divided at baseline into two equal portions using a hollenback carver in a buccal - lingual direction . the dentin samples were removed using a sterile dentin excavator , and immediately after removal , the dentin fragments were processed for morphological and mineral analysis . the cavity preparation involved only the removal of the carious tissue from the cavosurface margin to promote appropriate cavity sealing . in the gic group ( g1 ) , the pulpal floor of the cavity was covered with high - viscosity gic ( ketac molar easymix , 3 m espe , st . in the wax group ( g2 ) , a piece of utility wax ( lysanda , so paulo , sp , brazil ) approximately 1 mm thick was placed on the pulpal floor . all cavities were etched with 37% phosphoric acid gel ( villevie , joinville , sc , brazil ) , rinsed with water and slightly air dried . a two - step etch - and - rinse adhesive ( adper single bond 2 , 3 m espe ) was applied according to the manufacturer 's instructions , and the composite resin ( llis , fgm , joinville , sc , brazil ) was placed in slim increments . after the composite resin placement , a sealant ( fluroshield , dentsply , petrpolis , rj , brazil ) the light - curing process was performed with a quartz - tungsten - halogen - light under ramped curing ( dx turbo led 1200 , d - x , ribeiro preto , so paulo , brazil ) . the initial light intensity was 450 mw / cm , with an automatic increase to 1200 mw / cm after 10 s. sixty days after the procedure , the marginal integrity of the restorations was evaluated using the united states public health service criteria : ( 1 ) cavosurface marginal discoloration , ( 2 ) recurrent caries , ( 3 ) contour or loss of substance ( wear ) , ( 4 ) marginal integrity , and ( 5 ) surface texture . these variables were ranked with the following scores : alpha ( no defect clinically detectable , needing only a polish ) , bravo ( clinically acceptable , but repair is needed ) , and charlie ( clinically unacceptable , needs restoration replacement ) . dentin samples from the distal portion of the tooth were removed for morphological and mineral analysis in the same manner as described for the mesial portion . after the collection of the dentin samples in both periods , the teeth were restored with composite resin restorations . all of the clinical procedures were performed by a single operator ( eunice kuhn ) , a trained and experienced pediatric dentist . optimal cutoff limits of the lf device to detect in vivo occlusal caries lesion depth are as follows : 014 - sound teeth ; 1521 - enamel lesions ; 2237 - caries lesion in the outer half of the dentin ; and > 38 - caries lesion in the inner half of the dentin . three measurements on carious dentin in the mesial ( baseline measurement ) or distal portion ( 60 days after tooth restoration ) of the cavity were taken , and the mean value from each period was calculated to represent the individual tooth . dentin fragments from the mesial ( baseline ) and distal ( 60-day sample ) portions were stored into a 2% glutaraldehyde solution with a sodium phosphate buffer of 0.1 m ( ph 7.4 ) for 7 days , rinsed thoroughly with 50% sodium phosphate buffer ( 0.3 m ) and distilled water solution ( three 30-min rinses ) , and dehydrated in acetone at 30% , 50% , and 70% for 10 min , 90% for 20 min , 100% for 10 min , and 100% for 20 min . the weight percentages of calcium , phosphorus , and fluorine before and after tooth restoration were assessed at a magnification of 200 for 100 s. all of the microphotographs were analyzed by the same examiner ( ana claudia rodrigues chibinski ) , who was blinded to the group to which the specimen belonged . the characteristics evaluated were the presence of bacteria , the collagen network , and the mineralization of inter- and peri - tubular dentin . the software statistical package for social sciences ( ibm corporation , spss 17.0 , chicago , illinois , usa ) was used for statistical analysis with the significance level set at 0.05 . before submitting the data to statistical analysis , kolmogorov smirnov test was performed to assess the normality of the data , and bartlett 's test was used to verify if the assumption of equal variances was valid . as the data from the lf reading failed in both analysis of variance ( anova ) assumptions ( p < 0.05 ) , we used nonparametric statistics for data analysis . in each group , the lf readings before and after cavity sealing were compared by wilcoxon signed - rank test . in each period , the lf readings of the gic and wax groups were statistically analyzed with mann for the mineral data , the anova assumptions were valid ( p > 0.05 ) , and therefore , parametric statistics were employed . a paired t - test was used to compare the differences in wt% of calcium , phosphorus , and fluorine between the two study groups . of the 45 permanent molars at baseline , four teeth were not evaluated in the 60-day assessment . in g1 ( the gic group ) , the teeth were not evaluated due to pulp necrosis . in g2 ( the wax group ) , three students did not attend the 60-day recall , so we could not evaluate their teeth . in addition , after 60 days , one restoration was classified as charlie , so it was not included in the sample . no significant difference in lf readings was detected for the gic and wax groups in either period ( mann whitney u - test , p > 0.05 ) . significant reductions in lf means were detected between periods for both groups ( wilcoxon signed - rank test , p < 0.05 ) [ table 1 ] . means and standard deviations of laser fluorescence for both groups at baseline and in the 60 days follow - up baseline samples from both groups showed highly infected tissue and a clearly exposed collagen matrix [ figures 1 and 2 ] . after cavity sealing , fewer bacteria with a more compact arrangement of collagen fibers were seen in both groups [ figures 1 and 2 ] . a similar gain in calcium and phosphorus was detected for both groups ( t - test , p > 0.05 ) [ table 2 ] . an uptake of fluorine was significantly detected only for the gic group ( t - test , p = 0.032 ) [ table 2 ] . after restoration , we observed some dentinal tubules without microorganisms ( arrows ) for g1 and g2 , and a decrease in the bacterial contamination is evident although some microorganisms can still be seen in the intertubular dentin ( pointer ) the demineralized dentin tissue with exposed collagen fibers was observed at baseline for both groups . however , the remineralization process also occurred on g2 in peri- and inter - tubular dentin ( arrows ) means and standard deviations of the 60-day vs. baseline difference in the weight percentage of calcium , fluorine and phosphorus for both groups no significant difference in lf readings was detected for the gic and wax groups in either period ( mann whitney u - test , p > 0.05 ) . significant reductions in lf means were detected between periods for both groups ( wilcoxon signed - rank test , p < 0.05 ) [ table 1 ] . means and standard deviations of laser fluorescence for both groups at baseline and in the 60 days follow - up baseline samples from both groups showed highly infected tissue and a clearly exposed collagen matrix [ figures 1 and 2 ] . after cavity sealing , fewer bacteria with a more compact arrangement of collagen fibers were seen in both groups [ figures 1 and 2 ] . a similar gain in calcium and phosphorus was detected for both groups ( t - test , p > 0.05 ) [ table 2 ] . an uptake of fluorine was significantly detected only for the gic group ( t - test , p = 0.032 ) [ table 2 ] . after restoration , we observed some dentinal tubules without microorganisms ( arrows ) for g1 and g2 , and a decrease in the bacterial contamination is evident although some microorganisms can still be seen in the intertubular dentin ( pointer ) the demineralized dentin tissue with exposed collagen fibers was observed at baseline for both groups . however , the remineralization process also occurred on g2 in peri- and inter - tubular dentin ( arrows ) means and standard deviations of the 60-day vs. baseline difference in the weight percentage of calcium , fluorine and phosphorus for both groups the clinical success of conservative procedures ( step - wise excavation / indirect pulp capping ) is achieved when pulp vitality is maintained , and no adverse symptoms are reported after treatment . although this study was not pioneering research in demonstrating caries arrestment after dentin carious lesions sealing , few papers have compared a bioactive versus inert material as a liner . this article provided evidence that dentin reorganization and mineral changes were not dependent on the material placed in contact with the carious tissue , suggesting that the carious arrestment is a host - driven process rather than a material - induced process . the concept that dentin remineralization is a material - driven process was based on the benefits of the fluoride in the enamel remineralization . some authors believe that fluoride release from gic could favor the remineralization of carious dentin . however , if fluoride was necessary for dentin remineralization , we should have detected remineralization ( by increased percentage of calcium and phosphorus ) only in the gic group , and this was not observed . sealing the cavity isolates bacteria from the oral environment and active biofilm , arresting the carious process , and providing time for the defense mechanism from the pulp - dentin complex . this was possible because the carious tissue on the cavity floor was divided into two portions ( mesial and distal ) . the lower lf readings are associated with the reduction of the bacterial aggregates and their metabolic by - products such as protoporphyrin ix , mesoporphyrin , and coproporphyrin , but not the mineral content of the dentin . a possible explanation is that the residual metabolic products are still readable by an lf device even after cavity sealing . by no means has the present study suggested the placement of an inert material ( such as wax ) inside the cavity , which was used only as a negative control for research purposes . caries arrestment with dentin reorganization occurs regardless of the lining material placed in contact with the infected dentin .

renewable chemical fuels can be synthesized through solardriven electro , photo , and thermochemical splitting of co2 and h2o.1 the latter approach utilizes the entire spectrum of concentrated solar radiation as hightemperature process heat for the production of co and h2 ( syngas ) via metal oxide redox cycles.1b , 2 a critical drawback of this approach is the inert gas consumed to lower the partial pressure of oxygen ( po2 ) for shifting the thermodynamic equilibrium of the reduction step to lower temperatures.3 this , in turn , requires separation of o2 from the product gases for recycling the inert carrier gas and closing the material cycle.3a , 4 the separation of o2 has been a requirement in a variety of commercial applications such as oxycombustion , autothermal gasification of carbonaceous feedstock , and o2 removal to avoid catalyst passivation by o2 in fuel cells and when deoxygenating biofuels to make these more akin to petroleumderived fuels.5 industrially , o2 can be separated from air by pressure swing adsorption ( psa ) using zeolites and carbon molecular sieves , by ceramic mixed ionicelectronic conducting ( miec ) membranes,6 and by cryogenic distillation . psa and miec membranes can not produce highpurity inert gas,6b and separating o2 from gas mixtures at low po2 using membranes relies on a stripping gas with even lower po2 . these separation technologies further require an input of electrical work ranging from 100 to 350 kwh per metric ton o2,6 , 7 which penalizes the solartofuel energy conversion efficiencies . since solar thermochemical cycles inherently suffer from heat losses , it would be beneficial to utilize an oxygen separation technology driven by waste heat . thermochemical solidstate o2 separation ( tssos ) using metal oxide redox materials such as cu2o / cuo,3a , 8 mn3o4/mn2o3,8 and coo / co3o4 8 , 9 utilizes lowgrade process heat and does not require electricity . tssos has the potential to separate and concentrate o2 at low po2 via temperatureswing.3a the current stateoftheart tssos redox material , cu2o , has a maximum oxygen exchange capacity ( , i.e. , the difference in the oxygen nonstoichiometry between reducing and oxidizing conditions ) of about 200 mmol o2 per mol cu2o , exchanged at approximately 10 mol o 2 min g cu 2o when cycled between 11201450 k.3a we show below that cu2o can not be employed with lowgrade process heat at 600900 k. with the aim of augmenting the o2 exchange capacities and rates of tssos redox materials for a more energyefficient o2 separation process that utilizes lowgrade solar thermal energy at lower temperatures , such as waste heat from solar fuel production processes , we evaluate perovskites that offer high o2 conductivities and a stable crystal structure over a large range of oxygen nonstoichiometry.6a , c , 10 the o2 exchange capacity characterizes the tradeoff between high energy conversion efficiencies at low temperature during the endothermic reduction and high rates and extend of the oxygen separation process at high oxide reduction temperatures . for a perovskite with abo3 stoichiometry where a and b are metal cations in twelve and sixcoordinated interstices the tssos redox cycle can be represented by equations ( 1 ) and ( 2):(2 ) ( 1)equation image ( 2)equation image conceptually , o2 is stripped from a gas mixture at low po2 through oxidation of the perovskite at low temperatures . this yields as an output of the oxidation step an inert gas with a lowered po2 while concentrated o2 is evolved from the solid at an elevated temperature and increased po2 through partial reduction of the metal oxide . in principle , these reactions are analogous to the electrochemical oxygen reduction and evolution reactions ( orr / oer ) , where the bonding of o / oh or oh / ooh reaction intermediates to the catalyst surface controls the catalytic activity of the electrode surface.10 , 11 the ideal catalytic activity of a surface is determined by an intermediately strong bonding of the key reaction intermediates , which facilitates coverage of the surface with reactants and desorption of products from the surface , as described by the sabatier principle.12 analogously , we hypothesize that metal oxide redox materials for removal of o2 from gas mixtures with a lower po2 than the po2 during the extraction of o2 from the solid can be characterized with an intermediately strong binding of the lattice oxygen . to test this hypothesis , we screened the redox energetics of binary metal oxides across the periodic table using experimentbased thermochemical data.13 figure 1 a shows the free energy ( g ) of the oxidation and reduction reactions for 32 solid metal oxide and six metal / metal oxide pairs14 versus the thermochemical oxide stability . a ) free energy of the oxide oxidation at 600 k and oxide reduction at 900 k ( g rxn ) versus the enthalpy of the oxide reduction at 298 k ( h red ) . b ) the limiting free energy of the redox cycle ( g rxn , lim ) versus h red . the colored compositions were examined experimentally , with blue and red marked materials limited by the oxide reduction and oxidation , respectively , and purple marking materials that facilitate a redox tradeoff . c ) dftmodels of the oxidized and reduced srcoo3 and srcoo2.5 surface , representatively for strontium cobaltite . the analysis utilizes the enthalpy of the oxide reduction at room temperature as a descriptor1012 of the correlated reaction energetics , which is equivalent to the amount of energy required to break metal generally , either one of the two reactions is slightly more endergonic , thereby limiting the o2 exchange capacity . figure 1 b shows the limiting free energy of a redox cycle near the intersection of both correlations . as indicated by the volcanoshaped curve , the ideal redox material binds oxygen strongly enough to oxidize the oxide at relatively low temperatures stronger than the ag / ag2o reference but weakly enough to reduce the oxidized redox material at moderately higher temperatures the ideal metal oxide compositions are where these effects balance , located near the top of the volcano curve . ideally , this region corresponds to negative free energies for both reactions . for the temperatures chosen in our analysis , this can be achieved with rare materials such as rh2o / rho or toxic materials such as pbo / pb3o4.15 generally , the volcanolike shape of this correlation is due to the fact that the amount of energy absorbed for breaking metal oxygen bonds during the reduction step correlates with the amount of heat liberated when forming these bonds during the oxidation step . thus , as shown in figure s1 , the location of the volcanotop can be determined from only computing the reduction enthalpy as the entropy of o2 gas participating in either reaction is the same for all specific redox couples and as entropic contributions of the solids introduce significant deviation from these correlations only at significantly higher temperatures when approaching melting and boiling points . to tailor inexpensive and nontoxic metal oxides , we calculated the free energy of oxygen vacancy formation ( g v[o ] ) using density functional theory ( dft ) for twelve perovskites that have attracted attention for solidoxide fuel cells,6a , 10 air separation,6c and solarthermal applications.16 stoichiometric abo3(010 ) and oxygendeficient abo2.5(010 ) facets ( a = sr , ba , or la ; and b = mn , co , ni , or cu ) were modeled ( figure 1 c ) , using the gridbased projectoraugmented wave ( gpaw ) and atomic simulation environment ( ase ) electronicstructure code.17 figure 1 b shows the thermochemical stability and the reaction energetics as calculated from the scaling of g v[o ] and the redox energetics of the bulk oxides ( see the supporting information).18 the analysis predicts an ideal o2 exchange capacity for srcoo3 , relative to too strong and too weak oxygen binding for bamno3 and bacoo3 , respectively . we validated this descriptorbased design for metal oxide redox materials by means of dynamic o2 exchange experiments using srcoo3 , bamno3 , and bacoo3 , synthesized via the pecchini method3b and commercial ag2o and cu2o as reference materials . the composition and surface morphology of all solids were characterized using hightemperature xray diffraction ( htxrd ) and scanning electron microscopy ( sem ) . the o2 exchange capacity and exchange rates were determined by thermogravimetric analysis ( tga ) . figure 2 a and b display dynamic tga runs for srcoo2.95 , bacoo2.58 , bamno2.94 , ag2o , and cu2o ( initial stoichiometries ) that were cyclically reduced at 900 k and 0.2 bar o2 ( simulating air ) and oxidized at 600 k and 0.035 bar o2 ( simulating the composition of the gas phase from reducing ceria for solardriven splitting of co2 and h2o19 ) . as expected , cu2o and ag2o oxidize and reduce strongly to cuo and ag , respectively , essentially without exchanging o2 reversibly . compared to these reference materials , we find augmented o2 exchange capacities for the perovskites . srcoo2.95 reaches a maximum o2 exchange capacity of 440.012 mmol o2 per mol srcoo2.95 and a maximum o2 exchange rate of 12.10.003 mol o 2 min g srcoo 2.95 whereas bacoo2.58 and bamno2.94 perform at much lower capacities of 3.40.015 and 0.50.015 mmol o2 per mol of perovskite and lower exchange rates of 0.80.003 and 0.040.005 mol o 2 min gperovskite , respectively . as predicted by dft , the performance of bacoo3 and bamno3 appears limited by reoxidation and reduction , respectively . this theorybased screening of twelve perovskites identifies a wellknown composition , srcoo3 , that shows a significantly augmented performance for this novel application compared to the o2 exchange capacities and rates of the reference materials at the same conditions . additionally , with an o2 exchange rate of 12.1 mol o 2 min gperovskite , srcoo3 outperforms the stateoftheart cu2o / cuo cycle , which can not be used with lowgrade process heat at 600900 k and which has an o2 exchange rate of only 10 mol o 2 min g cu 2o ( for both , oxide reduction and oxidation ) at significantly higher and thereby economically less attractive temperatures of 11201450 k and comparable po2.3a dynamic o2 exchange : tga runs of a ) ag2o and cu2o and b ) srcoo3 , bacoo3 , and bamno3. c ) lattice constants of srcoo3 , bacoo3 , and bamno3 derived from htxrd analyses of oxide reduction at 0.2 bar po2 ( empty circles ) and oxide oxidation at 0.035 bar po2 ( filled circles ) . error bars are standard deviations within a 68 % confidence interval . to support that the tga data is indicative of reversible o2 exchange , figure 2 c shows the perovskite lattice constants computed using data from htxrd analysis . although all perovskites exhibit thermal expansion upon heating , only srcoo3 shows a major difference in the lattice expansion at varied po2 , which can be attributed to the formation and filling of o vacancies.20 this , along with the electronic structure trends and the tga analysis , suggests that srcoo3 is particularly suitable as a redox material for tssos . to understand how the oxide composition controls the o2 exchange , figure 3 a plots g v[o ] versus the dftcalculated bond length between the transition metal and the nearest o atom ( d b o ) at abo2.5(010 ) . generally , we observe stable o vacancies ( negative g v[o ] values ) correlating with large d b o values . this is in agreement with the lattice expansion shown in figure 2 c , as the lattice expansion due to a higher chemical potential for oxygen in the gas phase decreases the bonding of oxygen in the solid , which , in turn , increases the length of the metal oxygen bond . although the slope of this correlation is essentially due to the metal at the bsite interstices , the absolute value of d b o is governed by the metal at the a site , as demonstrated by the consistently higher d b o values of the ba versus the sr compounds however , the scatter of the correlation shown in figure 3 a suggests that the trends in the free energy of the o vacancy formation and in the metal oxygen bond length can not alone be rationalized with geometric arguments . correlation of g v[o ] with a ) d b o and b ) q o ( empty , lightgray , and darkgray symbols mark srbo2.5 , labo2.5 , and babo2.5 , whereas circles , squares , diamonds , and triangles mark acuo2.5 , anio2.5 , amno2.5 , and acoo2.5 ) . c ) charge density differences ( cdd ) of the marked surface after o vacancy formation relative to the stoichiometric surface and the reference gasphase o2 ( given at the height of the transition metal cation ) . figure 3 b plots g v[o ] versus the dftcalculated partial charge21 of oxygen ( q o ) . bacoo3 , for instance , accumulates less charge at the o anion than srcoo3 and bamno3 , which correlates with the facile reduction of bacoo3 . generally , g v[o ] scales with q o , with the slope corresponding approximately to the ionization energy of oxygen , 13.62 ev per electron22 and the intercept reflecting entropic contributions to g v[o ] and the reference chemical potential of oxygen in the gas phase ( see the supporting information ) . the quality of this linear correlation suggests that the trend in the free energy of the o vacancy formation is controlled by the enthalpy of breaking metal oxygen bonds , that is , by the quantity of electric charge transferred from the o atom to the lattice when forming the o vacancy . this is illustrated with figure 3 c , which shows the difference in the charge density distribution due to o vacancy formation at srcoo3(010 ) and bamno3(010 ) . although the partial charge of the o anion yielding the vacancy is approximately equal at both surfaces ( figure 3 b ) , the charge transfer from the bonding o 2p states to the 3d states of the transition metal is significantly higher at bamno3(010 ) relative to srcoo3 ( 010 ) . we note that this analysis describes the width of the 2p3d gap for 3d transition metals at the b site of abo3 perovskites . incorporating other metal cations into the b interstices , such as lanthanides with f states , may alter these trends due to a different entropycontrolled shape of the states that are accepting electrons when forming o vacancies . in summary , analogous to the enthalpy difference of bulk oxide reduction , we suggest that the charge transfer from o 2p to bsitemetal 3d states explains the higher o2 exchange capacity of srcoo3 versus bamno3 at the atomic scale . in accord with charge transfer controlling the formation of o vacancies at the oxide surface , our sabatier analysis employs the enthalpy of the bulk oxide reduction as a descriptor of the redox energetics . to demonstrate how this information can be used practically to predict the o2 exchange capacity of a metal oxide , we have determined the o2 exchange capacity for five metal oxides as the difference of the oxygen nonstoichiometry at equilibrium between o2 evolution at 900 k and 0.2 bar po2 and o2 fixation at 600 k and 0.035 bar po2 ( figure 4 ) . the plot shows that the o2 exchange capacity resembles the volcanoshaped trend of the limiting redox energetics . relative to this trend across three orders of magnitude are minor deviations , such as for bacoo3 , which are presumably due to differences in surface morphology , crystal structure , and the computational versus experimental nonstoichiometry ( see the supporting information ) . this demonstrates how computing the enthalpy of the oxide reduction from first principles allows predicting the o2 exchange capacity of metal oxides . predicting the o2 exchange capacity and the limiting free energy of solidstate o2 separation from the enthalpy of the oxide reduction at 298 k and 1 bar . although the present article focuses on the thermodynamics of o vacancy formation , the kinetics of conducting these vacancies from and to the surface are of equal importance when designing metal oxides for solidstate o2 separation . the defect chemistry of several perovskite families , including la2nio4+ with a relatively high mobility of o interstitials , has been investigated previously.23 in these perovskites excess oxygen is incorporated as interstitial o or o anions with anion frenkel pairs being the predominant intrinsic lattice defects.23 oxygen transport may be anisotropic23 or isotropic , such as in sr0.75y0.25coo2.625.24 a low frenkel energy may yield high o vacancy concentrations , in prbaco2o5.5 for instance,25 with an ordered sublattice of the a cations ensuring high oxygen ion mobility.25 similarly , dft was used previously to predict and understand oxygen conduction trends in metal oxides.18 these and other studies23 , 26 outline the prospects of an advanced understanding of oxygen conduction for designing advanced redox materials . based on the sabatier principle applied to the bonding of lattice oxygen atoms , we implemented a descriptorbased design principle for predicting the o2 exchange capacity of metal oxides and perovskites in particular . the computations were validated through dynamic o2 exchange experiments using ag2o , cu2o , and three perovskites and rationalized based on the compositiondependent bond geometry and charge transfer during the formation of o vacancies at the metal oxide surface . srcoo3 was identified as an ideal material for solardriven thermochemical separation of o2 . in a broader context , the presented principles may also aid the design of oxygen conductors for related applications , such as solidoxide fuel cells and air separation using dense ceramic membranes . thermochemical equilibrium calculations to guide the design of redox materials , the thermochemical equilibrium of binary bulk metal oxides , o2 , and their reduction products was determined at the specified po2 and temperatures from tabulated freeenergy data , with an absolute accuracy of 110 kj mol.13 per convention , negative free energy differences mark exergonic reactions . at the computed conditions , the correlations of g and h red for the metal oxide oxidation and reduction have average relative errors of 12.4 % and 17.6 % , respectively . these values increase with increasing temperature due to entropic contributions and indicate that the provided volcano plot could be employed for identifying materials active for thermochemical solidstate o2 separation . electronic structure calculations twelve perovskite surfaces were modeled using dft , performed with the gpaw code.17b , c exchangecorrelation interactions were treated by the revised perdew burke ernzerhof ( rpbe ) functional.27 atomic configurations were handled in ase.17a a fermi dirac smearing of 0.1 ev was used to achieve convergence , and the structure optimization results were extrapolated to 0 k. the linesearch broyden fletcher shanno ( bfgs ) algorithm was employed to optimize the atomic geometries until the maximum force was less than 0.05 ev . the utility of dft+u methods for surface calculations is not determined in general and was found unnecessary for many metal oxides.18 , 28 here , for all dft calculations the generalized gradient approximation ( gga ) was used without a hubbard u term as we found previously that the hubbard u correction did not improve the description of surface reactivity with the employed models.18 the cubic bulk structures of abo3 compositions consisted of one metal atom ( sr , ba , or la ) at the twelvecoordinated asite interstices , one metal atom ( mn , co , ni or cu ) at the sixcoordinated bsite interstices , and three oxygen atoms that were allowed to optimize their positions ( relax ) . the bulk structures had periodic boundary conditions in all directions and were modeled using a kpoint sampling of 444 . compositions containing mn , co , or ni were modeled using spinpolarized calculations and , to avoid reminiscent stress in the calculations , the lattice constants were chosen as the dftcalculated bulk lattice constants . table s1 provides a summary of the lattice constants and magnetic moments along with a discussion of the accuracy of the employed dft methods that predict the lattice constants within 0.363.89 % of experimental values . the aoterminated abo3(010 ) facet was chosen for modeling the perovskite surfaces as this facet was identified as being the thermodynamically most stable surface of cubic perovskites for various compositions.18 the surface models consisted of one upper abo3(010 ) layer that was allowed to relax and one lower abo3(010 ) layer constrained to the bulk geometry . the surfaces were periodically repeated in the directions parallel to the surface and were modeled with 10 of vacuum perpendicular to the surface . the partial charge density was determined for all atoms contained in the surface models through bader decomposition.21 to model the perovskite surfaces at different o vacancy concentrations , one third of the stoichiometric lattice oxygen in the upper surface layer was removed while the oxygen concentration in the lower surface layer was maintained . reduced and oxidized surface models with a2b2o5(010 ) and a2b2o6(010 ) stoichiometry , marked with the conventional abo3(010 ) and abo2.5(010 ) notation , respectively . the free energy of forming o vacancies ( g v[o ] ) at the surface was computed as follows [ eq . ( 3)]:18 , ( 3 ) ( 3)equation image where g v , g s , and go are the free energies of the perovskite surface with the o vacancies , the stoichiometric surface and the reference energy of the liberated lattice oxygen [ taken as the energy difference of stable h2o and h2 in the gas phase , see eq . ( 3 ) in the supporting information ] , such that negative free energies indicate exergonic reactions . the formation of o vacancies as computed corresponded to formation of one monolayer o vacancies equivalent to an oxygen nonstoichiometry of =0.5 in abo2.5 ( figure 1 c ) . details on converting the dftcomputed electronic energy to gibbs free energy at 298.15 k and 1.013 bar , the reference energies , and scaling relations18 to estimate the bulk formation energies are provided in the supporting information . the error of dftcomputed adsorption energies ( employed as a descriptor of surface reactivity , comparable to the energy of forming surface o vacancies in this work ) was estimated previously to be 0.08 ev.29 perovskite synthesis three perovskites , namely srcoo3 , bacoo3 , and bamno3 , were synthesized using a modified pecchini method , employing stoichiometric amounts of mn(no3)24 h2o ( alfa aesar , 98 % ) , sr(no3)2 ( alfa aesar , 98 % ) , co(no3)2 ( alfa aesar , 97.7 % ) , ba(no3)2 ( alfa aesar , 99 % ) , c2h6o ( alcosuisse , 96.1 % ) , and c6h8o7 ( fluka , 99.5 % ) . the solid products were ground using mortar and pestle , uniaxially pressed into pellets ( 10 metric tons , 6 and 25 mm in diameter ) , and sintered in air at 1473 k for 5 h ( srcoo3 and bacoo3 ) or in pure o2 at 1273 k for 48 h ( bamno3 ) . to achieve the desired oxidation state , the sintered srcoo3 was ground , fully immersed in naclo ( migros , < 5 % in h2o ) , washed with deionized water , and subsequently dried for at least 2 h at 473 k. ag2o ( merck , 99 % ) and cu2o ( johnson matthey alfa , 99.5 % ) were used as reference materials . solidstate analysis xrd and htxrd were performed in the bragg brentano geometry using cuk radiation ( 2080 2 , 0.06 min scan rate , 45 kv/20 ma output , panalytical / xpert mpd / dy636 , philips ) . the original oxygen content of the perovskites was srcoo2.95 , bacoo2.58 , and bamno2.94 before the redox cycling , as determined using tga ( sta 409/c/3 , netzsch ) of the complete reduction of the metal oxides in 5 % h2 in ar at 823 k and 1 bar . to estimate changes in the lattice constants , the perovskites were reduced by heating from 600 to 900 k in 100 k steps at 0.2 bar po2 and thereafter oxidized by cooling from 900 to 600 k in 100 k intervals at 0.035 bar po2 . the morphology of all materials was analyzed using sem ( 15 kv accelerating voltage , tm1000 , hitachi ) and is shown in detail in the supporting information . thermochemical cycling experiments starting materials ( 0.1 g ) were placed in an al2o3 crucible supported with an al2o3 rod on the microbalance of the tga ( 0.1 g ) . the materials were thereafter exposed to a gas flow ( constant flow rate of 200 ml min at 273 k and 1 bar ) with specified po2 , which was adjusted by mixing o2 ( 99.5 % , messer ) and n2 ( 99.999 % , carbagas ) using three electronic mass flow controllers ( mfc400 , netzsch ; accuracy 1 % , precision 1 ml min ) . the mass change of the samples was recorded during two consecutive redox cycles with oxide reduction at 900 k and 20 vol % o2 and oxide oxidation at 600 k and 3.5 vol % o2 , respectively ( 1 k ) . heating and cooling was performed at + 10 k min and 10 k min , respectively . to correct for buoyancy , blank runs were performed using the same measurement conditions employed for the experimental runs . the oxygen exchange capacity ( dimensionless ) was defined as the difference in the oxygen nonstoichiometry of the metal oxides after the oxide reduction ( red ) and after the oxide oxidation ( oxi):(4 ) ( 4)equation image where m red and m oxi are the metal oxide mass ( in g , determined using tga ) , after the oxide reduction , and after the oxide oxidation , and mo is the molar mass of oxygen ( in g mol).the uncertainties of the oxygen exchange capacities and rates were estimated using error propagation from the accuracies of the experimental analysis .

abstractseparation and concentration of o2 from gas mixtures is central to several sustainable energy technologies , such as solardriven synthesis of liquid hydrocarbon fuels from co2 , h2o , and concentrated sunlight . we introduce a rationale for designing metal oxide redox materials for oxygen separation through thermochemical pumping of o2 against a po2 gradient with lowgrade process heat . electronic structure calculations show that the activity of o vacancies in metal oxides pinpoints the ideal oxygen exchange capacity of perovskites . thermogravimetric analysis and hightemperature xray diffraction for srcoo3 , bacoo3 and bamno3 perovskites and ag2o and cu2o references confirm the predicted performance of srcoo3 , which surpasses the performance of stateoftheart cu2o at these conditions with an oxygen exchange capacity of 44 mmol o 2 mol srcoo 3 1 exchanged at 12.1 mol o 2 min1 g1 at 600900 k. the redox trends are understood due to lattice expansion and electronic charge transfer .

Introduction Results and Discussion Conclusions Experimental Section

renewable chemical fuels can be synthesized through solardriven electro , photo , and thermochemical splitting of co2 and h2o.1 the latter approach utilizes the entire spectrum of concentrated solar radiation as hightemperature process heat for the production of co and h2 ( syngas ) via metal oxide redox cycles.1b , 2 a critical drawback of this approach is the inert gas consumed to lower the partial pressure of oxygen ( po2 ) for shifting the thermodynamic equilibrium of the reduction step to lower temperatures.3 this , in turn , requires separation of o2 from the product gases for recycling the inert carrier gas and closing the material cycle.3a , 4 the separation of o2 has been a requirement in a variety of commercial applications such as oxycombustion , autothermal gasification of carbonaceous feedstock , and o2 removal to avoid catalyst passivation by o2 in fuel cells and when deoxygenating biofuels to make these more akin to petroleumderived fuels.5 industrially , o2 can be separated from air by pressure swing adsorption ( psa ) using zeolites and carbon molecular sieves , by ceramic mixed ionicelectronic conducting ( miec ) membranes,6 and by cryogenic distillation . psa and miec membranes can not produce highpurity inert gas,6b and separating o2 from gas mixtures at low po2 using membranes relies on a stripping gas with even lower po2 . since solar thermochemical cycles inherently suffer from heat losses , it would be beneficial to utilize an oxygen separation technology driven by waste heat . thermochemical solidstate o2 separation ( tssos ) using metal oxide redox materials such as cu2o / cuo,3a , 8 mn3o4/mn2o3,8 and coo / co3o4 8 , 9 utilizes lowgrade process heat and does not require electricity . tssos has the potential to separate and concentrate o2 at low po2 via temperatureswing.3a the current stateoftheart tssos redox material , cu2o , has a maximum oxygen exchange capacity ( , i.e. , the difference in the oxygen nonstoichiometry between reducing and oxidizing conditions ) of about 200 mmol o2 per mol cu2o , exchanged at approximately 10 mol o 2 min g cu 2o when cycled between 11201450 k.3a we show below that cu2o can not be employed with lowgrade process heat at 600900 k. with the aim of augmenting the o2 exchange capacities and rates of tssos redox materials for a more energyefficient o2 separation process that utilizes lowgrade solar thermal energy at lower temperatures , such as waste heat from solar fuel production processes , we evaluate perovskites that offer high o2 conductivities and a stable crystal structure over a large range of oxygen nonstoichiometry.6a , c , 10 the o2 exchange capacity characterizes the tradeoff between high energy conversion efficiencies at low temperature during the endothermic reduction and high rates and extend of the oxygen separation process at high oxide reduction temperatures . in principle , these reactions are analogous to the electrochemical oxygen reduction and evolution reactions ( orr / oer ) , where the bonding of o / oh or oh / ooh reaction intermediates to the catalyst surface controls the catalytic activity of the electrode surface.10 , 11 the ideal catalytic activity of a surface is determined by an intermediately strong bonding of the key reaction intermediates , which facilitates coverage of the surface with reactants and desorption of products from the surface , as described by the sabatier principle.12 analogously , we hypothesize that metal oxide redox materials for removal of o2 from gas mixtures with a lower po2 than the po2 during the extraction of o2 from the solid can be characterized with an intermediately strong binding of the lattice oxygen . to test this hypothesis , we screened the redox energetics of binary metal oxides across the periodic table using experimentbased thermochemical data.13 figure 1 a shows the free energy ( g ) of the oxidation and reduction reactions for 32 solid metal oxide and six metal / metal oxide pairs14 versus the thermochemical oxide stability . the analysis utilizes the enthalpy of the oxide reduction at room temperature as a descriptor1012 of the correlated reaction energetics , which is equivalent to the amount of energy required to break metal generally , either one of the two reactions is slightly more endergonic , thereby limiting the o2 exchange capacity . as indicated by the volcanoshaped curve , the ideal redox material binds oxygen strongly enough to oxidize the oxide at relatively low temperatures stronger than the ag / ag2o reference but weakly enough to reduce the oxidized redox material at moderately higher temperatures the ideal metal oxide compositions are where these effects balance , located near the top of the volcano curve . for the temperatures chosen in our analysis , this can be achieved with rare materials such as rh2o / rho or toxic materials such as pbo / pb3o4.15 generally , the volcanolike shape of this correlation is due to the fact that the amount of energy absorbed for breaking metal oxygen bonds during the reduction step correlates with the amount of heat liberated when forming these bonds during the oxidation step . to tailor inexpensive and nontoxic metal oxides , we calculated the free energy of oxygen vacancy formation ( g v[o ] ) using density functional theory ( dft ) for twelve perovskites that have attracted attention for solidoxide fuel cells,6a , 10 air separation,6c and solarthermal applications.16 stoichiometric abo3(010 ) and oxygendeficient abo2.5(010 ) facets ( a = sr , ba , or la ; and b = mn , co , ni , or cu ) were modeled ( figure 1 c ) , using the gridbased projectoraugmented wave ( gpaw ) and atomic simulation environment ( ase ) electronicstructure code.17 figure 1 b shows the thermochemical stability and the reaction energetics as calculated from the scaling of g v[o ] and the redox energetics of the bulk oxides ( see the supporting information).18 the analysis predicts an ideal o2 exchange capacity for srcoo3 , relative to too strong and too weak oxygen binding for bamno3 and bacoo3 , respectively . we validated this descriptorbased design for metal oxide redox materials by means of dynamic o2 exchange experiments using srcoo3 , bamno3 , and bacoo3 , synthesized via the pecchini method3b and commercial ag2o and cu2o as reference materials . the composition and surface morphology of all solids were characterized using hightemperature xray diffraction ( htxrd ) and scanning electron microscopy ( sem ) . the o2 exchange capacity and exchange rates were determined by thermogravimetric analysis ( tga ) . figure 2 a and b display dynamic tga runs for srcoo2.95 , bacoo2.58 , bamno2.94 , ag2o , and cu2o ( initial stoichiometries ) that were cyclically reduced at 900 k and 0.2 bar o2 ( simulating air ) and oxidized at 600 k and 0.035 bar o2 ( simulating the composition of the gas phase from reducing ceria for solardriven splitting of co2 and h2o19 ) . srcoo2.95 reaches a maximum o2 exchange capacity of 440.012 mmol o2 per mol srcoo2.95 and a maximum o2 exchange rate of 12.10.003 mol o 2 min g srcoo 2.95 whereas bacoo2.58 and bamno2.94 perform at much lower capacities of 3.40.015 and 0.50.015 mmol o2 per mol of perovskite and lower exchange rates of 0.80.003 and 0.040.005 mol o 2 min gperovskite , respectively . as predicted by dft , the performance of bacoo3 and bamno3 appears limited by reoxidation and reduction , respectively . additionally , with an o2 exchange rate of 12.1 mol o 2 min gperovskite , srcoo3 outperforms the stateoftheart cu2o / cuo cycle , which can not be used with lowgrade process heat at 600900 k and which has an o2 exchange rate of only 10 mol o 2 min g cu 2o ( for both , oxide reduction and oxidation ) at significantly higher and thereby economically less attractive temperatures of 11201450 k and comparable po2.3a dynamic o2 exchange : tga runs of a ) ag2o and cu2o and b ) srcoo3 , bacoo3 , and bamno3. c ) lattice constants of srcoo3 , bacoo3 , and bamno3 derived from htxrd analyses of oxide reduction at 0.2 bar po2 ( empty circles ) and oxide oxidation at 0.035 bar po2 ( filled circles ) . although all perovskites exhibit thermal expansion upon heating , only srcoo3 shows a major difference in the lattice expansion at varied po2 , which can be attributed to the formation and filling of o vacancies.20 this , along with the electronic structure trends and the tga analysis , suggests that srcoo3 is particularly suitable as a redox material for tssos . this is in agreement with the lattice expansion shown in figure 2 c , as the lattice expansion due to a higher chemical potential for oxygen in the gas phase decreases the bonding of oxygen in the solid , which , in turn , increases the length of the metal oxygen bond . although the slope of this correlation is essentially due to the metal at the bsite interstices , the absolute value of d b o is governed by the metal at the a site , as demonstrated by the consistently higher d b o values of the ba versus the sr compounds however , the scatter of the correlation shown in figure 3 a suggests that the trends in the free energy of the o vacancy formation and in the metal oxygen bond length can not alone be rationalized with geometric arguments . bacoo3 , for instance , accumulates less charge at the o anion than srcoo3 and bamno3 , which correlates with the facile reduction of bacoo3 . this is illustrated with figure 3 c , which shows the difference in the charge density distribution due to o vacancy formation at srcoo3(010 ) and bamno3(010 ) . incorporating other metal cations into the b interstices , such as lanthanides with f states , may alter these trends due to a different entropycontrolled shape of the states that are accepting electrons when forming o vacancies . in summary , analogous to the enthalpy difference of bulk oxide reduction , we suggest that the charge transfer from o 2p to bsitemetal 3d states explains the higher o2 exchange capacity of srcoo3 versus bamno3 at the atomic scale . in accord with charge transfer controlling the formation of o vacancies at the oxide surface , our sabatier analysis employs the enthalpy of the bulk oxide reduction as a descriptor of the redox energetics . to demonstrate how this information can be used practically to predict the o2 exchange capacity of a metal oxide , we have determined the o2 exchange capacity for five metal oxides as the difference of the oxygen nonstoichiometry at equilibrium between o2 evolution at 900 k and 0.2 bar po2 and o2 fixation at 600 k and 0.035 bar po2 ( figure 4 ) . the plot shows that the o2 exchange capacity resembles the volcanoshaped trend of the limiting redox energetics . relative to this trend across three orders of magnitude are minor deviations , such as for bacoo3 , which are presumably due to differences in surface morphology , crystal structure , and the computational versus experimental nonstoichiometry ( see the supporting information ) . this demonstrates how computing the enthalpy of the oxide reduction from first principles allows predicting the o2 exchange capacity of metal oxides . although the present article focuses on the thermodynamics of o vacancy formation , the kinetics of conducting these vacancies from and to the surface are of equal importance when designing metal oxides for solidstate o2 separation . the defect chemistry of several perovskite families , including la2nio4+ with a relatively high mobility of o interstitials , has been investigated previously.23 in these perovskites excess oxygen is incorporated as interstitial o or o anions with anion frenkel pairs being the predominant intrinsic lattice defects.23 oxygen transport may be anisotropic23 or isotropic , such as in sr0.75y0.25coo2.625.24 a low frenkel energy may yield high o vacancy concentrations , in prbaco2o5.5 for instance,25 with an ordered sublattice of the a cations ensuring high oxygen ion mobility.25 similarly , dft was used previously to predict and understand oxygen conduction trends in metal oxides.18 these and other studies23 , 26 outline the prospects of an advanced understanding of oxygen conduction for designing advanced redox materials . based on the sabatier principle applied to the bonding of lattice oxygen atoms , we implemented a descriptorbased design principle for predicting the o2 exchange capacity of metal oxides and perovskites in particular . the computations were validated through dynamic o2 exchange experiments using ag2o , cu2o , and three perovskites and rationalized based on the compositiondependent bond geometry and charge transfer during the formation of o vacancies at the metal oxide surface . in a broader context , the presented principles may also aid the design of oxygen conductors for related applications , such as solidoxide fuel cells and air separation using dense ceramic membranes . thermochemical equilibrium calculations to guide the design of redox materials , the thermochemical equilibrium of binary bulk metal oxides , o2 , and their reduction products was determined at the specified po2 and temperatures from tabulated freeenergy data , with an absolute accuracy of 110 kj mol.13 per convention , negative free energy differences mark exergonic reactions . these values increase with increasing temperature due to entropic contributions and indicate that the provided volcano plot could be employed for identifying materials active for thermochemical solidstate o2 separation . electronic structure calculations twelve perovskite surfaces were modeled using dft , performed with the gpaw code.17b , c exchangecorrelation interactions were treated by the revised perdew burke ernzerhof ( rpbe ) functional.27 atomic configurations were handled in ase.17a a fermi dirac smearing of 0.1 ev was used to achieve convergence , and the structure optimization results were extrapolated to 0 k. the linesearch broyden fletcher shanno ( bfgs ) algorithm was employed to optimize the atomic geometries until the maximum force was less than 0.05 ev . the utility of dft+u methods for surface calculations is not determined in general and was found unnecessary for many metal oxides.18 , 28 here , for all dft calculations the generalized gradient approximation ( gga ) was used without a hubbard u term as we found previously that the hubbard u correction did not improve the description of surface reactivity with the employed models.18 the cubic bulk structures of abo3 compositions consisted of one metal atom ( sr , ba , or la ) at the twelvecoordinated asite interstices , one metal atom ( mn , co , ni or cu ) at the sixcoordinated bsite interstices , and three oxygen atoms that were allowed to optimize their positions ( relax ) . ( 3)]:18 , ( 3 ) ( 3)equation image where g v , g s , and go are the free energies of the perovskite surface with the o vacancies , the stoichiometric surface and the reference energy of the liberated lattice oxygen [ taken as the energy difference of stable h2o and h2 in the gas phase , see eq . the formation of o vacancies as computed corresponded to formation of one monolayer o vacancies equivalent to an oxygen nonstoichiometry of =0.5 in abo2.5 ( figure 1 c ) . the error of dftcomputed adsorption energies ( employed as a descriptor of surface reactivity , comparable to the energy of forming surface o vacancies in this work ) was estimated previously to be 0.08 ev.29 perovskite synthesis three perovskites , namely srcoo3 , bacoo3 , and bamno3 , were synthesized using a modified pecchini method , employing stoichiometric amounts of mn(no3)24 h2o ( alfa aesar , 98 % ) , sr(no3)2 ( alfa aesar , 98 % ) , co(no3)2 ( alfa aesar , 97.7 % ) , ba(no3)2 ( alfa aesar , 99 % ) , c2h6o ( alcosuisse , 96.1 % ) , and c6h8o7 ( fluka , 99.5 % ) . the solid products were ground using mortar and pestle , uniaxially pressed into pellets ( 10 metric tons , 6 and 25 mm in diameter ) , and sintered in air at 1473 k for 5 h ( srcoo3 and bacoo3 ) or in pure o2 at 1273 k for 48 h ( bamno3 ) . to achieve the desired oxidation state , the sintered srcoo3 was ground , fully immersed in naclo ( migros , < 5 % in h2o ) , washed with deionized water , and subsequently dried for at least 2 h at 473 k. ag2o ( merck , 99 % ) and cu2o ( johnson matthey alfa , 99.5 % ) were used as reference materials . the original oxygen content of the perovskites was srcoo2.95 , bacoo2.58 , and bamno2.94 before the redox cycling , as determined using tga ( sta 409/c/3 , netzsch ) of the complete reduction of the metal oxides in 5 % h2 in ar at 823 k and 1 bar . the oxygen exchange capacity ( dimensionless ) was defined as the difference in the oxygen nonstoichiometry of the metal oxides after the oxide reduction ( red ) and after the oxide oxidation ( oxi):(4 ) ( 4)equation image where m red and m oxi are the metal oxide mass ( in g , determined using tga ) , after the oxide reduction , and after the oxide oxidation , and mo is the molar mass of oxygen ( in g mol).the uncertainties of the oxygen exchange capacities and rates were estimated using error propagation from the accuracies of the experimental analysis .

the original method to remove the epithelium before the excimer laser ablation was manual mechanical scraping , which was later enhanced by using an alcohol solution or brush . in 2003 , camellin proposed a new alcohol - assisted technique called laser - assisted subepithelial keratectomy ( lasek ) that enabled the epithelium to be preserved as a flap and reapplying it to the ocular surface after treatment . epithelial laser in situ keratomileusis ( epi - lasik ) is another method that uses an epithelial flap , but is performed with a microkeratome with a blunt oscillating blade . in the late 1990s , transepithelial photorefractive keratectomy ( prk ) was introduced where removal of the epithelium is carried out with laser phototherapeutic ablation followed by a laser refractive ablation of the stroma . this 2-step technique was not widely used due to the prolonged surgery time with the older generation of lasers , increased pain , and a lack of adjusted nomograms . newer generation of faster lasers and improved ablation algorithms and nomograms have over the years , allowed development of a new ( tprk ) variant of transepithelial prk . single - step transepithelial prk allows removing the epithelium and stroma in a single step with 1 ablation profile . this profile is calculated taking into account data from the literature estimating the central epithelial thickness of a normal cornea to be 55 and 65 m at 4 mm from the center , superimposed on the corneal wavefront guided aspheric ablation profiles . several studies have compared 2-step transepithelial prk to standard prk performed with alcohol or mechanical epithelial removal with variable results . tprk is a relatively new procedure and a limited number of publications are currently available . only 3 direct comparisons between a single - step tprk and conventional prk are published so far . meanwhile thus , there is a need for updated comparative evaluations based on a larger number of eyes . the aim of this study is to compare 3-month refractive results , predictability , safety , and efficacy of single - step transepithelial prk with alcohol - assisted prk ( aaprk ) when used to correct myopia and compound myopic astigmatism . control study comprised eyes that underwent either single - step transepithelial prk ( tprk ) or aaprk between august 2012 and april 2014 , at the oftalmika eye hospital , bydgoszcz , poland . before the procedure , each patient was adequately informed about the study as well as the risks and benefits of the surgery , and provided signed informed consent in accordance with the declaration of helsinki . inclusion criteria were as follows : age over 21 years , primary myopia or compound myopic astigmatism , preoperative manifest refraction spherical equivalent ( mrse ) within the range of 1.0 to 9.5 d , a stable refractive error for at least 12 months before the surgery , contact lens discontinuation for at least 3 weeks , estimated corneal stromal bed thickness of more than 300 m at the thinnest point . exclusion criteria were previous ocular surgery , any diagnosed ocular disease , a history of ocular trauma , irregular astigmatism on corneal topography , systemic disease that could affect corneal wound healing , and pregnancy . one patient developed retrobulbar neuritis of the left optic nerve 6 weeks after the surgery and this eye was excluded from the analysis . the choice of the procedure was based on patients preferences as tprk is a more expansive procedure . there are no significant differences in preoperative variables of patients in the tprk and aaprk groups , except the gender . patients who attended all visits , thus without any missing data , were included into analysis . demographics and preoperative variables of patients in the tprk and aaprk groups preoperative information on general and ocular medical history , contact lens wear , and medication use was obtained from each patient . the examination included uncorrected distance visual acuity ( udva ) , corrected distance visual acuity ( cdva ) , manifest and cycloplegic refraction , slit lamp biomicroscopy , tonometry , specular microscopy ( em-3000 , tomey , erlangen , germany ) , pupillometry , scheimpflug camera tomography ( sirius , schwind eye - tech - solutions gmbh , kleinostheim , germany ) , ocular aberrometry ( irx-3 , imagine eyes , paris , france ) , and fundus examination . all surgeries were performed with 6th - generation amaris excimer laser , version 750 s ( schwind eye - tech - solutions ) . the treatments were mostly aimed at emmetropia baring a few eyes with a target refraction of 0.25 d or 0.5 d ( nondominant eye in case of older patients ) . however , in statistical analysis and standardized graphs presented as results in this study , only eyes targeted plano ( plano target ) were taken it into account wherever necessary . before the surgery , proparacaine hydrochloride 0.5% drops ( alcaine , alcon , fort worth , tx ) were instilled 3 times within a 5-min interval . the cornea was exposed to a 20% ethyl alcohol solution for 30 s with the use of a well . subsequently , a superficial cut of the epithelium was made with either an 8.5- or 9.5-mm diameter trephine . the epithelium was mechanically debrided with a spatula . in the tprk group , where aspheric aberration - free transprk ablation algorithm was used ( schwind eye - tech - solutions ) , the epithelium was removed during laser ablation only from the area of the total ablation zone , which is the sum of the optical and transition zones . in both groups in all cases , 0.02% mitomycin c ( mmc ) mmc application was followed by generous irrigation of the eye with room temperature balanced solution . a bandage contact lens was applied ( acuvue oasis , j&j , new brunswick , tx ) for 7 days . the postoperative regimen included 0.3% tobramycin drops ( tobrexan , alcon , new brunswick , tx ) for 1 month , 0.1% diclofenac drops ( dicloabak , laboratoires thea , clermont - ferrand , france ) for 1 month , 0.15% hyaluronic acid drops ( biolan , penta arzneimittel , stulln , germany ) for 3 months , and 0.1% dexamethasone drops ( dexafree , laboratoires thea , stulln , germany ) 3 times daily for the first month , twice daily for the second month , and once daily for the third month . patients were instructed to visit the clinic for postoperative examinations after 1 day , 1 week , 1 month , and 3 months . examinations at 1 day , 1 week , and 1 month included udva , cdva , manifest refraction , tonometry , and slit lamp biomicroscopy . corneal haze was evaluated as proposed by fantes at al ( 0 = no haze ; 0.5 = trace haze on oblique illumination ; 1 = corneal cloudiness not interfering with the visibility of fine iris details ; 2 = mild effacement of fine iris details ; 3 and 4 = details of the lens and iris not discernible ) . at the last visit all ocular aberrations were measured for pupil diameter of 4 mm . moreover , immediately after the surgery and 1 week after we used a discrete , 10-category verbal rating scale ( vrs , 1no pain and 10the worst possible pain ) to evaluate pain level . three months after the surgery patients were asked about overall satisfaction with the surgery and ( high , moderate , low , not satisfied ) , and whether they would decide to have a surgery again ( yes , no ) . data were analyzed using datagraph - med version 4.20 d ( ingenieurbro pieger gmbh , wendelstein , germany ) , which is a relational database designed for refractive data analysis . statistical analysis was performed using statistica 10 software ( statsoft inc . , when numeric data were compared the z - test was used ( for distribution of mean preop mrse , mean preop mrs , mean preop mrc , and respective postoperative values ) . for the comparison of nominal data the chi - squared test ( for sex , follow - up rate , majority of complications , and satisfaction ) or fisher exact test ( for haze ) were used . for all tests , preoperative information on general and ocular medical history , contact lens wear , and medication use was obtained from each patient . the examination included uncorrected distance visual acuity ( udva ) , corrected distance visual acuity ( cdva ) , manifest and cycloplegic refraction , slit lamp biomicroscopy , tonometry , specular microscopy ( em-3000 , tomey , erlangen , germany ) , pupillometry , scheimpflug camera tomography ( sirius , schwind eye - tech - solutions gmbh , kleinostheim , germany ) , ocular aberrometry ( irx-3 , imagine eyes , paris , france ) , and fundus examination . all surgeries were performed with 6th - generation amaris excimer laser , version 750 s ( schwind eye - tech - solutions ) . the treatments were mostly aimed at emmetropia baring a few eyes with a target refraction of 0.25 d or 0.5 d ( nondominant eye in case of older patients ) . however , in statistical analysis and standardized graphs presented as results in this study , only eyes targeted plano ( plano target ) were taken it into account wherever necessary . before the surgery , proparacaine hydrochloride 0.5% drops ( alcaine , alcon , fort worth , tx ) were instilled 3 times within a 5-min interval . , the cornea was exposed to a 20% ethyl alcohol solution for 30 s with the use of a well . subsequently , a superficial cut of the epithelium was made with either an 8.5- or 9.5-mm diameter trephine . the epithelium was mechanically debrided with a spatula . in the tprk group , where aspheric aberration - free transprk ablation algorithm was used ( schwind eye - tech - solutions ) , the epithelium was removed during laser ablation only from the area of the total ablation zone , which is the sum of the optical and transition zones . in both groups in all cases , 0.02% mitomycin c ( mmc ) was applied for 2 min based on the standard protocol . mmc application was followed by generous irrigation of the eye with room temperature balanced solution . after the surgery , a bandage contact lens was applied ( acuvue oasis , j&j , new brunswick , tx ) for 7 days . the postoperative regimen included 0.3% tobramycin drops ( tobrexan , alcon , new brunswick , tx ) for 1 month , 0.1% diclofenac drops ( dicloabak , laboratoires thea , clermont - ferrand , france ) for 1 month , 0.15% hyaluronic acid drops ( biolan , penta arzneimittel , stulln , germany ) for 3 months , and 0.1% dexamethasone drops ( dexafree , laboratoires thea , stulln , germany ) 3 times daily for the first month , twice daily for the second month , and once daily for the third month . patients were instructed to visit the clinic for postoperative examinations after 1 day , 1 week , 1 month , and 3 months . examinations at 1 day , 1 week , and 1 month included udva , cdva , manifest refraction , tonometry , and slit lamp biomicroscopy . corneal haze was evaluated as proposed by fantes at al ( 0 = no haze ; 0.5 = trace haze on oblique illumination ; 1 = corneal cloudiness not interfering with the visibility of fine iris details ; 2 = mild effacement of fine iris details ; 3 and 4 = details of the lens and iris not discernible ) . at the last visit all ocular aberrations were measured for pupil diameter of 4 mm . moreover , immediately after the surgery and 1 week after we used a discrete , 10-category verbal rating scale ( vrs , 1no pain and 10the worst possible pain ) to evaluate pain level . three months after the surgery patients were asked about overall satisfaction with the surgery and ( high , moderate , low , not satisfied ) , and whether they would decide to have a surgery again ( yes , no ) . data were analyzed using datagraph - med version 4.20 d ( ingenieurbro pieger gmbh , wendelstein , germany ) , which is a relational database designed for refractive data analysis . statistical analysis was performed using statistica 10 software ( statsoft inc . , tulsa , ok ) . smirnov test . when numeric data were compared the z - test was used ( for distribution of mean preop mrse , mean preop mrs , mean preop mrc , and respective postoperative values ) . for the comparison of nominal data the chi - squared test ( for sex , follow - up rate , majority of complications , and satisfaction ) or fisher exact test ( for haze ) were used . for all tests , p < 0.05 was considered statistically significant . the mean ablation time was 44.35 13.68 s in the tprk group and 16.85 9.58 s in the aaprk group ( p < 0.001 ) , whereas mean time of the whole procedure , from introduction to removal of the lid speculum , was 165 24.62 s and 254 32.14 s , respectively ( p < 0.001 ) . in the tprk and aaprk group , respectively , the mean diameter of the optical zone was 6.96 0.45 and 7.11 0.43 mm ( p = 0.47 ) , and the transition zone was 1.39 0.50 and 1.29 0.63 mm ( p = 0.55 ) . the minimal estimated stromal residual thickness was 306 m among all the analyzed eyes ( 309 m in tprk group and 306 m in aaprk group ) . the 3-month follow - up rate was 82.1% in the tprk group ( n = 145 ) and 86.4% in the aaprk group ( n = 90 ) , because some of the patients were not able or not willing to attend all of the required visits . mean postoperative mrse was 0.14 0.26 d in the tprk group and 0.12 0.20 d in the aaprk group ( p = 0.9 ) . in the tprk group , the postoperative refractive spherical equivalent in 63% of eyes were within 0.13 d , 12% within + 0.14 to + 0.5 d , 24% within 0.14 to 0.5 d , and 1% within 0.5 and 1.0 d ; the respective values in the aaprk group were 70% , 4% , 26% , and 0% . refractive results , predictability , safety , and efficacy 3 months after the surgery are shown in figures 1 and 2 as standard graphs recommended for reporting refractive surgery outcomes . udva was 20/20 or better in 97% of eyes in the tprk group and 94% in the aaprk group ( p = 0.45 ) . in the tprk group , 13% of eyes lost 1 line of cdva and 30% gained a line or 2 . in the aaprk group , 21% of eyes lost 1 line of cdva and 31% gained a line or 2 ( p = 0.48 ) . comparison of uncorrected distance visual acuity ( a ) , change in corrected distance visual acuity ( b ) , and attempted vs achieved spherical equivalent ( c ) in single - step transepithelial photorefractive keratectomy ( tprk ; left panels ) and alcohol - assisted photorefractive keratectomy ( aaprk ; right panels ) groups . comparison of the spherical equivalent refractive accuracy ( d ) , refractive astigmatism ( e ) , and stability of spherical equivalent refraction ( f ) in single - step transepithelial photorefractive keratectomy ( tprk ; left panels ) and alcohol - assisted photorefractive keratectomy ( aaprk ; right panels ) groups . mean preoperative higher order rms for 4 mm pupil was 0.15 0.15 m in the tprk group and 0.17 0.21 m in the aaprk group ( p = 0.21 ) , and the postoperative values were 0.21 0.23 and 0.19 0.12 m ( p = 0.37 ) , respectively . the differences between the preoperative and postoperative higher - order rms were not significant for both groups ( p = 0.13 for tprk group and p = 0.27 for the prk group ) . the mean pain scores after the surgery were 4.78 2.65 in the tprk group and 4.59 2.85 in the aaprk group ( p = 0.85 ) . there were also no differences in pain intensity during first days after the surgery ( mean scores of 4.46 2.54 and 4.51 2.36 in the tprk and aaprk groups , respectively ; p = 0.86 ) . after tprk , 86.25% of patients declared high satisfaction with the surgery compared to 88.24% patients after aaprk ( p = 0.46 ) . the ratio for moderate satisfaction was 13.75% for tprk and 11.76% for aaprk , respectively ( p = 0.54 ) . the total postoperative complication rate was 19.31% in the tprk group and 14.44% in the aaprk group ( p = 0.13 ) . a slightly higher incidence of haze was detected with the slit lamp for the tprk group ( 13.79% ) , compared to the aaprk group ( 8.89% ) ; however , the results of the fisher exact test suggest that the proportions of patients falling in each subcategory within each group did not differ significantly ( p = 0.09 ) . the intensity of haze was also not statistically significantly different between groups and was at the 0.5 level in all but 2 eyes after tprk and 1 eye after aaprk in which the incidence of haze was evaluated at level 1 . during the follow up there was no statistically significant difference in the incidence of other postoperative complications , which included elevated iop in 1.38% of eyes after tprk and 2.22% after aaprk ; decreased visual acuity at night : 1.38% of eyes after tprk and 1.11% after aaprk ; more intensive dry eye symptoms in 2.76% of eyes after tprk and 2.22% after aaprk . no postoperative complications , such as epitheliopathy , delayed re - epithelialization or recurrent corneal erosion , were not reported to a level of clinical significance in our cohort . aspheric aberration - free ablation profile of single - step transepithelial prk ( transprk ) used in the study , has many implications over the standard aaprk procedures . the ablation profile is calculated estimating that the central epithelial thickness of a normal cornea is 55 and 65 m at 4 mm from the center . therefore , the epithelial thickness profile resembles a slight hyperopic treatment ( < 0.75 d ) and proper compensation helps to avoid hyperopic shift . the laser system is tuned to compensate for a difference in photoablative rates of the stroma and the epithelial tissue , which is approximately 20% higher in the epithelium . since 1 epithelial ablation algorithm is used for all eyes in tprk , regardless of the actual epithelial topometry , more stroma might be ablated than necessary in eyes with a thin epithelium , whereas in eyes with a thick epithelium the refractive part of the ablation might begin where there is still some epithelium left on the surface . in corneas with high toxicity , the epithelial thickness profile along the steepest meridian may differ from the thickness profile along the flattest meridian . moreover , in light of the studies by reinstein et al and kanellopoulos and asimellis the assumption that the epithelium thickness map is rotationally symmetrical may not be appropriate . by means of very high - frequency digital ultrasonography , reinstein et al found that the mean epithelial thickness at the corneal vertex was 53.4 4.6 m , and the average epithelial thickness map showed that the corneal epithelium was thicker inferiorly than superiorly ( 5.9 m at the 3-mm radius , p < 0.001 ) and thicker nasally than temporally ( 1.3 m at the 3-mm radius , p < 0.001 ) . the location of the thinnest epithelium was temporally displaced on average 0.33 mm , and 0.90 mm superiorly with reference to the corneal vertex . they used spectral - domain anterior - segment optical coherence tomography and measured a mean epithelial thickness at the pupil center of 53.28 3.34 m , superiorly 51.86 3.78 m , and inferiorly 53.81 3.44 m . both papers show high inter - individual variability of the central epithelial thickness and 3-dimensional epithelial maps . in theory , the above - mentioned findings may deteriorate the refractive results , predictability , safety , and efficacy of transprk ablations in comparison to the standard aaprk procedures . however , our clinical results analyzing largest material published so far , show the opposite . in our study in which the latest version of the tprk algorithm was used , we did not observe statistically significant differences between the tprk group and the aaprk group in terms of udva , cdva , and mrse , 3 months after the surgery . the correlation between attempted versus achieved mrse was very high in both groups with no statistically significant difference between the 2 groups . shortly after the procedure was introduced , luger et al , aslanides et al , and fadlallah et al evaluated relatively small cohorts and came to the similar conclusions ; in myopia and compound myopic astigmatism correction , the refractive and visual outcomes of single - step transepithelial prk are not different from those of conventional prk . however , luger et al reported that the postoperative mean spherical equivalent in tprk was slightly hyperopic at + 0.07 0.23 d. moreover , our results in both the tprk and aaprk groups were among the best achieved with surface ablation in terms of postoperative udva , postoperative refractive spherical equivalent , and refractive astigmatism . unequal preoperative epithelial thickness might potentially also be a source of higher - order aberrations , but we did not find statistically significant differences in the preop and postop higher - order rms between the groups . thus , the natural interindividual variability of epithelial thickness maps did not deteriorate the clinical results in the studied population in a noticeable way . one may expect that the epithelial map would have some similar features before and after the surgery , for example , the epithelium may be thicker inferiorly than superiorly and thicker nasally than temporally . reinstein et al and kanellopoulos and asimellis confirmed thickening of the epithelium centrally and progressively less thickening centrifugally across the central 6 mm after myopic correction . in both studies , the epithelial thickness was averaged within annular bands centered on the corneal vertex ; thus , these studies do not verify the above - mentioned hypothesis . another potential disadvantage of transepithelial prk is the higher total excimer laser energy load . in our study , mean ablation time was 163% longer in the transprk group ; however , the majority of the laser energy was delivered to the epithelium . excimer laser energy , among other effects , causes increase of the temperature of the stromal tissue , which is the main risk factor for haze formation , an inherent complication of excimer laser refractive surgery . after surface ablation , up to 52% of eyes develop some degree of corneal haze in the first months , with values most often reported are within the range of 5% to 20% . in our study population up to 3 months after the surgery , haze was more often detected in the tprk group ; however , the difference did not reach statistical significance ( 13.79% vs 8.89% , p = 0.09 ) . in all eyes , the intensity of haze was very low , in almost all cases at the 0.5 level , and the difference in haze intensity between groups was not statistically significant . the nonsignificant differences in haze intensity in the 2 groups could be attributed to the use of mmc . it must be accounted here that the use of mmc does not allow us to evaluate haze formation for the 2 techniques in an unbiased way . a weak point of our study , except lack of randomization , is that the haze was evaluated subjectively with the slit lamp by an unmasked examiner . in our opinion , the intensity of haze after transepithelial prk and the impact of mmc on haze formation need to be further studied , preferably with masked examiners and with tools that provide objective measurements , like optical densitometry . there are statistical more females in tprk group , which may be a source of bias especially in pain evaluation , as pain perception may be positively influenced by female gender . high - resolution spectral optical coherence tomography ( oct ) with speckle contrast reduction also failed to detect differences in the corneal healing processes after tprk and aaprk , except for the shorter time to cover the stroma with epithelium in the tprk group . the main reason explaining this observation is that the diameter of epithelial removal matches the total ablation zone in transepithelial prk treatments , decreasing the wound surface , and shortening the epithelial closure time . another advantage of tprk is reduced surgery time . in our study , the total surgery time was reduced by 35% in comparison to aaprk . aslanides et al and fadlallah et al reported decreased postoperative pain after the single - step transepithelial prk ; however , our results failed to confirm these findings . transepithelial approaches allow maximum correspondence between the corneal topography and the ablation profile , which may be especially useful in customized treatments of irregular corneal astigmatism . the topographic map corresponds more closely with the epithelial surface than with the stromal surface since the epithelium acts as a natural mask , thinning over stromal protrusions and thickening over excavations . several papers confirmed the value of customized , topography - guided transepithelial ablation in correcting irregular corneal astigmatism . however , in current transepithelial customized ablation profiles , a difference in photoablative rates of the stroma and the epithelial tissue can not be compensated precisely as long as an epithelial thickness map is not taken into account . in the future , the advent of a high - resolution oct technique could allow proper representation of the epithelial layer , potentially leading to improved customized epithelial ablations . in conclusion , single - step transepithelial prk and conventional prk performed on regular corneas produce very similar results 3 months after the surgery . these procedures are predictable , effective , and safe for correction of myopia and compound myopic astigmatism .

abstracttransepithelial photorefractive keratectomy ( tprk ) , where both the epithelium and stroma are removed in a single - step , is a relatively new procedure of laser refractive error correction . this study compares the 3-month results of myopia and compound myopic astigmatism correction by tprk or conventional alcohol - assisted prk ( aaprk).this prospective , nonrandomized , case control study recruited 148 consecutive patients ; 93 underwent tprk ( 173 eyes ) and 55 aaprk ( 103 eyes ) . refractive results , predictability , safety , and efficacy were evaluated during the 3-month follow - up . the main outcome measures were uncorrected distance visual acuity ( udva ) , corrected distance visual acuity ( cdva ) , and mean refractive spherical equivalent ( mrse).mean preoperative mrse was 4.30 1.72 d and 4.33 1.96 d , respectively ( p = 0.87 ) . the 3-month follow - up rate was 82.1% in the tprk group ( n = 145 ) and 86.4% in aaprk group ( n = 90 ) , p = 0.81 . postoperative udva was 20/20 or better in 97% and 94% of eyes , respectively ( p = 0.45 ) . in the tprk and aaprk groups , respectively , 13% and 21% of eyes lost 1 line of cdva , and 30% and 31% gained 1 or 2 lines ( p = 0.48 ) . mean postoperative mrse was 0.14 0.26 d in the tprk group and 0.12 0.20 d in the aaprk group ( p = 0.9 ) . the correlation between attempted versus achieved mrse was equally high in both groups.single-step transepithelial prk and conventional prk provide very similar results 3 months postoperatively . these procedures are predictable , effective , and safe for correction of myopia and compound myopic astigmatism .

INTRODUCTION METHODS Preoperative Examination Surgical Technique Postoperative Examinations Statistical Analysis RESULTS DISCUSSION

in 2003 , camellin proposed a new alcohol - assisted technique called laser - assisted subepithelial keratectomy ( lasek ) that enabled the epithelium to be preserved as a flap and reapplying it to the ocular surface after treatment . in the late 1990s , transepithelial photorefractive keratectomy ( prk ) was introduced where removal of the epithelium is carried out with laser phototherapeutic ablation followed by a laser refractive ablation of the stroma . newer generation of faster lasers and improved ablation algorithms and nomograms have over the years , allowed development of a new ( tprk ) variant of transepithelial prk . single - step transepithelial prk allows removing the epithelium and stroma in a single step with 1 ablation profile . tprk is a relatively new procedure and a limited number of publications are currently available . only 3 direct comparisons between a single - step tprk and conventional prk are published so far . meanwhile thus , there is a need for updated comparative evaluations based on a larger number of eyes . the aim of this study is to compare 3-month refractive results , predictability , safety , and efficacy of single - step transepithelial prk with alcohol - assisted prk ( aaprk ) when used to correct myopia and compound myopic astigmatism . control study comprised eyes that underwent either single - step transepithelial prk ( tprk ) or aaprk between august 2012 and april 2014 , at the oftalmika eye hospital , bydgoszcz , poland . before the procedure , each patient was adequately informed about the study as well as the risks and benefits of the surgery , and provided signed informed consent in accordance with the declaration of helsinki . inclusion criteria were as follows : age over 21 years , primary myopia or compound myopic astigmatism , preoperative manifest refraction spherical equivalent ( mrse ) within the range of 1.0 to 9.5 d , a stable refractive error for at least 12 months before the surgery , contact lens discontinuation for at least 3 weeks , estimated corneal stromal bed thickness of more than 300 m at the thinnest point . there are no significant differences in preoperative variables of patients in the tprk and aaprk groups , except the gender . demographics and preoperative variables of patients in the tprk and aaprk groups preoperative information on general and ocular medical history , contact lens wear , and medication use was obtained from each patient . the examination included uncorrected distance visual acuity ( udva ) , corrected distance visual acuity ( cdva ) , manifest and cycloplegic refraction , slit lamp biomicroscopy , tonometry , specular microscopy ( em-3000 , tomey , erlangen , germany ) , pupillometry , scheimpflug camera tomography ( sirius , schwind eye - tech - solutions gmbh , kleinostheim , germany ) , ocular aberrometry ( irx-3 , imagine eyes , paris , france ) , and fundus examination . in the tprk group , where aspheric aberration - free transprk ablation algorithm was used ( schwind eye - tech - solutions ) , the epithelium was removed during laser ablation only from the area of the total ablation zone , which is the sum of the optical and transition zones . in both groups in all cases , 0.02% mitomycin c ( mmc ) mmc application was followed by generous irrigation of the eye with room temperature balanced solution . the postoperative regimen included 0.3% tobramycin drops ( tobrexan , alcon , new brunswick , tx ) for 1 month , 0.1% diclofenac drops ( dicloabak , laboratoires thea , clermont - ferrand , france ) for 1 month , 0.15% hyaluronic acid drops ( biolan , penta arzneimittel , stulln , germany ) for 3 months , and 0.1% dexamethasone drops ( dexafree , laboratoires thea , stulln , germany ) 3 times daily for the first month , twice daily for the second month , and once daily for the third month . patients were instructed to visit the clinic for postoperative examinations after 1 day , 1 week , 1 month , and 3 months . examinations at 1 day , 1 week , and 1 month included udva , cdva , manifest refraction , tonometry , and slit lamp biomicroscopy . three months after the surgery patients were asked about overall satisfaction with the surgery and ( high , moderate , low , not satisfied ) , and whether they would decide to have a surgery again ( yes , no ) . data were analyzed using datagraph - med version 4.20 d ( ingenieurbro pieger gmbh , wendelstein , germany ) , which is a relational database designed for refractive data analysis . for the comparison of nominal data the chi - squared test ( for sex , follow - up rate , majority of complications , and satisfaction ) or fisher exact test ( for haze ) were used . the examination included uncorrected distance visual acuity ( udva ) , corrected distance visual acuity ( cdva ) , manifest and cycloplegic refraction , slit lamp biomicroscopy , tonometry , specular microscopy ( em-3000 , tomey , erlangen , germany ) , pupillometry , scheimpflug camera tomography ( sirius , schwind eye - tech - solutions gmbh , kleinostheim , germany ) , ocular aberrometry ( irx-3 , imagine eyes , paris , france ) , and fundus examination . the epithelium was mechanically debrided with a spatula . in the tprk group , where aspheric aberration - free transprk ablation algorithm was used ( schwind eye - tech - solutions ) , the epithelium was removed during laser ablation only from the area of the total ablation zone , which is the sum of the optical and transition zones . in both groups in all cases , 0.02% mitomycin c ( mmc ) was applied for 2 min based on the standard protocol . the postoperative regimen included 0.3% tobramycin drops ( tobrexan , alcon , new brunswick , tx ) for 1 month , 0.1% diclofenac drops ( dicloabak , laboratoires thea , clermont - ferrand , france ) for 1 month , 0.15% hyaluronic acid drops ( biolan , penta arzneimittel , stulln , germany ) for 3 months , and 0.1% dexamethasone drops ( dexafree , laboratoires thea , stulln , germany ) 3 times daily for the first month , twice daily for the second month , and once daily for the third month . patients were instructed to visit the clinic for postoperative examinations after 1 day , 1 week , 1 month , and 3 months . examinations at 1 day , 1 week , and 1 month included udva , cdva , manifest refraction , tonometry , and slit lamp biomicroscopy . three months after the surgery patients were asked about overall satisfaction with the surgery and ( high , moderate , low , not satisfied ) , and whether they would decide to have a surgery again ( yes , no ) . data were analyzed using datagraph - med version 4.20 d ( ingenieurbro pieger gmbh , wendelstein , germany ) , which is a relational database designed for refractive data analysis . for the comparison of nominal data the chi - squared test ( for sex , follow - up rate , majority of complications , and satisfaction ) or fisher exact test ( for haze ) were used . the mean ablation time was 44.35 13.68 s in the tprk group and 16.85 9.58 s in the aaprk group ( p < 0.001 ) , whereas mean time of the whole procedure , from introduction to removal of the lid speculum , was 165 24.62 s and 254 32.14 s , respectively ( p < 0.001 ) . in the tprk and aaprk group , respectively , the mean diameter of the optical zone was 6.96 0.45 and 7.11 0.43 mm ( p = 0.47 ) , and the transition zone was 1.39 0.50 and 1.29 0.63 mm ( p = 0.55 ) . the minimal estimated stromal residual thickness was 306 m among all the analyzed eyes ( 309 m in tprk group and 306 m in aaprk group ) . the 3-month follow - up rate was 82.1% in the tprk group ( n = 145 ) and 86.4% in the aaprk group ( n = 90 ) , because some of the patients were not able or not willing to attend all of the required visits . mean postoperative mrse was 0.14 0.26 d in the tprk group and 0.12 0.20 d in the aaprk group ( p = 0.9 ) . in the tprk group , the postoperative refractive spherical equivalent in 63% of eyes were within 0.13 d , 12% within + 0.14 to + 0.5 d , 24% within 0.14 to 0.5 d , and 1% within 0.5 and 1.0 d ; the respective values in the aaprk group were 70% , 4% , 26% , and 0% . refractive results , predictability , safety , and efficacy 3 months after the surgery are shown in figures 1 and 2 as standard graphs recommended for reporting refractive surgery outcomes . udva was 20/20 or better in 97% of eyes in the tprk group and 94% in the aaprk group ( p = 0.45 ) . in the tprk group , 13% of eyes lost 1 line of cdva and 30% gained a line or 2 . in the aaprk group , 21% of eyes lost 1 line of cdva and 31% gained a line or 2 ( p = 0.48 ) . comparison of uncorrected distance visual acuity ( a ) , change in corrected distance visual acuity ( b ) , and attempted vs achieved spherical equivalent ( c ) in single - step transepithelial photorefractive keratectomy ( tprk ; left panels ) and alcohol - assisted photorefractive keratectomy ( aaprk ; right panels ) groups . comparison of the spherical equivalent refractive accuracy ( d ) , refractive astigmatism ( e ) , and stability of spherical equivalent refraction ( f ) in single - step transepithelial photorefractive keratectomy ( tprk ; left panels ) and alcohol - assisted photorefractive keratectomy ( aaprk ; right panels ) groups . mean preoperative higher order rms for 4 mm pupil was 0.15 0.15 m in the tprk group and 0.17 0.21 m in the aaprk group ( p = 0.21 ) , and the postoperative values were 0.21 0.23 and 0.19 0.12 m ( p = 0.37 ) , respectively . the differences between the preoperative and postoperative higher - order rms were not significant for both groups ( p = 0.13 for tprk group and p = 0.27 for the prk group ) . the mean pain scores after the surgery were 4.78 2.65 in the tprk group and 4.59 2.85 in the aaprk group ( p = 0.85 ) . there were also no differences in pain intensity during first days after the surgery ( mean scores of 4.46 2.54 and 4.51 2.36 in the tprk and aaprk groups , respectively ; p = 0.86 ) . after tprk , 86.25% of patients declared high satisfaction with the surgery compared to 88.24% patients after aaprk ( p = 0.46 ) . the ratio for moderate satisfaction was 13.75% for tprk and 11.76% for aaprk , respectively ( p = 0.54 ) . the total postoperative complication rate was 19.31% in the tprk group and 14.44% in the aaprk group ( p = 0.13 ) . a slightly higher incidence of haze was detected with the slit lamp for the tprk group ( 13.79% ) , compared to the aaprk group ( 8.89% ) ; however , the results of the fisher exact test suggest that the proportions of patients falling in each subcategory within each group did not differ significantly ( p = 0.09 ) . during the follow up there was no statistically significant difference in the incidence of other postoperative complications , which included elevated iop in 1.38% of eyes after tprk and 2.22% after aaprk ; decreased visual acuity at night : 1.38% of eyes after tprk and 1.11% after aaprk ; more intensive dry eye symptoms in 2.76% of eyes after tprk and 2.22% after aaprk . aspheric aberration - free ablation profile of single - step transepithelial prk ( transprk ) used in the study , has many implications over the standard aaprk procedures . the laser system is tuned to compensate for a difference in photoablative rates of the stroma and the epithelial tissue , which is approximately 20% higher in the epithelium . by means of very high - frequency digital ultrasonography , reinstein et al found that the mean epithelial thickness at the corneal vertex was 53.4 4.6 m , and the average epithelial thickness map showed that the corneal epithelium was thicker inferiorly than superiorly ( 5.9 m at the 3-mm radius , p < 0.001 ) and thicker nasally than temporally ( 1.3 m at the 3-mm radius , p < 0.001 ) . the location of the thinnest epithelium was temporally displaced on average 0.33 mm , and 0.90 mm superiorly with reference to the corneal vertex . they used spectral - domain anterior - segment optical coherence tomography and measured a mean epithelial thickness at the pupil center of 53.28 3.34 m , superiorly 51.86 3.78 m , and inferiorly 53.81 3.44 m . in theory , the above - mentioned findings may deteriorate the refractive results , predictability , safety , and efficacy of transprk ablations in comparison to the standard aaprk procedures . in our study in which the latest version of the tprk algorithm was used , we did not observe statistically significant differences between the tprk group and the aaprk group in terms of udva , cdva , and mrse , 3 months after the surgery . the correlation between attempted versus achieved mrse was very high in both groups with no statistically significant difference between the 2 groups . shortly after the procedure was introduced , luger et al , aslanides et al , and fadlallah et al evaluated relatively small cohorts and came to the similar conclusions ; in myopia and compound myopic astigmatism correction , the refractive and visual outcomes of single - step transepithelial prk are not different from those of conventional prk . however , luger et al reported that the postoperative mean spherical equivalent in tprk was slightly hyperopic at + 0.07 0.23 d. moreover , our results in both the tprk and aaprk groups were among the best achieved with surface ablation in terms of postoperative udva , postoperative refractive spherical equivalent , and refractive astigmatism . thus , the natural interindividual variability of epithelial thickness maps did not deteriorate the clinical results in the studied population in a noticeable way . reinstein et al and kanellopoulos and asimellis confirmed thickening of the epithelium centrally and progressively less thickening centrifugally across the central 6 mm after myopic correction . in our study , mean ablation time was 163% longer in the transprk group ; however , the majority of the laser energy was delivered to the epithelium . excimer laser energy , among other effects , causes increase of the temperature of the stromal tissue , which is the main risk factor for haze formation , an inherent complication of excimer laser refractive surgery . after surface ablation , up to 52% of eyes develop some degree of corneal haze in the first months , with values most often reported are within the range of 5% to 20% . in our study population up to 3 months after the surgery , haze was more often detected in the tprk group ; however , the difference did not reach statistical significance ( 13.79% vs 8.89% , p = 0.09 ) . in all eyes , the intensity of haze was very low , in almost all cases at the 0.5 level , and the difference in haze intensity between groups was not statistically significant . in our opinion , the intensity of haze after transepithelial prk and the impact of mmc on haze formation need to be further studied , preferably with masked examiners and with tools that provide objective measurements , like optical densitometry . there are statistical more females in tprk group , which may be a source of bias especially in pain evaluation , as pain perception may be positively influenced by female gender . high - resolution spectral optical coherence tomography ( oct ) with speckle contrast reduction also failed to detect differences in the corneal healing processes after tprk and aaprk , except for the shorter time to cover the stroma with epithelium in the tprk group . the main reason explaining this observation is that the diameter of epithelial removal matches the total ablation zone in transepithelial prk treatments , decreasing the wound surface , and shortening the epithelial closure time . aslanides et al and fadlallah et al reported decreased postoperative pain after the single - step transepithelial prk ; however , our results failed to confirm these findings . in conclusion , single - step transepithelial prk and conventional prk performed on regular corneas produce very similar results 3 months after the surgery . these procedures are predictable , effective , and safe for correction of myopia and compound myopic astigmatism .

clinical and epidemiological studies have identified several factors that increase the risk of coronary heart disease and heart attack . these factors include gender , ageing , heredity , smoking , hypercholesterolemia , hypertension , physical inactivity , obesity and overweight , and diabetes mellitus . risk factors often occur in clusters and may build on one another , such as obesity leading to diabetes and elevated blood pressure . when grouped together , as in the metabolic syndrome , these factors lead to an even greater risk of coronary artery disease . special attention has been given to the effect of psychological stress , oral contraceptives , excessive alcohol intake , sleep apnea , elevated c - reactive protein levels , fibrinogen , homocysteine and lipoprotein and , finally the presence of an oxidative stress . sies has defined oxidative stress as an imbalance between oxidants ( e.g. , free radical species derived from oxygen ) and antioxidants in favour of the oxidants , leading to a disruption of redox signalling and/or molecular damage . a large number of studies have indicated that oxidative damages to proteins , lipids or dna resulting from an increased production of reactive oxygen species ( ros ) from pathological processes or external origins are involved in the development of cardiovascular diseases and cancer . to regulate ros production and fight against their deleterious effect , the organism responds with a large and complex battery of antioxidants including enzymes , proteins , iron chelators , low molecular weight compounds , trace elements , and antioxidants arising from our diet ; among them , vitamins c and e , carotenoids and polyphenols are particularly important . many epidemiological and clinical studies have also shown that the lower the antioxidant status , the higher the risk of developing cardiovascular diseases and cancer [ 313 ] . a few prospective , large - scale , european studies have shown a negative correlation between vitamin c plasma levels and all - cause or cardiovascular mortality . plasma vitamin c levels below the normal range 48.8 mg / l ( or 2350 m ) were found to double the risk of developing cardiovascular disease and cancer [ 1418 ] . for -carotene , the upper reference limit was estimated to be 0.22 mg / l ( or 0.4 m ) . factors like male gender , age , race , body mass index ( bmi ) , smoking , alcohol consumption , triacylglycerol concentration , and inadequate dietary antioxidant intakes contribute to lower plasma levels of antioxidants [ 1928 ] . to our knowledge , so far no study did investigate how lifestyle behaviours such as smoking , physical activity , and diet alone or in association contribute to reach critical vitamin c ( < 6 mg / l or 34.2 m ) and -carotene ( < 0.22 mg / l or 0.4 m ) levels . currently , an increasing number of general practitioners strive to assess the antioxidant status of their patients for prevention purposes . in this respect this prompted us to undertake a wide epidemiological study based on a sample of the belgian population . elan ( etude ligeoise sur les antioxydants ) is a cross - sectional epidemiological study conducted from march through july 2006 as a joint project between the university of lige , the university hospital of lige , and the public health services of the province of lige ( belgium ) . the province of lige , one of the 10 belgian provinces , has 1.040.297 inhabitants for a geographic area of 3862 km . a stratified random sample of 55 general practitioners working in the province was selected as follows : 21 ( 38% ) in urban environment , 15 ( 27% ) in semiurban , and 19 ( 35% ) in rural living environment . each physician was asked to recruit in his / her practice 20 presumably healthy volunteers aged 4060 years . exclusion criteria included intake of antioxidant supplementation and previous history of cardiovascular diseases , diabetesm or cancer . a total of 897 eligible subjects were finally enrolled in the study : 349 ( 39% ) men and 548 ( 61% ) women . the day before the examination visit , subjects fasted for at least 12 h and were not allowed to drink fruit juice and to perform physical activity . the day of the visit , information including age , height , weight , consumption of fruits and vegetable by means of a home - made questionnaire , and intake of alcohol and oral contraceptive pills was collected . the body mass index ( bmi ) smoking ( yes / no ) and physical activity ( inactive or active 2 - 3 times a week ) were also recorded . social status was classified in 7 categories , as follows : professionals ( i ) , managerial and technical occupations ( ii ) , manual ( iiia ) and nonmanual skilled workers ( iiib ) , partly skilled workers ( iv ) , retired ( va ) , and unemployed ( vb ) . all contacted volunteers received written information about the goal of the study and signed an informed consent form prior to enrolment . they were immediately centrifuged on site and plasma was frozen as aliquots on ice packs coming from a 80c freezer and placed in a refrigerating box . for the assay of vitamin c , 0.5 ml plasma was immediately transferred to ice - cold tubes containing 0.5 ml of 10% metaphosphoric acid . analyses were performed on the day of blood collection by a spectrophotometric method using the reduction of 2,6-dichlorophenolindophenol ( perkin elmer lambda 40 norwalk , usa , sensitivity : 2 mg / l , inter- and intra - cv : 4 and 6% ) . mg / l , inter- and intra - cv : 5.35 and 10.73% ) was determined by hplc procedure ( alliance waters , usa ) coupled with a diode array detector ( pda 2996 , waters , usa ) . both vitamin c and -carotene were analyzed in a routine way . independently of gender , our reference values , as published earlier on a population of 128 healthy subjects , were 6.218.8 mg / l ( 35.3107.1 m ) for vitamin c and 0.050.62 mg / l ( 0.091.15 m ) for -carotene [ 31 , 32 ] . results were expressed as means standard deviation ( sd ) for quantitative variables , while frequencies and proportions ( % ) were used for categorical variables . mean values between groups were compared by one - way analysis of variance or the kruskal - wallis nonparametric method if normality assumptions could not be fulfilled . correlation coefficients ( classical or spearman ) were calculated for measuring the association between two quantitative variables . logistic regression analysis was used to predict vitamin c and -carotene deficiency from risk factors . the association between deficiency and risk factors was measured by the odds ratio ( or ) and its 95% confidence interval . calculations were always carried out on the maximum number of data available . missing data were not replaced . results were considered to be significant at the 5% critical level ( p < .05 ) . data analysis was carried out using sas ( version 9.1 for windows ) and s - plus ( version 9.0 ) statistical packages . the gender - specific demographic , biometric , and clinical characteristics of the study population are given in table 1 . in the elan population , respectively 27% of women and 25% of men were smokers . height , weight , bmi , and both systolic and diastolic blood pressures were significantly higher in men than in women . the dietary intake of vitamin c and -carotene ( mg / day ) derived from fruits and vegetables consumption generally was significantly lower in men than in women ( p < .0001 ) . table 2 displays vitamin c and -carotene mean levels with respect to subject 's characteristics given in table 1 . as expected , vitamin c and -carotene values were lower by , respectively , 15 and 32% in men when compared to women ( p < .0001 ) . smokers had significantly decreased vitamin c and -carotene levels by 19% ( p < .0001 ) and 39% by contrast , regular physical activity was associated with a mean increase of 9% for vitamin c ( p < .0001 ) and 25% for -carotene ( p a positive relationship was demonstrated between the amount of daily fruit intake and both vitamin c and -carotene levels . the latter were , respectively , reduced by 27% and 46% ( p < .0001 ) in non consumers when compared to people eating 2 - 3 fruits / day or more . the group apple - pear - grapes - banana , kiwi , citrus fruits , orange , and all kinds of red fruits and strawberry had a positive influence on vitamin c. in contrast , none of the vegetables except endives had a positive effect on vitamin c ( data not shown ) . with respect to -carotene , apricot , the group apple - pear - grapes - banana , kiwi , citrus , mango , and orange contributed to improve the plasma level of -carotene but only asparagus , carrot , and tomato for vegetables ( data not shown ) . women taking oral contraceptive had significantly lower -carotene levels but vitamin c levels were unchanged . no relevant effect of blood pressure , intestinal disorders , environment , and age ( < and > 50 years ) has been evidenced on the plasma level of both studied antioxidants . figure 1 depicts the distribution of plasma vitamin c and -carotene levels in men and women . only 2.1% of the subjects had higher values than the upper reference value ( 18.8 mg / l ) . if a large majority ( 81.5% ) of participants were within the recommended values in vitamin c ( 6.218.8 mg / l ) , it should be noted that 16.4% of them were , however , either in a subdeficiency ( 10.3% ) or deficiency ( 6.1% ) status with respect to the cutoff value of 6 mg / l ( 34.2 m ) . as far as the -carotene is concerned , a small portion of the study subjects ( 7.0% ) had higher concentration in -carotene than the upper normal value ( 0.68 mg / l or 1.23 m ) and more than 90% of the elan cohort had a plasma -carotene concentration within the conventional and usual values ( 0.050.68 mg it is worth noting that 46.7% of the studied population had plasma values below the critical point of 0.22 mg / l ( 0.4 m ) . by logistic regression analysis applied to vitamin c data ( table 3 ) , it was found that male gender ( or = 1.70 ; p = .011 ) , smoking ( or = 2.84 ; p < .0001 ) , lack of physical activity ( or = 2.05 ; p = .0015 ) , and intake of less than 2 fruits / day ( or = 2.96 ; p = .0003 ) were significant risk factors of vitamin c deficiency . overweight ( or = 1.18 ; p = .42 ) and oc use for women ( or = 1.24 ; p = .61 ) were not significant . when adding the social status , the risk of vitamin c was significantly increased ( or = 1.34 ; p = .0044 ) , when being unemployed compared to the other professional categories . for -carotene deficiency , logistic regression analysis showed that male gender ( or = 2.67 ; p < .0001 ) , smoking ( or = 3.03 ; p < .0001 ) , overweight ( or = 2.28 ; p < .0001 ) , lack of physical activity ( or = 1.51 ; p = .0091 ) , intake of < 2 fruits / day ( or = 1.41 ; p = .045 ) , and oc use for women ( or = 4.67 ; p < table 4 examines the probability of vitamin c and -carotene deficiency ( i.e. , lower than the critical values ) with respect to an increasing number of risk factors . healthy habits consisting in no smoking , practising a regular physical activity , and eating at least two fruits per day resulted in a weak probability ( less than 5% ) to have plasma vitamin c below 6 by contrast , poor healthy living habits ( smoking , no physical activity , nonconsumption of fruits , and overweight ) significantly increased this probability up to 46.2% for men against 33.5% for women . when accounting for unemployment , these probabilities raised to 66.3% for men and 44.3% for women , respectively . it should also be noted that men were always associated with higher probabilities of vitamin c levels below 6 mg / l than women ( except those of social class iv and va ) smoking , practising no physical activity , and eating none fruits regardless of the combination of lifestyle behaviours . for -carotene , healthy habits ( nonsmoking , practising sport , and eating two or one fruits per day ) resulted in 13.7% and 29.7% of chance to get critical level in -carotene for women and men , respectively . smoking by itself significantly increased this probability but in a higher extent in men ( 56.1% ) than in women ( 32.4% ) . the combination of smoking , absence of regular physical activity , and any intake of fruits contributed to a high probability in men ( 73.2% ) and to a less extent in women ( 50.5% ) . the addition of overweight resulted in an increase of about 20% in women ( 69.9% ) and of only 13% in men ( 86.1% ) . finally , the worst situation ( 91.6% ) was observed for women by adding the intake of oral contraceptive pills . it should be noted that for both genders , all probabilities were largely higher than those derived for vitamin c risk of deficiency . the reference values for vitamin c established by our group are comprised between 6.2 mg / l ( 35.3 m ) and 18.8 mg / l ( 107.1 m ) . this is in agreement with other studies performed in different european populations [ 20 , 35 ] . in the work of rutkowski and grzegorczyk , ten ranges of concentrations given by medical handbooks and textbooks have been compared in detail with 15 parallel ranges taken from scientific papers , paying attention to their significant discrepancies . based on source values and basic statistical calculations , a reliable mean range of vitamin c normal concentrations " in blood plasma has been obtained : 6.314 mg / l ( 36.179.4 m ) . according to le grusse and watier , the critical range of accepted marginal vitamin c deficiency was between 3.5 and 6.2 mg / l ( 20 m35 m ) . for -carotene , we found the following normal range 0.050.62 mg / l ( 0.091.15 m ) which is in good agreement with the study of olmedilla et al . performed in five western european populations . with respect to gender , our mean values in antioxidants were in perfect agreement with those found in the reference french suvimax study ( vitamin c : men : 8.8 4.0 mg / l ; women : 10.6 5.5 mg / l ; p < .0001 ; -carotene : men : 0.22 0.16 mg / l ; women : 0.31 0.20 mg / l ; p < .0001 ) [ 20 , 38 ] . fruits and to a less extent vegetables are the primary dietary sources of both antioxidants . as confirmed in table 1 , it is recognized that women have dietary intakes richer in vitamin c and -carotene than men due to a higher intake of fruit and vegetables . as expected , our data clearly indicate that smoking was associated with a significant decrease of the mean plasma vitamin c ( 19% ) and -carotene ( 39% ) after adjustment for gender and all demographic variables described in table 1 . our observations were in good agreement with other reports [ 26 , 27 ] and , more particularly , the suvimax study performed on 3128 french men and women aged 3560 years . cigarette smoke contains a large number of free radicals species able to induce an oxidative stress on both the respiratory and circulatory systems with as consequence greater antioxidant depletion . after adjustment for all covariates , we also evidenced that a regular physical activity contributes to improve the vitamin c and -carotene levels . a simple explanation could be given by the fact that active people eat more fruits than inactive ones . southeast showing a significant association between fruit and vegetable consumption and physical activity ( p < we also evidenced a positive relationship between the frequency of fruit and the mean plasma level of vitamin c and -carotene . when compared to high consumers , people eating any fruit were characterized by plasma concentrations of both antioxidants which are extremely closed to the normal inferior value . by contrast , we were unable to evidence an association between antioxidant biomarkers and the consumption of vegetables . moreover , we also evidenced that only the intake of carrot and tomato among vegetables may significantly influence to a higher extent the plasma level of -carotene whilst a larger range of fruits were able to do it . it could be also possible that -carotene is a better predictor than -carotene as suggested in the european prospective investigation into cancer and nutrition ( epic study ) performed on a stratified random subsample of 3089 men and women . ideal bmi is the range of 2025 kg / m while a bmi of over 25 kg / m and 30 kg / m is , respectively associated with overweight and obesity . as described earlier [ 41 , 42 ] , we confirmed that a bmi > 25 kg / m resulted in a significant decrease by 31% of the plasma -carotene level . the use of oral contraceptives had a deep negative impact on the plasma level of -carotene . it has been speculated that estrogens induce an activation of the retinol binding protein , hence possibly increasing the conversion of -carotene into retinol . we have also shown that women taking oral contraceptives had higher oxidative damages to lipids than the others . it could be assumed that part of the antioxidant defences were more solicited in women using oral contraception to limit deleterious damages . neither blood pressure nor intestinal disorders ( which could explain a decrease in antioxidants due to possible malabsorption ) or environmental parameters had an influence on the mean plasma level of both antioxidants . based on large - scale epidemiological studies , it is now accepted that an alteration of antioxidant defences was significantly implicated in the development of several pathologies . however , if some variations of the mean plasma values may be evidenced with respect to lifestyle behaviours in all studies , no indication was ever given about the biological interpretation of these variations . therefore , the establishment of reference or usual values for antioxidants markers such vitamin c and -carotene is needed to detect abnormalities . as explained above , determination of normal ranges for both antioxidants has been achieved on a separate population of 128 healthy persons as earlier published [ 3133 ] . based on our reference values , we were able to detect that 16.4% of the whole elan population had non optimal plasma concentration in vitamin c ( < 6 mg / l or 34.2 m ) against 46.7% for -carotene ( < 0.22 mg / l or 0.4 m ) . at the light of the following but not exhaustive studies , the evidence of such abnormalities could therefore be of primordial interest as a preventive tool for health [ 44 , 45 ] . recently , langlois et al . proposed that plasma vitamin c should be considered as a predictor of cardiovascular disease in addition to being a classical nutritional biomarker . based on a large number of studies including the famous monica study [ 14 , 18 ] performed on 14 european populations , plasma cutoff levels ( 4.47.0 mg / l or 2540 m ) in vitamin c have been proposed , above which the risk for apparent cardiovascular events should decrease . in haemodialysis patients , the cutoff value of 5.66 mg / l ( 32 m ) was predictive of the appearance of adverse cardiovascular outcomes . in patients with peripheral arterial disease , the cutoff value of 4.9 mg / l ( 28 m ) was associated with increased levels of inflammation parameters . recently , myint et al . described in the european prospective investigation into cancer ( epic)-norfolk population that the relative risks for risk of stroke diminished inversely to the quartile of plasma vitamin c concentration as follows : 1.0 ( < 41 m or 7.27 mg / l ) , 0.83 ( 4153 m or 7.279.32 mg / l ) , 0.63 ( 5465 m or 9.5 or 11.44 mg / l ) , and 0.57 ( > 66 m or 11.6 , it was interesting to highlight that there was a continuous relation with mortality through the whole distribution of ascorbic acid concentrations . when compared to 3.66 mg / l , each 3.52 mg / l ( 20 m ) rise in plasma ascorbic acid concentration was associated with about a 20% reduction in risk of all - cause mortality ( p < .0001 ) , regardless of age , systolic blood pressure , blood cholesterol , cigarette smoking habit , diabetes , and supplement use . for -carotene , gey established that a plasma value below 0.22 mg / l ( 0.4 m ) was associated with an increased risk of developing cardiovascular diseases and cancer . among the elan population , 16.4% of the subjects presented a plasma vitamin c below the cutoff value of 6 mg / l ( 34.2 m ) and 46.7% a concentration in -carotene below the critical point of 0.22 mg / l ( 0.4 m ) . the availability of a statistical model for predicting the probability of getting a plasma value below the cutoff values of 6 mg / l ( 34.2 m ) for vitamin c and 0.22 mg / l ( 0.4 m ) for -carotene could be of interest for health prevention . cumulative probabilities were therefore presented instead of odd ratios to stress on the cumulative effect of the risk factors on low levels in both antioxidants . after adjustment for all covariates between them , table 3 clearly indicates that using such values rather than mean plasma value afforded better indications about the relationship between antioxidant biomarkers and lifestyle behaviours . a good base of healthy life could be nonsmoking , regular physical activity and eating more than 3 fruits / day . this resulted in a small probability to have inadequate plasma level of vitamin c. smoking and nonphysical activity significantly but moderately contributed to increase this probability for vitamin c for both genders . in contrast , the nonconsumption of fruits added to the parameters above produced a dramatic increase which was more pronounced in men ( 42.1% ) than in women ( 30% only for those belonging to social classes i , ii , iiia , and iiib ) . this suggests that the frequncy of fruit intake appears to be one of the most important regulators of the plasma concentration of vitamin c [ 50 , 51 ] with a more pronounced effect in men ( 65.3% ) having partial or no working activity . economical difficulties to buy fruits linked to a precarious social status can explain this last observation . as observed with the mean plasma values , no effect of bmi , intestinal disorders , and environment could be evidenced on the probability to get low plasma level in vitamin c. with respect to -carotene , it was quite interesting to note that despite a healthy way of life there was a significant risk ( 29.7% in men and 13.7% in women ) to get a value below 0.22 mg / l ( 0.4 m ) . malabsorption of this antioxidant could be a rationale explanation although we did not find any influence of intestinal disorders on the plasma level of -carotene . when compared to basal level and according to the different steps described in table , smoking contributes to a mean increase of 27% for men and 18% for women of getting critical plasma value . this is not surprising since the negative impact of smoking on antioxidant is well known . lack of physical activity was associated with a further but moderate increase of around 9% . this was less pronounced than those observed for vitamin c confirming that the latter is a better biomarker than -carotene of fruits intake . kg / m and oral contraceptives in women , significantly contributed to finally reach a high probability ( 86.1% for men and 91.6% for women ) to detect nonoptimal plasma value in -carotene . although representative of the province of lige , the study sample may not be considered as a national probability sample of the belgian population . moreover , we only focused our attention on people in the age range of 4060 years since we considered that lifestyle behaviours were well anchored in this population . it is clear that ageing ( > 60 years ) could also contribute to decrease the plasma level in antioxidants so that our observations in the elan population could be modified if taking this parameter in account . we only distinguished nonsmokers from both past and current smokers , this last one category being , however , in a large majority in the elan population . further , no question has been addressed with respect to environmental tobacco smoker which could possibly have a negative influence on plasma antioxidants . during our study , we also met some difficulties to integrate data about alcohol consumption due to a large underestimation of intake made by participants . as this parameter has been shown to reduce the level of plasma -carotene , it could partially explain the relatively high probability of getting a value below 0.22 mg / l ( 0.4 m ) even when observing optimal lifestyle behaviours ( no smoking , physical activity and eating more than 2 days a day ) . except for oral contraceptive , the influence of other drugs intake was not taken into consideration . to the best of our knowledge , the present study is the first one to address the relationship between plasma antioxidants and lifestyle behaviours in a belgian population . we have demonstrated that smoking regular physical activity and eating fruits were directly associated with the modulation of the mean plasma concentration of both vitamin c and -carotene . however , such variations , if well described in the literature , always remained within the normal or usual range of concentration . by using cutoff values associated with increased risk of developing cardiovascular diseases and cancer ( vitamin c < 0.22 mg / l or 0.4 m ) , we described how lifestyle factors alone or associated contribute to lower plasma concentrations . however , as vitamin c and -carotene are unstable constituents when not protected against air and light , a rigorous preanalytical sample handling and treatment ( immediate centrifugation , plasma precipitation and keeping the sample at 80c until analysis ) is required to interpret the data correctly . the elan ( etude ligeoise sur les antioxydants ) study was conducted from march through july 2006 as a joint project between the university of lige , the university hospital of lige , and the local health services of the province of lige ( belgium ) . sc j pincemail ( credec and dept of cardiovascular surgery ) were the main coordinators of the elan study . professor c charlier and professor jp chapelle ( laboratories of clinical biology ) allowed the analysis of -carotene while vitamin c determination was performed by mr jp cheramy - bien ( credec ) . g collette ( dept of general medicine ) allowed the recruitment of all general practitioners around the province of lige , belgium . sc s vanbelle ( dpt of medical informatics and biostatistics ) were involved in the statistical analysis of all data . all investigators critically revised the manuscript for the intellectual content and gave their final approval of the version to be published .

several factors , including fruit and vegetables intakes , have been shown to significantly influence the plasma concentrations of the two antioxidants vitamin c and -carotene . deficiency levels of 6 mg / l ( 34.2 m ) for vitamin c and of 0.22 mg / l ( 0.4 m ) for -carotene have been suggested below which cardiovascular risk might be increased . the present study performed on 897 presumably healthy subjects aged 4060 years aimed to examine how modifiable lifestyle factors may be related to vitamin c and/or -carotene deficiency . gender , smoking , lack of regular physical activity and of daily fruit consumption ( 2/day ) , and social status ( in particular , unemployment ) were found to be significant risk factors for vitamin c deficiency . for -carotene deficiency , the same factors were identified except social status ; moreover , overweight and oc use in women were also found to have a deleterious effect . for non exposed subjects , the probability of developing vitamin c deficiency was 4% in men and 2.4% in women . this probability increased to 66.3% for men and to 44.3% for women ( and even to 50.4% under oc use ) , when all risk factors were present . for -carotene deficiency , the corresponding probabilities were equal to 29.7% in men and 13.7% in women ( no risk factor present ) , and to 86.1% for men and 69.9% ( 91.6% for oc use ) for women ( all factors present ) , respectively .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusions Authors' Contribution

these factors include gender , ageing , heredity , smoking , hypercholesterolemia , hypertension , physical inactivity , obesity and overweight , and diabetes mellitus . to regulate ros production and fight against their deleterious effect , the organism responds with a large and complex battery of antioxidants including enzymes , proteins , iron chelators , low molecular weight compounds , trace elements , and antioxidants arising from our diet ; among them , vitamins c and e , carotenoids and polyphenols are particularly important . plasma vitamin c levels below the normal range 48.8 mg / l ( or 2350 m ) were found to double the risk of developing cardiovascular disease and cancer [ 1418 ] . for -carotene , the upper reference limit was estimated to be 0.22 mg / l ( or 0.4 m ) . factors like male gender , age , race , body mass index ( bmi ) , smoking , alcohol consumption , triacylglycerol concentration , and inadequate dietary antioxidant intakes contribute to lower plasma levels of antioxidants [ 1928 ] . to our knowledge , so far no study did investigate how lifestyle behaviours such as smoking , physical activity , and diet alone or in association contribute to reach critical vitamin c ( < 6 mg / l or 34.2 m ) and -carotene ( < 0.22 mg / l or 0.4 m ) levels . elan ( etude ligeoise sur les antioxydants ) is a cross - sectional epidemiological study conducted from march through july 2006 as a joint project between the university of lige , the university hospital of lige , and the public health services of the province of lige ( belgium ) . each physician was asked to recruit in his / her practice 20 presumably healthy volunteers aged 4060 years . the day before the examination visit , subjects fasted for at least 12 h and were not allowed to drink fruit juice and to perform physical activity . the body mass index ( bmi ) smoking ( yes / no ) and physical activity ( inactive or active 2 - 3 times a week ) were also recorded . social status was classified in 7 categories , as follows : professionals ( i ) , managerial and technical occupations ( ii ) , manual ( iiia ) and nonmanual skilled workers ( iiib ) , partly skilled workers ( iv ) , retired ( va ) , and unemployed ( vb ) . analyses were performed on the day of blood collection by a spectrophotometric method using the reduction of 2,6-dichlorophenolindophenol ( perkin elmer lambda 40 norwalk , usa , sensitivity : 2 mg / l , inter- and intra - cv : 4 and 6% ) . mg / l , inter- and intra - cv : 5.35 and 10.73% ) was determined by hplc procedure ( alliance waters , usa ) coupled with a diode array detector ( pda 2996 , waters , usa ) . both vitamin c and -carotene were analyzed in a routine way . independently of gender , our reference values , as published earlier on a population of 128 healthy subjects , were 6.218.8 mg / l ( 35.3107.1 m ) for vitamin c and 0.050.62 mg / l ( 0.091.15 m ) for -carotene [ 31 , 32 ] . logistic regression analysis was used to predict vitamin c and -carotene deficiency from risk factors . results were considered to be significant at the 5% critical level ( p < .05 ) . height , weight , bmi , and both systolic and diastolic blood pressures were significantly higher in men than in women . the dietary intake of vitamin c and -carotene ( mg / day ) derived from fruits and vegetables consumption generally was significantly lower in men than in women ( p < .0001 ) . table 2 displays vitamin c and -carotene mean levels with respect to subject 's characteristics given in table 1 . as expected , vitamin c and -carotene values were lower by , respectively , 15 and 32% in men when compared to women ( p < .0001 ) . smokers had significantly decreased vitamin c and -carotene levels by 19% ( p < .0001 ) and 39% by contrast , regular physical activity was associated with a mean increase of 9% for vitamin c ( p < .0001 ) and 25% for -carotene ( p a positive relationship was demonstrated between the amount of daily fruit intake and both vitamin c and -carotene levels . the group apple - pear - grapes - banana , kiwi , citrus fruits , orange , and all kinds of red fruits and strawberry had a positive influence on vitamin c. in contrast , none of the vegetables except endives had a positive effect on vitamin c ( data not shown ) . with respect to -carotene , apricot , the group apple - pear - grapes - banana , kiwi , citrus , mango , and orange contributed to improve the plasma level of -carotene but only asparagus , carrot , and tomato for vegetables ( data not shown ) . figure 1 depicts the distribution of plasma vitamin c and -carotene levels in men and women . only 2.1% of the subjects had higher values than the upper reference value ( 18.8 mg / l ) . if a large majority ( 81.5% ) of participants were within the recommended values in vitamin c ( 6.218.8 mg / l ) , it should be noted that 16.4% of them were , however , either in a subdeficiency ( 10.3% ) or deficiency ( 6.1% ) status with respect to the cutoff value of 6 mg / l ( 34.2 m ) . as far as the -carotene is concerned , a small portion of the study subjects ( 7.0% ) had higher concentration in -carotene than the upper normal value ( 0.68 mg / l or 1.23 m ) and more than 90% of the elan cohort had a plasma -carotene concentration within the conventional and usual values ( 0.050.68 mg it is worth noting that 46.7% of the studied population had plasma values below the critical point of 0.22 mg / l ( 0.4 m ) . by logistic regression analysis applied to vitamin c data ( table 3 ) , it was found that male gender ( or = 1.70 ; p = .011 ) , smoking ( or = 2.84 ; p < .0001 ) , lack of physical activity ( or = 2.05 ; p = .0015 ) , and intake of less than 2 fruits / day ( or = 2.96 ; p = .0003 ) were significant risk factors of vitamin c deficiency . overweight ( or = 1.18 ; p = .42 ) and oc use for women ( or = 1.24 ; p = .61 ) were not significant . when adding the social status , the risk of vitamin c was significantly increased ( or = 1.34 ; p = .0044 ) , when being unemployed compared to the other professional categories . for -carotene deficiency , logistic regression analysis showed that male gender ( or = 2.67 ; p < .0001 ) , smoking ( or = 3.03 ; p < .0001 ) , overweight ( or = 2.28 ; p < .0001 ) , lack of physical activity ( or = 1.51 ; p = .0091 ) , intake of < 2 fruits / day ( or = 1.41 ; p = .045 ) , and oc use for women ( or = 4.67 ; p < table 4 examines the probability of vitamin c and -carotene deficiency ( i.e. healthy habits consisting in no smoking , practising a regular physical activity , and eating at least two fruits per day resulted in a weak probability ( less than 5% ) to have plasma vitamin c below 6 by contrast , poor healthy living habits ( smoking , no physical activity , nonconsumption of fruits , and overweight ) significantly increased this probability up to 46.2% for men against 33.5% for women . when accounting for unemployment , these probabilities raised to 66.3% for men and 44.3% for women , respectively . it should also be noted that men were always associated with higher probabilities of vitamin c levels below 6 mg / l than women ( except those of social class iv and va ) smoking , practising no physical activity , and eating none fruits regardless of the combination of lifestyle behaviours . for -carotene , healthy habits ( nonsmoking , practising sport , and eating two or one fruits per day ) resulted in 13.7% and 29.7% of chance to get critical level in -carotene for women and men , respectively . smoking by itself significantly increased this probability but in a higher extent in men ( 56.1% ) than in women ( 32.4% ) . the combination of smoking , absence of regular physical activity , and any intake of fruits contributed to a high probability in men ( 73.2% ) and to a less extent in women ( 50.5% ) . the addition of overweight resulted in an increase of about 20% in women ( 69.9% ) and of only 13% in men ( 86.1% ) . finally , the worst situation ( 91.6% ) was observed for women by adding the intake of oral contraceptive pills . it should be noted that for both genders , all probabilities were largely higher than those derived for vitamin c risk of deficiency . the reference values for vitamin c established by our group are comprised between 6.2 mg / l ( 35.3 m ) and 18.8 mg / l ( 107.1 m ) . based on source values and basic statistical calculations , a reliable mean range of vitamin c normal concentrations " in blood plasma has been obtained : 6.314 mg / l ( 36.179.4 m ) . according to le grusse and watier , the critical range of accepted marginal vitamin c deficiency was between 3.5 and 6.2 mg / l ( 20 m35 m ) . for -carotene , we found the following normal range 0.050.62 mg / l ( 0.091.15 m ) which is in good agreement with the study of olmedilla et al . with respect to gender , our mean values in antioxidants were in perfect agreement with those found in the reference french suvimax study ( vitamin c : men : 8.8 4.0 mg / l ; women : 10.6 5.5 mg / l ; p < .0001 ; -carotene : men : 0.22 0.16 mg / l ; women : 0.31 0.20 mg / l ; p < .0001 ) [ 20 , 38 ] . as confirmed in table 1 , it is recognized that women have dietary intakes richer in vitamin c and -carotene than men due to a higher intake of fruit and vegetables . as expected , our data clearly indicate that smoking was associated with a significant decrease of the mean plasma vitamin c ( 19% ) and -carotene ( 39% ) after adjustment for gender and all demographic variables described in table 1 . our observations were in good agreement with other reports [ 26 , 27 ] and , more particularly , the suvimax study performed on 3128 french men and women aged 3560 years . after adjustment for all covariates , we also evidenced that a regular physical activity contributes to improve the vitamin c and -carotene levels . southeast showing a significant association between fruit and vegetable consumption and physical activity ( p < we also evidenced a positive relationship between the frequency of fruit and the mean plasma level of vitamin c and -carotene . when compared to high consumers , people eating any fruit were characterized by plasma concentrations of both antioxidants which are extremely closed to the normal inferior value . moreover , we also evidenced that only the intake of carrot and tomato among vegetables may significantly influence to a higher extent the plasma level of -carotene whilst a larger range of fruits were able to do it . it could be also possible that -carotene is a better predictor than -carotene as suggested in the european prospective investigation into cancer and nutrition ( epic study ) performed on a stratified random subsample of 3089 men and women . ideal bmi is the range of 2025 kg / m while a bmi of over 25 kg / m and 30 kg / m is , respectively associated with overweight and obesity . as described earlier [ 41 , 42 ] , we confirmed that a bmi > 25 kg / m resulted in a significant decrease by 31% of the plasma -carotene level . it could be assumed that part of the antioxidant defences were more solicited in women using oral contraception to limit deleterious damages . therefore , the establishment of reference or usual values for antioxidants markers such vitamin c and -carotene is needed to detect abnormalities . based on our reference values , we were able to detect that 16.4% of the whole elan population had non optimal plasma concentration in vitamin c ( < 6 mg / l or 34.2 m ) against 46.7% for -carotene ( < 0.22 mg / l or 0.4 m ) . at the light of the following but not exhaustive studies , the evidence of such abnormalities could therefore be of primordial interest as a preventive tool for health [ 44 , 45 ] . based on a large number of studies including the famous monica study [ 14 , 18 ] performed on 14 european populations , plasma cutoff levels ( 4.47.0 mg / l or 2540 m ) in vitamin c have been proposed , above which the risk for apparent cardiovascular events should decrease . in haemodialysis patients , the cutoff value of 5.66 mg / l ( 32 m ) was predictive of the appearance of adverse cardiovascular outcomes . in patients with peripheral arterial disease , the cutoff value of 4.9 mg / l ( 28 m ) was associated with increased levels of inflammation parameters . described in the european prospective investigation into cancer ( epic)-norfolk population that the relative risks for risk of stroke diminished inversely to the quartile of plasma vitamin c concentration as follows : 1.0 ( < 41 m or 7.27 mg / l ) , 0.83 ( 4153 m or 7.279.32 mg / l ) , 0.63 ( 5465 m or 9.5 or 11.44 mg / l ) , and 0.57 ( > 66 m or 11.6 , it was interesting to highlight that there was a continuous relation with mortality through the whole distribution of ascorbic acid concentrations . when compared to 3.66 mg / l , each 3.52 mg / l ( 20 m ) rise in plasma ascorbic acid concentration was associated with about a 20% reduction in risk of all - cause mortality ( p < .0001 ) , regardless of age , systolic blood pressure , blood cholesterol , cigarette smoking habit , diabetes , and supplement use . for -carotene , gey established that a plasma value below 0.22 mg / l ( 0.4 m ) was associated with an increased risk of developing cardiovascular diseases and cancer . among the elan population , 16.4% of the subjects presented a plasma vitamin c below the cutoff value of 6 mg / l ( 34.2 m ) and 46.7% a concentration in -carotene below the critical point of 0.22 mg / l ( 0.4 m ) . the availability of a statistical model for predicting the probability of getting a plasma value below the cutoff values of 6 mg / l ( 34.2 m ) for vitamin c and 0.22 mg / l ( 0.4 m ) for -carotene could be of interest for health prevention . cumulative probabilities were therefore presented instead of odd ratios to stress on the cumulative effect of the risk factors on low levels in both antioxidants . a good base of healthy life could be nonsmoking , regular physical activity and eating more than 3 fruits / day . this resulted in a small probability to have inadequate plasma level of vitamin c. smoking and nonphysical activity significantly but moderately contributed to increase this probability for vitamin c for both genders . in contrast , the nonconsumption of fruits added to the parameters above produced a dramatic increase which was more pronounced in men ( 42.1% ) than in women ( 30% only for those belonging to social classes i , ii , iiia , and iiib ) . this suggests that the frequncy of fruit intake appears to be one of the most important regulators of the plasma concentration of vitamin c [ 50 , 51 ] with a more pronounced effect in men ( 65.3% ) having partial or no working activity . as observed with the mean plasma values , no effect of bmi , intestinal disorders , and environment could be evidenced on the probability to get low plasma level in vitamin c. with respect to -carotene , it was quite interesting to note that despite a healthy way of life there was a significant risk ( 29.7% in men and 13.7% in women ) to get a value below 0.22 mg / l ( 0.4 m ) . when compared to basal level and according to the different steps described in table , smoking contributes to a mean increase of 27% for men and 18% for women of getting critical plasma value . lack of physical activity was associated with a further but moderate increase of around 9% . this was less pronounced than those observed for vitamin c confirming that the latter is a better biomarker than -carotene of fruits intake . kg / m and oral contraceptives in women , significantly contributed to finally reach a high probability ( 86.1% for men and 91.6% for women ) to detect nonoptimal plasma value in -carotene . moreover , we only focused our attention on people in the age range of 4060 years since we considered that lifestyle behaviours were well anchored in this population . as this parameter has been shown to reduce the level of plasma -carotene , it could partially explain the relatively high probability of getting a value below 0.22 mg / l ( 0.4 m ) even when observing optimal lifestyle behaviours ( no smoking , physical activity and eating more than 2 days a day ) . to the best of our knowledge , the present study is the first one to address the relationship between plasma antioxidants and lifestyle behaviours in a belgian population . we have demonstrated that smoking regular physical activity and eating fruits were directly associated with the modulation of the mean plasma concentration of both vitamin c and -carotene . by using cutoff values associated with increased risk of developing cardiovascular diseases and cancer ( vitamin c < 0.22 mg / l or 0.4 m ) , we described how lifestyle factors alone or associated contribute to lower plasma concentrations . however , as vitamin c and -carotene are unstable constituents when not protected against air and light , a rigorous preanalytical sample handling and treatment ( immediate centrifugation , plasma precipitation and keeping the sample at 80c until analysis ) is required to interpret the data correctly . the elan ( etude ligeoise sur les antioxydants ) study was conducted from march through july 2006 as a joint project between the university of lige , the university hospital of lige , and the local health services of the province of lige ( belgium ) . all investigators critically revised the manuscript for the intellectual content and gave their final approval of the version to be published .

major depressive disorder ( mdd ) is one of the most common psychiatric disorders , with a lifetime prevalence of 17% in the united states and 4% worldwide ( eaton et al . , 2008 , kessler et al . , 2005 ) . in terms of years lost to disability , mdd is considered one of the most disabling medical conditions and is predicted to become a leading contributor to the worldwide burden of disease ( mathers and loncar , 2006 ) . the majority of pharmacotherapies developed for the treatment of mdd target brain monoamine systems , primarily serotonin ( 5-ht ) , norepinephrine , and dopamine . the most common of these , the selective serotonin reuptake inhibitors ( ssris ) and selective norepinephrine reuptake inhibitors ( snris ) , comprise a large proportion of pharmaceutical sales and are considered first line treatments for mdd . unfortunately , an estimated 40% of patients fail to respond to these therapies ( cipriani et al . , 2009 , further insight into the neurobiological mechanisms underlying antidepressant response is needed for the development of more efficacious antidepressant regimens . the combination of genetic vulnerabilities and environmental factors , such as stress , are thought to be significant contributors to the onset of depression in humans ( charney and manji , 2004 ) . the likelihood of experiencing a depressive episode is greatly increased following a stressful life event or after accumulation of chronic minor stresses ( caspi et al . , 2003 , harkness and monroe , 2006 ) . moreover , many patients suffering from depression exhibit signs of dysfunctional hypothalamic - pituitary - adrenal ( hpa ) axis activity , as demonstrated by elevated basal cortisol levels and resistance to dexamethasone , an exogenous steroid that suppresses cortisol in healthy individuals ( gillespie and nemeroff , 2005 , pariante and miller , 2001 ) . interestingly , successful antidepressant treatment is often associated with restored suppression of hpa axis response ( schule , 2007 ) . together , these findings suggest a potential role of stress hormones , such as cortisol ( corticosterone ( cort ) in rodents ) , in the pathology and treatment of depression . cort produces its effects in the central nervous system via activation of glucocorticoid ( gr ) and mineralocorticoid ( mr ) receptors . though these receptors are ubiquitous throughout the brain , they are highly abundant in the hippocampus , where they provide crucial inhibitory feedback signals to the hpa axis ( jacobson and sapolsky , 1991 ; sapolsky et al . , 1984 ) . a reduction or absence of these inhibitory signals can promote hyperactivation of the axis and augmented secretion of glucocorticoids ( anacker et al . , 2011 , mcewen et al . , 2012 ) . in a healthy individual , elevated corticosteroid activity helps facilitate the physiological and behavioral adaptations required to appropriately respond to stressors and reinstate homeostasis . however , prolonged exposure to cort can inhibit the proliferation and survival of adult - born hippocampal neurons , which have been shown to play an important role in the behavioral and neuroendocrine components of stress responses in rodents ( gould and tanapat , 1999 ; snyder et al . , 2011 ) . conversely , chronic treatment of normal rodents with ssris , such as fluoxetine , increases hippocampal neurogenesis and neurotrophins such as brain derived neurotrophic factor ( bdnf ) ( duman and monteggia , 2006 , krishnan and nestler , 2008 , schmidt and duman , 2006 ) . increased hippocampal neurogenesis is associated with behavioral indications of antidepressant efficacy in rodents , such as reduced hyponeophagia in the novelty - induced hypophagia ( nih ) test and performance in the forced swim test ( dranovsky and hen , 2006 ) . not all strains of mice respond to the behavioral and neurogenic effects of antidepressant treatments . for example , normal c57bl/6 mice are unresponsive to the behavioral effects of chronic fluoxetine treatment , measured in the nih test , and do not exhibit increased hippocampal cell proliferation ( balu et al . although corticosteroids alone do not encompass all aspects of stress exposure ( belzung , 2014 ) , previous studies have shown that chronic cort exposure can induce a depressive - like motivational state in rodents that is similar to that produced by a chronic mild stress paradigm ( gourley et al . , 2008 ) . moreover , cort treatment alone is sufficient to alter molecular targets that are implicated in depression and antidepressant efficacy , such as hippocampal neurogenesis ( bilsland et al . , 2006 , ( 1997 ) , found that 4-day dexamethasone therapy significantly enhanced antidepressant response to ssris in treatment - resistant patients . therefore , we hypothesized that activation of stress circuitry might be important to reveal the behavioral and neurogenic effects of the ssri fluoxetine in c57bl/6 mice , a non - responsive mouse strain . in the current study we investigated the effects of exposure to commercial cort pellets for 21 days in augmenting fluoxetine 's behavioral and proliferative effects in c57bl/6 mice the results of this study showed that chronic fluoxetine produced behavioral effects in the nih test only in mice exposed to cort . furthermore , cort administration with fluoxetine co - treatment augmented hippocampal cell proliferation , an effect potentially mediated by alterations in hippocampal corticosteroid receptor expression . interestingly , analysis of plasma at the end of treatment revealed a paradoxical decrease in cort levels in animals treated with the pellets , suggesting that the cort pellets did not work as advertised . adrenalectomized animals implanted with cort pellets revealed a sharp drop in cort plasma levels by day 7 of treatment , indicating that this method of cort exposure produced transiently elevated , but not sustained , cort levels . nevertheless , these experiments revealed the important finding that cort exposure potentiates the behavioral and neurogenic effects of chronic fluoxetine administration in a mouse strain that is otherwise non - responsive to this antidepressant treatment . intact and adrenalectomized male c57bl/6j , 78 weeks old upon arrival , were purchased from jackson laboratories ( bar harbor , me ) . mice were housed in groups of 4 ( except for those used in the nih test whom were housed in pairs ) in polycarbonate cages and maintained on a 12 h light dark cycle ( lights on at 0700 h ) in a temperature ( 22 c)- and humidity - controlled environment . all experiments were approved by the institutional animal care and use committee at the university of pennsylvania . intact animals were implanted with cort pellets ( 10 mg ) or placebo pellets . beginning on day 7 of cort treatment , animals were dosed with either fluoxetine ( 5 mg / kg b.i.d . , i.p . ) or saline daily for the remaining 14 days of the experiment . cohort 1 : animals were tested in the nih and home cage test on the last two days of drug treatment ( n = 810 per group ) . cohort 2 : animals received a single injection of brdu on the last day of drug treatment and were sacrificed 24 h later . in these animals trunk blood was collected at time of sacrifice and analyzed for plasma cort levels ( n = 1519 per group ) . adrenalectomized animals were implanted with cort pellets ( 10 mg ) or placebo pellets and received chronic fluoxetine treatment as described in experiment 1 . all mice received additional cort replacement through the drinking water ( 25 g / ml in 0.9% saline ) to prevent the loss of electrolyte homeostasis ( funder , 2006 ) and eliminate the confounding effects of adrenalectomy alone on neurogenesis ( cameron and gould , 1994 ) . animals received a single injection of brdu on the last day of drug treatment and were sacrificed 24 h later . hippocampal tissue was dissected and analyzed for brdu positive cells ( n = 710 per group ) . intact animals were implanted with cort pellets ( 2.5 mg ) or placebo pellets and received chronic fluoxetine treatment as described in experiment 1 . animals received a single injection of brdu on the last day of drug treatment and were sacrificed 24 h later . hippocampal tissue was dissected and analyzed for brdu positive cells ( n = 910 per group ) . adrenalectomized animals were implanted with cort pellets ( 10 mg ) or placebo pellets and then sacrificed 1 , 7 , 14 , or 21 days after implantation . trunk blood was collected at time of sacrifice and analyzed for plasma cort levels ( n = 56 per group ) . cort pellets ( 2.5 mg and 10 mg , 21 day release , innovative research of america , sarasota , fl , usa ) were composed of a proprietary matrix of cholesterol , cellulose , lactose , phosphates and stearates designed to facilitate continuous and sustained diffusion of cort over a period of 21 days . fluoxetine hydrochloride ( 5 mg / kg ; anawa , zurich ) was dissolved in distilled water and delivered by intraperitoneal ( i.p . ) fluoxetine was administered twice daily because , due to its half - life , this dosing strategy results in relatively stable plasma levels ( hodes et al . , 2010 ) and occupation of brain serotonin transporters ( hirano et al . , 2004 ) . 5-bromo - deoxyuridine ( brdu ; roche applied sciences indianapolis , in ) was dissolved in warm saline at a dose of 200 mg / kg and administered i.p . in a volume of 10 ml / kg . mice were pair housed and trained to eat a palatable food ( three peanut butter chips presented in a small , clear petri dish ) in a home cage environment . animals were trained daily in 15-min sessions until they met the criteria of three consecutive days with approach latencies of 30 s or less . opaque , black , plastic dividers were placed inside each cage to separate the mice during training of home cage training sessions . mice were allowed to habituate to the dividers for 1 h before the start of the training session . once all animals had met criteria , training sessions were suspended and drug treatments were initiated . three days before novel testing all animals were re - exposed to the peanut butter chips through additional training sessions . for novel cage testing , peanut butter chips were presented in the center of an empty , clear polycarbonate cage ( 25.5 46 20 cm ) with bright lighting ( 60 w light bulb ) and scented with lemon ( 20% lemon joy solution ) . mice were placed into the test cage and the latency to approach during the 15-min test session was measured . flow cytometry is a frequently used method for analyzing newly dividing cells in the hippocampus . this method has been previously validated by our lab and others , and compared to results obtained from immunostaining ( balu et al . , 2009b ; bilsland et al . , 2006 , spoelgen et al . , brdu labeling was measured in cells displaying the nuclear marker 7-aminoactinomycin d ( 7-aad ) by flow cytometry as previously described ( balu et al . , 2009b ) . briefly , mice were decapitated 24 h following brdu injection , their brains quickly removed , and the hippocampus dissected . hippocampal tissue was manually minced , digested using an enzymatic mixture ( 1 mg / ml papain , roche applied science ; 0.1 m l - cysteine , sigma louis , mo ) , and then mechanically triturated to form a single cell suspension . cells were fixed , permeabilized , and stained using the fluorescein isothiocyanate ( fitc ) brdu flow kit ( bd biosciences , san jose , ca ) . data were collected on the same day using a bd facs canto system ( bd biosciences ) at the university of pennsylvania flow cytometry core facility . rna was extracted with trizol reagent ( gibco brl , life technologies , ny ) and purified using the rneasy mini kit ( qiagen , valencia , ca ) following the manufactures ' instructions . rna concentrations were measured and 300 ng/l rna was used as a template to synthesize c - dna using the superscript vilo c - dna synthesis kit ( invitrogen , carlsbad , ca , usa ) . all reactions were performed with a master mix of sybr green ( applied biosystems , austin , tx ) and 300 nm primers ( final concentration ) . quantitative real - time polymerase chain reactions ( qrt - pcr ) were run using the stratagene mx3000 and mxpro qpcr software . cycling parameters were as follows : 95 c for 10 min , 40 cycles at 95 c ( 30 s ) and 60 c ( 1 min ) , ending with a melting curve analysis to control for amplification . all reactions were performed in triplicate and the mean cycle threshold was used for analysis . the mrna levels of target genes were normalized to the house - keeping gene tata binding protein ( tbp ) using the 2 method . trunk blood was collected at time of sacrifice , which occurred between 8 and 10 am for all experiments . blood was stored in 0.5 ml heparin and centrifuged at 3000 rpm for 20 min . the amount of cort in the plasma from each sample was measured in duplicate by elisa following the manufactures instructions ( immunodiagnostic systems , fountain hills , az ) . intra - assay variability for the cort kit ranged from 5.9% to 7.0% , inter - assay variability ranged from 8.2 to 8.9% ; mean assay sensitivity was 0.17 ng / ml . one - way and two - way anova were performed to examine the significance of differences between treatments . significant overall main effects ( p < 0.05 ) or interactions showing a trend ( p < for all follow - up tests , p < 0.05 was considered statistically significant . mice were randomly assigned to either placebo or cort pellet exposure , and then further separated into either saline or fluoxetine treatment groups . as seen in fig . 1a , a two - way repeated measures anova revealed a significant interaction [ f(9,102 ) = 3.761 , p < 0.001 ] and main effect [ f(3,102 ) = 12.68 , p < 0.001 ] of time on body weight during drug treatment . placebo - treated animals exhibited significant weight gain by day 14 ( p < 0.05 ) . although cort - exposed animals failed to gain weight within the initial 7 days of treatment , animals subsequently treated with fluoxetine showed significant weight gain by day 21 ( p < 0.001 ) whereas saline treated animals continued to show inhibited weight gain . the overall change in body weight ( from day 1 to day 21 ) 1b illustrates a significant main effect of treatment with fluoxetine [ f(1 , 34 ) = 8.830 , p < 0.01 ] . the behavioral effects of cort and fluoxetine treatment were then measured in the nih test . exposure to a novel environment increased approach latency [ f(1,65 ) = 972.4 , p < 0.0001 ] and reduced the amount of food consumed [ f(1,68 ) = 136.0 , p < 0.0001 ] compared to home cage in all treatment groups . there was a significant interaction between cort exposure and fluoxetine treatment in approach latencies in the novel environment [ f(1,33 ) = 8.041 , p < 0.01 ] . cort - exposed animals treated with fluoxetine displayed significantly lower approach latencies compared to cort - exposed animals treated with saline . moreover , fluoxetine treatment had no effect on approach latency in placebo - treated animals in the novel environment ( fig . there were no significant differences in food consumption in the novel environment between drug treatment groups ( fig . cort treatment significantly reduced latency to approach [ f(1 , 33 ) = 4.772 , p < 0.05 ] and increased the amount of food consumed compared to placebo treated animals [ f(1 , 34 ) = 4.956 , p < 0.05 ] ( fig . 2c and d ) . in a separate cohort , animals received a single injection of brdu on the last day of drug treatment and were sacrificed 24 h later . additionally , trunk blood was collected at time of sacrifice and analyzed for plasma cort levels . as seen in fig . 3a , flow cytometric analysis of hippocampal tissue revealed that in placebo treated animals , fluoxetine had no effect on hippocampal cell proliferation . interestingly , cort - exposure significantly increased hippocampal cell proliferation compared to placebo treated animals [ f(1,59 ) = 50.87 , p < 0.001 ] . moreover , there was a significant interaction between cort exposure and fluoxetine treatment on neurogenesis [ f(1 , 59 ) = 6.702 , p < 0.05 ] . post - hoc multiple comparisons revealed that cort - exposed animals treated with fluoxetine displayed significantly higher hippocampal cell proliferation compared to cort - exposed animals treated with saline . analysis of circulating cort levels at the time of sacrifice revealed that exposure to cort pellets significantly reduced cort plasma levels in both saline and fluoxetine treated animals by approximately 50% when measured on day 21 [ f(1 , 66 ) = 36.06 , p < 0.001 ] ( fig . glucocorticoid ( gr ) and mineralocorticoid ( mr ) receptor transcription was examined in the hippocampus as a potential molecular mechanism underlying the cort - induced neurogenic response to fluoxetine in c57bl/6 mice . there was no significant effect of cort or fluoxetine on gr mrna expression ( fig . however , exposure to cort significantly reduced hippocampal mr mrna expression in both saline and fluoxetine treated animals [ f(1,68 ) = 4.276 , p < 0.05 ] ( fig . 3d ) . to investigate the mechanisms underlying the increase in hippocampal cell proliferation by cort pellets , adrenalectomized animals were used to examine the effects of 10 mg cort pellet exposure and fluoxetine treatment on hippocampal neurogenesis in the absence of adrenal feedback . there was a significant main effect of cort on cell proliferation [ f(1 , 30 ) = 5.298 , p < 0.05 ] and a trend towards an interaction [ f ( 1 , 30 ) = 3.372 p = 0.08 ] . as illustrated in fig . 4a , adrenalectomized cort - exposed animals treated with fluoxetine , but not saline , displayed a significant two - fold increase in cell proliferation compared to placebo treated animals ( p < 0.05 ) . we next examined whether a lower dose of cort pellet exposure combined with fluoxetine treatment would induce an increase in hippocampal neurogenesis in intact animals . there was a significant main effect of cort pellet exposure [ f(1 , 35 ) = 6.477 , p < 0.05 ] and a significant interaction between cort pellet exposure and fluoxetine treatment [ f(1 , 35 ) = 4.705 , p < 0.05 ] on hippocampal cell proliferation . as shown in fig . 4b , cort - exposed animals treated with saline exhibited a significant increase in cell proliferation compared to placebo treated animals , as in prior studies . in contrast , the lower dose of cort pellet was incapable of increasing hippocampal cell proliferation when combined with fluoxetine treatment . to determine whether 10 mg cort pellets maintain elevated plasma cort levels for the advertised duration , adrenalectomized animals were implanted with 10 mg cort pellets on day 0 and , cort plasma levels were assessed on day 1 , 7 , 14 , and 21 post - implantation . as shown in fig . 5 , plasma cort levels changed dramatically over time ( f(3,19 ) = 16.18 , p < 0.01 ) , and were no longer in the supraphysiological range by the seventh day of cort pellet exposure ( fig . 5 ) . activation of stress circuitry from implanted cort pellets produced behavioral and neurogenic effects from chronic fluoxetine treatment in a strain of mice that would otherwise have been unresponsive to the effects of the antidepressant . specifically , co - treatment of cort with fluoxetine significantly reduced the effects of novelty stress measured on approach behavior in the nih test and increased hippocampal cell proliferation . these two effects of antidepressant treatment have been linked together because the behavioral response to fluoxetine is blocked in mice that can not increase hippocampal cell proliferation ( sahay and hen , 2007 ) . although treatment with cort alone unexpectedly increased cell proliferation to a lesser extent , this effect was absent in adrenalectomized mice while the augmented combination treatment effect was preserved . measurement of plasma cort levels revealed that the cort pellet did not maintain elevated levels for more than a few days , even though it was expected to be active for 21 days , suggesting that the impact of the cort treatment was likely the after - effect resulting from the supraphysiological levels of acute exposure . overall , these findings reveal potential neurobiological mechanisms underlying effective antidepressant response in a unique model of treatment resistance . hyponeophagia , the unconditioned suppression of feeding in a novel environment , is a behavioral measure of stress that may be sensitive to the anxiolytic effects of chronic , but not acute , antidepressant treatment with ssris ( bechtholt et al . fluoxetine 's effect of reducing approach latency to food in a novel environment is abolished after focal irradiation of the hippocampus or genetic deletion of hippocampal precursor cells , indicating that hippocampal neurogenesis is a necessary component of this behavioral antidepressant response ( david et al . intriguingly , unlike other mouse strains , c57bl/6 mice did not exhibit reduced hyponeophagia or increased hippocampal neurogenesis following chronic fluoxetine treatment ( balu et al . , 2009a ) . however , in the present study , we showed that cort - exposure via pellet implantation induced a behavioral response to fluoxetine in the nih test in this unresponsive strain . moreover , cort - exposure in combination with fluoxetine treatment produced a robust increase in hippocampal neurogenesis that was not seen in placebo treated animals . although correlative , the increased behavioral response to chronic fluoxetine treatment in cort - treated mice could be attributed to heightened hippocampal cell proliferation . stress is a well - established robust inhibitor of adult neurogenesis ( gould and tanapat , 1999 , mcewen et al . , 2012 ) . similarly , cort exposure alone has been shown to be a negative regulator of hippocampal neurogenesis ( bilsland et al . , 2006 , brummelte and galea , 2010 , cameron and gould , 1994 , murray et al . , 2008 , reduced hippocampal cell proliferation typically coincides with increased plasma cort levels , signifying that circulating cort levels at the time of testing underlie cort - induced changes in proliferation ( wong and herbert , 2006 ) . paradoxically , we observed dramatically reduced plasma cort levels in all cort - exposed animals following the 21-day pellet treatment , while hippocampal cell proliferation was increased . the adrenals operate through an inhibitory feedback system in which increased circulating cort levels serve as a signal for reduced synthesis and secretion from the adrenals ( herman and cullinan , 1997 , sapolsky et al . , 1984 ) . cort pellet treatment may have increased internal negative feedback to the point of adrenal inactivation , resulting in reduced endogenous circulating cort levels and disinhibition of cell proliferation . to test for this we examined the effects of cort pellet and chronic fluoxetine co - treatment on hippocampal cell proliferation in adrenalectomized animals . notably , in the absence of adrenal feedback , cort - exposed animals treated with saline did not demonstrate increased neurogenesis whereas those given fluoxetine still exhibited a proliferative response . therefore , the mechanisms underlying the augmented neurogenic effect seen in combination treated animals can not be attributed to artifacts of adrenal feedback . it is important to note , however , that adrenalectomized animals in all treatment groups were supplemented with a low dose cort - treatment ( 25 g / ml ) delivered via drinking water . this mode of delivery produces small rhythmic changes in plasma cort levels , which have been shown to be necessary for fluoxetine - stimulated neurogenesis in rats ( huang and herbert , 2006 ) . it is possible , then , that rhythmic fluctuations in cort levels modulate sensitivity to the proliferative effects of fluoxetine . in spite of this , adrenalectomized placebo animals treated with fluoxetine did not exhibit increased proliferation , demonstrating that supplemental cort alone was not sufficient to induce a neurogenic response to fluoxetine . to determine whether a lower dose of cort could elicit a neurogenic response in the presence of fluoxetine without increasing proliferation on its own , we evaluated hippocampal cell proliferation in intact animals treated with 2.5 mg cort pellets . similar to the 10 mg cort pellet treatment , exposure to 2.5 mg cort pellet treatment produced elevated cell proliferation . however , there was no additional effect in the presence of fluoxetine , suggesting that this dose is sufficient to produce cort - induced increases in neurogenesis , but not sufficient to elicit an augmented proliferative response when combined with fluoxetine . ( 2009 ) who showed a low dose of 5 mg / kg / day cort treatment to be effective in reducing hippocampal cell proliferation alone and stimulating proliferation when paired with fluoxetine treatment in c57bl/6 mice . however , whereas the current study utilized a three - week cort pellet treatment , david and colleagues had cort delivered though drinking water and animals were treated for a substantially longer period of time ( 7 weeks ) . therefore , rhythmic low dose cort treatment over a longer period of time may be sufficient to increase neuronal sensitization to fluoxetine in this strain . altered corticosteroid activity can dysregulate the stress response system and enhance the risk of development of stress - related disorders ( groeneweg et al . , 2012 ) . on the other hand , synergistic interactions between hippocampal corticosteroid receptors and serotonergic signaling pathways may mediate the effects of cort exposure on enhancing the neurogenic responses to fluoxetine in c57bl/6 mice . for example , cort treatment has been shown to facilitate fluoxetine - induced enhancement of dopaminergic modulation at the mossy fiber synapse ( kobayashi et al . , 2013 ) . consistent with recent findings , cort treatment reduced hippocampal mr mrna expression while having no effect on gr mrna expression ( saenz del burgo et al . , 2013 ) , suggesting that mr mrna expression is more sensitive to the effects of cort exposure . this may be due to the fact that mrs exhibit 10-fold higher affinity for cort compared to grs ( joels et al . , 2008 ) . hippocampal mrs selectively contribute to neuronal stability and excitatory tone and have been shown to mediate behavioral reactivity to a novel environment ( berger et al . 1994 ) , hence changes in mr expression and function are likely to impact both hippocampal plasticity and associated behaviors . interestingly , in the current study , mr expression was similar between cort - exposed animals treated to either saline or fluoxetine treated , indicating that variations in expression alone can not explain the augmented behavioral and neurogenic responses seen in cort - exposed animals treated with fluoxetine . however , it is important to note that mr and gr expression exhibit a diurnal regulation that is modulated by circulating cort levels ( herman et al . , 1993 , holmes et al . , 1995 ) . since exogenous cort treatment has been reported to flatten the natural circadian rhythm of plasma cort ( leitch et al . , 2003 ) , cort pellet exposure could potentially alter rhythmic mr and gr occupancy throughout the day thus , the observed neurogenic effects of cort and fluoxetine treatment might be mediated by changes in circadian expression of mr and grs . a major caveat of this study is the lack of sustained cort release from the pellet treatment . cort pellets have been used to model chronically elevated cort levels , a physiological indicator of dysregulated hpa axis functioning and risk factor for the onset of mdd ( goodyer et al . , 2010 , owens et al . , 2014 ) . on the contrary , we found that cort plasma levels dropped precipitously between day 1 and day 7 of cort pellet treatment . rather than a sustained release , the cort pellets produced a rapid , but short - lived , elevation of cort during the initial days of exposure and then became inactive , resulting in cort levels falling to the normal physiological range at the time of the experimental studies . similar findings were reported in another study evaluating the performance of pellets designed to release cort for 7 days in birds . ( 2009 ) found that cort plasma levels peaked 12 days after pellet implantation and reached placebo levels by day 3 . the authors posited that the pellets , being originally designed for rodents , are not as effectively metabolized in other species . however , our data corroborate the findings that the cort pellets do not reliably produce sustained cort release for the indicated length of treatment . in light of this , slow - release cort pellets are not appropriate for modeling prolonged elevated cort levels . instead , these pellets may more closely model the effects of exposure to a strong acute stressor , as with post - traumatic stress disorder . interestingly , the initial surge in cort levels during the first few days of treatment was sufficient to induce long lasting molecular and behavioral changes in treated animals , suggesting that alterations in cort levels , not necessarily at a pathological level , can impact the efficacy of fluoxetine . in conclusion , this study found that exposure to exogenous cort increases behavioral and neurogenic sensitivity to chronic fluoxetine treatment in c57bl/6 mice , a typically non - responsive strain of mice . these data recapitulate the general findings that genetic background and environment play a fundamental role in antidepressant response . although slow - release cort pellets did not model the effects of sustained elevated cort exposure as anticipated , these studies effectively indicate that cort exposure is sufficient to reveal the anxiolytic and neuroplastic effects of chronic fluoxetine treatment in a typically unresponsive strain and could model an augmentation strategy for treatment - resistant patients . these findings implicate corticosteroid receptor activity and modulation as a potential variable in the stratification of antidepressant response in patients with mdd and possibly as a mediator of the effects of environmental stress on the effects of antidepressants .

both genetic background and pre - existing stress play critical roles in the effects of antidepressant drugs . the current studies showed this principal by demonstrating that exposure to the stress hormone corticosterone ( cort ) allowed behavioral and neurogenic effects to emerge following chronic treatment with fluoxetine of c57bl/6 mice , a strain ordinarily resistant to these effects . adult male mice were implanted subcutaneously with 21-day slow - release cort pellets ( 10 mg ) or placebo and then co - treated with 5 mg / kg fluoxetine ( b.i.d . , i.p . ) or saline for 14 days . animals were then assessed for approach behavior in the novelty - induced hypophagia ( nih ) test , hippocampal cell proliferation , corticosteroid receptor expression , and cort plasma levels . co - treatment of cort with fluoxetine significantly reduced approach behavior in the novel environment of the nih test and increased hippocampal cell proliferation whereas fluoxetine given alone was ineffective . cort given alone did not alter approach behavior in the novel environment and caused a smaller increase of cell proliferation . the cort effect was blocked by adrenalectomy and was likely due to increased adrenal feedback . cell proliferation in cort - treated animals was associated with reduced mineralocorticoid , but not glucocorticoid , receptor mrna expression . although the pellets were advertised to release cort for 21 days , plasma cort levels were increased at 1 day after implantation but were not sustained when measured at 7 days or longer intervals . nevertheless , the transient cort increase was sufficient to induce long - lasting behavioral and molecular changes when followed by fluoxetine treatment . these studies warrant further investigation into the role of glucocorticoids and environmental stress as adjunctive facilitators of the response to antidepressants , especially for treatment - resistant patients .

Introduction Materials and methods Results Discussion Conflict of interest

major depressive disorder ( mdd ) is one of the most common psychiatric disorders , with a lifetime prevalence of 17% in the united states and 4% worldwide ( eaton et al . in terms of years lost to disability , mdd is considered one of the most disabling medical conditions and is predicted to become a leading contributor to the worldwide burden of disease ( mathers and loncar , 2006 ) . together , these findings suggest a potential role of stress hormones , such as cortisol ( corticosterone ( cort ) in rodents ) , in the pathology and treatment of depression . a reduction or absence of these inhibitory signals can promote hyperactivation of the axis and augmented secretion of glucocorticoids ( anacker et al . however , prolonged exposure to cort can inhibit the proliferation and survival of adult - born hippocampal neurons , which have been shown to play an important role in the behavioral and neuroendocrine components of stress responses in rodents ( gould and tanapat , 1999 ; snyder et al . conversely , chronic treatment of normal rodents with ssris , such as fluoxetine , increases hippocampal neurogenesis and neurotrophins such as brain derived neurotrophic factor ( bdnf ) ( duman and monteggia , 2006 , krishnan and nestler , 2008 , schmidt and duman , 2006 ) . increased hippocampal neurogenesis is associated with behavioral indications of antidepressant efficacy in rodents , such as reduced hyponeophagia in the novelty - induced hypophagia ( nih ) test and performance in the forced swim test ( dranovsky and hen , 2006 ) . not all strains of mice respond to the behavioral and neurogenic effects of antidepressant treatments . for example , normal c57bl/6 mice are unresponsive to the behavioral effects of chronic fluoxetine treatment , measured in the nih test , and do not exhibit increased hippocampal cell proliferation ( balu et al . , 2006 , ( 1997 ) , found that 4-day dexamethasone therapy significantly enhanced antidepressant response to ssris in treatment - resistant patients . therefore , we hypothesized that activation of stress circuitry might be important to reveal the behavioral and neurogenic effects of the ssri fluoxetine in c57bl/6 mice , a non - responsive mouse strain . in the current study we investigated the effects of exposure to commercial cort pellets for 21 days in augmenting fluoxetine 's behavioral and proliferative effects in c57bl/6 mice the results of this study showed that chronic fluoxetine produced behavioral effects in the nih test only in mice exposed to cort . furthermore , cort administration with fluoxetine co - treatment augmented hippocampal cell proliferation , an effect potentially mediated by alterations in hippocampal corticosteroid receptor expression . interestingly , analysis of plasma at the end of treatment revealed a paradoxical decrease in cort levels in animals treated with the pellets , suggesting that the cort pellets did not work as advertised . adrenalectomized animals implanted with cort pellets revealed a sharp drop in cort plasma levels by day 7 of treatment , indicating that this method of cort exposure produced transiently elevated , but not sustained , cort levels . nevertheless , these experiments revealed the important finding that cort exposure potentiates the behavioral and neurogenic effects of chronic fluoxetine administration in a mouse strain that is otherwise non - responsive to this antidepressant treatment . mice were housed in groups of 4 ( except for those used in the nih test whom were housed in pairs ) in polycarbonate cages and maintained on a 12 h light dark cycle ( lights on at 0700 h ) in a temperature ( 22 c)- and humidity - controlled environment . intact animals were implanted with cort pellets ( 10 mg ) or placebo pellets . beginning on day 7 of cort treatment , animals were dosed with either fluoxetine ( 5 mg / kg b.i.d . , i.p . ) or saline daily for the remaining 14 days of the experiment . cohort 1 : animals were tested in the nih and home cage test on the last two days of drug treatment ( n = 810 per group ) . adrenalectomized animals were implanted with cort pellets ( 10 mg ) or placebo pellets and received chronic fluoxetine treatment as described in experiment 1 . intact animals were implanted with cort pellets ( 2.5 mg ) or placebo pellets and received chronic fluoxetine treatment as described in experiment 1 . adrenalectomized animals were implanted with cort pellets ( 10 mg ) or placebo pellets and then sacrificed 1 , 7 , 14 , or 21 days after implantation . cort pellets ( 2.5 mg and 10 mg , 21 day release , innovative research of america , sarasota , fl , usa ) were composed of a proprietary matrix of cholesterol , cellulose , lactose , phosphates and stearates designed to facilitate continuous and sustained diffusion of cort over a period of 21 days . fluoxetine hydrochloride ( 5 mg / kg ; anawa , zurich ) was dissolved in distilled water and delivered by intraperitoneal ( i.p . ) fluoxetine was administered twice daily because , due to its half - life , this dosing strategy results in relatively stable plasma levels ( hodes et al . 5-bromo - deoxyuridine ( brdu ; roche applied sciences indianapolis , in ) was dissolved in warm saline at a dose of 200 mg / kg and administered i.p . mice were allowed to habituate to the dividers for 1 h before the start of the training session . briefly , mice were decapitated 24 h following brdu injection , their brains quickly removed , and the hippocampus dissected . hippocampal tissue was manually minced , digested using an enzymatic mixture ( 1 mg / ml papain , roche applied science ; 0.1 m l - cysteine , sigma louis , mo ) , and then mechanically triturated to form a single cell suspension . the amount of cort in the plasma from each sample was measured in duplicate by elisa following the manufactures instructions ( immunodiagnostic systems , fountain hills , az ) . mice were randomly assigned to either placebo or cort pellet exposure , and then further separated into either saline or fluoxetine treatment groups . placebo - treated animals exhibited significant weight gain by day 14 ( p < 0.05 ) . although cort - exposed animals failed to gain weight within the initial 7 days of treatment , animals subsequently treated with fluoxetine showed significant weight gain by day 21 ( p < 0.001 ) whereas saline treated animals continued to show inhibited weight gain . the behavioral effects of cort and fluoxetine treatment were then measured in the nih test . exposure to a novel environment increased approach latency [ f(1,65 ) = 972.4 , p < 0.0001 ] and reduced the amount of food consumed [ f(1,68 ) = 136.0 , p < 0.0001 ] compared to home cage in all treatment groups . there was a significant interaction between cort exposure and fluoxetine treatment in approach latencies in the novel environment [ f(1,33 ) = 8.041 , p < 0.01 ] . cort - exposed animals treated with fluoxetine displayed significantly lower approach latencies compared to cort - exposed animals treated with saline . moreover , fluoxetine treatment had no effect on approach latency in placebo - treated animals in the novel environment ( fig . there were no significant differences in food consumption in the novel environment between drug treatment groups ( fig . cort treatment significantly reduced latency to approach [ f(1 , 33 ) = 4.772 , p < 0.05 ] and increased the amount of food consumed compared to placebo treated animals [ f(1 , 34 ) = 4.956 , p < 0.05 ] ( fig . additionally , trunk blood was collected at time of sacrifice and analyzed for plasma cort levels . 3a , flow cytometric analysis of hippocampal tissue revealed that in placebo treated animals , fluoxetine had no effect on hippocampal cell proliferation . interestingly , cort - exposure significantly increased hippocampal cell proliferation compared to placebo treated animals [ f(1,59 ) = 50.87 , p < 0.001 ] . post - hoc multiple comparisons revealed that cort - exposed animals treated with fluoxetine displayed significantly higher hippocampal cell proliferation compared to cort - exposed animals treated with saline . analysis of circulating cort levels at the time of sacrifice revealed that exposure to cort pellets significantly reduced cort plasma levels in both saline and fluoxetine treated animals by approximately 50% when measured on day 21 [ f(1 , 66 ) = 36.06 , p < 0.001 ] ( fig . glucocorticoid ( gr ) and mineralocorticoid ( mr ) receptor transcription was examined in the hippocampus as a potential molecular mechanism underlying the cort - induced neurogenic response to fluoxetine in c57bl/6 mice . there was no significant effect of cort or fluoxetine on gr mrna expression ( fig . however , exposure to cort significantly reduced hippocampal mr mrna expression in both saline and fluoxetine treated animals [ f(1,68 ) = 4.276 , p < 0.05 ] ( fig . to investigate the mechanisms underlying the increase in hippocampal cell proliferation by cort pellets , adrenalectomized animals were used to examine the effects of 10 mg cort pellet exposure and fluoxetine treatment on hippocampal neurogenesis in the absence of adrenal feedback . there was a significant main effect of cort on cell proliferation [ f(1 , 30 ) = 5.298 , p < 0.05 ] and a trend towards an interaction [ f ( 1 , 30 ) = 3.372 p = 0.08 ] . 4a , adrenalectomized cort - exposed animals treated with fluoxetine , but not saline , displayed a significant two - fold increase in cell proliferation compared to placebo treated animals ( p < 0.05 ) . we next examined whether a lower dose of cort pellet exposure combined with fluoxetine treatment would induce an increase in hippocampal neurogenesis in intact animals . there was a significant main effect of cort pellet exposure [ f(1 , 35 ) = 6.477 , p < 0.05 ] and a significant interaction between cort pellet exposure and fluoxetine treatment [ f(1 , 35 ) = 4.705 , p < 0.05 ] on hippocampal cell proliferation . 4b , cort - exposed animals treated with saline exhibited a significant increase in cell proliferation compared to placebo treated animals , as in prior studies . in contrast , the lower dose of cort pellet was incapable of increasing hippocampal cell proliferation when combined with fluoxetine treatment . to determine whether 10 mg cort pellets maintain elevated plasma cort levels for the advertised duration , adrenalectomized animals were implanted with 10 mg cort pellets on day 0 and , cort plasma levels were assessed on day 1 , 7 , 14 , and 21 post - implantation . 5 , plasma cort levels changed dramatically over time ( f(3,19 ) = 16.18 , p < 0.01 ) , and were no longer in the supraphysiological range by the seventh day of cort pellet exposure ( fig . activation of stress circuitry from implanted cort pellets produced behavioral and neurogenic effects from chronic fluoxetine treatment in a strain of mice that would otherwise have been unresponsive to the effects of the antidepressant . specifically , co - treatment of cort with fluoxetine significantly reduced the effects of novelty stress measured on approach behavior in the nih test and increased hippocampal cell proliferation . these two effects of antidepressant treatment have been linked together because the behavioral response to fluoxetine is blocked in mice that can not increase hippocampal cell proliferation ( sahay and hen , 2007 ) . although treatment with cort alone unexpectedly increased cell proliferation to a lesser extent , this effect was absent in adrenalectomized mice while the augmented combination treatment effect was preserved . measurement of plasma cort levels revealed that the cort pellet did not maintain elevated levels for more than a few days , even though it was expected to be active for 21 days , suggesting that the impact of the cort treatment was likely the after - effect resulting from the supraphysiological levels of acute exposure . hyponeophagia , the unconditioned suppression of feeding in a novel environment , is a behavioral measure of stress that may be sensitive to the anxiolytic effects of chronic , but not acute , antidepressant treatment with ssris ( bechtholt et al . fluoxetine 's effect of reducing approach latency to food in a novel environment is abolished after focal irradiation of the hippocampus or genetic deletion of hippocampal precursor cells , indicating that hippocampal neurogenesis is a necessary component of this behavioral antidepressant response ( david et al . intriguingly , unlike other mouse strains , c57bl/6 mice did not exhibit reduced hyponeophagia or increased hippocampal neurogenesis following chronic fluoxetine treatment ( balu et al . however , in the present study , we showed that cort - exposure via pellet implantation induced a behavioral response to fluoxetine in the nih test in this unresponsive strain . moreover , cort - exposure in combination with fluoxetine treatment produced a robust increase in hippocampal neurogenesis that was not seen in placebo treated animals . although correlative , the increased behavioral response to chronic fluoxetine treatment in cort - treated mice could be attributed to heightened hippocampal cell proliferation . , 2008 , reduced hippocampal cell proliferation typically coincides with increased plasma cort levels , signifying that circulating cort levels at the time of testing underlie cort - induced changes in proliferation ( wong and herbert , 2006 ) . paradoxically , we observed dramatically reduced plasma cort levels in all cort - exposed animals following the 21-day pellet treatment , while hippocampal cell proliferation was increased . cort pellet treatment may have increased internal negative feedback to the point of adrenal inactivation , resulting in reduced endogenous circulating cort levels and disinhibition of cell proliferation . to test for this we examined the effects of cort pellet and chronic fluoxetine co - treatment on hippocampal cell proliferation in adrenalectomized animals . notably , in the absence of adrenal feedback , cort - exposed animals treated with saline did not demonstrate increased neurogenesis whereas those given fluoxetine still exhibited a proliferative response . therefore , the mechanisms underlying the augmented neurogenic effect seen in combination treated animals can not be attributed to artifacts of adrenal feedback . it is important to note , however , that adrenalectomized animals in all treatment groups were supplemented with a low dose cort - treatment ( 25 g / ml ) delivered via drinking water . this mode of delivery produces small rhythmic changes in plasma cort levels , which have been shown to be necessary for fluoxetine - stimulated neurogenesis in rats ( huang and herbert , 2006 ) . it is possible , then , that rhythmic fluctuations in cort levels modulate sensitivity to the proliferative effects of fluoxetine . in spite of this , adrenalectomized placebo animals treated with fluoxetine did not exhibit increased proliferation , demonstrating that supplemental cort alone was not sufficient to induce a neurogenic response to fluoxetine . to determine whether a lower dose of cort could elicit a neurogenic response in the presence of fluoxetine without increasing proliferation on its own , we evaluated hippocampal cell proliferation in intact animals treated with 2.5 mg cort pellets . similar to the 10 mg cort pellet treatment , exposure to 2.5 mg cort pellet treatment produced elevated cell proliferation . however , there was no additional effect in the presence of fluoxetine , suggesting that this dose is sufficient to produce cort - induced increases in neurogenesis , but not sufficient to elicit an augmented proliferative response when combined with fluoxetine . ( 2009 ) who showed a low dose of 5 mg / kg / day cort treatment to be effective in reducing hippocampal cell proliferation alone and stimulating proliferation when paired with fluoxetine treatment in c57bl/6 mice . on the other hand , synergistic interactions between hippocampal corticosteroid receptors and serotonergic signaling pathways may mediate the effects of cort exposure on enhancing the neurogenic responses to fluoxetine in c57bl/6 mice . , 2013 ) , suggesting that mr mrna expression is more sensitive to the effects of cort exposure . this may be due to the fact that mrs exhibit 10-fold higher affinity for cort compared to grs ( joels et al . interestingly , in the current study , mr expression was similar between cort - exposed animals treated to either saline or fluoxetine treated , indicating that variations in expression alone can not explain the augmented behavioral and neurogenic responses seen in cort - exposed animals treated with fluoxetine . , 2003 ) , cort pellet exposure could potentially alter rhythmic mr and gr occupancy throughout the day thus , the observed neurogenic effects of cort and fluoxetine treatment might be mediated by changes in circadian expression of mr and grs . cort pellets have been used to model chronically elevated cort levels , a physiological indicator of dysregulated hpa axis functioning and risk factor for the onset of mdd ( goodyer et al . on the contrary , we found that cort plasma levels dropped precipitously between day 1 and day 7 of cort pellet treatment . rather than a sustained release , the cort pellets produced a rapid , but short - lived , elevation of cort during the initial days of exposure and then became inactive , resulting in cort levels falling to the normal physiological range at the time of the experimental studies . similar findings were reported in another study evaluating the performance of pellets designed to release cort for 7 days in birds . ( 2009 ) found that cort plasma levels peaked 12 days after pellet implantation and reached placebo levels by day 3 . in light of this , slow - release cort pellets are not appropriate for modeling prolonged elevated cort levels . instead , these pellets may more closely model the effects of exposure to a strong acute stressor , as with post - traumatic stress disorder . interestingly , the initial surge in cort levels during the first few days of treatment was sufficient to induce long lasting molecular and behavioral changes in treated animals , suggesting that alterations in cort levels , not necessarily at a pathological level , can impact the efficacy of fluoxetine . in conclusion , this study found that exposure to exogenous cort increases behavioral and neurogenic sensitivity to chronic fluoxetine treatment in c57bl/6 mice , a typically non - responsive strain of mice . these data recapitulate the general findings that genetic background and environment play a fundamental role in antidepressant response . although slow - release cort pellets did not model the effects of sustained elevated cort exposure as anticipated , these studies effectively indicate that cort exposure is sufficient to reveal the anxiolytic and neuroplastic effects of chronic fluoxetine treatment in a typically unresponsive strain and could model an augmentation strategy for treatment - resistant patients . these findings implicate corticosteroid receptor activity and modulation as a potential variable in the stratification of antidepressant response in patients with mdd and possibly as a mediator of the effects of environmental stress on the effects of antidepressants .

reliable estimates of population sizes of target species are of central importance to many conservation programs . density estimates provide baseline data on species abundance and allow the monitoring of population sizes , which is important in assessing the suitability of conservation strategies . however , few studies compare different survey methods and designs for large mammals under the same conditions , for instance , the same time of year and same location ( nijman and menken 2005 ; nomani et al . 2012 ; viquerat et al . such a comparison , however , can highlight strengths and weaknesses of the specific methods and thus provide useful information for conservation practitioners when deciding on the most suitable methods to deliver their conservation objectives . one of the most common population assessment methods for diurnal primates is line transect sampling ( buckland et al . this method is known to be accurate if a small number of key assumptions are met . best practice guidelines regarding survey design , field protocol , and analysis are available ( buckland et al . 2010 ) but several primatologists have discussed the possibility that this method is less efficient for some species , among them the members of the gibbon family hylobatidae ( nijman and menken 2005 ; whittaker 2005 ; and see discussion in waltert et al . 2008 ) . gibbons are small bodied arboreal apes found across eastern and southeastern asia , northwest india , and bangladesh . in comparison to other primates , gibbons have distinctive behavioural characteristics , including regular vocal displays ( bartlett 2007 ) . morning calls are thought to function as territory defense as well as to strengthen the pair bond ( cheyne et al . owing to their otherwise inconspicuous behavior and their tendency to use the upper canopy ( nijman 2001 ) , line transect surveys might fail to record 100 % of individuals near the transect line , resulting in underestimation of population densities if no action is taken to accommodate the violation of this key assumption ( buckland et al . triangulation ( also known as auditory sampling or fixed point counting ) has recently become a commonly applied method to estimate gibbon densities and has proved to be both successful and time efficient ( brockelman and ali 1987 ; brockelman and srikosamatara 1993 ; buckley et al . further , triangulation is seen as the most applicable method in hilly terrain ( nijman and menken 2005 ) . besides the obvious advantages , such as the fact that sampling does not affect gibbon behaviour toward the observer , triangulation can be performed within a relatively short time period and it is less destructive , as no transects need to be cut . it may , nevertheless , also introduce some sources of bias , e.g. , through the influence of weather conditions on singing behavior or by underestimation of distances to calling groups , the latter of which may result in overestimation of densities ( cheyne et al . in addition , calling rates and data on cluster ( groups or subgroups of primate social units ) size are needed , requiring long - term data or extra survey effort . triangulation alone does not ensure a sufficient number of sightings to estimate cluster size ( cheyne et al . here we compare line transect sampling with triangulation in estimating densities of hylobates klossii in the peleonan forest , a mixed evergreen rainforest with hilly terrain in the north of siberut island ( hadi et al . hylobates klossii is endemic to the mentawai archipelago and is listed as endangered by iucn ( whittaker and geissmann 2008 ) . if the two methods have comparable accuracy then the results for density estimates should not differ significantly . the mentawai archipelago is located 85135 km west of the island of sumatra , indonesia . siberut is the most northerly and largest island with an area of 4030 km ( whittaker 2006 ; whitten 1982a ) . close to the northern coast of siberut lies the peleonan forest , which consists of a 45 km section of primary lowland and swamp forest ( quinten et al . the maximum elevation is 190 m above sea level and the area is characterized by hills and ridges that are covered by primary mixed evergreen rain forest ( hadi et al . 2009 ) . pungut field station of the siberut conservation programme ( scp ) is located ( 101s and 9850e ) in the south of the peleonan forest . we conducted this study in a circular area of 10.7 km around the field station . the transect system around the station consists of 13 systematically placed transects each 13 km in length ( waltert et al . we conducted triangulation at four different survey sites covering the study area of the scp during 4 wk from september 8 until october 3 , 2010 . at each sample site although distance is not required for triangulation , we used the distance estimates to check that the same cluster was heard by two different listening posts . we checked distance estimates up to 900 m by moving from the listening post to the calling cluster using a global positioning system ( garmin gpsmap 60csx ) . we collected data from three listening posts situated in a triangle formation on elevated terrain . the distances between listening posts were 300500 m. we collected data on four consecutive days at each site at 04:3010:00 h. observers stayed at the listening posts until the gibbons had stopped singing for 30 min . we conducted line transect surveys between 06:3011:30 h and 15:3018:00 h from september 14 and november 26 , 2010 . protocols , season , survey times , surveyed transects , and data analysis followed those of the last survey in the study area in 2005 ( waltert et al . 2008 ) . we surveyed each of the 13 transects around the field station between six and eight times ( mean 7.23 , sd = 0.6 ) , resulting in a total survey effort of 155.5 km . we documented all detections of primates , recording species , cluster size , and the perpendicular distance from the transect line to the estimated center of the cluster using a laser range finder ( nikon 550 ) for distances > 10 m and a tape measure for distances < 10 m. we analyzed triangulation data following a triangulation protocol ( cheyne et al . the triangulation method usually uses only duet counts for gibbons to ensure that a reproductive gibbon cluster is recorded ( brockelman and srikosamatara 1993 ) . however , unlike most gibbon species , male and female hylobates klossii do not duet . instead males can be heard to sing between 01:00 and 13:00 h , but concentrate mainly on the hours before dawn , whereas females sing after dawn ( whitten 1982b ) . we used only clusters that were recorded after dawn , i.e. , those likely to be females , for analysis . we determined cluster size by mapping all sightings of gibbon clusters encountered during the entire study period . we assumed that individuals sighted > 300 m apart belonged to different gibbon clusters . for clusters encountered several times at the same location , we used the maximum number of individuals recorded as the size for that cluster . owing to time constraints , we could not assess calling rates , so we assumed that females call every 34 d ( whitten 1982b ) . we aimed for 3 d survey at each site , but unfortunately this turned out not to be possible , owing to time constraints and weather conditions in one site where we obtained only 2 survey days . distance to the cluster is noted to aid data analysis if there are two clusters singing on the same compass bearing . 2010 ) . where there is no conflict in number of clusters on any given compass bearing , the compass directions only are plotted and intersections used to indicate clusters . previous research on hylobates klossii estimated their home range to be 2035 ha ( whitten 1982a ) , giving a circle of diameter 250333 m. we , therefore , defined clusters > 300 m apart as different clusters . those closer than 300 m are likely to represent double counting where gibbons moved between calling bouts . we took the number of clusters heard per day and the total number of clusters heard from each listening post from the maps . we determined effective listening areas the areas where a minimum of two listening posts could have heard gibbon calls , within a radius of 1000 m around each post using arcgis 9.2 . we calculated density estimates using the following formula and correction factors , assuming that singing on successive days is independent ( brockelman and ali 1987 ; cheyne et al . 2008 ) . density estimate : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ d={n \left/ { { \left [ { p(m)\times e } \right ] } } \right . } $ $ \end{document } and correction factor : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p(m ) = 1\text{-- } { { \left [ { 1\text{--}\ p(1 ) } \right]}^m } , $ $ \end{document } where n refers to the cumulative total number of clusters heard in the effective listening area e at each site , p(m ) denotes the cumulative number of clusters singing in m days , p(1 ) refers to the mean of the proportion of clusters heard calling at each site ( once the data from all study days were combined ) . we calculated coefficients of variation ( cv ) using the formula cv = / , where denotes standard deviation and denotes mean . we calculated expected cluster size from a size - biased regression of ln[cluster size ] against estimated detection probability g(x ) . ideally surveys should be conducted using a systematic random design or a set of equally spaced transects randomly located in the survey region ( buckland et al . 2010 ) . at pungut , however , the trail system is oriented in a radial manner around the field station . to account for potential bias arising from over - coverage of the area directly surrounding the station , we stratified data in an inner circle with an area of 1.13 km ( radius = 0.6 km ) and an outer belt of 9.55 km . because cluster density estimates did not differ significantly between stratified data and nonstratified data ( z = 0.47 , p = 0.64 ) we present estimates from non - stratified data . we compared cluster and individual density estimates between the two survey methods using a two - tailed z - test . further , we compared cluster sizes including lone males with those excluding lone males , using a two - tailed z - test . further , we calculated resolution r ( minimum detectable change in population density ) from both methods by using the formula r = 2.77 * ( cv/100 ) for a significance level of 5 % and a statistical power of 50 % ( plumptre 2000 ) . the mentawai archipelago is located 85135 km west of the island of sumatra , indonesia . siberut is the most northerly and largest island with an area of 4030 km ( whittaker 2006 ; whitten 1982a ) . close to the northern coast of siberut lies the peleonan forest , which consists of a 45 km section of primary lowland and swamp forest ( quinten et al . the maximum elevation is 190 m above sea level and the area is characterized by hills and ridges that are covered by primary mixed evergreen rain forest ( hadi et al . 2009 ) . pungut field station of the siberut conservation programme ( scp ) is located ( 101s and 9850e ) in the south of the peleonan forest . we conducted this study in a circular area of 10.7 km around the field station . the transect system around the station consists of 13 systematically placed transects each 13 km in length ( waltert et al . we conducted triangulation at four different survey sites covering the study area of the scp during 4 wk from september 8 until october 3 , 2010 . at each sample site although distance is not required for triangulation , we used the distance estimates to check that the same cluster was heard by two different listening posts . we checked distance estimates up to 900 m by moving from the listening post to the calling cluster using a global positioning system ( garmin gpsmap 60csx ) . we collected data from three listening posts situated in a triangle formation on elevated terrain . the distances between listening posts were 300500 m. we collected data on four consecutive days at each site at 04:3010:00 h. observers stayed at the listening posts until the gibbons had stopped singing for 30 min . we conducted line transect surveys between 06:3011:30 h and 15:3018:00 h from september 14 and november 26 , 2010 . protocols , season , survey times , surveyed transects , and data analysis followed those of the last survey in the study area in 2005 ( waltert et al . 2008 ) . we surveyed each of the 13 transects around the field station between six and eight times ( mean 7.23 , sd = 0.6 ) , resulting in a total survey effort of 155.5 km . we documented all detections of primates , recording species , cluster size , and the perpendicular distance from the transect line to the estimated center of the cluster using a laser range finder ( nikon 550 ) for distances > 10 m and a tape measure for distances < 10 m. the triangulation method usually uses only duet counts for gibbons to ensure that a reproductive gibbon cluster is recorded ( brockelman and srikosamatara 1993 ) . however , unlike most gibbon species , male and female hylobates klossii do not duet . instead males can be heard to sing between 01:00 and 13:00 h , but concentrate mainly on the hours before dawn , whereas females sing after dawn ( whitten 1982b ) . we used only clusters that were recorded after dawn , i.e. , those likely to be females , for analysis . we determined cluster size by mapping all sightings of gibbon clusters encountered during the entire study period . we assumed that individuals sighted > 300 m apart belonged to different gibbon clusters . for clusters encountered several times at the same location , we used the maximum number of individuals recorded as the size for that cluster . owing to time constraints , we could not assess calling rates , so we assumed that females call every 34 d ( whitten 1982b ) . we aimed for 3 d survey at each site , but unfortunately this turned out not to be possible , owing to time constraints and weather conditions in one site where we obtained only 2 survey days . distance to the cluster is noted to aid data analysis if there are two clusters singing on the same compass bearing . this is a standard procedure ( cheyne et al . 2010 ) . where there is no conflict in number of clusters on any given compass bearing , the compass directions only are plotted and intersections used to indicate clusters . previous research on hylobates klossii estimated their home range to be 2035 ha ( whitten 1982a ) , giving a circle of diameter 250333 m. we , therefore , defined clusters > 300 m apart as different clusters . those closer than 300 m are likely to represent double counting where gibbons moved between calling bouts . we took the number of clusters heard per day and the total number of clusters heard from each listening post from the maps . we determined effective listening areas the areas where a minimum of two listening posts could have heard gibbon calls , within a radius of 1000 m around each post using arcgis 9.2 . we calculated density estimates using the following formula and correction factors , assuming that singing on successive days is independent ( brockelman and ali 1987 ; cheyne et al . 2008 ) . density estimate : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ d={n \left/ { { \left [ { p(m)\times e } \right ] } } \right . } $ $ \end{document } and correction factor : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p(m ) = 1\text{-- } { { \left [ { 1\text{--}\ p(1 ) } \right]}^m } , $ $ \end{document } where n refers to the cumulative total number of clusters heard in the effective listening area e at each site , p(m ) denotes the cumulative number of clusters singing in m days , p(1 ) refers to the mean of the proportion of clusters heard calling at each site ( once the data from all study days were combined ) . we calculated coefficients of variation ( cv ) using the formula cv = / , where denotes standard deviation and denotes mean . we calculated expected cluster size from a size - biased regression of ln[cluster size ] against estimated detection probability g(x ) . ideally surveys should be conducted using a systematic random design or a set of equally spaced transects randomly located in the survey region ( buckland et al . 2010 ) . at pungut , however , the trail system is oriented in a radial manner around the field station . to account for potential bias arising from over - coverage of the area directly surrounding the station , we stratified data in an inner circle with an area of 1.13 km ( radius = 0.6 km ) and an outer belt of 9.55 km . because cluster density estimates did not differ significantly between stratified data and nonstratified data ( z = 0.47 , p = 0.64 ) we present estimates from non - stratified data . we compared cluster and individual density estimates between the two survey methods using a two - tailed z - test . further , we compared cluster sizes including lone males with those excluding lone males , using a two - tailed z - test . further , we calculated resolution r ( minimum detectable change in population density ) from both methods by using the formula r = 2.77 * ( cv/100 ) for a significance level of 5 % and a statistical power of 50 % ( plumptre 2000 ) . triangulation resulted in a mean density estimate of 5.01 clusters / km ( 95 % ci : 3.16.9 ; table i ) . mean cluster size was estimated at 3.5 individuals / cluster excluding lone gibbons ( n = 27 , 95 % ci : 2.84.3 ) and 3.0 individuals / cluster ( n = 34 , 95 % ci : 2.33.7 ) including lone gibbons . those cluster sizes did not differ significantly from each other ( z = 0.19 , p = 0.85 ) . the individual density estimate was 17.5 individuals / km ( 95 % ci : 10.924.2 ) excluding lone males.table iparameters used to determine gibbon cluster densities at different sampling sites in the peleonan forest , siberut , indonesiasite numberclusters heardp(1 ) ( 95 % ci , cv)p(m)m ( days)e ( km)density ( clusters / km)1110.5 ( 0.141.14 , 0.14)0.7523.194.62170.34 ( 0.180.5 , 0.29)0.8143.236.53150.51 ( 0.170.86 , 0.27)0.8833.225.274100.47 ( 0.090.85 , 0.33)0.8533.213.67mean ( 95 % ci , cv)13.25 ( 7.9918.51 , 0.25)0.46 ( 0.330.58 , 0.17)0.82 ( 0.730.91 , 0.07)3 ( 1.704.30 , 0.27)3.21 ( 3.193.24 , 0.005)5.01 . ( 3.126.90 , 0.24)p(1 ) mean of proportion of clusters calling vs. the total number of clusters identified over m days with 95 % ci and cv ; p(m ) the cumulative number of cluster singing in m days ; e effective listening area at each site . mean values for all variables are given with 95 % ci and cv parameters used to determine gibbon cluster densities at different sampling sites in the peleonan forest , siberut , indonesia p(1 ) mean of proportion of clusters calling vs. the total number of clusters identified over m days with 95 % ci and cv ; p(m ) the cumulative number of cluster singing in m days ; e effective listening area at each site . mean values for all variables are given with 95 % ci and cv we recorded 101 observations of hylobates klossii during line transect sampling , including auditory and visual records . a subset of 62 observations from truncation distance data at 92 m provided a good model fit , as did truncation to 42 m ( fig . 1detection probability plot from line transect data ( histograms ) conducted for hylobates klossii between september 14 and november 26 , 2010 in the peleonan forest , siberut , indonesia , and fitted detection function for truncation widths w = 92 m ( a ) and w = 42 m ( b ) . detection probability plot from line transect data ( histograms ) conducted for hylobates klossii between september 14 and november 26 , 2010 in the peleonan forest , siberut , indonesia , and fitted detection function for truncation widths w = 92 m ( a ) and w = 42 m ( b ) . we estimated cluster size from observed clusters as 2.7 individuals / cluster ( 95 % ci : 2.33.2 individuals / cluster ) , cluster density as 5.5 clusters / km ( 95 % ci : 3.97.7/km ) and individual density as 14.89/km ( 95 % ci : 10.221.7/km ; table ii).table iiestimates , after data truncation , of encounter rate , cluster density , and expected cluster size , as well as detection probability ( probability of observing a cluster within w [ m ] from the transect line ) with 95 % ci for hylobates klossii from line transect sampling in the peleonan forest , siberut , indonesiamean encounter rate of clusters/ km ( 95 % ci , cv)0.40 ( 0.300.54 , 0.15)expected cluster size ( 95 % ci , cv)2.7 ( 2.33.2 , 0.09)mean cluster density / km ( 95 % ci , cv)5.5 ( 3.97.7 , 0.17)detection probability ( 95 % ci , cv)0.39 ( 0.330.46 , 0.08)truncation distance w [ m]92number of observations n62mean density , individuals / km ( 95 % ci , cv)14.9 ( 10.221.7 , 0.19)all estimates for truncation distance ( w ) from number of observations of clusters ( n ) . mean individual density estimates with 95 % ci and coefficient of variation cv estimates , after data truncation , of encounter rate , cluster density , and expected cluster size , as well as detection probability ( probability of observing a cluster within w [ m ] from the transect line ) with 95 % ci for hylobates klossii from line transect sampling in the peleonan forest , siberut , indonesia all estimates for truncation distance ( w ) from number of observations of clusters ( n ) . mean individual density estimates with 95 % ci and coefficient of variation cv there were no significant differences in cluster ( z = 0.32 , p = 0.75 ) nor in individual density estimates ( z = 0.53 , p = 0.6 ) between triangulation and line transect methods . thus , changes in cluster densities of r < 66 % at a significance level of 5 % with a statistical power of 50 % are unlikely to be detected using the triangulation method . for line transect surveys , changes in cluster densities are unlikely to be detected if r < 47 % at a significance level of 5 % with a statistical power of 50 % . we found no difference in our estimations of cluster or individual densities using triangulation and line transect sampling and therefore may assume similar accuracy from both methods . the true number of gibbons present in the area , however , is unknown , as is also the proportion of lone gibbons in the area , so we can not correct individual density estimates gained by triangulation for this potential bias ( cheyne et al . 2008 ) . we also do not know the levels of under - recording of lone gibbons and those close to observers . nevertheless , the detection probability plot showed a prominent shoulder when truncating to 42 m , suggesting no under - recording near the transect line ( fig . gibbon densities assessed by triangulation in our study area in 2003 resulted in density estimates of > 25 individuals / km ( whittaker 2005 ) , 50 % higher than estimates obtained from line transect surveys in 2005 ( waltert et al . 2008 ) . however , this difference is due mainly to an exceptionally high estimate of mean cluster size in the triangulation study ( mean cluster size 10 from n = 8 observations ; whittaker 2005 ) . our mean cluster size for all gibbon cluster sightings used for triangulation ( 3.5 individuals ) was similar to the mean cluster sizes in previous studies ( 3.4 individuals : tenaza 1975 ; 3.7 individuals : whitten 1982a ) . this suggests that more than eight sightings are needed to estimate cluster size ( perhaps n > 20 ) . if cluster size can not be estimated accurately , we suggest using cluster density estimates instead of individual density estimates to compare results from different studies or from different survey methods . although the two methods gave similar estimates of gibbon densities , line transect sampling resulted in more precise cluster density estimates , allowing better detection of changes over time . even if search effort is optimal , the line transect method is unlikely to detect changes of < 2530 % ( plumptre and cox 2006 ) whereas indirect methods , such as nest , dung , or cue surveys , are suggested to be unlikely to detect changes of < 3050 % ( buckland et al . 2001 ; plumptre 2000 ) . we conducted surveys over a relatively long time period ( triangulation 4 wk ; line transect surveys 10.5 wk ) ; nevertheless we found that only much larger changes in densities than the ones suggested for optimal searching effort could be detected using either method . as time and funding are often limiting factors in small conservation projects , it is important that neither method may detect small changes ( < 45 % ) in population densities even if search effort is relatively high . the recommended number of observations to detect population changes over time is 100 ( plumptre 2000 ) , which confirms that our line transect sampling effort is not optimal to observe changes . the likelihood of calling over all survey days should be at least 1 to detect all clusters present in the study area during triangulation . for example , a minimum of 5 consecutive days of sampling at each site are suggested for hylobates agilis ( obrien et al . female hylobates klossi sing every 34 d ( whitten 1982b ) , or even less frequently ( keith et al . thus , the 24 d we spent at each site might have led to an underestimation of cluster densities . overall , we conclude that triangulation can yield density estimates that are not significantly different from those given by line transect sampling in primary lowland rain forests . the method of choice will depend on the objectives of the study and should take into account the terrain . triangulation was effective in monitoring changes in population densities over time in hylobates albibarbis across kalimantan ( s. m. cheyne pers . comm . ) . however , we suggest that line transect sampling is preferable for studies that focus on monitoring relatively small changes in population densities over time , because of its more precise estimates . line transect sampling should also be chosen if a survey targets more than one species . if the objective of the study is to conduct rapid density estimates within a limited amount of time , in an area that is difficult to access , and/or if resources ( human , funding ) are limited , then triangulation might be more appropriate .

estimating population densities of key species is crucial for many conservation programs . density estimates provide baseline data and enable monitoring of population size . several different survey methods are available , and the choice of method depends on the species and study aims . few studies have compared the accuracy and efficiency of different survey methods for large mammals , particularly for primates . here we compare estimates of density and abundance of kloss gibbons ( hylobates klossii ) using two of the most common survey methods : line transect distance sampling and triangulation . line transect surveys ( survey effort : 155.5 km ) produced a total of 101 auditory and visual encounters and a density estimate of 5.5 gibbon clusters ( groups or subgroups of primate social units)/km2 . triangulation conducted from 12 listening posts during the same period revealed a similar density estimate of 5.0 clusters / km2 . coefficients of variation of cluster density estimates were slightly higher from triangulation ( 0.24 ) than from line transects ( 0.17 ) , resulting in a lack of precision in detecting changes in cluster densities of < 66 % for triangulation and < 47 % for line transect surveys at the 5 % significance level with a statistical power of 50 % . this case study shows that both methods may provide estimates with similar accuracy but that line transects can result in more precise estimates and allow assessment of other primate species . for a rapid assessment of gibbon density under time and financial constraints , the triangulation method also may be appropriate .

Introduction Methods Study Site Data Collection Data Analysis Results Discussion

reliable estimates of population sizes of target species are of central importance to many conservation programs . density estimates provide baseline data on species abundance and allow the monitoring of population sizes , which is important in assessing the suitability of conservation strategies . however , few studies compare different survey methods and designs for large mammals under the same conditions , for instance , the same time of year and same location ( nijman and menken 2005 ; nomani et al . such a comparison , however , can highlight strengths and weaknesses of the specific methods and thus provide useful information for conservation practitioners when deciding on the most suitable methods to deliver their conservation objectives . one of the most common population assessment methods for diurnal primates is line transect sampling ( buckland et al . best practice guidelines regarding survey design , field protocol , and analysis are available ( buckland et al . owing to their otherwise inconspicuous behavior and their tendency to use the upper canopy ( nijman 2001 ) , line transect surveys might fail to record 100 % of individuals near the transect line , resulting in underestimation of population densities if no action is taken to accommodate the violation of this key assumption ( buckland et al . , through the influence of weather conditions on singing behavior or by underestimation of distances to calling groups , the latter of which may result in overestimation of densities ( cheyne et al . in addition , calling rates and data on cluster ( groups or subgroups of primate social units ) size are needed , requiring long - term data or extra survey effort . here we compare line transect sampling with triangulation in estimating densities of hylobates klossii in the peleonan forest , a mixed evergreen rainforest with hilly terrain in the north of siberut island ( hadi et al . if the two methods have comparable accuracy then the results for density estimates should not differ significantly . pungut field station of the siberut conservation programme ( scp ) is located ( 101s and 9850e ) in the south of the peleonan forest . we conducted triangulation at four different survey sites covering the study area of the scp during 4 wk from september 8 until october 3 , 2010 . at each sample site although distance is not required for triangulation , we used the distance estimates to check that the same cluster was heard by two different listening posts . we collected data from three listening posts situated in a triangle formation on elevated terrain . the distances between listening posts were 300500 m. we collected data on four consecutive days at each site at 04:3010:00 h. observers stayed at the listening posts until the gibbons had stopped singing for 30 min . we conducted line transect surveys between 06:3011:30 h and 15:3018:00 h from september 14 and november 26 , 2010 . protocols , season , survey times , surveyed transects , and data analysis followed those of the last survey in the study area in 2005 ( waltert et al . we surveyed each of the 13 transects around the field station between six and eight times ( mean 7.23 , sd = 0.6 ) , resulting in a total survey effort of 155.5 km . we documented all detections of primates , recording species , cluster size , and the perpendicular distance from the transect line to the estimated center of the cluster using a laser range finder ( nikon 550 ) for distances > 10 m and a tape measure for distances < 10 m. we analyzed triangulation data following a triangulation protocol ( cheyne et al . the triangulation method usually uses only duet counts for gibbons to ensure that a reproductive gibbon cluster is recorded ( brockelman and srikosamatara 1993 ) . however , unlike most gibbon species , male and female hylobates klossii do not duet . we determined cluster size by mapping all sightings of gibbon clusters encountered during the entire study period . for clusters encountered several times at the same location , we used the maximum number of individuals recorded as the size for that cluster . distance to the cluster is noted to aid data analysis if there are two clusters singing on the same compass bearing . previous research on hylobates klossii estimated their home range to be 2035 ha ( whitten 1982a ) , giving a circle of diameter 250333 m. we , therefore , defined clusters > 300 m apart as different clusters . density estimate : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ d={n \left/ { { \left [ { p(m)\times e } \right ] } } \right . } we calculated coefficients of variation ( cv ) using the formula cv = / , where denotes standard deviation and denotes mean . at pungut , however , the trail system is oriented in a radial manner around the field station . to account for potential bias arising from over - coverage of the area directly surrounding the station , we stratified data in an inner circle with an area of 1.13 km ( radius = 0.6 km ) and an outer belt of 9.55 km . because cluster density estimates did not differ significantly between stratified data and nonstratified data ( z = 0.47 , p = 0.64 ) we present estimates from non - stratified data . we compared cluster and individual density estimates between the two survey methods using a two - tailed z - test . further , we calculated resolution r ( minimum detectable change in population density ) from both methods by using the formula r = 2.77 * ( cv/100 ) for a significance level of 5 % and a statistical power of 50 % ( plumptre 2000 ) . we conducted triangulation at four different survey sites covering the study area of the scp during 4 wk from september 8 until october 3 , 2010 . at each sample site although distance is not required for triangulation , we used the distance estimates to check that the same cluster was heard by two different listening posts . we collected data from three listening posts situated in a triangle formation on elevated terrain . the distances between listening posts were 300500 m. we collected data on four consecutive days at each site at 04:3010:00 h. observers stayed at the listening posts until the gibbons had stopped singing for 30 min . we conducted line transect surveys between 06:3011:30 h and 15:3018:00 h from september 14 and november 26 , 2010 . protocols , season , survey times , surveyed transects , and data analysis followed those of the last survey in the study area in 2005 ( waltert et al . we surveyed each of the 13 transects around the field station between six and eight times ( mean 7.23 , sd = 0.6 ) , resulting in a total survey effort of 155.5 km . we documented all detections of primates , recording species , cluster size , and the perpendicular distance from the transect line to the estimated center of the cluster using a laser range finder ( nikon 550 ) for distances > 10 m and a tape measure for distances < 10 m. the triangulation method usually uses only duet counts for gibbons to ensure that a reproductive gibbon cluster is recorded ( brockelman and srikosamatara 1993 ) . we determined cluster size by mapping all sightings of gibbon clusters encountered during the entire study period . for clusters encountered several times at the same location , we used the maximum number of individuals recorded as the size for that cluster . distance to the cluster is noted to aid data analysis if there are two clusters singing on the same compass bearing . where there is no conflict in number of clusters on any given compass bearing , the compass directions only are plotted and intersections used to indicate clusters . previous research on hylobates klossii estimated their home range to be 2035 ha ( whitten 1982a ) , giving a circle of diameter 250333 m. we , therefore , defined clusters > 300 m apart as different clusters . $ $ \end{document } and correction factor : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p(m ) = 1\text{-- } { { \left [ { 1\text{--}\ p(1 ) } \right]}^m } , $ $ \end{document } where n refers to the cumulative total number of clusters heard in the effective listening area e at each site , p(m ) denotes the cumulative number of clusters singing in m days , p(1 ) refers to the mean of the proportion of clusters heard calling at each site ( once the data from all study days were combined ) . we calculated coefficients of variation ( cv ) using the formula cv = / , where denotes standard deviation and denotes mean . at pungut , however , the trail system is oriented in a radial manner around the field station . to account for potential bias arising from over - coverage of the area directly surrounding the station , we stratified data in an inner circle with an area of 1.13 km ( radius = 0.6 km ) and an outer belt of 9.55 km . because cluster density estimates did not differ significantly between stratified data and nonstratified data ( z = 0.47 , p = 0.64 ) we present estimates from non - stratified data . we compared cluster and individual density estimates between the two survey methods using a two - tailed z - test . further , we calculated resolution r ( minimum detectable change in population density ) from both methods by using the formula r = 2.77 * ( cv/100 ) for a significance level of 5 % and a statistical power of 50 % ( plumptre 2000 ) . triangulation resulted in a mean density estimate of 5.01 clusters / km ( 95 % ci : 3.16.9 ; table i ) . the individual density estimate was 17.5 individuals / km ( 95 % ci : 10.924.2 ) excluding lone males.table iparameters used to determine gibbon cluster densities at different sampling sites in the peleonan forest , siberut , indonesiasite numberclusters heardp(1 ) ( 95 % ci , cv)p(m)m ( days)e ( km)density ( clusters / km)1110.5 ( 0.141.14 , 0.14)0.7523.194.62170.34 ( 0.180.5 , 0.29)0.8143.236.53150.51 ( 0.170.86 , 0.27)0.8833.225.274100.47 ( 0.090.85 , 0.33)0.8533.213.67mean ( 95 % ci , cv)13.25 ( 7.9918.51 , 0.25)0.46 ( 0.330.58 , 0.17)0.82 ( 0.730.91 , 0.07)3 ( 1.704.30 , 0.27)3.21 ( 3.193.24 , 0.005)5.01 . ( 3.126.90 , 0.24)p(1 ) mean of proportion of clusters calling vs. the total number of clusters identified over m days with 95 % ci and cv ; p(m ) the cumulative number of cluster singing in m days ; e effective listening area at each site . mean values for all variables are given with 95 % ci and cv parameters used to determine gibbon cluster densities at different sampling sites in the peleonan forest , siberut , indonesia p(1 ) mean of proportion of clusters calling vs. the total number of clusters identified over m days with 95 % ci and cv ; p(m ) the cumulative number of cluster singing in m days ; e effective listening area at each site . mean values for all variables are given with 95 % ci and cv we recorded 101 observations of hylobates klossii during line transect sampling , including auditory and visual records . 1detection probability plot from line transect data ( histograms ) conducted for hylobates klossii between september 14 and november 26 , 2010 in the peleonan forest , siberut , indonesia , and fitted detection function for truncation widths w = 92 m ( a ) and w = 42 m ( b ) . detection probability plot from line transect data ( histograms ) conducted for hylobates klossii between september 14 and november 26 , 2010 in the peleonan forest , siberut , indonesia , and fitted detection function for truncation widths w = 92 m ( a ) and w = 42 m ( b ) . we estimated cluster size from observed clusters as 2.7 individuals / cluster ( 95 % ci : 2.33.2 individuals / cluster ) , cluster density as 5.5 clusters / km ( 95 % ci : 3.97.7/km ) and individual density as 14.89/km ( 95 % ci : 10.221.7/km ; table ii).table iiestimates , after data truncation , of encounter rate , cluster density , and expected cluster size , as well as detection probability ( probability of observing a cluster within w [ m ] from the transect line ) with 95 % ci for hylobates klossii from line transect sampling in the peleonan forest , siberut , indonesiamean encounter rate of clusters/ km ( 95 % ci , cv)0.40 ( 0.300.54 , 0.15)expected cluster size ( 95 % ci , cv)2.7 ( 2.33.2 , 0.09)mean cluster density / km ( 95 % ci , cv)5.5 ( 3.97.7 , 0.17)detection probability ( 95 % ci , cv)0.39 ( 0.330.46 , 0.08)truncation distance w [ m]92number of observations n62mean density , individuals / km ( 95 % ci , cv)14.9 ( 10.221.7 , 0.19)all estimates for truncation distance ( w ) from number of observations of clusters ( n ) . mean individual density estimates with 95 % ci and coefficient of variation cv estimates , after data truncation , of encounter rate , cluster density , and expected cluster size , as well as detection probability ( probability of observing a cluster within w [ m ] from the transect line ) with 95 % ci for hylobates klossii from line transect sampling in the peleonan forest , siberut , indonesia all estimates for truncation distance ( w ) from number of observations of clusters ( n ) . mean individual density estimates with 95 % ci and coefficient of variation cv there were no significant differences in cluster ( z = 0.32 , p = 0.75 ) nor in individual density estimates ( z = 0.53 , p = 0.6 ) between triangulation and line transect methods . thus , changes in cluster densities of r < 66 % at a significance level of 5 % with a statistical power of 50 % are unlikely to be detected using the triangulation method . for line transect surveys , changes in cluster densities are unlikely to be detected if r < 47 % at a significance level of 5 % with a statistical power of 50 % . we found no difference in our estimations of cluster or individual densities using triangulation and line transect sampling and therefore may assume similar accuracy from both methods . the true number of gibbons present in the area , however , is unknown , as is also the proportion of lone gibbons in the area , so we can not correct individual density estimates gained by triangulation for this potential bias ( cheyne et al . gibbon densities assessed by triangulation in our study area in 2003 resulted in density estimates of > 25 individuals / km ( whittaker 2005 ) , 50 % higher than estimates obtained from line transect surveys in 2005 ( waltert et al . however , this difference is due mainly to an exceptionally high estimate of mean cluster size in the triangulation study ( mean cluster size 10 from n = 8 observations ; whittaker 2005 ) . our mean cluster size for all gibbon cluster sightings used for triangulation ( 3.5 individuals ) was similar to the mean cluster sizes in previous studies ( 3.4 individuals : tenaza 1975 ; 3.7 individuals : whitten 1982a ) . if cluster size can not be estimated accurately , we suggest using cluster density estimates instead of individual density estimates to compare results from different studies or from different survey methods . although the two methods gave similar estimates of gibbon densities , line transect sampling resulted in more precise cluster density estimates , allowing better detection of changes over time . even if search effort is optimal , the line transect method is unlikely to detect changes of < 2530 % ( plumptre and cox 2006 ) whereas indirect methods , such as nest , dung , or cue surveys , are suggested to be unlikely to detect changes of < 3050 % ( buckland et al . we conducted surveys over a relatively long time period ( triangulation 4 wk ; line transect surveys 10.5 wk ) ; nevertheless we found that only much larger changes in densities than the ones suggested for optimal searching effort could be detected using either method . as time and funding are often limiting factors in small conservation projects , it is important that neither method may detect small changes ( < 45 % ) in population densities even if search effort is relatively high . the recommended number of observations to detect population changes over time is 100 ( plumptre 2000 ) , which confirms that our line transect sampling effort is not optimal to observe changes . thus , the 24 d we spent at each site might have led to an underestimation of cluster densities . overall , we conclude that triangulation can yield density estimates that are not significantly different from those given by line transect sampling in primary lowland rain forests . the method of choice will depend on the objectives of the study and should take into account the terrain . triangulation was effective in monitoring changes in population densities over time in hylobates albibarbis across kalimantan ( s. m. cheyne pers . however , we suggest that line transect sampling is preferable for studies that focus on monitoring relatively small changes in population densities over time , because of its more precise estimates . if the objective of the study is to conduct rapid density estimates within a limited amount of time , in an area that is difficult to access , and/or if resources ( human , funding ) are limited , then triangulation might be more appropriate .

it is important to understand the mechanism of the oxidatively generated damage to guanines in dna . the reason is that the damage causes mutations and cancer , and it is an obstacle to use dna as a molecular device.(1 ) in the case of the damage due to a one - electron oxidant , the reactions occur as follows . it then migrates to a nearby guanine base , because a guanine base has the lowest ionization potential ( ip ) among the dna bases . the trapped hole escapes to other guanine bases or triggers the chemical reactions which cause the damage to the guanine . it was found that the longer guanine run in a duplex dna is damaged more than the shorter ones . it was also reported that the attachment of a phenyl group to a guanine suppresses the damage not only at the phenylated guanine but also at nearby guanines in the duplex dna.(18 ) to understand the mechanism underlying these experimental results , we need to know how the hole transfer reactions occur in dna . one of the important parameters determining the charge transfer rate is the free energy difference between the reactant and the product . thus , in the present work , we theoretically analyzed the ip of duplex dna corresponding to the free energy of the transferring hole on the duplex dna . since the above experiments were carried out in the presence of a solvent ( water ) , we took into account the solvent ; this turned out to be crucial to understanding the above experimental results . one of our goals is to resolve the discrepancy between the theoretical results and the experimental one;(21 ) these results are about the free energy dependence of the transferring hole on the length of the guanine runs . the ips ( corresponding to the free energy of the hole ) obtained by the ab initio hartreefock ( hf ) molecular orbital ( mo ) calculations are smaller for the longer guanine run . these calculations were performed for the dna oligomers without a backbone in the gas phase . this result of the ip is in qualitative agreement with the experimental results as follows . if the free energy of the hole on the longer guanine run is smaller , then the probability of the hole to be there is larger and thus the longer one is damaged more . however , the calculated ip difference between g and gg ( ggg ) of 0.47 ( 0.68 ) ev is too large compared to the corresponding free energy difference of 0.052 ( 0.077 ) ev obtained from the time - resolved data for dna oligomers in water.(21 ) that is , if the previously calculated value was close to the real value , then the hole transfer from the longer guanine run to the shorter one hardly occurred and thus the distribution of the damage in the dna would become considerably different from the real one . here , we neglect the entropy difference in the free energy difference between the reactant and the product of the hole transfer reaction by assuming that the potential functions of the reactant and the product are harmonic around the equilibrium structures and their second derivatives are similar . another question to be answered in the present paper is about the effect of the chemical modifications of dna oligomers on the hole transfer reactions . when a neutral functional group is attached to a guanine in a dna oligomer in water , this removal was found to reduce the free energy of the hole on the guanine.(22 ) this might sound paradoxical . that is , since a polar solvent usually reduces the free energies of ions , the removal of nearby solvent molecules might be expected to increase the free energy of the hole it will turn out that this mechanism also explains the discrepancy mentioned in the previous paragraph . the free energy reduction of the hole mentioned above was confirmed by the ab initio and the semiempirical hf calculations for various neutral functional groups , i.e. , the phenyl , the benzyl , and the tert - butyl groups with the two structures of the typical b - dna structures.(22 ) this free energy reduction was observed even when the phenyl group attached was replaced by an artificial h2 cluster mimicking the solvent - accessible surface of the phenyl group . thus , it is considered that this free energy reduction is due to the removal of the solvent molecules.(22 ) this result agrees with the experimental one(18 ) that the damage to guanines near the phenylated guanine is suppressed as follows . the phenyl group attachment to a guanine reduces the free energy of the hole on it , and thus , the hole is trapped there ; therefore , the guanines near the phenylated guanine are less damaged . we briefly review the relevant works on the electrostatic interaction and the solvent effects on dna , since they turned out to play crucial roles in the present cases . the electrostatic potential in dna has been studied for many years in order to understand its reaction with other molecules . it was pointed out that the effect of the electrostatic interaction on the ip of a guanine doublet is important.(25 ) as for the solvent effects , the hydration effect on the ip of the small fragments of dna , namely , the watsoncrick ( wc ) base pairs , nucleotides , and a phosphorylated dinucleotide , was investigated . recently , the solvent effects on the much - larger - sized dna were investigated . these studies showed the importance of the solvent effects on dna . in the present work , we calculated and analyzed the ip of the duplex dna by taking into account the solvent , where this ip corresponds to the free energy of the hole on the duplex dna . on the basis of the same mechamism , we answer the above two questions as follows . since the electrostatic interaction between the hole and a nucleotide pair in the duplex dna is attractive , the ip of the longer guanine run is smaller than that of the shorter one . however , this attractive interaction is reduced in water , which results in the small difference of the ip between the longer guanine run and the shorter one in water . this mechanism explains why the theoretical results without taking into account the solvent were too large compared to the experimental one.(21 ) the effect of the chemical modification is understood as follows . a neutral functional group attached to a guanine in a duplex dna in water expels the nearby water molecules which reduce the electrostatic interaction . thus , the attractive electrostatic interaction stabilizing the hole on the guanine increases and the hole is stabilized . this is why the free energy of the hole decreases when nearby water molecules are removed . we calculated the ip of the duplex dna oligomers in the gas phase and in water as follows . we used the two structures of the typical b - dna structures , namely , the 55th(37 ) and the 4th(38 ) fiber models , to ensure that the result is independent of the structure deviation within the b - dna structure . their global structures are similar ; the helical twists are 36 for both of the two models and the rises are 3.39 and 3.38 for the 55th and 4th fiber models , respectively . these model structures are determined by x - ray diffraction , and thus , the coordinates of the h atoms are missing . therefore , we added these coordinates and optimized them using the electronic structure calculations described in the next paragraph . we calculated these optimized coordinates for dna oligomers , which are in the neutral state in vacuum and in water , and in the ionic state in water . we calculated the electronic structures using the austin model 1 ( am1 ) hamiltonian(39 ) implemented in the mopac2002 v1.0 program.(40 ) ( we did not use the linear scaling calculation program mozyme . ) we included the solvent as the dielectric continuum , namely , the conductor - like screening model ( cosmo ) with the dielectric constant of water , 78.4 , and the vdw parameters optimized for the density functional theory.(42 ) these optimized parameters are expected to produce better results than the default ones implemented in the software;(43 ) this expectation was confirmed in the case of the ip of the nucleosides.(22 ) we calculated the ip as the difference between the total energies after and before one electron is removed . for these calculations there was no spin contamination in the case of an even number of electrons . in the case of an odd number of electrons , the spin contamination was less than about 3% and thus the error in the energy due to the spin contamination was calculated to be less than a few tenths of an ev . as for the ip in water , we calculated it for dna oligomers in the neutral state and the ionic state ; in the latter state , the phosphate backbones are completely ionized . the actual ip in water is considered to be between these ips , since the ionic ( neutral ) state corresponds to the case when the counterions are far away from ( very close to ) the dna oligomers.(22 ) as for the validity of the method , we calculated the relative ips of the nucleosides in water ( the relative ops ) , and compared them to the experimental result.(44 ) the root - mean - square ( rms ) deviation between the computational and the experimental result(44 ) was 0.14 v ( table 1 ) , which is similar to the experimental error.(44 ) in addition , the method described here was successfully used to calculate the ips and the electron affinities of nucleic acid bases in the gas phase , where the rms deviations between the computational results and the experimental ones are 0.12 and 0.20 ev , respectively . the ips calculated by the method include the reorganization energies due to the relaxation of the solvent and h atoms of the solute but not that due to the relaxation of the heavy atoms of the solute . our conclusions are not affected by this approximation , as will be discussed in section . the solvation energy for the solvent change acetonitrile / h2o is calculated to be 0.02 ev using the born equation,(44 ) and thus , the correction to the relative op for the solvent change is negligible . in order to interpret the ips obtained by the electronic structure calculations , we used the net charges and dipoles of atoms and the electrostatic interaction calculated from them . the charge distribution of the hole is defined as the difference in the charge distributions between after and before the removal of an electron . we calculated the ip of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] ( figure 1a ) ; we found that the ip in the gas phase significantly decreases with the increasing number of base pairs , n , whereas the ip in water ( or op ) only slightly does ( figure 1b ) . the decrease in the ip in the gas phase from n = 1 to 3 is more than about 1 ev , but that in water is less than 0.2 ev . the latter result is consistent with the experimental result for the dna oligomers in water.(21 ) the difference between the ip decreases in the gas phase and in water is much larger than the rms deviation ( 0.14 v ) between the computational and the experimental results obtained in section . the values of the ips in the gas phase rapidly decrease for n 3 and converge for n 5 or 6 . about 90% of the charges of the hole is in a certain guanine and 5% is in the sugar covalently bonded to this guanine both in the gas phase and in water . we confirmed the above results using the two structures of the typical b - dna structures . note that the actual ip in water is between the solid and dashed lines , as described in section . ( a ) the structure of the duplex dna d[5-(g)6 - 3]d[3-(c)6 - 5 ] . ( b ) the ip of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] as a function of the number n of base pairs for the 55th fiber model structure and the 4th fiber model structure , as indicated . solid line with closed circles : the dna oligomer in the neutral state in water . dashed line with closed diamonds : the one in the ionic state in water . dotdashed line with closed squares : the one without a backbone in the gas phase . we calculated the ip of the dna oligomers , d(5-aagaa-3)d(3-ttctt-5 ) and d(5-aggga-3)d(3-tccct-5 ) , where the lengths of the guanine runs are different but the total number of base pairs is fixed ; we again found that the ip difference between the two sequences in water , 0.11 ( 0.10 ) ev , is smaller than that in the gas phase , 0.21 ( 0.15 ) ev , for the 55th fiber model ( the 4th fiber model ) . as shown in figure 2 , the ips calculated above ( the closed symbols ) are close to the electrostatic energy of the hole both in the gas phase ( the open triangles ) and in water ( the open circles ) . it is also shown that this energy in the gas phase decreases more strongly when we replace the charge distribution of the dna oligomer in the gas phase by that in the neutral state in water ( open inverted triangles ) . we define the electrostatic energy of the hole as the electrostatic interaction between the hole and all atoms except for the atoms of the guanine where the hole is localized plus a constant ; this constant is such that the value of this electrostatic energy coincides with the ip in the gas phase for n = 1 . in the case of the dna oligomer in water , we added the electrostatic interaction between the hole and the water ; we obtained this interaction as the dielectric energy,(49 ) which is considered to be the free energy but not the energy . we neglected the charge distribution of the hole outside the guanine where the hole is localized . we used the charge distribution after the removal of an electron as the charges which interact with the hole . we confirmed the above results using the two structures of the typical b - dna structures . comparison of the electrostatic energy of the hole ( defined in the text ) with the ip of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] as a function of the number n of base pairs for the 55th fiber model structure and the 4th fiber model structure , as indicated . the symbols and lines are the same as those in figure 1b except that the open symbols denote the electrostatic energy of the hole . open inverted triangles : the dna oligomer in the gas phase but with the charge distribution of the dna oligomer in the neutral state in water . to understand the electrostatic interactions in the dna oligomers , we calculated the charge distributions of the dna oligomer d[5-(g)53]d[3-(c)5 - 5 ] in the neutral state in the gas phase and in water ( figure 3 ) ; we found that the bases and sugars have negative partial charges and the phosphates have positive partial charges both in the gas phase and in water ( figure 4 ) . ( the phosphate backbones in the neutral state are not ionized . ) about 90% of the charges of the hole is in a certain guanine both in the gas phase and in water , as described earlier . we observed the same features in other sequences of the dna oligomers ( not shown ) . charge distributions in the dna oligomer d[5-(g)5 - 3]d[3-(c)5 - 5 ] in the neutral state in the gas phase and in water , as indicated . g , c , s , and p denote guanine , cytosine , sugar , and a part of the phosphate , respectively . simplified charge distributions in the dna oligomer d[5-(g)5 - 3]d[3-(c)5 - 5 ] in the neutral state in the gas phase and in water , as indicated . same as in figure 3 except that the net charges of the fragments are shown . the fragment from which the largest fraction of an electron is removed is shown by the bold fonts and bold lines . the net charge of the fragment after removing an electron is indicated by an arrow . the fragments of sugars and bases are divided at the n9c1 bond for guanines and the n1c1 bond for cytosines . the fragments of the phosphates and sugars are divided at the po5 and po3 bonds . we calculated the ip of the dna oligomers with other sequences , where a guanine is embedded in the center of the contiguous adenines or thymines , and obtained the same result with that of d[5-(g)n-3]d[3-(c)n-5 ] ( figure 5 ) . that is , the ip significantly decreases with the increasing n in the gas phase , whereas it does only slightly in water . the values of the ip decreases are slightly smaller than the corresponding values of the contiguous guanines in figure 1b , both in the gas phase and in water . ip of the dna oligomers as a function of the number n of base pairs , where a guanine is embedded in the center of the contiguous adenines or thymines . ( a ) n = 1 , 2 , 3 , 4 , 5 , and 6 are for d(5-g-3)d(3-c-5 ) , d(5-ag-3)d(3-tc-5 ) , d(5-aga-3)d(3-tct-5 ) , d(5-aaga-3)d(3-ttct-5 ) , d(5-aagaa-3)d(3-ttctt-5 ) , and d(5-aaagaa-3)d(3-tttctt-5 ) , respectively . ( b ) same as in figure 5a except that a and t are exchanged in the sequences of the dna oligomers . to understand the dependence of the obtained ips on n , we calculated the ip of the system made of two gc base pairs ( the inset in figure 6 ) as a function of the rise r ; we found that the ip of this system ( the solid line in figure 6 ) is close to the ip of the single base pair plus the electrostatic interaction between the hole and the base pair where the hole does not reside ( the dashed line in the same figure ) . the values of the ip rapidly increase for r 5 and converge for r 10 . we used the method described in section except that we removed the sugarphosphate backbones ( except the c1 atom ) from the structure of the dna oligomer d(5-gg-3)d(3-cc-5 ) , capped the dangling bonds with the h atoms , and optimized the coordinates of h atoms for single base pairs . that is , the structure of the system in the cationic ( neutral ) state is made of the optimized structure of the gc base pair of the 5-side in the cationic ( neutral ) state and that of the gc base pair of the 3-side in the neutral state . we calculated the electrostatic interaction between the hole and the base pair where the hole does not reside ; this interaction is the electrostatic interaction between one base pair in the cationic state and the other in the neutral state , minus that between the two base pairs both in the neutral state . more than 97% of the charges of the hole is in the guanine of the 5-side in the calculated electronic structures of the system . ip of the two gc base pairs as a function of the rise r. the inset shows the structure of the two gc base pairs . solid line : ip of the system made of the two gc base pairs . dashed line : ip of the single gc base pair plus the electrostatic interaction between the hole and the base pair where the hole does not reside ( defined in the text ) . to understand the reduction in the ip decrease with the increasing n due to the solvent , we developed simple models of dna oligomers ( figure 7a ) ; we showed that these models reproduce the ips of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] ( figure 7b ) . in this figure , the open symbols are for the simple models and the others are the same as those in figure 1b . a duplex dna oligomer is modeled as a box with the simplified charge distribution of a duplex dna oligomer in the neutral state ( the hole is localized on a certain guanine ) and the water is replaced by a conductor ( figure 7a ) . the reason is that the dielectric constant of water is large ( 78.4 ) and the solvation energies of the dielectric continuum scale as ( 1)/( + x ) with the dielectric constant , where 0 x 2.(50 ) ( a ) charge distributions of the simple models for dna pentamers with and without backbones , as indicated . one electron is removed from the filled circle marked by the outer circle , when the dna is oxidized . the size of the box in the x- and y - directions is 18 . charge distributions for dna oligomers with different numbers of base pairs are defined in the same way as for the pentamers . ( b ) comparison of the ips of the simple models with those obtained by the mo calculations in section for the 55th fiber model structure ( figure 1b ) . the symbols and lines are the same as those in figure 1b except that the open symbols are for the simple models of the dna oligomers in the gas phase , those in water in the neutral state , and those without a backbone in the gas phase , as indicated . to discuss the obtained ips , we calculated the vertical ip of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] ( figure 8) , and obtained a similar result with that of the ip ( figure 1b ) . that is , the vertical ip more significantly decreases with the increasing n in vacuum than in water . since , by definition , the vertical ip does not include the reorganization energy , we obtained the vertical ip as the energy level of the highest occupied molecular orbital ( homo ) with the opposite sign using the restricted hf ( rhf ) method with koopmans theorem . the vertical ip of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] as a function of the number n of base pairs . we calculated the ip of the modified nucleotide - containing dna oligomer , d[5-t1a2g3g4t5a6 - 3]d[3-a12t11c10c9a8t7 - 5 ] , where a phenyl group is attached to g3 ( figure 9a ) ; we found that the attachment of a phenyl group reduces the ip in water but not in the gas phase ( figure 9b ) . for the phenylated dna oligomer in water , the hole is always localized on the phenylated guanine g3 . we used the dna sequence of the six base pairs around the phenylated guanine in the experiment.(18 ) we used the method described in section except that we optimized the coordinates of the phenyl group atoms by the electronic structure calculations . as for a single base , the op of n - phenyldeoxyguanosine g relative to that of deoxyguanosine was 0.03 v in the experiment,(18 ) and the calculated one is 0.09 v , where the geometries are fully optimized . in the experiment,(18 ) the solvent was dimethylformamide ( dmf ) ; the solvation energy for the solvent change dmf / h2o is calculated to be 0.02 ev using the born equation and thus the correction to the relative op for the solvent change is negligible . ( a ) structure of the modified nucleotide - containing dna oligomer , d[5-t1a2g3g4t5a6 - 3]d[3-a12t11c10c9a8t7 - 5 ] , where a phenyl group is attached to g3 . the dna sequence is the sequence of the six base pairs around the phenylated guanine in the experiment.(18 ) ( b ) the effect of the phenyl group attachment on the ip of the dna oligomer in the gas phase , in the neutral state in water , and in the ionic state in water , for the 55th fiber model structure and the 4th fiber model structure , as indicated . the ips before and after a phenyl group is attached are indicated by g and g , respectively . figure 1b shows that the large decrease ( in the order of 1 ev ) of the ip in the gas phase with the increasing dna length is reduced to about a tenth of an ev in water . this reduction in the ip decrease is due to the solvent , since the computational error is less than about a tenth of an ev ( section ) . we can now understand why the calculated ip difference between g and gg ( ggg ) ( in the order of 1 ev ) is much larger than the experimental one ( in the order of 0.1 ev).(21 ) this is because the solvent is not included in the calculations . in the present work , we used the two structures of the typical b - dna structures , i.e. , the 55th(37 ) and the 4th(38 ) fiber models . these models do not include the sequence dependence of the dna structure ; namely , the structure parameters of a base are independent of nearby bases . since figure 1b shows that the general features of the ips are similar for both structures , we consider that the effect of the structure deviation within the b - dna structure on the ip is minor and so is that of the sequence dependence of the dna structure . from figure 2 , it is clear that the decrease of the electrostatic energy of the hole causes the ip decrease with the increasing number of base pairs , n. since the electrostatic energy of the hole decreases with the increasing n in figure 2 , the electrostatic interaction between the hole and a nucleotide pair is attractive in a duplex dna ; this nucleotide pair includes the two phosphates bonded to it . we now understand the obtained dependence of the ips on n as follows . because of the attractive interaction between the hole and a nucleotide pair , the ip in the gas phase decreases with the increasing n. this attractive interaction is reduced in water , and so is the ip decrease with the increasing n. when we replace the charge distribution of the dna oligomer in the gas phase by that in the neutral state in water , the electrostatic energy of the hole in the gas phase decreases more strongly ( figure 2 ) . the reason is that the polarization of the dna oligomer is induced in water ( figure 4 ) . from the simplified charge distribution of the dna oligomer in figure 4 , we understand why the electrostatic interaction between the hole and a nucleotide pair is attractive in the duplex dna ; this nucleotide pair includes the two phosphates bonded to it . the reason is that the hole is on a guanine and thus it is closer to nucleobases and sugars , which have negative partial charges , compared to phosphates , which have positive partial charges . we consider that the simplified charge distribution can be used to understand the above reason because the hole is delocalized over a guanine base and the rough electrostatic potential is smoothed . the ip in the gas phase decreases more strongly than that without a backbone ( figure 1b ) , because the positive partial charges are separated more from the hole for the dna oligomer with backbones compared to that without . in water , the ips of the dna oligomers in the ionic states are smaller than those in the neutral states ( figure 1b ) . this is because the pn s , which have positive partial charges in the neutral state of dna oligomers , become almost neutral in the ionic state of the dna oligomers and the repulsive interaction between pn s and the hole is reduced in the ionic state . the charge distribution of the dna oligomer in figure 4 is in accord with the result that the electrostatic potential around the guanine is lower in the interior than at the end of the sequence.(51 ) figure 5 shows that the similar dependence of the ip is observed also for the different sequences . we consider that the reason is the same as that for the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] , since the charge distribution has the same features , as mentioned above . figure 6 shows that the attractive electrostatic interaction between the hole and a base pair causes the ip dependence of the rise r ; this supports that this attractive interaction causes the dependence of the ip on n in figure 1b . the ip in figure 6 rapidly increases for r 5 and converges for r 10 . from this result , we can understand that the ips in the gas phase in figure 1b rapidly decrease for n 3 and converge for n 5 or 6 by considering that the hole is usually in the middle of the dna oligomer and the rise in the duplex dna is 3.4 . the simple models of dna oligomers ( figure 7a ) illustrate that water molecules around the duplex dna ( but not between the base pairs ) reduce the electrostatic interaction in the duplex dna ( figure 7b ) . as shown in figure 8 , the vertical ip more significantly decreases with the increasing n in vacuum than in water , similarly to the ip in figure 1 . as for the dielectric shielding , when we use koopmans theorem , the hole is not shielded by the solvent , but other charges are shielded , and thus , the attractive electrostatic interaction between them is reduced in water . thus , this reduction in the attractive electrostatic interaction leads to the result that the vertical ip in water is larger than that in the gas phase . because of the reorganization energy , the ips in water are more than a few ev smaller than the corresponding vertical ips . the result of the ips include the electronic energy reduction due to the charge redistribution in dna after an electron is removed . however , that of the vertical ip does not . this is because we used koopmans theorem to calculate the vertical ip . we also observed a slightly smaller decrease in the vertical ip in the gas phase with the increasing n and the slightly larger one in water compared to the corresponding ips . the reorganization energy due to the relaxation of the dna atoms and the electronic energy reduction mentioned above increase with the increasing n and these are included in the ips but not in the vertical ips . thus , the ip in the gas phase decreases slightly more than the corresponding vertical ip . as for dna in water , when n is increased , a part of the dna replaces a part of the solvent , where the dielectric constant of the former is smaller than that of the latter , and thus , the total reorganization energy decreases . therefore , the ip in water decreases less than the corresponding vertical ip . since we did not include the reorganization energy due to the relaxation of the heavy atoms of dna , the above decrease in the reorganization energy with the increasing n was overestimated . thus , the decrease in the exact ip with the increasing n should be between those of the ip and the vertical ip obtained in the present work . as shown in figure 9 , when the neutral functional group is attached to the guanine in the duplex dna in water , the free energy of the hole on the guanine is reduced ( but not in the gas phase ) . this result is counterintuitive , since the attached group expels the nearby water molecules , which are usually expected to stabilize ions . thus , when nearby water molecules are removed , the attractive electrostatic interaction stabilizing the hole increases . as for a single base , guanosine , the calculated op of n - phenyldeoxyguanosine g relative to that of deoxyguanosine is small and positive , i.e. , 0.09 v , which is close to the experimental value of 0.03 ev.(18 ) this means that the free energy of the hole ( or op ) is not reduced by attaching the phenyl group . the reason is that there is no other bases and sugars which could attractively interact with the hole in the case of a single base . this result of the free energy reduction agrees with the previous one.(22 ) the difference between the former and the latter is that the former includes the reorganization energy due to the relaxation of the solvent but the latter does not . the stabilization of the guanine radical cation by the attached phenyl group similarly affects its chemical reactions , namely , the hydration and deprotonation processes,(52 ) as follows . this stabilization makes the possibility that the hole injected is on the phenylated guanine greater and that for guanines nearby less . thus , the quantum yields of both of the chemical reactions of the nearby guanines decrease . in addition to that , this stabilization reduces the free energy of the initial state for both of the chemical reactions of the phenylated guanine radical cation . this result agrees with the experimental result that the damage to the guanines near the phenylated guanine is suppressed.(18 ) here , we have to be careful about the chemical yield data in the experiment(18 ) as follows . in this experiment , thus , the product of the deprotonation was detected , but the products of the hydration processes , namely , 8-oxo-7,8-dihydroguanine ( 8-oxogua ) and 2,6-diamino-4-hydroxy-5-formanido phyrimidine ( fapygua ) were not . the ratio of these two kinds of damage is sequence dependent,(53 ) and 8-oxogua , which is the main product of the hydration process , is highly susceptible to secondary one - electron oxidation reactions . however , if the probability of the damage of one kind is larger ( or smaller ) at a site , then so is that for the other kind,(53 ) and this feature is not changed by the secondary one - electron oxidation reactions . thus , if the probability of the damage of one kind is smaller at a site , then so is the other . the miscellaneous points about the stabilization of the guanine radical cation by the attached phenyl group are as follows . according to the above mechanism , the ip of the modified nucleotide - containing dna oligomer in the neutral state in water is supposed to be between the ip of the dna not modified in the neutral state in water and that in the gas phase . however , it is not , as shown in figure 9 . one of the reasons is that when the nearby water molecules are removed , the ip of the modified nucleotide - containing dna oligomer in the neutral state in water approaches not simply the ip of the dna in the gas phase but that when the dna is polarized by water . in water , the ip of the modified nucleotide - containing dna oligomer in the ionic state is smaller than that in the neutral state . the reason is the same as that for the dna oligomers which are not modified . there are the computational results obtained by neglecting the solvent , where the ip dependences on the dna sequence are smaller than those obtained by the ab initio hf mo calculations . for example , the calculated ip differences between the dna oligomers , 5-aga-3 and 5-ggg-3 , are 0.10.2 ev , which are closer to the corresponding experimental result , 0.077 ev.(21 ) however , in the case of the different sequences , 5-tgt-3 and 5-ggg-3 , the calculated ip differences are 0.30.5 ev , which are larger than the value(58 ) ( less than 0.1 ev ) calculated from the experimental results . in these computations , the semiempirical nddo - g method(56 ) and the density functional theory(57 ) were used . in both of them , the backbones of the dna oligomers were neglected ; this makes the ip dependence on the dna sequence smaller ( figure 1b ) . in the latter,(57 ) the charge distribution of the hole was calculated from the homo obtained by diagonalizing the reduced hamiltonian with the elements i|h|j , where h is the kohnsham ( ks ) hamiltonian , i and j are homos of individual nucleobases , and the total system is made of the stacked three base pairs without a backbone . however , the vertical ionization potential was obtained not as the energy of the homo of the total system but as the diagonal element of the reduced hamiltonian at the middle guanine site in the base pairs . the reason was not described . about 90% of the charges of the hole is always in a certain guanine in our calculations . the inclusion of the neglected reorganization energy due to the relaxation of the heavy atoms of dna will further localize the hole and not change the situation . the damage observed in the experiments is rather localized on a certain guanine in the g run . this fact also supports that the hole is localized . by neglecting the reorganization energy and the solvent effects , more than 95% of the charges of the hole is in a certain guanine for the stacked guanine bases and 70% is in a certain guanine for the stacked gc base pairs.(19 ) more than 80% is in a middle guanine for the stacked three ( n = 3 ) gc base pairs with backbones , but it is delocalized over two guanines for n = 4.(51 ) the reason for the difference in the extent of the localization between these results and ours is mainly that the reorganization energy was neglected in these calculations . the calculated ip of the stacked ga is smaller than that of g in the gas phase , and the difference between them is reduced in water.(27 ) this result is similar to that in figure 1 and is understood in the same way . the calculated vertical ips of the dna tetramers with 65 water molecules are larger than those in the gas phase by a few ev,(34 ) where the b - dna structure model is different from the ones used here . this result is similar to that in figure 8 and is understood in the same way . thus , it supports that the effect of the structure deviation within the b - dna structure on the ip is minor and so is that of the sequence dependence of the dna structure . it was discussed that the discrepancy between the theories and the experiment on the ip of the guanine run is reduced by taking into account the solvent effects;(29 ) in this work , the previously calculated ips(19 ) were used as the values for the hole fully delocalized over the guanine run and this delocalization was assumed to be the origin of the ip dependence on n in vacuum . however , more than 95% of the hole is localized on a certain guanine in the results(19 ) used there , and the origin of the ip dependence on n is not the delocalization of the hole , as described above . if the origin of the ip dependence on n was the delocalization of the hole , the average of the homo and homo-1 of the guanine doublet was close to the homo of the single guanine . we examined the ionization potential ( ip ) corresponding to the free energy of a hole on a duplex dna using the am1 method(39 ) with cosmo . the electrostatic interaction between the hole and a nucleotide pair in the duplex dna is attractive . due to this attractive interaction , the ip in the gas phase significantly decreases with the increasing number of base pairs of the dna oligomer . on the other hand , this attractive electrostatic interaction is reduced in water . the guanine runs , this is the reason why this ip dependence calculated by neglecting the solvent(19 ) was much larger than that obtained from the time - resolved data.(21 ) including the solvent makes this ip dependence consistent with the experimental result . as for the effect of the chemical modifications of dna , when a neutral functional group is attached to a guanine in a dna oligomer in water , nearby water molecules are removed ; this removal was found to reduce the free energy of the hole on the guanine.(22 ) one might naively have expected the opposite case , since a polar solvent usually stabilizes ions . when some water molecules are removed , the attractive electrostatic interaction stabilizing the hole increases , and thus , the hole is stabilized . in order to design the hole energetics by a chemical modification of dna ,

we examined the ionization potential ( ip ) corresponding to the free energy of a hole on duplex dna by semiempirical molecular orbital theory with a continuum solvent model . as for the contiguous guanines ( a guanine run ) , we found that the ip in the gas phase significantly decreases with the increasing number of nucleotide pairs of the guanine run , whereas the ip in water ( op , oxidation potential ) only slightly does . the latter result is consistent with the experimental result for dna oligomers in water . this decrease in the ip is mainly due to the attractive electrostatic interaction between the hole and a nucleotide pair in the duplex dna . this interaction is reduced in water , which results in the small decrease in the ip in water . this mechanism explains the discrepancy between the experimental result and the previous computational results obtained by neglecting the solvent . as for the chemically modified guanine , the previous work showed that the removal of some solvent ( water ) molecules due to the attachment of a neutral functional group to a guanine in a duplex dna stabilizes the hole on the guanine . one might naively have expected the opposite case , since a polar solvent usually stabilizes ions . this mechanism also explains this unexpected stabilization of a hole as follows . when some water molecules are removed , the attractive electrostatic interaction stabilizing the hole increases , and thus , the hole is stabilized . in order to design the hole energetics by a chemical modification of dna , this mechanism has to be taken into account and can be used .

Introduction Methods Results Discussion Conclusions

it was also reported that the attachment of a phenyl group to a guanine suppresses the damage not only at the phenylated guanine but also at nearby guanines in the duplex dna. thus , in the present work , we theoretically analyzed the ip of duplex dna corresponding to the free energy of the transferring hole on the duplex dna . since the above experiments were carried out in the presence of a solvent ( water ) , we took into account the solvent ; this turned out to be crucial to understanding the above experimental results . one of our goals is to resolve the discrepancy between the theoretical results and the experimental one;(21 ) these results are about the free energy dependence of the transferring hole on the length of the guanine runs . the ips ( corresponding to the free energy of the hole ) obtained by the ab initio hartreefock ( hf ) molecular orbital ( mo ) calculations are smaller for the longer guanine run . if the free energy of the hole on the longer guanine run is smaller , then the probability of the hole to be there is larger and thus the longer one is damaged more . however , the calculated ip difference between g and gg ( ggg ) of 0.47 ( 0.68 ) ev is too large compared to the corresponding free energy difference of 0.052 ( 0.077 ) ev obtained from the time - resolved data for dna oligomers in water. here , we neglect the entropy difference in the free energy difference between the reactant and the product of the hole transfer reaction by assuming that the potential functions of the reactant and the product are harmonic around the equilibrium structures and their second derivatives are similar . another question to be answered in the present paper is about the effect of the chemical modifications of dna oligomers on the hole transfer reactions . when a neutral functional group is attached to a guanine in a dna oligomer in water , this removal was found to reduce the free energy of the hole on the guanine. that is , since a polar solvent usually reduces the free energies of ions , the removal of nearby solvent molecules might be expected to increase the free energy of the hole it will turn out that this mechanism also explains the discrepancy mentioned in the previous paragraph . thus , it is considered that this free energy reduction is due to the removal of the solvent molecules. the phenyl group attachment to a guanine reduces the free energy of the hole on it , and thus , the hole is trapped there ; therefore , the guanines near the phenylated guanine are less damaged . we briefly review the relevant works on the electrostatic interaction and the solvent effects on dna , since they turned out to play crucial roles in the present cases . (25 ) as for the solvent effects , the hydration effect on the ip of the small fragments of dna , namely , the watsoncrick ( wc ) base pairs , nucleotides , and a phosphorylated dinucleotide , was investigated . in the present work , we calculated and analyzed the ip of the duplex dna by taking into account the solvent , where this ip corresponds to the free energy of the hole on the duplex dna . since the electrostatic interaction between the hole and a nucleotide pair in the duplex dna is attractive , the ip of the longer guanine run is smaller than that of the shorter one . however , this attractive interaction is reduced in water , which results in the small difference of the ip between the longer guanine run and the shorter one in water . a neutral functional group attached to a guanine in a duplex dna in water expels the nearby water molecules which reduce the electrostatic interaction . thus , the attractive electrostatic interaction stabilizing the hole on the guanine increases and the hole is stabilized . this is why the free energy of the hole decreases when nearby water molecules are removed . we calculated the ip of the duplex dna oligomers in the gas phase and in water as follows . we calculated these optimized coordinates for dna oligomers , which are in the neutral state in vacuum and in water , and in the ionic state in water . we included the solvent as the dielectric continuum , namely , the conductor - like screening model ( cosmo ) with the dielectric constant of water , 78.4 , and the vdw parameters optimized for the density functional theory. in the case of an odd number of electrons , the spin contamination was less than about 3% and thus the error in the energy due to the spin contamination was calculated to be less than a few tenths of an ev . as for the ip in water , we calculated it for dna oligomers in the neutral state and the ionic state ; in the latter state , the phosphate backbones are completely ionized . (22 ) as for the validity of the method , we calculated the relative ips of the nucleosides in water ( the relative ops ) , and compared them to the experimental result. (44 ) in addition , the method described here was successfully used to calculate the ips and the electron affinities of nucleic acid bases in the gas phase , where the rms deviations between the computational results and the experimental ones are 0.12 and 0.20 ev , respectively . the solvation energy for the solvent change acetonitrile / h2o is calculated to be 0.02 ev using the born equation,(44 ) and thus , the correction to the relative op for the solvent change is negligible . we calculated the ip of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] ( figure 1a ) ; we found that the ip in the gas phase significantly decreases with the increasing number of base pairs , n , whereas the ip in water ( or op ) only slightly does ( figure 1b ) . the decrease in the ip in the gas phase from n = 1 to 3 is more than about 1 ev , but that in water is less than 0.2 ev . the latter result is consistent with the experimental result for the dna oligomers in water. (21 ) the difference between the ip decreases in the gas phase and in water is much larger than the rms deviation ( 0.14 v ) between the computational and the experimental results obtained in section . about 90% of the charges of the hole is in a certain guanine and 5% is in the sugar covalently bonded to this guanine both in the gas phase and in water . we calculated the ip of the dna oligomers , d(5-aagaa-3)d(3-ttctt-5 ) and d(5-aggga-3)d(3-tccct-5 ) , where the lengths of the guanine runs are different but the total number of base pairs is fixed ; we again found that the ip difference between the two sequences in water , 0.11 ( 0.10 ) ev , is smaller than that in the gas phase , 0.21 ( 0.15 ) ev , for the 55th fiber model ( the 4th fiber model ) . as shown in figure 2 , the ips calculated above ( the closed symbols ) are close to the electrostatic energy of the hole both in the gas phase ( the open triangles ) and in water ( the open circles ) . we define the electrostatic energy of the hole as the electrostatic interaction between the hole and all atoms except for the atoms of the guanine where the hole is localized plus a constant ; this constant is such that the value of this electrostatic energy coincides with the ip in the gas phase for n = 1 . in the case of the dna oligomer in water , we added the electrostatic interaction between the hole and the water ; we obtained this interaction as the dielectric energy,(49 ) which is considered to be the free energy but not the energy . comparison of the electrostatic energy of the hole ( defined in the text ) with the ip of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] as a function of the number n of base pairs for the 55th fiber model structure and the 4th fiber model structure , as indicated . to understand the electrostatic interactions in the dna oligomers , we calculated the charge distributions of the dna oligomer d[5-(g)53]d[3-(c)5 - 5 ] in the neutral state in the gas phase and in water ( figure 3 ) ; we found that the bases and sugars have negative partial charges and the phosphates have positive partial charges both in the gas phase and in water ( figure 4 ) . about 90% of the charges of the hole is in a certain guanine both in the gas phase and in water , as described earlier . we calculated the ip of the dna oligomers with other sequences , where a guanine is embedded in the center of the contiguous adenines or thymines , and obtained the same result with that of d[5-(g)n-3]d[3-(c)n-5 ] ( figure 5 ) . that is , the ip significantly decreases with the increasing n in the gas phase , whereas it does only slightly in water . the values of the ip decreases are slightly smaller than the corresponding values of the contiguous guanines in figure 1b , both in the gas phase and in water . to understand the dependence of the obtained ips on n , we calculated the ip of the system made of two gc base pairs ( the inset in figure 6 ) as a function of the rise r ; we found that the ip of this system ( the solid line in figure 6 ) is close to the ip of the single base pair plus the electrostatic interaction between the hole and the base pair where the hole does not reside ( the dashed line in the same figure ) . we calculated the electrostatic interaction between the hole and the base pair where the hole does not reside ; this interaction is the electrostatic interaction between one base pair in the cationic state and the other in the neutral state , minus that between the two base pairs both in the neutral state . dashed line : ip of the single gc base pair plus the electrostatic interaction between the hole and the base pair where the hole does not reside ( defined in the text ) . to understand the reduction in the ip decrease with the increasing n due to the solvent , we developed simple models of dna oligomers ( figure 7a ) ; we showed that these models reproduce the ips of the dna oligomer d[5-(g)n-3]d[3-(c)n-5 ] ( figure 7b ) . a duplex dna oligomer is modeled as a box with the simplified charge distribution of a duplex dna oligomer in the neutral state ( the hole is localized on a certain guanine ) and the water is replaced by a conductor ( figure 7a ) . the symbols and lines are the same as those in figure 1b except that the open symbols are for the simple models of the dna oligomers in the gas phase , those in water in the neutral state , and those without a backbone in the gas phase , as indicated . we calculated the ip of the modified nucleotide - containing dna oligomer , d[5-t1a2g3g4t5a6 - 3]d[3-a12t11c10c9a8t7 - 5 ] , where a phenyl group is attached to g3 ( figure 9a ) ; we found that the attachment of a phenyl group reduces the ip in water but not in the gas phase ( figure 9b ) . (18 ) ( b ) the effect of the phenyl group attachment on the ip of the dna oligomer in the gas phase , in the neutral state in water , and in the ionic state in water , for the 55th fiber model structure and the 4th fiber model structure , as indicated . figure 1b shows that the large decrease ( in the order of 1 ev ) of the ip in the gas phase with the increasing dna length is reduced to about a tenth of an ev in water . from figure 2 , it is clear that the decrease of the electrostatic energy of the hole causes the ip decrease with the increasing number of base pairs , n. since the electrostatic energy of the hole decreases with the increasing n in figure 2 , the electrostatic interaction between the hole and a nucleotide pair is attractive in a duplex dna ; this nucleotide pair includes the two phosphates bonded to it . because of the attractive interaction between the hole and a nucleotide pair , the ip in the gas phase decreases with the increasing n. this attractive interaction is reduced in water , and so is the ip decrease with the increasing n. when we replace the charge distribution of the dna oligomer in the gas phase by that in the neutral state in water , the electrostatic energy of the hole in the gas phase decreases more strongly ( figure 2 ) . from the simplified charge distribution of the dna oligomer in figure 4 , we understand why the electrostatic interaction between the hole and a nucleotide pair is attractive in the duplex dna ; this nucleotide pair includes the two phosphates bonded to it . the ip in the gas phase decreases more strongly than that without a backbone ( figure 1b ) , because the positive partial charges are separated more from the hole for the dna oligomer with backbones compared to that without . in water , the ips of the dna oligomers in the ionic states are smaller than those in the neutral states ( figure 1b ) . this is because the pn s , which have positive partial charges in the neutral state of dna oligomers , become almost neutral in the ionic state of the dna oligomers and the repulsive interaction between pn s and the hole is reduced in the ionic state . figure 6 shows that the attractive electrostatic interaction between the hole and a base pair causes the ip dependence of the rise r ; this supports that this attractive interaction causes the dependence of the ip on n in figure 1b . from this result , we can understand that the ips in the gas phase in figure 1b rapidly decrease for n 3 and converge for n 5 or 6 by considering that the hole is usually in the middle of the dna oligomer and the rise in the duplex dna is 3.4 . the simple models of dna oligomers ( figure 7a ) illustrate that water molecules around the duplex dna ( but not between the base pairs ) reduce the electrostatic interaction in the duplex dna ( figure 7b ) . as shown in figure 8 , the vertical ip more significantly decreases with the increasing n in vacuum than in water , similarly to the ip in figure 1 . as for the dielectric shielding , when we use koopmans theorem , the hole is not shielded by the solvent , but other charges are shielded , and thus , the attractive electrostatic interaction between them is reduced in water . thus , this reduction in the attractive electrostatic interaction leads to the result that the vertical ip in water is larger than that in the gas phase . we also observed a slightly smaller decrease in the vertical ip in the gas phase with the increasing n and the slightly larger one in water compared to the corresponding ips . the reorganization energy due to the relaxation of the dna atoms and the electronic energy reduction mentioned above increase with the increasing n and these are included in the ips but not in the vertical ips . thus , the ip in the gas phase decreases slightly more than the corresponding vertical ip . as for dna in water , when n is increased , a part of the dna replaces a part of the solvent , where the dielectric constant of the former is smaller than that of the latter , and thus , the total reorganization energy decreases . since we did not include the reorganization energy due to the relaxation of the heavy atoms of dna , the above decrease in the reorganization energy with the increasing n was overestimated . thus , the decrease in the exact ip with the increasing n should be between those of the ip and the vertical ip obtained in the present work . as shown in figure 9 , when the neutral functional group is attached to the guanine in the duplex dna in water , the free energy of the hole on the guanine is reduced ( but not in the gas phase ) . thus , when nearby water molecules are removed , the attractive electrostatic interaction stabilizing the hole increases . (22 ) the difference between the former and the latter is that the former includes the reorganization energy due to the relaxation of the solvent but the latter does not . according to the above mechanism , the ip of the modified nucleotide - containing dna oligomer in the neutral state in water is supposed to be between the ip of the dna not modified in the neutral state in water and that in the gas phase . one of the reasons is that when the nearby water molecules are removed , the ip of the modified nucleotide - containing dna oligomer in the neutral state in water approaches not simply the ip of the dna in the gas phase but that when the dna is polarized by water . there are the computational results obtained by neglecting the solvent , where the ip dependences on the dna sequence are smaller than those obtained by the ab initio hf mo calculations . by neglecting the reorganization energy and the solvent effects , more than 95% of the charges of the hole is in a certain guanine for the stacked guanine bases and 70% is in a certain guanine for the stacked gc base pairs. the calculated ip of the stacked ga is smaller than that of g in the gas phase , and the difference between them is reduced in water. the calculated vertical ips of the dna tetramers with 65 water molecules are larger than those in the gas phase by a few ev,(34 ) where the b - dna structure model is different from the ones used here . it was discussed that the discrepancy between the theories and the experiment on the ip of the guanine run is reduced by taking into account the solvent effects;(29 ) in this work , the previously calculated ips(19 ) were used as the values for the hole fully delocalized over the guanine run and this delocalization was assumed to be the origin of the ip dependence on n in vacuum . however , more than 95% of the hole is localized on a certain guanine in the results(19 ) used there , and the origin of the ip dependence on n is not the delocalization of the hole , as described above . we examined the ionization potential ( ip ) corresponding to the free energy of a hole on a duplex dna using the am1 method(39 ) with cosmo . the electrostatic interaction between the hole and a nucleotide pair in the duplex dna is attractive . due to this attractive interaction , the ip in the gas phase significantly decreases with the increasing number of base pairs of the dna oligomer . on the other hand , this attractive electrostatic interaction is reduced in water . as for the effect of the chemical modifications of dna , when a neutral functional group is attached to a guanine in a dna oligomer in water , nearby water molecules are removed ; this removal was found to reduce the free energy of the hole on the guanine. (22 ) one might naively have expected the opposite case , since a polar solvent usually stabilizes ions . when some water molecules are removed , the attractive electrostatic interaction stabilizing the hole increases , and thus , the hole is stabilized . in order to design the hole energetics by a chemical modification of dna ,

spinal muscular atrophy ( sma ) is characterized by motor neuron degeneration with muscular atrophy , paralysis , and an attenuated lifespan . sma exhibits an autosomal recessive pattern of inheritance with an incidence of 1 in 6,00010,000 newborns and a carrier frequency of about 1 : 35 [ 2 , 3 ] . based on age of onset and achievement of motor milestones , sma has been subdivided into four clinical types : severe ( type i ; werdnig - hoffmann disease ) , intermediate ( type ii ) , mild ( type iii ; kugelberg - welander disease ) , and adult forms . most sma patients harbor deletions , mutations , or conversions of the telomeric copy of the survival motor neuron gene ( smn1 ) [ 5 , 6 ] . the centromeric smn gene ( smn2 ) is present in all sma patients , but is unable to compensate for the smn1 gene defect as the primary transcript of smn2 gene is defectively spliced [ 5 , 6 ] . since there is an inverted correlation between the amount of smn protein and disease severity [ 7 , 8 ] , smn has been the most important therapeutic target for development of sma treatment [ 9 , 10 ] . however , some smn independent targets and therapeutic strategies have been demonstrated to have the potential to benefit sma [ 1120 ] . although most are still under investigation , these nonclassical therapies might provide an adjunctive method for future sma therapy . although pathogenesis of sma has been investigated extensively , some of the detailed disease mechanisms are still not fully understood . the smn is a 38-kda protein expressed in both the cytoplasm and nucleus of all cells . smn serves as a chaperone in the assembly of spliceosome precursors by combining small nuclear rna ( snrna ) molecules with sm proteins to generate small nuclear ribonucleoproteins ( snrnps ) [ 22 , 23 ] . the snrnp assembly activity is dramatically reduced in spinal cord from sma model mice and the degree of snrnp assembly impairment correlates with disease severity . evidence shows that smn is also involved in the stabilization and maturation of the neuromuscular junction and the transportation of axonal mrnas in motor neurons [ 2527 ] . smn - deficient motor neurons exhibit severe defects in clustering voltage - gated calcium channels in axonal growth cones . an alteration of calcium channel distribution might influence neurotransmitter release , causing dysfunction and immaturation of neuromuscular junction [ 25 , 28 ] . in addition , the smn protein can form granules that are transported and associated with -actin mrna in neuronal processes . the close relationship of smn and -actin has further demonstrated that motor neurons derived from sma model mice have shortened axons and small growth cones , which are also deficient in -actin mrna and protein . therefore , smn has a function in maintaining proper neuronal machinery via assistance in splicing process and establishing adequate communication between the muscles and nerves at the motor end plate through stabilization of the neuromuscular junction . the loss of maintenance and communication might thus trigger the cascade of events that probably results in motor neuron death . sma mouse models have been generated through mouse smn knockout and human smn2 transgenic methods [ 8 , 31 ] . these mice reveal spinal motor neuron degeneration , muscle atrophy , and impaired motor performances similar to sma patients . the disease severity of these sma mice is also inversely correlated to the copy number of the smn2 transgenes [ 8 , 31 ] . neuromuscular junction can form and function normally prior to the postnatal onset of disease . afterward , abnormal neurofilament accumulation and functional disruption at the neuromuscular junction become evident . alongside these morphological and functional changes at the neuromuscular junction , studies on the spinal cord of sma mice showed an apparent failure of expression of genes that cluster in postnatal developmental pathways . subsequently , through still unknown mechanisms , motor neurons degenerate in spinal anterior horn regions probably through cell apoptosis [ 16 , 34 ] , and muscle atrophy and motor dysfunction become apparent . recently , congenital heart defects have been recognized as additional important phenotypes especially in type i sma patients , including atrial septal defects , dilated right ventricle , and ventricular septal defects . the histological studies in sma model mice also showed that cardiac remodeling starts at the embryonic stage in the severe sma mice while motor neurons are not yet visibly affected at this stage . after birth , there is progressive cardiac fibrosis , which may result from oxidative stress . sma mice also suffer from severe bradyarrhythmia characterized by progressive heart block and impaired ventricular depolarization , which may be related to defective sympathetic innervation . notably , systemic restoration of smn expression is able to diminish the cardiac defects accompanied with prolonged lifespan , implying that cardiac abnormalities are playing a critical role on sma pathogenesis [ 38 , 39 ] . since smn levels generally correlate with disease severity in sma patients and mouse models [ 7 , 8 , 31 , 40 ] , smn is the best therapeutic target for development of sma treatment . various strategies to increase the smn levels have been tested in sma mouse models and some of them have even showed promising beneficial effects [ 9 , 10 ] . until now , none of them have been demonstrated to be consistently robust or produce continual benefits in sma patients . these therapeutic strategies are divided into small molecules , antisense oligonucleotides ( aso ) , and viral vector - mediated gene therapy . all sma patients have at least one copy of the smn2 gene , providing an opportunity for manipulation of the smn2 gene expression . the mode - of - action for a potential sma therapy using small molecules mainly includes restoration of the smn2 splicing pattern , activating the smn2 promoter , and extending the half - life of smn mrna or protein . the potential drugs include histone deacetylase ( hdac ) inhibitors such as sodium butyrate , phenylbutyrate , valproic acid ( vpa ) , trichostatin a , saha , and lbh589 , as well as hydroxyuria , sodium vanadate , aclarubicin , indoprofen , bortezomib , and aminoglycosides , such as tobramycin , amikacin , tc007 , and g418 . since there are still no drugs that have shown consistent benefits in clinical trials [ 55 , 56 ] , finding an effective treatment with distinct therapeutic mechanisms , such as smn independent targets , is necessary for future sma therapy . among these small molecules , vpa is the drug being studied most extensively and has been used in patients with epilepsy and bipolar disorders for decades . vpa treatment increased levels of smn transcripts and protein in fibroblasts derived from sma patients through upregulation of serine / arginine - rich ( sr ) proteins , which are involved in regulating smn2 exon 7 recruitment [ 58 , 59 ] . autophagy , the degradation of cytosolic components in lysosomes , maintains neuronal homeostasis ; its dysfunction has been linked to various neurodegenerative diseases , possibly including sma . vpa is also an autophagy enhancer , which activated autophagic pathways and attenuated rotenone - induced toxicity in sh - sy5y cells . in addition , vpa upregulates some antiapoptotic factors such as bcl-2 and bcl - xl , perhaps via activation of erk44/42 [ 43 , 62 , 63 ] . probably through multiple therapeutic effects , vpa reduced motor neuron degeneration , muscle atrophy , and motor dysfunction in sma mice [ 43 , 64 ] , and a small group of sma patients showed obvious improvement in muscle strength after daily vpa treatment [ 65 , 66 ] . despite these encouraging results , large clinical trials did not confirm the beneficial effects of vpa in sma patients [ 6769 ] . these disappointing outcomes may contribute to different pharmacokinetics and bioavailability between rodents and humans as well as dose - limiting intolerance and drug adverse effects . in addition , the responses of vpa treatment showed intrapatient and interpatient variability in the study using fibroblasts and lymphoblasts from sma patients , probably indicating that tissue and individual factors may affect the vpa effects with unknown reasons . using aso to inhibit the splicing silencer for smn2 exon 7 leads to restoration of the normal smn2 splicing pattern . the effects of aso were further improved through the incorporation of a binding platform with aso for recruitment of sr protein to the smn2 exon 7 region . these bifunctional asos were able to achieve nearly 100% exon 7 inclusion and enhance smn expression up to 2- to 3-fold in cell - based assays . injection of aso into cerebroventricles elicited a robust induction of smn protein in the brain and throughout the spinal cord and extended the lifespan of sma mice . a recent study demonstrated that systemic delivery of aso resulted in dramatic prolongation of lifespan in sma mice and the effects were much better than those with intracerebroventricular delivery of aso ( median survival , 108 versus 16 days ) . these findings suggest that aso therapy has great potential in this field and extra - cns targeting is required to rescue the sma phenotype . however , another similar study showed different results that early intracerebroventricular delivery of aso had a better outcome than intravenous aso delivery , which suggests that therapeutic methods for aso treatment still need further investigation and optimization . direct injection of adeno - associated viral vector serotype 8 ( aav8 ) carrying smn into both cerebroventricles and upper lumbar spinal cord of sma mice showed a robust increase in lifespan by 880% with less motor neuron degeneration and abnormal architectures of neuromuscular junction . however , augmented smn is expressed in thoracolumbar regions , but sparse in the cervical cord , which may suggest poor diffusion of aav in subarachnoid space . in contrast , intravenous aav serotype 9 ( aav9 ) injection has shown success in affecting widespread gene delivery in entire spinal cord . intravenous injection of aav9 carrying human codon - optimized smn1 at postnatal day 1 recovered most motor function , neuromuscular physiology , and lifespan in sma mice . notably , postnatal day 1 treatment resulted in the maximal transduction of the motor neurons , while postnatal day 10 treatment led to glia - predominant transduction . this shift in cell type specificity was probably because of the closure of the blood brain barrier that occurs within the first week of life in neonatal mice . when the blood brain barrier is mature and patent , virions are probably not able to penetrate out of vessels smoothly to access motor neurons , but only encounter the endothelial wrappings of astrocyte end feet . since blood brain barrier likely matures in as early as human neonatal period , the aav9 transduction efficacy should further be tested in nonhuman primates of different ages to identify the optimal temporal window for future therapy . insulin - like growth factor-1 ( igf-1 ) is a trophic factor mainly secreted by the liver and circulates at high levels in the bloodstream . igf-1 is a key molecule involved in normal brain growth and function and may have a neuroprotective effect by inhibiting neuronal death in huntington 's disease and spinocerebellar ataxia [ 81 , 82 ] . igf1-null mice show some phenotypic similarity to sma mice , such as small size and generalized muscle dystrophy , with most of them dying at birth . notably , serum igf-1 level was decreased in sma mice , and systemic increase of smn expression using the aso strategy in sma mice was accompanied with restoration of serum igf-1 to normal levels . interestingly , mrna levels of igf - binding protein , acid labile subunit ( igfals ) , but not igf-1 , was reduced in sma mice . igfals binds to igf-1 and igf - binding protein 3 to form a stable ternary complex , extending the half - life of igf-1 from 10 minutes to 12 hours . therefore , the low serum igf-1 level in sma mice is likely related to downregulation of igfals , and igf-1 may be one of the factors that contribute to the pathogenesis of sma . igf-1 treatment has been shown to improve disease phenotypes in rodent models of motor neuron diseases such as amyotrophic lateral sclerosis ( als ) and spinal and bulbar muscular atrophy ( sbma ) . for sma , transgenic expression of igf-1 in skeletal muscle of sma mice resulted in an increase in myofiber size and a modest improvement in median survival . delivery of a plasmid dna vector encoding igf-1 by intracerebroventricular injection into newborn sma mice also increased body mass and provided a modest improvement in median survival . however , intracerebellar viral delivery of igf-1 reduced motor neuron degeneration , but did not improve motor function in the mildly affected sma mice . therefore , the effects of igf-1 and igfals - related therapy using different treatment strategies in sma still require further investigation . these cells express ciliary neurotrophic factor ( cntf ) , and lack of cntf expression strongly reduces terminal sprouting and motor unit size . in a mouse model of als , the depletion of synaptic vesicles precedes the loss of synapses ; cntf could prevent the depletion of synaptic vesicles and thus maintain function of neuromuscular junctions . cntf treatment using cntf - secreting stem cells or by local cntf injection into skeletal muscle led to better maintenance of peripheral motor axons in a mouse mutant , progressive motor neuronopathy ( pmn ) [ 91 , 92 ] . in a severe type of sma mice , in contrast , the architecture and function of neuromuscular junctions in heterozygous smn ( + / ) mice are relatively preserved , despite some loss of spinal motor neurons . however , completed knockout of cntf in heterozygous smn ( + / ) mice reduces the sprouting responses of the nerve terminals accompanied with reduced muscle strength . these results imply that cntf may be able to compensate loss of motor neurons by sprouting from remaining motor axon terminals so that neuromuscular endplates remain innervated ; cntf may thus guide the way for new therapies for sma . although systemic cntf treatment elicited severe adverse effects including fever and cachexia in als patients , muscle or cns targeting cntf therapy might offer a chance to reduce these side effects and show benefits in sma . cntf and cardiotrophin-1 ( ct-1 ) are both members of the il-6 family , which bind a common receptor complex requiring leukemia inhibitory factor receptor ( lifr ) and gp130 . ct-1 is essential in normal motor neuron development and is also able to support long - term survival of motor neurons as demonstrated in culture cells and rats with axotomy . in addition , overexpression of ct-1 in pmn and als mice both significantly delayed disease onset , reduced degeneration of motor neurons and axons , and preserved the terminal innervation of skeletal muscles [ 97 , 98 ] . for sma mice , intramuscular injection of adenoviral vector expressing ct-1 , even at very low doses , prolonged survival , delayed motor defects , and diminished motor axonal degeneration and aberrant synaptic terminals . although most of studies regarding ct-1 are focused on diseases in the cardiovascular system , ct-1 might still be a valuable therapeutic agent for motor neuron diseases through neurotrophic effects . degeneration of spinal motor neurons in sma is mediated in part through apoptosis [ 16 , 34 ] . in the bcl-2 family , bcl - xl and bax are important regulators of cell death in the nervous system when cells have matured . bcl - xl is an antiapoptotic member of the bcl-2 family and acts by inhibiting proapoptotic members of the bcl-2 family through heterodimerization . bcl - xl was downregulated in sma patients and model mice [ 17 , 100 ] . bcl - xl overexpression can protect against motor neuron death in cultured primary motor neurons and embryonic motor neurons with smn knockdown . interestingly , bcl - xl overexpression in sma mice reduced motor neuron degeneration , preserved motor function , and prolonged lifespan without changes in smn expression levels . bax knockout sma mice had milder disease severity and longer lifespan with less spinal neuronal degeneration than sma littermates with wild - type bax genes . therefore , effects of bcl - xl and bax may not be simply through apoptotic pathways , but through unknown mechanisms to salvage neural function in sma . the ratio of bcl - xl / bax is thus another attractive target , where the potential to increase bcl - xl and decrease bax expression may be of benefit to sma patients . riluzole , a 2-aminobenzothiazole , is the only disease - modifying therapy available for als . although riluzole is known to modulate excitatory neurotransmission mainly through inhibition of glutamate release , the precise neuroprotective mechanisms remain largely speculative . in sma mice , however , a small phase i clinical trial , enrolling 7 riluzole - treated and 3 placebo - treated type i sma infants , demonstrated no significant differences in survival and the change in motor abilities after riluzole treatment . nevertheless , further analysis showed that 3 patients in the riluzole group presented an unusual disease course and were still alive at the age of 30 to 64 months . the pharmacokinetics of riluzole in sma patients has recently been investigated , and the long - term benefits of riluzole still warrant large clinical trials for sma patients . gabapentin is a gaba analogue and has been used clinically for patients with seizures and neuropathic pain for more than 10 years . gabapentin could also have a neuroprotective action in part by reducing the pool of releasable glutamate in neurons , thereby diminishing the excitotoxicity potential [ 109 , 110 ] . although gabapentin treatment showed marginal reduction in disease progression in a phase ii clinical trial for als patients , the following phase iii clinical trial did not reveal significant benefits after gabapentin treatment for 9 months . for sma , there was no difference between the gabapentin and placebo groups in any outcome measure including changes in muscle strength , pulmonary function , or motor functional rating scale after 12 months of treatment . however , another clinical trial which enrolled 120 type ii and iii sma patients showed a significant improvement in muscle strength of legs at both 6 and 12 months after gabapentin treatment . meta - analysis of these two trials did not successfully demonstrate the beneficial effects of gabapentin in sma . 2-adrenergic agonist , such as salbutamol ( albuterol in the united states ) , enhanced muscle strength in aged rats , human healthy volunteers , and some pathological conditions [ 117 , 118 ] . in a pilot clinical trial , thirteen type ii or iii sma patients receiving salbutamol for 6 months showed significant increase in myometry , forced vital capacity , and lean body mass . a further larger trial enrolling 23 type ii sma patients consistently got similar results that functional scores were better after daily salbutamol treatment for 6 or 12 months . notably , the drug did not produce any major side effects [ 119 , 120 ] . the mechanism of action of 2-adrenergic agonists on human skeletal muscles to enhance muscle strength is not completely understood . interestingly , salbutamol also promoted exon 7 inclusion in smn2 transcripts and thus increased levels of full - length transcripts of smn2 in sma fibroblasts . in sma patients , daily salbutamol significantly and consistently increased smn2 full - length transcript levels in peripheral leukocytes , and the response was directly proportional to smn2 gene copy number . considering bifunctional therapeutic effects and safety of salbutamol , large randomized double - blinded placebo - controlled clinical trials are mandatory . myostatin is a member of the tgf- family and functions as a potent negative regulator of muscle growth . inhibition of myostatin increases muscle mass and strength in wild - type rodents and improves the pathophysiology of a mouse model for muscular dystrophy [ 124 , 125 ] . follistatin is a cystine - rich glycoprotein , which binds to and inhibits several tgf- family members , including myostatin . follistatin delivered by intramuscular injection of recombinant viral vectors increased muscle mass in mouse models of both als and duchenne muscular dystrophy [ 127 , 128 ] . since sma also features diffuse muscle atrophy , inhibition of myostatin may also be a therapeutic strategy . intraperitoneal injection of recombinant follistatin in sma model mice increased muscle mass , improved motor function , and prolonged lifespan by 30% without changes in smn protein levels in spinal cord and muscles . however , other studies detected no phenotypic alteration in transgenic overexpression of follistatin or ablation of myostatin in sma mice [ 129 , 130 ] . the reason for this discrepancy is unclear and the effects of follistatin for sma treatment still need further validation . although sma - affected siblings usually develop similar disease severity in terms of their age at onset and the progression of disease , a small proportion of individuals with homozygous smn1 mutation are fully asymptomatic despite carrying an identical number of smn2 copies as their affected siblings , suggesting the influence of modifier genes [ 132 , 133 ] . the first potential smn - independent disease modifier , plastin-3 , was recently identified from six sma - discordant families with eight fully asymptomatic females who had inherited the same smn1 and smn2 alleles as their affected siblings . increased levels of plastin-3 were also found to correlate with a mild sma phenotype in female patients , independently of smn protein levels [ 18 , 134 ] . plastin-3 , an actin binding protein , is a regulator of actin filament organization and is expressed in almost all solid tissues , including the human brain , spinal cord , and muscles . plastin-3 colocalizes with smn in granules throughout motor neuron axons , and plastin-3 protein levels are reduced in brain and spinal cord of an sma mouse model [ 18 , 135 ] . in smn - depleted neuronal pc12 cells and primary mouse motor neuron cultures derived from sma mice , notably , overexpression of plastin-3 or its orthologues also led to diminishment of axon defects and disease severity in smn depleted zebrafish embryos , drosophila , and c. elegans [ 18 , 136 ] . smn has been shown to moderate and restrict the negative function of profilin iia on actin polymerization . profilin iia is another actin binding protein , and knockdown of profilin iia results in stimulation of neurite outgrowth , while overexpression of profilin iia reduces neurite number and size . knockout of profilin iia in sma model mice was able to restore abnormal low plastin-3 levels . however , the phenotype of these sma mice was not ameliorated despite the depletion of profilin iia and restoration of plastin-3 levels , which suggests that other components of actin dynamics are also critically affected in sma . although some questions need to be answered , such as the mechanisms behind plastin-3 in sma and effects of plastin-3 upregulation in sma mouse models , plastin-3 may become an important smn - independent therapeutic target for sma in the future . the candidate plasticity - related gene 15 ( cpg15 ) is highly expressed in the developing ventral spinal cord and can promote motor axon branching and neuromuscular synapse formation [ 139 , 140 ] . cpg15 mrna colocalizes with smn protein in axons and is locally translated in growth cones . hud is a neuron - specific rna - binding protein and also an interacting partner of smn [ 141143 ] . cpg15 may be an mrna target for the smn - hud complex and smn deficiency reduced cpg15 mrna levels in neurons . most importantly , cpg15 overexpression partially recovered from motor axonal deficits in zebrafish with smn deficiency . therefore , cpg15 appears to be a crucial downstream effecter of smn in neurons and may serve as a modifier of sma disease by regulating axon extension and axon terminal differentiation . rho - kinase signaling is a major regulatory pathway of actin dynamics , and rho - kinase activation is associated with dendritic simplification , and reduced spine length and density . rho - kinase activity is upregulated in smn - depleted pc12 cells and sma model mice [ 145 , 146 ] . the migratory capacity of the u87 mg astroglioma cells was attenuated by knockdown of smn through abnormal activation of rho - kinase pathway . normally , smn binds to profilin iia to form complexes , and rho - kinase may phosphorylate profilin iia . through competition between smn and rho - kinase for binding to profilin iia , smn deficiency results in a decrease in smn - profilin iia complexes and stronger interaction of profilin iia with rho - kinase . therefore , rho - kinase inhibition might be able to correct the effect of smn reduction in sma to achieve an adequate ratio of de-/phosphorylated profilin iia . notably , treatment of sma model mice with rho - kinase inhibitor y-27632 or fasudil led to a significant prolongation in survival , improvement in integrity of neuromuscular junction , and increase in muscle fiber size without altered smn expression or increase in the number of spinal motor neurons [ 146 , 149 ] . since fasudil has been successfully applied in many clinical trials for other neurological and vascular diseases based on its neuroprotection , vasodilatation , and immune modulation effects , the results of fasudil therapeutic studies for sma patients are anticipated . a diagnosis of sma is usually made following a patient 's initial presentation of muscle weakness , at which there would be substantial spinal motor neuron loss . both smn dependent and independent treatments described above could only prevent disease progression , but not regain lost motor neurons , while stem cell therapy might provide a possibility for cell replacement . fetal - derived neural stem cells ( nscs ) are able to self - renew and are multipotent with the capacity of producing neurons ( including motor neurons ) , astrocytes , and oligodendrocytes . nscs can be isolated from mouse embryonic spinal cords and differentiated toward a motor neuron cell fate by priming with retinoic acid and sonic hedgehog . intrathecal injection of these primed nscs in nmd mice , another model of motor neuron disease , resulted in improvement of abnormal phenotypes and extension of survival . in addition , nscs derived from human fetal spinal cord delayed disease onset and prolonged lifespan after being transplanted directly into spinal cord of als mice [ 153 , 154 ] . in a severe type of sma mouse model , intrathecal injection at postnatal day 1 with primed nscs derived from mouse embryonic spinal cord also promoted motor neuron survival , improved motor function , and prolonged lifespan . although some grafted cells expressed motor neuron markers , there was no direct evidence suggesting that the beneficial effects resulting from the formation of functional motor units by the transplanted cells . transplantation of undifferentiating nscs also showed a significant increase in survival of sma mice , although not as efficient as the effects of nscs primed into a motor neuron fate . therefore , the observed benefits of nscs in sma model mice were likely related to trophic support . although fetal - derived nsc transplantation in sma mice showed promising effects , their derivation from a spinal cord source impedes further clinical implementation because of ethical and technical issues . on the other hand , embryonic stem cells might be easier to obtain and are also able to differentiate in vitro and in vivo into nscs and a motor neuron fate . intraspinal grafting of embryonic stem cell - derived motor neurons resulted in a significant improvement in motor behaviors in the als rat . for sma , embryonic stem cell - derived nscs transplanted intrathecally in sma model mice migrated to spinal anterior horn and improved motor function and lifespan . although the grafted stem cells integrated appropriately into the parenchyma , and expressed both neuron- and motor neuron - specific markers , there was again no evidence of newly generated motor neuron outgrowth to the muscles . in one previous study , a boy with ataxia telangiectasia received intracerebellar and intrathecal injection of human fetal nscs . four years later , he was diagnosed with a donor - derived multifocal brain glioneuronal neoplasm . to increase the differentiation rate of embryonic stem cells into nscs before transplantation , the above sma study used drug - selectable embryonic stem cell lines that ganciclovir and g418 have been applied for selection against undifferentiated embryonic stem cells and for neuroepithelial cells , respectively . usage of these drug - selectable stem cells not only promoted transplantation safety , but also produced superior treatment results as compared to using wild - type embryonic stem cells . since the first report on reprogramming of mouse fibroblasts into so - called induced pluripotent stem ( ips ) cells by the expression of oct3/4 , sox2 , c - myc , and klf4 in 2006 , reprogramming of human somatic cells to a pluripotent state was achieved using similar approaches [ 160 , 161 ] . the ips cells can be differentiated into cells of endodermal , mesodermal , or ectodermal origin , and further lineage restriction can obtain specific neural subtypes or astrocytes . recently , ips cells have been successfully generated from fibroblasts of sma patients [ 162 , 163 ] . the sma - specific ips cells exhibited a reduced capacity to form motor neurons and an abnormality in neurite outgrowth that ectopic smn expression rescued these abnormal phenotypes . these ips cells provide a novel opportunity in disease modeling for investigating sma pathogenesis and can be used in screening novel compounds for sma treatment . the use of fetal - derived cells or embryonic stem cells for transplantation is hurdled by problems of availability , the possibility of immune rejection , and ethics . in contrast , the source of ips cells is unlimited , and ips cells can be transplanted autologously . transplantation of normal neurons derived from ips cells reduced abnormal phenotypes in a murine model of parkinson 's disease . notably , when ips cell - derived neural precursor cells from a patient with parkinson 's disease were transplanted into the striatum of a parkinson 's disease rat model , the donor cells differentiated into dopaminergic neurons , survived in the rodent brain for several months , and reduced the abnormal motor asymmetry . for autologous ips cell transplantation in sma , ips - derived neural precursor cells or motor neurons should be pretreated to express a high level of smn before transplantation . until now in various neurological disorders , many diseases , such as parkinson 's disease , epilepsy , and multiple sclerosis , are treated clinically with multiple drugs in combination to enhance the therapeutic effects . motor neurons may also require additional support to optimally respond to smn - based treatment . in the past two decades , there has been tremendous progress in sma regarding genetics , pathophysiology , and therapeutics . some useful strategies to enhance smn expression have been developed , and some novel smn - independent therapeutic targets have been discovered . while smn acts to modulate and correct the neuromuscular junction for functional improvement , smn - independent targets could play a role of extension in the survival of motor neurons and reduce the influence of smn depletion in axonal dynamics . the two currently available stem cell transplantation studies for sma have only demonstrated benefits likely with trophic support without evidence of functional cell replacement [ 19 , 20 ] . to generate functional motor units , the grafted stem cells should be able to differentiate into motor neurons , appropriately project the axons a long distance toward corresponding muscles , and form functional synapses within neuromuscular junctions . in a virus - induced rat model of motor neuron degeneration , mouse embryonic stem cell - derived motor neurons transplanted into spinal cord could survive , extend axons , form functional motor units , and promote recovery from paralysis [ 166 , 167 ] . the successful development of motor units in the above studies may result from a combination approach , which includes administration of dibutyryl - camp , rolipram , cyclosporine , and glial cell line - derived neurotrophic factors to promote motor neuron survival , circumvent myelin repulsion , prevent immune rejection , and enhance axonal outgrowth , respectively . therefore , cell replacement therapy using stem cells for sma is not totally impossible ; however , there is still much to be accomplished in cell therapy before being applied clinically to treat motor neuron diseases .

spinal muscular atrophy ( sma ) is an autosomal recessive neurodegenerative disorder , leading to progressive muscle weakness , atrophy , and sometimes premature death . sma is caused by mutation or deletion of the survival motor neuron-1 ( smn1 ) gene . an effective treatment does not presently exist . since the severity of the sma phenotype is inversely correlated with expression levels of smn , the smn - encoded protein , smn is the most important therapeutic target for development of an effective treatment for sma . in recent years , numerous smn independent targets and therapeutic strategies have been demonstrated to have potential roles in sma treatment . for example , some neurotrophic , antiapoptotic , and myotrophic factors are able to promote survival of motor neurons or improve muscle strength shown in sma mouse models or clinical trials . plastin-3 , cpg15 , and a rho - kinase inhibitor regulate axonal dynamics and might reduce the influences of smn depletion in disarrangement of neuromuscular junction . stem cell transplantation in sma model mice resulted in improvement of motor behaviors and extension of survival , likely from trophic support . although most therapies are still under investigation , these nonclassical treatments might provide an adjunctive method for future sma therapy .

1. Introduction 2. Disease Mechanisms 3. SMN Dependent Therapy 4. SMN Independent Targets and Treatment 5. Conclusions

spinal muscular atrophy ( sma ) is characterized by motor neuron degeneration with muscular atrophy , paralysis , and an attenuated lifespan . sma exhibits an autosomal recessive pattern of inheritance with an incidence of 1 in 6,00010,000 newborns and a carrier frequency of about 1 : 35 [ 2 , 3 ] . based on age of onset and achievement of motor milestones , sma has been subdivided into four clinical types : severe ( type i ; werdnig - hoffmann disease ) , intermediate ( type ii ) , mild ( type iii ; kugelberg - welander disease ) , and adult forms . most sma patients harbor deletions , mutations , or conversions of the telomeric copy of the survival motor neuron gene ( smn1 ) [ 5 , 6 ] . since there is an inverted correlation between the amount of smn protein and disease severity [ 7 , 8 ] , smn has been the most important therapeutic target for development of sma treatment [ 9 , 10 ] . however , some smn independent targets and therapeutic strategies have been demonstrated to have the potential to benefit sma [ 1120 ] . although most are still under investigation , these nonclassical therapies might provide an adjunctive method for future sma therapy . although pathogenesis of sma has been investigated extensively , some of the detailed disease mechanisms are still not fully understood . the snrnp assembly activity is dramatically reduced in spinal cord from sma model mice and the degree of snrnp assembly impairment correlates with disease severity . evidence shows that smn is also involved in the stabilization and maturation of the neuromuscular junction and the transportation of axonal mrnas in motor neurons [ 2527 ] . smn - deficient motor neurons exhibit severe defects in clustering voltage - gated calcium channels in axonal growth cones . an alteration of calcium channel distribution might influence neurotransmitter release , causing dysfunction and immaturation of neuromuscular junction [ 25 , 28 ] . the close relationship of smn and -actin has further demonstrated that motor neurons derived from sma model mice have shortened axons and small growth cones , which are also deficient in -actin mrna and protein . therefore , smn has a function in maintaining proper neuronal machinery via assistance in splicing process and establishing adequate communication between the muscles and nerves at the motor end plate through stabilization of the neuromuscular junction . sma mouse models have been generated through mouse smn knockout and human smn2 transgenic methods [ 8 , 31 ] . these mice reveal spinal motor neuron degeneration , muscle atrophy , and impaired motor performances similar to sma patients . the disease severity of these sma mice is also inversely correlated to the copy number of the smn2 transgenes [ 8 , 31 ] . subsequently , through still unknown mechanisms , motor neurons degenerate in spinal anterior horn regions probably through cell apoptosis [ 16 , 34 ] , and muscle atrophy and motor dysfunction become apparent . the histological studies in sma model mice also showed that cardiac remodeling starts at the embryonic stage in the severe sma mice while motor neurons are not yet visibly affected at this stage . notably , systemic restoration of smn expression is able to diminish the cardiac defects accompanied with prolonged lifespan , implying that cardiac abnormalities are playing a critical role on sma pathogenesis [ 38 , 39 ] . since smn levels generally correlate with disease severity in sma patients and mouse models [ 7 , 8 , 31 , 40 ] , smn is the best therapeutic target for development of sma treatment . various strategies to increase the smn levels have been tested in sma mouse models and some of them have even showed promising beneficial effects [ 9 , 10 ] . until now , none of them have been demonstrated to be consistently robust or produce continual benefits in sma patients . these therapeutic strategies are divided into small molecules , antisense oligonucleotides ( aso ) , and viral vector - mediated gene therapy . the mode - of - action for a potential sma therapy using small molecules mainly includes restoration of the smn2 splicing pattern , activating the smn2 promoter , and extending the half - life of smn mrna or protein . since there are still no drugs that have shown consistent benefits in clinical trials [ 55 , 56 ] , finding an effective treatment with distinct therapeutic mechanisms , such as smn independent targets , is necessary for future sma therapy . vpa treatment increased levels of smn transcripts and protein in fibroblasts derived from sma patients through upregulation of serine / arginine - rich ( sr ) proteins , which are involved in regulating smn2 exon 7 recruitment [ 58 , 59 ] . probably through multiple therapeutic effects , vpa reduced motor neuron degeneration , muscle atrophy , and motor dysfunction in sma mice [ 43 , 64 ] , and a small group of sma patients showed obvious improvement in muscle strength after daily vpa treatment [ 65 , 66 ] . despite these encouraging results , large clinical trials did not confirm the beneficial effects of vpa in sma patients [ 6769 ] . a recent study demonstrated that systemic delivery of aso resulted in dramatic prolongation of lifespan in sma mice and the effects were much better than those with intracerebroventricular delivery of aso ( median survival , 108 versus 16 days ) . these findings suggest that aso therapy has great potential in this field and extra - cns targeting is required to rescue the sma phenotype . direct injection of adeno - associated viral vector serotype 8 ( aav8 ) carrying smn into both cerebroventricles and upper lumbar spinal cord of sma mice showed a robust increase in lifespan by 880% with less motor neuron degeneration and abnormal architectures of neuromuscular junction . intravenous injection of aav9 carrying human codon - optimized smn1 at postnatal day 1 recovered most motor function , neuromuscular physiology , and lifespan in sma mice . notably , postnatal day 1 treatment resulted in the maximal transduction of the motor neurons , while postnatal day 10 treatment led to glia - predominant transduction . when the blood brain barrier is mature and patent , virions are probably not able to penetrate out of vessels smoothly to access motor neurons , but only encounter the endothelial wrappings of astrocyte end feet . notably , serum igf-1 level was decreased in sma mice , and systemic increase of smn expression using the aso strategy in sma mice was accompanied with restoration of serum igf-1 to normal levels . interestingly , mrna levels of igf - binding protein , acid labile subunit ( igfals ) , but not igf-1 , was reduced in sma mice . therefore , the low serum igf-1 level in sma mice is likely related to downregulation of igfals , and igf-1 may be one of the factors that contribute to the pathogenesis of sma . igf-1 treatment has been shown to improve disease phenotypes in rodent models of motor neuron diseases such as amyotrophic lateral sclerosis ( als ) and spinal and bulbar muscular atrophy ( sbma ) . for sma , transgenic expression of igf-1 in skeletal muscle of sma mice resulted in an increase in myofiber size and a modest improvement in median survival . therefore , the effects of igf-1 and igfals - related therapy using different treatment strategies in sma still require further investigation . in a mouse model of als , the depletion of synaptic vesicles precedes the loss of synapses ; cntf could prevent the depletion of synaptic vesicles and thus maintain function of neuromuscular junctions . in a severe type of sma mice , in contrast , the architecture and function of neuromuscular junctions in heterozygous smn ( + / ) mice are relatively preserved , despite some loss of spinal motor neurons . these results imply that cntf may be able to compensate loss of motor neurons by sprouting from remaining motor axon terminals so that neuromuscular endplates remain innervated ; cntf may thus guide the way for new therapies for sma . ct-1 is essential in normal motor neuron development and is also able to support long - term survival of motor neurons as demonstrated in culture cells and rats with axotomy . in addition , overexpression of ct-1 in pmn and als mice both significantly delayed disease onset , reduced degeneration of motor neurons and axons , and preserved the terminal innervation of skeletal muscles [ 97 , 98 ] . for sma mice , intramuscular injection of adenoviral vector expressing ct-1 , even at very low doses , prolonged survival , delayed motor defects , and diminished motor axonal degeneration and aberrant synaptic terminals . degeneration of spinal motor neurons in sma is mediated in part through apoptosis [ 16 , 34 ] . bcl - xl is an antiapoptotic member of the bcl-2 family and acts by inhibiting proapoptotic members of the bcl-2 family through heterodimerization . interestingly , bcl - xl overexpression in sma mice reduced motor neuron degeneration , preserved motor function , and prolonged lifespan without changes in smn expression levels . the pharmacokinetics of riluzole in sma patients has recently been investigated , and the long - term benefits of riluzole still warrant large clinical trials for sma patients . for sma , there was no difference between the gabapentin and placebo groups in any outcome measure including changes in muscle strength , pulmonary function , or motor functional rating scale after 12 months of treatment . 2-adrenergic agonist , such as salbutamol ( albuterol in the united states ) , enhanced muscle strength in aged rats , human healthy volunteers , and some pathological conditions [ 117 , 118 ] . interestingly , salbutamol also promoted exon 7 inclusion in smn2 transcripts and thus increased levels of full - length transcripts of smn2 in sma fibroblasts . in sma patients , daily salbutamol significantly and consistently increased smn2 full - length transcript levels in peripheral leukocytes , and the response was directly proportional to smn2 gene copy number . intraperitoneal injection of recombinant follistatin in sma model mice increased muscle mass , improved motor function , and prolonged lifespan by 30% without changes in smn protein levels in spinal cord and muscles . the reason for this discrepancy is unclear and the effects of follistatin for sma treatment still need further validation . the first potential smn - independent disease modifier , plastin-3 , was recently identified from six sma - discordant families with eight fully asymptomatic females who had inherited the same smn1 and smn2 alleles as their affected siblings . increased levels of plastin-3 were also found to correlate with a mild sma phenotype in female patients , independently of smn protein levels [ 18 , 134 ] . plastin-3 , an actin binding protein , is a regulator of actin filament organization and is expressed in almost all solid tissues , including the human brain , spinal cord , and muscles . plastin-3 colocalizes with smn in granules throughout motor neuron axons , and plastin-3 protein levels are reduced in brain and spinal cord of an sma mouse model [ 18 , 135 ] . in smn - depleted neuronal pc12 cells and primary mouse motor neuron cultures derived from sma mice , notably , overexpression of plastin-3 or its orthologues also led to diminishment of axon defects and disease severity in smn depleted zebrafish embryos , drosophila , and c. elegans [ 18 , 136 ] . profilin iia is another actin binding protein , and knockdown of profilin iia results in stimulation of neurite outgrowth , while overexpression of profilin iia reduces neurite number and size . knockout of profilin iia in sma model mice was able to restore abnormal low plastin-3 levels . however , the phenotype of these sma mice was not ameliorated despite the depletion of profilin iia and restoration of plastin-3 levels , which suggests that other components of actin dynamics are also critically affected in sma . although some questions need to be answered , such as the mechanisms behind plastin-3 in sma and effects of plastin-3 upregulation in sma mouse models , plastin-3 may become an important smn - independent therapeutic target for sma in the future . cpg15 may be an mrna target for the smn - hud complex and smn deficiency reduced cpg15 mrna levels in neurons . therefore , cpg15 appears to be a crucial downstream effecter of smn in neurons and may serve as a modifier of sma disease by regulating axon extension and axon terminal differentiation . rho - kinase signaling is a major regulatory pathway of actin dynamics , and rho - kinase activation is associated with dendritic simplification , and reduced spine length and density . rho - kinase activity is upregulated in smn - depleted pc12 cells and sma model mice [ 145 , 146 ] . the migratory capacity of the u87 mg astroglioma cells was attenuated by knockdown of smn through abnormal activation of rho - kinase pathway . normally , smn binds to profilin iia to form complexes , and rho - kinase may phosphorylate profilin iia . through competition between smn and rho - kinase for binding to profilin iia , smn deficiency results in a decrease in smn - profilin iia complexes and stronger interaction of profilin iia with rho - kinase . therefore , rho - kinase inhibition might be able to correct the effect of smn reduction in sma to achieve an adequate ratio of de-/phosphorylated profilin iia . notably , treatment of sma model mice with rho - kinase inhibitor y-27632 or fasudil led to a significant prolongation in survival , improvement in integrity of neuromuscular junction , and increase in muscle fiber size without altered smn expression or increase in the number of spinal motor neurons [ 146 , 149 ] . since fasudil has been successfully applied in many clinical trials for other neurological and vascular diseases based on its neuroprotection , vasodilatation , and immune modulation effects , the results of fasudil therapeutic studies for sma patients are anticipated . a diagnosis of sma is usually made following a patient 's initial presentation of muscle weakness , at which there would be substantial spinal motor neuron loss . both smn dependent and independent treatments described above could only prevent disease progression , but not regain lost motor neurons , while stem cell therapy might provide a possibility for cell replacement . fetal - derived neural stem cells ( nscs ) are able to self - renew and are multipotent with the capacity of producing neurons ( including motor neurons ) , astrocytes , and oligodendrocytes . intrathecal injection of these primed nscs in nmd mice , another model of motor neuron disease , resulted in improvement of abnormal phenotypes and extension of survival . in a severe type of sma mouse model , intrathecal injection at postnatal day 1 with primed nscs derived from mouse embryonic spinal cord also promoted motor neuron survival , improved motor function , and prolonged lifespan . therefore , the observed benefits of nscs in sma model mice were likely related to trophic support . although fetal - derived nsc transplantation in sma mice showed promising effects , their derivation from a spinal cord source impedes further clinical implementation because of ethical and technical issues . on the other hand , embryonic stem cells might be easier to obtain and are also able to differentiate in vitro and in vivo into nscs and a motor neuron fate . intraspinal grafting of embryonic stem cell - derived motor neurons resulted in a significant improvement in motor behaviors in the als rat . for sma , embryonic stem cell - derived nscs transplanted intrathecally in sma model mice migrated to spinal anterior horn and improved motor function and lifespan . to increase the differentiation rate of embryonic stem cells into nscs before transplantation , the above sma study used drug - selectable embryonic stem cell lines that ganciclovir and g418 have been applied for selection against undifferentiated embryonic stem cells and for neuroepithelial cells , respectively . since the first report on reprogramming of mouse fibroblasts into so - called induced pluripotent stem ( ips ) cells by the expression of oct3/4 , sox2 , c - myc , and klf4 in 2006 , reprogramming of human somatic cells to a pluripotent state was achieved using similar approaches [ 160 , 161 ] . these ips cells provide a novel opportunity in disease modeling for investigating sma pathogenesis and can be used in screening novel compounds for sma treatment . the use of fetal - derived cells or embryonic stem cells for transplantation is hurdled by problems of availability , the possibility of immune rejection , and ethics . in contrast , the source of ips cells is unlimited , and ips cells can be transplanted autologously . notably , when ips cell - derived neural precursor cells from a patient with parkinson 's disease were transplanted into the striatum of a parkinson 's disease rat model , the donor cells differentiated into dopaminergic neurons , survived in the rodent brain for several months , and reduced the abnormal motor asymmetry . for autologous ips cell transplantation in sma , ips - derived neural precursor cells or motor neurons should be pretreated to express a high level of smn before transplantation . motor neurons may also require additional support to optimally respond to smn - based treatment . in the past two decades , there has been tremendous progress in sma regarding genetics , pathophysiology , and therapeutics . some useful strategies to enhance smn expression have been developed , and some novel smn - independent therapeutic targets have been discovered . while smn acts to modulate and correct the neuromuscular junction for functional improvement , smn - independent targets could play a role of extension in the survival of motor neurons and reduce the influence of smn depletion in axonal dynamics . the two currently available stem cell transplantation studies for sma have only demonstrated benefits likely with trophic support without evidence of functional cell replacement [ 19 , 20 ] . to generate functional motor units , the grafted stem cells should be able to differentiate into motor neurons , appropriately project the axons a long distance toward corresponding muscles , and form functional synapses within neuromuscular junctions . in a virus - induced rat model of motor neuron degeneration , mouse embryonic stem cell - derived motor neurons transplanted into spinal cord could survive , extend axons , form functional motor units , and promote recovery from paralysis [ 166 , 167 ] . the successful development of motor units in the above studies may result from a combination approach , which includes administration of dibutyryl - camp , rolipram , cyclosporine , and glial cell line - derived neurotrophic factors to promote motor neuron survival , circumvent myelin repulsion , prevent immune rejection , and enhance axonal outgrowth , respectively . therefore , cell replacement therapy using stem cells for sma is not totally impossible ; however , there is still much to be accomplished in cell therapy before being applied clinically to treat motor neuron diseases .

the past two decades have witnessed major strides in the treatment of several pediatric and adult cancers , particularly with the use of multiagent chemotherapy , radiation therapy , and recently , monoclonal antibodies . nevertheless , a subset of these patients will develop resistance to these modalities , leaving few treatment options with curative potential . in addition , patients with high risk metastatic disease continue to have dismal treatment outcomes , despite these advances . therefore , for patients with relapsed , therapy refractory disease and tumors at high risk for recurrence , new treatment strategies are desperately needed . over the past two decades the success in using adoptive cellular immunotherapy to fight viral infections following allogeneic stem cell transplantation has encouraged some groups to focus their efforts on the infusion of cancer antigen specific , or otherwise activated , t lymphocytes.1,2 there is a long history of clinical investigation with cancer vaccines for a variety of malignant solid tumors . the recognition that dendritic cells ( dc ) play a key role in antigen presentation led to several groups using dc pulsed with cancer relevant antigens , while other groups have used whole tumor antigens or human leukocyte antigen ( hla ) restricted epitopes.3,4 several different antigens have been targeted in these strategies , most notably the cancer testis ( ct ) antigens . these tumor proteins are of interest since they are expressed on several malignant solid tumors , as well as some leukemias , and have a restricted pattern of expression , thereby limiting the possibility of an immune response directed against normal host tissues . these antigens can also be epigenetically upregulated on tumors following exposure to demethylating chemotherapy agents , potentially making tumors more susceptible to killing by antigen - specific t cells that have been stimulated following a ct antigen vaccine . in this review we will summarize past studies which target these antigens and future directions in ct antigen - based immunotherapy . an improved understanding of cellular immunology has helped to facilitate the rational design of cancer immunotherapy strategies . while conventional therapy such as chemotherapy and radiation are useful for the majority of patients , the use of these modalities alone may be insufficient for patients with relapsed cancer or for those who initially present with advanced disease . chemotherapy often has limited efficacy in patients with relapsed disease , for whom intensification of conventional therapy to overcome drug resistance can lead to significant morbidity . immunotherapy can specifically target , or in general , modulate cellular immune responses against cancer proteins and has the potential to provide long - lasting responses . adoptive transfer of autologous in vitro generated and expanded effector t cells is one such effective method . initial studies in adoptive immunotherapy were performed in the allogeneic stem cell transplant setting to fight serious , potentially life - threatening viral infections , such as cytomegalovirus and epstein barr virus . while adoptive immunotherapy has been largely successful against several viral infections57 this approach has had limited success against cancer . the precursor frequency of cancer antigen - specific cells is very low , and the expansion of these cells requires multiple stimulations . in addition , the low avidity of expanded t cells against cancer antigens and the short life span of adoptively transferred effector t cells are practical limitations of adoptive immunotherapy . several strategies have been developed to overcome these challenges , such as the use of chimeric antigen receptors,8 t cells genetically engineered to express t cell receptors ( tcrs ) with high affinity and specificity,9,10 and bispecific antibodies to promote t cell recognition of tumors.11 several immune evasion mechanisms pose major obstacles for the practical application of immunotherapy against cancer . tumor cells can evade the immune system by ( a ) downregulating the expression of major histocompatibility complex ( mhc ) class i and class ii molecules that are required for antigen presentation to t cells ; ( b ) downregulating costimulatory molecules , such as cd80 and cd86 , which are required for optimal activation of t cells ; ( c ) upregulating coinhibitory molecules , such as cytotoxic t - lymphocyte antigen 4 ( ctla-4 ) ligands and programmed cell death ligand 1 ( pdl-1 ) , on tumor cells;10 and ( d ) recruiting regulatory t cells ( tregs ) that produce immunosuppressive cytokines at the tumor site . for example , high expression of ctla-4 has been correlated with increased t cell dysfunction in melanoma patients.12 ctla-4 and programmed cell death 1 ( pd-1 ) are expressed on activated t cells and contribute to t cell exhaustion . the upregulation and ligation of ctla-4/pd-1 ( on t cells ) with ctla-4 ligands and pdl-1 ( on tumor cells ) dampens effector t cell activation and negatively attenuates adaptive immune responses.13 researchers have developed strategies to overcome the immunosuppressive tumor microenvironment by blocking the inhibitory pathways.14 therefore antibodies blocking ctla-4 or pd-1 on t cells can prevent the inhibitory signals typically transmitted through these receptors and prevent effector cells from entering into the exhaustion phase , thereby extending the life and function of activated t cells . it seems logical to combine genetically targeted therapies / adoptive immunotherapy with negative regulatory blockade to minimize the chances of tumor resistance and escape . accordingly , treg depletion followed by pd-1/pdl-1 blockade has shown some efficacy in the treatment of acute myeloid leukemia ( aml).15 in a phase i clinical trial of antibody - mediated pd-1 blockade , an objective response ( complete response [ cr ] or partial response [ pr ] ) was observed in those with non - small - cell lung cancer ( [ nsclc ] 18% ) , melanoma ( 28% ) , and renal cell cancer ( [ rcc ] 27%).16 a similar phase i trial using antibody - mediated blockade of pdl-1 induced durable tumor regression and prolonged stabilization of disease in patients with advanced cancers.17 a study has evaluated the contributions of ctla-4 blockade on effector t cells and treg populations in a mouse model of melanoma.18 it revealed that ctla-4 blockade on effector cells significantly improves tumor protection while blockade of tregs completely fails to enhance antitumor responses , and a concomitant blockade of both effector and tregs leads to maximal antitumor activity . ctla-4 blockade with ipilimumab ( an anti - ctla-4 antibody ) has resulted in some clinical responses in patients with melanoma , ovarian cancer , prostate cancer , and rcc.19 a phase iii trial showed that ipilimumab , when given with or without a glycoprotein ( gp)100 peptide vaccine , improved the overall survival to 10 months when compared to 6.4 months with gp100 alone in patients with metastatic melanoma.20 several phase ii studies suggest that ipilimumab is effective in patients with melanoma and brain metastases.21,22 in a phase ii trial of ipilimumab plus fotemustine in 86 patients with advanced melanoma , of whom 20 patients had asymptomatic brain metastases at baseline . 40 of 86 ( 46.5% ) patients in the study population achieved disease control similar to 10 of 20 patients ( 50% ) with brain metastases.23 furthermore , ipilimumab when combined with decarbazine improved the overall survival to 47% when compared to decarbazine alone ( 36%).24 these results suggest that blocking the immune checkpoints can improve overall survival in cancer patients . the success of a cancer vaccine is dependent on the ability of a patient to mount a primary or memory immune response against cancer antigens used in the vaccine . thus far , the majority of cancer vaccine studies have focused on patients with relapsed or therapy refractory disease , but there is a growing interest on the potential to use this approach to prevent relapse in patients who are at high risk for recurrence . three main types of cancer vaccines that have been used in previous studies , including cellular vaccines , largely consist of dcs pulsed with cancer relevant antigens or tumor cell lysates , protein- or peptide - based vaccines , and vector - based vaccines where plasmid dna and viral / bacterial / yeast vectors are used to deliver tumor - specific antigens.25 potential problems with using whole cell lysates , peptides , or plasmid dna approaches include the immunogenicity of the vaccine , the majority of cancer reactive t cells exist in low numbers and are difficult to expand , and that most tumors have developed multiple means to evade the immune system . adjuvants can be used to enhance vaccine immunogenicity and thereby increase the likelihood of eliciting a t cell response . granulocyte - macrophage colony - stimulating factor ( gm - csf ) has been used as an adjuvant in several types of tumors including melanoma , colorectal carcinoma , rcc , and lymphoma . for example , an idiotypic protein vaccine together with gm - csf resulted in complete molecular remission ( by polymerase chain reaction [ pcr ] ) in 8 of 11 lymphoma patients and tumor - specific cytotoxic cd4 + and cd8 + cells were found in 95% of the patients.26 dcs play a central role in initiating antitumor responses by activating innate and adaptive immune cells . different dc subsets express distinct toll - like receptors ( tlrs ) , such as tlrs 1 to 8 , and upon stimulation , upregulate costimulatory molecules , pro - inflammatory cytokines , and chemokines which can assist in priming tumor - specific t cells . therefore , different types of tlr agonists have been used as adjuvants along with dc - based vaccines in treating glioblastoma , breast cancer , melanoma , rcc , and leukemia.27 a list of clinical trials using dc as therapeutic vaccines has been detailed in a comprehensive review of cancer immunotherapy with these antigen presenting cells.28 an ideal tumor antigen for immunotherapy should be ( a ) expressed specifically on tumor cells and not on healthy cells , ( b ) stably and homogenously expressed on all / majority of tumor cells , ( c ) vital for the existence of cancer cells , and ( d ) targeted by tumor antigen - specific cytotoxic t lymphocytes.29 identification of such tumor antigens would enhance the success of cancer vaccines . ct antigens are tumor proteins with a restricted pattern of expression , generally limited to germ cell and trophoblast tissue , but are also expressed in various human cancers . their stable and specific expression on tumor cells and lack of expression on normal tissues make them an attractive target for cancer immunotherapy . based on the frequency of ct antigen expression , chen et al30 and caballero and chen31 classified certain types of cancers including melanoma , ovarian cancer , lung cancer , and bladder cancer as ct - rich tumors ; rcc , colorectal cancer , and lymphoma / leukemia as ct - poor tumors ; and breast cancer , bladder cancer , and prostate cancer as ct - intermediate tumors . ct antigens are divided into two groups : ct - x ( encoded on x chromosome ) and non - x ct antigens . an excellent review by simpson et al summarizes the characteristics and functions of these two types of ct antigens.29 until 2004 , there were around 40 ct antigens identified,33 but by 2012 , the number of ct antigens identified had increased to 110.32 our review will focus mainly on melanoma antigen family ( mage)-a1 , mage - a3 , and new york esophageal squamous cell carcinoma ( ny - eso-1 ) , three of the initially identified and most widely studied ct antigens in melanoma . mage - a1 and mage - a3 are members of the mage gene family that are expressed on male germ line cells and placenta , as well as in melanoma , bladder cancer , breast cancer , prostate cancer , and nsclc.33 ny - eso-1 is another ct antigen found on several tumors , including in ovarian cancer , lung cancer , melanoma , as well as some sarcomas and neuroblastomas.34 expression rates of mage - a1 and mage - a3 were 53.7% and 36.6% , respectively , in ovarian cancer.35 several mage - a1 peptides restricted to individual hla alleles have been reported in healthy donors.3638 the frequency of expression of mage - a1 and ny - eso-1 in bladder cancer versus liver cancer was 22% and 80% versus 80% and 29% , respectively.33 in pharyngeal tumors , mage - a and ny - eso-1 were detectable in 70% and 33.3% of tumors , respectively.39 in nsclc patients , the expression of ny - eso-1 was only 8.3%,40 while its expression in synovial sarcoma was 80%,41 and its expression was 100% in myxoid / round cell liposarcoma patients.42 screening neuroblastoma cell lines for these antigens by reverse transcriptase - pcr ( rt - pcr ) has revealed that 44% are positive for mage - a1 , 21% for mage - a3 , and 30%82% for ny - eso-1 , and immunohistochemical analysis has shown a good correlation between gene and protein expression.43 in addition , in neuroblastoma , a higher level of ny - eso-1 expression has been reported in patients with later stage disease.44 the frequency of mage - a1 expression increased from 20% ( in primary tumors ) to 51% with advanced disease ( in distant metastases ) , while ny - eso-1 expression remained at 45% , regardless of stage of disease in melanoma patients.45 in malignant gammopathies , the expression pattern of mage - a1 , mage - a3 , and ny - eso-1 was heterogeneous , and the expression of these antigens was greater in patients with stage iii extramedullary plasmacytoma or high risk myeloma relative to low risk disease groups.46 this indicates that levels of expression of ct antigens vary depending upon the type of cancer and the stage of a patient s disease , with many tumors having increased expression of ct antigens upon progression / relapse.4547 the expression of ct antigens on tumors has been correlated with the presence of ct antigen - specific b and t cell responses . studies in adult patients have demonstrated that mage - a1 and mage - a3 specific t cells are present and can be augmented with a vaccine , or by stimulation of these t cells in culture.4851 there is also a correlation between the detection of mage - a3 specific cd8 t cells and regression of tumors in melanoma patients.52 mage - specific cd8 t cell responses have been reported in aml patients.53 in adult t cell leukemia / lymphoma cells , ny - eso-1 and mage - a3 were expressed in 61.4% and 31.6% of cells , respectively . this study detected ny - eso-1 specific antibodies in 11.6% , and ny - eso-1 specific cd8 t cell responses in 55.6% , of adult t cell leukemia / lymphoma patients.54 another study demonstrated cd8 t cell responses in 10 of 11 patients with ny - eso-1 positive melanoma who had ny - eso-1 antibodies , but not in patients with ny - eso-1 negative tumors or those lacking antibodies.55,56 there has also been a report on the detection of interferon- ( ifn- ) producing ny - eso-1 specific t cells in neuroblastoma patients.45 these studies indicate that mage - a1 , mage - a3 , and ny - eso-1 are immunogenic and capable of eliciting t and b cell responses . clinical trials have been reported using dc - based vaccines , whole protein vaccines , or hla restricted epitopes for mage - a1 and mage - a3 positive malignancies . chianese - bullock et al gave vaccines consisting of mage - a1 , mage - a10 , and gp100 peptides with gm - csf and incomplete freund s adjuvant to patients with stage iib to iv melanoma.49 there were increases in mage - a1 specific ifn- production postvaccination , and cytotoxic t lymphocyte ( ctl ) from these patients lysed tumor cells expressing mage - a1 . mackensen et al reported on the results of a mage - a1 and mage - a3 peptide loaded dc vaccine in 14 melanoma patients.57 clinical and immunologic responses were seen in two patients , and increased melanoma peptide specific immune responses were seen in four patients.57 thurner et al reported the use of mage - a3 peptide pulsed mature dc at doses of 3 10 dc per vaccine , given at 14 day intervals.51 significant expansion of mage - a3 specific cd8 cytotoxic t cells was induced in 8 of 11 patients , with regression of individual metastases in 6 of 11 patients . the ongoing clinical trials with ct antigens , mage - a1 , mage - a3 , and ny - eso-1 are presented in table 1 . the majority of clinical trials with ny - eso-1 tumor vaccines have used either individual hla restricted epitopes or whole protein , with or without adjuvants . most of these studies have demonstrated enhancement of t and b cell responses to this antigen postvaccination . some of the initial clinical trials with ny - eso-1 peptide vaccines used hla - a2 restricted peptides , and demonstrated that cd8 t cell responses can be expanded postvaccination.56,58,59 bender et al used an hla - a2 restricted ny - eso-1 peptide for vaccination , and reported that three of nine seronegative patients developed cd8 t cell responses.60 one study used full length ny - eso-1 protein with the iscomatrix adjuvant in 46 patients with fully resected , ny - eso-1 positive tumors.61 these investigators found high titer antibody responses , as well as cd4 and cd8 t cell responses , against a wide range of ny - eso-1 epitopes postvaccination . there was improved survival , with only two of 19 relapses in the group receiving adjuvant and protein , in comparison with nine of 16 relapses in the group receiving protein alone . upon further evaluation , persisting anti - ny - eso-1 immunity was detected in ten of 14 recipients who had previously received vaccine with iscomatrix adjuvant , while immunity only persisted in three of 14 recipients who received vaccine alone.62 a major focus of research during the past two decades has been to identify methods to overcome the mechanisms used by tumors to evade the immune system . different approaches including conventional therapy , molecular - targeted therapy , and immunotherapy have been combined in an attempt to improve clinical outcomes . this includes using chemotherapy and blockade of immune checkpoints,20,63,64 cancer vaccines and radiation therapy,65 cancer vaccines and chemotherapy,66,67 cancer vaccines and molecular - targeted agents,68 and molecular - targeted agents and blockade of immune checkpoints.69 current available combinations of immunotherapy and molecular - targeted therapy for cancer treatment are summarized in a review by vanneman and dranoff.70 depletion of tregs in combination with a cancer vaccine is another approach . tregs can be depleted by using anti - cd25 monoclonal antibodies71,72 and studies show that chemotherapy agents such as cyclophosphamide can deplete / suppress tregs.73,74 among the different approaches available , we will focus our discussion on combining immunotherapy ( using ct antigens ) and chemotherapy , especially on the use of decitabine ( [ dac ] 5-aza-2-deoxycytidine ) , a demethylating chemotherapeutic agent that epigenetically upregulates the expression of ct antigens , and review how ct antigens have been targeted in clinical trials . the success of immunotherapy is largely dependent on the recognition of cancer cells expressing ct antigens by antigen - specific t cells , and this is dependent on antigen expression in the context of mhc class i and class ii molecules . in cancer cells , hypermethylation of promoters leads to the downregulation of expression of ct antigens75 and mhc molecules,76 which are required for antigen presentation and recognition by antigen - specific cytotoxic t cells . since not all tumors express ct antigens , one way to upregulate the expression of ct antigens and mhc molecules , and enhance tumor cell killing by antigen - specific cytotoxic t lymphocytes , would be to reverse hypermethylation by using demethylating agents . dac is a potent inhibitor of dna methylation , and the doses associated with the demethylating action of dac are much lower than those required for cytotoxicity.7780 several groups have demonstrated that demethylating agents , such as dac , upregulate the expression of mage - a1 , mage - a3 , and ny - eso-1 in a number of tumor cell lines,8184 potentially making these tumors more susceptible to mage - a1 , mage - a3 , and ny - eso-1 mediated killing . there have been several in vitro studies showing the effects of demethylating chemotherapy on the expression of ct antigens . one study demonstrated that the use of dac could result in the restoration of mhc class i and mage antigens on melanoma cells.85 another group demonstrated that the treatment of ovarian cancer cell lines with dac resulted in the upregulation of mage - a1 and mage - a3 expression , as well as mhc class i molecules.81 sigalotti et al treated 33 patients with aml or myelodysplastic syndrome ( mds ) with dac , and measured the expression of several ct antigens by rt - pcr.86 in 31 of 33 patients who had no ct antigen expression prior to treatment , de novo expression of mage - a1 and ny - eso-1 was observed in all but one patient 15 days after treatment . weber et al demonstrated that mage - a1 expression was upregulated on several malignant melanoma cell lines following exposure to dac,83 and other studies have demonstrated that dac can increase the expression of ny - eso-1 on malignant glioma cell lines.87,88 our group recently demonstrated that the majority of neuroblastoma cell lines had increased expression of mage - a1 , mage - a3 , and ny - eso-1 , on both a molecular and protein level , after 5 days exposure to dac , and that this effect was associated with enhanced tumor cell killing by ct antigen specific ctl.89 upregulation of ct antigens and enhanced killing of tumor cells following treatment with dac by ct antigen specific t cells suggests that immunotherapy using ct antigens in combination with dac can be a potential strategy to treat relapsed patients . our ongoing phase i clinical trial combining dac and a dc vaccine targeting mage - a1 , mage - a3 , and ny - eso-1 for patients with relapsed neuroblastoma demonstrated a complete response in our first patient . the clinical outcome was correlated with a robust increase in the number of mage - a3 specific cd8 and cd4 t cells , and the patient remains disease free 1 year following his vaccination.90 this study indicates that a combination of demethylation - based chemotherapy followed by vaccine formulations containing ct antigens can elicit antigen - specific immune responses , potentially leading to an intensified antitumor effect . clinical trials are currently underway using genetically engineered ny - eso-1 specific t cells for patients with synovial sarcoma , tcrs specific for magea3/a6/b18 or ny - eso-1/l antigen family member ( lage ) for patients with ovarian cancer , and tcrs specific for mage - a3 and ny - eso-1 for patients with melanoma . adoptive transfer of autologous t cells transduced with tcr directed against ny - eso-1 has shown an objective clinical response in 4 of 6 patients with synovial cell sarcoma and in 5 of 11 patients with melanoma.91 this study demonstrated a partial response lasting 18 months in 1 of 6 patients with synovial cell sarcoma and a complete regression , that lasted over 12 months , in 2 of 11 patients with melanoma . ct antigens are ideal targets for immunotherapy and success of ct antigen based immunotherapy is largely dependent on the recognition of cancer cells expressing ct antigens by antigen - specific t cells . combination therapy that includes a combination of different immunotherapeutic modalities , or combination of immunotherapy with dac and/or other chemotherapy / irradiation , or both could overcome the obstacles related to effective antitumor immunity . such a combination therapy should primarily target upregulation of ct antigen expression and pro - apoptotic molecules on tumor cells , enhance the expression of mhc class i and class ii molecules and costimulatory molecules on antigen presenting cells , and downregulate the expression of coinhibitory molecules on the surface of t cells . a combination therapy using agents to target all three types of cells could result in an antitumor immune response , and further studies addressing issues of cell dosage , timing , and necessary sequence of agents used could improve clinical outcomes .

the identification of cancer testis ( ct ) antigens has been an important advance in determining potential targets for cancer immunotherapy . multiple previous studies have shown that ct antigen vaccines , using both peptides and dendritic cell vaccines , can elicit clinical and immunologic responses in several different tumors . this review details the expression of melanoma antigen family a , 1 ( mage - a1 ) , melanoma antigen family a , 3 ( mage - a3 ) , and new york esophageal squamous cell carcinoma-1 ( ny - eso-1 ) in various malignancies , and presents our current understanding of ct antigen based immunotherapy .

Introduction Cancer immunotherapy Cancer vaccines and immunotherapy Cancer testis antigens Combination therapy Conclusion

the recognition that dendritic cells ( dc ) play a key role in antigen presentation led to several groups using dc pulsed with cancer relevant antigens , while other groups have used whole tumor antigens or human leukocyte antigen ( hla ) restricted epitopes.3,4 several different antigens have been targeted in these strategies , most notably the cancer testis ( ct ) antigens . in this review we will summarize past studies which target these antigens and future directions in ct antigen - based immunotherapy . an improved understanding of cellular immunology has helped to facilitate the rational design of cancer immunotherapy strategies . the precursor frequency of cancer antigen - specific cells is very low , and the expansion of these cells requires multiple stimulations . tumor cells can evade the immune system by ( a ) downregulating the expression of major histocompatibility complex ( mhc ) class i and class ii molecules that are required for antigen presentation to t cells ; ( b ) downregulating costimulatory molecules , such as cd80 and cd86 , which are required for optimal activation of t cells ; ( c ) upregulating coinhibitory molecules , such as cytotoxic t - lymphocyte antigen 4 ( ctla-4 ) ligands and programmed cell death ligand 1 ( pdl-1 ) , on tumor cells;10 and ( d ) recruiting regulatory t cells ( tregs ) that produce immunosuppressive cytokines at the tumor site . for example , high expression of ctla-4 has been correlated with increased t cell dysfunction in melanoma patients.12 ctla-4 and programmed cell death 1 ( pd-1 ) are expressed on activated t cells and contribute to t cell exhaustion . accordingly , treg depletion followed by pd-1/pdl-1 blockade has shown some efficacy in the treatment of acute myeloid leukemia ( aml).15 in a phase i clinical trial of antibody - mediated pd-1 blockade , an objective response ( complete response [ cr ] or partial response [ pr ] ) was observed in those with non - small - cell lung cancer ( [ nsclc ] 18% ) , melanoma ( 28% ) , and renal cell cancer ( [ rcc ] 27%).16 a similar phase i trial using antibody - mediated blockade of pdl-1 induced durable tumor regression and prolonged stabilization of disease in patients with advanced cancers.17 a study has evaluated the contributions of ctla-4 blockade on effector t cells and treg populations in a mouse model of melanoma.18 it revealed that ctla-4 blockade on effector cells significantly improves tumor protection while blockade of tregs completely fails to enhance antitumor responses , and a concomitant blockade of both effector and tregs leads to maximal antitumor activity . ctla-4 blockade with ipilimumab ( an anti - ctla-4 antibody ) has resulted in some clinical responses in patients with melanoma , ovarian cancer , prostate cancer , and rcc.19 a phase iii trial showed that ipilimumab , when given with or without a glycoprotein ( gp)100 peptide vaccine , improved the overall survival to 10 months when compared to 6.4 months with gp100 alone in patients with metastatic melanoma.20 several phase ii studies suggest that ipilimumab is effective in patients with melanoma and brain metastases.21,22 in a phase ii trial of ipilimumab plus fotemustine in 86 patients with advanced melanoma , of whom 20 patients had asymptomatic brain metastases at baseline . thus far , the majority of cancer vaccine studies have focused on patients with relapsed or therapy refractory disease , but there is a growing interest on the potential to use this approach to prevent relapse in patients who are at high risk for recurrence . three main types of cancer vaccines that have been used in previous studies , including cellular vaccines , largely consist of dcs pulsed with cancer relevant antigens or tumor cell lysates , protein- or peptide - based vaccines , and vector - based vaccines where plasmid dna and viral / bacterial / yeast vectors are used to deliver tumor - specific antigens.25 potential problems with using whole cell lysates , peptides , or plasmid dna approaches include the immunogenicity of the vaccine , the majority of cancer reactive t cells exist in low numbers and are difficult to expand , and that most tumors have developed multiple means to evade the immune system . granulocyte - macrophage colony - stimulating factor ( gm - csf ) has been used as an adjuvant in several types of tumors including melanoma , colorectal carcinoma , rcc , and lymphoma . different dc subsets express distinct toll - like receptors ( tlrs ) , such as tlrs 1 to 8 , and upon stimulation , upregulate costimulatory molecules , pro - inflammatory cytokines , and chemokines which can assist in priming tumor - specific t cells . therefore , different types of tlr agonists have been used as adjuvants along with dc - based vaccines in treating glioblastoma , breast cancer , melanoma , rcc , and leukemia.27 a list of clinical trials using dc as therapeutic vaccines has been detailed in a comprehensive review of cancer immunotherapy with these antigen presenting cells.28 an ideal tumor antigen for immunotherapy should be ( a ) expressed specifically on tumor cells and not on healthy cells , ( b ) stably and homogenously expressed on all / majority of tumor cells , ( c ) vital for the existence of cancer cells , and ( d ) targeted by tumor antigen - specific cytotoxic t lymphocytes.29 identification of such tumor antigens would enhance the success of cancer vaccines . their stable and specific expression on tumor cells and lack of expression on normal tissues make them an attractive target for cancer immunotherapy . based on the frequency of ct antigen expression , chen et al30 and caballero and chen31 classified certain types of cancers including melanoma , ovarian cancer , lung cancer , and bladder cancer as ct - rich tumors ; rcc , colorectal cancer , and lymphoma / leukemia as ct - poor tumors ; and breast cancer , bladder cancer , and prostate cancer as ct - intermediate tumors . an excellent review by simpson et al summarizes the characteristics and functions of these two types of ct antigens.29 until 2004 , there were around 40 ct antigens identified,33 but by 2012 , the number of ct antigens identified had increased to 110.32 our review will focus mainly on melanoma antigen family ( mage)-a1 , mage - a3 , and new york esophageal squamous cell carcinoma ( ny - eso-1 ) , three of the initially identified and most widely studied ct antigens in melanoma . mage - a1 and mage - a3 are members of the mage gene family that are expressed on male germ line cells and placenta , as well as in melanoma , bladder cancer , breast cancer , prostate cancer , and nsclc.33 ny - eso-1 is another ct antigen found on several tumors , including in ovarian cancer , lung cancer , melanoma , as well as some sarcomas and neuroblastomas.34 expression rates of mage - a1 and mage - a3 were 53.7% and 36.6% , respectively , in ovarian cancer.35 several mage - a1 peptides restricted to individual hla alleles have been reported in healthy donors.3638 the frequency of expression of mage - a1 and ny - eso-1 in bladder cancer versus liver cancer was 22% and 80% versus 80% and 29% , respectively.33 in pharyngeal tumors , mage - a and ny - eso-1 were detectable in 70% and 33.3% of tumors , respectively.39 in nsclc patients , the expression of ny - eso-1 was only 8.3%,40 while its expression in synovial sarcoma was 80%,41 and its expression was 100% in myxoid / round cell liposarcoma patients.42 screening neuroblastoma cell lines for these antigens by reverse transcriptase - pcr ( rt - pcr ) has revealed that 44% are positive for mage - a1 , 21% for mage - a3 , and 30%82% for ny - eso-1 , and immunohistochemical analysis has shown a good correlation between gene and protein expression.43 in addition , in neuroblastoma , a higher level of ny - eso-1 expression has been reported in patients with later stage disease.44 the frequency of mage - a1 expression increased from 20% ( in primary tumors ) to 51% with advanced disease ( in distant metastases ) , while ny - eso-1 expression remained at 45% , regardless of stage of disease in melanoma patients.45 in malignant gammopathies , the expression pattern of mage - a1 , mage - a3 , and ny - eso-1 was heterogeneous , and the expression of these antigens was greater in patients with stage iii extramedullary plasmacytoma or high risk myeloma relative to low risk disease groups.46 this indicates that levels of expression of ct antigens vary depending upon the type of cancer and the stage of a patient s disease , with many tumors having increased expression of ct antigens upon progression / relapse.4547 the expression of ct antigens on tumors has been correlated with the presence of ct antigen - specific b and t cell responses . studies in adult patients have demonstrated that mage - a1 and mage - a3 specific t cells are present and can be augmented with a vaccine , or by stimulation of these t cells in culture.4851 there is also a correlation between the detection of mage - a3 specific cd8 t cells and regression of tumors in melanoma patients.52 mage - specific cd8 t cell responses have been reported in aml patients.53 in adult t cell leukemia / lymphoma cells , ny - eso-1 and mage - a3 were expressed in 61.4% and 31.6% of cells , respectively . this study detected ny - eso-1 specific antibodies in 11.6% , and ny - eso-1 specific cd8 t cell responses in 55.6% , of adult t cell leukemia / lymphoma patients.54 another study demonstrated cd8 t cell responses in 10 of 11 patients with ny - eso-1 positive melanoma who had ny - eso-1 antibodies , but not in patients with ny - eso-1 negative tumors or those lacking antibodies.55,56 there has also been a report on the detection of interferon- ( ifn- ) producing ny - eso-1 specific t cells in neuroblastoma patients.45 these studies indicate that mage - a1 , mage - a3 , and ny - eso-1 are immunogenic and capable of eliciting t and b cell responses . clinical trials have been reported using dc - based vaccines , whole protein vaccines , or hla restricted epitopes for mage - a1 and mage - a3 positive malignancies . chianese - bullock et al gave vaccines consisting of mage - a1 , mage - a10 , and gp100 peptides with gm - csf and incomplete freund s adjuvant to patients with stage iib to iv melanoma.49 there were increases in mage - a1 specific ifn- production postvaccination , and cytotoxic t lymphocyte ( ctl ) from these patients lysed tumor cells expressing mage - a1 . mackensen et al reported on the results of a mage - a1 and mage - a3 peptide loaded dc vaccine in 14 melanoma patients.57 clinical and immunologic responses were seen in two patients , and increased melanoma peptide specific immune responses were seen in four patients.57 thurner et al reported the use of mage - a3 peptide pulsed mature dc at doses of 3 10 dc per vaccine , given at 14 day intervals.51 significant expansion of mage - a3 specific cd8 cytotoxic t cells was induced in 8 of 11 patients , with regression of individual metastases in 6 of 11 patients . the ongoing clinical trials with ct antigens , mage - a1 , mage - a3 , and ny - eso-1 are presented in table 1 . the majority of clinical trials with ny - eso-1 tumor vaccines have used either individual hla restricted epitopes or whole protein , with or without adjuvants . some of the initial clinical trials with ny - eso-1 peptide vaccines used hla - a2 restricted peptides , and demonstrated that cd8 t cell responses can be expanded postvaccination.56,58,59 bender et al used an hla - a2 restricted ny - eso-1 peptide for vaccination , and reported that three of nine seronegative patients developed cd8 t cell responses.60 one study used full length ny - eso-1 protein with the iscomatrix adjuvant in 46 patients with fully resected , ny - eso-1 positive tumors.61 these investigators found high titer antibody responses , as well as cd4 and cd8 t cell responses , against a wide range of ny - eso-1 epitopes postvaccination . upon further evaluation , persisting anti - ny - eso-1 immunity was detected in ten of 14 recipients who had previously received vaccine with iscomatrix adjuvant , while immunity only persisted in three of 14 recipients who received vaccine alone.62 a major focus of research during the past two decades has been to identify methods to overcome the mechanisms used by tumors to evade the immune system . tregs can be depleted by using anti - cd25 monoclonal antibodies71,72 and studies show that chemotherapy agents such as cyclophosphamide can deplete / suppress tregs.73,74 among the different approaches available , we will focus our discussion on combining immunotherapy ( using ct antigens ) and chemotherapy , especially on the use of decitabine ( [ dac ] 5-aza-2-deoxycytidine ) , a demethylating chemotherapeutic agent that epigenetically upregulates the expression of ct antigens , and review how ct antigens have been targeted in clinical trials . the success of immunotherapy is largely dependent on the recognition of cancer cells expressing ct antigens by antigen - specific t cells , and this is dependent on antigen expression in the context of mhc class i and class ii molecules . in cancer cells , hypermethylation of promoters leads to the downregulation of expression of ct antigens75 and mhc molecules,76 which are required for antigen presentation and recognition by antigen - specific cytotoxic t cells . since not all tumors express ct antigens , one way to upregulate the expression of ct antigens and mhc molecules , and enhance tumor cell killing by antigen - specific cytotoxic t lymphocytes , would be to reverse hypermethylation by using demethylating agents . dac is a potent inhibitor of dna methylation , and the doses associated with the demethylating action of dac are much lower than those required for cytotoxicity.7780 several groups have demonstrated that demethylating agents , such as dac , upregulate the expression of mage - a1 , mage - a3 , and ny - eso-1 in a number of tumor cell lines,8184 potentially making these tumors more susceptible to mage - a1 , mage - a3 , and ny - eso-1 mediated killing . there have been several in vitro studies showing the effects of demethylating chemotherapy on the expression of ct antigens . one study demonstrated that the use of dac could result in the restoration of mhc class i and mage antigens on melanoma cells.85 another group demonstrated that the treatment of ovarian cancer cell lines with dac resulted in the upregulation of mage - a1 and mage - a3 expression , as well as mhc class i molecules.81 sigalotti et al treated 33 patients with aml or myelodysplastic syndrome ( mds ) with dac , and measured the expression of several ct antigens by rt - pcr.86 in 31 of 33 patients who had no ct antigen expression prior to treatment , de novo expression of mage - a1 and ny - eso-1 was observed in all but one patient 15 days after treatment . weber et al demonstrated that mage - a1 expression was upregulated on several malignant melanoma cell lines following exposure to dac,83 and other studies have demonstrated that dac can increase the expression of ny - eso-1 on malignant glioma cell lines.87,88 our group recently demonstrated that the majority of neuroblastoma cell lines had increased expression of mage - a1 , mage - a3 , and ny - eso-1 , on both a molecular and protein level , after 5 days exposure to dac , and that this effect was associated with enhanced tumor cell killing by ct antigen specific ctl.89 upregulation of ct antigens and enhanced killing of tumor cells following treatment with dac by ct antigen specific t cells suggests that immunotherapy using ct antigens in combination with dac can be a potential strategy to treat relapsed patients . our ongoing phase i clinical trial combining dac and a dc vaccine targeting mage - a1 , mage - a3 , and ny - eso-1 for patients with relapsed neuroblastoma demonstrated a complete response in our first patient . the clinical outcome was correlated with a robust increase in the number of mage - a3 specific cd8 and cd4 t cells , and the patient remains disease free 1 year following his vaccination.90 this study indicates that a combination of demethylation - based chemotherapy followed by vaccine formulations containing ct antigens can elicit antigen - specific immune responses , potentially leading to an intensified antitumor effect . clinical trials are currently underway using genetically engineered ny - eso-1 specific t cells for patients with synovial sarcoma , tcrs specific for magea3/a6/b18 or ny - eso-1/l antigen family member ( lage ) for patients with ovarian cancer , and tcrs specific for mage - a3 and ny - eso-1 for patients with melanoma . adoptive transfer of autologous t cells transduced with tcr directed against ny - eso-1 has shown an objective clinical response in 4 of 6 patients with synovial cell sarcoma and in 5 of 11 patients with melanoma.91 this study demonstrated a partial response lasting 18 months in 1 of 6 patients with synovial cell sarcoma and a complete regression , that lasted over 12 months , in 2 of 11 patients with melanoma . ct antigens are ideal targets for immunotherapy and success of ct antigen based immunotherapy is largely dependent on the recognition of cancer cells expressing ct antigens by antigen - specific t cells . such a combination therapy should primarily target upregulation of ct antigen expression and pro - apoptotic molecules on tumor cells , enhance the expression of mhc class i and class ii molecules and costimulatory molecules on antigen presenting cells , and downregulate the expression of coinhibitory molecules on the surface of t cells .

advances in noninvasive technologies in cardiology are growing and improving both the diagnosis of heart disease and the guidance of therapy . there are currently many emerging technologies , which are being used in the alternative and complementary medicine practice , including computer meridian diagnostics and virtual scanning . suffice to note that the body is a dynamic system in which reaction kinetics are affected by the reaction conditions . in particular , visual defects are associated with heart condition[35 ] . associated with the defects are antioxidant activities[68 ] , which in turn are associated to adenosine triphosphate ( atp ) metabolism . changes to the levels of proteins and reactive substrates in the body cause the release of ultra weak light or chemiluminescence . examples of substrates and/or products of reactions that involve chemiluminescence include antioxidants and atp respectively . the concept of stress is related to the complexity of the interactions between the cells , organs and systems of the body . the constancy , homeostasis or steady - state condition means that any tendency towards change is either automatically resisted by a feedback or feedforward response . two of the adrenal hormones involved in the neuro - endocrine response to stress are catecholamines and glucocorticoids . the catecholamines decrease insulin production and increase glucagon 's release , which culminate in increased glucose level in the blood vis - - vis glucose metabolism . normal human physiological processes are strongly dependent on glucose metabolism ( i.e. the biochemical process ) for the generation of energy . glucose metabolism is a cell level ( glycolysis and hexose monophosphate shunt pathways ) activity that inherently generates free radicals ( oxidants ) . when a free radical reacts with a molecule , a new radical is always formed . the new radical readily reacts with another molecule to produce yet another radical , and this continues exponentially until the radicals react with a chain breaking molecule ( antioxidant ) , which will result in the formation of a stable product . typical examples of antioxidants are vitamins c and e. others are glutathione , coenzyme - q10 and carotenoid . there is a paradox that many antioxidants including vitamins c and e are potentially toxic . recalling the famous chemistry slogan atoms can neither be created nor destroyed , an antioxidant vitamin that has finished a much needed reaction is not destroyed . it is transformed into a reaction product or intermediate , which has pro - oxidant toxic potentials and therefore requires recycling back to its original state . in good health , there are co - antioxidants in a delicately balanced or closed circuit interaction ( fig . closed - circuit nature of co - antioxidants interaction network - vitamin e regeneration system . keys : aa = ascorbic acid , coq = oxidized coenzyme - q10 , coq - h2 = coenzyme - q10 , cvd = cardiovascular disease , dha = dehydro - ascorbic acid ( free radical ) , gr = glutathione reductase , gsh = reduced glutathione , gssg = oxidized glutathione , loo = lipid peroxyl radical , looh = lipid peroxide , os = oxidative stress , rc = mitochondrial respiratory chain , toh = tocopherol ( vitamin e ) , to = tocopheroxyl radical . a major part of neuroendocrine system involved in stress adaptation is the hypothalamus - pituitary - adrenalin axis ( hpaa ) . when there is stress , there is release of adrenal hormones and associated with this is increased glucose metabolism . if the stress condition is prolonged ( chronic ) , there will also be chronic increase in glucose metabolism ( similar to an undiagnosed diabetic state ) . this leads to more - than - normal - level production of free radicals and by default , an alteration in the body 's biochemical makeup . among the obvious alterations in biochemical makeup specifically , when oxidative stress involves the red blood cells , the biochemical alterations drive the physiology as in the flow diagram ( fig 2 ) . the figure illustrates how stress ( such as chronic anxiety ) can cause hypertension and increased heart rate en route cardiological diseases . it is known that antioxidant activities involve reactions with proteins with concomitant emission of ultra weak light and are increased in obstructive sleep apnoea states . it is also known that oxidants contribute to the exacerbation of sleep apnoea , particularly including the cardiovascular complications . at this juncture , it is pertinent to note at least the following half - a - dozen points : biochemical changes associated with disease or drug substances influence perception of color.changes to the levels of proteins and reactive substrates in the body cause the release of ultra - weak light albeit chemiluminescence.the emissions are measurable luminescence as index of oxidative stress.oxidative stress as a common event that can lead to emission of chemiluminescence has been indicated in studies on erythrocytes . further support is provided by studies indicating that emission can be attenuated or increased by addition of antioxidants or reactive oxygen species respectively[2426].atp is the ultimate of glucose metabolism . the associated increase in free radicals is involved in chemiluminescence when a high energy state intermediate changes to a lower energy state . above all , alteration in atp level is correlated to photon chemiluminescence.photon emission affects color or heat perception and intensity and this property is utilized in ct scan . therefore , abnormal level of emissions arising from biochemical processes during disease conditions could affect color vision . changes to the levels of proteins and reactive substrates in the body cause the release of ultra - weak light albeit chemiluminescence . oxidative stress as a common event that can lead to emission of chemiluminescence has been indicated in studies on erythrocytes . further support is provided by studies indicating that emission can be attenuated or increased by addition of antioxidants or reactive oxygen species respectively[2426 ] . the associated increase in free radicals is involved in chemiluminescence when a high energy state intermediate changes to a lower energy state . above all , alteration in atp level is correlated to photon chemiluminescence . photon emission affects color or heat perception and intensity and this property is utilized in ct scan . therefore , abnormal level of emissions arising from biochemical processes during disease conditions could affect color vision . what this article brings to fore is that there is a non - invasive cognitive test procedure , virtual scanning technology , which utilizes the chemiluminescence arising from biochemical processes to assess and manage stress - related cardiological conditions . the biochemical basis of the technology has not been previously explained , except as implied in our papers[2932 ] . this article furthers explanation that oxidative stress effect of luminescence on cognition and colour perception is possibly the biochemical basis of virtual scanning technology . the concept of stress is related to the complexity of the interactions between the cells , organs and systems of the body . the constancy , homeostasis or steady - state condition means that any tendency towards change is either automatically resisted by a feedback or feedforward response . two of the adrenal hormones involved in the neuro - endocrine response to stress are catecholamines and glucocorticoids . the catecholamines decrease insulin production and increase glucagon 's release , which culminate in increased glucose level in the blood vis - - vis glucose metabolism . normal human physiological processes are strongly dependent on glucose metabolism ( i.e. the biochemical process ) for the generation of energy . glucose metabolism is a cell level ( glycolysis and hexose monophosphate shunt pathways ) activity that inherently generates free radicals ( oxidants ) . when a free radical reacts with a molecule , a new radical is always formed . the new radical readily reacts with another molecule to produce yet another radical , and this continues exponentially until the radicals react with a chain breaking molecule ( antioxidant ) , which will result in the formation of a stable product . typical examples of antioxidants are vitamins c and e. others are glutathione , coenzyme - q10 and carotenoid . there is a paradox that many antioxidants including vitamins c and e are potentially toxic . recalling the famous chemistry slogan atoms can neither be created nor destroyed , an antioxidant vitamin that has finished a much needed reaction is not destroyed . it is transformed into a reaction product or intermediate , which has pro - oxidant toxic potentials and therefore requires recycling back to its original state . in good health , there are co - antioxidants in a delicately balanced or closed circuit interaction ( fig . closed - circuit nature of co - antioxidants interaction network - vitamin e regeneration system . keys : aa = ascorbic acid , coq = oxidized coenzyme - q10 , coq - h2 = coenzyme - q10 , cvd = cardiovascular disease , dha = dehydro - ascorbic acid ( free radical ) , gr = glutathione reductase , gsh = reduced glutathione , gssg = oxidized glutathione , loo = lipid peroxyl radical , looh = lipid peroxide , os = oxidative stress , rc = mitochondrial respiratory chain , toh = tocopherol ( vitamin e ) , to = tocopheroxyl radical . a major part of neuroendocrine system involved in stress adaptation is the hypothalamus - pituitary - adrenalin axis ( hpaa ) . when there is stress , there is release of adrenal hormones and associated with this is increased glucose metabolism . if the stress condition is prolonged ( chronic ) , there will also be chronic increase in glucose metabolism ( similar to an undiagnosed diabetic state ) . this leads to more - than - normal - level production of free radicals and by default , an alteration in the body 's biochemical makeup . among the obvious alterations in biochemical makeup specifically , when oxidative stress involves the red blood cells , the biochemical alterations drive the physiology as in the flow diagram ( fig 2 ) . the figure illustrates how stress ( such as chronic anxiety ) can cause hypertension and increased heart rate en route cardiological diseases . it is known that antioxidant activities involve reactions with proteins with concomitant emission of ultra weak light and are increased in obstructive sleep apnoea states . it is also known that oxidants contribute to the exacerbation of sleep apnoea , particularly including the cardiovascular complications . at this juncture , it is pertinent to note at least the following half - a - dozen points : biochemical changes associated with disease or drug substances influence perception of color.changes to the levels of proteins and reactive substrates in the body cause the release of ultra - weak light albeit chemiluminescence.the emissions are measurable luminescence as index of oxidative stress.oxidative stress as a common event that can lead to emission of chemiluminescence has been indicated in studies on erythrocytes . further support is provided by studies indicating that emission can be attenuated or increased by addition of antioxidants or reactive oxygen species respectively[2426].atp is the ultimate of glucose metabolism . the associated increase in free radicals is involved in chemiluminescence when a high energy state intermediate changes to a lower energy state . above all , alteration in atp level is correlated to photon chemiluminescence.photon emission affects color or heat perception and intensity and this property is utilized in ct scan . therefore , abnormal level of emissions arising from biochemical processes during disease conditions could affect color vision . changes to the levels of proteins and reactive substrates in the body cause the release of ultra - weak light albeit chemiluminescence . oxidative stress as a common event that can lead to emission of chemiluminescence has been indicated in studies on erythrocytes . further support is provided by studies indicating that emission can be attenuated or increased by addition of antioxidants or reactive oxygen species respectively[2426 ] . the associated increase in free radicals is involved in chemiluminescence when a high energy state intermediate changes to a lower energy state . above all , alteration in atp level is correlated to photon chemiluminescence . photon emission affects color or heat perception and intensity and this property is utilized in ct scan . therefore , abnormal level of emissions arising from biochemical processes during disease conditions could affect color vision . what this article brings to fore is that there is a non - invasive cognitive test procedure , virtual scanning technology , which utilizes the chemiluminescence arising from biochemical processes to assess and manage stress - related cardiological conditions . the biochemical basis of the technology has not been previously explained , except as implied in our papers[2932 ] . this article furthers explanation that oxidative stress effect of luminescence on cognition and colour perception is possibly the biochemical basis of virtual scanning technology . the concept of stress is related to the complexity of the interactions between the cells , organs and systems of the body . the constancy , homeostasis or steady - state condition means that any tendency towards change is either automatically resisted by a feedback or feedforward response . two of the adrenal hormones involved in the neuro - endocrine response to stress are catecholamines and glucocorticoids . the catecholamines decrease insulin production and increase glucagon 's release , which culminate in increased glucose level in the blood vis - - vis glucose metabolism . normal human physiological processes are strongly dependent on glucose metabolism ( i.e. the biochemical process ) for the generation of energy . glucose metabolism is a cell level ( glycolysis and hexose monophosphate shunt pathways ) activity that inherently generates free radicals ( oxidants ) . when a free radical reacts with a molecule , a new radical is always formed . the new radical readily reacts with another molecule to produce yet another radical , and this continues exponentially until the radicals react with a chain breaking molecule ( antioxidant ) , which will result in the formation of a stable product . typical examples of antioxidants are vitamins c and e. others are glutathione , coenzyme - q10 and carotenoid . there is a paradox that many antioxidants including vitamins c and e are potentially toxic . recalling the famous chemistry slogan atoms can neither be created nor destroyed , an antioxidant vitamin that has finished a much needed reaction is not destroyed . it is transformed into a reaction product or intermediate , which has pro - oxidant toxic potentials and therefore requires recycling back to its original state . in good health , there are co - antioxidants in a delicately balanced or closed circuit interaction ( fig . closed - circuit nature of co - antioxidants interaction network - vitamin e regeneration system . keys : aa = ascorbic acid , coq = oxidized coenzyme - q10 , coq - h2 = coenzyme - q10 , cvd = cardiovascular disease , dha = dehydro - ascorbic acid ( free radical ) , gr = glutathione reductase , gsh = reduced glutathione , gssg = oxidized glutathione , loo = lipid peroxyl radical , looh = lipid peroxide , os = oxidative stress , rc = mitochondrial respiratory chain , toh = tocopherol ( vitamin e ) , to = tocopheroxyl radical . a major part of neuroendocrine system involved in stress adaptation is the hypothalamus - pituitary - adrenalin axis ( hpaa ) . when there is stress , there is release of adrenal hormones and associated with this is increased glucose metabolism . if the stress condition is prolonged ( chronic ) , there will also be chronic increase in glucose metabolism ( similar to an undiagnosed diabetic state ) . this leads to more - than - normal - level production of free radicals and by default , an alteration in the body 's biochemical makeup . among the obvious alterations in biochemical makeup are increased atp generation and reduction in cellular antioxidant levels . specifically , when oxidative stress involves the red blood cells , the biochemical alterations drive the physiology as in the flow diagram ( fig 2 ) . the figure illustrates how stress ( such as chronic anxiety ) can cause hypertension and increased heart rate en route cardiological diseases . it is known that antioxidant activities involve reactions with proteins with concomitant emission of ultra weak light and are increased in obstructive sleep apnoea states . it is also known that oxidants contribute to the exacerbation of sleep apnoea , particularly including the cardiovascular complications . at this juncture , it is pertinent to note at least the following half - a - dozen points : biochemical changes associated with disease or drug substances influence perception of color.changes to the levels of proteins and reactive substrates in the body cause the release of ultra - weak light albeit chemiluminescence.the emissions are measurable luminescence as index of oxidative stress.oxidative stress as a common event that can lead to emission of chemiluminescence has been indicated in studies on erythrocytes . further support is provided by studies indicating that emission can be attenuated or increased by addition of antioxidants or reactive oxygen species respectively[2426].atp is the ultimate of glucose metabolism . the associated increase in free radicals is involved in chemiluminescence when a high energy state intermediate changes to a lower energy state . above all , alteration in atp level is correlated to photon chemiluminescence.photon emission affects color or heat perception and intensity and this property is utilized in ct scan . therefore , abnormal level of emissions arising from biochemical processes during disease conditions could affect color vision . changes to the levels of proteins and reactive substrates in the body cause the release of ultra - weak light albeit chemiluminescence . oxidative stress as a common event that can lead to emission of chemiluminescence has been indicated in studies on erythrocytes . further support is provided by studies indicating that emission can be attenuated or increased by addition of antioxidants or reactive oxygen species respectively[2426 ] . the associated increase in free radicals is involved in chemiluminescence when a high energy state intermediate changes to a lower energy state . above all , alteration in atp level is correlated to photon chemiluminescence . photon emission affects color or heat perception and intensity and this property is utilized in ct scan . therefore , abnormal level of emissions arising from biochemical processes during disease conditions could affect color vision . what this article brings to fore is that there is a non - invasive cognitive test procedure , virtual scanning technology , which utilizes the chemiluminescence arising from biochemical processes to assess and manage stress - related cardiological conditions . the biochemical basis of the technology has not been previously explained , except as implied in our papers[2932 ] . this article furthers explanation that oxidative stress effect of luminescence on cognition and colour perception is possibly the biochemical basis of virtual scanning technology . it may be likened to other emission - based technologies such as ct scan , magnetic resonance , or ultrasound , but there is a difference . other technologies involve the body 's interaction with , and response to , some exogenous agents being inputted to the system as source of emission . virtual scanning is based upon the body 's inherent biochemical processes as source of light emission and defects in color perception . the cognitive test procedure , which is presented earlier in this volume , the computer then processes the patient 's response and shows the results on the screen . the red signals report the extent of the pathology whilst the blue signal reports the extent of the body 's natural compensatory response ( fig . signals below 10-units are pre - symptomatic whilst those above 10-units are symptomatic . a typical virtual scanning report indicating cardiological problem . formatted comments included in the report compensatory signal : 5/0 ischemic heart disease expressed pathological signals ( from left to right ) : 15/31 angina pectoris , 7/17 cardiosclerosis , 7/17 chronic fatigue , 3/31 cardiac insufficiency , 0/24 cardiac myopathy the diagnosis procedure ends with a recommendation of patient treatment strategy and first , the area ( organ , system ) where correction is necessary and features that are subject to correction are identified . the virtual scanning light therapy involves the presentation of flash lights of uniquely selected colors , shapes and frequencies . the emission is transmitted at delta frequencies to the autonomic nervous system by the computer through his eyes . the third stage of the therapeutic procedure is where healing signals are sensed , as the patient 's system ( vision ) responds , through the monitor . the brain establishes synchronized neural function and hence synchronized function of the autonomic nervous system and physiological systems thereby restoring its normal physiological stability and functions . the principle is referred variously as brain - wave coherence , brain - wave entrainment , or photic stimulation amongst others . one session of treatment takes average of 30 minutes and one module of therapy comprises typically 24 - 36 sessions . other treatment options of virtual scanning technology ( not emphasized in this article ) are nutrition , exercise and relaxation . it may be likened to other emission - based technologies such as ct scan , magnetic resonance , or ultrasound , but there is a difference . other technologies involve the body 's interaction with , and response to , some exogenous agents being inputted to the system as source of emission . virtual scanning is based upon the body 's inherent biochemical processes as source of light emission and defects in color perception . the cognitive test procedure , which is presented earlier in this volume , the computer then processes the patient 's response and shows the results on the screen . the red signals report the extent of the pathology whilst the blue signal reports the extent of the body 's natural compensatory response ( fig . signals below 10-units are pre - symptomatic whilst those above 10-units are symptomatic . a typical virtual scanning report indicating cardiological problem . formatted comments included in the report compensatory signal : 5/0 ischemic heart disease expressed pathological signals ( from left to right ) : 15/31 angina pectoris , 7/17 cardiosclerosis , 7/17 chronic fatigue , 3/31 cardiac insufficiency , 0/24 cardiac myopathy the diagnosis procedure ends with a recommendation of patient treatment strategy and first , the area ( organ , system ) where correction is necessary and features that are subject to correction are identified . the virtual scanning light therapy involves the presentation of flash lights of uniquely selected colors , shapes and frequencies . the emission is transmitted at delta frequencies to the autonomic nervous system by the computer through his eyes . the third stage of the therapeutic procedure is where healing signals are sensed , as the patient 's system ( vision ) responds , through the monitor . the brain establishes synchronized neural function and hence synchronized function of the autonomic nervous system and physiological systems thereby restoring its normal physiological stability and functions . the principle is referred variously as brain - wave coherence , brain - wave entrainment , or photic stimulation amongst others . one session of treatment takes average of 30 minutes and one module of therapy comprises typically 24 - 36 sessions . other treatment options of virtual scanning technology ( not emphasized in this article ) are nutrition , exercise and relaxation . it may be likened to other emission - based technologies such as ct scan , magnetic resonance , or ultrasound , but there is a difference . other technologies involve the body 's interaction with , and response to , some exogenous agents being inputted to the system as source of emission . virtual scanning is based upon the body 's inherent biochemical processes as source of light emission and defects in color perception . the cognitive test procedure , which is presented earlier in this volume , the computer then processes the patient 's response and shows the results on the screen . the red signals report the extent of the pathology whilst the blue signal reports the extent of the body 's natural compensatory response ( fig . signals below 10-units are pre - symptomatic whilst those above 10-units are symptomatic . a typical virtual scanning report indicating cardiological problem . formatted comments included in the report compensatory signal : 5/0 ischemic heart disease expressed pathological signals ( from left to right ) : 15/31 angina pectoris , 7/17 cardiosclerosis , 7/17 chronic fatigue , 3/31 cardiac insufficiency , 0/24 cardiac myopathy the diagnosis procedure ends with a recommendation of patient treatment strategy and the standard therapy involve a basic concept of three stages . first , the area ( organ , system ) where correction is necessary and features that are subject to correction are identified . the virtual scanning light therapy involves the presentation of flash lights of uniquely selected colors , shapes and frequencies . the emission is transmitted at delta frequencies to the autonomic nervous system by the computer through his eyes . the third stage of the therapeutic procedure is where healing signals are sensed , as the patient 's system ( vision ) responds , through the monitor . the brain establishes synchronized neural function and hence synchronized function of the autonomic nervous system and physiological systems thereby restoring its normal physiological stability and functions . the principle is referred variously as brain - wave coherence , brain - wave entrainment , or photic stimulation amongst others . one session of treatment takes average of 30 minutes and one module of therapy comprises typically 24 - 36 sessions . other treatment options of virtual scanning technology ( not emphasized in this article ) are nutrition , exercise and relaxation . we have explained that metabolic and physiological interactions such as glucose metabolism and hpaa , respectively , enable the generation of reactive oxygen species and consequential existence of oxidative stress . the feedback and feedforward responses to oxidative stress reactions form the biochemical basis of pathological processes and systemic instability including cardiological problems ( fig . the chemiluminescence arising from oxidative stress are perceived and memorized as characteristic of different disease conditions according to colour perception and intensity and attenuated by flash light using virtual scanning technology . perhaps , the question would be is this applicable to cardiological diseases? light is conducted within the body along the acupuncture meridians , and clinical benefits of light therapy have been known . what is yet unknown are the biochemical and/or metabolic basis of the light that is conducted within the body and the basis of therapeutic applicability in cardiology . we had posited that the biochemical basis of signal employed by virtual scanning technology is antioxidant activities . we postulate that oxidative stress induced chemiluminescence and susceptibility to flash light therapy is the basis of virtual scanning technology . furthermore , we rationalize the applicability to cardiological problems to be based on the attenuating effect of light on oxidative stress . our postulation is based on the available itemized knowledge on flash light therapy in relation to cardiological problems : the level of blood glucose is a factor in virtual scanning technology . increased glucose metabolism is implicated in stress and cardiology ( fig . 2).light therapy affects cortisol rhythm.low level ( infrared ) light therapy attenuates hyperglycaemia - induced oxidative stress and enhances the antioxidant protection system.flash light therapy attenuates venous cannulation pain.the concept of selective photothermolysis is applicable in vascular lesions management.atherothrombosis can be cleared , at least in part , by flash light therapy by the principle of laser thrombolysis . 2 ) . light therapy affects cortisol rhythm . low level ( infrared ) light therapy attenuates hyperglycaemia - induced oxidative stress and enhances the antioxidant protection system . atherothrombosis can be cleared , at least in part , by flash light therapy by the principle of laser thrombolysis . therefore , given the available knowledge listed above , there is credible evidence that flash light therapy is applicable in the management of cardiological problems . however , validation research would be required to enable incorporation of any new technology such as virtual scanning into conventional clinical practice . this is being incorporated in another closely - related emerging technology , neuropattern , which is based on the argument on neurobehavioral medicine and stress - related disorders that manipulation of the brain by photostimulation technologies can be applied in clinical management . while neuropattern technology acknowledges cortisol rhythm without recourse to light therapy , virtual scanning uses light therapy without recourse to laboratory assessment of cortisol . therefore , except for reason of availability and cost effectiveness , salivary cortisol test can be employed to monitor and validate therapeutic outcome in virtual scanning . based on fig . this would include blood glucose level as well as vitamin c , vitamin e and whole blood viscosity . it would be expected that increased blood glucose level , antioxidant imbalance and increased whole blood viscosity will be observed at baseline stress ; and normalized after virtual scanning therapy . this hypothesis is in agreement with the implication of oxidative stress and blood flow / shear stress that are involved in several processes of atherogenesis . the association between the triage of ( i ) biological stress , ( ii ) cardiology , and ( iii ) cortisol level is known . this paper articulates that color perception ( i ) is influenced by biological stress , ( ii ) is useful for the management of cardiological problems and ( iii ) can be monitored and validated using cortisol level or the suggested panel of associated biochemical indices , which are centered on oxidative stress . especially , it is known that oxidative stress is a common mediator of apoptosis and cardiac damage and that antioxidant as adjuvant therapy is effective in improving parameters of cardiac function . what this article contributes is a basis to use blood glucose level , vitamin c , vitamin e and whole blood viscosity as a laboratory panel to validate the technology for possible incorporation , as well as monitor the technology 's treatment outcome in clinical practice . it has been argued that virtual scanning technology lacks clear link between plausible mechanism and the theory proposed .

background : virtual scanning is one of the emerging technologies in complementary medicine practice . the diagnostic principle is hinged on perception and ultra weak light emission , while the treatment options associated with it includes diet , flash light , exercise and relaxation . however , a mechanism that links the diagnostic and treatment principles has yet to be elucidated.aims:the objective here is to further explanation of oxidative stress concept as the biochemical basis of the technology.materials and methods : using available literature and basic science textbook , the function of the hypothalamus - pituitary - adrenalin axis as neuro - endocrine physiological system that is strongly linked to the rate of alterations in biochemical processes through to cardiovascular complications is articulated.results:the hypothesis brings to fore the potential of using the alterations in biochemical processes associated with cognition as tool to validate the virtual scanning technology for possible incorporation into clinical practice . or vice versa to use virtual scanning technology to determine the chemiluminescence - related biochemical changes resulting from pathologies that could benefit from relaxation , light therapy , exercise and antioxidant nutrition.conclusions:this article advances the applicability of cognitive test procedure for indication of the disease(s ) affecting heart function . the implication for some laboratory indices that are already available in clinical practice is highlighted . investigation of this hypothesis will help provide clear link between plausible mechanism and the theory proposed .

Introduction Biochemistry of brain-heart connection: biochemical alterations during stress None Oxidative stress is biochemical basis of biological stress Brain and heart connection Implication for colour perception The Virtual Scanning None Principle Therapeutic Applicability to cardiology: Does light therapy attenuate cardiology problems? Suggestions of laboratory indices for validation research Conclusions

advances in noninvasive technologies in cardiology are growing and improving both the diagnosis of heart disease and the guidance of therapy . there are currently many emerging technologies , which are being used in the alternative and complementary medicine practice , including computer meridian diagnostics and virtual scanning . in particular , visual defects are associated with heart condition[35 ] . associated with the defects are antioxidant activities[68 ] , which in turn are associated to adenosine triphosphate ( atp ) metabolism . changes to the levels of proteins and reactive substrates in the body cause the release of ultra weak light or chemiluminescence . the concept of stress is related to the complexity of the interactions between the cells , organs and systems of the body . two of the adrenal hormones involved in the neuro - endocrine response to stress are catecholamines and glucocorticoids . when a free radical reacts with a molecule , a new radical is always formed . keys : aa = ascorbic acid , coq = oxidized coenzyme - q10 , coq - h2 = coenzyme - q10 , cvd = cardiovascular disease , dha = dehydro - ascorbic acid ( free radical ) , gr = glutathione reductase , gsh = reduced glutathione , gssg = oxidized glutathione , loo = lipid peroxyl radical , looh = lipid peroxide , os = oxidative stress , rc = mitochondrial respiratory chain , toh = tocopherol ( vitamin e ) , to = tocopheroxyl radical . a major part of neuroendocrine system involved in stress adaptation is the hypothalamus - pituitary - adrenalin axis ( hpaa ) . when there is stress , there is release of adrenal hormones and associated with this is increased glucose metabolism . among the obvious alterations in biochemical makeup specifically , when oxidative stress involves the red blood cells , the biochemical alterations drive the physiology as in the flow diagram ( fig 2 ) . it is known that antioxidant activities involve reactions with proteins with concomitant emission of ultra weak light and are increased in obstructive sleep apnoea states . it is also known that oxidants contribute to the exacerbation of sleep apnoea , particularly including the cardiovascular complications . at this juncture , it is pertinent to note at least the following half - a - dozen points : biochemical changes associated with disease or drug substances influence perception of color.changes to the levels of proteins and reactive substrates in the body cause the release of ultra - weak light albeit chemiluminescence.the emissions are measurable luminescence as index of oxidative stress.oxidative stress as a common event that can lead to emission of chemiluminescence has been indicated in studies on erythrocytes . above all , alteration in atp level is correlated to photon chemiluminescence.photon emission affects color or heat perception and intensity and this property is utilized in ct scan . therefore , abnormal level of emissions arising from biochemical processes during disease conditions could affect color vision . changes to the levels of proteins and reactive substrates in the body cause the release of ultra - weak light albeit chemiluminescence . oxidative stress as a common event that can lead to emission of chemiluminescence has been indicated in studies on erythrocytes . photon emission affects color or heat perception and intensity and this property is utilized in ct scan . therefore , abnormal level of emissions arising from biochemical processes during disease conditions could affect color vision . what this article brings to fore is that there is a non - invasive cognitive test procedure , virtual scanning technology , which utilizes the chemiluminescence arising from biochemical processes to assess and manage stress - related cardiological conditions . the biochemical basis of the technology has not been previously explained , except as implied in our papers[2932 ] . this article furthers explanation that oxidative stress effect of luminescence on cognition and colour perception is possibly the biochemical basis of virtual scanning technology . the concept of stress is related to the complexity of the interactions between the cells , organs and systems of the body . two of the adrenal hormones involved in the neuro - endocrine response to stress are catecholamines and glucocorticoids . the biochemical process ) for the generation of energy . when a free radical reacts with a molecule , a new radical is always formed . keys : aa = ascorbic acid , coq = oxidized coenzyme - q10 , coq - h2 = coenzyme - q10 , cvd = cardiovascular disease , dha = dehydro - ascorbic acid ( free radical ) , gr = glutathione reductase , gsh = reduced glutathione , gssg = oxidized glutathione , loo = lipid peroxyl radical , looh = lipid peroxide , os = oxidative stress , rc = mitochondrial respiratory chain , toh = tocopherol ( vitamin e ) , to = tocopheroxyl radical . a major part of neuroendocrine system involved in stress adaptation is the hypothalamus - pituitary - adrenalin axis ( hpaa ) . when there is stress , there is release of adrenal hormones and associated with this is increased glucose metabolism . among the obvious alterations in biochemical makeup specifically , when oxidative stress involves the red blood cells , the biochemical alterations drive the physiology as in the flow diagram ( fig 2 ) . it is known that antioxidant activities involve reactions with proteins with concomitant emission of ultra weak light and are increased in obstructive sleep apnoea states . it is also known that oxidants contribute to the exacerbation of sleep apnoea , particularly including the cardiovascular complications . at this juncture , it is pertinent to note at least the following half - a - dozen points : biochemical changes associated with disease or drug substances influence perception of color.changes to the levels of proteins and reactive substrates in the body cause the release of ultra - weak light albeit chemiluminescence.the emissions are measurable luminescence as index of oxidative stress.oxidative stress as a common event that can lead to emission of chemiluminescence has been indicated in studies on erythrocytes . therefore , abnormal level of emissions arising from biochemical processes during disease conditions could affect color vision . changes to the levels of proteins and reactive substrates in the body cause the release of ultra - weak light albeit chemiluminescence . oxidative stress as a common event that can lead to emission of chemiluminescence has been indicated in studies on erythrocytes . photon emission affects color or heat perception and intensity and this property is utilized in ct scan . therefore , abnormal level of emissions arising from biochemical processes during disease conditions could affect color vision . what this article brings to fore is that there is a non - invasive cognitive test procedure , virtual scanning technology , which utilizes the chemiluminescence arising from biochemical processes to assess and manage stress - related cardiological conditions . the biochemical basis of the technology has not been previously explained , except as implied in our papers[2932 ] . this article furthers explanation that oxidative stress effect of luminescence on cognition and colour perception is possibly the biochemical basis of virtual scanning technology . the concept of stress is related to the complexity of the interactions between the cells , organs and systems of the body . two of the adrenal hormones involved in the neuro - endocrine response to stress are catecholamines and glucocorticoids . the biochemical process ) for the generation of energy . when a free radical reacts with a molecule , a new radical is always formed . keys : aa = ascorbic acid , coq = oxidized coenzyme - q10 , coq - h2 = coenzyme - q10 , cvd = cardiovascular disease , dha = dehydro - ascorbic acid ( free radical ) , gr = glutathione reductase , gsh = reduced glutathione , gssg = oxidized glutathione , loo = lipid peroxyl radical , looh = lipid peroxide , os = oxidative stress , rc = mitochondrial respiratory chain , toh = tocopherol ( vitamin e ) , to = tocopheroxyl radical . a major part of neuroendocrine system involved in stress adaptation is the hypothalamus - pituitary - adrenalin axis ( hpaa ) . when there is stress , there is release of adrenal hormones and associated with this is increased glucose metabolism . among the obvious alterations in biochemical makeup are increased atp generation and reduction in cellular antioxidant levels . specifically , when oxidative stress involves the red blood cells , the biochemical alterations drive the physiology as in the flow diagram ( fig 2 ) . it is known that antioxidant activities involve reactions with proteins with concomitant emission of ultra weak light and are increased in obstructive sleep apnoea states . it is also known that oxidants contribute to the exacerbation of sleep apnoea , particularly including the cardiovascular complications . at this juncture , it is pertinent to note at least the following half - a - dozen points : biochemical changes associated with disease or drug substances influence perception of color.changes to the levels of proteins and reactive substrates in the body cause the release of ultra - weak light albeit chemiluminescence.the emissions are measurable luminescence as index of oxidative stress.oxidative stress as a common event that can lead to emission of chemiluminescence has been indicated in studies on erythrocytes . above all , alteration in atp level is correlated to photon chemiluminescence.photon emission affects color or heat perception and intensity and this property is utilized in ct scan . therefore , abnormal level of emissions arising from biochemical processes during disease conditions could affect color vision . changes to the levels of proteins and reactive substrates in the body cause the release of ultra - weak light albeit chemiluminescence . oxidative stress as a common event that can lead to emission of chemiluminescence has been indicated in studies on erythrocytes . photon emission affects color or heat perception and intensity and this property is utilized in ct scan . therefore , abnormal level of emissions arising from biochemical processes during disease conditions could affect color vision . what this article brings to fore is that there is a non - invasive cognitive test procedure , virtual scanning technology , which utilizes the chemiluminescence arising from biochemical processes to assess and manage stress - related cardiological conditions . the biochemical basis of the technology has not been previously explained , except as implied in our papers[2932 ] . this article furthers explanation that oxidative stress effect of luminescence on cognition and colour perception is possibly the biochemical basis of virtual scanning technology . other technologies involve the body 's interaction with , and response to , some exogenous agents being inputted to the system as source of emission . virtual scanning is based upon the body 's inherent biochemical processes as source of light emission and defects in color perception . the cognitive test procedure , which is presented earlier in this volume , the computer then processes the patient 's response and shows the results on the screen . the red signals report the extent of the pathology whilst the blue signal reports the extent of the body 's natural compensatory response ( fig . a typical virtual scanning report indicating cardiological problem . formatted comments included in the report compensatory signal : 5/0 ischemic heart disease expressed pathological signals ( from left to right ) : 15/31 angina pectoris , 7/17 cardiosclerosis , 7/17 chronic fatigue , 3/31 cardiac insufficiency , 0/24 cardiac myopathy the diagnosis procedure ends with a recommendation of patient treatment strategy and first , the area ( organ , system ) where correction is necessary and features that are subject to correction are identified . the virtual scanning light therapy involves the presentation of flash lights of uniquely selected colors , shapes and frequencies . the emission is transmitted at delta frequencies to the autonomic nervous system by the computer through his eyes . the third stage of the therapeutic procedure is where healing signals are sensed , as the patient 's system ( vision ) responds , through the monitor . the brain establishes synchronized neural function and hence synchronized function of the autonomic nervous system and physiological systems thereby restoring its normal physiological stability and functions . the principle is referred variously as brain - wave coherence , brain - wave entrainment , or photic stimulation amongst others . other treatment options of virtual scanning technology ( not emphasized in this article ) are nutrition , exercise and relaxation . other technologies involve the body 's interaction with , and response to , some exogenous agents being inputted to the system as source of emission . virtual scanning is based upon the body 's inherent biochemical processes as source of light emission and defects in color perception . the cognitive test procedure , which is presented earlier in this volume , the computer then processes the patient 's response and shows the results on the screen . the red signals report the extent of the pathology whilst the blue signal reports the extent of the body 's natural compensatory response ( fig . a typical virtual scanning report indicating cardiological problem . formatted comments included in the report compensatory signal : 5/0 ischemic heart disease expressed pathological signals ( from left to right ) : 15/31 angina pectoris , 7/17 cardiosclerosis , 7/17 chronic fatigue , 3/31 cardiac insufficiency , 0/24 cardiac myopathy the diagnosis procedure ends with a recommendation of patient treatment strategy and first , the area ( organ , system ) where correction is necessary and features that are subject to correction are identified . the virtual scanning light therapy involves the presentation of flash lights of uniquely selected colors , shapes and frequencies . the emission is transmitted at delta frequencies to the autonomic nervous system by the computer through his eyes . the third stage of the therapeutic procedure is where healing signals are sensed , as the patient 's system ( vision ) responds , through the monitor . the brain establishes synchronized neural function and hence synchronized function of the autonomic nervous system and physiological systems thereby restoring its normal physiological stability and functions . the principle is referred variously as brain - wave coherence , brain - wave entrainment , or photic stimulation amongst others . other treatment options of virtual scanning technology ( not emphasized in this article ) are nutrition , exercise and relaxation . other technologies involve the body 's interaction with , and response to , some exogenous agents being inputted to the system as source of emission . virtual scanning is based upon the body 's inherent biochemical processes as source of light emission and defects in color perception . the cognitive test procedure , which is presented earlier in this volume , the computer then processes the patient 's response and shows the results on the screen . the red signals report the extent of the pathology whilst the blue signal reports the extent of the body 's natural compensatory response ( fig . formatted comments included in the report compensatory signal : 5/0 ischemic heart disease expressed pathological signals ( from left to right ) : 15/31 angina pectoris , 7/17 cardiosclerosis , 7/17 chronic fatigue , 3/31 cardiac insufficiency , 0/24 cardiac myopathy the diagnosis procedure ends with a recommendation of patient treatment strategy and the standard therapy involve a basic concept of three stages . first , the area ( organ , system ) where correction is necessary and features that are subject to correction are identified . the virtual scanning light therapy involves the presentation of flash lights of uniquely selected colors , shapes and frequencies . the third stage of the therapeutic procedure is where healing signals are sensed , as the patient 's system ( vision ) responds , through the monitor . the brain establishes synchronized neural function and hence synchronized function of the autonomic nervous system and physiological systems thereby restoring its normal physiological stability and functions . the principle is referred variously as brain - wave coherence , brain - wave entrainment , or photic stimulation amongst others . other treatment options of virtual scanning technology ( not emphasized in this article ) are nutrition , exercise and relaxation . we have explained that metabolic and physiological interactions such as glucose metabolism and hpaa , respectively , enable the generation of reactive oxygen species and consequential existence of oxidative stress . the feedback and feedforward responses to oxidative stress reactions form the biochemical basis of pathological processes and systemic instability including cardiological problems ( fig . the chemiluminescence arising from oxidative stress are perceived and memorized as characteristic of different disease conditions according to colour perception and intensity and attenuated by flash light using virtual scanning technology . perhaps , the question would be is this applicable to cardiological diseases? what is yet unknown are the biochemical and/or metabolic basis of the light that is conducted within the body and the basis of therapeutic applicability in cardiology . we had posited that the biochemical basis of signal employed by virtual scanning technology is antioxidant activities . we postulate that oxidative stress induced chemiluminescence and susceptibility to flash light therapy is the basis of virtual scanning technology . furthermore , we rationalize the applicability to cardiological problems to be based on the attenuating effect of light on oxidative stress . our postulation is based on the available itemized knowledge on flash light therapy in relation to cardiological problems : the level of blood glucose is a factor in virtual scanning technology . 2).light therapy affects cortisol rhythm.low level ( infrared ) light therapy attenuates hyperglycaemia - induced oxidative stress and enhances the antioxidant protection system.flash light therapy attenuates venous cannulation pain.the concept of selective photothermolysis is applicable in vascular lesions management.atherothrombosis can be cleared , at least in part , by flash light therapy by the principle of laser thrombolysis . light therapy affects cortisol rhythm . low level ( infrared ) light therapy attenuates hyperglycaemia - induced oxidative stress and enhances the antioxidant protection system . atherothrombosis can be cleared , at least in part , by flash light therapy by the principle of laser thrombolysis . therefore , given the available knowledge listed above , there is credible evidence that flash light therapy is applicable in the management of cardiological problems . however , validation research would be required to enable incorporation of any new technology such as virtual scanning into conventional clinical practice . this is being incorporated in another closely - related emerging technology , neuropattern , which is based on the argument on neurobehavioral medicine and stress - related disorders that manipulation of the brain by photostimulation technologies can be applied in clinical management . while neuropattern technology acknowledges cortisol rhythm without recourse to light therapy , virtual scanning uses light therapy without recourse to laboratory assessment of cortisol . therefore , except for reason of availability and cost effectiveness , salivary cortisol test can be employed to monitor and validate therapeutic outcome in virtual scanning . it would be expected that increased blood glucose level , antioxidant imbalance and increased whole blood viscosity will be observed at baseline stress ; and normalized after virtual scanning therapy . this hypothesis is in agreement with the implication of oxidative stress and blood flow / shear stress that are involved in several processes of atherogenesis . this paper articulates that color perception ( i ) is influenced by biological stress , ( ii ) is useful for the management of cardiological problems and ( iii ) can be monitored and validated using cortisol level or the suggested panel of associated biochemical indices , which are centered on oxidative stress . especially , it is known that oxidative stress is a common mediator of apoptosis and cardiac damage and that antioxidant as adjuvant therapy is effective in improving parameters of cardiac function . what this article contributes is a basis to use blood glucose level , vitamin c , vitamin e and whole blood viscosity as a laboratory panel to validate the technology for possible incorporation , as well as monitor the technology 's treatment outcome in clinical practice . it has been argued that virtual scanning technology lacks clear link between plausible mechanism and the theory proposed .

since the advent of nanotechnology , nanoscale particle - based sensors have attracted tremendous attention from scientists , because of their unique optical and electrical properties.14 localized surface plasmon resonance ( lspr ) , which is recognized as one of the special optical properties of noble metallic nanoparticles ( eg , silver or gold ) , is generated when the incident photon frequency is resonant with the collective oscillation of conduction electrons.5 the lspr biosensor , a novel type of optical fiber - based biosensor , uses an optical fiber or optical fiber bundle to transform biological recognition information into analytically useful signals in the lspr spectrum , and has been proven to be an effective platform for detection techniques.5,6 the sensing principle is based on its sensitivity to local refractive index changes near the nanoparticle surface induced by biomolecular interactions.58 the applicability of this nanobiosensor has been studied in many fields , such as drug screening , medical diagnostics , and environmental monitoring , and has become a hot research topic all over the world.913 the detection of biotin - streptavidin and microalbumin in patients urine using the proposed domestic lspr biosensor has been reported previously , without quantitative analysis.14,15 however , to date , this biosensor has not been widely utilized in the field of gynecological oncology . ovarian cancer is one of the most common malignancies of the female reproductive system . according to the american cancer society , ovarian cancer accounts for about 4% of cancers occurring in women , but ranks fourth among the cancer - related deaths in women , because most cases are unfortunately diagnosed at an advanced stage.16 currently , ca125 is the only biomarker of ovarian cancer that is most widely and routinely used in clinical practice . however , the clinical use of ca125 as a marker for early detection is severely restricted , because it is elevated in only half of early - stage ovarian cancers and is elevated frequently in many benign gynecological diseases , such as endometriosis , ovarian cysts , and pelvic inflammation.17,18 recently , the human epididymis secretory protein 4 ( he4 ) , which is a novel biomarker for ovarian cancer , has been widely studied and used in the early diagnosis of ovarian cancer . reportedly , he4 is highly sensitive to early ovarian cancer and can be used in combination with ca125 , offering the best method of differential diagnosis in ovarian cancer and other pelvic masses.1921 currently used approaches for detection of he4 are the enzyme - linked immunosorbent assay ( elisa ) and the chemiluminescent immunoassay ( clia ) . although it is one of the most mature methods for protein detection used in the last three decades and is considered the gold standard , elisa still has certain shortcomings in terms of the long assay time required , the indirect detection format , and the need for multiple washing steps.22 clia also has some disadvantages , including the large volume of the analysis instrument , high cost , and special labeling requirements . thus , a rapid , label - free , simple , low - cost , and portable protocol for detecting he4 is urgently required . in the present work , the lspr biosensor developed was utilized based on silver nanoparticles for the direct detection of the he4 biomarker in blood samples from patients with ovarian cancer . under the optimum conditions , he4 in both buffer and human serum samples based on current information , this study is the first to investigate the lspr system for the detection of he4 . the study is also the first to discuss and analyze in detail the detection limit , linear range , and regeneration of the proposed homemade lspr sensor . 11-mercaptoundecanoic acid and bovine serum albumin were purchased from sigma - aldrich ( st louis , mo ) . n - hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide hydrochloride ( edc ) were purchased from aladdin ( shanghai , china ) . mouse monoclonal anti - he4 antibody ( anti - he4 ) and standard he4 were obtained from abnova ( taiwan , china ) . an he4 elisa kit was obtained from fujirebio diagnostics inc ( malvern , pa ) . ultrapure water ( 18.3 m/cm ) used for the preparation of all solutions was obtained from millipore co ( boston , ma ) . the human serum specimens were collected from west china second university hospital ( chengdu , china ) . written informed consents were not obtained , because these samples were from leftover blood samples in routine blood tests , and this study did not cause any harm to the patients . sera were isolated through the centrifugation of whole blood samples at 2000 rpm for 20 minutes , and subsequently kept frozen at 80c until analysis . the integrated lspr biosensor used in this work was a custom system built on - site , as previously described in detail.14,23 the silver nanochip was fabricated using the nanosphere lithography method . the peak wavelength of the lspr extinction spectrum ( max ) excited by the silver nanoparticles was measured and recorded using an ultraviolet - visible spectroscope ( sciencetech 9055 , sciencetech , ottawa , canada ) with a charge - coupled device detector ( koan electro - optics co , shanghai , china).14 the entire measurement process could be described as follows . the white light emerging from an optical fiber bundle and the transmitted light coupled into the detection probe of the optical fiber bundle provided the incident light . the nanochip was placed perpendicular to the incident light , which was taken using the ultraviolet - visible spectrometer ranging from 400 nm to 800 nm at room temperature in air.14 all the extinction spectra could be calculated through a software program ( ocean optics , dunedin , fl ) and directly displayed on the screen of the computer . the shift toward longer wavelengths , defined as a red shift , was indicated as ( + ) , whereas the shift toward shorter wavelengths , defined as a blue shift , was indicated as ( ) . the relative wavelength shift , namely max , was used to monitor the binding of target analytes.13 the experimental setup is illustrated in figure 1 . functionalization is a multistep process that prepares the lspr nanosensor for biodetection events ( figure 2 ) . first , the silver nanochip was incubated in 1 mm 11-mercaptoundecanoic acid solution ( in ethanol ) for 12 hours to form a self - assembled monolayer on the slice surface , after which the nanochip was washed thoroughly with ethanol and dried at room temperature . the nanochip was then immersed in 75 mm edc/15 mm n - hydroxysuccinimide solution for 2 hours to activate the carboxyl groups of the self - assembled monolayer , which reacts with amino groups of antibodies to form amides . subsequently , 50 l of anti - he4 solution at 10 g / ml was spotted on the self - assembled monolayer - modified surface and overnight incubation at 4c followed . the anti - he4 immobilized surface was immersed in 1 m ethanolamine solution ( ph 8.5 ) for 30 minutes to deactivate the unreacted esters , after which the surface was washed with phosphate - buffered solution ( ph 7.4 ) and dried . the different concentrations of standard he4 ( 1 pm to 0.1 m ) and the patient samples were incubated on the functionalized lspr chip for 40 minutes , followed by a thorough rinsing with phosphate - buffered solution containing 0.05% tween-20 to dissociate the nonspecific binding . each value was averaged from three parallel experiments . a statistical evaluation of the correlation of the lspr and elisa methods was performed and computed using a software program ( origin 8.0 , originlab corporation , northampton , ma ) . 11-mercaptoundecanoic acid and bovine serum albumin were purchased from sigma - aldrich ( st louis , mo ) . n - hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide hydrochloride ( edc ) were purchased from aladdin ( shanghai , china ) . mouse monoclonal anti - he4 antibody ( anti - he4 ) and standard he4 were obtained from abnova ( taiwan , china ) . an he4 elisa kit was obtained from fujirebio diagnostics inc ( malvern , pa ) . ultrapure water ( 18.3 m/cm ) used for the preparation of all solutions was obtained from millipore co ( boston , ma ) . the human serum specimens were collected from west china second university hospital ( chengdu , china ) . written informed consents were not obtained , because these samples were from leftover blood samples in routine blood tests , and this study did not cause any harm to the patients . sera were isolated through the centrifugation of whole blood samples at 2000 rpm for 20 minutes , and subsequently kept frozen at 80c until analysis . the integrated lspr biosensor used in this work was a custom system built on - site , as previously described in detail.14,23 the silver nanochip was fabricated using the nanosphere lithography method . the peak wavelength of the lspr extinction spectrum ( max ) excited by the silver nanoparticles was measured and recorded using an ultraviolet - visible spectroscope ( sciencetech 9055 , sciencetech , ottawa , canada ) with a charge - coupled device detector ( koan electro - optics co , shanghai , china).14 the entire measurement process could be described as follows . the white light emerging from an optical fiber bundle and the transmitted light coupled into the detection probe of the optical fiber bundle provided the incident light . the nanochip was placed perpendicular to the incident light , which was taken using the ultraviolet - visible spectrometer ranging from 400 nm to 800 nm at room temperature in air.14 all the extinction spectra could be calculated through a software program ( ocean optics , dunedin , fl ) and directly displayed on the screen of the computer . the shift toward longer wavelengths , defined as a red shift , was indicated as ( + ) , whereas the shift toward shorter wavelengths , defined as a blue shift , was indicated as ( ) . the relative wavelength shift , namely max , was used to monitor the binding of target analytes.13 the experimental setup is illustrated in figure 1 . functionalization is a multistep process that prepares the lspr nanosensor for biodetection events ( figure 2 ) . first , the silver nanochip was incubated in 1 mm 11-mercaptoundecanoic acid solution ( in ethanol ) for 12 hours to form a self - assembled monolayer on the slice surface , after which the nanochip was washed thoroughly with ethanol and dried at room temperature . the nanochip was then immersed in 75 mm edc/15 mm n - hydroxysuccinimide solution for 2 hours to activate the carboxyl groups of the self - assembled monolayer , which reacts with amino groups of antibodies to form amides . subsequently , 50 l of anti - he4 solution at 10 g / ml was spotted on the self - assembled monolayer - modified surface and overnight incubation at 4c followed . the anti - he4 immobilized surface was immersed in 1 m ethanolamine solution ( ph 8.5 ) for 30 minutes to deactivate the unreacted esters , after which the surface was washed with phosphate - buffered solution ( ph 7.4 ) and dried . in the detection stage , the different concentrations of standard he4 ( 1 pm to 0.1 m ) and the patient samples were incubated on the functionalized lspr chip for 40 minutes , followed by a thorough rinsing with phosphate - buffered solution containing 0.05% tween-20 to dissociate the nonspecific binding . each value was averaged from three parallel experiments . a statistical evaluation of the correlation of the lspr and elisa methods was performed and computed using a software program ( origin 8.0 , originlab corporation , northampton , ma ) . the lspr spectra of the nanobiosensor in each processing step are shown in figure 3 . before modification , the lspr max of the bare silver nanochip a representative lspr max of the silver nanochip after modification with 11-mercaptoundecanoic acid was 619.85 nm with a corresponding lspr max of + 27.27 nm ( figure 3b ) . after anti - he4 immobilization , the lspr max shifted to 630.97 nm , with an additional 11.12 nm red shift ( figure 3c ) . after incubation in 500 pm he4 , the lspr wavelength shifted to + 14.48 nm , showing a max of 645.45 nm ( figure 3d ) . this experimental evidence clearly showed that he4 in the buffer solution was detected successfully by the lspr biosensor . the optical characteristics of the nanosensor are notably based on the wavelength shift of the absorbance peak , max . according to mie theory , therefore , the change in the local refractive index that accompanies the molecular binding can be sensed by the nanoparticles , and the quantitative detection of targets can be achieved by monitoring the max when the analytes are bound to the nanoparticles.10 figure 4 shows a calibration curve of the nanosensor constructed by measuring the lspr wavelength shifts after exposure of the anti - he4 attachment surface to the he4 standard solutions of concentrations ranging from 1 pm to 0.1 m under optimal conditions . as seen from the data , the lspr max values increased stepwise with increasing he4 concentrations . like many immunoassays , the curve is sigmoid rather than linear.25 in addition , a good linear relationship between the lspr shifts and the logarithm of the he4 concentration could be fitted to the experimental points from 10 pm to 10,000 pm ( inset of figure 4 ) . the linear regression equation was lspr ( nm ) = 3.72 log [ he4 ] ( m ) + 47.37 , with a linear correlation coefficient ( r ) of 0.997 . this linear range is broader than that of the commercial he4 elisa kit ( 15 pm to 900 pm ) , indicating that it is capable of testing the samples without predilution which could not be confirmed through a routine elisa . given that the noise level is defined as the standard deviation of the blank ( n = 12 ) , the limit of detection , defined as the analyte concentration corresponding to a signal - to - noise ratio of three ( about 5 nm ) , was estimated to be 4 pm . this limit of detection was more than sufficient for analysis of he4 in serum where normal values of he4 are considered to be less than 150 pm.26 this detection limit is a little better than that obtained using the elisa method for he4 ( 15 pm ) . therefore , the lspr biosensor had good analytical performance for the detection of he4 , and was comparable with elisa . several control experiments were designed to ensure that the results of figure 4 were not disturbed by nonspecific adsorption . squamous cell carcinoma ( scc ) antigen , another tumor marker and bovine serum albumin , the most abundant protein component in blood serum were chosen as interferences . the functionalized biosensor was incubated with a solution of 500 pm scc or bovine serum albumin . the same concentration of he4 was also introduced to the nanosensor surface in the absence of anti - he4 . thus , the nonspecific binding of protein molecules on the self - assembled monolayer - covered nanosensor was found to be relatively low . the results indicated that the selectivity of the lspr biosensor based on the highly specific antigen - antibody reaction and surface passivation with ethanolamine was excellent . the precision within and between batches is an important factor in the practical application of the biosensor.25 the intrarun precision was tested at two he4 concentration levels ( 500 pm and 5000 pm ) using the lspr biosensors of the same batch for five continuous measurements within one day . the interrun precision was tested similarly with five biosensors , which were selected randomly from five batches . the results in table 1 suggest that the nanobiosensor showed acceptable precision and reproducibility . following completion of the antigen - antibody reaction , the nanochips were regenerated via exposure to 8 m urea solution , and then washed with ultrapure water . the reproducibility of responses from the identical biosensor and from different biosensors was evaluated by measuring 500 pm standard he4 solution , and the coefficient of variation for the lspr shift was obtained . after performing the corresponding he4 incubation with the freshly regenerated biosensor four times , a coefficient of variation of 14.7% was obtained , as shown in table 2 . this value indicates that the anti - he4-modified sensor can provide reliable detection of he4 . the biosensor stored in the refrigerator at 4c was regenerated and observed every week for one month . after one , 2 , 3 , and 4 weeks , the extent of response for 500 pm he4 dropped by 2.30% , 7.21% , 9.66% , and 17.09% , respectively , compared with the initial signal . this drop in response seems to be related to gradual deactivation of the anti - he4 antibody . the silver surface is susceptible to oxidative damage , which could directly affect the stability of the lspr sensor . however , the nanochips were stable in air after chemical modification through to the end of the 4-week study . a series of ten human serum samples , five from the ovarian cancer group and five from the control group , were screened for serum he4 levels using the proposed lspr sensor and an elisa kit . according to the literature , the cutoff value for he4 with 98% specificity is 150 pm.26 therefore , a sample was defined positive when its concentration was more than 150 pm . the five ovarian cancer samples with red shifts ranging from 11 nm to 17 nm were shown to be positive . meanwhile , red shifts less than 10.5 nm were observed in the negative controls . thus , consistent results were achieved between the lspr and elisa methods in terms of semiquantitative analysis . based on a t - test , differences in he4 levels between the ovarian cancer group and the control group detected were statistically significant ( p < 0.05 ) using both lspr and elisa . furthermore , the lspr sensor could specifically distinguish between ovarian cancer and the negative controls without the need for labeling in response to he4 binding in the sera tested . the lspr biosensor clearly has good specificity that can not be disrupted by other proteins or components in serum . the concentration of the ten samples was found to be within the range of 11.39911.16 pm , which was also within the linear range of the lspr sensor . a comparison between the two methods of analysis was also done using the regression line method.27 the results are shown in figure 5 . the analytical curve was calibrated using the correlation equation , ie , y = 83.88x 677.07 , with a correlation coefficient of 0.926 . the results indicate that the lspr biosensor could serve as a good alternative to the laborious and time - consuming elisa method for direct detection of he4 . moreover , the technological barrier regarding transfer of bench research to clinical application was overcome to a certain extent . in terms of product commercialization , serum he4 can be detected in real time using an antibody - coated lspr sensor within 40 minutes without predilution , thereby reducing the chance of potential procedural errors . the lspr spectra of the nanobiosensor in each processing step are shown in figure 3 . before modification , the lspr max of the bare silver nanochip a representative lspr max of the silver nanochip after modification with 11-mercaptoundecanoic acid was 619.85 nm with a corresponding lspr max of + 27.27 nm ( figure 3b ) . after anti - he4 immobilization , the lspr max shifted to 630.97 nm , with an additional 11.12 nm red shift ( figure 3c ) . after incubation in 500 pm he4 , the lspr wavelength shifted to + 14.48 nm , showing a max of 645.45 nm ( figure 3d ) . this experimental evidence clearly showed that he4 in the buffer solution was detected successfully by the lspr biosensor . the optical characteristics of the nanosensor are notably based on the wavelength shift of the absorbance peak , max . according to mie theory , therefore , the change in the local refractive index that accompanies the molecular binding can be sensed by the nanoparticles , and the quantitative detection of targets can be achieved by monitoring the max when the analytes are bound to the nanoparticles.10 figure 4 shows a calibration curve of the nanosensor constructed by measuring the lspr wavelength shifts after exposure of the anti - he4 attachment surface to the he4 standard solutions of concentrations ranging from 1 pm to 0.1 m under optimal conditions . as seen from the data , the lspr max values increased stepwise with increasing he4 concentrations . like many immunoassays , the curve is sigmoid rather than linear.25 in addition , a good linear relationship between the lspr shifts and the logarithm of the he4 concentration could be fitted to the experimental points from 10 pm to 10,000 pm ( inset of figure 4 ) . the linear regression equation was lspr ( nm ) = 3.72 log [ he4 ] ( m ) + 47.37 , with a linear correlation coefficient ( r ) of 0.997 . this linear range is broader than that of the commercial he4 elisa kit ( 15 pm to 900 pm ) , indicating that it is capable of testing the samples without predilution which could not be confirmed through a routine elisa . given that the noise level is defined as the standard deviation of the blank ( n = 12 ) , the limit of detection , defined as the analyte concentration corresponding to a signal - to - noise ratio of three ( about 5 nm ) , was estimated to be 4 pm . this limit of detection was more than sufficient for analysis of he4 in serum where normal values of he4 are considered to be less than 150 pm.26 this detection limit is a little better than that obtained using the elisa method for he4 ( 15 pm ) . therefore , the lspr biosensor had good analytical performance for the detection of he4 , and was comparable with elisa . several control experiments were designed to ensure that the results of figure 4 were not disturbed by nonspecific adsorption . all the experiments were implemented in triplicate . squamous cell carcinoma ( scc ) antigen , another tumor marker and bovine serum albumin , the most abundant protein component in blood serum were chosen as interferences . the functionalized biosensor was incubated with a solution of 500 pm scc or bovine serum albumin . the same concentration of he4 was also introduced to the nanosensor surface in the absence of anti - he4 . thus , the nonspecific binding of protein molecules on the self - assembled monolayer - covered nanosensor was found to be relatively low . the results indicated that the selectivity of the lspr biosensor based on the highly specific antigen - antibody reaction and surface passivation with ethanolamine was excellent . the precision within and between batches is an important factor in the practical application of the biosensor.25 the intrarun precision was tested at two he4 concentration levels ( 500 pm and 5000 pm ) using the lspr biosensors of the same batch for five continuous measurements within one day . the interrun precision was tested similarly with five biosensors , which were selected randomly from five batches . the results in table 1 suggest that the nanobiosensor showed acceptable precision and reproducibility . following completion of the antigen - antibody reaction , the nanochips were regenerated via exposure to 8 m urea solution , and then washed with ultrapure water . the reproducibility of responses from the identical biosensor and from different biosensors was evaluated by measuring 500 pm standard he4 solution , and the coefficient of variation for the lspr shift was obtained . after performing the corresponding he4 incubation with the freshly regenerated biosensor four times , a coefficient of variation of 14.7% was obtained , as shown in table 2 . this value indicates that the anti - he4-modified sensor can provide reliable detection of he4 . the biosensor stored in the refrigerator at 4c was regenerated and observed every week for one month . after one , 2 , 3 , and 4 weeks , the extent of response for 500 pm he4 dropped by 2.30% , 7.21% , 9.66% , and 17.09% , respectively , compared with the initial signal . this drop in response seems to be related to gradual deactivation of the anti - he4 antibody . the silver surface is susceptible to oxidative damage , which could directly affect the stability of the lspr sensor . however , the nanochips were stable in air after chemical modification through to the end of the 4-week study . a series of ten human serum samples , five from the ovarian cancer group and five from the control group , were screened for serum he4 levels using the proposed lspr sensor and an elisa kit . according to the literature , the cutoff value for he4 with 98% specificity is 150 pm.26 therefore , a sample was defined positive when its concentration was more than 150 pm . the five ovarian cancer samples with red shifts ranging from 11 nm to 17 nm were shown to be positive . meanwhile , red shifts less than 10.5 nm were observed in the negative controls . thus , consistent results were achieved between the lspr and elisa methods in terms of semiquantitative analysis . based on a t - test , differences in he4 levels between the ovarian cancer group and the control group detected were statistically significant ( p < 0.05 ) using both lspr and elisa . furthermore , the lspr sensor could specifically distinguish between ovarian cancer and the negative controls without the need for labeling in response to he4 binding in the sera tested . the lspr biosensor clearly has good specificity that can not be disrupted by other proteins or components in serum . the concentration of the ten samples was found to be within the range of 11.39911.16 pm , which was also within the linear range of the lspr sensor . a comparison between the two methods of analysis was also done using the regression line method.27 the results are shown in figure 5 . the analytical curve was calibrated using the correlation equation , ie , y = 83.88x 677.07 , with a correlation coefficient of 0.926 . the results indicate that the lspr biosensor could serve as a good alternative to the laborious and time - consuming elisa method for direct detection of he4 . moreover , the technological barrier regarding transfer of bench research to clinical application was overcome to a certain extent . in terms of product commercialization , serum he4 can be detected in real time using an antibody - coated lspr sensor within 40 minutes without predilution , thereby reducing the chance of potential procedural errors . ideal biosensors should be rapid , sensitive , specific , label - free , stable , reproducible , cheap , portable , and easy to operate . 28 the lspr technique has many of these characteristics , making this method comparable with other immunoassay techniques . the lspr biosensor has significant advantages in terms of label - free biomarker detection , a rapid test time , and in a direct assay format unlike the traditional immunoassay approaches , such as elisa . compared with chemiluminescence analysis and current commercial surface plasmon resonance sensors , the lspr sensor has outstanding features , including miniaturization , portability , and low cost . a custom - built lspr system was used for the first time in medical diagnostics in the field of gynecological oncology . the experiments described in this study demonstrate that a label - free lspr technique could serve as a very effective alternative to the label - based conventional elisa method . furthermore , direct detection of protein targets in human serum , which retains the native specific properties of antibodies or proteins without complicated procedures , makes the lspr method a very attractive strategy for cancer biomarker studies . future studies need to be performed to construct a serum calibration curve . a large , randomized , case - controlled clinical study can be used to evaluate the applicability of this biosensor in medical diagnostics for tumors such as ovarian cancer . the lspr biosensor is anticipated to be a promising platform for point - of - service medical diagnostics and should rival the commercially available instruments .

backgrounddetection of the human epididymis secretory protein 4 ( he4 ) biomarker plays an important role in the early diagnosis of ovarian cancer . this study aimed to develop a novel localized surface plasmon resonance ( lspr ) biosensor for detecting he4 in blood samples from patients with ovarian cancer.methodssilver nanoparticles were fabricated using a nanosphere lithography method . the anti - he4 antibody as a probe , which can distinctly recognize he4 , was assembled onto the nanochip surface using an amine coupling method . detection was based on the shift in the extinction maximum of the lspr spectrum before and after the he4-anti - he4 antibody reaction . these nanobiosensors were applied to detect he4 in human serum samples and compare them using an enzyme - linked immunosorbent assay.resultstests relating to the detection of he4 demonstrated that the lspr - based biosensor featured a fast detection speed , good specificity , effective reproducibility , and long - term stability . the linear range for lspr was between 10 pm and 10,000 pm , with a detection limit of 4 pm . an excellent correlation between lspr and enzyme - linked immunosorbent assay results was observed in human serum.conclusionthis study is the first clinical diagnostic application of the lspr biosensor in ovarian cancer . the lspr biosensor , a rapid , low - cost , label - free and portable screening tool , can serve as a very effective alternative for the clinical serological diagnosis of ovarian cancer .

Introduction Materials and methods Materials Patients and samples Preparation of LSPR biosensor and experimental setup Functionalization of LSPR biosensor Detection of HE4 by LSPR and ELISA Results and discussion LSPR response to immobilization process Calibration curve Selectivity, precision, regeneration, and stability tests Comparison of results from the LSPR system and ELISA Conclusion

since the advent of nanotechnology , nanoscale particle - based sensors have attracted tremendous attention from scientists , because of their unique optical and electrical properties.14 localized surface plasmon resonance ( lspr ) , which is recognized as one of the special optical properties of noble metallic nanoparticles ( eg , silver or gold ) , is generated when the incident photon frequency is resonant with the collective oscillation of conduction electrons.5 the lspr biosensor , a novel type of optical fiber - based biosensor , uses an optical fiber or optical fiber bundle to transform biological recognition information into analytically useful signals in the lspr spectrum , and has been proven to be an effective platform for detection techniques.5,6 the sensing principle is based on its sensitivity to local refractive index changes near the nanoparticle surface induced by biomolecular interactions.58 the applicability of this nanobiosensor has been studied in many fields , such as drug screening , medical diagnostics , and environmental monitoring , and has become a hot research topic all over the world.913 the detection of biotin - streptavidin and microalbumin in patients urine using the proposed domestic lspr biosensor has been reported previously , without quantitative analysis.14,15 however , to date , this biosensor has not been widely utilized in the field of gynecological oncology . according to the american cancer society , ovarian cancer accounts for about 4% of cancers occurring in women , but ranks fourth among the cancer - related deaths in women , because most cases are unfortunately diagnosed at an advanced stage.16 currently , ca125 is the only biomarker of ovarian cancer that is most widely and routinely used in clinical practice . however , the clinical use of ca125 as a marker for early detection is severely restricted , because it is elevated in only half of early - stage ovarian cancers and is elevated frequently in many benign gynecological diseases , such as endometriosis , ovarian cysts , and pelvic inflammation.17,18 recently , the human epididymis secretory protein 4 ( he4 ) , which is a novel biomarker for ovarian cancer , has been widely studied and used in the early diagnosis of ovarian cancer . reportedly , he4 is highly sensitive to early ovarian cancer and can be used in combination with ca125 , offering the best method of differential diagnosis in ovarian cancer and other pelvic masses.1921 currently used approaches for detection of he4 are the enzyme - linked immunosorbent assay ( elisa ) and the chemiluminescent immunoassay ( clia ) . although it is one of the most mature methods for protein detection used in the last three decades and is considered the gold standard , elisa still has certain shortcomings in terms of the long assay time required , the indirect detection format , and the need for multiple washing steps.22 clia also has some disadvantages , including the large volume of the analysis instrument , high cost , and special labeling requirements . thus , a rapid , label - free , simple , low - cost , and portable protocol for detecting he4 is urgently required . in the present work , the lspr biosensor developed was utilized based on silver nanoparticles for the direct detection of the he4 biomarker in blood samples from patients with ovarian cancer . under the optimum conditions , he4 in both buffer and human serum samples based on current information , this study is the first to investigate the lspr system for the detection of he4 . the study is also the first to discuss and analyze in detail the detection limit , linear range , and regeneration of the proposed homemade lspr sensor . mouse monoclonal anti - he4 antibody ( anti - he4 ) and standard he4 were obtained from abnova ( taiwan , china ) . the human serum specimens were collected from west china second university hospital ( chengdu , china ) . written informed consents were not obtained , because these samples were from leftover blood samples in routine blood tests , and this study did not cause any harm to the patients . the integrated lspr biosensor used in this work was a custom system built on - site , as previously described in detail.14,23 the silver nanochip was fabricated using the nanosphere lithography method . the peak wavelength of the lspr extinction spectrum ( max ) excited by the silver nanoparticles was measured and recorded using an ultraviolet - visible spectroscope ( sciencetech 9055 , sciencetech , ottawa , canada ) with a charge - coupled device detector ( koan electro - optics co , shanghai , china).14 the entire measurement process could be described as follows . the white light emerging from an optical fiber bundle and the transmitted light coupled into the detection probe of the optical fiber bundle provided the incident light . the nanochip was placed perpendicular to the incident light , which was taken using the ultraviolet - visible spectrometer ranging from 400 nm to 800 nm at room temperature in air.14 all the extinction spectra could be calculated through a software program ( ocean optics , dunedin , fl ) and directly displayed on the screen of the computer . the shift toward longer wavelengths , defined as a red shift , was indicated as ( + ) , whereas the shift toward shorter wavelengths , defined as a blue shift , was indicated as ( ) . the nanochip was then immersed in 75 mm edc/15 mm n - hydroxysuccinimide solution for 2 hours to activate the carboxyl groups of the self - assembled monolayer , which reacts with amino groups of antibodies to form amides . subsequently , 50 l of anti - he4 solution at 10 g / ml was spotted on the self - assembled monolayer - modified surface and overnight incubation at 4c followed . the anti - he4 immobilized surface was immersed in 1 m ethanolamine solution ( ph 8.5 ) for 30 minutes to deactivate the unreacted esters , after which the surface was washed with phosphate - buffered solution ( ph 7.4 ) and dried . a statistical evaluation of the correlation of the lspr and elisa methods was performed and computed using a software program ( origin 8.0 , originlab corporation , northampton , ma ) . mouse monoclonal anti - he4 antibody ( anti - he4 ) and standard he4 were obtained from abnova ( taiwan , china ) . written informed consents were not obtained , because these samples were from leftover blood samples in routine blood tests , and this study did not cause any harm to the patients . the integrated lspr biosensor used in this work was a custom system built on - site , as previously described in detail.14,23 the silver nanochip was fabricated using the nanosphere lithography method . the peak wavelength of the lspr extinction spectrum ( max ) excited by the silver nanoparticles was measured and recorded using an ultraviolet - visible spectroscope ( sciencetech 9055 , sciencetech , ottawa , canada ) with a charge - coupled device detector ( koan electro - optics co , shanghai , china).14 the entire measurement process could be described as follows . the white light emerging from an optical fiber bundle and the transmitted light coupled into the detection probe of the optical fiber bundle provided the incident light . the nanochip was placed perpendicular to the incident light , which was taken using the ultraviolet - visible spectrometer ranging from 400 nm to 800 nm at room temperature in air.14 all the extinction spectra could be calculated through a software program ( ocean optics , dunedin , fl ) and directly displayed on the screen of the computer . the shift toward longer wavelengths , defined as a red shift , was indicated as ( + ) , whereas the shift toward shorter wavelengths , defined as a blue shift , was indicated as ( ) . the nanochip was then immersed in 75 mm edc/15 mm n - hydroxysuccinimide solution for 2 hours to activate the carboxyl groups of the self - assembled monolayer , which reacts with amino groups of antibodies to form amides . subsequently , 50 l of anti - he4 solution at 10 g / ml was spotted on the self - assembled monolayer - modified surface and overnight incubation at 4c followed . the anti - he4 immobilized surface was immersed in 1 m ethanolamine solution ( ph 8.5 ) for 30 minutes to deactivate the unreacted esters , after which the surface was washed with phosphate - buffered solution ( ph 7.4 ) and dried . in the detection stage , the different concentrations of standard he4 ( 1 pm to 0.1 m ) and the patient samples were incubated on the functionalized lspr chip for 40 minutes , followed by a thorough rinsing with phosphate - buffered solution containing 0.05% tween-20 to dissociate the nonspecific binding . a statistical evaluation of the correlation of the lspr and elisa methods was performed and computed using a software program ( origin 8.0 , originlab corporation , northampton , ma ) . before modification , the lspr max of the bare silver nanochip a representative lspr max of the silver nanochip after modification with 11-mercaptoundecanoic acid was 619.85 nm with a corresponding lspr max of + 27.27 nm ( figure 3b ) . after anti - he4 immobilization , the lspr max shifted to 630.97 nm , with an additional 11.12 nm red shift ( figure 3c ) . this experimental evidence clearly showed that he4 in the buffer solution was detected successfully by the lspr biosensor . the optical characteristics of the nanosensor are notably based on the wavelength shift of the absorbance peak , max . according to mie theory , therefore , the change in the local refractive index that accompanies the molecular binding can be sensed by the nanoparticles , and the quantitative detection of targets can be achieved by monitoring the max when the analytes are bound to the nanoparticles.10 figure 4 shows a calibration curve of the nanosensor constructed by measuring the lspr wavelength shifts after exposure of the anti - he4 attachment surface to the he4 standard solutions of concentrations ranging from 1 pm to 0.1 m under optimal conditions . like many immunoassays , the curve is sigmoid rather than linear.25 in addition , a good linear relationship between the lspr shifts and the logarithm of the he4 concentration could be fitted to the experimental points from 10 pm to 10,000 pm ( inset of figure 4 ) . the linear regression equation was lspr ( nm ) = 3.72 log [ he4 ] ( m ) + 47.37 , with a linear correlation coefficient ( r ) of 0.997 . given that the noise level is defined as the standard deviation of the blank ( n = 12 ) , the limit of detection , defined as the analyte concentration corresponding to a signal - to - noise ratio of three ( about 5 nm ) , was estimated to be 4 pm . this limit of detection was more than sufficient for analysis of he4 in serum where normal values of he4 are considered to be less than 150 pm.26 this detection limit is a little better than that obtained using the elisa method for he4 ( 15 pm ) . therefore , the lspr biosensor had good analytical performance for the detection of he4 , and was comparable with elisa . the same concentration of he4 was also introduced to the nanosensor surface in the absence of anti - he4 . the results indicated that the selectivity of the lspr biosensor based on the highly specific antigen - antibody reaction and surface passivation with ethanolamine was excellent . the precision within and between batches is an important factor in the practical application of the biosensor.25 the intrarun precision was tested at two he4 concentration levels ( 500 pm and 5000 pm ) using the lspr biosensors of the same batch for five continuous measurements within one day . following completion of the antigen - antibody reaction , the nanochips were regenerated via exposure to 8 m urea solution , and then washed with ultrapure water . the reproducibility of responses from the identical biosensor and from different biosensors was evaluated by measuring 500 pm standard he4 solution , and the coefficient of variation for the lspr shift was obtained . this value indicates that the anti - he4-modified sensor can provide reliable detection of he4 . this drop in response seems to be related to gradual deactivation of the anti - he4 antibody . the silver surface is susceptible to oxidative damage , which could directly affect the stability of the lspr sensor . however , the nanochips were stable in air after chemical modification through to the end of the 4-week study . a series of ten human serum samples , five from the ovarian cancer group and five from the control group , were screened for serum he4 levels using the proposed lspr sensor and an elisa kit . meanwhile , red shifts less than 10.5 nm were observed in the negative controls . based on a t - test , differences in he4 levels between the ovarian cancer group and the control group detected were statistically significant ( p < 0.05 ) using both lspr and elisa . furthermore , the lspr sensor could specifically distinguish between ovarian cancer and the negative controls without the need for labeling in response to he4 binding in the sera tested . the lspr biosensor clearly has good specificity that can not be disrupted by other proteins or components in serum . the concentration of the ten samples was found to be within the range of 11.39911.16 pm , which was also within the linear range of the lspr sensor . the results indicate that the lspr biosensor could serve as a good alternative to the laborious and time - consuming elisa method for direct detection of he4 . before modification , the lspr max of the bare silver nanochip a representative lspr max of the silver nanochip after modification with 11-mercaptoundecanoic acid was 619.85 nm with a corresponding lspr max of + 27.27 nm ( figure 3b ) . after anti - he4 immobilization , the lspr max shifted to 630.97 nm , with an additional 11.12 nm red shift ( figure 3c ) . after incubation in 500 pm he4 , the lspr wavelength shifted to + 14.48 nm , showing a max of 645.45 nm ( figure 3d ) . this experimental evidence clearly showed that he4 in the buffer solution was detected successfully by the lspr biosensor . the optical characteristics of the nanosensor are notably based on the wavelength shift of the absorbance peak , max . according to mie theory , therefore , the change in the local refractive index that accompanies the molecular binding can be sensed by the nanoparticles , and the quantitative detection of targets can be achieved by monitoring the max when the analytes are bound to the nanoparticles.10 figure 4 shows a calibration curve of the nanosensor constructed by measuring the lspr wavelength shifts after exposure of the anti - he4 attachment surface to the he4 standard solutions of concentrations ranging from 1 pm to 0.1 m under optimal conditions . like many immunoassays , the curve is sigmoid rather than linear.25 in addition , a good linear relationship between the lspr shifts and the logarithm of the he4 concentration could be fitted to the experimental points from 10 pm to 10,000 pm ( inset of figure 4 ) . the linear regression equation was lspr ( nm ) = 3.72 log [ he4 ] ( m ) + 47.37 , with a linear correlation coefficient ( r ) of 0.997 . given that the noise level is defined as the standard deviation of the blank ( n = 12 ) , the limit of detection , defined as the analyte concentration corresponding to a signal - to - noise ratio of three ( about 5 nm ) , was estimated to be 4 pm . this limit of detection was more than sufficient for analysis of he4 in serum where normal values of he4 are considered to be less than 150 pm.26 this detection limit is a little better than that obtained using the elisa method for he4 ( 15 pm ) . therefore , the lspr biosensor had good analytical performance for the detection of he4 , and was comparable with elisa . the same concentration of he4 was also introduced to the nanosensor surface in the absence of anti - he4 . the results indicated that the selectivity of the lspr biosensor based on the highly specific antigen - antibody reaction and surface passivation with ethanolamine was excellent . the precision within and between batches is an important factor in the practical application of the biosensor.25 the intrarun precision was tested at two he4 concentration levels ( 500 pm and 5000 pm ) using the lspr biosensors of the same batch for five continuous measurements within one day . following completion of the antigen - antibody reaction , the nanochips were regenerated via exposure to 8 m urea solution , and then washed with ultrapure water . the reproducibility of responses from the identical biosensor and from different biosensors was evaluated by measuring 500 pm standard he4 solution , and the coefficient of variation for the lspr shift was obtained . this value indicates that the anti - he4-modified sensor can provide reliable detection of he4 . this drop in response seems to be related to gradual deactivation of the anti - he4 antibody . the silver surface is susceptible to oxidative damage , which could directly affect the stability of the lspr sensor . a series of ten human serum samples , five from the ovarian cancer group and five from the control group , were screened for serum he4 levels using the proposed lspr sensor and an elisa kit . meanwhile , red shifts less than 10.5 nm were observed in the negative controls . based on a t - test , differences in he4 levels between the ovarian cancer group and the control group detected were statistically significant ( p < 0.05 ) using both lspr and elisa . furthermore , the lspr sensor could specifically distinguish between ovarian cancer and the negative controls without the need for labeling in response to he4 binding in the sera tested . the lspr biosensor clearly has good specificity that can not be disrupted by other proteins or components in serum . the concentration of the ten samples was found to be within the range of 11.39911.16 pm , which was also within the linear range of the lspr sensor . the analytical curve was calibrated using the correlation equation , ie , y = 83.88x 677.07 , with a correlation coefficient of 0.926 . the results indicate that the lspr biosensor could serve as a good alternative to the laborious and time - consuming elisa method for direct detection of he4 . ideal biosensors should be rapid , sensitive , specific , label - free , stable , reproducible , cheap , portable , and easy to operate . the lspr biosensor has significant advantages in terms of label - free biomarker detection , a rapid test time , and in a direct assay format unlike the traditional immunoassay approaches , such as elisa . compared with chemiluminescence analysis and current commercial surface plasmon resonance sensors , the lspr sensor has outstanding features , including miniaturization , portability , and low cost . a custom - built lspr system was used for the first time in medical diagnostics in the field of gynecological oncology . the experiments described in this study demonstrate that a label - free lspr technique could serve as a very effective alternative to the label - based conventional elisa method . furthermore , direct detection of protein targets in human serum , which retains the native specific properties of antibodies or proteins without complicated procedures , makes the lspr method a very attractive strategy for cancer biomarker studies .

today , clinical research is strongly regulated with the aim of ensuring that clinical trials are well designed and conducted in an ethical manner . in europe , several guidelines , directives and regulations pertaining to clinical trials have been released by the european parliament , council and commission [ 13 ] . one in particular , directive 2001/20/ec , establishes specific provisions regarding the conduct of clinical trials of medicinal products on human subjects and recognises the principles of good clinical practice . in line with this , many medicines have been developed based on the results of well - designed , randomised , controlled clinical trials , and these treatments are currently used in a range of patient groups , offering considerable clinical and economic utility in healthcare . that said , however , it is widely acknowledged that not all patients respond in the same way to a given therapy and , as a result , the current methodological approach will require updating in future to take account of individual patient characteristics . for many adult and some childhood conditions , biomarkers that reliably reflect inter - individual variability in disease expression and that also correlate with or predict outcomes have already been discovered among the genes , mrna , proteins and metabolites found in the body . polymorphisms in the human leukocyte antigen ( hla ) , for example , have been identified as risk factors for severe adverse drug reactions ( adrs ) : these include the association between hla - b*1502 and carbamazepine - induced skin necrolysis and stevens johnson syndrome , as well as the association between hla - b*5701 and hypersensitivity reactions to the hiv drug , abacavir [ 5 , 6 ] . screening for both of these alleles is recommended before starting the respective treatment and , in the case of hla - b*5701 , screening prior to abacavir initiation has been suggested to produce cost savings [ 6 , 7 ] . in future , it is expected that such biomarkers will be detected more readily thanks to the new high - throughput technologies and powerful analytical approaches employed within the fields of pharmacogenomics , proteomics , metabolomics and other so - called omics. this type of genetic knowledge may ultimately be included within the drug development process where biomarkers will serve as identifiers of treatment response . as a result , it will become important to include population screening early in the clinical trial development phase in order to establish which subpopulations receiving an experimental drug are most / least likely to benefit from , or to experience adverse reactions associated with , that treatment . refinement of this approach will lead to more targeted patient recruitment and should reduce the likelihood of new therapies failing at the clinical trial stage . most importantly , it should reduce the risk of unsuccessful treatments or undesired secondary adverse effects in clinical practice . whether pharmacodiagnostic ( theranostic ) applications can simplify the selection of drugs for clinical testing , and help to develop more focused clinical trials that provide enhanced knowledge about the effects of a drug in humans within shorter time frames , are currently being explored by various organisations such as the national institutes of health ( nih ) . the use of biomarkers as indicators of genetic factors affecting drug response is of particular interest for children . historically , despite the need for treatments for conditions affecting children , for ethical , economic and methodological reasons , there have been few clinical trials in young populations [ 9 , 10 ] . as a result , off - label drug use in paediatric patients has been relatively common [ 9 , 11 , 12 ] . an example of this is the fact that metabolic capacity is different in children and adults . hepatic glucoronidation activity of the udp - glucuronosyltransferase ( ugt ) family of enzymes that are needed for the elimination of certain xenobiotic and endogenous substances , for instance , is lower in children than adults which may have implications for drug detoxification . ugt activity towards bilirubin at birth , for example , is approximately 1% of the level observed in adults , although activity increases rapidly and reaches levels equivalent to those in adults 14 weeks after birth . total ugt activity has been reported to develop to adult levels by 20 months of age ; however , that age at which enzymes reach adult levels of activity may not be universally indicative of in vivo drug clearance . this study showed that intrinsic hepatic clearance of the antipsychotic drug trifluoperazine did not appear to reach adult levels until 18.9 years of age despite ugt being maximally active well before this time . these types of infancy / childhood - specific developmental changes are clearly complex and likely underlie some of the variability in drug response seen in neonates and children . failing to take the potential impact of these differences on drug action into account places children at risk of adrs . in recent years there has in fact been a rise in the number of paediatric drug safety and efficacy studies , as well as changes in drug labelling for young patients that show unique paediatric doses are often necessary [ 17 , 18 ] . furthermore , some diseases that occur in children either do not develop in adults or have a different effect / prevalence in adults , making it necessary to develop drugs specifically for children . despite the clear need for individualised medicine for paediatric patient populations , we still know very little about developmental pharmacogenomics and there have been few applications of biomarkers in the management of paediatric disease . results of a literature search conducted by the task force in europe for drug development for the young ( teddy ) european network of excellence demonstrated that throughout the world more than 50% of pharmacogenomic / pharmacogenetic research activities in children during the last 10 years were related to predisposition , which means the investigation of the correlation between genetic traits and the probability of or susceptibility for a given pathology or disease . these types of exploratory studies provide little or no insight into mechanisms of disease or drug action , and their results can not be used to improve medical practice or to support therapeutic solutions . only a small number of polymorphisms that are linked to therapeutic response have been studied in children and , even when studies demonstrate an impact on treatment response , their use in clinical practice remains limited . this is highlighted by the fact that very few fda drug label amendments for pharmacogenomic testing refer to paediatric populations . a major consideration that has arisen from analyses of publications on pharmacogenetics and pharmacogenomics is that studies often show contradictory or inconsistent results . this is particularly the case for studies in paediatric populations which , by definition , generally have limited patient numbers . a systematic review and field synopsis of pharmacogenetic studies in general that was conducted in 2009 suggested that the limited translation of pharmacogenetic research into practice may be explained by the preponderance of reviews over primary research , small sample sizes , a mainly candidate gene approach , the use of surrogate markers , an excess of nominally positive or truly positive associations and paucity of meta - analyses . the small sample sizes , coupled with the more frequent evaluation of common rather than rare variants , the use of surrogate outcome measures rather than more clinically relevant outcomes , and subgroup analysis with multiple hypothesis testing , suggest that only a proportion of the positive associations reported are in fact genuine . clear analysis of the situation is further complicated by the use of different research methods across studies , the lack of both standardised outcome measures and study replication , and the low level of consideration given to potential covariates such as co - morbidities . other investigators have similarly raised concerns about the need to improve the quality of studies and prevent false - positive associations . in conclusion , while the new omics techniques offer great promise for the identification of biomarkers of drug response , these studies are subject to many of the same pitfalls that apply in general to paediatric and other clinical trials . notwithstanding the limited experience and considerations above , some exciting examples of the usefulness of biomarkers in clinical practice can be derived from recent published literature . childhood disorders in which pharmacogenetic studies have most commonly been conducted include acute lymphoblastic leukaemia , attention deficit hyperactivity disorder , growth hormone deficiency , juvenile idiopathic arthritis , atopic dermatitis and asthma ( see [ 19 , 22 , 23 ] for reviews ) . a good example of a childhood condition where there will be benefits from the use of pharmacogenetics is acute lymphoblastic leukaemia ( all ) . current treatment for patients with all results in event - free survival greater than 75% for most patients ; however , approximately 25% fail to respond to treatment . one of the reasons for treatment failure is the development of toxicity due to the absence or presence of only low levels of enzymes that metabolise chemotherapies . thiopurines ( azathioprine , mercaptopurine and thioguanine ) , for example , are often used to treat all and their safety has been shown to be related to thiopurine methyltransferase ( tpmt ) genotype . genotypes resulting in reduced tpmt enzyme activity have been shown to be associated with dose - related side effects / toxic events to thiopurines in children with all [ 2527 ] . thus , tpmt genotype may be used to help choose the appropriate dose of thiopurine for an individual child . in the us , the drug labels for azathioprine and 6-mercaptopurine now give the fda recommendation for tpmt testing . although this biomarker is not yet reliably assessed in children within europe , because tpmt - deficiency can now be easily detected , this knowledge should lead to improvements in the safety of treatment in children receiving thiopurine chemotherapy . the recent publication of clinical pharmacogenetics implementation consortium guidelines for tpmt genotyping and thiopurine dosing may also facilitate the routine incorporation of tpmt genotyping in clinical practice for paediatric patients receiving these compounds . there are several pharmacogenomic / pharmacogenetic networks already in existence that aim to help collect , coordinate or communicate information about genetic determinants of drug response . in europe , for example , there is the european network for pharmacogenetics ( http://www.epr-network.org/ ) , in the us there is the nih pharmacogenomics research network ( http://www.nigms.nih.gov/initiatives/pgrn ) , in canada , the canadian pharmacogenomics network for drug safety ( cpnds ) , and there is also the international hapmap project ( http://hapmap.ncbi.nlm.nih.gov/ ) that represents a partnership of researchers and funding agencies from several countries . each of these networks is distinct ; however , the activities of the cpnds illustrate how the establishment of networks may play an important role in the development of personalised medicine . the cpnds is an active surveillance network that collects information on adrs , and operates predominantly within canadian paediatric teaching hospitals [ 9 , 29 ] . the aim of the network is to improve drug safety by identifying genetic markers that can predict which patients are likely to experience specific serious adrs , as well as those who may experience decreased therapeutic response to a drug . to help determine the role of genetic variations in the development of adrs , the network has created a database of clinical adrs and a bio - bank of dna and other samples . among other examples , this approach has already proven useful by contributing to the development of a research protocol to validate reports of a potential association between genetic variations in enzymes in the cyp and ugt families in breastfeeding women taking codeine and the development of codeine - induced central nervous system ( cns ) depression in their babies . the development of databases containing information about genomic positions and bio - banks containing tissue samples from large numbers of individuals also make it possible to conduct genome - wide association studies [ 19 , 31 ] . rather than selecting candidate genes , this type of investigation can look at thousands of single nucleotide polymorphisms across the genome in one investigation which may represent an important approach for studying complex , common diseases where multiple genetic variations may contribute to drug response . as mentioned previously , despite the fact that many biomarkers are currently available for use within the clinical research setting , few have been integrated into paediatric clinical practice . in addition , the validation process of such markers in children remains an issue . as a consequence , it is important to encourage the establishment of paediatric bio - banks , as well as the connection of existing bio - banks through intelligent informatics platforms in order to share data , integrate results and promote the use of biomarkers in drug development and clinical trials . furthermore , the uptake of biomarkers within the drug development process requires investment and research on the part of the pharmaceutical industry . as of 2007 , very few pharmaceutical companies indicated ongoing pharmacogenomic or pharmacogenetic - related research in children . however , more recently an investigation performed by tufts suggests a growing interest and investment in this area . their report shows that 12%50% of current clinical pipelines of the companies interviewed involve personalised medicines . thus , although at present paediatric drug development and utilisation can not benefit fully from pharmacogenetic / pharmacogenomic findings there is hope for the future . particular actions that have to be undertaken in order to reduce the existing gaps include : the identification of the most promising research approaches for use in paediatric populations from among those based on omics sciences; the development of model - based methodologies for clinical trials in children based on the use of omics-derived biomarkers; the initiation and completion of pilot studies designed with the goal of developing paediatric personalised medicines in a specific sector such as oncology , respiratory disease , neurodegenerative disease or infective disease . the identification of the most promising research approaches for use in paediatric populations from among those based on omics sciences ; the development of model - based methodologies for clinical trials in children based on the use of omics-derived biomarkers ; the initiation and completion of pilot studies designed with the goal of developing paediatric personalised medicines in a specific sector such as oncology , respiratory disease , neurodegenerative disease or infective disease . the development of personalised medicine is of relevance for all paediatric patient groups , and in particular for those populations that are generally deprived of drugs when the traditional clinical trial approach is applied . examples of populations for whom the personalised medicine approach will be particularly important are neonates and children with rare diseases . in the european union ( eu ) , a rare disease is defined as one that affects fewer than five people per 10,000 . most of these conditions are genetically inherited and , importantly , many develop during childhood . although individually uncommon , collectively there are thousands of rare diseases , making them an important group of disorders that affect a large number of patients throughout europe . in the context that we have so far identified somewhere in the region of 5,0008,000 distinct rare diseases , between 27 and 36 million people in the eu could be affected ( 6%8% of the population ) , making the management of these conditions an important issue . for these reasons , the eu takes the position that rare diseases are a serious public health concern and should be a priority in eu health and research programmes . although neurodegenerative diseases are most prevalent in the elderly , in rare cases they also affect individuals early in life . in children , neurodegeneration leads to severe mental retardation and premature death with devastating consequences on their immediate environment . these conditions also have relatively high costs for society . in total , there are thought to be about 600 different genetically inherited metabolic diseases , approximately 400 of which involve the cns with differing degrees of severity and rates of degeneration . lysosomal storage diseases ( lsds ) are a subgroup of approximately 40 of these metabolic disorders that affect about one in 7,500 newborns [ 34 , 35 ] , although more recently a combined incidence as high as one in 5,000 newborns has been suggested . the proportion of patients diagnosed with different lsds in australia between 1980 and 1996 is shown in fig . 1 . these conditions are caused by the lack of certain ( lysosomal ) enzymes or lysosome components , thus preventing the complete degradation of macromolecules and the recycling of their components . as a result of the failure to degrade these molecules , intermediate degradation products begin to accumulate within the cells , affecting the function of lysosomes and other cellular organelles . the accumulation of these proteins starts immediately after birth and continues throughout life , often altering the structure and impairing the function of several organs , including the cns ( fig . 2 ) . cns pathology causes mental retardation and progressive neurodegeneration that ultimately ends in early death of these young patients [ 3741 ] . fig . 1proportion of patients diagnosed with different lysosomal storage diseases ( lsds ) in australia between 1980 and 1996 . more than ten patients were identified for 18 of the 27 lsds diagnosed during this period . based on data from meikle et al . . images include brain scans showing : a atrophy with enlarged virchow robin spaces and ventricles , b communicating hydrocephalus , and c spinal cord compression at the cervical junctions . other images show d corneal clouding , e larynx thickening , f valvular alterations due to the accumulation of undegraded substrates , and g i dysostosis multiplex of back bone , hand and long bones proportion of patients diagnosed with different lysosomal storage diseases ( lsds ) in australia between 1980 and 1996 . more than ten patients were identified for 18 of the 27 lsds diagnosed during this period . based on data from meikle et al . . mps i , mucopolysaccharidosis type i impact of the accumulation of undegraded proteins in lysosomal storage diseases . images include brain scans showing : a atrophy with enlarged virchow robin spaces and ventricles , other images show d corneal clouding , e larynx thickening , f valvular alterations due to the accumulation of undegraded substrates , and g i dysostosis multiplex of back bone , hand and long bones our understanding of the pathogenesis of the lsds has grown dramatically over the last 1020 years and there is already a strong community supporting research and scientific developments in this area , which includes physicians , researchers , patient advocates and industry . thanks largely to advances in cellular and molecular biology , and the incentives for drug development provided by the introduction of the orphan drug regulation in europe , we are now moving closer to being able to provide therapies for neurological signs and symptoms in patients with these conditions . the advanced state of knowledge of the mechanisms of action of lsds , in particular , is allowing the elucidation of features of neurodegenerative diseases that will most likely also prove to be useful in the study of more prevalent neurodegenerative diseases that predominantly affect the elderly ( e.g. alzheimer s and parkinson s diseases ) . furthermore , secondary events that lead to neurodegeneration ( e.g. brain inflammation , alteration of intracellular trafficking , impairment of autophagy , or oxidative stress ) are common to both paediatric and adult neurodegenerative diseases ( for a review see ) therefore , at least some of the observations relative to loss and recovery of brain plasticity in paediatric neurodegeneration can be extrapolated to adult disorders , and findings will provide unique insights into the pathophysiology and restorative capacities of neurodegenerative diseases in general . in fact , suppressing the primary cause of neurodegeneration ( e.g. by supplementing the missing enzyme ) in a young brain has the potential to maximise benefit as the brain retains considerable plasticity at this stage of development . there are several factors that make the lsds good candidates for pharmacogenetic studies and personalised medicine . the first is that the genetic basis of most of these conditions is well defined ; they are monogenic disorders and many disease - causing mutations have already been characterised . although these mutations are often private , they all result in reduced enzyme levels and storage to varying degrees . despite a current general lack of awareness of these conditions among physicians which can lead to delayed diagnosis , there is the potential for pre - symptomatic diagnosis and , in many cases , prenatal diagnosis [ 4244 ] . this means that once biomarkers that predict therapeutic response have been identified , it will be possible to initiate the appropriate treatment at an early stage of disease . furthermore , for many of the lsds there is some overlap in the pathways that are involved in the degradation of the proteins that accumulate , making it likely that any biomarkers discovered may apply in more than one condition . another factor is that patients show considerable phenotypic variability ( even those with the same underlying genetic mutation ) and there is similarly variation between individuals in terms of response to therapy . at present , many different therapeutic approaches have been proposed including replacement of the missing enzyme ( enzyme replacement therapy , ert ) , reduction of levels of the enzyme substrate ( substrate reduction therapy , srt ) , the use of chemical chaperones to stabilise existing but unstable enzyme ( chaperone therapy ) , bone marrow transplantation , and gene therapies [ 4547 ] ( fig . 3 ) . based on current knowledge , predicting which patients will respond to which treatment remains a challenge . 3routes of intervention for the treatment of patients with lysosomal storage diseases routes of intervention for the treatment of patients with lysosomal storage diseases in order to make personalised medicine for children with rare neurological disorders a reality it is necessary to refine the techniques used to ensure that therapy gains access to the cns . although it is known which enzyme deficiency causes each of the lsds , supplying a correct version of the affected lysosomal enzyme intravenously only allows for modification of the natural history of these diseases in the peripheral organs ( e.g. liver , spleen , joints , etc . ) . the reason for this is that the therapeutic enzyme molecules used in ert are too large to cross the blood brain - barrier which protects the brain by allowing only those molecules required naturally by the cns to pass into the brain from the peripheral circulation . proposed methods to circumvent the blood brain - barrier in patients with lsds include the direct injection into the brain of either the recombinant enzyme itself or of gene transfer vehicles able to induce the synthesis of the enzyme in genetically deficient brain cells . another approach is to inhibit enzymes early in the biosynthetic pathway of the accumulating protein(s ) to reduce the number of molecules that require degradation in the lysosome . small pharmaceutical molecules that are capable of crossing the blood brain barrier are used to achieve this . in cases where the affected lysosomal enzyme is present but does not function because it is unstable , small pharmaceutical molecules known as chemical chaperones that can cross the blood brain - barrier may be used to stabilise the enzyme , thus at least partially restoring its function . as the process of neurodegeneration observed in lsds is characterised by secondary events such as neuroinflammation , glutamatergic activation and oxidative stress , the use of small molecules with anti - inflammatory and/or neuroprotective properties may also be a useful adjunctive approach to treat lsds . to date , the utility of most of these approaches has already been demonstrated in animal models . looking to the future , there is also a need to establish and validate biomarkers that will allow us to understand both disease pathophysiology and which patients are most likely to respond to the different therapies that are available . although we are currently able to quantify the stored protein material and to assess changes in storage in response to treatment , in general , these variables have not proved to be reliable biomarkers for the lsds . other candidate biomarkers are already under investigation . in the future , the availability of biomarkers other than the stored material , will allow the choice of the most appropriate therapy ( ert , chaperone therapy or srt ) when taking into account patient phenotype and expected disease severity and course of progression . once a therapy is developed that can slow or prevent the progression of neurodegeneration , it will become extremely important to facilitate early detection of the genetic defect by newborn screening to enable early intervention . the development of new tools / technologies to diagnose patients and to predict disease severity and progression , as well as to assess new therapies , can be achieved only through collaboration between existing stakeholders . the first steps towards establishing a community to focus efforts on developing therapies for rare neurological diseases of childhood were taken in december 2010 at a meeting held at the european parliament in brussels . this meeting , which was organised by the brains for brain foundation [ b4b ( www.brains4brain.eu ) ] , the european brain council ( ebc ) , the lysosomal storage disease ( lsd ) patient collaborative and the veneto region , and sponsored by member of the european parliament , mrs amalia sartori ( veneto , italy ) , brought together stakeholders from 13 eu countries , as well as the usa and brazil . those registered for the meeting included 22 political representatives and members of 17 scientific organisations , 14 european family organisations , 10 biotechnology companies and over 40 european universities . the organisers called for a new initiative to coordinate the efforts of all existing groups in europe with the shared goal of improving the treatment and care of patients with rare neurological disorders . as commented in a recent editorial , there is a need for harmonised interaction between all stakeholders to enable the further development of personalised medicine . in line with this , the present project will unite all interested parties , who are working to increase the visibility , recognition and awareness of rare neurological disorders in order to facilitate early diagnosis of these conditions ; to promote and facilitate partnership and collaboration between physicians , researchers , patient advocates , carers , policy- and decision - makers and industry ; to encourage and support research and the translation of scientific breakthroughs into clinical practice ; to contribute to the establishment of a standard of care for patients with rare neurological disorders which is agreed across europe ; and to ensure equity of access to diagnosis , treatment and care . achieving better and more predictable treatment outcomes offers benefits in terms of reducing unnecessary treatment and side effects and may ultimately lead to more cost effective healthcare . in order to progress the development of personalised medicine it is necessary for these benefits to be clearly demonstrated and for there to be buy - in and investment from all stakeholders , including the pharmaceutical industry , regulatory authorities and payers . the increasing incorporation of biomarkers within product pipelines is a sign of the growing interest of the pharmaceutical industry in this area . nevertheless , there is still work to do in order to realise the potential of this approach and there remains a need for more examples that demonstrate proof of concept in clinical practice .

the development of personalised medicine is of considerable importance for paediatric patient populations , and represents a move away from the use of treatment dosages based on experience with the same compounds in adults . currently , however , we know little about developmental pharmacogenomics and , although many biomarkers are available for clinical research use , there have been few applications in the management of paediatric diseases . this paper reviews where we are in the journey towards achieving paediatric personalised medicine and describes a group of diseases requiring such an approach . the personalised medicine approach is particularly relevant for the treatment of rare childhood diseases , and the group of life - threatening neurological disorders known as lysosomal storage diseases represents a potential study population . the genetic bases of these disorders are generally well defined , there is the potential for diagnosis at birth or prenatally , and there are a range of therapeutic options available or under development .

Introduction The use of biomarkers in paediatrics What is available and what is needed to develop personalised medicines? Choice of a model disease to study personalised medicine in children: the need for personalised medicine for children with rare neurological diseases Concluding remarks and outlook

today , clinical research is strongly regulated with the aim of ensuring that clinical trials are well designed and conducted in an ethical manner . in line with this , many medicines have been developed based on the results of well - designed , randomised , controlled clinical trials , and these treatments are currently used in a range of patient groups , offering considerable clinical and economic utility in healthcare . that said , however , it is widely acknowledged that not all patients respond in the same way to a given therapy and , as a result , the current methodological approach will require updating in future to take account of individual patient characteristics . polymorphisms in the human leukocyte antigen ( hla ) , for example , have been identified as risk factors for severe adverse drug reactions ( adrs ) : these include the association between hla - b*1502 and carbamazepine - induced skin necrolysis and stevens johnson syndrome , as well as the association between hla - b*5701 and hypersensitivity reactions to the hiv drug , abacavir [ 5 , 6 ] . screening for both of these alleles is recommended before starting the respective treatment and , in the case of hla - b*5701 , screening prior to abacavir initiation has been suggested to produce cost savings [ 6 , 7 ] . this type of genetic knowledge may ultimately be included within the drug development process where biomarkers will serve as identifiers of treatment response . whether pharmacodiagnostic ( theranostic ) applications can simplify the selection of drugs for clinical testing , and help to develop more focused clinical trials that provide enhanced knowledge about the effects of a drug in humans within shorter time frames , are currently being explored by various organisations such as the national institutes of health ( nih ) . the use of biomarkers as indicators of genetic factors affecting drug response is of particular interest for children . historically , despite the need for treatments for conditions affecting children , for ethical , economic and methodological reasons , there have been few clinical trials in young populations [ 9 , 10 ] . hepatic glucoronidation activity of the udp - glucuronosyltransferase ( ugt ) family of enzymes that are needed for the elimination of certain xenobiotic and endogenous substances , for instance , is lower in children than adults which may have implications for drug detoxification . ugt activity towards bilirubin at birth , for example , is approximately 1% of the level observed in adults , although activity increases rapidly and reaches levels equivalent to those in adults 14 weeks after birth . total ugt activity has been reported to develop to adult levels by 20 months of age ; however , that age at which enzymes reach adult levels of activity may not be universally indicative of in vivo drug clearance . failing to take the potential impact of these differences on drug action into account places children at risk of adrs . in recent years there has in fact been a rise in the number of paediatric drug safety and efficacy studies , as well as changes in drug labelling for young patients that show unique paediatric doses are often necessary [ 17 , 18 ] . furthermore , some diseases that occur in children either do not develop in adults or have a different effect / prevalence in adults , making it necessary to develop drugs specifically for children . despite the clear need for individualised medicine for paediatric patient populations , we still know very little about developmental pharmacogenomics and there have been few applications of biomarkers in the management of paediatric disease . results of a literature search conducted by the task force in europe for drug development for the young ( teddy ) european network of excellence demonstrated that throughout the world more than 50% of pharmacogenomic / pharmacogenetic research activities in children during the last 10 years were related to predisposition , which means the investigation of the correlation between genetic traits and the probability of or susceptibility for a given pathology or disease . these types of exploratory studies provide little or no insight into mechanisms of disease or drug action , and their results can not be used to improve medical practice or to support therapeutic solutions . only a small number of polymorphisms that are linked to therapeutic response have been studied in children and , even when studies demonstrate an impact on treatment response , their use in clinical practice remains limited . this is particularly the case for studies in paediatric populations which , by definition , generally have limited patient numbers . a systematic review and field synopsis of pharmacogenetic studies in general that was conducted in 2009 suggested that the limited translation of pharmacogenetic research into practice may be explained by the preponderance of reviews over primary research , small sample sizes , a mainly candidate gene approach , the use of surrogate markers , an excess of nominally positive or truly positive associations and paucity of meta - analyses . the small sample sizes , coupled with the more frequent evaluation of common rather than rare variants , the use of surrogate outcome measures rather than more clinically relevant outcomes , and subgroup analysis with multiple hypothesis testing , suggest that only a proportion of the positive associations reported are in fact genuine . clear analysis of the situation is further complicated by the use of different research methods across studies , the lack of both standardised outcome measures and study replication , and the low level of consideration given to potential covariates such as co - morbidities . in conclusion , while the new omics techniques offer great promise for the identification of biomarkers of drug response , these studies are subject to many of the same pitfalls that apply in general to paediatric and other clinical trials . a good example of a childhood condition where there will be benefits from the use of pharmacogenetics is acute lymphoblastic leukaemia ( all ) . one of the reasons for treatment failure is the development of toxicity due to the absence or presence of only low levels of enzymes that metabolise chemotherapies . genotypes resulting in reduced tpmt enzyme activity have been shown to be associated with dose - related side effects / toxic events to thiopurines in children with all [ 2527 ] . in the us , the drug labels for azathioprine and 6-mercaptopurine now give the fda recommendation for tpmt testing . although this biomarker is not yet reliably assessed in children within europe , because tpmt - deficiency can now be easily detected , this knowledge should lead to improvements in the safety of treatment in children receiving thiopurine chemotherapy . the recent publication of clinical pharmacogenetics implementation consortium guidelines for tpmt genotyping and thiopurine dosing may also facilitate the routine incorporation of tpmt genotyping in clinical practice for paediatric patients receiving these compounds . there are several pharmacogenomic / pharmacogenetic networks already in existence that aim to help collect , coordinate or communicate information about genetic determinants of drug response . in europe , for example , there is the european network for pharmacogenetics ( http://www.epr-network.org/ ) , in the us there is the nih pharmacogenomics research network ( http://www.nigms.nih.gov/initiatives/pgrn ) , in canada , the canadian pharmacogenomics network for drug safety ( cpnds ) , and there is also the international hapmap project ( http://hapmap.ncbi.nlm.nih.gov/ ) that represents a partnership of researchers and funding agencies from several countries . each of these networks is distinct ; however , the activities of the cpnds illustrate how the establishment of networks may play an important role in the development of personalised medicine . to help determine the role of genetic variations in the development of adrs , the network has created a database of clinical adrs and a bio - bank of dna and other samples . among other examples , this approach has already proven useful by contributing to the development of a research protocol to validate reports of a potential association between genetic variations in enzymes in the cyp and ugt families in breastfeeding women taking codeine and the development of codeine - induced central nervous system ( cns ) depression in their babies . the development of databases containing information about genomic positions and bio - banks containing tissue samples from large numbers of individuals also make it possible to conduct genome - wide association studies [ 19 , 31 ] . as mentioned previously , despite the fact that many biomarkers are currently available for use within the clinical research setting , few have been integrated into paediatric clinical practice . as a consequence , it is important to encourage the establishment of paediatric bio - banks , as well as the connection of existing bio - banks through intelligent informatics platforms in order to share data , integrate results and promote the use of biomarkers in drug development and clinical trials . thus , although at present paediatric drug development and utilisation can not benefit fully from pharmacogenetic / pharmacogenomic findings there is hope for the future . particular actions that have to be undertaken in order to reduce the existing gaps include : the identification of the most promising research approaches for use in paediatric populations from among those based on omics sciences; the development of model - based methodologies for clinical trials in children based on the use of omics-derived biomarkers; the initiation and completion of pilot studies designed with the goal of developing paediatric personalised medicines in a specific sector such as oncology , respiratory disease , neurodegenerative disease or infective disease . the identification of the most promising research approaches for use in paediatric populations from among those based on omics sciences ; the development of model - based methodologies for clinical trials in children based on the use of omics-derived biomarkers ; the initiation and completion of pilot studies designed with the goal of developing paediatric personalised medicines in a specific sector such as oncology , respiratory disease , neurodegenerative disease or infective disease . the development of personalised medicine is of relevance for all paediatric patient groups , and in particular for those populations that are generally deprived of drugs when the traditional clinical trial approach is applied . examples of populations for whom the personalised medicine approach will be particularly important are neonates and children with rare diseases . most of these conditions are genetically inherited and , importantly , many develop during childhood . although individually uncommon , collectively there are thousands of rare diseases , making them an important group of disorders that affect a large number of patients throughout europe . in the context that we have so far identified somewhere in the region of 5,0008,000 distinct rare diseases , between 27 and 36 million people in the eu could be affected ( 6%8% of the population ) , making the management of these conditions an important issue . for these reasons , the eu takes the position that rare diseases are a serious public health concern and should be a priority in eu health and research programmes . although neurodegenerative diseases are most prevalent in the elderly , in rare cases they also affect individuals early in life . in total , there are thought to be about 600 different genetically inherited metabolic diseases , approximately 400 of which involve the cns with differing degrees of severity and rates of degeneration . lysosomal storage diseases ( lsds ) are a subgroup of approximately 40 of these metabolic disorders that affect about one in 7,500 newborns [ 34 , 35 ] , although more recently a combined incidence as high as one in 5,000 newborns has been suggested . these conditions are caused by the lack of certain ( lysosomal ) enzymes or lysosome components , thus preventing the complete degradation of macromolecules and the recycling of their components . the accumulation of these proteins starts immediately after birth and continues throughout life , often altering the structure and impairing the function of several organs , including the cns ( fig . cns pathology causes mental retardation and progressive neurodegeneration that ultimately ends in early death of these young patients [ 3741 ] . 1proportion of patients diagnosed with different lysosomal storage diseases ( lsds ) in australia between 1980 and 1996 . other images show d corneal clouding , e larynx thickening , f valvular alterations due to the accumulation of undegraded substrates , and g i dysostosis multiplex of back bone , hand and long bones proportion of patients diagnosed with different lysosomal storage diseases ( lsds ) in australia between 1980 and 1996 . based on data from meikle et al . mps i , mucopolysaccharidosis type i impact of the accumulation of undegraded proteins in lysosomal storage diseases . images include brain scans showing : a atrophy with enlarged virchow robin spaces and ventricles , other images show d corneal clouding , e larynx thickening , f valvular alterations due to the accumulation of undegraded substrates , and g i dysostosis multiplex of back bone , hand and long bones our understanding of the pathogenesis of the lsds has grown dramatically over the last 1020 years and there is already a strong community supporting research and scientific developments in this area , which includes physicians , researchers , patient advocates and industry . thanks largely to advances in cellular and molecular biology , and the incentives for drug development provided by the introduction of the orphan drug regulation in europe , we are now moving closer to being able to provide therapies for neurological signs and symptoms in patients with these conditions . the advanced state of knowledge of the mechanisms of action of lsds , in particular , is allowing the elucidation of features of neurodegenerative diseases that will most likely also prove to be useful in the study of more prevalent neurodegenerative diseases that predominantly affect the elderly ( e.g. brain inflammation , alteration of intracellular trafficking , impairment of autophagy , or oxidative stress ) are common to both paediatric and adult neurodegenerative diseases ( for a review see ) therefore , at least some of the observations relative to loss and recovery of brain plasticity in paediatric neurodegeneration can be extrapolated to adult disorders , and findings will provide unique insights into the pathophysiology and restorative capacities of neurodegenerative diseases in general . by supplementing the missing enzyme ) in a young brain has the potential to maximise benefit as the brain retains considerable plasticity at this stage of development . there are several factors that make the lsds good candidates for pharmacogenetic studies and personalised medicine . the first is that the genetic basis of most of these conditions is well defined ; they are monogenic disorders and many disease - causing mutations have already been characterised . despite a current general lack of awareness of these conditions among physicians which can lead to delayed diagnosis , there is the potential for pre - symptomatic diagnosis and , in many cases , prenatal diagnosis [ 4244 ] . furthermore , for many of the lsds there is some overlap in the pathways that are involved in the degradation of the proteins that accumulate , making it likely that any biomarkers discovered may apply in more than one condition . another factor is that patients show considerable phenotypic variability ( even those with the same underlying genetic mutation ) and there is similarly variation between individuals in terms of response to therapy . at present , many different therapeutic approaches have been proposed including replacement of the missing enzyme ( enzyme replacement therapy , ert ) , reduction of levels of the enzyme substrate ( substrate reduction therapy , srt ) , the use of chemical chaperones to stabilise existing but unstable enzyme ( chaperone therapy ) , bone marrow transplantation , and gene therapies [ 4547 ] ( fig . based on current knowledge , predicting which patients will respond to which treatment remains a challenge . 3routes of intervention for the treatment of patients with lysosomal storage diseases routes of intervention for the treatment of patients with lysosomal storage diseases in order to make personalised medicine for children with rare neurological disorders a reality it is necessary to refine the techniques used to ensure that therapy gains access to the cns . although it is known which enzyme deficiency causes each of the lsds , supplying a correct version of the affected lysosomal enzyme intravenously only allows for modification of the natural history of these diseases in the peripheral organs ( e.g. another approach is to inhibit enzymes early in the biosynthetic pathway of the accumulating protein(s ) to reduce the number of molecules that require degradation in the lysosome . in cases where the affected lysosomal enzyme is present but does not function because it is unstable , small pharmaceutical molecules known as chemical chaperones that can cross the blood brain - barrier may be used to stabilise the enzyme , thus at least partially restoring its function . as the process of neurodegeneration observed in lsds is characterised by secondary events such as neuroinflammation , glutamatergic activation and oxidative stress , the use of small molecules with anti - inflammatory and/or neuroprotective properties may also be a useful adjunctive approach to treat lsds . to date , the utility of most of these approaches has already been demonstrated in animal models . looking to the future , there is also a need to establish and validate biomarkers that will allow us to understand both disease pathophysiology and which patients are most likely to respond to the different therapies that are available . although we are currently able to quantify the stored protein material and to assess changes in storage in response to treatment , in general , these variables have not proved to be reliable biomarkers for the lsds . in the future , the availability of biomarkers other than the stored material , will allow the choice of the most appropriate therapy ( ert , chaperone therapy or srt ) when taking into account patient phenotype and expected disease severity and course of progression . once a therapy is developed that can slow or prevent the progression of neurodegeneration , it will become extremely important to facilitate early detection of the genetic defect by newborn screening to enable early intervention . the development of new tools / technologies to diagnose patients and to predict disease severity and progression , as well as to assess new therapies , can be achieved only through collaboration between existing stakeholders . this meeting , which was organised by the brains for brain foundation [ b4b ( www.brains4brain.eu ) ] , the european brain council ( ebc ) , the lysosomal storage disease ( lsd ) patient collaborative and the veneto region , and sponsored by member of the european parliament , mrs amalia sartori ( veneto , italy ) , brought together stakeholders from 13 eu countries , as well as the usa and brazil . those registered for the meeting included 22 political representatives and members of 17 scientific organisations , 14 european family organisations , 10 biotechnology companies and over 40 european universities . the organisers called for a new initiative to coordinate the efforts of all existing groups in europe with the shared goal of improving the treatment and care of patients with rare neurological disorders . as commented in a recent editorial , there is a need for harmonised interaction between all stakeholders to enable the further development of personalised medicine . in line with this , the present project will unite all interested parties , who are working to increase the visibility , recognition and awareness of rare neurological disorders in order to facilitate early diagnosis of these conditions ; to promote and facilitate partnership and collaboration between physicians , researchers , patient advocates , carers , policy- and decision - makers and industry ; to encourage and support research and the translation of scientific breakthroughs into clinical practice ; to contribute to the establishment of a standard of care for patients with rare neurological disorders which is agreed across europe ; and to ensure equity of access to diagnosis , treatment and care . in order to progress the development of personalised medicine it is necessary for these benefits to be clearly demonstrated and for there to be buy - in and investment from all stakeholders , including the pharmaceutical industry , regulatory authorities and payers . nevertheless , there is still work to do in order to realise the potential of this approach and there remains a need for more examples that demonstrate proof of concept in clinical practice .

spinal cord injuries ( scis ) cause substantial social , economic , and health burdens . in the majority of cases , the spinal cord is not completely severed and thus some fiber tracts and segmental spinal cord circuits remain intact , which determine the preserved functions and provide the basis for functional restoration . in incomplete sci persons , recovery of sensorimotor function increases progressively during the first year , with reorganization of sensory and motor cortices to lead to recovery of function and maladaptive behavior . in para- and tetraplegic patients , the cortical hand area was expanded towards the cortical leg area and was different based on the lesion level . further , in paraplegic patients the representation of the nonimpaired upper limb muscles was modified showing an increased activation in the corresponding primary motor cortex ( m1 ) , in the parietal cortex , supplementary motor area , and cerebellum . an fmri study in rats showed that after midthoracic spinal cord transection , deafferented hindlimb territories in s1 exhibited responses to electrical stimulation of the unaffected forepaw , presumably mediated by both spinothalamic and dorsal column nuclei pathways . evidence suggests that functional plasticity of motor cortical representations is mediated by an anatomical framework of preexisting projections that transverse representation borders . in addition to spontaneous reorganization of the brain after sci , spinal cord circuitries have the capacity to alter their structure and function with motor training , as supported by the physiological leg muscle activation patterns observed after locomotor training in spinalized animals [ 914 ] . body weight - supported treadmill training ( bwstt ) is a therapeutic approach in which a person with sci steps on a motorized treadmill while some body weight is removed through an upper body harness and repetitive rhythmic leg movement patterns are promoted either through manual assistance provided by therapists or through a robotic exoskeleton system . evidence that supports this intervention has been derived largely from studies conducted in spinalized animals [ 1619 ] . specifically , treadmill training increases axonal regrowth and collateral sprouting proximal to the lesion site in mice , phosphorylation of erk1/2 in the motor cortex as well as the spinal cord injury area , expression of brain - derived neurotrophic factor ( bdnf ) in the spinal cord , ameliorates muscle atrophy in moderate contused sci rats , and alters properties of spinal motor neurons . these changes are only a small representation of activity - dependent plasticity located at the synaptic terminals of a variety of systems , that involves physiological , structural , and biochemical changes ( see more in [ 25 , 26 ] ) . in humans , bwstt improves lower extremity motor scores , increases the amplitude of muscle activity in the ankle extensors during the stance phase of walking , and improves walking ability and clinical outcome measures [ 2731 ] . a recent single - blind , randomized clinical trial involving bwstt with manual assistance , stimulation , over - ground training with stimulation and treadmill training with robotic assistance showed improvements in walking speed and distance . walking speed was not significantly different between groups , but distance gains were greatest with overground walking training . further , lower extremity motor scores increased in all groups , regardless the type of intervention . based on the aforementioned findings , it is apparent that bwstt contributes to restoration of locomotion . because remodeling of neuronal circuits as a result of plasticity occurs at multiple sites of the central nervous system [ 8 , 32 ] restoration of movement after training is anticipated to be the result of neural reorganization that occurs simultaneously in supraspinal and spinal cord circuits . the aim of this paper is to focus on the corticospinal neural plasticity after locomotor training in sci . the corticospinal tract is the most direct pathway between the cerebral cortex and spinal cord with corticospinal axons monosynaptically synapsing onto spinal motor neurons . even though neurons of the motor cortex are not required for simple locomotion , they exhibit a profound step - related frequency modulation in the cat [ 3335 ] . this modulation is driven by a combination of signals from the spinal central pattern generators and sensory afferent feedback reflex mechanisms that support interlimb coordination . the modulation of motor cortex neurons is necessary for accurate stepping on uneven terrain when adjustments of the limb trajectory are required to overstep an obstacle or to place the foot on a definite spot on the ground [ 3739 ] . however , pyramidal tract stimulation evokes disynaptic excitatory postsynaptic potentials ( epsps ) in flexor motor neurons that are much bigger in the locomotor state than in the resting state , which are rhythmically modulated so that the facilitation occurs in the flexor - active phase . while the spinal cord of vertebrates possesses the neural structures for genesis of the locomotor rhythm [ 4143 ] and the spinal pattern generator plays a decisive role in the recovery of locomotion after incomplete sci , lesions of the dorsolateral funiculi at thoracic t13 level in the cat induced long - term deficits on the locomotor pattern , supporting that the corticospinal tract plays a prominent role in the neural control of locomotion . the involvement of supraspinal neural control in human walking can be assessed by a variety of techniques utilized in isolation or in combination , including electroencephalography ( eeg ) , electromyography ( emg ) , transcranial magnetic and electric stimulation ( tms and tes ) , and neuroimaging [ 45 , 46 ] . single - photon emission tomography and near - infrared spectroscopic topography have shown that the sensorimotor and supplementary motor cortices are activated during real and imagined locomotion [ 47 , 48 ] , while the prefrontal and premotor cortices were involved in adapting the locomotor speed on the treadmill . a recent study postulated a significant coupling between eeg recordings over the leg motor area and emg from the tibialis anterior ( ta ) muscle in the frequency band of 2440 hz prior to heel strike during the swing phase of walking , supporting a cortical involvement in human gait function . ( time ( cross - correlation ) and frequency ( coherence ) domain techniques for the detection of coupling between signals provide an analytical framework from which functional coupling between localized cortical activity ( measured by meg or eeg ) and motor output ( emg ) can be identified in human subjects . ) a single stimulus of tms produces a synchronized discharge of cortical interneurons and pyramidal neurons that travel down the corticospinal tract . epidural electrodes in the spinal cord detect several waves following tms , termed direct ( d ) and indirect ( i ) waves . i waves originate in the motor cortex most likely through activation of corticocortical projections onto corticospinal neurons , while d waves are thought to result from direct depolarization of the initial axon segment of the corticospinal neuron . recordings from the peripheral muscles demonstrate compound muscle action potentials known as motor evoked potentials ( meps ) , which are a summation of multiple motor units depolarizing in response to d and i waves arriving onto the spinal motor neurons . however , the mep amplitude is not a reliable measure of corticospinal excitability . this is because tms - induced action potentials in cortical axons spread transynaptically to many other neurons that activate different descending pathways which are differently regulated during human movement . further , in order to support cortical excitability changes based on alterations of mep amplitude due to motor plasticity , both need to be mediated by the same motor neurons and caused exclusively by direct monosynaptic projections from the motor cortex without any contamination through indirect interneuronal relays . the peaks in the peristimulus time histogram of the discharge probability of motor units induced by tms have the same duration as those induced by ia stimulation , and thus there is ample time for nonmonosynaptic effects to influence the mep amplitude as is the case for the h - reflex [ 57 , 58 ] . lastly , because meps are facilitated on average 12 ms before the reaction time to contraction during which antagonists are concomitantly facilitated by subcortical circuits [ 59 , 60 ] , it is apparent that they are sensitive to the excitability state of spinal -motor neurons and interneurons . the aforementioned limitations can be counteracted by reducing the tms intensity below the mep threshold . direct recordings in awake human subjects have shown that tms at subthreshold mep intensities , which does not evoke any descending corticospinal volleys , depresses the mep evoked by a subsequent suprathreshold tms and the emg activity of ankle extensor muscles during the stance phase of walking , while the ta ongoing emg activity is facilitated at a short - latency at early swing phase . at subthreshold tms intensities the excitability of spinal motor neurons at short latencies is influenced by intracortical inhibitory circuits and mechanisms [ 62 , 64 ] , including but not limited to intracortical and interhemispheric inhibition [ 6570 ] , that in turn influence soleus or ta coupled corticomotoneuronal cells . these findings support that cortical excitability changes can be assessed in awake humans and that cortical cells with direct motoneuronal connections change their excitability during human walking . corticospinal drive of human locomotion is further addressed in sections 3 and 4 , whereas the cortical control of spinal reflex and interneuronal circuits is discussed . various training protocols in uninjured subjects induce reorganization of corticospinal actions on lumbosacral motor neurons . for example , balance training decreased the ta and soleus mep amplitudes , while 32 min voluntary ankle dorsi- and plantar - flexion training increased the ta mep amplitude regardless of the stimulation intensity level . repeated visuomotor skill training increased the maximal mep and decreased the stimulation intensity needed to evoke an mep , while opposite results were obtained after strength training , suggesting that reorganization of corticospinal actions on lumbosacral motor neurons depends on the type of training . in motor incomplete sci subjects at rest , meps are either absent or very small in amplitude with prolonged latencies , which are considered signs of impaired transmission of the fastest conducting corticospinal neurons [ 7477 ] . further , the absent or small ta meps prevail in sci persons with increased foot drop . further , the peak coherence in the 10 to 20 hz frequency band and synchronization within a narrow time band between paired ta emg recordings taken during the swing phase were absent during the swing phase and were positively correlated to the degree of foot drop . because coherence in the frequency and time domain reflects the common synaptic drive , which may be corticospinal in origin , behavioral deficits in ambulatory sci persons are driven by impaired corticospinal excitability . reorganization of corticospinal actions with training in neurological disorders has been shown in few studies . in 4 male sci subjects with tetraparesis , fmri showed a greater activation in sensorimotor cortical and cerebellar regions following 36 bwstt sessions consistent with the changes observed in the activation patterns of both hemispheres in poststroke subjects after 4 weeks of bwstt . three - to-5 month bwstt enhanced the mep amplitude in 9 out of 13 muscles tested , increased the maximal mep , and changed the slope of the mep input - output curve in the majority of sci subjects tested while seated . furthermore , in incomplete sci participants whom their locomotor function improved following treadmill training , the coherence ( 2440 hz ) of emg activity , which is thought to indicate a common drive from corticospinal inputs , between antagonist muscles acting at the knee joint was increased and remained unaltered in participants that the locomotor ability was not improved . the lower - frequency coherence ( 518 hz ) , which is thought to contain common synaptic drive from spinal inputs , remained unchanged in both groups . one person ( 49 yo female , 5 years after - injury ) with an american spinal injury association ( asia ) impairment scale ( ais ) d at thoracic 57 received 60 bwstt sessions ( 1 h / day ; 5 days / week ) with a robotic exoskeleton device ( lokomat ) . before training , the patient stepped at 0.5 m / s with 50% body weight support ( bws ) , and after training the patient stepped at 0.89 m / s with 20% bws . electrophysiological tests , illustrated as a schema in figure 1 , were conducted before and after training in the same patient while seated as well as during bws assisted stepping . data presented in this paper are original , have not been published elsewhere , and are from the same patient . all experimental procedures were approved by the institutional review board of the northwestern university irb committee and were conducted in compliance with the 1964 declaration of helsinki . the ta meps evoked at 1.3 ta mep threshold during assisted stepping before and after training are shown in figure 2 . ( the ta mep threshold was established with the subject standing at equivalent bws levels to that utilized during stepping . during stepping , ta meps were evoked randomly at different phases of the step cycle every 3 to 5 steps based on the signal from the ipsilateral foot switch . the step cycle of the right leg was divided into 16 equal time windows or bins . ) before training , the ta mep amplitude was increased during early swing ( bins 1013 ) when compared to that observed at midstance ( bins 35 ) , but an mep was not evocable from mid stance ( bin 6 ) until swing phase initiation ( bin 9 ) . after training , the ta mep amplitude increased significantly compared to that observed before training and was modulated in a phase - dependent pattern ; that is , it was progressively depressed during the stance phase ( bins 17 ) and was facilitated during the swing phase ( bins 914 ) ( figure 2 ) . this ta mep modulation pattern during assisted stepping is consistent with that reported in uninjured subjects , which is generally increased when the muscle from which it is recorded is active and small when the antagonist muscle is active [ 8284 ] . although the findings reported in figure 2 are from a single case , the altered mep modulation pattern supports the notion that locomotor training alters the efficacy of corticospinal descending motor volleys synapsing with ta spinal motor neurons in a manner that supports a physiologic gait pattern . it is apparent that more studies are needed on the neuronal mechanisms mediating improvement of locomotor function after training in spinal lesions of different segmental levels and types , in order that currently available rehabilitation strategies are optimized . the spinal cord constitutes the final common pathway for segmental and supraspinal pathways underlying motor behavior . electrical stimulation of a mixed peripheral nerve at low intensities activates primary ( ia ) afferent axons which synapse in the spinal cord . alpha motor neurons activated monosynaptically by ia afferent volleys induce a synchronized reflex response known as the hoffmann-(h- ) reflex , which is the electrical analogue of the monosynaptic stretch reflex . when the h - reflex is used as a test reflex , the effects of conditioning volleys from other afferents or descending tracts on the motoneuron pool and synaptic actions from different sources in health and disease can be assessed [ 85 , 86 ] . cortical control of spinal reflex circuits has been extensively investigated in awake humans by means of tms . subthreshold tms produces a short - latency inhibition on the soleus h - reflex followed by a period of facilitation [ 56 , 8789 ] with subjects at rest . in contrast , the ta h - reflex is facilitated at an early conditioning - test ( c - t ) interval . superficial peroneal or sural nerve stimulation potentiates the presumably monosynaptic facilitation of the ta h - reflex evoked by brain stimulation . the cortical modulation of the soleus h - reflex depends largely on the position of the ankle joint , with subthreshold tms to induce an early long - lasting facilitation or depression of the soleus h - reflex during tonic plantar flexion and dorsiflexion , respectively [ 87 , 91 ] . similar findings have been reported for pyramidal monkeys , cats , and baboons during which cortical inhibition predominated on the soleus and gastrocnemius monosynaptic reflex , while cortical facilitation influenced largely flexor motor neurons [ 92 , 93 ] . it should be noted , however , that a single cortical d wave could produce changes in segmental motor neurons in the primates but not in the cat that required d and i waves or multiple d - waves . in addition to the h - reflex , the ta long - latency ( or m3 ) ankle stretch reflex was facilitated when the mep arrived in the spinal cord at the same time . however , subthreshold tms intensities delivered 5585 ms prior to the m3 depressed the long - latency ta stretch reflex . because the long - latency response was reduced in size following subthreshold tms while the short - latency response remained unchanged [ 95 , 96 ] , it provides evidence that the long - latency stretch reflex is mediated in part by a transcortical path that can be affected by subthreshold tms . during human walking , subthreshold tms induces a short - latency , presumably monosynaptic , facilitation of the soleus h - reflex followed by a long - lasting inhibition . because potentiation of ta meps was synchronized with the peak ta ankle stretch reflex , corticospinal pathways are partly involved in the generation of spinal stretch reflexes during human walking [ 97 , 98 ] . in human sci , the conditioned h - reflex profile by subthreshold tms varied significantly based on the ais scores [ 99 , 100 ] . in patients with severe paralysis ( ais a - b ) an early or late soleus h - reflex facilitation by tms was absent , suggesting for a nonphysiological interaction between descending inputs and spinal reflex excitability in patients with spastic paraparesis . persistent changes in h- or stretch reflex amplitudes may be regarded as signs of learning and plasticity as a result of training , which have been shown after various training protocols . for example , 30 min ankle cocontraction training decreased the ratio of maximal h - reflex versus maximal m wave ( hmax / mmax ) and improved motor performance defined as the difference between the maximum and minimum torque displacements within 1 min . the soleus h - reflex amplitude was enhanced after 3 week isometric maximal plantar flexion training when measured at 20% and 60% of maximal voluntary contraction ( mvc ) , with similar results to be reported after 14 week of resistance training that involved heavy weight - lifting exercises for the leg muscles with reflexes measured during maximal isometric ramp contractions . in contrast , 18 sessions eccentric strength training of the plantar flexor muscles for a 7 week period increased the hmax / mmax ratio during eccentric mvc but not during isometric or concentric contractions , suggesting that spinal reflex excitability is adjusted based on the type of exercise training protocol . nonetheless , the aforementioned changes in h - reflex amplitude can result from modifications of interneuronal circuits interposed in the spinal pathway or by changes on the strength of descending pathways , since the latter is potent regulator of spinal reflex circuits behavior [ 105107 ] . this is supported by the failed operant conditioning of the h - reflex in rats when the corticospinal tract was transected at the spinal cord level [ 108 , 109 ] . limited evidence exists on plastic changes of the cortical control of spinal reflexes after locomotor training in neurological disorders . forty bwstt sessions in 29 patients with incomplete sci reestablished the tms - induced long - latency soleus h - reflex facilitation with subjects at rest . it should be noted that bws improves the efficacy of the sensorimotor cortex function , decreases the ta mep threshold , and increases the map size for the ta in both hemispheres of stroke patients . nonetheless , when tms effects on spinal reflexes are assessed with patients at a resting state , it can not be assumed that corticospinal changes due to training are transferrable at a locomotor state and thus be functional relevant . this is largely based on that ( 1 ) short - latency ankle or quadriceps extensor reflexes ( h- or stretch reflexes ) are modulated in a phase - dependent pattern in uninjured subjects [ 113115 ] , ( 2 ) the phase - dependent modulation of these reflexes is affected substantially in individuals with an sci [ 115117 ] , and ( 3 ) cortical control constitutes one of the sources for the phasic patterned reflex excitability during human walking . in figure 3(a ) , the soleus h - reflex recorded during bws assisted stepping according to methods described in detail [ 115 , 118 , 119 ] , before and after 60 bwstt sessions , is indicated for the same patient whose ta mep modulation pattern was described in figure 2 . after 60 bwstt sessions , the maximal reflex excitability shifted , with respect to the step cycle phase , from mid- to early stance ( bins 13 ) , while a maintained h - reflex excitability commonly observed throughout the stance phase in uninjured subjects was absent before and after training ( figure 3(a ) ) . however , after 60 bwstt sessions the soleus h - reflex amplitude increased during the late swing phase ( bins 1216 ) , consistent to a reflex behavior observed in some control subjects . the effects of subthreshold tms on the soleus h - reflex at a c - t interval of 1-ms during bws assisted stepping are indicated as a function of the step cycle before and after 60 bwstt sessions in figure 3(b ) . it is clear that , after 60 bwstt sessions , subthreshold tms affected substantially the soleus h - reflex during the stance phase resulting in a progressive increase of the soleus h - reflex amplitude . the soleus h - reflex amplitude was maintained throughout the stance phase ( compare bins 18 in figures 3(a ) and 3(b ) ) . modifications in synaptic actions of cortical inhibitory circuits exerted on soleus motor neurons might be the source of these changes since the phasic soleus h - reflex excitability during bws assisted stepping with or without leg assistance by a robotic exoskeleton remains unaltered [ 115 , 118 ] . one of the spinal interneuronal circuits with paramount contribution to the neural control of movement is that of disynaptic reciprocal ia inhibition . reciprocal inhibition refers to an automatic antagonist motor neuron inhibition when an agonist muscle contracts . following an sci , the reciprocal inhibition is either reduced or replaced by reciprocal facilitation [ 121124 ] leading to coactivation of antagonist ankle muscles , spasticity , and poor movement performance . regulation of locomotion by reflexly mediated spinal circuits that integrate sensory inputs is well established . the contribution of muscle afferents mediating information about the amplitude and rate of muscle stretch is easily recognized by the phase - dependent modulation of short - latency spinal reflexes during walking . the short - latency soleus and quadriceps extensor reflexes ( stretch , tendon , or h - reflex ) in humans are modulated in a way that promotes bipedal gait . the ankle stretch and soleus h - reflexes increase progressively from mid- to late stance in parallel with the soleus emg activity and are significantly depressed or abolished during the swing phase of gait [ 113115 , 125 ] . a phase - dependent modulation has been demonstrated for the ankle stretch reflex in the high decerebrate mesencephalic cat . the soleus h - reflex depression during the swing phase in humans has been partly ascribed to reciprocal ia inhibition exerted from common peroneal nerve group i afferents on soleus motor neurons , which is regulated in a similar manner to that reported in animals and corresponds largely to absent reciprocal inhibition in the stance phase and maximal in the swing phase [ 127 , 128 ] . during fictive locomotion in cats without brainstem connections , simultaneous extracellular recordings from ia inhibitory interneurons and intracellular recordings from lumbar motor neurons revealed that hyperpolarization of soleus motor neurons coincided with activity of ia inhibitory interneurons [ 129 , 130 ] . ia inhibitory interneurons were rhythmically active due to periodic excitation and not due to periodic inhibition by other spinal inhibitory interneurons . recent evidence obtained from spinalized animals verified that reciprocal ia inhibition contributes to hyperpolarization of motor neurons during the inactive ( flexion ) phase of locomotion . animal studies through intracellular recordings provided a detailed knowledge of the pathway and integration of segmental and supraspinal convergence at the interneuronal level [ 132135 ] with volleys in the corticospinal tract to exert an excitatory effect over ia inhibitory interneurons . in monkeys , intracortical stimulation revealed that the same interneurons mediate the disynaptic inhibition of motor neurons evoked by corticospinal fibers and the disynaptic inhibition of motor neurons evoked by group ia afferents of antagonist muscles . further , motor neurons and ia inhibitory interneurons were activated in parallel by supraspinal centers in order to secure a coordinated contraction of agonists and relaxation of antagonists [ 138 , 139 ] . descending control of reciprocal inhibition in particular , the reciprocal inhibition exerted from common peroneal nerve group i afferents on soleus motor neurons was observed 50 ms before the onset of ta emg activity . further , when subjects attempted to dorsiflex the ankle after the common peroneal nerve was blocked with a local anesthetic a strong soleus h - reflex depression was still evident . the test h - reflex facilitation , induced by tes applied to the scalp below the intensity needed to produce a motor response , was quickly terminated by subsequent arrivals of ipsps at the motor neurons . these ipsps might be produced by activity in ia inhibitory interneurons , which in monkeys receive monosynaptic tract projections . single subthreshold tes reduced the inhibition of the flexor carpi radialis h - reflex evoked by radial nerve stimulation at a latency compatible with a monosynaptic or disynaptic corticospinal projection to ia inhibitory interneurons . descending facilitation of ia inhibitory interneurons has also been documented for the human leg [ 87 , 89 ] . findings on the reorganization of reciprocal inhibition as a result of motor training in health and disease are limited . stimulation of the common peroneal nerve with a train of 10 pulses at 100 hz with and without motor cortex stimulation potentiated reciprocal inhibition in control subjects . reciprocal inhibition was potentiated after 12 sessions of ankle dorsiflexion strength training when measured at the onset of ankle dorsiflexion but remained unchanged when measured with subjects at rest . in figure 4 , the mean amplitude of the soleus h - reflex conditioned by stimulation of common peroneal nerve group i afferents at a c - t interval of 3 ms and established according to methods outlined in detail , which represents the amount of reciprocal inhibition ( rci ) , before and after 60 bwstt sessions with subject seated ( same patient for data previously described in figures 2 and 3 is indicated as a percentage of the control h - reflex ) . further , the soleus h - reflex conditioned by subthreshold tms at a c - t interval of 1 ms and the reciprocal inhibition conditioned by subthreshold tms ( c - t intervals : 3 and 1 ms , resp . ) as a percentage of the control h - reflex is indicated . it is apparent that locomotor training reestablished the reciprocal inhibition exerted from flexor group i afferents on soleus motor neurons , potentiated the soleus h - reflex depression following subthreshold tms , and potentiated the reciprocal inhibition conditioned by subthreshold tms , consistent with findings reported in uninjured subjects ( see figure 4 in ) . the net effects of subthreshold tms on the reciprocal inhibition during bws - assisted stepping before and after 60 bwstt sessions are indicated in figure 5 for the same patient . the net effects ( or net modulation ) were estimated at each bin of the step cycle based on the equation ( d - c)-(b - a ) whereas a is the test soleus h - reflex ( baseline soleus h - reflex modulation pattern during stepping ) , b is the soleus h - reflex conditioned by subthreshold tms , c is the soleus h - reflex conditioned by common peroneal nerve stimulation ( i.e. , reciprocal inhibition ) , and d is the reciprocal inhibition conditioned by subthreshold tms . positive values indicate potentiation of reciprocal inhibition and negative values indicate attenuation of reciprocal inhibition . locomotor training contributed significantly to attenuation of reciprocal inhibition exerted from ankle flexor afferents to extensor motor neurons during the stance phase . most importantly , potentiation of reciprocal inhibition at swing phase initiation ( i.e. , bin 9 in figure 5 ) was evident . adaptation of cortical control of reciprocal inhibition after locomotor training supports that changes of corticospinal neuronal pathways interacting with ia interneurons are possible in people with a chronic sci , although altered corticospinal interactions with other spinal inhibitory interneurons , such as renshaw cells and presynaptic inhibitory interneurons , can not be excluded [ 85 , 148 , 149 ] . sci changes the human body homeostasis leading to myriad changes of multiple systems . in most cases , the spinal cord is not completely severed and thus some fiber tracts and segmental spinal cord circuits remain intact . based on the plastic capabilities of the central nervous system , it is apparent that the adult lesioned motor system reorganization occurs spontaneously after an injury and after training . electrophysiological studies have shown that bwstt increases the mep amplitude , changes the common drive of antagonist muscles from corticospinal inputs with subjects seated , and alters the ta mep modulation pattern during bws assisted stepping . further , bwstt reestablished the tms - induced long - latency soleus h - reflex facilitation and potentiated the short - latency soleus h - reflex depression following subthreshold tms with subjects at rest , while cortical modulation of the soleus h - reflex during stepping changed significantly . lastly , bwstt changed the cortical control of reciprocal inhibition during bws assisted stepping in a manner that promotes bipedal gait . these findings support the notion that improvements in locomotor function from treadmill training are mediated , in part , by changes in the corticospinal drive of spinal reflex circuits , spinal interneuronal circuits , and output of leg muscles during walking . plasticity in the brain and spinal cord underlying restoration of lost function can be driven by appropriately designed interventions [ 150 , 151 ] . development of such interventions depends largely on gaining a detailed understanding of the underlying neural mechanisms that support restoration of motor function . based on this brief paper it is clear that there is a need for translational neuroscience research in order that the neural mechanisms underlying restoration of lost voluntary motor function are outlined based on specific clinical cases . this body of knowledge will contribute significantly to the development of new rehabilitation strategies and/or optimization of the currently available strategies , and to patient - orientated rehabilitation protocols promoting evidence - based rehabilitation .

spinal lesions substantially impair ambulation , occur generally in young and otherwise healthy individuals , and result in devastating effects on quality of life . restoration of locomotion after damage to the spinal cord is challenging because axons of the damaged neurons do not regenerate spontaneously . body - weight - supported treadmill training ( bwstt ) is a therapeutic approach in which a person with a spinal cord injury ( sci ) steps on a motorized treadmill while some body weight is removed through an upper body harness . bwstt improves temporal gait parameters , muscle activation patterns , and clinical outcome measures in persons with sci . these changes are likely the result of reorganization that occurs simultaneously in supraspinal and spinal cord neural circuits . this paper will focus on the cortical control of human locomotion and motor output , spinal reflex circuits , and spinal interneuronal circuits and how corticospinal control is reorganized after locomotor training in people with sci . based on neurophysiological studies , it is apparent that corticospinal plasticity is involved in restoration of locomotion after training . however , the neural mechanisms underlying restoration of lost voluntary motor function are not well understood and translational neuroscience research is needed so patient - orientated rehabilitation protocols to be developed .

1. Introduction 2. Cortical Control of Locomotion 3. Cortical Control of Spinal Reflex Circuits 4. Spinal Interneuronal Inhibitory Circuits: Reciprocal Ia Inhibition 5. Conclusion 6. Perspective

spinal cord injuries ( scis ) cause substantial social , economic , and health burdens . in the majority of cases , the spinal cord is not completely severed and thus some fiber tracts and segmental spinal cord circuits remain intact , which determine the preserved functions and provide the basis for functional restoration . in para- and tetraplegic patients , the cortical hand area was expanded towards the cortical leg area and was different based on the lesion level . further , in paraplegic patients the representation of the nonimpaired upper limb muscles was modified showing an increased activation in the corresponding primary motor cortex ( m1 ) , in the parietal cortex , supplementary motor area , and cerebellum . an fmri study in rats showed that after midthoracic spinal cord transection , deafferented hindlimb territories in s1 exhibited responses to electrical stimulation of the unaffected forepaw , presumably mediated by both spinothalamic and dorsal column nuclei pathways . in addition to spontaneous reorganization of the brain after sci , spinal cord circuitries have the capacity to alter their structure and function with motor training , as supported by the physiological leg muscle activation patterns observed after locomotor training in spinalized animals [ 914 ] . body weight - supported treadmill training ( bwstt ) is a therapeutic approach in which a person with sci steps on a motorized treadmill while some body weight is removed through an upper body harness and repetitive rhythmic leg movement patterns are promoted either through manual assistance provided by therapists or through a robotic exoskeleton system . specifically , treadmill training increases axonal regrowth and collateral sprouting proximal to the lesion site in mice , phosphorylation of erk1/2 in the motor cortex as well as the spinal cord injury area , expression of brain - derived neurotrophic factor ( bdnf ) in the spinal cord , ameliorates muscle atrophy in moderate contused sci rats , and alters properties of spinal motor neurons . these changes are only a small representation of activity - dependent plasticity located at the synaptic terminals of a variety of systems , that involves physiological , structural , and biochemical changes ( see more in [ 25 , 26 ] ) . in humans , bwstt improves lower extremity motor scores , increases the amplitude of muscle activity in the ankle extensors during the stance phase of walking , and improves walking ability and clinical outcome measures [ 2731 ] . based on the aforementioned findings , it is apparent that bwstt contributes to restoration of locomotion . because remodeling of neuronal circuits as a result of plasticity occurs at multiple sites of the central nervous system [ 8 , 32 ] restoration of movement after training is anticipated to be the result of neural reorganization that occurs simultaneously in supraspinal and spinal cord circuits . the aim of this paper is to focus on the corticospinal neural plasticity after locomotor training in sci . the corticospinal tract is the most direct pathway between the cerebral cortex and spinal cord with corticospinal axons monosynaptically synapsing onto spinal motor neurons . even though neurons of the motor cortex are not required for simple locomotion , they exhibit a profound step - related frequency modulation in the cat [ 3335 ] . the modulation of motor cortex neurons is necessary for accurate stepping on uneven terrain when adjustments of the limb trajectory are required to overstep an obstacle or to place the foot on a definite spot on the ground [ 3739 ] . while the spinal cord of vertebrates possesses the neural structures for genesis of the locomotor rhythm [ 4143 ] and the spinal pattern generator plays a decisive role in the recovery of locomotion after incomplete sci , lesions of the dorsolateral funiculi at thoracic t13 level in the cat induced long - term deficits on the locomotor pattern , supporting that the corticospinal tract plays a prominent role in the neural control of locomotion . the involvement of supraspinal neural control in human walking can be assessed by a variety of techniques utilized in isolation or in combination , including electroencephalography ( eeg ) , electromyography ( emg ) , transcranial magnetic and electric stimulation ( tms and tes ) , and neuroimaging [ 45 , 46 ] . single - photon emission tomography and near - infrared spectroscopic topography have shown that the sensorimotor and supplementary motor cortices are activated during real and imagined locomotion [ 47 , 48 ] , while the prefrontal and premotor cortices were involved in adapting the locomotor speed on the treadmill . ( time ( cross - correlation ) and frequency ( coherence ) domain techniques for the detection of coupling between signals provide an analytical framework from which functional coupling between localized cortical activity ( measured by meg or eeg ) and motor output ( emg ) can be identified in human subjects . ) epidural electrodes in the spinal cord detect several waves following tms , termed direct ( d ) and indirect ( i ) waves . i waves originate in the motor cortex most likely through activation of corticocortical projections onto corticospinal neurons , while d waves are thought to result from direct depolarization of the initial axon segment of the corticospinal neuron . however , the mep amplitude is not a reliable measure of corticospinal excitability . further , in order to support cortical excitability changes based on alterations of mep amplitude due to motor plasticity , both need to be mediated by the same motor neurons and caused exclusively by direct monosynaptic projections from the motor cortex without any contamination through indirect interneuronal relays . the peaks in the peristimulus time histogram of the discharge probability of motor units induced by tms have the same duration as those induced by ia stimulation , and thus there is ample time for nonmonosynaptic effects to influence the mep amplitude as is the case for the h - reflex [ 57 , 58 ] . lastly , because meps are facilitated on average 12 ms before the reaction time to contraction during which antagonists are concomitantly facilitated by subcortical circuits [ 59 , 60 ] , it is apparent that they are sensitive to the excitability state of spinal -motor neurons and interneurons . corticospinal drive of human locomotion is further addressed in sections 3 and 4 , whereas the cortical control of spinal reflex and interneuronal circuits is discussed . further , the absent or small ta meps prevail in sci persons with increased foot drop . further , the peak coherence in the 10 to 20 hz frequency band and synchronization within a narrow time band between paired ta emg recordings taken during the swing phase were absent during the swing phase and were positively correlated to the degree of foot drop . in 4 male sci subjects with tetraparesis , fmri showed a greater activation in sensorimotor cortical and cerebellar regions following 36 bwstt sessions consistent with the changes observed in the activation patterns of both hemispheres in poststroke subjects after 4 weeks of bwstt . three - to-5 month bwstt enhanced the mep amplitude in 9 out of 13 muscles tested , increased the maximal mep , and changed the slope of the mep input - output curve in the majority of sci subjects tested while seated . furthermore , in incomplete sci participants whom their locomotor function improved following treadmill training , the coherence ( 2440 hz ) of emg activity , which is thought to indicate a common drive from corticospinal inputs , between antagonist muscles acting at the knee joint was increased and remained unaltered in participants that the locomotor ability was not improved . one person ( 49 yo female , 5 years after - injury ) with an american spinal injury association ( asia ) impairment scale ( ais ) d at thoracic 57 received 60 bwstt sessions ( 1 h / day ; 5 days / week ) with a robotic exoskeleton device ( lokomat ) . before training , the patient stepped at 0.5 m / s with 50% body weight support ( bws ) , and after training the patient stepped at 0.89 m / s with 20% bws . electrophysiological tests , illustrated as a schema in figure 1 , were conducted before and after training in the same patient while seated as well as during bws assisted stepping . data presented in this paper are original , have not been published elsewhere , and are from the same patient . during stepping , ta meps were evoked randomly at different phases of the step cycle every 3 to 5 steps based on the signal from the ipsilateral foot switch . the step cycle of the right leg was divided into 16 equal time windows or bins . ) after training , the ta mep amplitude increased significantly compared to that observed before training and was modulated in a phase - dependent pattern ; that is , it was progressively depressed during the stance phase ( bins 17 ) and was facilitated during the swing phase ( bins 914 ) ( figure 2 ) . although the findings reported in figure 2 are from a single case , the altered mep modulation pattern supports the notion that locomotor training alters the efficacy of corticospinal descending motor volleys synapsing with ta spinal motor neurons in a manner that supports a physiologic gait pattern . it is apparent that more studies are needed on the neuronal mechanisms mediating improvement of locomotor function after training in spinal lesions of different segmental levels and types , in order that currently available rehabilitation strategies are optimized . the spinal cord constitutes the final common pathway for segmental and supraspinal pathways underlying motor behavior . electrical stimulation of a mixed peripheral nerve at low intensities activates primary ( ia ) afferent axons which synapse in the spinal cord . alpha motor neurons activated monosynaptically by ia afferent volleys induce a synchronized reflex response known as the hoffmann-(h- ) reflex , which is the electrical analogue of the monosynaptic stretch reflex . when the h - reflex is used as a test reflex , the effects of conditioning volleys from other afferents or descending tracts on the motoneuron pool and synaptic actions from different sources in health and disease can be assessed [ 85 , 86 ] . cortical control of spinal reflex circuits has been extensively investigated in awake humans by means of tms . the cortical modulation of the soleus h - reflex depends largely on the position of the ankle joint , with subthreshold tms to induce an early long - lasting facilitation or depression of the soleus h - reflex during tonic plantar flexion and dorsiflexion , respectively [ 87 , 91 ] . similar findings have been reported for pyramidal monkeys , cats , and baboons during which cortical inhibition predominated on the soleus and gastrocnemius monosynaptic reflex , while cortical facilitation influenced largely flexor motor neurons [ 92 , 93 ] . it should be noted , however , that a single cortical d wave could produce changes in segmental motor neurons in the primates but not in the cat that required d and i waves or multiple d - waves . in addition to the h - reflex , the ta long - latency ( or m3 ) ankle stretch reflex was facilitated when the mep arrived in the spinal cord at the same time . however , subthreshold tms intensities delivered 5585 ms prior to the m3 depressed the long - latency ta stretch reflex . because potentiation of ta meps was synchronized with the peak ta ankle stretch reflex , corticospinal pathways are partly involved in the generation of spinal stretch reflexes during human walking [ 97 , 98 ] . in human sci , the conditioned h - reflex profile by subthreshold tms varied significantly based on the ais scores [ 99 , 100 ] . in patients with severe paralysis ( ais a - b ) an early or late soleus h - reflex facilitation by tms was absent , suggesting for a nonphysiological interaction between descending inputs and spinal reflex excitability in patients with spastic paraparesis . the soleus h - reflex amplitude was enhanced after 3 week isometric maximal plantar flexion training when measured at 20% and 60% of maximal voluntary contraction ( mvc ) , with similar results to be reported after 14 week of resistance training that involved heavy weight - lifting exercises for the leg muscles with reflexes measured during maximal isometric ramp contractions . in contrast , 18 sessions eccentric strength training of the plantar flexor muscles for a 7 week period increased the hmax / mmax ratio during eccentric mvc but not during isometric or concentric contractions , suggesting that spinal reflex excitability is adjusted based on the type of exercise training protocol . nonetheless , the aforementioned changes in h - reflex amplitude can result from modifications of interneuronal circuits interposed in the spinal pathway or by changes on the strength of descending pathways , since the latter is potent regulator of spinal reflex circuits behavior [ 105107 ] . this is supported by the failed operant conditioning of the h - reflex in rats when the corticospinal tract was transected at the spinal cord level [ 108 , 109 ] . limited evidence exists on plastic changes of the cortical control of spinal reflexes after locomotor training in neurological disorders . it should be noted that bws improves the efficacy of the sensorimotor cortex function , decreases the ta mep threshold , and increases the map size for the ta in both hemispheres of stroke patients . nonetheless , when tms effects on spinal reflexes are assessed with patients at a resting state , it can not be assumed that corticospinal changes due to training are transferrable at a locomotor state and thus be functional relevant . this is largely based on that ( 1 ) short - latency ankle or quadriceps extensor reflexes ( h- or stretch reflexes ) are modulated in a phase - dependent pattern in uninjured subjects [ 113115 ] , ( 2 ) the phase - dependent modulation of these reflexes is affected substantially in individuals with an sci [ 115117 ] , and ( 3 ) cortical control constitutes one of the sources for the phasic patterned reflex excitability during human walking . in figure 3(a ) , the soleus h - reflex recorded during bws assisted stepping according to methods described in detail [ 115 , 118 , 119 ] , before and after 60 bwstt sessions , is indicated for the same patient whose ta mep modulation pattern was described in figure 2 . after 60 bwstt sessions , the maximal reflex excitability shifted , with respect to the step cycle phase , from mid- to early stance ( bins 13 ) , while a maintained h - reflex excitability commonly observed throughout the stance phase in uninjured subjects was absent before and after training ( figure 3(a ) ) . the effects of subthreshold tms on the soleus h - reflex at a c - t interval of 1-ms during bws assisted stepping are indicated as a function of the step cycle before and after 60 bwstt sessions in figure 3(b ) . it is clear that , after 60 bwstt sessions , subthreshold tms affected substantially the soleus h - reflex during the stance phase resulting in a progressive increase of the soleus h - reflex amplitude . one of the spinal interneuronal circuits with paramount contribution to the neural control of movement is that of disynaptic reciprocal ia inhibition . following an sci , the reciprocal inhibition is either reduced or replaced by reciprocal facilitation [ 121124 ] leading to coactivation of antagonist ankle muscles , spasticity , and poor movement performance . descending control of reciprocal inhibition in particular , the reciprocal inhibition exerted from common peroneal nerve group i afferents on soleus motor neurons was observed 50 ms before the onset of ta emg activity . the test h - reflex facilitation , induced by tes applied to the scalp below the intensity needed to produce a motor response , was quickly terminated by subsequent arrivals of ipsps at the motor neurons . single subthreshold tes reduced the inhibition of the flexor carpi radialis h - reflex evoked by radial nerve stimulation at a latency compatible with a monosynaptic or disynaptic corticospinal projection to ia inhibitory interneurons . findings on the reorganization of reciprocal inhibition as a result of motor training in health and disease are limited . stimulation of the common peroneal nerve with a train of 10 pulses at 100 hz with and without motor cortex stimulation potentiated reciprocal inhibition in control subjects . in figure 4 , the mean amplitude of the soleus h - reflex conditioned by stimulation of common peroneal nerve group i afferents at a c - t interval of 3 ms and established according to methods outlined in detail , which represents the amount of reciprocal inhibition ( rci ) , before and after 60 bwstt sessions with subject seated ( same patient for data previously described in figures 2 and 3 is indicated as a percentage of the control h - reflex ) . further , the soleus h - reflex conditioned by subthreshold tms at a c - t interval of 1 ms and the reciprocal inhibition conditioned by subthreshold tms ( c - t intervals : 3 and 1 ms , resp . ) it is apparent that locomotor training reestablished the reciprocal inhibition exerted from flexor group i afferents on soleus motor neurons , potentiated the soleus h - reflex depression following subthreshold tms , and potentiated the reciprocal inhibition conditioned by subthreshold tms , consistent with findings reported in uninjured subjects ( see figure 4 in ) . the net effects of subthreshold tms on the reciprocal inhibition during bws - assisted stepping before and after 60 bwstt sessions are indicated in figure 5 for the same patient . the net effects ( or net modulation ) were estimated at each bin of the step cycle based on the equation ( d - c)-(b - a ) whereas a is the test soleus h - reflex ( baseline soleus h - reflex modulation pattern during stepping ) , b is the soleus h - reflex conditioned by subthreshold tms , c is the soleus h - reflex conditioned by common peroneal nerve stimulation ( i.e. , reciprocal inhibition ) , and d is the reciprocal inhibition conditioned by subthreshold tms . adaptation of cortical control of reciprocal inhibition after locomotor training supports that changes of corticospinal neuronal pathways interacting with ia interneurons are possible in people with a chronic sci , although altered corticospinal interactions with other spinal inhibitory interneurons , such as renshaw cells and presynaptic inhibitory interneurons , can not be excluded [ 85 , 148 , 149 ] . in most cases , the spinal cord is not completely severed and thus some fiber tracts and segmental spinal cord circuits remain intact . based on the plastic capabilities of the central nervous system , it is apparent that the adult lesioned motor system reorganization occurs spontaneously after an injury and after training . lastly , bwstt changed the cortical control of reciprocal inhibition during bws assisted stepping in a manner that promotes bipedal gait . these findings support the notion that improvements in locomotor function from treadmill training are mediated , in part , by changes in the corticospinal drive of spinal reflex circuits , spinal interneuronal circuits , and output of leg muscles during walking . plasticity in the brain and spinal cord underlying restoration of lost function can be driven by appropriately designed interventions [ 150 , 151 ] . development of such interventions depends largely on gaining a detailed understanding of the underlying neural mechanisms that support restoration of motor function . based on this brief paper it is clear that there is a need for translational neuroscience research in order that the neural mechanisms underlying restoration of lost voluntary motor function are outlined based on specific clinical cases . this body of knowledge will contribute significantly to the development of new rehabilitation strategies and/or optimization of the currently available strategies , and to patient - orientated rehabilitation protocols promoting evidence - based rehabilitation .

the new zealand census - mortality study is a population - wide cohort study , in which the cohort consists of the entire 1996 resident population ( n=3 732 000 ) and the outcome of interest is mortality . for this analysis , records from the 1996 census were anonymously linked to 3 years of subsequent mortality data , creating a cohort study of the new zealand population followed up for 3 years.19 20 in new zealand , urban areas are defined as cities having a population of at least 30 000 people ; about half of the approximately 2000 census areas are classified as urban . because the definition of urban areas in new zealand includes small towns and suburbs , many urban census areas have low levels of air pollution . the method of record linkage has been described in detail elsewhere.19 briefly , probabilistic record linkage is a process used to link two files of records , in which records in one file have a corresponding record in the other file . records from the first file are compared with records from the second file in order to find matching record pairs ( ie , two records belonging to the same individual ) . census and mortality records were linked using date of birth ( day , month and year as separate matching variables ) , country of birth , sex , ethnicity and address of usual residence . linkage was restricted to individuals aged 74 years or below at the time of the census . the proportion of mortality records linked overall was 78%,20 and varied by sex , age , ethnicity and the deprivation index.19 weights were therefore applied to adjust for linkage bias , by strata of sex , age , ethnicity , deprivation , rurality and cause of death.21 for example , if 20 of 30 maori men who died aged 4564 years and living in moderately deprived ( see below ) rural areas of new zealand were linked to a census record , each of the 20 linked records received a weight of 1.5 ( 30/20 ) . air pollution monitoring data are typically only available for a few sites and may not be representative of exposure in other areas . detailed atmospheric dispersion modelling can be used to provide estimates of air pollution exposure for small areas , but these models are difficult to apply to large areas due to data and computer processing limitations . the method of air pollution exposure assessment has been described in detail elsewhere.22 briefly , the approach to modelling long - term average pm10 was as follows . atmospheric dispersion modelling results were available for one city ( christchurch , population 300 000 ) . these data were assumed to be representative of the spatial pattern of annual average pm10 exposures for small areas ( census area units ) in christchurch.23 data on meteorological variables and indicators of air pollutant emissions for these areas were used as predictors of pm10 exposure in christchurch , using regression models . the predictors for the regression models were : census data on domestic heating ; estimates of industrial emissions ; and vehicle kilometres travelled within small areas . because data for these predictor variables were available for all of new zealand , we were able to extrapolate the empirical results for the christchurch regression model to urban census area units throughout the country . the resulting exposure estimates agreed well with multiyear averages of pm10 based on routine monitoring data for urban centres ( 19952001 ) ( n=43 ; r=0.87 ) . while we acknowledge that there may be considerable exposure misclassification , we assume that the pm10 estimates are representative of long - term average spatial patterns of exposure during the 1990s . ideally , for analyses with the new zealand census mortality study data we would have used the continuous pm10 estimates for individual census areas . however , because the new zealand census mortality study data contain census information at unit record level , access to the data is restricted and additional data can only be added if confidentiality is not compromised as a result . assigning continuous pm10 estimates at census area level would have permitted many census areas to be uniquely identified . in order to maintain confidentiality , it was necessary to aggregate the pm10 estimates into quintiles before they could be merged with the census data . quintiles of exposure were calculated based on the estimates for individual census areas . for this purpose , we assigned a pm10 estimate of 0 g / m to rural census areas where pm10 estimates were unavailable . the average pm10 level for all new zealand census areas was 8.3 g / m ( sd 8.4 g / m ) and the cut - off values between pm10 quintiles were 0.0 , 0.5 , 12.5 and 15.4 g / m . individuals were assigned to quintiles of pm10 exposure depending on their census area of residence on census night . in order to avoid bias affecting analyses in smaller rural areas due to migration to cities following the development of disease , we restricted the analyses to the urban population . following restriction to the urban population , the lowest two quintiles of exposure had relatively few observations ( table 1 ) . for this reason , and given the small variation in pm levels between quintiles 1 and 2 , we combined the two lowest quintiles of pm10 to produce four categories in total . the estimated mean pm10 levels for these categories were 0.1 , 7 , 14 and 19 g / m ( long - term averages ) . weighted counts , for deaths occurring among the census respondents with complete data . ethnicity and socioeconomic position are strong predictors of mortality in new zealand , and potential confounders of the association of air pollution with mortality . we assigned each respondent to a mutually exclusive ethnic group using a prioritisation system commonly used in new zealand : maori , if any one of the responses was maori ; pacific , if any one response was pacific but not maori ; and the remainder non - maori non - pacific ( mostly new zealand european ) . smoking status was reported in the census using the categories : never smoker , ex - smoker , or current smoker.24 socioeconomic position was characterised as : total household income , with adjustment for the number of children and adults in the household to allow for economies of scale , log - transformed having first set all values of less than nz$1000 to equal nz$1000;20 highest educational qualification ( higher than school , school , or none ) ; and neighbourhood deprivation measured by the nzdep index ( in quintiles).25 this index of deprivation within small geographical areas was calculated using census data on socioeconomic characteristics ( eg , car access , tenure and receipt of benefits ) at aggregations of approximately 100 people , and assigned to mortality data by use of address . as climate is correlated with pm10 , and associated with mortality ( independently of pm10 ) , temperature has the potential to confound air pollution effects . therefore , we also included estimates of long - term average minimum temperature ( in addition to the above sociodemographic factors ) in the models , also in quintiles based on place of residence . these data were derived from an interpolated temperature surface.26 mean values for the minimum temperature quintiles were 0.2 , 2 , 4 , 5 and 7c . logistic regression analyses were conducted to investigate associations between all - cause and cause - specific mortality rates and average exposure to pm10 , with control for confounding by age , sex , ethnicity , social deprivation , income , education , smoking history and average minimum temperature . as death is a rare outcome over 3 years for the age groups included in the analysis , logistic regression results differ very little from those from either poisson or cox proportional hazards modelling . initial models incorporated pm10 categories as dummy variables , and given a reasonably linear dose response , final models used the estimated mean pm10 for these four categories ( see above ) . in order to facilitate comparison with overseas findings , , age was included as a linear plus a squared term , and the income variable was natural - log transformed . logistic regression model including all variables listed as independent variables ( all deaths , all ethnicities : n= 1 065 645 ) * rounded ; for logit form of model , constant 6.528 ( 7.036 to 6.020 ) . any association of air pollution with mortality might be modified by social and environmental factors , due to different vulnerability or intensity of exposure to air pollution . finally , we conducted a sensitivity analysis by restricting the dataset for analysis to those people who had lived in the same geographical unit at the 1991 census as they did at the time of exposure assignment ( 1996 ) in this cohort study . this restriction should reduce exposure misclassification by excluding those people who have not been exposed to the same level of pm10 for at least 5 years . the method of record linkage has been described in detail elsewhere.19 briefly , probabilistic record linkage is a process used to link two files of records , in which records in one file have a corresponding record in the other file . records from the first file are compared with records from the second file in order to find matching record pairs ( ie , two records belonging to the same individual ) . census and mortality records were linked using date of birth ( day , month and year as separate matching variables ) , country of birth , sex , ethnicity and address of usual residence . linkage was restricted to individuals aged 74 years or below at the time of the census . the proportion of mortality records linked overall was 78%,20 and varied by sex , age , ethnicity and the deprivation index.19 weights were therefore applied to adjust for linkage bias , by strata of sex , age , ethnicity , deprivation , rurality and cause of death.21 for example , if 20 of 30 maori men who died aged 4564 years and living in moderately deprived ( see below ) rural areas of new zealand were linked to a census record , each of the 20 linked records received a weight of 1.5 ( 30/20 ) . air pollution monitoring data are typically only available for a few sites and may not be representative of exposure in other areas . detailed atmospheric dispersion modelling can be used to provide estimates of air pollution exposure for small areas , but these models are difficult to apply to large areas due to data and computer processing limitations . the method of air pollution exposure assessment has been described in detail elsewhere.22 briefly , the approach to modelling long - term average pm10 was as follows . atmospheric dispersion modelling results were available for one city ( christchurch , population 300 000 ) . these data were assumed to be representative of the spatial pattern of annual average pm10 exposures for small areas ( census area units ) in christchurch.23 data on meteorological variables and indicators of air pollutant emissions for these areas were used as predictors of pm10 exposure in christchurch , using regression models . the predictors for the regression models were : census data on domestic heating ; estimates of industrial emissions ; and vehicle kilometres travelled within small areas . because data for these predictor variables were available for all of new zealand , we were able to extrapolate the empirical results for the christchurch regression model to urban census area units throughout the country . the resulting exposure estimates agreed well with multiyear averages of pm10 based on routine monitoring data for urban centres ( 19952001 ) ( n=43 ; r=0.87 ) . while we acknowledge that there may be considerable exposure misclassification , we assume that the pm10 estimates are representative of long - term average spatial patterns of exposure during the 1990s . ideally , for analyses with the new zealand census mortality study data we would have used the continuous pm10 estimates for individual census areas . however , because the new zealand census mortality study data contain census information at unit record level , access to the data is restricted and additional data can only be added if confidentiality is not compromised as a result . assigning continuous pm10 estimates at census area level would have permitted many census areas to be uniquely identified . in order to maintain confidentiality , it was necessary to aggregate the pm10 estimates into quintiles before they could be merged with the census data . quintiles of exposure were calculated based on the estimates for individual census areas . for this purpose , we assigned a pm10 estimate of 0 g / m to rural census areas where pm10 estimates were unavailable . the average pm10 level for all new zealand census areas was 8.3 g / m ( sd 8.4 g / m ) and the cut - off values between pm10 quintiles were 0.0 , 0.5 , 12.5 and 15.4 g / m . individuals were assigned to quintiles of pm10 exposure depending on their census area of residence on census night . in order to avoid bias affecting analyses in smaller rural areas due to migration to cities following the development of disease following restriction to the urban population , the lowest two quintiles of exposure had relatively few observations ( table 1 ) . for this reason , and given the small variation in pm levels between quintiles 1 and 2 , we combined the two lowest quintiles of pm10 to produce four categories in total . the estimated mean pm10 levels for these categories were 0.1 , 7 , 14 and 19 g / m ( long - term averages ) . weighted counts , for deaths occurring among the census respondents with complete data . ethnicity and socioeconomic position are strong predictors of mortality in new zealand , and potential confounders of the association of air pollution with mortality . we assigned each respondent to a mutually exclusive ethnic group using a prioritisation system commonly used in new zealand : maori , if any one of the responses was maori ; pacific , if any one response was pacific but not maori ; and the remainder non - maori non - pacific ( mostly new zealand european ) . smoking status was reported in the census using the categories : never smoker , ex - smoker , or current smoker.24 socioeconomic position was characterised as : total household income , with adjustment for the number of children and adults in the household to allow for economies of scale , log - transformed having first set all values of less than nz$1000 to equal nz$1000;20 highest educational qualification ( higher than school , school , or none ) ; and neighbourhood deprivation measured by the nzdep index ( in quintiles).25 this index of deprivation within small geographical areas was calculated using census data on socioeconomic characteristics ( eg , car access , tenure and receipt of benefits ) at aggregations of approximately 100 people , and assigned to mortality data by use of address . as climate is correlated with pm10 , and associated with mortality ( independently of pm10 ) , temperature has the potential to confound air pollution effects . therefore , we also included estimates of long - term average minimum temperature ( in addition to the above sociodemographic factors ) in the models , also in quintiles based on place of residence . these data were derived from an interpolated temperature surface.26 mean values for the minimum temperature quintiles were 0.2 , 2 , 4 , 5 and 7c . logistic regression analyses were conducted to investigate associations between all - cause and cause - specific mortality rates and average exposure to pm10 , with control for confounding by age , sex , ethnicity , social deprivation , income , education , smoking history and average minimum temperature . as death is a rare outcome over 3 years for the age groups included in the analysis , logistic regression results differ very little from those from either poisson or cox proportional hazards modelling . initial models incorporated pm10 categories as dummy variables , and given a reasonably linear dose response , final models used the estimated mean pm10 for these four categories ( see above ) . in order to facilitate comparison with overseas findings , we report results for adults aged 3074 years . in final models , age was included as a linear plus a squared term , and the income variable was natural - log transformed . all other covariates logistic regression model including all variables listed as independent variables ( all deaths , all ethnicities : n= 1 065 645 ) * rounded ; for logit form of model , constant 6.528 ( 7.036 to 6.020 ) . any association of air pollution with mortality might be modified by social and environmental factors , due to different vulnerability or intensity of exposure to air pollution . finally , we conducted a sensitivity analysis by restricting the dataset for analysis to those people who had lived in the same geographical unit at the 1991 census as they did at the time of exposure assignment ( 1996 ) in this cohort study . this restriction should reduce exposure misclassification by excluding those people who have not been exposed to the same level of pm10 for at least 5 years . when pm10 was modelled using dummy variables , there was an approximately linear increase in mortality with increasing average pm10 exposure ( figure 1 ) . three models are shown : ( 1 ) adjusting for sex , age and ethnicity on all observations ( n=1 364 454 ) ; ( 2 ) the same model , but restricted to observations with non - missing data on other covariates ( n=1 065 645 ; a test of any selection bias compared with model 1 ) ; ( 3 ) fully adjusted model ( n=1 065 645 ; a test of possible confounding compared with model 2 ) . there is no meaningful difference between models 1 and 2 , suggesting no selection bias when restricting analyses to those with full data on covariates . adjusting for potential confounders in model 3 attenuated the or for all non - referent groups , although the relative differences between quintiles 3 , 4 and 5 were not much reduced . that is , adjusting for confounders mostly closed the gap between the referent group and quintile 3 . or and 95% ci of all - cause mortality for people living in the three non - referent pm10 categories , compared with quintiles 1 and 2 combined . model is for sexes combined , restricted to adults aged 3074 years on census night , urban population , with covariates as follows : model 1 : age , sex , ethnicity , all data , n=1 364 454 ; model 2 : age , sex , ethnicity , data with non - missing values for covariates in model 3 , n=1 065 645 ; model 3 : age , sex , ethnicity , deprivation , income , education , smoking , temperature , n=1 065 645 . considering the preferred model 3 , the or increased monotonically and linearly with increasing pm10 level , and the 95% ci for the two highest quintiles excluded 1.0 . given the approximately linear association of pm10 with mortality described above , subsequent models incorporated pm10 as a linear term , using the average pm10 for each category ) ( tables 2 and 3 ) . table 2 shows the same model as in figure 1 , except for the continuous treatment of pm10 . the or of 1.007 corresponds to a 1 unit increase in pm10 , which equates to an increase of 7% ( 95% ci 3% to 10% ) in the odds of all - cause mortality in adults ( aged between age 30 and 74 years at census ) per 10 g / m increase in long - term average pm10 exposure . findings by subgroups of ethnicity and cause of death ( model 3 , fully adjusted ) note : models included persons dying from the specified causes ( coded 1 ) and people presumed alive at the end of follow - up ( coded 0 ) , but excluded persons dying from other causes . other deaths are all those deaths ( including unspecified ) that are not included among lung cancer , respiratory , cardiovascular disease or accidents . we found stronger effects of pm10 among people who lived in the same census area in 1991 ( at the time of the previous census ) , 8% ( 4% to 12% ) per 10 g / m increase in pm10 . by cause of death , the association was similar for all natural causes , 7% ( 3% to 10% ) , but substantially stronger for respiratory deaths ( including lung cancers ) , 14% ( 5% to 23% ) and for lung cancers , 16% ( 4% to 29% ) . for cardiovascular disease , 6% ( 1% to 12% ) and for other and unspecified causes of death , 5% ( 1% to 10% ) , the association was marginally significant ; while for accidental deaths and injuries , the association was non - significant , 4% ( 9% to 20% ) ( table 3 ) . considering interaction with social variables , there was an apparently stronger association of pm10 with all - cause mortality among maori , 20% ( 7% to 33% ) . however , the 95% ci for europeans overlapped that for maori , 7% ( 3% to 10% ) . a wald test for interaction between ethnicity and pm10 was not significant ( p=0.12 ) . there were no statistically significant interactions with age , sex , income , deprivation , educational status or average temperature . mortality was lower among people living in warmer census areas , and the difference between the lowest and the highest quintile of annual average temperature was statistically significant . using linked mortality and census data , we report a significant positive association between estimated long - term exposure to air pollution ( pm10 ) and mortality in new zealand urban areas . this setting includes approximately 75% of the new zealand population , who are exposed to relatively low levels of pm10 compared with other countries . the results persist after controlling for plausible confounders , including multiple measures of socioeconomic position and smoking . most of this misclassification was probably non - differential by mortality risk ( meaning we have probably significantly underestimated the true strength of association ) . there is potential differential misclassification of pm10 by mortality risk in our study , because our assessment was based on modelling in one city using proxies , including domestic heating , estimates of industrial emissions and vehicle kilometres travelled within small areas . if these proxies are not such reliable predictors of pm10 in other cities , and are ( say ) correlated with socioeconomic position , then it may be that our pm10 estimates are also capturing aspects of socioeconomic exposure . however , the fact that an association remained after extensive control of socioeconomic factors , including individual level income and education , makes this an unlikely explanation of the results . the use of modelled estimates of pm10 exposure will tend to smooth the data and reduce the resulting ci it is possible that less healthy people might migrate towards health services ( or other service amenities ) that happen to be in more polluted areas a form of reverse causation or endogeneity . however , we think this is unlikely to be an important factor as new zealand cities are relatively small ( maximum 1.4 million ) , and most suburbs in new zealand 's main cities have relatively good access to hospitals . the odds of all - cause mortality in adults ( aged between 30 and 74 years at census ) increased by 7% ( 95% ci 3% to 10% ) per 10 g / m increase in average pm10 exposure . our observations are consistent with an increasing number of studies of long - term exposure to particulate matter and mortality.310 1318 the original us six cities study reported an adjusted mortality rate ratio of 1.27 ( 95% ci 1.08 to 1.48 ) for the most polluted compared with the least polluted city , corresponding to 18.2 and 46.5 g / m pm10 , respectively3equivalent to an increase in mortality of approximately 10% per 10 g / m pm10 . in the us nurses health study , there was a 16% ( 5% to 28% ) increase in all - cause mortality per 10 g / m pm10.14 it is not yet clear to what extent the heterogeneity in reported dose response in those studies is related to differences in the accuracy of exposure measurement , to differences in the toxicity of complex mixtures of pollutants at differing levels of exposure , and/or differences in the sensitivity of exposed populations . recent studies use the more specific measure pm2.5 ( particulate matter with an aerodynamic diameter less than 2.5 m ) rather than pm10 , whereas several european studies use black smoke or total suspended particulates as the exposure measure . the association between particulate air pollution exposure and mortality is usually found to be strongest for finer fractions , such that the dose response for pm2.5 is greater than for pm10 , which in turn is greater than for total suspended particulates . an extended follow - up of the us six cities study reported a 14% ( 6% to 22% ) increase in mortality per 10 g / m pm2.5,9 whereas the american cancer society study reported 6% ( 2% to 11% ) and the nurses health study 26% ( 2% to 54% ) , while coarse particulate matter exposure ( pm102.5 ) was not associated with an increase in mortality in that study.27 an analysis based on electoral wards in the uk found a 1.3% ( 1% to 1.6% ) increase in all - cause mortality per 10 g / m increase in black smoke.10 in 18 regions in france , the paarc study reported a 7% ( 3% to 10% ) increase per 10 g / m increase in black smoke and a 5% ( 2% to 8% ) increase for total suspended particulates.7 in the netherlands , there was a 5% ( 0% to 10% ) increase in mortality per 10 g / m increase in black smoke . our study assessed the association of pm10 and mortality over 3 years . in the us nurses health study , mortality was most strongly associated with average pm10 exposures in the 24 months before death . in the uk , the association , although weak , was stronger for exposure in the 4 years before death . however , a re - analysis of the american cancer society study found no clear effect of exposure period.17 a priori , we hypothesised that the association of pm10 with mortality in our study would be stronger for a cohort restricted to those census respondents who lived in the same census area at the time of the 1991 census . we also hypothesised that the association of pm10 with mortality would be stronger for cardiorespiratory deaths . our results were consistent with these a priori hypotheses , strengthening the ability to make causal inference . there is some evidence , most consistently in studies with individual measurements of social factors , that more deprived populations are particularly sensitive to air pollution effects.5 11 12 28 29 our ability to detect a true difference by ethnicity in sensitivity to air pollution was limited by the relatively small maori population . although not significant , the difference in our central effect estimates for european and maori was substantial : 7% versus 20% . this might reflect a higher prevalence of pre - existing cardiorespiratory disease among maori , or a difference in the toxicity of air pollution to which different ethnic groups are typically exposed . we found no other suggestion of interaction of social factors with pm10 in the association with mortality . in this longitudinal study we report an association of pm10 with mortality , consistent with that reported elsewhere , in a country with low levels of air pollution . the major limitation of our study is the probable misclassification of the pm10 exposure . on balance , this means we have probably underestimated the strength of association . the study design has several strengths , including national population coverage and good control of confounding . we found that the association was , as hypothesised a priori , stronger for cardiorespiratory deaths and people with less residential mobility . relatively few cohort studies of the health effects of urban air pollution have been published , particularly outside north america and europe . we report here findings from a longitudinal study in new zealand , based on the national census . there is evidence , most consistently in studies with individual measurement of social factors , that more deprived populations are particularly sensitive to air pollution effects . we found an association of pm10 with mortality in a country with relatively low levels of air pollution . there was some evidence that maori may have greater susceptibility to life - shortening effects of air pollution .

backgroundfew cohort studies of the health effects of urban air pollution have been published . there is evidence , most consistently in studies with individual measurement of social factors , that more deprived populations are particularly sensitive to air pollution effects.methodsrecords from the 1996 new zealand census were anonymously and probabilistically linked to mortality data , creating a cohort study of the new zealand population followed up for 3 years . there were 1.06 million adults living in urban areas for which data were available on all covariates . estimates of exposure to air pollution ( measured as particulate matter with an aerodynamic diameter less than 10 m , pm10 ) were available for census area units from a previous land use regression study . logistic regression analyses were conducted to investigate associations between cause - specific mortality rates and average exposure to pm10 in urban areas , with control for confounding by age , sex , ethnicity , social deprivation , income , education , smoking history and ambient temperature.resultsthe odds of all - cause mortality in adults ( aged 3074 years at census ) increased by 7% per 10 g / m3 increase in average pm10 exposure ( 95% ci 3% to 10% ) and 20% per 10 g / m3 among maori , but with wide ci ( 7% to 33% ) . associations were stronger for respiratory and lung cancer deaths.conclusionsan association of pm10 with mortality is reported in a country with relatively low levels of air pollution . the major limitation of the study is the probable misclassification of pm10 exposure . on balance , this means the strength of association was probably underestimated . the apparently greater association among maori might be due to different levels of co - morbidity .

Methods Record linkage Air pollution exposure estimation Results Discussion Conclusion

the new zealand census - mortality study is a population - wide cohort study , in which the cohort consists of the entire 1996 resident population ( n=3 732 000 ) and the outcome of interest is mortality . for this analysis , records from the 1996 census were anonymously linked to 3 years of subsequent mortality data , creating a cohort study of the new zealand population followed up for 3 years.19 20 in new zealand , urban areas are defined as cities having a population of at least 30 000 people ; about half of the approximately 2000 census areas are classified as urban . because the definition of urban areas in new zealand includes small towns and suburbs , many urban census areas have low levels of air pollution . census and mortality records were linked using date of birth ( day , month and year as separate matching variables ) , country of birth , sex , ethnicity and address of usual residence . the proportion of mortality records linked overall was 78%,20 and varied by sex , age , ethnicity and the deprivation index.19 weights were therefore applied to adjust for linkage bias , by strata of sex , age , ethnicity , deprivation , rurality and cause of death.21 for example , if 20 of 30 maori men who died aged 4564 years and living in moderately deprived ( see below ) rural areas of new zealand were linked to a census record , each of the 20 linked records received a weight of 1.5 ( 30/20 ) . air pollution monitoring data are typically only available for a few sites and may not be representative of exposure in other areas . detailed atmospheric dispersion modelling can be used to provide estimates of air pollution exposure for small areas , but these models are difficult to apply to large areas due to data and computer processing limitations . the method of air pollution exposure assessment has been described in detail elsewhere.22 briefly , the approach to modelling long - term average pm10 was as follows . atmospheric dispersion modelling results were available for one city ( christchurch , population 300 000 ) . these data were assumed to be representative of the spatial pattern of annual average pm10 exposures for small areas ( census area units ) in christchurch.23 data on meteorological variables and indicators of air pollutant emissions for these areas were used as predictors of pm10 exposure in christchurch , using regression models . because data for these predictor variables were available for all of new zealand , we were able to extrapolate the empirical results for the christchurch regression model to urban census area units throughout the country . ideally , for analyses with the new zealand census mortality study data we would have used the continuous pm10 estimates for individual census areas . however , because the new zealand census mortality study data contain census information at unit record level , access to the data is restricted and additional data can only be added if confidentiality is not compromised as a result . assigning continuous pm10 estimates at census area level would have permitted many census areas to be uniquely identified . the average pm10 level for all new zealand census areas was 8.3 g / m ( sd 8.4 g / m ) and the cut - off values between pm10 quintiles were 0.0 , 0.5 , 12.5 and 15.4 g / m . individuals were assigned to quintiles of pm10 exposure depending on their census area of residence on census night . ethnicity and socioeconomic position are strong predictors of mortality in new zealand , and potential confounders of the association of air pollution with mortality . we assigned each respondent to a mutually exclusive ethnic group using a prioritisation system commonly used in new zealand : maori , if any one of the responses was maori ; pacific , if any one response was pacific but not maori ; and the remainder non - maori non - pacific ( mostly new zealand european ) . smoking status was reported in the census using the categories : never smoker , ex - smoker , or current smoker.24 socioeconomic position was characterised as : total household income , with adjustment for the number of children and adults in the household to allow for economies of scale , log - transformed having first set all values of less than nz$1000 to equal nz$1000;20 highest educational qualification ( higher than school , school , or none ) ; and neighbourhood deprivation measured by the nzdep index ( in quintiles).25 this index of deprivation within small geographical areas was calculated using census data on socioeconomic characteristics ( eg , car access , tenure and receipt of benefits ) at aggregations of approximately 100 people , and assigned to mortality data by use of address . as climate is correlated with pm10 , and associated with mortality ( independently of pm10 ) , temperature has the potential to confound air pollution effects . logistic regression analyses were conducted to investigate associations between all - cause and cause - specific mortality rates and average exposure to pm10 , with control for confounding by age , sex , ethnicity , social deprivation , income , education , smoking history and average minimum temperature . any association of air pollution with mortality might be modified by social and environmental factors , due to different vulnerability or intensity of exposure to air pollution . finally , we conducted a sensitivity analysis by restricting the dataset for analysis to those people who had lived in the same geographical unit at the 1991 census as they did at the time of exposure assignment ( 1996 ) in this cohort study . census and mortality records were linked using date of birth ( day , month and year as separate matching variables ) , country of birth , sex , ethnicity and address of usual residence . the proportion of mortality records linked overall was 78%,20 and varied by sex , age , ethnicity and the deprivation index.19 weights were therefore applied to adjust for linkage bias , by strata of sex , age , ethnicity , deprivation , rurality and cause of death.21 for example , if 20 of 30 maori men who died aged 4564 years and living in moderately deprived ( see below ) rural areas of new zealand were linked to a census record , each of the 20 linked records received a weight of 1.5 ( 30/20 ) . air pollution monitoring data are typically only available for a few sites and may not be representative of exposure in other areas . detailed atmospheric dispersion modelling can be used to provide estimates of air pollution exposure for small areas , but these models are difficult to apply to large areas due to data and computer processing limitations . the method of air pollution exposure assessment has been described in detail elsewhere.22 briefly , the approach to modelling long - term average pm10 was as follows . atmospheric dispersion modelling results were available for one city ( christchurch , population 300 000 ) . these data were assumed to be representative of the spatial pattern of annual average pm10 exposures for small areas ( census area units ) in christchurch.23 data on meteorological variables and indicators of air pollutant emissions for these areas were used as predictors of pm10 exposure in christchurch , using regression models . because data for these predictor variables were available for all of new zealand , we were able to extrapolate the empirical results for the christchurch regression model to urban census area units throughout the country . ideally , for analyses with the new zealand census mortality study data we would have used the continuous pm10 estimates for individual census areas . however , because the new zealand census mortality study data contain census information at unit record level , access to the data is restricted and additional data can only be added if confidentiality is not compromised as a result . assigning continuous pm10 estimates at census area level would have permitted many census areas to be uniquely identified . the average pm10 level for all new zealand census areas was 8.3 g / m ( sd 8.4 g / m ) and the cut - off values between pm10 quintiles were 0.0 , 0.5 , 12.5 and 15.4 g / m . individuals were assigned to quintiles of pm10 exposure depending on their census area of residence on census night . in order to avoid bias affecting analyses in smaller rural areas due to migration to cities following the development of disease following restriction to the urban population , the lowest two quintiles of exposure had relatively few observations ( table 1 ) . ethnicity and socioeconomic position are strong predictors of mortality in new zealand , and potential confounders of the association of air pollution with mortality . we assigned each respondent to a mutually exclusive ethnic group using a prioritisation system commonly used in new zealand : maori , if any one of the responses was maori ; pacific , if any one response was pacific but not maori ; and the remainder non - maori non - pacific ( mostly new zealand european ) . smoking status was reported in the census using the categories : never smoker , ex - smoker , or current smoker.24 socioeconomic position was characterised as : total household income , with adjustment for the number of children and adults in the household to allow for economies of scale , log - transformed having first set all values of less than nz$1000 to equal nz$1000;20 highest educational qualification ( higher than school , school , or none ) ; and neighbourhood deprivation measured by the nzdep index ( in quintiles).25 this index of deprivation within small geographical areas was calculated using census data on socioeconomic characteristics ( eg , car access , tenure and receipt of benefits ) at aggregations of approximately 100 people , and assigned to mortality data by use of address . as climate is correlated with pm10 , and associated with mortality ( independently of pm10 ) , temperature has the potential to confound air pollution effects . logistic regression analyses were conducted to investigate associations between all - cause and cause - specific mortality rates and average exposure to pm10 , with control for confounding by age , sex , ethnicity , social deprivation , income , education , smoking history and average minimum temperature . as death is a rare outcome over 3 years for the age groups included in the analysis , logistic regression results differ very little from those from either poisson or cox proportional hazards modelling . any association of air pollution with mortality might be modified by social and environmental factors , due to different vulnerability or intensity of exposure to air pollution . finally , we conducted a sensitivity analysis by restricting the dataset for analysis to those people who had lived in the same geographical unit at the 1991 census as they did at the time of exposure assignment ( 1996 ) in this cohort study . when pm10 was modelled using dummy variables , there was an approximately linear increase in mortality with increasing average pm10 exposure ( figure 1 ) . three models are shown : ( 1 ) adjusting for sex , age and ethnicity on all observations ( n=1 364 454 ) ; ( 2 ) the same model , but restricted to observations with non - missing data on other covariates ( n=1 065 645 ; a test of any selection bias compared with model 1 ) ; ( 3 ) fully adjusted model ( n=1 065 645 ; a test of possible confounding compared with model 2 ) . or and 95% ci of all - cause mortality for people living in the three non - referent pm10 categories , compared with quintiles 1 and 2 combined . model is for sexes combined , restricted to adults aged 3074 years on census night , urban population , with covariates as follows : model 1 : age , sex , ethnicity , all data , n=1 364 454 ; model 2 : age , sex , ethnicity , data with non - missing values for covariates in model 3 , n=1 065 645 ; model 3 : age , sex , ethnicity , deprivation , income , education , smoking , temperature , n=1 065 645 . given the approximately linear association of pm10 with mortality described above , subsequent models incorporated pm10 as a linear term , using the average pm10 for each category ) ( tables 2 and 3 ) . the or of 1.007 corresponds to a 1 unit increase in pm10 , which equates to an increase of 7% ( 95% ci 3% to 10% ) in the odds of all - cause mortality in adults ( aged between age 30 and 74 years at census ) per 10 g / m increase in long - term average pm10 exposure . findings by subgroups of ethnicity and cause of death ( model 3 , fully adjusted ) note : models included persons dying from the specified causes ( coded 1 ) and people presumed alive at the end of follow - up ( coded 0 ) , but excluded persons dying from other causes . we found stronger effects of pm10 among people who lived in the same census area in 1991 ( at the time of the previous census ) , 8% ( 4% to 12% ) per 10 g / m increase in pm10 . by cause of death , the association was similar for all natural causes , 7% ( 3% to 10% ) , but substantially stronger for respiratory deaths ( including lung cancers ) , 14% ( 5% to 23% ) and for lung cancers , 16% ( 4% to 29% ) . for cardiovascular disease , 6% ( 1% to 12% ) and for other and unspecified causes of death , 5% ( 1% to 10% ) , the association was marginally significant ; while for accidental deaths and injuries , the association was non - significant , 4% ( 9% to 20% ) ( table 3 ) . considering interaction with social variables , there was an apparently stronger association of pm10 with all - cause mortality among maori , 20% ( 7% to 33% ) . however , the 95% ci for europeans overlapped that for maori , 7% ( 3% to 10% ) . there were no statistically significant interactions with age , sex , income , deprivation , educational status or average temperature . using linked mortality and census data , we report a significant positive association between estimated long - term exposure to air pollution ( pm10 ) and mortality in new zealand urban areas . this setting includes approximately 75% of the new zealand population , who are exposed to relatively low levels of pm10 compared with other countries . most of this misclassification was probably non - differential by mortality risk ( meaning we have probably significantly underestimated the true strength of association ) . there is potential differential misclassification of pm10 by mortality risk in our study , because our assessment was based on modelling in one city using proxies , including domestic heating , estimates of industrial emissions and vehicle kilometres travelled within small areas . if these proxies are not such reliable predictors of pm10 in other cities , and are ( say ) correlated with socioeconomic position , then it may be that our pm10 estimates are also capturing aspects of socioeconomic exposure . however , the fact that an association remained after extensive control of socioeconomic factors , including individual level income and education , makes this an unlikely explanation of the results . the use of modelled estimates of pm10 exposure will tend to smooth the data and reduce the resulting ci it is possible that less healthy people might migrate towards health services ( or other service amenities ) that happen to be in more polluted areas a form of reverse causation or endogeneity . the odds of all - cause mortality in adults ( aged between 30 and 74 years at census ) increased by 7% ( 95% ci 3% to 10% ) per 10 g / m increase in average pm10 exposure . our observations are consistent with an increasing number of studies of long - term exposure to particulate matter and mortality.310 1318 the original us six cities study reported an adjusted mortality rate ratio of 1.27 ( 95% ci 1.08 to 1.48 ) for the most polluted compared with the least polluted city , corresponding to 18.2 and 46.5 g / m pm10 , respectively3equivalent to an increase in mortality of approximately 10% per 10 g / m pm10 . in the us nurses health study , there was a 16% ( 5% to 28% ) increase in all - cause mortality per 10 g / m pm10.14 it is not yet clear to what extent the heterogeneity in reported dose response in those studies is related to differences in the accuracy of exposure measurement , to differences in the toxicity of complex mixtures of pollutants at differing levels of exposure , and/or differences in the sensitivity of exposed populations . recent studies use the more specific measure pm2.5 ( particulate matter with an aerodynamic diameter less than 2.5 m ) rather than pm10 , whereas several european studies use black smoke or total suspended particulates as the exposure measure . the association between particulate air pollution exposure and mortality is usually found to be strongest for finer fractions , such that the dose response for pm2.5 is greater than for pm10 , which in turn is greater than for total suspended particulates . an extended follow - up of the us six cities study reported a 14% ( 6% to 22% ) increase in mortality per 10 g / m pm2.5,9 whereas the american cancer society study reported 6% ( 2% to 11% ) and the nurses health study 26% ( 2% to 54% ) , while coarse particulate matter exposure ( pm102.5 ) was not associated with an increase in mortality in that study.27 an analysis based on electoral wards in the uk found a 1.3% ( 1% to 1.6% ) increase in all - cause mortality per 10 g / m increase in black smoke.10 in 18 regions in france , the paarc study reported a 7% ( 3% to 10% ) increase per 10 g / m increase in black smoke and a 5% ( 2% to 8% ) increase for total suspended particulates.7 in the netherlands , there was a 5% ( 0% to 10% ) increase in mortality per 10 g / m increase in black smoke . our study assessed the association of pm10 and mortality over 3 years . however , a re - analysis of the american cancer society study found no clear effect of exposure period.17 a priori , we hypothesised that the association of pm10 with mortality in our study would be stronger for a cohort restricted to those census respondents who lived in the same census area at the time of the 1991 census . we also hypothesised that the association of pm10 with mortality would be stronger for cardiorespiratory deaths . there is some evidence , most consistently in studies with individual measurements of social factors , that more deprived populations are particularly sensitive to air pollution effects.5 11 12 28 29 our ability to detect a true difference by ethnicity in sensitivity to air pollution was limited by the relatively small maori population . this might reflect a higher prevalence of pre - existing cardiorespiratory disease among maori , or a difference in the toxicity of air pollution to which different ethnic groups are typically exposed . we found no other suggestion of interaction of social factors with pm10 in the association with mortality . in this longitudinal study we report an association of pm10 with mortality , consistent with that reported elsewhere , in a country with low levels of air pollution . the major limitation of our study is the probable misclassification of the pm10 exposure . on balance , this means we have probably underestimated the strength of association . we found that the association was , as hypothesised a priori , stronger for cardiorespiratory deaths and people with less residential mobility . relatively few cohort studies of the health effects of urban air pollution have been published , particularly outside north america and europe . we report here findings from a longitudinal study in new zealand , based on the national census . there is evidence , most consistently in studies with individual measurement of social factors , that more deprived populations are particularly sensitive to air pollution effects . we found an association of pm10 with mortality in a country with relatively low levels of air pollution . there was some evidence that maori may have greater susceptibility to life - shortening effects of air pollution .

the oregon health authority conducts active , laboratory - based surveillance for all cases of nts infection . physicians and laboratories are required by law to report laboratory - confirmed and clinically suspected cases of salmonellosis to the patient s local health department ; reports should contain the patient s date of birth , sex , diagnosis , date of symptom onset , date of specimen collection , and laboratory test results . all salmonella isolates are forwarded to the oregon state public health laboratory ( osphl ) , where they are serotyped . local health department officials interview patients about hospitalization , clinical outcomes , additional demographic information , and exposure history for the 7 days before illness onset . risk - factor questions ask about specific travel , human , animal , and high - risk food exposures . international travel was considered a risk factor only if it had taken place in the 30 days before illness onset . patients with recurrent infection or multiple salmonella isolates ( of same serotype within a plausible time frame for the original infection ) are interviewed only once , at the time of initial illness onset . during 20042009 , the population of oregon was 3.63.8 million persons , which is 1.2% of the us population ( 21,22 ) . the surveillance system in oregon is estimated to capture > 99% of laboratory - confirmed cases of salmonellosis ; however , for every 1 case confirmed , an estimated 25 additional cases are not detected ( 2 ) . for 2004 and 2005 , all confirmed isolates were forwarded to the oregon state university veterinary diagnostic laboratory for susceptibility testing . from 2006 through 2009 , susceptibility testing was performed by osphl . all isolates were tested by using broth microdilution to determine mics for the following 10 antimicrobial agents : ampicillin , ceftriaxone , chloramphenicol , ciprofloxacin , gentamicin , nalidixic acid , nitrofurantoin , sulfamethoxazole , tetracycline , and trimethoprim / sulfamethoxazole . susceptibilities were determined according to clinical and laboratory standards institute interpretative criteria ( 23 ) . to ascertain cephalosporin resistance , osphl tested isolates for ceftriaxone susceptibility ; and the oregon state university veterinary diagnostic laboratory tested for susceptibility to cefuroxime and cephalothin by using analogous broth microdilution methods . isolates were included in analyses only if they were cultured from specific specimens , such as feces , urine , or blood or other normally sterile tissues ( i.e. , cerebrospinal fluid ) . cir was defined as resistance to at least 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole ( 13 ) . we used the cochran - armitage test for trend to analyze nts case data for 20042009 to determine whether the proportion of salmonella isolates with cir increased significantly . demographic and exposure risk factors , specifically international travel , were evaluated as risk factors for acquisition of a resistant isolate . the 9 most common salmonella serotypes were fixed ( included in all models regardless whether they met the p<0.05 level of significance ) in all analyses , and remaining serotypes were grouped as all other . serotype enterditis is the most frequently isolated serotype in oregon and was therefore used as the referent for all comparisons . we sought to evaluate whether resistance was associated with increased disease severity , including hospitalization and invasive infection . invasive infection was defined as isolation of salmonella from a normally sterile body site or tissue , such as blood ( 13 ) . multiple logistic regression models were constructed with variables that were significant at the p<0.25 level in unadjusted analyses . salmonella serotype and patient race , age , and year of illness onset were fixed in all models . other variables were given further consideration according to disease severity or relevance for external validity . predictor variables significant at p<0.05 were retained in the final model , and adjusted log odds ratios ( aors ) were calculated . model fit was assessed by using the hosmer lemeshow goodness - of - fit test . all analyses were performed by using sas version 9.2 ( sas institute inc . , cary , nc , usa ) . because this study involved more extensive analysis only of data collected routinely as part of public health surveillance , it was not considered human subjects research . the oregon health authority conducts active , laboratory - based surveillance for all cases of nts infection . physicians and laboratories are required by law to report laboratory - confirmed and clinically suspected cases of salmonellosis to the patient s local health department ; reports should contain the patient s date of birth , sex , diagnosis , date of symptom onset , date of specimen collection , and laboratory test results . all salmonella isolates are forwarded to the oregon state public health laboratory ( osphl ) , where they are serotyped . local health department officials interview patients about hospitalization , clinical outcomes , additional demographic information , and exposure history for the 7 days before illness onset . risk - factor questions ask about specific travel , human , animal , and high - risk food exposures . international travel was considered a risk factor only if it had taken place in the 30 days before illness onset . patients with recurrent infection or multiple salmonella isolates ( of same serotype within a plausible time frame for the original infection ) are interviewed only once , at the time of initial illness onset . during 20042009 , the population of oregon was 3.63.8 million persons , which is 1.2% of the us population ( 21,22 ) . the surveillance system in oregon is estimated to capture > 99% of laboratory - confirmed cases of salmonellosis ; however , for every 1 case confirmed , an estimated 25 additional cases are not detected ( 2 ) . for 2004 and 2005 , all confirmed isolates were forwarded to the oregon state university veterinary diagnostic laboratory for susceptibility testing . from 2006 through 2009 , all isolates were tested by using broth microdilution to determine mics for the following 10 antimicrobial agents : ampicillin , ceftriaxone , chloramphenicol , ciprofloxacin , gentamicin , nalidixic acid , nitrofurantoin , sulfamethoxazole , tetracycline , and trimethoprim / sulfamethoxazole . susceptibilities were determined according to clinical and laboratory standards institute interpretative criteria ( 23 ) . to ascertain cephalosporin resistance , osphl tested isolates for ceftriaxone susceptibility ; and the oregon state university veterinary diagnostic laboratory tested for susceptibility to cefuroxime and cephalothin by using analogous broth microdilution methods . isolates were included in analyses only if they were cultured from specific specimens , such as feces , urine , or blood or other normally sterile tissues ( i.e. , cerebrospinal fluid ) . cir was defined as resistance to at least 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole ( 13 ) . we used the cochran - armitage test for trend to analyze nts case data for 20042009 to determine whether the proportion of salmonella isolates with cir increased significantly . demographic and exposure risk factors , specifically international travel , were evaluated as risk factors for acquisition of a resistant isolate . the 9 most common salmonella serotypes were fixed ( included in all models regardless whether they met the p<0.05 level of significance ) in all analyses , and remaining serotypes were grouped as all other . serotype enterditis is the most frequently isolated serotype in oregon and was therefore used as the referent for all comparisons . we sought to evaluate whether resistance was associated with increased disease severity , including hospitalization and invasive infection . invasive infection was defined as isolation of salmonella from a normally sterile body site or tissue , such as blood ( 13 ) . multiple logistic regression models were constructed with variables that were significant at the p<0.25 level in unadjusted analyses . salmonella serotype and patient race , age , and year of illness onset were fixed in all models . other variables were given further consideration according to disease severity or relevance for external validity . predictor variables significant at p<0.05 were retained in the final model , and adjusted log odds ratios ( aors ) were calculated . model fit was assessed by using the hosmer lemeshow goodness - of - fit test . all analyses were performed by using sas version 9.2 ( sas institute inc . , cary , nc , usa ) . because this study involved more extensive analysis only of data collected routinely as part of public health surveillance , it was not considered human subjects research . from 2004 through 2009 , a total of 2,255 laboratory - confirmed cases of nontyphoidal salmonellosis were reported in oregon . in accordance with oregon law , 2,153 isolates were forwarded to osphl , and 2,127 ( 98.8% of all nts isolates ) were cultured from a specific source and had antimicrobial drug susceptibility testing information ( figure ) . of these isolates , 26 ( 1.2% ) were obtained through a nonspecific source , such as lesions or sputum , and were excluded from analysis . culture - confirmed salmonellosis cases ascertained by statewide active surveillance and included in analyses , oregon , usa , 20042009 . the most common salmonella serotypes detected were enteritidis ( 18.4% ) , typhimurium ( 14.3% ) , heidelberg ( 8.2% ) , typhimurium var . cases that were part of identified outbreaks represented 24.1% of the cohort ; the remaining 75.9% were considered sporadic cases . the median age of patients was 29 years ( interquartile range 951 years ) , and 53.1% of patients were female . information about race was not available for 9% ; of patients for whom race was known , 91.8% were white and 9.2% were not white . similarly , information about ethnicity was not available for 10.5% of patients . among patients for whom isolates from 1,213 ( 57% ) patients were susceptible to all antimicrobial drugs screened ( pansusceptible ) , and isolates from 347 ( 16.3% ) had cir ( table 1 ) . of the 2,127 patients , 412 ( 19.4% ) were hospitalized and 110 ( 5.2% ) had invasive disease . * cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . the proportion of isolates that were pansusceptible significantly decreased from 69.5% in 2004 to 53.6% in 2009 ( p<0.01 ) . stratification by serotype revealed that cir increased among the 3 most common serotypes : enteritidis ( 3% to 8% , p = 0.02 ) , typhimurium ( 19% to 34% , p = 0.03 ) , and heidelberg ( 6% to 30% , p<0.01 ) . significant increases were identified for resistance to ciprofloxacin ( p<0.05 ) , nalidixic acid ( p<0.01 ) , sulfamethoxazole ( p<0.01 ) , tetracycline ( p<0.01 ) , and trimethoprim / sulfamethoxazole ( p<0.01 ) . we suspected that these findings were confounded by serotype and therefore used logistic regression to model the odds of acquiring a resistant infection for each of the clinically important antimicrobial drugs ( ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole ) as well as cir . serotype - adjusted log odds ratios were generated with year of infection entered as a discrete continuous variable . after adjusting for serotype , we found that with each subsequent year , patients were 30% more likely to acquire an infection that was resistant to quinolones ( nalidixic acid or ciprofloxacin ) and trimethoprim / sulfamethoxazole ( table 2 ) . we also found that with each year , odds of acquiring an infection with cir increased by 13% . * multiple logistic regression analysis of 2,127 isolates . cir was associated with hospitalization ( odds ratio [ or ] 1.5 , 95% ci 1.12.0 ) . this association was preserved after adjustment for serotype , patient age , patient race , and year ( aor 1.7 , 95% ci 1.22.1 ) . for patients with cir infections , odds of invasive infection were increased , although not significantly , according to unadjusted or adjusted analyses ( or 1.4 , 95% ci 0.92.2 and aor 1.5 , 95% ci 0.92.5 , respectively ) . of the 2,127 patients included in the previous analyses , 1,813 ( 84.2% of all oregon patients with nts ) were interviewed . for 305 ( 16.8% ) of these patients , isolates had cir , and for the remaining 1,508 ( 83.2% ) , isolates were susceptible to all clinically important antimicrobial drugs ( figure ) . of the 1,508 isolates susceptible to clinically important drugs , 1,002 ( 55.3% ) were susceptible to all drugs screened and 506 ( 27.9% ) were resistant to at least 1 non - cir drug . according to the unadjusted analysis , several serotypes were more likely than the referent serotype , enteritidis , to be resistant to > 1 clinically important antimicrobial drug ( table 3 ) . patient sex , race , age , and ethnicity were not significantly associated with resistance . * multiple logistic regression analysis of 1,813 patients ; cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . cir was not associated with any other demographic risk factors or high - risk food or animal exposures . however when international travel was examined by individual countries or applicable united nations region , cir was significantly associated with travel to southeast asia ( 24 ) . associations of resistance with travel to mexico and eastern asia also approached significance ( table 4 ) . the most common travel destinations in asia where resistant infections were acquired were thailand , china , and malaysia / indonesia . on the basis of these findings , we analyzed international travel by 3 destinations : central america , including mexico ( 135 patients ) , eastern and southeast asia ( 46 patients ) , and all other international travel destinations ( 77 patients ) . * cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . patients who were part of identified outbreak clusters were significantly less likely than patients with sporadic infections to have a resistant infection ( or 0.5 , 95% ci 0.40.8 ) . during our study period , 131 outbreaks ( 406 cases ) occurred , and for 25 of these outbreaks a causative vehicle was successfully identified . to assess whether oversampling of cases from outbreak clusters could explain this association , we first restricted cases to 1 isolate per outbreak where a causative vehicle was implicated while retaining all cases from outbreaks for which a vehicle was not implicated ( 302 cases ) . second , we further restricted cases to 1 isolate per outbreak , regardless whether a vehicle was identified ( 131 cases ) . in each of these analyses the magnitude , direction , and significance of the association was preserved ( or 0.6 , 95% ci 0.40.8 and or 0.5 , 95% ci 0.30.9 , respectively ) , suggesting that oversampling could not have explained this association . furthermore , 53.6% of outbreaks had intra - outbreak cases for which the antimicrobial drug susceptibility profiles of the isolates differed . the resultant main - effects model included the fixed variables of serotype , patient age , year of onset , and patient race , along with travel to eastern or southeast asia , and outbreak association ( table 3 ) . we hypothesized that effect modification occurred between the variables of outbreak cases and travel to eastern or southeast asia , as well as between travel to eastern or southeast asia , serotypes , and outbreaks . the association between resistance and travel to eastern or southeast asia was preserved after exclusion of all outbreak - associated cases ( aor 4.6 , 95% ci 2.39.4 ; table 5 ) . similarly , the association between resistance and outbreak - associated cases was preserved after exclusion of patients with a history of international travel ( aor 0.5 , 95% ci 0.40.8 ; table 6 ) . therefore , these results suggest that inclusion of patients with a history of travel to asia , as well as patients with outbreak - associated infections for the main analysis , was appropriate . * multiple logistic regression analysis . cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole.clinically important resistance . patients with a history of recent travel to eastern or southeast asia were > 5 times more likely to acquire a cir infection than were patients with no history of recent international travel . the most common serotypes acquired among persons with a history of travel to asia were enteriditis ( n = 13 , 54% cir ) , typhimurium ( n = 5 , 60% cir ) , newport ( n = 4 , 25% cir ) , i 4 , 5 , 12:i:- ( n = 4 , 50% cir ) , stanley ( n = 3 , 33% cir ) , and typhimurium var . copenhagen ( n = 2 , 100% cir ) . patients with outbreak - associated infections were half as likely as those with sporadic infections to have cir ( table 3 ) . to identify risk factors for resistance to individual antimicrobial drugs , we constructed models with each of the clinically important antimicrobial drugs . travel to eastern or southeast asia was significantly associated with resistance to ampicillin , quinolones ( nalidixic acid or ciprofloxacin ) , and trimethoprim / sulfamethoxazole ( table 7 ) . only individual serotypes were associated with resistance to cephalosporins or gentamicin , and no other risk factors were significantly associated with resistance to ampicillin , quinolones , or trimethoprim / sulfamethoxazole . * multiple logistic regression analysis for 1,813 patients , comparing odds of resistance for those with a history of travel to asia with those with no history of international travel . adjusted by serotype , year , patient age , patient race , and outbreak status . from 2004 through 2009 , a total of 2,255 laboratory - confirmed cases of nontyphoidal salmonellosis were reported in oregon . in accordance with oregon law , 2,153 isolates were forwarded to osphl , and 2,127 ( 98.8% of all nts isolates ) were cultured from a specific source and had antimicrobial drug susceptibility testing information ( figure ) . of these isolates , 26 ( 1.2% ) were obtained through a nonspecific source , such as lesions or sputum , and were excluded from analysis . culture - confirmed salmonellosis cases ascertained by statewide active surveillance and included in analyses , oregon , usa , 20042009 . the most common salmonella serotypes detected were enteritidis ( 18.4% ) , typhimurium ( 14.3% ) , heidelberg ( 8.2% ) , typhimurium var . cases that were part of identified outbreaks represented 24.1% of the cohort ; the remaining 75.9% were considered sporadic cases . the median age of patients was 29 years ( interquartile range 951 years ) , and 53.1% of patients were female . information about race was not available for 9% ; of patients for whom race was known , 91.8% were white and 9.2% were not white . similarly , information about ethnicity was not available for 10.5% of patients . among patients for whom isolates from 1,213 ( 57% ) patients were susceptible to all antimicrobial drugs screened ( pansusceptible ) , and isolates from 347 ( 16.3% ) had cir ( table 1 ) . of the 2,127 patients , 412 ( 19.4% ) were hospitalized and 110 ( 5.2% ) had invasive disease . * cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . the proportion of isolates that were pansusceptible significantly decreased from 69.5% in 2004 to 53.6% in 2009 ( p<0.01 ) . stratification by serotype revealed that cir increased among the 3 most common serotypes : enteritidis ( 3% to 8% , p = 0.02 ) , typhimurium ( 19% to 34% , p = 0.03 ) , and heidelberg ( 6% to 30% , p<0.01 ) . significant increases were identified for resistance to ciprofloxacin ( p<0.05 ) , nalidixic acid ( p<0.01 ) , sulfamethoxazole ( p<0.01 ) , tetracycline ( p<0.01 ) , and trimethoprim / sulfamethoxazole ( p<0.01 ) . we suspected that these findings were confounded by serotype and therefore used logistic regression to model the odds of acquiring a resistant infection for each of the clinically important antimicrobial drugs ( ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole ) as well as cir . serotype - adjusted log odds ratios were generated with year of infection entered as a discrete continuous variable . after adjusting for serotype , we found that with each subsequent year , patients were 30% more likely to acquire an infection that was resistant to quinolones ( nalidixic acid or ciprofloxacin ) and trimethoprim / sulfamethoxazole ( table 2 ) . we also found that with each year , odds of acquiring an infection with cir increased by 13% . * multiple logistic regression analysis of 2,127 isolates . cir was associated with hospitalization ( odds ratio [ or ] 1.5 , 95% ci 1.12.0 ) . this association was preserved after adjustment for serotype , patient age , patient race , and year ( aor 1.7 , 95% ci 1.22.1 ) . for patients with cir infections , odds of invasive infection were increased , although not significantly , according to unadjusted or adjusted analyses ( or 1.4 , 95% ci 0.92.2 and aor 1.5 , 95% ci 0.92.5 , respectively ) . of the 2,127 patients included in the previous analyses , 1,813 ( 84.2% of all oregon patients with nts ) were interviewed . for 305 ( 16.8% ) of these patients , isolates had cir , and for the remaining 1,508 ( 83.2% ) , isolates were susceptible to all clinically important antimicrobial drugs ( figure ) . of the 1,508 isolates susceptible to clinically important drugs , 1,002 ( 55.3% ) were susceptible to all drugs screened and 506 ( 27.9% ) were resistant to at least 1 non - cir drug . according to the unadjusted analysis , several serotypes were more likely than the referent serotype , enteritidis , to be resistant to > 1 clinically important antimicrobial drug ( table 3 ) . patient sex , race , age , and ethnicity were not significantly associated with resistance . * multiple logistic regression analysis of 1,813 patients ; cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . cir was not associated with any other demographic risk factors or high - risk food or animal exposures . however when international travel was examined by individual countries or applicable united nations region , cir was significantly associated with travel to southeast asia ( 24 ) . associations of resistance with travel to mexico and eastern asia also approached significance ( table 4 ) . the most common travel destinations in asia where resistant infections were acquired were thailand , china , and malaysia / indonesia . on the basis of these findings , we analyzed international travel by 3 destinations : central america , including mexico ( 135 patients ) , eastern and southeast asia ( 46 patients ) , and all other international travel destinations ( 77 patients ) . * cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . patients who were part of identified outbreak clusters were significantly less likely than patients with sporadic infections to have a resistant infection ( or 0.5 , 95% ci 0.40.8 ) . during our study period , 131 outbreaks ( 406 cases ) occurred , and for 25 of these outbreaks a causative vehicle was successfully identified . to assess whether oversampling of cases from outbreak clusters could explain this association , we first restricted cases to 1 isolate per outbreak where a causative vehicle was implicated while retaining all cases from outbreaks for which a vehicle was not implicated ( 302 cases ) . second , we further restricted cases to 1 isolate per outbreak , regardless whether a vehicle was identified ( 131 cases ) . in each of these analyses the magnitude , direction , and significance of the association was preserved ( or 0.6 , 95% ci 0.40.8 and or 0.5 , 95% ci 0.30.9 , respectively ) , suggesting that oversampling could not have explained this association . furthermore , 53.6% of outbreaks had intra - outbreak cases for which the antimicrobial drug susceptibility profiles of the isolates differed . the resultant main - effects model included the fixed variables of serotype , patient age , year of onset , and patient race , along with travel to eastern or southeast asia , and outbreak association ( table 3 ) . we hypothesized that effect modification occurred between the variables of outbreak cases and travel to eastern or southeast asia , as well as between travel to eastern or southeast asia , serotypes , and outbreaks . the association between resistance and travel to eastern or southeast asia was preserved after exclusion of all outbreak - associated cases ( aor 4.6 , 95% ci 2.39.4 ; table 5 ) . similarly , the association between resistance and outbreak - associated cases was preserved after exclusion of patients with a history of international travel ( aor 0.5 , 95% ci 0.40.8 ; table 6 ) . therefore , these results suggest that inclusion of patients with a history of travel to asia , as well as patients with outbreak - associated infections for the main analysis , was appropriate . * cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole.clinically important resistance . patients with a history of recent travel to eastern or southeast asia were > 5 times more likely to acquire a cir infection than were patients with no history of recent international travel . the most common serotypes acquired among persons with a history of travel to asia were enteriditis ( n = 13 , 54% cir ) , typhimurium ( n = 5 , 60% cir ) , newport ( n = 4 , 25% cir ) , i 4 , 5 , 12:i:- ( n = 4 , 50% cir ) , stanley ( n = 3 , 33% cir ) , and typhimurium var . patients with outbreak - associated infections were half as likely as those with sporadic infections to have cir ( table 3 ) . to identify risk factors for resistance to individual antimicrobial drugs , we constructed models with each of the clinically important antimicrobial drugs . travel to eastern or southeast asia was significantly associated with resistance to ampicillin , quinolones ( nalidixic acid or ciprofloxacin ) , and trimethoprim / sulfamethoxazole ( table 7 ) . only individual serotypes were associated with resistance to cephalosporins or gentamicin , and no other risk factors were significantly associated with resistance to ampicillin , quinolones , or trimethoprim / sulfamethoxazole . * multiple logistic regression analysis for 1,813 patients , comparing odds of resistance for those with a history of travel to asia with those with no history of international travel . adjusted by serotype , year , patient age , patient race , and outbreak status . we found that nts infections were more likely to have cir with each subsequent year of our study . in oregon during 20042009 , the proportion of isolates susceptible to all antimicrobial drugs significantly decreased . such travel was specifically associated with resistance to ampicillin , quinolones , and trimethoprim / sulfamethoxazole . isolates from patients who were part of identified outbreak clusters were significantly less likely to be resistant , suggesting that resistance estimates based on outbreak cases alone may underestimate the true level of resistance . our analysis was performed by using salmonella susceptibility data from a surveillance system that captures 100% of confirmed infections , has antimicrobial drug susceptibility information for > 95% of confirmed cases , and includes exposure histories for > 84% of patients . this study is strengthened by having collected data on several known and potential confounders before the drug - susceptibility profiles were known . our study design complements a previous national antimicrobial resistance monitoring system / foodnet study that analyzed antimicrobial drug resistance and increased disease severity ( 13 ) . however , we determined where resistant infections were acquired ( exposures ) and patient outcomes associated with resistant infections by using an entire population . the ability to integrate resistance and serotype data with case - specific demographic and risk - factor data improves the generalizability and plausibility of our study and provides population - level risk estimates ( 1420 ) . widespread quinolone resistance in southeast asia has been reported ( 26 ) ; a better understanding of global use of antimicrobial drugs might suggest where resistant salmonellae are prevalent . examination of serotype profiles among patients who had traveled to eastern or southeast asia and multivariate analyses adjusted for serotype provided strong evidence that the increased resistance in this region is widespread and not specifically attributable to a single serotype or regional serotype differences . we confirm the results of varma et al . and lee et al . , who found antimicrobial drug resistance to be associated with increased likelihood of hospitalization ( 13,25 ) . more severe infections can lead to treatment failure , sepsis , meningitis , and even death . if resistance to clinically important antimicrobial drugs continues to increase by 13% per year , as our data suggest , we can expect more severe illnesses , hospitalizations , and deaths , along with the accompanying higher economic costs . the association between resistance and outbreak cases persisted after restricting the data in unadjusted and adjusted analyses . the lack of effect modification between outbreak cases and a history of travel to asia in the multiple logistic regression modeling suggests that this finding is independent of travel . resistant isolates might be less infectious and therefore less likely to cause recognizable outbreaks . alternatively , however , this exposure would be expected to confound the observed associations nondifferentially , thereby resulting in lower point estimates . reporting lags could have delayed risk - factor interviews , resulting in nondifferential recall bias . this bias would not be expected to explain the association between resistance and international travel and would ultimately lead to underestimation of the true effect size . case ascertainment among persons with a history of travel to eastern or southeast asia could have been biased . this bias could have affected our analyses if more severe illness developed in travelers with resistant infections , who were more likely to seek health care or be reported than were travelers without resistant infections . however , according to a subanalysis , not presented here , we found that patients who traveled to eastern or southeast asia were less likely to be hospitalized than were those who had not recently traveled internationally ( or 0.4 , 95% ci 0.21.2 ) . this finding might be explained by a healthy traveler effect ; thus , the association between resistance and travel to eastern and southeast asia can not be explained by biased case ascertainment ( 29 ) . both laboratories were licensed and were using clinical and laboratory standards institute standardized methods , suggesting that this bias , if present , would be minimal and could not explain the observed associations . this study demonstrates that antimicrobial drug resistance among nts is increasing and has clinical and public health implications . when considering antimicrobial drug therapy , providers should evaluate patient travel history and salmonella serotype . our results highlight the need for enhanced domestic surveillance for antimicrobial drug resistance and suggest a need for increased prudence regarding the use of antimicrobial drugs .

to evaluate trends in and risk factors for acquisition of antimicrobial - drug resistant nontyphoidal salmonella infections , we searched oregon surveillance data for 20042009 for all culture - confirmed cases of salmonellosis . we defined clinically important resistance ( cir ) as decreased susceptibility to ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . of 2,153 cases , 2,127 ( 99% ) nontyphoidal salmonella isolates were obtained from a specific source ( e.g. , feces , urine , blood , or other normally sterile tissue ) and had been tested for drug susceptibility . among these , 347 ( 16% ) isolates had cir . the odds of acquiring cir infection significantly increased each year . hospitalization was more likely for patients with than without cir infections . among patients with isolates that had been tested , we analyzed data from 1,813 ( 84% ) who were interviewed . travel to eastern or southeast asia was associated with increased cir . isolates associated with outbreaks were less likely to have cir . future surveillance activities should evaluate resistance with respect to international travel .

Methods Reportable Infectious Disease Surveillance in Oregon Antimicrobial Drug Susceptibility Testing Analyses Results Descriptive Epidemiology and Resistance Trends Risk Factors Discussion

physicians and laboratories are required by law to report laboratory - confirmed and clinically suspected cases of salmonellosis to the patient s local health department ; reports should contain the patient s date of birth , sex , diagnosis , date of symptom onset , date of specimen collection , and laboratory test results . patients with recurrent infection or multiple salmonella isolates ( of same serotype within a plausible time frame for the original infection ) are interviewed only once , at the time of initial illness onset . the surveillance system in oregon is estimated to capture > 99% of laboratory - confirmed cases of salmonellosis ; however , for every 1 case confirmed , an estimated 25 additional cases are not detected ( 2 ) . all isolates were tested by using broth microdilution to determine mics for the following 10 antimicrobial agents : ampicillin , ceftriaxone , chloramphenicol , ciprofloxacin , gentamicin , nalidixic acid , nitrofurantoin , sulfamethoxazole , tetracycline , and trimethoprim / sulfamethoxazole . to ascertain cephalosporin resistance , osphl tested isolates for ceftriaxone susceptibility ; and the oregon state university veterinary diagnostic laboratory tested for susceptibility to cefuroxime and cephalothin by using analogous broth microdilution methods . isolates were included in analyses only if they were cultured from specific specimens , such as feces , urine , or blood or other normally sterile tissues ( i.e. cir was defined as resistance to at least 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole ( 13 ) . we used the cochran - armitage test for trend to analyze nts case data for 20042009 to determine whether the proportion of salmonella isolates with cir increased significantly . demographic and exposure risk factors , specifically international travel , were evaluated as risk factors for acquisition of a resistant isolate . we sought to evaluate whether resistance was associated with increased disease severity , including hospitalization and invasive infection . invasive infection was defined as isolation of salmonella from a normally sterile body site or tissue , such as blood ( 13 ) . the oregon health authority conducts active , laboratory - based surveillance for all cases of nts infection . physicians and laboratories are required by law to report laboratory - confirmed and clinically suspected cases of salmonellosis to the patient s local health department ; reports should contain the patient s date of birth , sex , diagnosis , date of symptom onset , date of specimen collection , and laboratory test results . the surveillance system in oregon is estimated to capture > 99% of laboratory - confirmed cases of salmonellosis ; however , for every 1 case confirmed , an estimated 25 additional cases are not detected ( 2 ) . from 2006 through 2009 , all isolates were tested by using broth microdilution to determine mics for the following 10 antimicrobial agents : ampicillin , ceftriaxone , chloramphenicol , ciprofloxacin , gentamicin , nalidixic acid , nitrofurantoin , sulfamethoxazole , tetracycline , and trimethoprim / sulfamethoxazole . isolates were included in analyses only if they were cultured from specific specimens , such as feces , urine , or blood or other normally sterile tissues ( i.e. cir was defined as resistance to at least 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole ( 13 ) . we used the cochran - armitage test for trend to analyze nts case data for 20042009 to determine whether the proportion of salmonella isolates with cir increased significantly . demographic and exposure risk factors , specifically international travel , were evaluated as risk factors for acquisition of a resistant isolate . we sought to evaluate whether resistance was associated with increased disease severity , including hospitalization and invasive infection . invasive infection was defined as isolation of salmonella from a normally sterile body site or tissue , such as blood ( 13 ) . from 2004 through 2009 , a total of 2,255 laboratory - confirmed cases of nontyphoidal salmonellosis were reported in oregon . in accordance with oregon law , 2,153 isolates were forwarded to osphl , and 2,127 ( 98.8% of all nts isolates ) were cultured from a specific source and had antimicrobial drug susceptibility testing information ( figure ) . culture - confirmed salmonellosis cases ascertained by statewide active surveillance and included in analyses , oregon , usa , 20042009 . among patients for whom isolates from 1,213 ( 57% ) patients were susceptible to all antimicrobial drugs screened ( pansusceptible ) , and isolates from 347 ( 16.3% ) had cir ( table 1 ) . * cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . significant increases were identified for resistance to ciprofloxacin ( p<0.05 ) , nalidixic acid ( p<0.01 ) , sulfamethoxazole ( p<0.01 ) , tetracycline ( p<0.01 ) , and trimethoprim / sulfamethoxazole ( p<0.01 ) . we suspected that these findings were confounded by serotype and therefore used logistic regression to model the odds of acquiring a resistant infection for each of the clinically important antimicrobial drugs ( ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole ) as well as cir . after adjusting for serotype , we found that with each subsequent year , patients were 30% more likely to acquire an infection that was resistant to quinolones ( nalidixic acid or ciprofloxacin ) and trimethoprim / sulfamethoxazole ( table 2 ) . we also found that with each year , odds of acquiring an infection with cir increased by 13% . cir was associated with hospitalization ( odds ratio [ or ] 1.5 , 95% ci 1.12.0 ) . for patients with cir infections , odds of invasive infection were increased , although not significantly , according to unadjusted or adjusted analyses ( or 1.4 , 95% ci 0.92.2 and aor 1.5 , 95% ci 0.92.5 , respectively ) . of the 2,127 patients included in the previous analyses , 1,813 ( 84.2% of all oregon patients with nts ) were interviewed . for 305 ( 16.8% ) of these patients , isolates had cir , and for the remaining 1,508 ( 83.2% ) , isolates were susceptible to all clinically important antimicrobial drugs ( figure ) . according to the unadjusted analysis , several serotypes were more likely than the referent serotype , enteritidis , to be resistant to > 1 clinically important antimicrobial drug ( table 3 ) . * multiple logistic regression analysis of 1,813 patients ; cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . cir was not associated with any other demographic risk factors or high - risk food or animal exposures . however when international travel was examined by individual countries or applicable united nations region , cir was significantly associated with travel to southeast asia ( 24 ) . associations of resistance with travel to mexico and eastern asia also approached significance ( table 4 ) . on the basis of these findings , we analyzed international travel by 3 destinations : central america , including mexico ( 135 patients ) , eastern and southeast asia ( 46 patients ) , and all other international travel destinations ( 77 patients ) . * cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . patients who were part of identified outbreak clusters were significantly less likely than patients with sporadic infections to have a resistant infection ( or 0.5 , 95% ci 0.40.8 ) . the resultant main - effects model included the fixed variables of serotype , patient age , year of onset , and patient race , along with travel to eastern or southeast asia , and outbreak association ( table 3 ) . we hypothesized that effect modification occurred between the variables of outbreak cases and travel to eastern or southeast asia , as well as between travel to eastern or southeast asia , serotypes , and outbreaks . the association between resistance and travel to eastern or southeast asia was preserved after exclusion of all outbreak - associated cases ( aor 4.6 , 95% ci 2.39.4 ; table 5 ) . similarly , the association between resistance and outbreak - associated cases was preserved after exclusion of patients with a history of international travel ( aor 0.5 , 95% ci 0.40.8 ; table 6 ) . cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole.clinically important resistance . patients with a history of recent travel to eastern or southeast asia were > 5 times more likely to acquire a cir infection than were patients with no history of recent international travel . the most common serotypes acquired among persons with a history of travel to asia were enteriditis ( n = 13 , 54% cir ) , typhimurium ( n = 5 , 60% cir ) , newport ( n = 4 , 25% cir ) , i 4 , 5 , 12:i:- ( n = 4 , 50% cir ) , stanley ( n = 3 , 33% cir ) , and typhimurium var . patients with outbreak - associated infections were half as likely as those with sporadic infections to have cir ( table 3 ) . to identify risk factors for resistance to individual antimicrobial drugs , we constructed models with each of the clinically important antimicrobial drugs . travel to eastern or southeast asia was significantly associated with resistance to ampicillin , quinolones ( nalidixic acid or ciprofloxacin ) , and trimethoprim / sulfamethoxazole ( table 7 ) . only individual serotypes were associated with resistance to cephalosporins or gentamicin , and no other risk factors were significantly associated with resistance to ampicillin , quinolones , or trimethoprim / sulfamethoxazole . * multiple logistic regression analysis for 1,813 patients , comparing odds of resistance for those with a history of travel to asia with those with no history of international travel . from 2004 through 2009 , a total of 2,255 laboratory - confirmed cases of nontyphoidal salmonellosis were reported in oregon . in accordance with oregon law , 2,153 isolates were forwarded to osphl , and 2,127 ( 98.8% of all nts isolates ) were cultured from a specific source and had antimicrobial drug susceptibility testing information ( figure ) . culture - confirmed salmonellosis cases ascertained by statewide active surveillance and included in analyses , oregon , usa , 20042009 . among patients for whom isolates from 1,213 ( 57% ) patients were susceptible to all antimicrobial drugs screened ( pansusceptible ) , and isolates from 347 ( 16.3% ) had cir ( table 1 ) . * cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . we suspected that these findings were confounded by serotype and therefore used logistic regression to model the odds of acquiring a resistant infection for each of the clinically important antimicrobial drugs ( ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole ) as well as cir . after adjusting for serotype , we found that with each subsequent year , patients were 30% more likely to acquire an infection that was resistant to quinolones ( nalidixic acid or ciprofloxacin ) and trimethoprim / sulfamethoxazole ( table 2 ) . we also found that with each year , odds of acquiring an infection with cir increased by 13% . for patients with cir infections , odds of invasive infection were increased , although not significantly , according to unadjusted or adjusted analyses ( or 1.4 , 95% ci 0.92.2 and aor 1.5 , 95% ci 0.92.5 , respectively ) . of the 2,127 patients included in the previous analyses , 1,813 ( 84.2% of all oregon patients with nts ) were interviewed . for 305 ( 16.8% ) of these patients , isolates had cir , and for the remaining 1,508 ( 83.2% ) , isolates were susceptible to all clinically important antimicrobial drugs ( figure ) . according to the unadjusted analysis , several serotypes were more likely than the referent serotype , enteritidis , to be resistant to > 1 clinically important antimicrobial drug ( table 3 ) . * multiple logistic regression analysis of 1,813 patients ; cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . however when international travel was examined by individual countries or applicable united nations region , cir was significantly associated with travel to southeast asia ( 24 ) . on the basis of these findings , we analyzed international travel by 3 destinations : central america , including mexico ( 135 patients ) , eastern and southeast asia ( 46 patients ) , and all other international travel destinations ( 77 patients ) . * cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . patients who were part of identified outbreak clusters were significantly less likely than patients with sporadic infections to have a resistant infection ( or 0.5 , 95% ci 0.40.8 ) . the resultant main - effects model included the fixed variables of serotype , patient age , year of onset , and patient race , along with travel to eastern or southeast asia , and outbreak association ( table 3 ) . we hypothesized that effect modification occurred between the variables of outbreak cases and travel to eastern or southeast asia , as well as between travel to eastern or southeast asia , serotypes , and outbreaks . the association between resistance and travel to eastern or southeast asia was preserved after exclusion of all outbreak - associated cases ( aor 4.6 , 95% ci 2.39.4 ; table 5 ) . similarly , the association between resistance and outbreak - associated cases was preserved after exclusion of patients with a history of international travel ( aor 0.5 , 95% ci 0.40.8 ; table 6 ) . * cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole . cir , clinically important resistance to > 1 of the following : ampicillin , ceftriaxone , ciprofloxacin , gentamicin , or trimethoprim / sulfamethoxazole.clinically important resistance . patients with a history of recent travel to eastern or southeast asia were > 5 times more likely to acquire a cir infection than were patients with no history of recent international travel . patients with outbreak - associated infections were half as likely as those with sporadic infections to have cir ( table 3 ) . to identify risk factors for resistance to individual antimicrobial drugs , we constructed models with each of the clinically important antimicrobial drugs . travel to eastern or southeast asia was significantly associated with resistance to ampicillin , quinolones ( nalidixic acid or ciprofloxacin ) , and trimethoprim / sulfamethoxazole ( table 7 ) . only individual serotypes were associated with resistance to cephalosporins or gentamicin , and no other risk factors were significantly associated with resistance to ampicillin , quinolones , or trimethoprim / sulfamethoxazole . * multiple logistic regression analysis for 1,813 patients , comparing odds of resistance for those with a history of travel to asia with those with no history of international travel . we found that nts infections were more likely to have cir with each subsequent year of our study . such travel was specifically associated with resistance to ampicillin , quinolones , and trimethoprim / sulfamethoxazole . isolates from patients who were part of identified outbreak clusters were significantly less likely to be resistant , suggesting that resistance estimates based on outbreak cases alone may underestimate the true level of resistance . our analysis was performed by using salmonella susceptibility data from a surveillance system that captures 100% of confirmed infections , has antimicrobial drug susceptibility information for > 95% of confirmed cases , and includes exposure histories for > 84% of patients . however , we determined where resistant infections were acquired ( exposures ) and patient outcomes associated with resistant infections by using an entire population . examination of serotype profiles among patients who had traveled to eastern or southeast asia and multivariate analyses adjusted for serotype provided strong evidence that the increased resistance in this region is widespread and not specifically attributable to a single serotype or regional serotype differences . , who found antimicrobial drug resistance to be associated with increased likelihood of hospitalization ( 13,25 ) . resistant isolates might be less infectious and therefore less likely to cause recognizable outbreaks . case ascertainment among persons with a history of travel to eastern or southeast asia could have been biased . this bias could have affected our analyses if more severe illness developed in travelers with resistant infections , who were more likely to seek health care or be reported than were travelers without resistant infections . however , according to a subanalysis , not presented here , we found that patients who traveled to eastern or southeast asia were less likely to be hospitalized than were those who had not recently traveled internationally ( or 0.4 , 95% ci 0.21.2 ) . this finding might be explained by a healthy traveler effect ; thus , the association between resistance and travel to eastern and southeast asia can not be explained by biased case ascertainment ( 29 ) .

this study was approved by the institutional review board of the duke university health system , and all subjects participated with full informed consent . a group of 14 ophthalmic surgeons - in - training were recruited from duke university ophthalmology residents . the subjects were randomized into two groups balanced by level of training and surgical experience : with one group assigned to work with the ability to view the mi - oct ( mi - oct+ ) and the other without ( mi - oct ) such guidance ( fig . a total of three first - year , two second - year , and two third - year residents were randomized to each group ( n = 14 total ) . all residents within the same year had equivalent wet laboratory experience prior to the commencement of the study . fourteen ophthalmology residents were stratified according to the year of training and randomized to either the mi - oct or mi - oct+ group ( a ) . they were asked to perform four surgical maneuvers , including simple corneal pass at 50% and 90% depth , repair corneal laceration at 90% depth , and create triplanar clear - cornea wound on porcine eyes . the mi - oct residents initially operated without mi - oct guidance , whereas the mi - oct+ residents operated under mi - oct feedback ( trial 1 ) . the same maneuvers were repeated by both groups of residents without mi - oct feedback ( trial 2 ) . finally , the mi - oct group crossed over and repeated all the maneuvers with mi - oct guidance ( trial 3 ) ( a , b ) . ( c ) wet laboratory setup , which included operating microscope with custom - designed , ss the subjects were shown a standardized powerpoint slide set to introduce the wet laboratory , surgical maneuvers , instrumentation , the operating microscope , and mi - oct . freshly enucleated porcine eyes obtained from a local slaughterhouse and stored at 4c were used for the wet laboratory within 18 hours to minimize corneal edema . the residents were asked to perform a corneal suture pass at 50% thickness , a corneal suture pass at 90% thickness , repair a linear full - thickness corneal laceration with suture at 90% thickness , and construct a triplanar clear - corneal cataract - type incision on the porcine eye ( fig . 1 ) . the corneal suture passes and laceration repair were with 10 - 0 nylon suture on a spatulated needle ( ethicon , somerville , nj , usa ) , whereas the clear - corneal wound was constructed using a standard 2.75-mm disposable keratome for cataract surgery ( alcon , fort worth , tx , usa ) . in the trials involving mi - oct feedback , the residents could ask for oct at any point during the surgical maneuver . the oct feedback entailed obtaining scans in any orientation anywhere within surgical field as directed by the surgeon . at that point , based on the feedback , the resident could rectify the maneuver ( i.e. , back out the needle pass and go deeper or shallower ) based on the mi - oct finding for up to three trial attempts . in the trials not involving mi - oct feedback , the entire procedure was performed using only the operating microscope . for both , however , intraoperative oct was used to obtain images used for grading the maneuver at the end of the procedure . for depth - based maneuvers , an intraoperative oct image perpendicular to the needle and when visible along the suture tract was obtained after each completed pass to allow subsequent grading of the depth of the pass ( figs . 1 , 2 ) . the depth scores of the individual attempts were averaged and analyzed for statistically significant differences between the mi - oct+ and mi - oct groups . for the clear - corneal incision , a b - scan oriented along the radial axis of the wound was acquired at the conclusion of the maneuver and used for grading . close - up view of the mi - oct display options is demonstrated in a resident surgeons had a choice to view oct display data of the surgical field during each maneuver in ( a ) a 3d view demonstrating the cornea ( white arrow ) and anterior chamber ( red arrow ) , ( b ) cross - sectional b - scan view , or ( c ) as a surface volume projection . three consecutive trials were performed : trial 1 , each resident performed the above surgical maneuvers with ( mi - oct+ group ) or without ( mi - oct group ) mi - oct feedback based on the randomized assignment ; trial 2 , the same maneuvers were repeated by each surgeon using only the microscope for viewing ( no mi - oct guidance for either group ) to test the persistence of effect in the mi - oct+ group compared with the mi - oct group ; and trial 3 , the same maneuvers were repeated by each surgeon in the mi - oct group with intraoperative oct feedback . this crossover was designed as an internal control to ensure that any observed differences between the group performance in trials 1 and 2 were not due to confounding variables such as innate surgical ability . the mi - oct scanner used was previously published and utilized an external display for image viewing . briefly , a customized mechanical enclosure allowed integration of the mi - oct into an ophthalmic surgical microscope ( leica microsystems , inc . , buffalo grove , il , usa ) . a dichroic mirror folded the oct beam path onto the surgical microscope path to allow unobstructed surgical viewing with simultaneous ( ss - oct ) imaging . the final surgical microscope objective lens was shared between the two systems and ensured that both systems were parfocal . the ss mi - oct system used a ( ss - oct ) engine utilizing a swept - frequency source centered at 1040 nm with a sweep bandwidth of 100 nm and a sweep rate of 100 khz ( axsun , billerca , ma , usa ) . this resulted in a measured system signal - to - noise ratio ( snr ) of 99 db , and axial and lateral resolutions of 7.8 and 15 m , respectively . the imaging protocol consisted of 500 a - lines / b - scan and 100 b - scans / volume with an average of 510 volumes per second . custom gpu - accelerated software enabled real - time acquisition , processing , and simultaneous display during surgery of a continuously updated and optimized oct b - scan , top - down retinal view , and volumetric rendering on a computer screen adjacent to the operating microscope . the site of user - specified b - scan of interest in the volume was selected by an assistant using a mouse ; at that site , b - scans were averaged 5 times to enhance visualization . to grade the suture placement , custom software was created using matlab ( mathworks , natick , ma , usa ) . on averaged b - scans at the point of maximal depth of the needle and suture passes , the surface of the corneal endothelium and epithelium were marked by a masked trained grader . two - dimensional correction of the selected b - scan was performed to correct for image distortions due to refraction as previously described . the corneal thickness at the suture pass was defined as the shortest distance between the epithelial and endothelial layers that included the position of the surgical needle or suture . the percent depth was calculated as the ratio between the distance from the epithelium to the needle / suture and the distance between the epithelium and endothelium ( corneal thickness ) . in some corneal laceration cases , jagged edges and protrusions occurred in the epithelium at the edge of the laceration . in these b - scans , the epithelial segmentation was fit to a higher - order polynomial to accurately follow these high frequency features . refraction correction was performed as described previously , except where a significant portion of the corneal tissue was missing in the laceration . in those cases , the best - approximated orthogonal path through the cornea was segmented manually in these instances , and the shortest linear path was found between the endothelium and epithelium . for clear - corneal incision geometry assessment , volume and averaged b - scans parallel to the direction of the keratome path were deidentified , shuffled , and reviewed by two masked expert graders . graders determined the geometry of the clear corneal incisions on a categorical scale ( 3 = triplanar wound , 2 = biplanar wound , 1 = monoplanar wound , 0 = unable to determine geometry of the wound ) by comparison with standard images for each grade . the scores of the individual graders were averaged for each scan , and the agreement ( statistic ) between the graders was computed . all subjects were given an anonymous survey to determine their subjective experience ( supplementary tables ) . the mi - oct+ group was asked to rank their agreement with statements evaluating their comfort in performing simple corneal passes , repairing the corneal laceration , and constructing the clear corneal incision under direct mi - oct guidance . the mi - oct group was asked to rank their agreement with statements evaluating their comfort in performing simple corneal passes , repair of corneal laceration , and construction of clear corneal wound without direct mi - oct feedback on a 1 to 5 numerical scale . they were then asked to rerank their performance of the same maneuvers with direct mi - oct feedback at the completion of trial 3 when they were finally provided with mi - oct guidance . both groups were asked to rank their overall experience of using mi - oct , as well as their likelihood of using mi - oct in their future practice on a 1 to 5 numerical scale . in both groups , the subjective score of 1 represented the lowest confidence score , whereas a score of 5 represented the highest confidence score . the composite scores of the depth - based maneuvers were computed and expressed as box - plots using graphpad prism software ( graphpad , la jolla , ca , usa ) . the statistical comparisons conducted across the mi - oct+ and mi - oct groups were performed unpaired , using a wilcoxon rank sum test . thus , comparisons between mi - oct and mi - oct+ group scores for trial 1 ( mi - oct+ versus mi - oct ) , trial 2 ( both groups without mi - oct ) , and trial 3 ( mi - oct group repeating maneuvers using mi - oct ) were performed using the wilcoxon rank sum test of differences between medians . the statistical comparisons within a group ( either mi - oct or mi - oct+ ) were performed using the wilcoxon signed rank test of median differences . the wilcoxon signed rank test was used to ascertain , in a paired fashion , whether there was a statistically significant difference in scores within the mi - oct group when they only had the microscope alone ( trials 1 and 2 ) and when they had guidance from mi - oct ( trial 3 ) . similarly , the wilcoxon signed rank test was used to compare , in a paired fashion , within the mi - oct+ group whether there was a statistically significant difference in scores when they had the mi - oct guidance first compared with their performance without the mi - oct afterward . all comparisons of the medians using the wilcoxon signed rank test and wilcoxon rank sum test with a p value of < 0.05 were deemed statistically significant . a group of 14 ophthalmic surgeons - in - training were recruited from duke university ophthalmology residents . the subjects were randomized into two groups balanced by level of training and surgical experience : with one group assigned to work with the ability to view the mi - oct ( mi - oct+ ) and the other without ( mi - oct ) such guidance ( fig . a total of three first - year , two second - year , and two third - year residents were randomized to each group ( n = 14 total ) . all residents within the same year had equivalent wet laboratory experience prior to the commencement of the study . fourteen ophthalmology residents were stratified according to the year of training and randomized to either the mi - oct or mi - oct+ group ( a ) . they were asked to perform four surgical maneuvers , including simple corneal pass at 50% and 90% depth , repair corneal laceration at 90% depth , and create triplanar clear - cornea wound on porcine eyes . the mi - oct residents initially operated without mi - oct guidance , whereas the mi - oct+ residents operated under mi - oct feedback ( trial 1 ) . the same maneuvers were repeated by both groups of residents without mi - oct feedback ( trial 2 ) . finally , the mi - oct group crossed over and repeated all the maneuvers with mi - oct guidance ( trial 3 ) ( a , b ) . ( c ) wet laboratory setup , which included operating microscope with custom - designed , ss the subjects were shown a standardized powerpoint slide set to introduce the wet laboratory , surgical maneuvers , instrumentation , the operating microscope , and mi - oct . freshly enucleated porcine eyes obtained from a local slaughterhouse and stored at 4c were used for the wet laboratory within 18 hours to minimize corneal edema . the residents were asked to perform a corneal suture pass at 50% thickness , a corneal suture pass at 90% thickness , repair a linear full - thickness corneal laceration with suture at 90% thickness , and construct a triplanar clear - corneal cataract - type 1 ) . the corneal suture passes and laceration repair were with 10 - 0 nylon suture on a spatulated needle ( ethicon , somerville , nj , usa ) , whereas the clear - corneal wound was constructed using a standard 2.75-mm disposable keratome for cataract surgery ( alcon , fort worth , tx , usa ) . in the trials involving mi - oct feedback , the residents could ask for oct at any point during the surgical maneuver . the oct feedback entailed obtaining scans in any orientation anywhere within surgical field as directed by the surgeon . at that point , based on the feedback , the resident could rectify the maneuver ( i.e. , back out the needle pass and go deeper or shallower ) based on the mi - oct finding for up to three trial attempts . in the trials not involving mi - oct feedback , the entire procedure was performed using only the operating microscope . for both , however , intraoperative oct was used to obtain images used for grading the maneuver at the end of the procedure . for depth - based maneuvers , an intraoperative oct image perpendicular to the needle and when visible along the suture tract was obtained after each completed pass to allow subsequent grading of the depth of the pass ( figs . 1 , 2 ) . the depth scores of the individual attempts were averaged and analyzed for statistically significant differences between the mi - oct+ and mi - oct groups . for the clear - corneal incision , a b - scan oriented along the radial axis of the wound was acquired at the conclusion of the maneuver and used for grading . close - up view of the mi - oct display options is demonstrated in a resident surgeons had a choice to view oct display data of the surgical field during each maneuver in ( a ) a 3d view demonstrating the cornea ( white arrow ) and anterior chamber ( red arrow ) , ( b ) cross - sectional b - scan view , or ( c ) as a surface volume projection . three consecutive trials were performed : trial 1 , each resident performed the above surgical maneuvers with ( mi - oct+ group ) or without ( mi - oct group ) mi - oct feedback based on the randomized assignment ; trial 2 , the same maneuvers were repeated by each surgeon using only the microscope for viewing ( no mi - oct guidance for either group ) to test the persistence of effect in the mi - oct+ group compared with the mi - oct group ; and trial 3 , the same maneuvers were repeated by each surgeon in the mi - oct group with intraoperative oct feedback . this crossover was designed as an internal control to ensure that any observed differences between the group performance in trials 1 and 2 were not due to confounding variables such as innate surgical ability . the mi - oct scanner used was previously published and utilized an external display for image viewing . briefly , a customized mechanical enclosure allowed integration of the mi - oct into an ophthalmic surgical microscope ( leica microsystems , inc . , buffalo grove , il , usa ) . a dichroic mirror folded the oct beam path onto the surgical microscope path to allow unobstructed surgical viewing with simultaneous ( ss - oct ) imaging . the final surgical microscope objective lens was shared between the two systems and ensured that both systems were parfocal . the ss mi - oct system used a ( ss - oct ) engine utilizing a swept - frequency source centered at 1040 nm with a sweep bandwidth of 100 nm and a sweep rate of 100 khz ( axsun , billerca , ma , usa ) . this resulted in a measured system signal - to - noise ratio ( snr ) of 99 db , and axial and lateral resolutions of 7.8 and 15 m , respectively . the imaging protocol consisted of 500 a - lines / b - scan and 100 b - scans / volume with an average of 510 volumes per second . custom gpu - accelerated software enabled real - time acquisition , processing , and simultaneous display during surgery of a continuously updated and optimized oct b - scan , top - down retinal view , and volumetric rendering on a computer screen adjacent to the operating microscope . the site of user - specified b - scan of interest in the volume was selected by an assistant using a mouse ; at that site , b - scans were averaged 5 times to enhance visualization . to grade the suture placement , custom software was created using matlab ( mathworks , natick , ma , usa ) . on averaged b - scans at the point of maximal depth of the needle and suture passes , the surface of the corneal endothelium and epithelium were marked by a masked trained grader . two - dimensional correction of the selected b - scan was performed to correct for image distortions due to refraction as previously described . the corneal thickness at the suture pass was defined as the shortest distance between the epithelial and endothelial layers that included the position of the surgical needle or suture . the percent depth was calculated as the ratio between the distance from the epithelium to the needle / suture and the distance between the epithelium and endothelium ( corneal thickness ) . in some corneal laceration cases , jagged edges and protrusions occurred in the epithelium at the edge of the laceration . in these b - scans , the epithelial segmentation was fit to a higher - order polynomial to accurately follow these high frequency features . refraction correction was performed as described previously , except where a significant portion of the corneal tissue was missing in the laceration . in those cases , the best - approximated orthogonal path through the cornea was segmented manually in these instances , and the shortest linear path was found between the endothelium and epithelium . for clear - corneal incision geometry assessment , volume and averaged b - scans parallel to the direction of the keratome path were deidentified , shuffled , and reviewed by two masked expert graders . graders determined the geometry of the clear corneal incisions on a categorical scale ( 3 = triplanar wound , 2 = biplanar wound , 1 = monoplanar wound , 0 = unable to determine geometry of the wound ) by comparison with standard images for each grade . the scores of the individual graders were averaged for each scan , and the agreement ( statistic ) between the graders was computed . all subjects were given an anonymous survey to determine their subjective experience ( supplementary tables ) . the mi - oct+ group was asked to rank their agreement with statements evaluating their comfort in performing simple corneal passes , repairing the corneal laceration , and constructing the clear corneal incision under direct mi - oct guidance . the mi - oct group was asked to rank their agreement with statements evaluating their comfort in performing simple corneal passes , repair of corneal laceration , and construction of clear corneal wound without direct mi - oct feedback on a 1 to 5 numerical scale . they were then asked to rerank their performance of the same maneuvers with direct mi - oct feedback at the completion of trial 3 when they were finally provided with mi - oct guidance . both groups were asked to rank their overall experience of using mi - oct , as well as their likelihood of using mi - oct in their future practice on a 1 to 5 numerical scale . in both groups , the subjective score of 1 represented the lowest confidence score , whereas a score of 5 represented the highest confidence score . the composite scores of the depth - based maneuvers were computed and expressed as box - plots using graphpad prism software ( graphpad , la jolla , ca , usa ) . the statistical comparisons conducted across the mi - oct+ and mi - oct groups were performed unpaired , using a wilcoxon rank sum test . thus , comparisons between mi - oct and mi - oct+ group scores for trial 1 ( mi - oct+ versus mi - oct ) , trial 2 ( both groups without mi - oct ) , and trial 3 ( mi - oct group repeating maneuvers using mi - oct ) were performed using the wilcoxon rank sum test of differences between medians . the statistical comparisons within a group ( either mi - oct or mi - oct+ ) were performed using the wilcoxon signed rank test of median differences . the wilcoxon signed rank test was used to ascertain , in a paired fashion , whether there was a statistically significant difference in scores within the mi - oct group when they only had the microscope alone ( trials 1 and 2 ) and when they had guidance from mi - oct ( trial 3 ) . similarly , the wilcoxon signed rank test was used to compare , in a paired fashion , within the mi - oct+ group whether there was a statistically significant difference in scores when they had the mi - oct guidance first compared with their performance without the mi - oct afterward . all comparisons of the medians using the wilcoxon signed rank test and wilcoxon rank sum test with a p value of < 0.05 were deemed statistically significant . figure 3a demonstrates a representative orthogonal b - scan of a corneal needle pass at 50% depth ( arrow ) . when asked to perform corneal passes at 50% depth in trial 1 , residents operating without mi - oct feedback ( mi - oct group ) on average placed the pass at 39.4% depth ( sd = 13% , median = 35% ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ group ) on average placed the pass at 54.6% depth ( sd = 5% , median = 56% ) . 3b ) . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the mi - oct group on average placed the pass at 37.6% depth ( sd = 7% , median = 38% ) , whereas mi - oct+ on average placed the pass at 51.0% depth ( sd = 7% , median = 51% ) , which was also significantly different ( p = 0.009 ; fig . 3b ) . to ensure that the observed difference was not due to inferior surgical ability in the mi - oct group , these subjects were asked to perform the same maneuver with mi - oct feedback . with mi - oct feedback , there was a significant difference ( p = 0.016 ) in the performance of the mi - oct residents with an average of 53% depth ( sd = 5% , median = 55% ) compared with their earlier performance without mi - oct in trial 2 with an average of 37.6 % ( sd = 7% , median = 38% ) . corneal passes with the goal depth of 50% corneal thickness were performed by mi - oct and mi - oct+ group residents . a representative cross - sectional b - scan orthogonal to the direction of corneal needle pass is shown , and the hyperreflective dot ( circle ) represents an attempted pass at 50% corneal depth ( a ) . the average score of the residents in each group and the median depth scores are compared within the individual groups using paired analysis with the wilcoxon signed rank test , with relevant comparisons demonstrating statistical significance indicated at p < 0.05 ( b ) . for the corneal pass at 90% depth , a representative b - scan of this maneuver is shown in figure 4a . when asked to perform this maneuver in trial 1 , residents operating without mi - oct feedback ( mi - oct ) on average placed the pass at 49.1% depth ( sd = 14% , median = 43.0 ) , in contrast to 85.5% depth ( sd = 5% , median = 87% ) for the mi - oct+ group . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the average was 46.4% depth ( sd = 12% , median = 41% ) for the mi - oct group and 81.5% depth ( sd = 9% , median = 79% ) for the mi - oct+ group , which was also a statistically significant difference between the mi - oct+ and mi - oct groups without the use of the microscope ( p = 0.002 ; fig . the mi - oct+ group had no significant difference in depth performance between trials 1 and 2 with and without mi - oct ( median difference = 0.03 , p = 0.812 ) . in trial 3 , the mi - oct group repeated the surgical maneuver with mi - oct feedback , with the average corneal pass at 84.1% depth ( sd = 9% , median = 85% ) ; using the wilcoxon signed rank test , nonparametric testing for paired data , the mi - oct group 's performance with and without mi - oct was compared , and noted to be significantly different ( median difference = 0.34 , p = 0.016 ) . corneal passes with goal depth of 90% corneal thicknesses were performed by mi - oct and mi - oct+ group residents . a representative cross - sectional b - scan obtained orthogonal to the direction of needle pass demonstrates a hyperreflective dot ( circle ) at the goal of 90% corneal depth ( a ) . the average score of each resident was plotted , and the median depth scores were compared within individual groups using the wilcoxon signed rank test , with relevant comparisons demonstrating statistical significance indicated at p < 0.05 ( b ) . figure 5a demonstrates a representative orthogonal b - scan of a corneal needle pass at 90% depth ( arrow ) through the laceration . while performing this maneuver in trial 1 , residents operating without mi - oct feedback ( mi - oct ) on average placed the pass at 55.5% depth ( sd = 14% , median = 53% ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ ) on average placed the laceration pass at 81.5% depth ( sd = 3% , median = 82% ) . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the mi - oct group on average placed the pass at 50.6% depth ( sd = 15% , median = 49% ) , whereas the mi - oct+ group on average placed the pass at 73.3% depth ( sd = 12% , median = 74% ) , which was also statistically significant ( p = 0.025 ; fig . notably , the mi - oct+ group , which first operated with direct mi - oct guidance , continued to perform well in the second trial once mi - oct guidance was removed . there was no significant difference in their performance ( median difference = 0.07 , p = 0.11 ) . in trial 3 , the mi - oct group repeated the surgical maneuvers with mi - oct feedback , resulting in an average 83.8% suture pass depth ( sd = 5% , median = 83% ) , which was significant compared with their earlier performance without mi - oct in trial 2 with paired analysis through the wilcoxon signed ranks test ( p = 0.016 ) . fresh porcine corneas were used to construct vertical linear corneal laceration using a 15-blade scalpel . the corneal laceration repair with 10 - 0 nylon suture was performed by mi - oct and mi - oct+ group residents . a representative cross - sectional b - scan was obtained orthogonal to the direction of needle pass and demonstrates a jagged break in corneal integrity consistent with corneal laceration ( arrow ) and a hyperreflective dot ( circle ) at goal depth of 90% corneal thickness ( a ) . the average score of each resident was plotted , and the median depth scores compared across the groups using the wilcoxon signed rank test , with relevant comparisons demonstrating statistical significance indicated at p < 0.05 ( b ) . the agreement between the expert graders in scoring corneal incision geometry on a scale of 0 to 3 was determined to not be significantly different ( weighted = 0.6381 ) . figures 6a and 6b demonstrate two representative b - scans of the constructed wounds . when asked to create a triplanar incision in trial 1 , residents operating without mi - oct feedback ( mi - oct group ) on average constructed a corneal wound with a mean score of 1.82 planes ( sd = 0.67 , median = 1.75 ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ group ) had a mean score of 2.14 planes ( sd = 0.46 , median = 2.00 ) . when both groups were asked to repeat this maneuver without mi - oct in trial 2 , the mi - oct group on average constructed wounds with a mean score of 2.41 planes ( sd = 0.65 , median = 2.75 ) , whereas the mi - oct+ group had a mean score of 2.38 planes ( sd = 0.53 , median = 2.50 ) , which was also not statistically significant ( p = 0.521 ) . in trial 3 , the mi - oct group was given an opportunity to repeat the surgical maneuver with mi - oct feedback and on average received a mean score of 2.21 ( sd = 0.86 , median = 2.64 ) , which was not significantly different than this group 's performance in trial 2 ( p = 0.437 ) . clear corneal incisions were created in porcine eyes by mi - oct and mi - oct+ group residents using 2.75-mm surgical keratomes . although the mi - oct+ resident could view the mi - oct throughout and at the completion of the maneuver , a representative cross - sectional b - scan longitudinal to the length of the wound was obtained at the completion of each maneuver ( a , b ) for postmaneuver grading of the wound profile visible from two separate representative trials ( arrows ) . all incisions viewed on such b - scans were assigned a grade of 0 to 3 by two expert graders masked to study group . last , qualitative and quantitative surgeon 's subjective feedback was obtained from the residents following completion of the study maneuvers via an administered survey ( fig . only the mi - oct+ group had the direct use of mi - oct ; the mi - oct+ group reported higher levels of comfort compared with the mi - oct group , in performing simple cornea suture passes ( p = 0.007 ) and cornea laceration repair ( p = 0.028 ) , but this trend was not observed for creation of triplanar corneal wound . at the end of the training , when both mi - oct and mi - oct+ groups had experienced the direct use of mi - oct , there was no statistically significant difference between the groups ' subjective experience of mi - oct impact on their ability to perform those same procedures . in total , both mi - oct and mi - oct+ residents on average rated the overall experience of using mi - oct feedback for anterior segment surgical maneuvers as helpful ( score of 4 ) or very helpful ( score of 5 ) , reporting a mean score 4.57 ( sd = 0.53 ) for the mi - oct group and 4.86 ( sd = 0.38 ) , and a median of 5 for both groups on a 1 to 5 scale . ( score 4 on 15 scale ) to use mi - oct in their future practice as a result of mi - oct experience in this study . the survey administered to each resident surgeon at the conclusion of initial training and end of the study ranked their subjective experience on a 1 to 5 numerical scale . the mean subjective score with sd and minimum , maximum , and median responses to the survey questions the comparison between the mi - oct group and the mi - oct+ groups was done using the wilcoxon rank sum test of difference between medians . figure 3a demonstrates a representative orthogonal b - scan of a corneal needle pass at 50% depth ( arrow ) . when asked to perform corneal passes at 50% depth in trial 1 , residents operating without mi - oct feedback ( mi - oct group ) on average placed the pass at 39.4% depth ( sd = 13% , median = 35% ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ group ) on average placed the pass at 54.6% depth ( sd = 5% , median = 56% ) . 3b ) . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the mi - oct group on average placed the pass at 37.6% depth ( sd = 7% , median = 38% ) , whereas mi - oct+ on average placed the pass at 51.0% depth ( sd = 7% , median = 51% ) , which was also significantly different ( p = 0.009 ; fig . 3b ) . to ensure that the observed difference was not due to inferior surgical ability in the mi - oct group , these subjects were asked to perform the same maneuver with mi - oct feedback . with mi - oct feedback , there was a significant difference ( p = 0.016 ) in the performance of the mi - oct residents with an average of 53% depth ( sd = 5% , median = 55% ) compared with their earlier performance without mi - oct in trial 2 with an average of 37.6 % ( sd = 7% , median = 38% ) . corneal passes with the goal depth of 50% corneal thickness were performed by mi - oct and mi - oct+ group residents . a representative cross - sectional b - scan orthogonal to the direction of corneal needle pass is shown , and the hyperreflective dot ( circle ) represents an attempted pass at 50% corneal depth ( a ) . the average score of the residents in each group and the median depth scores are compared within the individual groups using paired analysis with the wilcoxon signed rank test , with relevant comparisons demonstrating statistical significance indicated at p < 0.05 ( b ) . for the corneal pass at 90% depth , a representative b - scan of this maneuver is shown in figure 4a . when asked to perform this maneuver in trial 1 , residents operating without mi - oct feedback ( mi - oct ) on average placed the pass at 49.1% depth ( sd = 14% , median = 43.0 ) , in contrast to 85.5% depth ( sd = 5% , median = 87% ) for the mi - oct+ group . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the average was 46.4% depth ( sd = 12% , median = 41% ) for the mi - oct group and 81.5% depth ( sd = 9% , median = 79% ) for the mi - oct+ group , which was also a statistically significant difference between the mi - oct+ and mi - oct groups without the use of the microscope ( p = 0.002 ; fig . the mi - oct+ group had no significant difference in depth performance between trials 1 and 2 with and without mi - oct ( median difference = 0.03 , p = 0.812 ) . in trial 3 , the mi - oct group repeated the surgical maneuver with mi - oct feedback , with the average corneal pass at 84.1% depth ( sd = 9% , median = 85% ) ; using the wilcoxon signed rank test , nonparametric testing for paired data , the mi - oct group 's performance with and without mi - oct was compared , and noted to be significantly different ( median difference = 0.34 , p = 0.016 ) . corneal passes with goal depth of 90% corneal thicknesses were performed by mi - oct and mi - oct+ group residents . a representative cross - sectional b - scan obtained orthogonal to the direction of needle pass demonstrates a hyperreflective dot ( circle ) at the goal of 90% corneal depth ( a ) . the average score of each resident was plotted , and the median depth scores were compared within individual groups using the wilcoxon signed rank test , with relevant comparisons demonstrating statistical significance indicated at p < 0.05 ( b ) . figure 5a demonstrates a representative orthogonal b - scan of a corneal needle pass at 90% depth ( arrow ) through the laceration . while performing this maneuver in trial 1 , residents operating without mi - oct feedback ( mi - oct ) on average placed the pass at 55.5% depth ( sd = 14% , median = 53% ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ ) on average placed the laceration pass at 81.5% depth ( sd = 3% , median = 82% ) . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the mi - oct group on average placed the pass at 50.6% depth ( sd = 15% , median = 49% ) , whereas the mi - oct+ group on average placed the pass at 73.3% depth ( sd = 12% , median = 74% ) , which was also statistically significant ( p = 0.025 ; fig . notably , the mi - oct+ group , which first operated with direct mi - oct guidance , continued to perform well in the second trial once mi - oct guidance was removed . there was no significant difference in their performance ( median difference = 0.07 , p = 0.11 ) . in trial 3 , the mi - oct group repeated the surgical maneuvers with mi - oct feedback , resulting in an average 83.8% suture pass depth ( sd = 5% , median = 83% ) , which was significant compared with their earlier performance without mi - oct in trial 2 with paired analysis through the wilcoxon signed ranks test ( p = 0.016 ) . fresh porcine corneas were used to construct vertical linear corneal laceration using a 15-blade scalpel . the corneal laceration repair with 10 - 0 nylon suture was performed by mi - oct and mi - oct+ group residents . a representative cross - sectional b - scan was obtained orthogonal to the direction of needle pass and demonstrates a jagged break in corneal integrity consistent with corneal laceration ( arrow ) and a hyperreflective dot ( circle ) at goal depth of 90% corneal thickness ( a ) . the average score of each resident was plotted , and the median depth scores compared across the groups using the wilcoxon signed rank test , with relevant comparisons demonstrating statistical significance indicated at p < 0.05 ( b ) . figure 3a demonstrates a representative orthogonal b - scan of a corneal needle pass at 50% depth ( arrow ) . when asked to perform corneal passes at 50% depth in trial 1 , residents operating without mi - oct feedback ( mi - oct group ) on average placed the pass at 39.4% depth ( sd = 13% , median = 35% ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ group ) on average placed the pass at 54.6% depth ( sd = 5% , median = 56% ) . 3b ) . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the mi - oct group on average placed the pass at 37.6% depth ( sd = 7% , median = 38% ) , whereas mi - oct+ on average placed the pass at 51.0% depth ( sd = 7% , median = 51% ) , which was also significantly different ( p = 0.009 ; fig . 3b ) . to ensure that the observed difference was not due to inferior surgical ability in the mi - oct group , these subjects were asked to perform the same maneuver with mi - oct feedback . with mi - oct feedback , there was a significant difference ( p = 0.016 ) in the performance of the mi - oct residents with an average of 53% depth ( sd = 5% , median = 55% ) compared with their earlier performance without mi - oct in trial 2 with an average of 37.6 % ( sd = 7% , median = 38% ) . corneal passes with the goal depth of 50% corneal thickness were performed by mi - oct and mi - oct+ group residents . a representative cross - sectional b - scan orthogonal to the direction of corneal needle pass is shown , and the hyperreflective dot ( circle ) represents an attempted pass at 50% corneal depth ( a ) . the average score of the residents in each group and the median depth scores are compared within the individual groups using paired analysis with the wilcoxon signed rank test , with relevant comparisons demonstrating statistical significance indicated at p < 0.05 ( b ) . for the corneal pass at 90% depth , a representative b - scan of this maneuver is shown in figure 4a . when asked to perform this maneuver in trial 1 , residents operating without mi - oct feedback ( mi - oct ) on average placed the pass at 49.1% depth ( sd = 14% , median = 43.0 ) , in contrast to 85.5% depth ( sd = 5% , median = 87% ) for the mi - oct+ group . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the average was 46.4% depth ( sd = 12% , median = 41% ) for the mi - oct group and 81.5% depth ( sd = 9% , median = 79% ) for the mi - oct+ group , which was also a statistically significant difference between the mi - oct+ and mi - oct groups without the use of the microscope ( p = 0.002 ; fig . the mi - oct+ group had no significant difference in depth performance between trials 1 and 2 with and without mi - oct ( median difference = 0.03 , p = 0.812 ) . in trial 3 , the mi - oct group repeated the surgical maneuver with mi - oct feedback , with the average corneal pass at 84.1% depth ( sd = 9% , median = 85% ) ; using the wilcoxon signed rank test , nonparametric testing for paired data , the mi - oct group 's performance with and without mi - oct was compared , and noted to be significantly different ( median difference = 0.34 , p = 0.016 ) . corneal passes with goal depth of 90% corneal thicknesses were performed by mi - oct and mi - oct+ group residents . a representative cross - sectional b - scan obtained orthogonal to the direction of needle pass demonstrates a hyperreflective dot ( circle ) at the goal of 90% corneal depth ( a ) . the average score of each resident was plotted , and the median depth scores were compared within individual groups using the wilcoxon signed rank test , with relevant comparisons demonstrating statistical significance indicated at p < 0.05 ( b ) . figure 5a demonstrates a representative orthogonal b - scan of a corneal needle pass at 90% depth ( arrow ) through the laceration . while performing this maneuver in trial 1 , residents operating without mi - oct feedback ( mi - oct ) on average placed the pass at 55.5% depth ( sd = 14% , median = 53% ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ ) on average placed the laceration 5b ) . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the mi - oct group on average placed the pass at 50.6% depth ( sd = 15% , median = 49% ) , whereas the mi - oct+ group on average placed the pass at 73.3% depth ( sd = 12% , median = 74% ) , which was also statistically significant ( p = 0.025 ; fig . notably , the mi - oct+ group , which first operated with direct mi - oct guidance , continued to perform well in the second trial once mi - oct guidance was removed . there was no significant difference in their performance ( median difference = 0.07 , p = 0.11 ) . in trial 3 , the mi - oct group repeated the surgical maneuvers with mi - oct feedback , resulting in an average 83.8% suture pass depth ( sd = 5% , median = 83% ) , which was significant compared with their earlier performance without mi - oct in trial 2 with paired analysis through the wilcoxon signed ranks test ( p = 0.016 ) . fresh porcine corneas were used to construct vertical linear corneal laceration using a 15-blade scalpel . the corneal laceration repair with 10 - 0 nylon suture was performed by mi - oct and mi - oct+ group residents . a representative cross - sectional b - scan was obtained orthogonal to the direction of needle pass and demonstrates a jagged break in corneal integrity consistent with corneal laceration ( arrow ) and a hyperreflective dot ( circle ) at goal depth of 90% corneal thickness ( a ) . the average score of each resident was plotted , and the median depth scores compared across the groups using the wilcoxon signed rank test , with relevant comparisons demonstrating statistical significance indicated at p < 0.05 ( b ) . the agreement between the expert graders in scoring corneal incision geometry on a scale of 0 to 3 was determined to not be significantly different ( weighted = 0.6381 ) . figures 6a and 6b demonstrate two representative b - scans of the constructed wounds . when asked to create a triplanar incision in trial 1 , residents operating without mi - oct feedback ( mi - oct group ) on average constructed a corneal wound with a mean score of 1.82 planes ( sd = 0.67 , median = 1.75 ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ group ) had a mean score of 2.14 planes ( sd = 0.46 , median = 2.00 ) . this difference was not statistically significant ( p = 0.244 ) . when both groups were asked to repeat this maneuver without mi - oct in trial 2 , the mi - oct group on average constructed wounds with a mean score of 2.41 planes ( sd = 0.65 , median = 2.75 ) , whereas the mi - oct+ group had a mean score of 2.38 planes ( sd = 0.53 , median = 2.50 ) , which was also not statistically significant ( p = 0.521 ) . in trial 3 , the mi - oct group was given an opportunity to repeat the surgical maneuver with mi - oct feedback and on average received a mean score of 2.21 ( sd = 0.86 , median = 2.64 ) , which was not significantly different than this group 's performance in trial 2 ( p = 0.437 ) . clear corneal incisions were created in porcine eyes by mi - oct and mi - oct+ group residents using 2.75-mm surgical keratomes . although the mi - oct+ resident could view the mi - oct throughout and at the completion of the maneuver , a representative cross - sectional b - scan longitudinal to the length of the wound was obtained at the completion of each maneuver ( a , b ) for postmaneuver grading of the wound profile visible from two separate representative trials ( arrows ) . all incisions viewed on such b - scans were assigned a grade of 0 to 3 by two expert graders masked to study group . last , qualitative and quantitative surgeon 's subjective feedback was obtained from the residents following completion of the study maneuvers via an administered survey ( fig . 7 ) . after completing the first portion of the study , only the mi - oct+ group had the direct use of mi - oct ; the mi - oct+ group reported higher levels of comfort compared with the mi - oct group , in performing simple cornea suture passes ( p = 0.007 ) and cornea laceration repair ( p = 0.028 ) , but this trend was not observed for creation of triplanar corneal wound . at the end of the training , when both mi - oct and mi - oct+ groups had experienced the direct use of mi - oct , there was no statistically significant difference between the groups ' subjective experience of mi - oct impact on their ability to perform those same procedures . in total , both mi - oct and mi - oct+ residents on average rated the overall experience of using mi - oct feedback for anterior segment surgical maneuvers as helpful ( score of 4 ) or very helpful ( score of 5 ) , reporting a mean score 4.57 ( sd = 0.53 ) for the mi - oct group and 4.86 ( sd = 0.38 ) , and a median of 5 for both groups on a 1 to 5 scale . ( score 4 on 15 scale ) to use mi - oct in their future practice as a result of mi - oct experience in this study . the survey administered to each resident surgeon at the conclusion of initial training and end of the study ranked their subjective experience on a 1 to 5 numerical scale . the mean subjective score with sd and minimum , maximum , and median responses to the survey questions are summarized below . the comparison between the mi - oct group and the mi - oct+ groups was done using the wilcoxon rank sum test of difference between medians . in this randomized prospective study , we demonstrated that mi - oct feedback results in enhanced surgical performance of ophthalmology residents and improved anatomic outcomes in select anterior segment surgical maneuvers in model eyes . the residents performing depth - based maneuvers ( simple corneal passes at 50% and 90% depth , and repair of corneal laceration at 90% depth ) with direct mi - oct feedback outperformed their counterparts who were operating with only the microscope alone . interestingly , the enhanced performance persisted even when direct mi - oct assistance was removed , which suggests that mi - oct can allow for sustained learning of surgical skill even when mi - oct feedback is not subsequently available . we also demonstrated a favorable subjective experience of the trainee surgeons in using mi - oct through a surgeon - administered survey . on average , most residents ' reported that their comfort in performing study maneuvers increased as a result of using mi - oct , and residents ranked the mi - oct experience as either very helpful or helpful , while reporting on average being more likely to use mi - oct in their future practice . overall , the results of this study suggest that mi - oct feedback can directly impact surgical outcomes , and for the first time , demonstrates its potential utility in surgical training of ophthalmic residents . not all surgical maneuvers were amenable to effective mi - oct feedback under the current study methodology . in our study , we found no effect of mi - oct feedback on corneal incision geometry . we attributed this to the metal keratome shadowing large portions of the oct image , which limited feedback while the incision was being created , and the use of an external display of the oct images , which was adjacent to , but not within the oculars . both of these limited the surgeon to assessing the wound geometry after , but not readily during the performance of the surgical maneuver . development of oct heads - up display through the operating microscope eyepieces and an oct - compatible keratome may aid in the utility of direct mi - oct guidance for construction of cataract incisions and other similar maneuvers in the future . the survey questions were used to obtain general feedback and were not provided before and after testing . a more standard validated questionnaire would be useful in future assessment of imaging as a training tool . other limitations of our study include the small sample size and a single study site . validation of the utility of mi - oct with a larger number of ophthalmology residents and fellows at other programs , and expanding the range of surgical maneuvers for which mi - oct may be useful in ophthalmic surgical training , such as posterior segment surgery , would be important future steps . such feedback may also be useful in ophthalmic surgeon self - assessment using a similar wet laboratory setup or with comparable model eye materials ( e.g. , kitaro kit ; fci ophthalmics , pembroke , ma , usa ) . in addition , a natural future evolution from our study would be for potential integration of mi - oct feedback for future training of residents and fellows in patient surgical treatment , such as during early experience in repair of corneal lacerations , open globes , and corneal suturing ( e.g. , penetrating keratoplasty ) . determining whether improved anatomic outcomes with mi - oct feedback in resident - performed and early career surgery will translate into improved visual outcomes , shorter operative time , and faster visual rehabilitation in patients would be important to justify significant cost of acquiring this technology for training or for use during routine surgery . clinical assessment from oct imaging of the eye has revolutionized diagnosis and management of many ophthalmic conditions and is now an indispensable part of practice of almost all ophthalmic subspecialties . although relatively recently introduced in the operating room , the histology - level feedback that intraoperative imaging provides during ophthalmic surgery holds the potential of a similar disruptive change in the surgical practice of ophthalmology . multiple studies to date have reported the use of intraoperative oct in anterior and posterior segment surgery . recent us food and drug administration approval of microscope - integrated sd - oct rescan 700 ( carl zeiss meditec , dublin , ca , usa ) will allow for access to microscope integrated , heads - up display oct viewing during live surgery in the united states . recently published 2-year results of a prospective evaluation of microscope - mounted sd - oct used at pauses in 275 anterior segment and 256 posterior segment ophthalmic surgeries . in this single center study , the use of mm - oct ( which required moving the microscope to the side ) resulted in a mean delay of 4.9 minutes while altering intraoperative surgeon 's decision making in more than 40% of surgical cases . the current study demonstrates that mi - oct feedback can not only guide a surgeon 's intraoperative decision making , but also has the potential to improve surgical performance , enhance anatomic outcomes , and be used as a training tool that is directly translatable to the operating room for use in live human surgery . as intraoperative oct technology continues to evolve , so will our understanding of its potential , which will allow for its increasing creative applications in many aspects of ophthalmic surgery .

purposethe integration of swept - source optical coherence tomography ( ss - oct ) into the operating microscope enables real - time , tissue - level three - dimensional ( 3d ) imaging to aid in ophthalmic microsurgery . in this prospective randomized controlled study , we evaluated the impact of ss microscope - integrated oct ( mi - oct ) on ophthalmology residents ' performance of ophthalmic microsurgical maneuvers.methodsfourteen ophthalmology residents from a single institution were stratified by year of training and randomized to perform four anterior segment surgical maneuvers on porcine eyes with ( mi - oct+ ) or without ( mi - oct ) direct intraoperative oct guidance . subsequently , both groups repeated the same maneuvers without mi - oct feedback to test whether initial mi - oct experience affected subsequent surgical performance . finally , the mi - oct group was crossed over and allowed to repeat the same maneuvers with direct mi - oct guidance . each resident completed a survey at the completion of the study.resultswith direct mi - oct feedback , residents demonstrated enhanced performance in depth - based anterior segment maneuvers ( corneal suture passes at 50% and 90% depth and corneal laceration repair ) compared with the residents operating without mi - oct . microscope - integrated oct+ residents continued to outperform the controls when both groups subsequently operated without mi - oct . for clear corneal wound geometry , there was no statistically significant effect of mi - oct as applied in this study . overall , the resident surgeons rated their subjective experience of using mi - oct very favorably.conclusionsmicroscope-integrated oct feedback enhances performance of ophthalmology residents in select anterior segment surgical maneuvers . microscope - integrated oct represents a valuable tool in the surgical education of ophthalmology residents .

Methods Subjects, Surgical Maneuvers, and Randomization Microscope-Integrated OCT Setup Depth-Based Maneuvers Grading Corneal Wound Grading Surgeon's Survey Statistics Results Depth-Based Maneuvers Corneal Pass at 50% Depth Target. Corneal Pass at 90% Depth Target. Repair of Corneal Laceration With 90% Depth Target. Corneal Incision Geometry Subjective Impression of MI-OCT Experience Discussion Supplementary Material

the subjects were randomized into two groups balanced by level of training and surgical experience : with one group assigned to work with the ability to view the mi - oct ( mi - oct+ ) and the other without ( mi - oct ) such guidance ( fig . fourteen ophthalmology residents were stratified according to the year of training and randomized to either the mi - oct or mi - oct+ group ( a ) . they were asked to perform four surgical maneuvers , including simple corneal pass at 50% and 90% depth , repair corneal laceration at 90% depth , and create triplanar clear - cornea wound on porcine eyes . finally , the mi - oct group crossed over and repeated all the maneuvers with mi - oct guidance ( trial 3 ) ( a , b ) . three consecutive trials were performed : trial 1 , each resident performed the above surgical maneuvers with ( mi - oct+ group ) or without ( mi - oct group ) mi - oct feedback based on the randomized assignment ; trial 2 , the same maneuvers were repeated by each surgeon using only the microscope for viewing ( no mi - oct guidance for either group ) to test the persistence of effect in the mi - oct+ group compared with the mi - oct group ; and trial 3 , the same maneuvers were repeated by each surgeon in the mi - oct group with intraoperative oct feedback . they were then asked to rerank their performance of the same maneuvers with direct mi - oct feedback at the completion of trial 3 when they were finally provided with mi - oct guidance . the subjects were randomized into two groups balanced by level of training and surgical experience : with one group assigned to work with the ability to view the mi - oct ( mi - oct+ ) and the other without ( mi - oct ) such guidance ( fig . fourteen ophthalmology residents were stratified according to the year of training and randomized to either the mi - oct or mi - oct+ group ( a ) . three consecutive trials were performed : trial 1 , each resident performed the above surgical maneuvers with ( mi - oct+ group ) or without ( mi - oct group ) mi - oct feedback based on the randomized assignment ; trial 2 , the same maneuvers were repeated by each surgeon using only the microscope for viewing ( no mi - oct guidance for either group ) to test the persistence of effect in the mi - oct+ group compared with the mi - oct group ; and trial 3 , the same maneuvers were repeated by each surgeon in the mi - oct group with intraoperative oct feedback . they were then asked to rerank their performance of the same maneuvers with direct mi - oct feedback at the completion of trial 3 when they were finally provided with mi - oct guidance . when asked to perform corneal passes at 50% depth in trial 1 , residents operating without mi - oct feedback ( mi - oct group ) on average placed the pass at 39.4% depth ( sd = 13% , median = 35% ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ group ) on average placed the pass at 54.6% depth ( sd = 5% , median = 56% ) . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the mi - oct group on average placed the pass at 37.6% depth ( sd = 7% , median = 38% ) , whereas mi - oct+ on average placed the pass at 51.0% depth ( sd = 7% , median = 51% ) , which was also significantly different ( p = 0.009 ; fig . with mi - oct feedback , there was a significant difference ( p = 0.016 ) in the performance of the mi - oct residents with an average of 53% depth ( sd = 5% , median = 55% ) compared with their earlier performance without mi - oct in trial 2 with an average of 37.6 % ( sd = 7% , median = 38% ) . when asked to perform this maneuver in trial 1 , residents operating without mi - oct feedback ( mi - oct ) on average placed the pass at 49.1% depth ( sd = 14% , median = 43.0 ) , in contrast to 85.5% depth ( sd = 5% , median = 87% ) for the mi - oct+ group . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the average was 46.4% depth ( sd = 12% , median = 41% ) for the mi - oct group and 81.5% depth ( sd = 9% , median = 79% ) for the mi - oct+ group , which was also a statistically significant difference between the mi - oct+ and mi - oct groups without the use of the microscope ( p = 0.002 ; fig . in trial 3 , the mi - oct group repeated the surgical maneuver with mi - oct feedback , with the average corneal pass at 84.1% depth ( sd = 9% , median = 85% ) ; using the wilcoxon signed rank test , nonparametric testing for paired data , the mi - oct group 's performance with and without mi - oct was compared , and noted to be significantly different ( median difference = 0.34 , p = 0.016 ) . while performing this maneuver in trial 1 , residents operating without mi - oct feedback ( mi - oct ) on average placed the pass at 55.5% depth ( sd = 14% , median = 53% ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ ) on average placed the laceration pass at 81.5% depth ( sd = 3% , median = 82% ) . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the mi - oct group on average placed the pass at 50.6% depth ( sd = 15% , median = 49% ) , whereas the mi - oct+ group on average placed the pass at 73.3% depth ( sd = 12% , median = 74% ) , which was also statistically significant ( p = 0.025 ; fig . notably , the mi - oct+ group , which first operated with direct mi - oct guidance , continued to perform well in the second trial once mi - oct guidance was removed . in trial 3 , the mi - oct group repeated the surgical maneuvers with mi - oct feedback , resulting in an average 83.8% suture pass depth ( sd = 5% , median = 83% ) , which was significant compared with their earlier performance without mi - oct in trial 2 with paired analysis through the wilcoxon signed ranks test ( p = 0.016 ) . when asked to create a triplanar incision in trial 1 , residents operating without mi - oct feedback ( mi - oct group ) on average constructed a corneal wound with a mean score of 1.82 planes ( sd = 0.67 , median = 1.75 ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ group ) had a mean score of 2.14 planes ( sd = 0.46 , median = 2.00 ) . when both groups were asked to repeat this maneuver without mi - oct in trial 2 , the mi - oct group on average constructed wounds with a mean score of 2.41 planes ( sd = 0.65 , median = 2.75 ) , whereas the mi - oct+ group had a mean score of 2.38 planes ( sd = 0.53 , median = 2.50 ) , which was also not statistically significant ( p = 0.521 ) . in trial 3 , the mi - oct group was given an opportunity to repeat the surgical maneuver with mi - oct feedback and on average received a mean score of 2.21 ( sd = 0.86 , median = 2.64 ) , which was not significantly different than this group 's performance in trial 2 ( p = 0.437 ) . only the mi - oct+ group had the direct use of mi - oct ; the mi - oct+ group reported higher levels of comfort compared with the mi - oct group , in performing simple cornea suture passes ( p = 0.007 ) and cornea laceration repair ( p = 0.028 ) , but this trend was not observed for creation of triplanar corneal wound . at the end of the training , when both mi - oct and mi - oct+ groups had experienced the direct use of mi - oct , there was no statistically significant difference between the groups ' subjective experience of mi - oct impact on their ability to perform those same procedures . in total , both mi - oct and mi - oct+ residents on average rated the overall experience of using mi - oct feedback for anterior segment surgical maneuvers as helpful ( score of 4 ) or very helpful ( score of 5 ) , reporting a mean score 4.57 ( sd = 0.53 ) for the mi - oct group and 4.86 ( sd = 0.38 ) , and a median of 5 for both groups on a 1 to 5 scale . when asked to perform corneal passes at 50% depth in trial 1 , residents operating without mi - oct feedback ( mi - oct group ) on average placed the pass at 39.4% depth ( sd = 13% , median = 35% ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ group ) on average placed the pass at 54.6% depth ( sd = 5% , median = 56% ) . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the mi - oct group on average placed the pass at 37.6% depth ( sd = 7% , median = 38% ) , whereas mi - oct+ on average placed the pass at 51.0% depth ( sd = 7% , median = 51% ) , which was also significantly different ( p = 0.009 ; fig . with mi - oct feedback , there was a significant difference ( p = 0.016 ) in the performance of the mi - oct residents with an average of 53% depth ( sd = 5% , median = 55% ) compared with their earlier performance without mi - oct in trial 2 with an average of 37.6 % ( sd = 7% , median = 38% ) . when asked to perform this maneuver in trial 1 , residents operating without mi - oct feedback ( mi - oct ) on average placed the pass at 49.1% depth ( sd = 14% , median = 43.0 ) , in contrast to 85.5% depth ( sd = 5% , median = 87% ) for the mi - oct+ group . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the average was 46.4% depth ( sd = 12% , median = 41% ) for the mi - oct group and 81.5% depth ( sd = 9% , median = 79% ) for the mi - oct+ group , which was also a statistically significant difference between the mi - oct+ and mi - oct groups without the use of the microscope ( p = 0.002 ; fig . in trial 3 , the mi - oct group repeated the surgical maneuver with mi - oct feedback , with the average corneal pass at 84.1% depth ( sd = 9% , median = 85% ) ; using the wilcoxon signed rank test , nonparametric testing for paired data , the mi - oct group 's performance with and without mi - oct was compared , and noted to be significantly different ( median difference = 0.34 , p = 0.016 ) . while performing this maneuver in trial 1 , residents operating without mi - oct feedback ( mi - oct ) on average placed the pass at 55.5% depth ( sd = 14% , median = 53% ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ ) on average placed the laceration pass at 81.5% depth ( sd = 3% , median = 82% ) . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the mi - oct group on average placed the pass at 50.6% depth ( sd = 15% , median = 49% ) , whereas the mi - oct+ group on average placed the pass at 73.3% depth ( sd = 12% , median = 74% ) , which was also statistically significant ( p = 0.025 ; fig . notably , the mi - oct+ group , which first operated with direct mi - oct guidance , continued to perform well in the second trial once mi - oct guidance was removed . in trial 3 , the mi - oct group repeated the surgical maneuvers with mi - oct feedback , resulting in an average 83.8% suture pass depth ( sd = 5% , median = 83% ) , which was significant compared with their earlier performance without mi - oct in trial 2 with paired analysis through the wilcoxon signed ranks test ( p = 0.016 ) . when asked to perform corneal passes at 50% depth in trial 1 , residents operating without mi - oct feedback ( mi - oct group ) on average placed the pass at 39.4% depth ( sd = 13% , median = 35% ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ group ) on average placed the pass at 54.6% depth ( sd = 5% , median = 56% ) . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the mi - oct group on average placed the pass at 37.6% depth ( sd = 7% , median = 38% ) , whereas mi - oct+ on average placed the pass at 51.0% depth ( sd = 7% , median = 51% ) , which was also significantly different ( p = 0.009 ; fig . with mi - oct feedback , there was a significant difference ( p = 0.016 ) in the performance of the mi - oct residents with an average of 53% depth ( sd = 5% , median = 55% ) compared with their earlier performance without mi - oct in trial 2 with an average of 37.6 % ( sd = 7% , median = 38% ) . when asked to perform this maneuver in trial 1 , residents operating without mi - oct feedback ( mi - oct ) on average placed the pass at 49.1% depth ( sd = 14% , median = 43.0 ) , in contrast to 85.5% depth ( sd = 5% , median = 87% ) for the mi - oct+ group . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the average was 46.4% depth ( sd = 12% , median = 41% ) for the mi - oct group and 81.5% depth ( sd = 9% , median = 79% ) for the mi - oct+ group , which was also a statistically significant difference between the mi - oct+ and mi - oct groups without the use of the microscope ( p = 0.002 ; fig . in trial 3 , the mi - oct group repeated the surgical maneuver with mi - oct feedback , with the average corneal pass at 84.1% depth ( sd = 9% , median = 85% ) ; using the wilcoxon signed rank test , nonparametric testing for paired data , the mi - oct group 's performance with and without mi - oct was compared , and noted to be significantly different ( median difference = 0.34 , p = 0.016 ) . while performing this maneuver in trial 1 , residents operating without mi - oct feedback ( mi - oct ) on average placed the pass at 55.5% depth ( sd = 14% , median = 53% ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ ) on average placed the laceration 5b ) . when both groups were asked to repeat this corneal pass without mi - oct in trial 2 , the mi - oct group on average placed the pass at 50.6% depth ( sd = 15% , median = 49% ) , whereas the mi - oct+ group on average placed the pass at 73.3% depth ( sd = 12% , median = 74% ) , which was also statistically significant ( p = 0.025 ; fig . notably , the mi - oct+ group , which first operated with direct mi - oct guidance , continued to perform well in the second trial once mi - oct guidance was removed . in trial 3 , the mi - oct group repeated the surgical maneuvers with mi - oct feedback , resulting in an average 83.8% suture pass depth ( sd = 5% , median = 83% ) , which was significant compared with their earlier performance without mi - oct in trial 2 with paired analysis through the wilcoxon signed ranks test ( p = 0.016 ) . when asked to create a triplanar incision in trial 1 , residents operating without mi - oct feedback ( mi - oct group ) on average constructed a corneal wound with a mean score of 1.82 planes ( sd = 0.67 , median = 1.75 ) , whereas residents operating with direct mi - oct feedback ( mi - oct+ group ) had a mean score of 2.14 planes ( sd = 0.46 , median = 2.00 ) . when both groups were asked to repeat this maneuver without mi - oct in trial 2 , the mi - oct group on average constructed wounds with a mean score of 2.41 planes ( sd = 0.65 , median = 2.75 ) , whereas the mi - oct+ group had a mean score of 2.38 planes ( sd = 0.53 , median = 2.50 ) , which was also not statistically significant ( p = 0.521 ) . in trial 3 , the mi - oct group was given an opportunity to repeat the surgical maneuver with mi - oct feedback and on average received a mean score of 2.21 ( sd = 0.86 , median = 2.64 ) , which was not significantly different than this group 's performance in trial 2 ( p = 0.437 ) . after completing the first portion of the study , only the mi - oct+ group had the direct use of mi - oct ; the mi - oct+ group reported higher levels of comfort compared with the mi - oct group , in performing simple cornea suture passes ( p = 0.007 ) and cornea laceration repair ( p = 0.028 ) , but this trend was not observed for creation of triplanar corneal wound . at the end of the training , when both mi - oct and mi - oct+ groups had experienced the direct use of mi - oct , there was no statistically significant difference between the groups ' subjective experience of mi - oct impact on their ability to perform those same procedures . in total , both mi - oct and mi - oct+ residents on average rated the overall experience of using mi - oct feedback for anterior segment surgical maneuvers as helpful ( score of 4 ) or very helpful ( score of 5 ) , reporting a mean score 4.57 ( sd = 0.53 ) for the mi - oct group and 4.86 ( sd = 0.38 ) , and a median of 5 for both groups on a 1 to 5 scale . in this randomized prospective study , we demonstrated that mi - oct feedback results in enhanced surgical performance of ophthalmology residents and improved anatomic outcomes in select anterior segment surgical maneuvers in model eyes . the residents performing depth - based maneuvers ( simple corneal passes at 50% and 90% depth , and repair of corneal laceration at 90% depth ) with direct mi - oct feedback outperformed their counterparts who were operating with only the microscope alone .

chemotherapy - induced nausea and vomiting ( cinv ) associated with highly emetogenic chemotherapy ( hec ) adversely affects the quality of life of patients ; it is especially challenging to manage in the delayed phase ( 24120 hours after chemotherapy)1 and affects chemotherapy compliance.2,3 hec includes chemotherapeutic agents with the potential to cause emesis in > 90% of patients in the absence of prophylaxis.4 for patients receiving hec , antiemesis guidelines from the american society of clinical oncology ( asco ) , the national comprehensive cancer network ( nccn ) , the european society of medical oncology ( esmo ) , and multinational association of supportive care in cancer ( mascc ) recommend the use of a three - drug regimen consisting of a 5-hydroxytryptamine type 3 ( 5-ht3 ) receptor antagonist ( ra ) , a neurokinin 1 ( nk-1 ) ra , and a corticosteroid.3,5,6 despite these comprehensive treatment guidelines , there is still an unmet clinical need for improved prevention of delayed cinv in patients receiving hec regimens.7,8 anthracycline and cyclophosphamide ( ac)based regimens represent a distinct class of emetogenic chemotherapy . previously classified as moderately emetogenic chemotherapy ( mec ) according to the hesketh emetogenicity criteria , developed in 1997,4 ac - based regimens were subsequently reclassified as hec in the asco 2011 emetogenicity guidelines to recognize their high emetogenic risk.9 updated mascc and esmo guidelines have also recommended classification of ac - based regimens as hec and recommend the above - mentioned three - drug regimen for cinv prevention in this setting.5 ac - based regimens are most commonly administered to women with breast cancer , a population at higher risk for cinv , based on both female gender and typically younger age.1015 therefore , cinv in this population is influenced by both chemotherapy- and patient - related risk factors . cinv prevention in patients receiving ac - based hec6 is challenging and remains an urgent therapeutic need . granisetron , a first - generation serotonin ( 5-ht3 ) ra , is commonly used to treat cinv but has a short half - life ( 9 hours).16 apf530 is a new formulation of 2% granisetron in a viscous bioerodible biochronomer tri(ethylene glycol ) poly(orthoester ) ( teg - poe ) polymer.17 following subcutaneous ( sc ) administration of apf530 in the upper arm or abdomen , the polymer undergoes slow , controlled hydrolysis , maintaining therapeutic concentrations of granisetron for 5 days.18,19 a single dose of apf530 ( granisetron 10 mg ) provides extended release of granisetron for the prevention of both acute ( 024 hours ) and delayed ( 24120 hours ) cinv.17 in a phase iii noninferiority trial , apf530 ( 500 mg sc ) was noninferior to palonosetron ( 0.25 mg iv ) in the control of acute cinv in patients receiving mec or hec and in the prevention of delayed cinv in patients receiving mec.17,20 apf530 is approved by the us food and drug administration ( fda ) for use in conjunction with other antiemetics to prevent acute or delayed cinv following initial or repeat courses of mec or ac combination regimens.21 the phase iii modified absorption of granisetron in the prevention of cinv ( magic ) trial compared delayed - phase complete response ( cr ; no emesis [ vomit or retch ] and no rescue medication use ) achieved by using apf530 with that using ondansetron , each in a three - drug regimen with an nk-1 ra and dexamethasone . ondansetron , the active comparator , has been used in large - scale cinv studies as the positive comparator for other 5-ht3 ras22,23 and is indicated for the prevention of nausea and vomiting associated with initial and repeat courses of emetogenic cancer chemotherapy , including high - dose cisplatin.24 in magic , the apf530 arm demonstrated superior cr versus ondansetron in delayed cinv following hec ( 64.7% vs 56.6% ; p=0.014 ; 8% absolute improvement).25 apf530 is the first and only 5-ht3 ra to demonstrate superiority over another 5-ht3 ra in a three - drug versus three - drug pivotal phase iii efficacy trial . this exploratory post hoc analysis evaluated the efficacy and safety of an apf530 regimen versus an ondansetron regimen in magic trial patients who received ac - based chemotherapy regimens . details of the magic trial design and methodology have been presented previously,25 whereas a brief overview is presented in this report . this prospective , multicenter , randomized , double - blind , double - dummy , parallel - group phase iii trial was conducted at 77 sites ( see table s1 for the list of study investigators list ) in the united states ( clinicaltrials.gov identifier : nct02106494 ) . the protocol was approved by the institutional review board at all sites and conducted according to the international conference on harmonisation e6 good clinical practice guidelines and the declaration of helsinki . access to the data for this post hoc analysis was provided by heron therapeutics , inc . adult patients who had histologically or cytologically confirmed malignancy and were scheduled to receive their first cycle of single - day hec , according to asco 2011 emetogenicity criteria,9 were enrolled . eligible patients had an eastern cooperative oncology group ( ecog ) performance status of 0 or 1 . patients with current or prior significant cardiac disease , including qt interval prolongation , were excluded . patients were randomly assigned 1:1 to receive either apf530 500 mg sc ( granisetron 10 mg ) and ondansetron placebo iv or ondansetron 0.15 mg / kg iv ( to a maximum of 16 mg ) and apf530 placebo sc ( containing the biochronomer teg - poe vehicle ) ; stratification was by planned cisplatin 50 mg / m ( yes / no ) . in addition , all patients received fosaprepitant 150 mg iv and dexamethasone 12 mg iv on day 1 and were scheduled to receive dexamethasone 8 mg orally once daily on day 2 and twice daily on days 3 and 4 . the primary objective was to demonstrate the superiority of apf530 500 mg sc in achieving delayed - phase cr , compared with ondansetron 0.15 mg / kg iv , in patients receiving hec in cycle 1 . secondary end points included cr in the overall ( 0120 hours ) phase and two , more stringent end points that measure additional effects on nausea : complete control ( cc ; cr and no more than mild nausea ) in delayed and overall phases . other end points included cr and cc in the acute phase and total response ( tr ; cr and no nausea ) in acute , delayed , and overall phases ; the number of nausea episodes and rescue medication use , results for which were based on observed data without imputation for missing data , were also included . patients recorded daily , up to 120 hours following chemotherapy , the number of nausea , retching and/or vomiting episodes , and instances of rescue medication use ; these data were used to evaluate the above - mentioned efficacy measures . response rates were compared using 95% confidence intervals for treatment differences using a modified intent - to - treat ( mitt ) population ( all patients who received hec and the study drug and had postbaseline efficacy measures ) ; p - values between treatment arms were based on the chi - square test . this post hoc analysis was conducted on the subgroup of patients who received an ac - containing hec regimen ; however , the study was not powered to detect statistically significant treatment differences between the arms with this subgroup . safety was assessed by adverse events , physical examinations , vital signs , and clinical laboratory values in the safety population , comprising all patients who received the study drug . the type of treatment - emergent adverse event ( teae ) and its duration , severity , and relationship to the study drug were evaluated . teaes , serious adverse events ( saes ) , treatment - related teaes , and treatment - related saes were assessed . teaes were defined as adverse events that either began within 8 days of study drug administration or prior to and increased in severity within 8 days of study drug administration . treatment - related teaes included teaes with possible , probable , or definite relationship to study drug treatment or events with unknown or missing causality . severity of teaes was graded by the national cancer institute common terminology criteria for adverse events version 4.03 . all injection site reactions ( isrs ) were collected by patient diary entries in addition to investigators evaluation . the severity of most isrs was based on prespecified criteria of size and appearance only , rather than functional impairment . of the 942 patients randomized in the entire study between march 31 , 2014 , and may 15 , 2015 , 600 patients received ac - based hec ( apf530 arm , 296 ; ondansetron arm , 304 ) ( figure 1 ) . of the 296 randomized patients in the apf530 arm , 3 discontinued prior to treatment ( 1 due to a protocol violation , 1 due to withdrawn consent , and 1 due to other reason ) ; of the 304 randomized patients in the ondansetron arm , 1 discontinued prior to treatment because of an adverse event . the safety population consisted of 293 patients in the apf530 arm and 303 patients in the ondansetron arm . of the 293 patients in the apf530 arm safety population , 2 discontinued the study prior to day 6 ( 1 due to an adverse event and 1 due to other reason ) . among the 303 patients in the ondansetron arm safety population , 5 were excluded from the mitt population : 2 discontinued the study without postbaseline efficacy data ( 1 due to protocol violation and 1 due to an adverse event ) and 3 completed the study , but 2 had no postbaseline efficacy data and 1 had improper study medication ( figure 1 ) . all patients in the apf530 arm mitt population completed the study ; among the ondansetron arm mitt population , 2 patients discontinued the study because of a protocol violation ( figure 1 ) . of the 902 patients in the mitt population of the entire study , 589 ( 65% ) received ac - based hec ( apf530 arm , n=291 ; ondansetron arm , n=298 ) . baseline demographics were generally balanced between the treatment arms in the ac subgroup ( table 1 ) . most patients in the ac subgroup were female ( apf530 , 99.3% ; ondansetron , 98.3% ) , white ( apf530 , 80.1% ; ondansetron , 77.9% ) , and had an ecog performance status of 0 ( apf530 , 83.8% ; ondansetron , 79.9% ) . the mean age of patients in the apf530 arm was 54.1 years and that in the ondansetron arm was 53.8 years . the most common ac - based chemotherapy regimen in both treatment arms was cyclophosphamide < 1500 mg / m and doxorubicin ( apf530 arm , 87.3% ; ondansetron arm , 89.3% ) ( table 2 ) . in the ac subgroup , delayed - phase cr was numerically higher in the apf530 arm versus the ondansetron arm , approaching statistical significance ( 63.6% vs 56.0% ; p=0.062 ) ( table 3 ) . similarly , in the overall phase , a trend favoring the apf530 arm versus the ondansetron arm was observed , although it was not statistically significant . no appreciable efficacy difference was observed in the acute phase in the apf530 arm compared with the ondansetron arm ( table 3 ) . for cc and tr , numerically higher rates were observed in the apf530 arm versus the ondansetron arm in the delayed and overall phases , although the treatment differences were not statistically significant . minor differences were observed in cc and tr rates in the acute phase ( table 3 ) . a numerically higher proportion of patients in the apf530 arm versus the ondansetron arm reported no rescue medication use in the delayed ( 68.9% vs 61.7% ; p=0.069 ) and overall phases ( 63.3% vs 56.9% ; p=0.116 ) ( table 4 ) . the proportion of patients with no rescue medication use was numerically higher in the apf530 arm compared with the ondansetron arm across all phases ( figure s1 ) . clinical results also favored the apf530 arm versus the ondansetron arm in the proportion of patients reporting no nausea in the delayed and overall phases ( table 5 ) . the apf530 regimen was generally well tolerated in this ac subgroup ; no new safety signals were identified ( table 6 ) . most patients experienced at least one teae ( apf530 arm , 93.5% ; ondansetron arm , 91.1% ) . excluding isrs , the most frequently reported teaes were fatigue , constipation , nausea , and headache , occurring with a similar frequency in each treatment arm . serious teaes were experienced by 4.1% of the patients in the apf530 arm and 2.0% of the patients in the ondansetron arm ; no teaes led to death . excluding isrs , the most common treatment - related teaes in the apf530 and ondansetron arms were constipation ( 8.2% vs 5.9% , respectively ) and headache ( 7.2% vs 5.9% , respectively ) . a treatment - related sae occurred in one patient in the apf530 arm ( 0.3% , injection - site infection ) 14 days after apf530 administration and recovered within 9 days with antibiotic use . a treatment - related sae occurred in one patient in the ondansetron arm ( 0.3% , dehydration ) and subsequently resolved . the most frequently reported teaes were isrs ( table 6 ) , which occurred in 66.9% of patients in the apf530 arm and 60.7% in the ondansetron arm . the severity of most isrs was based on prespecified criteria of size and appearance only , rather than functional impairment . these were generally mild or moderate , most resolved by the end of the study , and no isr led to death or study discontinuation . of the 942 patients randomized in the entire study between march 31 , 2014 , and may 15 , 2015 , 600 patients received ac - based hec ( apf530 arm , 296 ; ondansetron arm , 304 ) ( figure 1 ) . of the 296 randomized patients in the apf530 arm , 3 discontinued prior to treatment ( 1 due to a protocol violation , 1 due to withdrawn consent , and 1 due to other reason ) ; of the 304 randomized patients in the ondansetron arm , 1 discontinued prior to treatment because of an adverse event . the safety population consisted of 293 patients in the apf530 arm and 303 patients in the ondansetron arm . of the 293 patients in the apf530 arm safety population , 2 discontinued the study prior to day 6 ( 1 due to an adverse event and 1 due to other reason ) . among the 303 patients in the ondansetron arm safety population , 5 were excluded from the mitt population : 2 discontinued the study without postbaseline efficacy data ( 1 due to protocol violation and 1 due to an adverse event ) and 3 completed the study , but 2 had no postbaseline efficacy data and 1 had improper study medication ( figure 1 ) . all patients in the apf530 arm mitt population completed the study ; among the ondansetron arm mitt population , 2 patients discontinued the study because of a protocol violation ( figure 1 ) . of the 902 patients in the mitt population of the entire study , 589 ( 65% ) received ac - based hec ( apf530 arm , n=291 ; ondansetron arm , n=298 ) . baseline demographics were generally balanced between the treatment arms in the ac subgroup ( table 1 ) . most patients in the ac subgroup were female ( apf530 , 99.3% ; ondansetron , 98.3% ) , white ( apf530 , 80.1% ; ondansetron , 77.9% ) , and had an ecog performance status of 0 ( apf530 , 83.8% ; ondansetron , 79.9% ) . the mean age of patients in the apf530 arm was 54.1 years and that in the ondansetron arm was 53.8 years . the most common ac - based chemotherapy regimen in both treatment arms was cyclophosphamide < 1500 mg / m and doxorubicin ( apf530 arm , 87.3% ; ondansetron arm , 89.3% ) ( table 2 ) . in the ac subgroup , delayed - phase cr was numerically higher in the apf530 arm versus the ondansetron arm , approaching statistical significance ( 63.6% vs 56.0% ; p=0.062 ) ( table 3 ) . similarly , in the overall phase , a trend favoring the apf530 arm versus the ondansetron arm was observed , although it was not statistically significant . no appreciable efficacy difference was observed in the acute phase in the apf530 arm compared with the ondansetron arm ( table 3 ) . for cc and tr , numerically higher rates were observed in the apf530 arm versus the ondansetron arm in the delayed and overall phases , although the treatment differences were not statistically significant . minor differences were observed in cc and tr rates in the acute phase ( table 3 ) . a numerically higher proportion of patients in the apf530 arm versus the ondansetron arm reported no rescue medication use in the delayed ( 68.9% vs 61.7% ; p=0.069 ) and overall phases ( 63.3% vs 56.9% ; p=0.116 ) ( table 4 ) . the proportion of patients with no rescue medication use was numerically higher in the apf530 arm compared with the ondansetron arm across all phases ( figure s1 ) . clinical results also favored the apf530 arm versus the ondansetron arm in the proportion of patients reporting no nausea in the delayed and overall phases ( table 5 ) . the apf530 regimen was generally well tolerated in this ac subgroup ; no new safety signals were identified ( table 6 ) . most patients experienced at least one teae ( apf530 arm , 93.5% ; ondansetron arm , 91.1% ) . excluding isrs , the most frequently reported teaes were fatigue , constipation , nausea , and headache , occurring with a similar frequency in each treatment arm . serious teaes were experienced by 4.1% of the patients in the apf530 arm and 2.0% of the patients in the ondansetron arm ; no teaes led to death . excluding isrs , the most common treatment - related teaes in the apf530 and ondansetron arms were constipation ( 8.2% vs 5.9% , respectively ) and headache ( 7.2% vs 5.9% , respectively ) . a treatment - related sae occurred in one patient in the apf530 arm ( 0.3% , injection - site infection ) 14 days after apf530 administration and recovered within 9 days with antibiotic use . a treatment - related sae occurred in one patient in the ondansetron arm ( 0.3% , dehydration ) and subsequently resolved . the most frequently reported teaes were isrs ( table 6 ) , which occurred in 66.9% of patients in the apf530 arm and 60.7% in the ondansetron arm . the severity of most isrs was based on prespecified criteria of size and appearance only , rather than functional impairment . these were generally mild or moderate , most resolved by the end of the study , and no isr led to death or study discontinuation . the use of ac - based chemotherapy remains highly prevalent because of its effectiveness in patients with breast cancer and the relatively high breast cancer incidence.2629 placebo - controlled studies have shown that this combination induces emesis in a sufficient number of patients to warrant its reclassification , in 2011 , from mec to hec . the magic trial compared apf530 versus ondansetron in the context of a guideline - recommended three - drug regimen in cinv prevention following hec regimens , including ac . the use of ondansetron was appropriate because it has been used in pivotal cinv studies as the active comparator for other 5-ht3 ras22,23 and is indicated for the prevention of nausea and vomiting associated with initial and repeat courses of emetogenic cancer chemotherapy , including high - dose cisplatin.24 no previous pivotal efficacy trial involving patients receiving ac - based regimens has compared two 5-ht3 ras within a three - drug regimen . the magic trial design was consistent with current antiemesis guideline recommendations and was conducted in us community practices , so the results are likely to be representative of this clinical practice setting . in contrast to other hec , ac - based regimens are administered mostly to younger female patients ; both female sex and young age constitute patient - related cinv risk factors.30,31 accordingly , in the magic trial , most patients receiving ac - based regimens were female and aged < 55 years , and so at higher risk for cinv.31,32 the interaction of high emetogenicity of ac - based regimens with patient - related risk factors highlights the distinct cinv prevention needs in this patient group . in two large phase iii trials , cr rates were lower in patients receiving ac - based than in those receiving non ac - based regimens across all phases , suggesting greater cinv prevention challenges associated with ac - based regimens.13,33 furthermore , patients receiving hec or mec regimens continue to experience delayed - phase cinv , highlighting the need for new therapies.34 although this exploratory post hoc ac subgroup analysis was not powered for statistical significance , trends favoring the apf530 versus the ondansetron arm in delayed and overall phases were observed in multiple measures of cinv control : cr , cc , tr , rescue medication use , and proportion of patients with no nausea . the biochronomer technology underlying apf530 provides sustained therapeutic granisetron concentrations across 5 days following a single apf530 injection and was expected to provide better delayed - phase cinv control than ondansetron in the magic trial . the lack of any appreciable differences in efficacy in the acute phase is consistent with the trial design . acknowledging the limits of cross - trial comparisons , the control arm of the magic ac subgroup showed results consistent with those from a previous study in patients with breast cancer who received ac - based regimens.15 similar to findings in the entire study population , apf530 was generally well tolerated in this patient subgroup receiving ac - based hec , and no new safety signals were observed.25 the incidences of isrs were similar in both the arms , and most resolved by the end of the study . consistent with the treatment - related teaes observed with other 5-ht3 ras,35 constipation and headache were the most commonly reported teaes with apf530 . trial results suggest that apf530 is a safe and effective cinv management option in this particularly challenging clinical setting . consequently , the fda approved apf530 for the prevention of both acute and delayed cinv following initial and repeat courses of mec or ac combination regimens.21 the findings of the present study from the ac subgroup are concordant with the superior delayed - phase cr rate observed in the entire magic study population.25 the superiority of apf530 versus ondansetron , in the presence of an nk-1 ra and dexamethasone , in achieving delayed - phase cr in the magic trial patients suggests that apf530 provided benefit in addition to that of the nk-1 ra . overall , these results demonstrate the benefit of the extended - release design of the apf530 formulation , whereby a single sc dose provides therapeutic granisetron concentrations for 5 days.18 limitations of this analysis include being an exploratory post hoc analysis with a relatively small number of patients in each arm . in addition , use of the double - dummy design resulted in an increased incidence of isrs in the ondansetron arm due to the presence of the viscous teg - poe vehicle in the apf530 placebo injection . in a recently published randomized , double - blind phase iii trial , olanzapine was compared with placebo , when added to a three - drug regimen of 5-ht3 ra , nk-1 ra , and dexamethasone in patients receiving cisplatin or ac - based hec.36 in that study , the addition of olanzapine resulted in a significant improvement in nausea control . it will therefore be of interest to determine whether the addition of olanzapine to a three - drug regimen of apf530 may similarly further improve cinv control in patients receiving ac - based regimens . the magic trial demonstrated the superiority of apf530 versus ondansetron , each in a guideline - recommended three - drug regimen with fosaprepitant and dexamethasone , in the prevention of delayed - phase cinv following hec . the findings of this post hoc subgroup analysis in patients receiving ac - based hec and at a high risk of experiencing cinv are consistent with the results from the entire study population , particularly in control of delayed cinv . the apf530 regimen was generally well tolerated in the ac subgroup , as in the entire population . apf530 , in conjunction with other anti - emetics , is approved for preventing cinv in both acute and delayed phases in patients receiving initial or repeat courses of mec or ac - based regimens.21 thus , apf530 may be a convenient antiemetic option for female patients with breast cancer receiving ac - based chemotherapy , a population in which preventing nausea and vomiting is particularly difficult .

backgroundapf530 , a novel extended - release granisetron injection , was superior to ondansetron in a guideline - recommended three - drug regimen in preventing delayed - phase chemotherapy - induced nausea and vomiting ( cinv ) among patients receiving highly emetogenic chemotherapy ( hec ) in the double - blind phase iii modified absorption of granisetron in the prevention of cinv ( magic ) trial.patients and methodsthis magic post hoc analysis evaluated cinv prevention efficacy and safety of apf530 versus ondansetron , each with fosaprepitant and dexamethasone , in patient subgroup receiving an anthracycline plus cyclophosphamide ( ac ) regimen . patients were randomized 1:1 to apf530 500 mg subcutaneously ( granisetron 10 mg ) or ondansetron 0.15 mg / kg intravenously ( iv ) ( 16 mg ) ; stratification was by planned cisplatin 50 mg / m2 ( yes / no ) . patients were to receive fosaprepitant 150 mg iv and dexamethasone 12 mg iv on day 1 , then dexamethasone 8 mg orally once daily on day 2 and twice daily on days 3 and 4 . patients were mostly younger females ( apf530 arm , mean age 54.1 years , female , 99.3% ; ondansetron arm , 53.8 years , female 98.3% ) . the primary end point was delayed - phase ( > 24120 hours ) complete response ( cr).resultsapf530 versus ondansetron regimens achieved numerically better cinv control in delayed and overall ( 0120 hours ) phases for cr , complete control , total response , rescue medication use , and proportion with no nausea . apf530 trends are consistent with the overall population , although not statistically superior given the underpowered ac subgroup analysis . the apf530 regimen in this population was generally well tolerated , with safety comparable to that of the overall population.conclusionapf530 plus fosaprepitant and dexamethasone effectively prevented cinv among patients receiving ac - based hec , a large subgroup in whom cinv control has traditionally been challenging .

Introduction Patients and methods Results Patient disposition and characteristics Efficacy analyses Safety analyses Discussion Conclusion

chemotherapy - induced nausea and vomiting ( cinv ) associated with highly emetogenic chemotherapy ( hec ) adversely affects the quality of life of patients ; it is especially challenging to manage in the delayed phase ( 24120 hours after chemotherapy)1 and affects chemotherapy compliance.2,3 hec includes chemotherapeutic agents with the potential to cause emesis in > 90% of patients in the absence of prophylaxis.4 for patients receiving hec , antiemesis guidelines from the american society of clinical oncology ( asco ) , the national comprehensive cancer network ( nccn ) , the european society of medical oncology ( esmo ) , and multinational association of supportive care in cancer ( mascc ) recommend the use of a three - drug regimen consisting of a 5-hydroxytryptamine type 3 ( 5-ht3 ) receptor antagonist ( ra ) , a neurokinin 1 ( nk-1 ) ra , and a corticosteroid.3,5,6 despite these comprehensive treatment guidelines , there is still an unmet clinical need for improved prevention of delayed cinv in patients receiving hec regimens.7,8 anthracycline and cyclophosphamide ( ac)based regimens represent a distinct class of emetogenic chemotherapy . previously classified as moderately emetogenic chemotherapy ( mec ) according to the hesketh emetogenicity criteria , developed in 1997,4 ac - based regimens were subsequently reclassified as hec in the asco 2011 emetogenicity guidelines to recognize their high emetogenic risk.9 updated mascc and esmo guidelines have also recommended classification of ac - based regimens as hec and recommend the above - mentioned three - drug regimen for cinv prevention in this setting.5 ac - based regimens are most commonly administered to women with breast cancer , a population at higher risk for cinv , based on both female gender and typically younger age.1015 therefore , cinv in this population is influenced by both chemotherapy- and patient - related risk factors . cinv prevention in patients receiving ac - based hec6 is challenging and remains an urgent therapeutic need . granisetron , a first - generation serotonin ( 5-ht3 ) ra , is commonly used to treat cinv but has a short half - life ( 9 hours).16 apf530 is a new formulation of 2% granisetron in a viscous bioerodible biochronomer tri(ethylene glycol ) poly(orthoester ) ( teg - poe ) polymer.17 following subcutaneous ( sc ) administration of apf530 in the upper arm or abdomen , the polymer undergoes slow , controlled hydrolysis , maintaining therapeutic concentrations of granisetron for 5 days.18,19 a single dose of apf530 ( granisetron 10 mg ) provides extended release of granisetron for the prevention of both acute ( 024 hours ) and delayed ( 24120 hours ) cinv.17 in a phase iii noninferiority trial , apf530 ( 500 mg sc ) was noninferior to palonosetron ( 0.25 mg iv ) in the control of acute cinv in patients receiving mec or hec and in the prevention of delayed cinv in patients receiving mec.17,20 apf530 is approved by the us food and drug administration ( fda ) for use in conjunction with other antiemetics to prevent acute or delayed cinv following initial or repeat courses of mec or ac combination regimens.21 the phase iii modified absorption of granisetron in the prevention of cinv ( magic ) trial compared delayed - phase complete response ( cr ; no emesis [ vomit or retch ] and no rescue medication use ) achieved by using apf530 with that using ondansetron , each in a three - drug regimen with an nk-1 ra and dexamethasone . ondansetron , the active comparator , has been used in large - scale cinv studies as the positive comparator for other 5-ht3 ras22,23 and is indicated for the prevention of nausea and vomiting associated with initial and repeat courses of emetogenic cancer chemotherapy , including high - dose cisplatin.24 in magic , the apf530 arm demonstrated superior cr versus ondansetron in delayed cinv following hec ( 64.7% vs 56.6% ; p=0.014 ; 8% absolute improvement).25 apf530 is the first and only 5-ht3 ra to demonstrate superiority over another 5-ht3 ra in a three - drug versus three - drug pivotal phase iii efficacy trial . this exploratory post hoc analysis evaluated the efficacy and safety of an apf530 regimen versus an ondansetron regimen in magic trial patients who received ac - based chemotherapy regimens . this prospective , multicenter , randomized , double - blind , double - dummy , parallel - group phase iii trial was conducted at 77 sites ( see table s1 for the list of study investigators list ) in the united states ( clinicaltrials.gov identifier : nct02106494 ) . patients were randomly assigned 1:1 to receive either apf530 500 mg sc ( granisetron 10 mg ) and ondansetron placebo iv or ondansetron 0.15 mg / kg iv ( to a maximum of 16 mg ) and apf530 placebo sc ( containing the biochronomer teg - poe vehicle ) ; stratification was by planned cisplatin 50 mg / m ( yes / no ) . in addition , all patients received fosaprepitant 150 mg iv and dexamethasone 12 mg iv on day 1 and were scheduled to receive dexamethasone 8 mg orally once daily on day 2 and twice daily on days 3 and 4 . the primary objective was to demonstrate the superiority of apf530 500 mg sc in achieving delayed - phase cr , compared with ondansetron 0.15 mg / kg iv , in patients receiving hec in cycle 1 . secondary end points included cr in the overall ( 0120 hours ) phase and two , more stringent end points that measure additional effects on nausea : complete control ( cc ; cr and no more than mild nausea ) in delayed and overall phases . other end points included cr and cc in the acute phase and total response ( tr ; cr and no nausea ) in acute , delayed , and overall phases ; the number of nausea episodes and rescue medication use , results for which were based on observed data without imputation for missing data , were also included . of the 942 patients randomized in the entire study between march 31 , 2014 , and may 15 , 2015 , 600 patients received ac - based hec ( apf530 arm , 296 ; ondansetron arm , 304 ) ( figure 1 ) . of the 296 randomized patients in the apf530 arm , 3 discontinued prior to treatment ( 1 due to a protocol violation , 1 due to withdrawn consent , and 1 due to other reason ) ; of the 304 randomized patients in the ondansetron arm , 1 discontinued prior to treatment because of an adverse event . of the 293 patients in the apf530 arm safety population , 2 discontinued the study prior to day 6 ( 1 due to an adverse event and 1 due to other reason ) . all patients in the apf530 arm mitt population completed the study ; among the ondansetron arm mitt population , 2 patients discontinued the study because of a protocol violation ( figure 1 ) . of the 902 patients in the mitt population of the entire study , 589 ( 65% ) received ac - based hec ( apf530 arm , n=291 ; ondansetron arm , n=298 ) . most patients in the ac subgroup were female ( apf530 , 99.3% ; ondansetron , 98.3% ) , white ( apf530 , 80.1% ; ondansetron , 77.9% ) , and had an ecog performance status of 0 ( apf530 , 83.8% ; ondansetron , 79.9% ) . the mean age of patients in the apf530 arm was 54.1 years and that in the ondansetron arm was 53.8 years . the most common ac - based chemotherapy regimen in both treatment arms was cyclophosphamide < 1500 mg / m and doxorubicin ( apf530 arm , 87.3% ; ondansetron arm , 89.3% ) ( table 2 ) . in the ac subgroup , delayed - phase cr was numerically higher in the apf530 arm versus the ondansetron arm , approaching statistical significance ( 63.6% vs 56.0% ; p=0.062 ) ( table 3 ) . similarly , in the overall phase , a trend favoring the apf530 arm versus the ondansetron arm was observed , although it was not statistically significant . no appreciable efficacy difference was observed in the acute phase in the apf530 arm compared with the ondansetron arm ( table 3 ) . for cc and tr , numerically higher rates were observed in the apf530 arm versus the ondansetron arm in the delayed and overall phases , although the treatment differences were not statistically significant . a numerically higher proportion of patients in the apf530 arm versus the ondansetron arm reported no rescue medication use in the delayed ( 68.9% vs 61.7% ; p=0.069 ) and overall phases ( 63.3% vs 56.9% ; p=0.116 ) ( table 4 ) . the proportion of patients with no rescue medication use was numerically higher in the apf530 arm compared with the ondansetron arm across all phases ( figure s1 ) . clinical results also favored the apf530 arm versus the ondansetron arm in the proportion of patients reporting no nausea in the delayed and overall phases ( table 5 ) . the apf530 regimen was generally well tolerated in this ac subgroup ; no new safety signals were identified ( table 6 ) . most patients experienced at least one teae ( apf530 arm , 93.5% ; ondansetron arm , 91.1% ) . serious teaes were experienced by 4.1% of the patients in the apf530 arm and 2.0% of the patients in the ondansetron arm ; no teaes led to death . of the 942 patients randomized in the entire study between march 31 , 2014 , and may 15 , 2015 , 600 patients received ac - based hec ( apf530 arm , 296 ; ondansetron arm , 304 ) ( figure 1 ) . of the 296 randomized patients in the apf530 arm , 3 discontinued prior to treatment ( 1 due to a protocol violation , 1 due to withdrawn consent , and 1 due to other reason ) ; of the 304 randomized patients in the ondansetron arm , 1 discontinued prior to treatment because of an adverse event . of the 293 patients in the apf530 arm safety population , 2 discontinued the study prior to day 6 ( 1 due to an adverse event and 1 due to other reason ) . all patients in the apf530 arm mitt population completed the study ; among the ondansetron arm mitt population , 2 patients discontinued the study because of a protocol violation ( figure 1 ) . of the 902 patients in the mitt population of the entire study , 589 ( 65% ) received ac - based hec ( apf530 arm , n=291 ; ondansetron arm , n=298 ) . most patients in the ac subgroup were female ( apf530 , 99.3% ; ondansetron , 98.3% ) , white ( apf530 , 80.1% ; ondansetron , 77.9% ) , and had an ecog performance status of 0 ( apf530 , 83.8% ; ondansetron , 79.9% ) . the mean age of patients in the apf530 arm was 54.1 years and that in the ondansetron arm was 53.8 years . the most common ac - based chemotherapy regimen in both treatment arms was cyclophosphamide < 1500 mg / m and doxorubicin ( apf530 arm , 87.3% ; ondansetron arm , 89.3% ) ( table 2 ) . in the ac subgroup , delayed - phase cr was numerically higher in the apf530 arm versus the ondansetron arm , approaching statistical significance ( 63.6% vs 56.0% ; p=0.062 ) ( table 3 ) . similarly , in the overall phase , a trend favoring the apf530 arm versus the ondansetron arm was observed , although it was not statistically significant . no appreciable efficacy difference was observed in the acute phase in the apf530 arm compared with the ondansetron arm ( table 3 ) . for cc and tr , numerically higher rates were observed in the apf530 arm versus the ondansetron arm in the delayed and overall phases , although the treatment differences were not statistically significant . a numerically higher proportion of patients in the apf530 arm versus the ondansetron arm reported no rescue medication use in the delayed ( 68.9% vs 61.7% ; p=0.069 ) and overall phases ( 63.3% vs 56.9% ; p=0.116 ) ( table 4 ) . the proportion of patients with no rescue medication use was numerically higher in the apf530 arm compared with the ondansetron arm across all phases ( figure s1 ) . clinical results also favored the apf530 arm versus the ondansetron arm in the proportion of patients reporting no nausea in the delayed and overall phases ( table 5 ) . the apf530 regimen was generally well tolerated in this ac subgroup ; no new safety signals were identified ( table 6 ) . most patients experienced at least one teae ( apf530 arm , 93.5% ; ondansetron arm , 91.1% ) . serious teaes were experienced by 4.1% of the patients in the apf530 arm and 2.0% of the patients in the ondansetron arm ; no teaes led to death . the magic trial compared apf530 versus ondansetron in the context of a guideline - recommended three - drug regimen in cinv prevention following hec regimens , including ac . the use of ondansetron was appropriate because it has been used in pivotal cinv studies as the active comparator for other 5-ht3 ras22,23 and is indicated for the prevention of nausea and vomiting associated with initial and repeat courses of emetogenic cancer chemotherapy , including high - dose cisplatin.24 no previous pivotal efficacy trial involving patients receiving ac - based regimens has compared two 5-ht3 ras within a three - drug regimen . in contrast to other hec , ac - based regimens are administered mostly to younger female patients ; both female sex and young age constitute patient - related cinv risk factors.30,31 accordingly , in the magic trial , most patients receiving ac - based regimens were female and aged < 55 years , and so at higher risk for cinv.31,32 the interaction of high emetogenicity of ac - based regimens with patient - related risk factors highlights the distinct cinv prevention needs in this patient group . in two large phase iii trials , cr rates were lower in patients receiving ac - based than in those receiving non ac - based regimens across all phases , suggesting greater cinv prevention challenges associated with ac - based regimens.13,33 furthermore , patients receiving hec or mec regimens continue to experience delayed - phase cinv , highlighting the need for new therapies.34 although this exploratory post hoc ac subgroup analysis was not powered for statistical significance , trends favoring the apf530 versus the ondansetron arm in delayed and overall phases were observed in multiple measures of cinv control : cr , cc , tr , rescue medication use , and proportion of patients with no nausea . the biochronomer technology underlying apf530 provides sustained therapeutic granisetron concentrations across 5 days following a single apf530 injection and was expected to provide better delayed - phase cinv control than ondansetron in the magic trial . acknowledging the limits of cross - trial comparisons , the control arm of the magic ac subgroup showed results consistent with those from a previous study in patients with breast cancer who received ac - based regimens.15 similar to findings in the entire study population , apf530 was generally well tolerated in this patient subgroup receiving ac - based hec , and no new safety signals were observed.25 the incidences of isrs were similar in both the arms , and most resolved by the end of the study . consequently , the fda approved apf530 for the prevention of both acute and delayed cinv following initial and repeat courses of mec or ac combination regimens.21 the findings of the present study from the ac subgroup are concordant with the superior delayed - phase cr rate observed in the entire magic study population.25 the superiority of apf530 versus ondansetron , in the presence of an nk-1 ra and dexamethasone , in achieving delayed - phase cr in the magic trial patients suggests that apf530 provided benefit in addition to that of the nk-1 ra . overall , these results demonstrate the benefit of the extended - release design of the apf530 formulation , whereby a single sc dose provides therapeutic granisetron concentrations for 5 days.18 limitations of this analysis include being an exploratory post hoc analysis with a relatively small number of patients in each arm . in addition , use of the double - dummy design resulted in an increased incidence of isrs in the ondansetron arm due to the presence of the viscous teg - poe vehicle in the apf530 placebo injection . in a recently published randomized , double - blind phase iii trial , olanzapine was compared with placebo , when added to a three - drug regimen of 5-ht3 ra , nk-1 ra , and dexamethasone in patients receiving cisplatin or ac - based hec.36 in that study , the addition of olanzapine resulted in a significant improvement in nausea control . it will therefore be of interest to determine whether the addition of olanzapine to a three - drug regimen of apf530 may similarly further improve cinv control in patients receiving ac - based regimens . the magic trial demonstrated the superiority of apf530 versus ondansetron , each in a guideline - recommended three - drug regimen with fosaprepitant and dexamethasone , in the prevention of delayed - phase cinv following hec . the findings of this post hoc subgroup analysis in patients receiving ac - based hec and at a high risk of experiencing cinv are consistent with the results from the entire study population , particularly in control of delayed cinv . the apf530 regimen was generally well tolerated in the ac subgroup , as in the entire population . apf530 , in conjunction with other anti - emetics , is approved for preventing cinv in both acute and delayed phases in patients receiving initial or repeat courses of mec or ac - based regimens.21 thus , apf530 may be a convenient antiemetic option for female patients with breast cancer receiving ac - based chemotherapy , a population in which preventing nausea and vomiting is particularly difficult .

few countries have traditionally regarded alcohol as a public health issue to the same extent as the nordic countries . as a result , the alcohol policies of particularly sweden , finland and norway have been very restrictive , the goal being to limit not only alcohol consumption among problem drinkers but also the overall level of drinking . the most important tools for keeping alcohol consumption ( total volume consumed ) at a low level have been extensive restrictions on physical availability and high alcohol prices . to make these tools efficient , sweden has also applied strict restrictions on the amount of alcohol that can be brought back privately from abroad , but these restrictions have been repealed since sweden joined the european union ( eu ) in 1995 ( table 1 ) . table 1changes in swedish alcohol quotas from other eu countries since membership in the eutime periodspritsfortified winetable winestrong beer1 january 1995 - 1 l spirits or 3 l fortified wine5 l15 l1 july 2000 - 1 l3 l20 l24 l1 january 2001 - 1 l6 l26 l32 l1 january 2002 - 2 l6 l26 l32 l1 january 2003 - 5 l6 l52 l64 l1 january 2004- ( eu - quota)free import for personal use ( indicative level : 10 l)free import for personal use ( indicative level : 20 l)free import for personal use ( indicative level : 90 l)free import for personal use ( indicative level : 110 l)source : commissions of the board 2000/44/eg , ministry of finance and the website of swedish customs ( http://www.tullverket.se ) . table was first published in leifman and gustafsson.quotas refer to the amount ( in litres ) that can be brought into sweden by travellers aged 20 years without any swedish alcohol tax being paid . changes in swedish alcohol quotas from other eu countries since membership in the eu source : commissions of the board 2000/44/eg , ministry of finance and the website of swedish customs ( http://www.tullverket.se ) . quotas refer to the amount ( in litres ) that can be brought into sweden by travellers aged 20 years without any swedish alcohol tax being paid . with higher import quotas , raising alcohol taxes was no longer regarded as efficient owing to the risk of substitution effects , i.e. more alcohol would be purchased abroad instead . furthermore , availability was increasing in practice , especially among residents in southern sweden who live close to low - tax countries like germany and denmark , from which most of the privately imported alcohol comes . earlier studies have also shown that especially southern sweden has a high rate of private imports and that such imports account for almost half of all alcohol consumed in this part of sweden . therefore , it was assumed that the amount of alcohol consumed in this region would increase when denmark , on 1 october 2003 , decreased its tax on spirits by 45%which was equivalent to a 25% decrease in the price of the cheaper brands of spirits and when sweden shortly thereafter , on 1 january 2004 , increased its alcohol import quotas to the levels required by the eu ( i.e. indicative levels of when import can be considered to be for one s own use ) . thus , although the decrease in price did not actually take place in sweden , the already high market share for privately imported alcohol in southern sweden and the short distance to denmark suggested that the changes were equivalent to a price reduction in southern sweden . a recent study based on large repeated cross - sectional surveys was able to establish that travellers imports did increase substantially in southern sweden in 2004 . however , surveys measuring actual drinking did not find any increase in alcohol consumption or alcohol - related social problems . for instance , a comparison between 2003 and 2004 did not show any increase in volume of alcohol consumed in any region in sweden , neither in the repeated cross - sectional samples for the total population nor in the longitudinal data . a study using a longer follow - up period did not change this conclusion , and no change was found in southern sweden when data up until 2006 were analysed . in accordance with the results on consumption , alcohol - related social problems in southern sweden did not increase as expected . thus , the results from population surveys did not verify that alcohol consumption increased following these changes in alcohol policy . as these unexpected results are based on survey data , with their limitations ( i.e. in reaching heavy drinkers ) , further analyses on data less affected by such problems ( i.e. various forms of register data ) are warranted . in the present study , the number of hospital admissions for acute alcohol poisonings and the number of police - reported violent assaults and drunk - driving incidents were used to assess the impact of lower taxes in denmark and higher swedish import quotas . analyses from other nordic countries suggest that an analysis of this kind of register data can give new insights compared with studies based on survey data . in finland , the raised quotas for private alcohol import were shown to have a great effect on heavier drinkers , among whom there was a striking increase in alcohol - related deaths between 2003 and 2004 . herttua , mkel and martikainen studied the effect of the reduction in price following the tax decrease in finland in 2004 and also found an increase in alcohol - related mortality ( mainly for chronic causes and particularly for liver diseases ) after the change . in a study using interrupted time - series analysis , koski et al . found an increase in alcohol - positive sudden deaths related to the tax cut on alcohol . other sources of information in finland have supported these findings as well , stating that substance abusers have been shown to be in poorer health after the changes and that intoxicated persons have been taken into custody more often . as for denmark , time - series analysis performed on violent assaults and hospitalizations for acute alcohol intoxication from january 2000 through december 2005 showed an increase in hospitalizations due to acute alcohol intoxication after the danish tax reduction in 2003 among individuals 15 years of age , but did not find such an increase in the total population or for violent assaults . for sweden , no study using register data has yet been carried out to examine the effects on alcohol - related harm of the changes in private import quotas or the reduction in the danish alcohol tax in 2003 . according to a study of the relatively modest increase in travellers allowances of wine and beer in 1995 , no increases in alcohol - related mortality , traffic accidents or reported criminal violence were found . the aim of this article is thus to study whether the abolishment of alcohol import quotas in 2004 and the danish reduction in spirits tax 3 months earlier had any effect on alcohol - related harm in southern sweden , using register data on alcohol poisonings , drunk driving and assaults as indicators of alcohol - related harm ( more details are presented in the methods section ) . as the hospitalization data on alcohol poisonings are available by age , we will also test the possibility of age - specific effects in order to replicate a similar study based on danish data . our starting point is that the effect may be stronger in age groups , mainly middle - aged people , known to travel and import most of the alcohol . furthermore , a recent analysis of the impact of the increasing allowances and tax reduction in finland suggested that the increase in alcohol - related mortality was basically seen in the age group 5069 years . the selection of alcohol poisoning , drunk driving and assault as indicators of increasing harm in the analysis was justified by the following arguments . alcohol poisonings and drunk driving are by definition alcohol - related , and it is well established that a high proportion of perpetrators as well as victims of violent assaults are intoxicated . in addition , all three indicators have been found to have a positive association with sales at systembolaget [ the swedish alcohol retail monopoly stores ] as well as with estimates of travellers alcohol imports in southern sweden . furthermore , data are available on a monthly basis , thus providing a sufficient number of data points for a meaningful time - series analysis as well as allowing us to come as close as possible in time to the specific changes . it was also considered important to have indicators that respond rapidly to an increase in drinking , thus ensuring that the problem of lagged effects will play a minor role . alcohol poisonings were measured as hospitalizations in which alcohol poisoning was the main or secondary diagnosis . these data are recorded in the swedish hospital discharge records and cover all public in - patient care in sweden ( for more information on this register , see http://www.sos.se/epc/english/pareng.htm ) . the validity of the data is considered to be high , and the quality is continuously being evaluated . the selection of icd codes ( 10th revision ) that represent a case of alcohol poisoning was guided by the criteria set up by the swedish board of health and welfare and includes statistics on alcohol - related crimes were collected from the swedish national council for crime prevention ( br ) . police - reported assaults include deadly violence , attempted murder , assaults and rape including aggravated cases . data on police - reported drunk driving offences do not include driving while under the influence of drugs . it should be noted that some of these crimes do not lead to a sentence ; this share accounts for only a few percentage points each year . as noted in the descriptive figure for violence ( figure 2 ) , there was a peak in june to july 2001 in southern sweden that was caused by riots in gothenburg ( southern sweden ) in relation to an eu meeting at this time . the series was therefore corrected using the linear interpolation command in statistical package for social sciences , version 16.0 , for these months . monthly data covering the period january 2000 until december 2007 were analysed using interrupted time - series analysis , more specifically the technique developed by box and jenkins , often referred to as arima models , in this case arima impact analyses . intervention components ( dummy variables ) were constructed to test whether there was any impact of the two interventions that could not be accounted for by normal fluctuations and long - term trends within the harm series . the first dummy took the value 0 before the tax change and 1 afterwards , and similarly the second dummy took the value 0 before the quota change and 1 afterwards . the final model controlled for changes in harm in northern sweden as well as for two earlier changes in private import quotas of alcohol on 1 january 2002 and 2003 ( table 1 ) using dummies computed as for the other quota dummy in arima modelling , data need to be stationary , which was assessed using the dickey fuller test . most series showed seasonal peaking ( every 12th lag ) with gradual attenuation , which indicates that the series requires seasonal differencing for stationarity . with a seasonal differencing of yt , defined as yt yt12 , the relationship between the changes in the same month but during the following years are analysed rather than the relationship between the raw series yt and yt + 1 , one month compared with the following month during the same year . furthermore , the method requires that the noise term ( including other etiological factors ) be allowed to have a temporal structure . the structure of the noise term ( n ) was modelled and estimated in terms of the autoregressive parameters ( ars ) or moving average parameters ( mas ) . the model and noise specification are specified by the expression ( p , d , q ) ( p , d , q ) , the first parenthesis referring to the regular noise modelling and the second to the seasonality component . the order of the ars and mas is indicated by p and q , respectively , and the order of differencing is indicated by d. the corresponding symbols for the seasonal parameters are p , d and q. the residuals of the estimated model should be white noise , which means that there is no structure of autocorrelation in the noise term . other factors , in addition to the danish tax cut and the increasing import quotas , are likely to affect both alcohol consumption and harm rates in southern sweden , i.e. general trends in purchasing power and travelling habits in sweden . there may also exist national trends in police enforcement and health policies that affected the selected harm indicators . therefore , we used corresponding harm rates in northern sweden as controls in our analysis , the assumption being that the long distance to the danish and german borders would mean little effect of the reforms and that we would still capture the impact of other relevant factors . this idea was supported in separate analyses by site ( without using a control site ) , where no effects of the interventions were found for northern sweden ( results not shown ) . it should be noted , however , that the findings for southern sweden were little affected by the inclusion of northern sweden as a control in the analysis and that the main results remained without this control variable ( analysis not reported here ) . the following specification was used in the final model : where ht symbolizes the harm indicator in the southern site at time t , i.e. by month . this means that the estimates of b1 b5 can be expressed as change in percentage by applying the formula : 100[exp(b ) 1 ] . t is the dummy variable for the danish tax change in 2003 , and q1q3 represents the swedish quota change in 2004 and the two previous quota changes in sweden in 2002 and 2003 . the graphs ( figures 13 ) reveal an increase in alcohol poisonings as well as in the number of reported violent crimes in both northern and southern sweden , but no obvious increase in the number of drunk - driving cases for the period january 2000 until december 2007 . with respect to alcohol poisonings , the development during the pre - intervention period was similar in both regions , but between 2003 and 2006 , the rates increased more in southern sweden than in the northern regions . in 2007 , however , the rates were once again more similar . figure 1descriptive figure over the trends in number of alcohol poisonings in southern and northern sweden . figure covers the period january 2000 to december 2007 figure 2descriptive figure over the trends in number of reported violent assaults in southern and northern sweden ( for south , this original series was corrected with linear interpolation ) . figure covers the period january 2000 to december 2007 figure 3descriptive figure over the trends in number of reported drunk driving in southern and northern sweden . figure covers the period january 2000 to december 2007 descriptive figure over the trends in number of alcohol poisonings in southern and northern sweden . figure covers the period january 2000 to december 2007 descriptive figure over the trends in number of reported violent assaults in southern and northern sweden ( for south , this original series was corrected with linear interpolation ) . figure covers the period january 2000 to december 2007 descriptive figure over the trends in number of reported drunk driving in southern and northern sweden . figure covers the period january 2000 to december 2007 to test whether the two interventions had any effect on these kinds of harm , a series of interrupted time - series analyses were performed . the findings from the final model controlling for harm in northern sweden and earlier quota changes are presented in table 2 . table 2estimated effects of danish tax - cut in october 2003 and increased alcohol quotas in january 2004 on alcohol poisonings , violent crimes and drunk drivingoutputestimatesep - value95% cibox - youngp - valuealcohol poisoning(1,0,0 ) ( 1,1,0,12)6.150.91tax change 030.0310.0440.4800.0550.118quota change 040.100**0.0390.0090.0250.176violent crime(0,0,0 ) ( 1,1,0,12)14.290.28tax change 030.0370.0310.2370.0980.024quota change 040.0250.0260.3470.0270.076drunk driving(0,0,0 ) ( 0,1,1,12)10.800.55tax change 030.0090.0490.8560.1060.088quota change 040.0310.0490.5290.0650.126alcohol poisoning 1019 years(0,0,0 ) ( 0,1,1,12)7.920.79 tax change 030.0000.1450.9980.2850.284 quota change 040.1380.1440.3380.1440.421 2029 years(0,0,0)14.080.30 tax change 030.0760.1820.6750.4340.281 quota change 040.1210.1770.4930.2260.469 3049 years(0,0,1 ) ( 1,1,0,12)17.230.14 tax change 030.1060.0920.2490.0740.285 quota change 040.0500.0850.5560.1170.218 5069 years(0,0,0 ) ( 1,1,0,12)11.170.51 tax change 030.0130.0620.8390.1100.135 quota change 040.122 * 0.0580.0370.0080.236 70 years(1,0,0)13.450.34 tax change 030.2860.3550.4210.4110.982 quota change 040.0110.3490.9750.6720.694ci : confidence interval ; se : standard error . semi - logarithmic arima models estimated on monthly data january 2000 to december 2007 for southern sweden , controlling for changes in the harm rates in northern sweden and quota changes of 2002 and 2003.for alcohol poisonings , trends are also estimated by age.*p < 0.05.**p < 0.01 . estimated effects of danish tax - cut in october 2003 and increased alcohol quotas in january 2004 on alcohol poisonings , violent crimes and drunk driving ci : confidence interval ; se : standard error . semi - logarithmic arima models estimated on monthly data january 2000 to december 2007 for southern sweden , controlling for changes in the harm rates in northern sweden and quota changes of 2002 and 2003 . for alcohol poisonings , the only effect revealed in all these arima models was the effect of the traveller s allowance change in 2004 on alcohol poisonings in southern sweden . after applying the conversion formula to the estimate of 0.10 in the final model , we note that the intervention effect of the increased import quotas was followed by a 10.5% increase in the number of hospitalizations due to alcohol poisoning . however , the tax decrease in denmark was not shown to have affected the different kinds of harm , and none of the interventions had any impact on violent crime or drunk driving . a further analysis showed that there was a strong correlation ( rxy = 0.76 ) between the estimates of the quota change and the tax decrease in southern sweden , which produced uncertain results when both variables were estimated in the same model . thus , we can not rule out that the tax decrease , too , was part of the effect found for the increase in private import quotas of alcohol . an increase in alcohol poisonings due to the change in the traveller s allowance in 2004 was not observed among all age groups ( table 2 ) , but was in fact only observed among those aged 5069 years . the changes in quotas implied a 13% increase in alcohol poisonings in southern sweden in this group . all models were satisfactory with respect to the residual autocorrelation ; thus the auto correlations were close to zero , indicating that the time series are random ( white noise ) . it should be noted that the semi - logarithmic model specification applied here assumes multiplicative effects of the two policy changes . as this may not necessarily be the case , the analyses were also performed using linear models , which , however , did not change the outcome . the danish decrease in spirits tax in 2003 and the abolishment of swedish alcohol import quotas on alcohol in 2004 suggested that alcohol consumption and alcohol - related harm would increase , especially in the southern parts of sweden . this expectation has not been verified in previous studies using survey data , and the aim of the present study was to investigate the same issue by analysing various register data using an interrupted time - series approach . hospitalization for acute alcohol poisoning and police - reported cases of drunk driving and violent assaults were used as indicators of alcohol - related harm . overall , the results of the present analyses did not allow for any definite conclusion as to whether alcohol - related harm increased in southern sweden . on the one hand , an increase in acute alcohol poisonings was found after the increase in import quotas , particularly among those aged 5069 years , but on the other hand , no increase was found in violent assaults or in drunk driving . thus , the findings with respect to assaults and drunk driving supported previous findings based on survey data and the conclusion that problems had not increased , whereas the increase in alcohol - related harm ( alcohol poisonings ) in southern sweden suggests that some problems may have worsened , at least among middle - aged people in southern sweden . one interpretation of the results is simply that some problems increased whereas some did not and that these harm indicators measure specific problems rather than alcohol - related harm in general . thus , drunk driving and alcohol - related violence were not affected by the increase in travellers imports allowances , because these crimes are primarily committed by younger people who are less affected by lower import quotas . only about 10 and 20% of the perpetrators of assault and drunk drivers are > 50 years of age , whereas the corresponding figure for those hospitalized for alcohol poisoning is 50% ( according to data from the swedish council for crime prevention ) . thus , the finding that the travellers imports of alcohol are most common among people > 50 years of age could explain some part of the difference , although further studies are needed to test this idea . it is also worth noting that there are some similarities with finnish experiences related to their abolishment of import quotas and reduction of alcohol taxes in 2004 . an analysis by herttua found no impact on assaults after the changes , but an increase in alcohol - related hospitalizations , which was largest in the age group 5069 years . thus the increases in both countries can be linked to older heavy drinkers , who are known to be less well represented in surveys . our findings also have some similarities with a study from denmark using the same time - series approach . this study examined changes in alcohol - related harm in denmark between 2003 and 2005 after changes in alcohol policies were introduced and found no increase in violent assaults . on the other hand , an increase in alcohol poisonings was revealed but only among those aged 15 years . although the overall findings from these countries indicate an increase in harms , the results vary by subgroup , suggesting that the effects of this kind of increase in availability may be influenced by cultural differences and that findings for one country should not automatically be generalized to other countries . in this specific case , it is also important to consider that the danish and finnish changes were domestic , whereas in sweden a trip abroad was required to take advantage of the new possibilities to buy cheaper alcohol . naturally , one major limitation of this kind of study , which uses a quasi - experimental design , is that the intervention and control groups have not been randomly selected . thus , there may have been large systematic initial differences between southern and northern sweden , meaning that the latter was a poor control site . the higher level of drinking and harm in the southern site are two possible examples . however , in the present study , we had to identify a region affected to a lesser degree by the major interventions of interest : the danish tax decrease and the abolishment of import quotas . although certainly not a perfect control area , the northern parts of sweden were the best choice available for fulfilling these criteria and , as previously mentioned , no effect on harm in northern sweden could be established in relation to these reforms . in conclusion , because previous survey analyses have not revealed any increase in alcohol - related problems in southern sweden , the present findings highlight the importance of using various data sources when studying policy changes and their potential impacts on alcohol - related harm . it also stresses the fact that various data sources are able to cover different segments of the population and that choosing one over the other may result in missing an existing effect . additionally , the results presented here raise important questions about the association between changes in availability and alcohol - related harms in sweden today . us national institute on alcohol abuse and alcoholism [ r01 aa014879 ] , as part of the study effects of major changes in alcohol availability . conflict of interest : none declared . key messageswhen denmark decreased their tax on spirits and sweden shortly thereafter abolished its private import quotas on alcohol to unlimited levels if it was for private consumption , it was assumed that people in the southern parts of sweden would increase their imports of alcohol . because private imports already corresponded to 50% of the total alcohol consumption among swedes in the south , it was expected that per capita alcohol consumption in this region would increase , and thus also the alcohol - related harms.the present results in this study did not allow for any definite conclusion regarding whether alcohol - related harm increased in southern sweden . using an interrupted time - series analysis , an increase in alcohol poisonings was found in relation to the repeal of the alcohol import quotas , but not in relation to the danish tax decrease . similarly , no increase was found in violent assaults or drunk driving.it is concluded that the results raise important questions about the association between changes in availability and alcohol - related harms in sweden today . when denmark decreased their tax on spirits and sweden shortly thereafter abolished its private import quotas on alcohol to unlimited levels if it was for private consumption , it was assumed that people in the southern parts of sweden would increase their imports of alcohol . because private imports already corresponded to 50% of the total alcohol consumption among swedes in the south , it was expected that per capita alcohol consumption in this region would increase , and thus also the alcohol - related harms.the present results in this study did not allow for any definite conclusion regarding whether alcohol - related harm increased in southern sweden . using an interrupted time - series analysis , an increase in alcohol poisonings was found in relation to the repeal of the alcohol import quotas , but not in relation to the danish tax decrease . similarly , no increase was found in violent assaults or drunk driving.it is concluded that the results raise important questions about the association between changes in availability and alcohol - related harms in sweden today . when denmark decreased their tax on spirits and sweden shortly thereafter abolished its private import quotas on alcohol to unlimited levels if it was for private consumption , it was assumed that people in the southern parts of sweden would increase their imports of alcohol . because private imports already corresponded to 50% of the total alcohol consumption among swedes in the south , it was expected that per capita alcohol consumption in this region would increase , and thus also the alcohol - related harms . the present results in this study did not allow for any definite conclusion regarding whether alcohol - related harm increased in southern sweden . using an interrupted time - series analysis , an increase in alcohol poisonings was found in relation to the repeal of the alcohol import quotas , but not in relation to the danish tax decrease . it is concluded that the results raise important questions about the association between changes in availability and alcohol - related harms in sweden today .

background denmark decreased its tax on spirits by 45% on 1 october 2003 . shortly thereafter , on 1 january 2004 , sweden increased its import quotas of privately imported alcohol , allowing travellers to bring in much larger amounts of alcohol from other european union countries . although these changes were assumed to increase alcohol - related harm in sweden , particularly among people living close to denmark , analyses based on survey data collected before and after these changes have not supported this assumption . the present article tests whether alcohol - related harm in southern sweden was affected by these changes by analysing other indicators of alcohol - related harm , e.g. harm recorded in different kinds of registers.methods interrupted time - series analysis was performed with monthly data on cases of hospitalization due to acute alcohol poisoning , number of reported violent assaults and drunk driving for the years 200007 in southern sweden using the northern parts of sweden as a control and additionally controlling for two earlier major changes in quotas.results the findings were not consistent with respect to whether alcohol - related harm increased in southern sweden after the decrease in danish spirits tax and the increase in swedish alcohol import quotas . on the one hand , an increase in acute alcohol poisonings was found , particularly in the 5069 years age group , on the other hand , no increase was found in violent assaults and drunk driving.conclusions the present results raise important questions about the association between changes in availability and alcohol - related harms . more research using other methodological approaches and data is needed to obtain a comprehensive picture of what actually happened in southern sweden .

Introduction Methods Results Discussion Funding

to make these tools efficient , sweden has also applied strict restrictions on the amount of alcohol that can be brought back privately from abroad , but these restrictions have been repealed since sweden joined the european union ( eu ) in 1995 ( table 1 ) . table 1changes in swedish alcohol quotas from other eu countries since membership in the eutime periodspritsfortified winetable winestrong beer1 january 1995 - 1 l spirits or 3 l fortified wine5 l15 l1 july 2000 - 1 l3 l20 l24 l1 january 2001 - 1 l6 l26 l32 l1 january 2002 - 2 l6 l26 l32 l1 january 2003 - 5 l6 l52 l64 l1 january 2004- ( eu - quota)free import for personal use ( indicative level : 10 l)free import for personal use ( indicative level : 20 l)free import for personal use ( indicative level : 90 l)free import for personal use ( indicative level : 110 l)source : commissions of the board 2000/44/eg , ministry of finance and the website of swedish customs ( http://www.tullverket.se ) . changes in swedish alcohol quotas from other eu countries since membership in the eu source : commissions of the board 2000/44/eg , ministry of finance and the website of swedish customs ( http://www.tullverket.se ) . furthermore , availability was increasing in practice , especially among residents in southern sweden who live close to low - tax countries like germany and denmark , from which most of the privately imported alcohol comes . therefore , it was assumed that the amount of alcohol consumed in this region would increase when denmark , on 1 october 2003 , decreased its tax on spirits by 45%which was equivalent to a 25% decrease in the price of the cheaper brands of spirits and when sweden shortly thereafter , on 1 january 2004 , increased its alcohol import quotas to the levels required by the eu ( i.e. thus , although the decrease in price did not actually take place in sweden , the already high market share for privately imported alcohol in southern sweden and the short distance to denmark suggested that the changes were equivalent to a price reduction in southern sweden . however , surveys measuring actual drinking did not find any increase in alcohol consumption or alcohol - related social problems . for instance , a comparison between 2003 and 2004 did not show any increase in volume of alcohol consumed in any region in sweden , neither in the repeated cross - sectional samples for the total population nor in the longitudinal data . a study using a longer follow - up period did not change this conclusion , and no change was found in southern sweden when data up until 2006 were analysed . in accordance with the results on consumption , alcohol - related social problems in southern sweden did not increase as expected . as these unexpected results are based on survey data , with their limitations ( i.e. in the present study , the number of hospital admissions for acute alcohol poisonings and the number of police - reported violent assaults and drunk - driving incidents were used to assess the impact of lower taxes in denmark and higher swedish import quotas . analyses from other nordic countries suggest that an analysis of this kind of register data can give new insights compared with studies based on survey data . in finland , the raised quotas for private alcohol import were shown to have a great effect on heavier drinkers , among whom there was a striking increase in alcohol - related deaths between 2003 and 2004 . herttua , mkel and martikainen studied the effect of the reduction in price following the tax decrease in finland in 2004 and also found an increase in alcohol - related mortality ( mainly for chronic causes and particularly for liver diseases ) after the change . in a study using interrupted time - series analysis , koski et al . found an increase in alcohol - positive sudden deaths related to the tax cut on alcohol . as for denmark , time - series analysis performed on violent assaults and hospitalizations for acute alcohol intoxication from january 2000 through december 2005 showed an increase in hospitalizations due to acute alcohol intoxication after the danish tax reduction in 2003 among individuals 15 years of age , but did not find such an increase in the total population or for violent assaults . for sweden , no study using register data has yet been carried out to examine the effects on alcohol - related harm of the changes in private import quotas or the reduction in the danish alcohol tax in 2003 . according to a study of the relatively modest increase in travellers allowances of wine and beer in 1995 , no increases in alcohol - related mortality , traffic accidents or reported criminal violence were found . the aim of this article is thus to study whether the abolishment of alcohol import quotas in 2004 and the danish reduction in spirits tax 3 months earlier had any effect on alcohol - related harm in southern sweden , using register data on alcohol poisonings , drunk driving and assaults as indicators of alcohol - related harm ( more details are presented in the methods section ) . furthermore , a recent analysis of the impact of the increasing allowances and tax reduction in finland suggested that the increase in alcohol - related mortality was basically seen in the age group 5069 years . the selection of alcohol poisoning , drunk driving and assault as indicators of increasing harm in the analysis was justified by the following arguments . alcohol poisonings and drunk driving are by definition alcohol - related , and it is well established that a high proportion of perpetrators as well as victims of violent assaults are intoxicated . in addition , all three indicators have been found to have a positive association with sales at systembolaget [ the swedish alcohol retail monopoly stores ] as well as with estimates of travellers alcohol imports in southern sweden . furthermore , data are available on a monthly basis , thus providing a sufficient number of data points for a meaningful time - series analysis as well as allowing us to come as close as possible in time to the specific changes . these data are recorded in the swedish hospital discharge records and cover all public in - patient care in sweden ( for more information on this register , see http://www.sos.se/epc/english/pareng.htm ) . the selection of icd codes ( 10th revision ) that represent a case of alcohol poisoning was guided by the criteria set up by the swedish board of health and welfare and includes statistics on alcohol - related crimes were collected from the swedish national council for crime prevention ( br ) . monthly data covering the period january 2000 until december 2007 were analysed using interrupted time - series analysis , more specifically the technique developed by box and jenkins , often referred to as arima models , in this case arima impact analyses . the final model controlled for changes in harm in northern sweden as well as for two earlier changes in private import quotas of alcohol on 1 january 2002 and 2003 ( table 1 ) using dummies computed as for the other quota dummy in arima modelling , data need to be stationary , which was assessed using the dickey fuller test . other factors , in addition to the danish tax cut and the increasing import quotas , are likely to affect both alcohol consumption and harm rates in southern sweden , i.e. it should be noted , however , that the findings for southern sweden were little affected by the inclusion of northern sweden as a control in the analysis and that the main results remained without this control variable ( analysis not reported here ) . t is the dummy variable for the danish tax change in 2003 , and q1q3 represents the swedish quota change in 2004 and the two previous quota changes in sweden in 2002 and 2003 . the graphs ( figures 13 ) reveal an increase in alcohol poisonings as well as in the number of reported violent crimes in both northern and southern sweden , but no obvious increase in the number of drunk - driving cases for the period january 2000 until december 2007 . with respect to alcohol poisonings , the development during the pre - intervention period was similar in both regions , but between 2003 and 2006 , the rates increased more in southern sweden than in the northern regions . figure 1descriptive figure over the trends in number of alcohol poisonings in southern and northern sweden . figure covers the period january 2000 to december 2007 figure 2descriptive figure over the trends in number of reported violent assaults in southern and northern sweden ( for south , this original series was corrected with linear interpolation ) . figure covers the period january 2000 to december 2007 figure 3descriptive figure over the trends in number of reported drunk driving in southern and northern sweden . figure covers the period january 2000 to december 2007 descriptive figure over the trends in number of alcohol poisonings in southern and northern sweden . figure covers the period january 2000 to december 2007 descriptive figure over the trends in number of reported violent assaults in southern and northern sweden ( for south , this original series was corrected with linear interpolation ) . figure covers the period january 2000 to december 2007 descriptive figure over the trends in number of reported drunk driving in southern and northern sweden . figure covers the period january 2000 to december 2007 to test whether the two interventions had any effect on these kinds of harm , a series of interrupted time - series analyses were performed . the findings from the final model controlling for harm in northern sweden and earlier quota changes are presented in table 2 . table 2estimated effects of danish tax - cut in october 2003 and increased alcohol quotas in january 2004 on alcohol poisonings , violent crimes and drunk drivingoutputestimatesep - value95% cibox - youngp - valuealcohol poisoning(1,0,0 ) ( 1,1,0,12)6.150.91tax change 030.0310.0440.4800.0550.118quota change 040.100**0.0390.0090.0250.176violent crime(0,0,0 ) ( 1,1,0,12)14.290.28tax change 030.0370.0310.2370.0980.024quota change 040.0250.0260.3470.0270.076drunk driving(0,0,0 ) ( 0,1,1,12)10.800.55tax change 030.0090.0490.8560.1060.088quota change 040.0310.0490.5290.0650.126alcohol poisoning 1019 years(0,0,0 ) ( 0,1,1,12)7.920.79 tax change 030.0000.1450.9980.2850.284 quota change 040.1380.1440.3380.1440.421 2029 years(0,0,0)14.080.30 tax change 030.0760.1820.6750.4340.281 quota change 040.1210.1770.4930.2260.469 3049 years(0,0,1 ) ( 1,1,0,12)17.230.14 tax change 030.1060.0920.2490.0740.285 quota change 040.0500.0850.5560.1170.218 5069 years(0,0,0 ) ( 1,1,0,12)11.170.51 tax change 030.0130.0620.8390.1100.135 quota change 040.122 * 0.0580.0370.0080.236 70 years(1,0,0)13.450.34 tax change 030.2860.3550.4210.4110.982 quota change 040.0110.3490.9750.6720.694ci : confidence interval ; se : standard error . semi - logarithmic arima models estimated on monthly data january 2000 to december 2007 for southern sweden , controlling for changes in the harm rates in northern sweden and quota changes of 2002 and 2003.for alcohol poisonings , trends are also estimated by age. estimated effects of danish tax - cut in october 2003 and increased alcohol quotas in january 2004 on alcohol poisonings , violent crimes and drunk driving ci : confidence interval ; se : standard error . semi - logarithmic arima models estimated on monthly data january 2000 to december 2007 for southern sweden , controlling for changes in the harm rates in northern sweden and quota changes of 2002 and 2003 . after applying the conversion formula to the estimate of 0.10 in the final model , we note that the intervention effect of the increased import quotas was followed by a 10.5% increase in the number of hospitalizations due to alcohol poisoning . however , the tax decrease in denmark was not shown to have affected the different kinds of harm , and none of the interventions had any impact on violent crime or drunk driving . a further analysis showed that there was a strong correlation ( rxy = 0.76 ) between the estimates of the quota change and the tax decrease in southern sweden , which produced uncertain results when both variables were estimated in the same model . thus , we can not rule out that the tax decrease , too , was part of the effect found for the increase in private import quotas of alcohol . an increase in alcohol poisonings due to the change in the traveller s allowance in 2004 was not observed among all age groups ( table 2 ) , but was in fact only observed among those aged 5069 years . the changes in quotas implied a 13% increase in alcohol poisonings in southern sweden in this group . the danish decrease in spirits tax in 2003 and the abolishment of swedish alcohol import quotas on alcohol in 2004 suggested that alcohol consumption and alcohol - related harm would increase , especially in the southern parts of sweden . this expectation has not been verified in previous studies using survey data , and the aim of the present study was to investigate the same issue by analysing various register data using an interrupted time - series approach . hospitalization for acute alcohol poisoning and police - reported cases of drunk driving and violent assaults were used as indicators of alcohol - related harm . overall , the results of the present analyses did not allow for any definite conclusion as to whether alcohol - related harm increased in southern sweden . on the one hand , an increase in acute alcohol poisonings was found after the increase in import quotas , particularly among those aged 5069 years , but on the other hand , no increase was found in violent assaults or in drunk driving . thus , the findings with respect to assaults and drunk driving supported previous findings based on survey data and the conclusion that problems had not increased , whereas the increase in alcohol - related harm ( alcohol poisonings ) in southern sweden suggests that some problems may have worsened , at least among middle - aged people in southern sweden . one interpretation of the results is simply that some problems increased whereas some did not and that these harm indicators measure specific problems rather than alcohol - related harm in general . thus , drunk driving and alcohol - related violence were not affected by the increase in travellers imports allowances , because these crimes are primarily committed by younger people who are less affected by lower import quotas . an analysis by herttua found no impact on assaults after the changes , but an increase in alcohol - related hospitalizations , which was largest in the age group 5069 years . this study examined changes in alcohol - related harm in denmark between 2003 and 2005 after changes in alcohol policies were introduced and found no increase in violent assaults . on the other hand , an increase in alcohol poisonings was revealed but only among those aged 15 years . although the overall findings from these countries indicate an increase in harms , the results vary by subgroup , suggesting that the effects of this kind of increase in availability may be influenced by cultural differences and that findings for one country should not automatically be generalized to other countries . however , in the present study , we had to identify a region affected to a lesser degree by the major interventions of interest : the danish tax decrease and the abolishment of import quotas . although certainly not a perfect control area , the northern parts of sweden were the best choice available for fulfilling these criteria and , as previously mentioned , no effect on harm in northern sweden could be established in relation to these reforms . in conclusion , because previous survey analyses have not revealed any increase in alcohol - related problems in southern sweden , the present findings highlight the importance of using various data sources when studying policy changes and their potential impacts on alcohol - related harm . additionally , the results presented here raise important questions about the association between changes in availability and alcohol - related harms in sweden today . key messageswhen denmark decreased their tax on spirits and sweden shortly thereafter abolished its private import quotas on alcohol to unlimited levels if it was for private consumption , it was assumed that people in the southern parts of sweden would increase their imports of alcohol . because private imports already corresponded to 50% of the total alcohol consumption among swedes in the south , it was expected that per capita alcohol consumption in this region would increase , and thus also the alcohol - related harms.the present results in this study did not allow for any definite conclusion regarding whether alcohol - related harm increased in southern sweden . using an interrupted time - series analysis , an increase in alcohol poisonings was found in relation to the repeal of the alcohol import quotas , but not in relation to the danish tax decrease . similarly , no increase was found in violent assaults or drunk driving.it is concluded that the results raise important questions about the association between changes in availability and alcohol - related harms in sweden today . when denmark decreased their tax on spirits and sweden shortly thereafter abolished its private import quotas on alcohol to unlimited levels if it was for private consumption , it was assumed that people in the southern parts of sweden would increase their imports of alcohol . because private imports already corresponded to 50% of the total alcohol consumption among swedes in the south , it was expected that per capita alcohol consumption in this region would increase , and thus also the alcohol - related harms.the present results in this study did not allow for any definite conclusion regarding whether alcohol - related harm increased in southern sweden . using an interrupted time - series analysis , an increase in alcohol poisonings was found in relation to the repeal of the alcohol import quotas , but not in relation to the danish tax decrease . similarly , no increase was found in violent assaults or drunk driving.it is concluded that the results raise important questions about the association between changes in availability and alcohol - related harms in sweden today . when denmark decreased their tax on spirits and sweden shortly thereafter abolished its private import quotas on alcohol to unlimited levels if it was for private consumption , it was assumed that people in the southern parts of sweden would increase their imports of alcohol . because private imports already corresponded to 50% of the total alcohol consumption among swedes in the south , it was expected that per capita alcohol consumption in this region would increase , and thus also the alcohol - related harms . the present results in this study did not allow for any definite conclusion regarding whether alcohol - related harm increased in southern sweden . using an interrupted time - series analysis , an increase in alcohol poisonings was found in relation to the repeal of the alcohol import quotas , but not in relation to the danish tax decrease . it is concluded that the results raise important questions about the association between changes in availability and alcohol - related harms in sweden today .

sodium - ion batteries ( nibs ) are of great interest as a complementary technology to lithium - ion batteries in applications such as grid - storage , where cost and sustainability are of greater importance than high energy density . however , graphite , the anode of choice for lithium - ion batteries , shows almost no capacity for sodium , meaning that the identification of suitable alternative anode materials is a major technological challenge . alloying anodes consisting of metals or metalloids that form alloys with sodium are of great interest due to their low operating voltage and high gravimetric capacities , reversible capacities of 480 , 350 , 500 , and 580 mahg having been reported for pb , ge , sn and sb , respectively . the performance of antimony at high rates sets it apart from other alloying materials ; micrometric antimony can retain a capacity of 580 mahg at a rate of c/6 and 520 mahg at a rate of 3/4 c , something that has not been reported for lead , germanium or tin elemental anodes in nibs . despite the advantages of alloying anodes , there are still major issues that need to be addressed if these systems are going to be used in real applications . for example , the large volume expansions that take place on sodiation can lead to harmful mechanical processes and side - reactions that cause capacity fade with cycling . this can be somewhat mediated by careful electrode formulation or nanocompositing the material with carbon . more fundamental , however , is a lack of understanding of the phases that are formed within the battery and the transformation between them , which stems from the formation of highly reactive and often amorphous intermediate phases . structural elucidation of these amorphous phases is challenging for conventional crystallographic methods , especially in complex systems where there is coexistence of amorphous and crystalline phases ; the latter can overwhelm the analysis and obscure information about the relatively weakly scattering amorphous phases . therefore , in order to obtain a full understanding of the electrochemical mechanism it is necessary to develop analyses that are able to extract the structure of these amorphous phases . in the case of antimony , numerous details of the ( de)sodiation processes remain a mystery ; the first sodiation is dominated by a single process at 500 mv , but subsequent ( de)sodiations have strikingly different electrochemical profiles , suggesting that the structural transformations on the first and second sodiation are significantly different . this raises questions about the origin of this effect as well as what the structure of the electrode at the top of charge is , as this effectively becomes the active the intermediate phases that are formed during cycling are also unknown ; x - ray diffraction ( xrd ) studies confirm that crystalline na3sb ( c - na3sb ) is formed at the end of both the first and second sodiation , but other than the formation of this crystalline phase , the electrode is amorphous for large sections of the first sodiation and all of the subsequent ( de)sodiation processes , meaning xrd is unable to probe their structure . there is no clear evidence from xrd that nasb , the only other sodium antimonide in the known na sb phase diagram is formed at any stage in the ( de)sodiation process . probed thin film electrodes in the first sodiation and first desodiation using sb mossbauer spectroscopy and proposed that sb environments similar to those found in nasb are formed during both processes . however , the close similarity between the isomeric shifts of the nasb structure and those arising from a combination of sb and na3sb do not allow for an unequivocal conclusion about the nature of this intermediate phase . given these complexities , it is clear that further structural elucidation is required in the intermediate regions of the ( de)sodiation of antimony , with a focus on techniques that are able to isolate and characterize any amorphous or disordered intermediates and track their interconversion , especially in the presence of additional crystalline phases . pair distribution function ( pdf ) analysis uses bragg scattering and diffuse scattering in parallel , meaning that while any long - range order that is present in the material is taken into account , it is also possible to characterize structures with deviations from long - range order , or phases that lack long - range order altogether . the method has recently found application in characterization of battery electrodes both ex situ and during operation . operando measurements , where scattering data is collected during battery operation , have particular advantages over post - mortem analysis as they increase the consistency between data sets and circumvent issues of air contamination , which can complicate analysis ; this is particularly relevant to the very air sensitive alloy samples considered here . in addition to these factors , operando measurements can capture the formation of metastable species that may form in the battery but may relax before post - mortem analysis is carried out . visual examination of the pdf can give direct structural information , but additional quantitative information can be extracted by using atomistic modeling approaches , such as real - space rietveld , reverse monte carlo methods and empirical potential structure refinements . however , one of the challenges of modeling the pdf of battery systems is the separation of amorphous and crystalline phases when present simultaneously . to obtain unambiguous interpretation , it is necessary to add additional chemical information about the number and nature of phases . furthermore , while x - ray pdf is highly sensitive to the components of the electrode that are strongly scattering in this case meaning that changes to the antimony connectivity during sodium insertion are very distinct , we present a pair distribution function and high - resolution na magic - angle spinning solid - state nuclear magnetic resonance spectroscopy ( mas ssnmr ) study of antimony anodes for sodium - ion batteries . we focus on using in - depth analysis of pdf data , constrained by chemical information from na ssnmr experiments referenced to synthesized model compounds of the two known sodium - antimonide compounds nasb and na3sb , in order to isolate amorphous and crystalline phases present in the electrode , and understand their transformations during the ( de)sodiation process . through this approach , we are able to propose structural origins for each of the distinct electrochemical signatures , and rationalize the different electrochemical profile observed on the second sodiation process . stoichiometric ratios of sb powder ( sigma - aldrich , 99.99% ) and sodium metal ( sigma - aldrich , > 99% ) were ball milled using a hardened stainless steel ball - mill jar in a planetary ball mill pm 100 under ar atmosphere . an active - milling time of 60 min was sufficient to obtain a complete reaction of sb and na . the formation of nasb and na3sb was confirmed using xrd ( see section 5 , supporting information , figure s10 ) . antimony powder ( 200 mesh , 99.99% , sigma - aldrich ) was ball milled under argon for 30 min using a spex 8000 m high - energy ball mill . electrodes were formulated by forming an aqueous slurry using a 70:18:12 ratio of sb , carboxymethylcellulose ( cmc ) ( ds = 0.7 , mw = 250 000 , sigma - aldrich ) , superp carbon ( timcal ) . the slurry was ball - milled in a retsch pm100 plenary ball - mill for 1 h , before being cast on a glass slide using a 150 m doctor blade . the film was dried for 16 h in air , and dried at 110 c under a vacuum for 2 h. ex situ pdf samples were packed into 0.0485 in . diameter kapton capillaries ( cole - parmer ) , and sealed with epoxy in an argon atmosphere . cells were assembled using na - metal ( sigma , > 99% ) as the counter electrode , 1 m napf6 ( 99.5 + , sigma ) in pc ( anhydrous , sigma - aldrich ) as the electrolyte and a glass - fiber separator ( whatman , gf / a ) in an argon atmosphere glovebox . cells were cycled galvanostatically at a rate of c/20 ( based on mass of antimony ) in the range of 2.5 to 0 v , where c/20 corresponds to the insertion of 3 na per sb in 20 h. x - ray total scattering data were collected using 45 min intervals , corresponding to the insertion of 0.1125 na , with an exposure time of 180 s. data were collected for the first sodiation , first desodiation , and second sodiation . the cycling rate and number of discharge x - ray scattering data were collected at 11-id - b at the advanced photon source , argonne national laboratory using an x - ray energy of 86.7 kev ( = 0.1430 ) and an amorphous silicon area detector ( perkinelmer ) to obtain data to large momentum transfer values . data were integrated using the program fit2d , standard corrections ( background , compton scattering , detector effects ) were applied , and the data fourier transformed ( qmax = 18 ) to obtain g(r ) using the software pdfgetx2 . structural models were refined against pdf data using pdfgui , using previously published structures from the icsd database . for the refinement of any crystalline phases present within the electrode , starting values for the structural models were taken from the refinement of model compounds for sb and na3sb . if the r - factor ( rw ) for the refinement was unsatisfactory , an additional minor phase was added and the relative scale parameters were refined . when the minor phase was present in small quantities ( when the scale factor was less than 10% of the scale factor of the phase when present as a single - phase electrode , i.e. , at the start of sodiation for c - sb or the end of sodiation for c - na3sb ) thermal parameters were fixed to values representative of those in regions where the phase was the major constituent of the electrode . a spherical particle envelope was used to model the particle size and/or length scale of ordering in areas where phases were present with limited correlations . rw values from the final refinements are quoted to give an idea of the attainable fit and to allow the competing models to be compared . rw for pdf refinements is generally higher than those typically found an xrd rietveld refinement , which reflects the fact that the functions being fit are different , with g(r ) being more sensitive to the local atomic structure . rw remains , however , useful as a measure of goodness - of - fit when comparing models fit to pdf data . to extract information about additional phases with limited correlation lengths , a refinement for the crystalline phases present was performed at high - r ( 2050 ) using the values for low - r peak sharpening fixed to values determined from refinements where each the crystalline phase was dominant . the refinement was extended to the full r - range ( 250 ) keeping all parameters fixed to examine the contribution of the crystalline phase to the whole pdf . the contribution of amorphous components was assessed from the residual ( g(r)experiment g(r)model ) of this refinement . from the onset of desodiation , the pdfs obtained have a small contribution from sodium metal , due to additional texture on the counter electrode that could not be accounted for during background subtraction . this was modeled as an additional phase with fixed unit cell parameters and thermal parameters . the contribution of this phase to the pdf as a function of sodiation ( figure s15 ) was found to be small in all data sets compared to the contribution from naxsb phases . additional amorphous phases were added to the refinements for the first sodiation process using a model that was refined against data from the second sodiation ( at the end of the d2-c process ) , where the amorphous phase is assumed to be phase - pure . in these multiphase refinements , the sodiation level , defined here as the number of sodium atoms per antimony was calculated from structural data by considering the distribution of antimony between the phases present ( as output from refinements in pdfgui ) and the number of sodiums per antimony in each phase ( e.g. , 0 for c - sb , 3 for na3sb , 2.5 for a - na3xsb ) . for the electrochemical measurements , the number of na per sb was calculated from the current that was passed ; 0.1125 na per sb were added between each data set at the current used . details of the calculations for the sodiation level of naxsb phases are shown in the supporting information . sb / c / cmc films ( 612 mg ) were assembled in 2032 coin cells using a fiber - glass separator ( whatman ) and sodium metal ( sigma - aldrich , > 99% ) as the counter electrode . 1 m napf6 ( sigma - aldrich , > 99.5% ) in pc ( sigma - aldrich , anhydrous ) was used as the electrolyte . cells were cycled at a rate of c/20 using an arbin instruments electrochemical cycler , to points of interest in the electrochemical curve . the batteries were dissembled inside an argon glovebox , and the active material washed with dimethylcarbonate ( sigma - aldrich , anhydrous , > 99% ) in order to remove excess electrolyte from the sample . samples were dried under a vacuum , before being packed into kel - f inserts placed in 4 mm zirconia rotors and closed with kel - f caps inside an argon glovebox . though the formation of decomposition products of naxsb phases ( e.g. , naoh ) was minimized by the careful handling of materials in an argon atmosphere , it is possible that small quantities remain . na solid - state nmr data were collected on a bruker aviii-700 spectrometer working at a na larmor frequency of 185.2 mhz . rotors were spun at magic - angle spinning ( mas ) rates between 5 khz and 10 khz . spectra were acquired using a pulse - acquire sequence , with a 90 pulse duration of 2.75 s and a recycle interval of 5 s. between 2000 and 10 000 transients were coadded for each spectrum , depending on the mass of the sample in the rotor , and level of sodiation . spectra in the plots are normalized with respect to the sample weight and the number of scans used in the experiment . na chemical shifts prior to the study of sb battery anodes , structural characterization was carried out for crystalline nasb and na3sb reference materials . na3sb ( space group 194 , p63/mmc ) is formed of hexagonal layers of alternating na and sb atoms with additional sodium ions residing between the layers ( figure 1 , top left ) . there are two sodium environments in a 1:2 ratio ; sodium within the hexagonal layers sits in a trigonal environment , and sodium between the layers is in a tetrahedral environment . nasb ( space group 14 , p21/c ) is formed of interlinked helical chains of sb and na running parallel to the b - axis ( figure 1 , bottom left ) . ( a ) left : unit cell of na3sb ( top ) and 1 2 1 unit cells of nasb showing the helical chains of antimony ( bottom ) . ( b ) middle : na nmr spectra of synthesized na3sb at 268 k ( top ) , na3sb at 298 k ( middle ) and nasb ( bottom ) , all recorded at 10 khz mas with an external field of 16.4 t. chemical shifts of major isotropic resonances are marked . * = spinning sidebands . ( c ) right : least - squared refinement of na3sb ( top ) and nasb ( bottom ) structures against ex situ pdf data . the difference between the experimental and model pdfs is shown by the black line , offset for clarity . a 7% sb impurity ( by mass ) na nmr spectra for the two model compounds are shown in figure 1 ( middle ) . for na3sb , the spectrum recorded at 298 k shows a sharp feature with a poorly resolved shoulder . in a second spectrum recorded at 268 k , the observed features are resolved into a sharp resonance at 52 ppm together with a clearly defined quadrupolar line shape centered around 65 ppm . fitting of the two resonances yields nmr parameters iso = 52 ppm , cq = 0 mhz , q = 0.15 , ( ascribed to the tetrahedral na1 site between the layers ) and iso = 83 ppm , cq = 4.6 mhz , q = 0.06 ( ascribed to the trigonal na2 site within the layers ) . the fit of simulated peaks to the experimental spectrum for na3sb is shown in figure s12 and table s5 . the large temperature dependence observed in the spectra ( figure s13 ) indicates that there is significant exchange between the two sodium sites in the structure , occurring even at room temperature ; simulations of the na nmr spectra allow the exchange frequency between sodium sites to be estimated as around 12 khz at room temperature . the na nmr spectrum of nasb exhibits two partially resolved features at 18 and 22 ppm . a multiple - quantum mas ( mqmas ) spectrum ( figure s14 ) confirms that these correspond to two sites , consistent with the two crystallographic sites present in the nasb structure , both with very small quadrupolar parameters . the na nmr results demonstrate that the sb - connectivity has a clear effect on the na shift , something that has been observed in lithium - alloy electrodes . in the case of na sb , a change from sb sb chains ( as in nasb ) to units of isolated sb ions ( as in na3sb ) moves the resonances to higher chemical shift . the opposite trend is observed in lithium - alloys ( li si , li ge , li sb ) , highlighting potential differences in the electronic structure of sodium phases . the shifts observed for these reference compounds will be used to help interpret the spectra for electrochemically sodiated naxsb phases . real - space least - squared refinements using the na3sb and nasb structures reported by brauer et al . and cromer et al . show a good match to the experimental pdf data for these compounds indicating that there is a good agreement of the local and long - range structure . of note , is the large value of the u33 thermal displacement parameter obtained from the refinement for the na2 ( table s4 ) , the sodium - ions within the hexagonal layers of na3sb , corresponding to displacements in the c - direction , normal to the hexagonal layers of the material . this implies that there is some disorder in the height / position of the in plane sodium ions , which may come about through the significant site exchange observed by na nmr . in order to assess the contribution of sodium within the cmc binder and conductive carbon to the na nmr spectra for the battery samples , na nmr experiments for the pristine electrode and for sample of super p and cmc in the ratio 40:60 discharged to 0 v with the same effective current as the antimony samples were performed ( figure s16 ) . the cmc binder in the pristine electrode gives a broad resonance between 40 and 20 ppm . the spectra for the discharged super p carbon / cmc sample is dominated by this resonance and by resonances between 0 and 20 ppm from sodium contained in the solid - electrolyte interphase ( sei ) , on the surface of the electrode , and that reacts with the conductive carbon additive , as well as from any residual electrolyte that was not removed by washing . the galvanostatic ( constant current ) electrochemical profile and the differential capacity ( dq / dv ) for antimony , shown in figure 2 , are similar to those reported previously . the first sodiation process is dominated by a flat plateau at around 500 mv ( s1-a ) before a voltage drop to 0 v ( s1-b ) , corresponding to the addition of slightly more than 3 na per sb . during desodiation , approximately 2.5 na per sb are removed from the electrode , corresponding to 82% of the theoretical capacity . the desodiation processes shows a significant voltage hysteresis and features three processes : a distinct plateau at 800 mv ( d1-a ) followed by a sloping process at 850950 mv ( d1-b ) and a sharp rise to 2 v ( d1-c ) . subsequent sodiations show three processes ; a short plateau at 750650 mv ( s2-a ) , a sloping process to 450 mv ( s2-b ) and at least one additional plateau at 450 mv ( s2-c ) , before dropping to 0 v ( s2-d ) . the relatively large sb - particle size means that no significant capacity from the reaction of surface antimony oxides , which is expected to occur primarily above 500 mv , is observed . clean model system for studying only the antimony phase transformations , but is also highly relevant to nanostructured antimony ( e.g. , nanoparticles or sb / c composites ) where similar electrochemical processes are observed beyond the first sodiation . note that the chief difference between nanostructured antimony and bulk antimony in the first ( de)sodiation cycle is the additional irreversible capacity , which can be attributed to increased sei formation on the higher surface area electrodes , and the increased quantities of surface antimony oxides . electrochemical data for subsequent cycles for bulk antimony is similar and has been reported previously . top : ( de)sodiation curves obtained for antimony vs sodium metal cycled at a rate of c/20 in the voltage range of 2.5 to 0 v. the different electrochemical processes are labeled and these labels referred to subsequently in the text . the different electrochemical processes are marked with a notation that is used throughout the subsequent text . ex situ na mas ssnmr provides insight into the sodium local environments as a function of ( de)sodiation ( figure 3 , left ) . at a low sodiation level of 0.6 na per sb the na nmr spectra are dominated by resonances between 40 and 20 ppm from sodium within the binder , the conductive carbon and the sei as discussed in section 3.1 . on further sodiation ( > 1 na per sb ) , an additional broad peak is observed in the na nmr , centered around 37 ppm . it is clear that this shift is different to those observed for both nasb and na3sb , and indicates that the sodium exists in environments where the antimony connectivity is intermediate between nasb and na3sb . during the second half of s1-a , the resonance at 37 ppm grows and shifts gradually on increasing sodiation to 42 ppm . additional weak resonances between 50 and 80 ppm are observed , corresponding to the two sites of na3sb , clearly indicating the coexistence of multiple naxsb phases in this region . during s1-b , the additional phase is converted to c - na3sb ; the 3742 ppm resonance becomes weaker and the c - na3sb resonances dominate the na nmr spectra . the peaks from c - na3sb show a similar broadening to the model na3sb compound indicating that similar na exchange processes are present within the electrode . the final nominal composition of the sample at the end of sodiation ( eos ) is na3.375sb , indicating that 0.375 na has been consumed in side reactions to form the sei . ( a ) ex situ na nmr spectra ( normalized ) of cycled sb electrodes at the states of charge . spectra were recorded at 10 khz mas with an external field of 16.4 t. chemical shifts of major isotropic resonances are marked . the shaded region marks where resonances from sodium within the cmc binder , the sei and the conductive carbon are dominant . * mark spinning sidebands . number ( # ) of sodiums per antimony is labeled next to each spectra , based on the calculations outlined in the supporting information , eos = end of sodiation , eod = end of desodiation . ( b ) discharge charge curves obtained for sb during the in situ pdf measurements . ( c ) selected pdfs obtained during the first discharge , first charge and second discharge cycles by fourier transforming the total scattering x - ray data . the colors of the curves correspond to the colors of the points on the electrochemical curve in ( b ) where the samples were extracted for nmr / pdf analyses . on desodiation , na nmr resonances from c - na3sb lose intensity during d1-a . the resonance at 37 ppm from a - na3xsb does not reappear , confirming that a different reaction pathway is taken on desodiation . instead , a very broad resonance centered at 27 ppm appears , indicative of sodium being within a phase with a higher sb sb connectivity than na3sb , and ruling out the formation of nasb . at the end of desodiation , the only strong peaks in the na nmr spectra originate from sodium with the conductive carbon , the sei and the cmc binder . approximately 1 sodium per antimony remains in the electrode at the end of desodiation ( eod ) , at least some of which are likely present in the sei ( approximately 0.375 na per sb based on the additional capacity observed on sodiation ) . however , based on the consistency of capacity measurements between electrode formulations and experimental setups in this study and previous studies ( table s1 ) , it is likely that at least some of the sodium remains within the connected naxsb phases of the electrode , but in concentrations too low to give rise to distinct resonances in the region dominated by sodium contained in the sei and the cmc binder . certainly , the na nmr spectra indicate that very little c - na3sb remains in the electrode at the end of desodiation , indicating that significant loss of electrical contact does not take place during the d-1a / b processes , suggesting that if any sodium antimonide remains , it does so within the naxsb phases . during the second sodiation , both intermediate species and c - na3sb are reformed ; the resonance at 27 ppm is the dominant naxsb phase at the end of s2-a , and grows in intensity during s2-b . during s2-c resonances at both 37 ppm and from c - na3sb appear , and at the end of the second sodiation , these results demonstrate that sodium environments consistent with c - nasb are not formed during ( de)sodiation ; instead two other naxsb intermediates species , characterized by broad na resonances at 37 ppm and 27 ppm are formed . in the following sections , the structure and interconversion of these phases is analyzed in the context of operando pdf measurements , constrained by the information from these na nmr spectra . pdfs obtained as a function of sodiation are shown in figure 3 ( right ) , alongside the electrochemical profile for the galvanostatic ( constant current ) measurement during the in situ pdf measurements ( figure 3 , middle ) . least - squares refinement of the structure of crystalline hexagonal antimony ( c - sb ) against pdf data for the pristine electrode shows a good fit ( figure s17 , table s3 ) . sb bonds within the puckered hexagonal layers ; a longer correlation at 3.3 is a signature of the weaker between - layer correlations . no attempt was made to add contributions from the cmc binder or conductive carbon to the refinement , because these are weak x - ray scatterers with limited correlations lengths , so are unlikely to contribute strongly to the pdf . no significant peaks are observed in the residual from the one - phase pdf refinement for the pristine material , confirming that the contribution of other electrode components is minimal . early in s1-a , up to a sodiation level of 0.5 na per sb , the pdf shows relatively little change , confirming the utilization of sodium inserted in this region in the formation of light species in the sei or reaction with surface species and the conductive carbon to which the pdf is relatively insensitive . upon further sodiation , the large observed changes to the pdf are an indication that the structure of the electrode undergoes drastic changes , as is expected for an alloying mechanism . the high - r peaks lose intensity as the long - range structure of crystalline sb is broken down but strong peaks remain at low - r indicating that some degree of local - order remains . while the amount of crystalline material present in the material decreases during s1-a , those present can be modeled well through a two - phase refinement using the c - sb and c - na3sb structure against pdf data in the distance range of 2050 , i.e. , the distance where the crystalline phases will dominate ; good fits are obtained in this distance range for all data sets with no significant peaks remaining in the residual , indicating no further crystalline phases are formed , in agreement with previous studies . during s1-a , c - sb is the dominant crystalline phase present up to a na : sb ratio of approximately 1.7:1 , albeit with a steady decrease in the scale factor for this phase ( figure s18 ) ; no significant quantity of c - na3sb is present ( table s7 ) . the absence of sodium - containing crystalline phases results in a large discrepancy between the level of sodiation determined from electrochemical measurements and that calculated from the phase fractions of the crystalline phases present ( figure 4 , top ) meaning additional amorphous phases must account for a significant amount of the sodium present in the electrode . ( a ) comparison of the sodiation calculated from electrochemical measurements and from the phase fractions determined from least - squares refinements of pdf data . blue triangles show the sodiation calculated from a two - phase fit using c - sb and c - na3sb in the distance range ( 2050 ) ; green crosses show the sodiation calculated from a two - phase fit using c - sb and c - na3sb in the distance range ( 250 ) ; red circles show the sodiation calculated from a three - phase refinement using c - sb , c - na3sb and a - na3xsb . bottom : real - space least - squares refinements against pdf data at an electrode stoichiometry of na2.36sb during the first sodiation . ( b ) a two phase refinement in the range 250 . ( c ) a constrained refinement where the values obtained during a refinement in the distance range 2050 were fixed for the refinement in the full distance range . ( d ) three phase refinement using c - sb , c - na3sb and a - na3xsb ( x = 0.5 ) . for all refinements , experimental data is shown in gray circles , the fit to the data in orange , and the residual in gray ( raw ) or black ( r - averaged over termination ripples ) . details of the calculations used to estimate error bars on the sodiation levels are shown in the supporting information . when two - phase refinements are extended to the full distance range ( 250 ) , the fit is unsatisfactory ( figure 4(b ) ) ; at low - r there is significant mismatch between the model and the pdf data , and the fit at high - r is worse compared to the refinements against high - r pdf data ( table s6 ) , providing further evidence for the presence of additional phases in the system . in order to extract the pdf for these amorphous phases , the structural parameters for refinements at high - r ( 2050 ) were fixed and used to constrain a full r - range ( 250 ) refinement against the pdf data . the residual ( g(r)experiment g(r)model ) from these constrained refinements were then extracted as a function of sodiation level and represent the pdf of the amorphous phase . the differential pdf for the amorphous phase was calculated by subtracting the residual for the pristine electrode from subsequent residuals . peaks in this residual pdf correspond to the additional interactions within the amorphous phase(s ) in the system when sodium is added to the electrode . strong peaks are observed at 3.1 and 5.4 , along with broader peaks out to 20 , but no high - r peaks are seen ( figure 5 , figure 6a ) . the positions of the low - r peaks are close to the na sb and sb sb nearest correlations in c - na3sb ( the pdf for both of these phases is shown in figure 6a ) , suggesting local environments similar to c - na3sb are formed during s1-a , but without the long - range correlations present in a crystalline material . this is consistent with large peak width of the na nmr spectra in this region , indicating a highly disordered structure in which the sodium is likely to have a range of coordination numbers and geometries . pdfs for the a - na3xsb intermediate formed during s1-a as a function of sodiation . phase fractions for the crystalline phases are obtained from a two phase ( c - sb and c - na3sb ) refinements against data at high - r ( 2050 ) . these phase fractions were then fixed for refinements against data over the full r - range . the residual of these refinements represents the additional amorphous phases present in the electrode . the electrochemical profile of the first sodiation is shown below ; the color of the curve corresponds to the point on the electrochemical curve designated by the dot of that color . ( a ) pdfs for the amorphous phases formed during ( de)sodiation of antimony extracted from experimental data : a - na1.0sb extracted from the amorphous component of the pdf at the end of d1-b ; a - na1.7sb extracted from the amorphous component of the pdf at the end of d1-a ; a - na3xsb is the pdf extracted from the end of s2-c . distinguishing distances for each of the phases are marked on the pdfs and calculated pdfs for c - sb and c - na3sb ( scaled by 0.5 ) are shown for comparison . ( b ) an sb sb offset dumbbell arrangement giving rise to peaks in the same positions as the pdf for a - na1.7sb . ( c ) comparison the arrangement of sb in c - na3sb ( left ) and c - sb ( right ) looking down the c - axis ; sodium is shown in orange , antimony is in blue . light blue atoms lie in one plane , dark blue lie offset in another plane . a comparison between the sb and na3sb structure ( figure 6c ) shows that the puckered hexagonal arrangement of sb atoms is retained in na3sb , with the insertion of additional sodium both between the sb atoms within layers and between the layers , representing a kinetically facile pathway for sodiation . therefore , we propose that the amorphous structure formed initially on sodiation is related to these other structures , with the sb - interlayer distances reduced compared to c - na3sb , probably due to vacancies between the layers . on the basis of the na nmr shift , we propose that this phase referred to herein as a - na3xsb is undersodiated compared to c - na3sb . using data collected at a total electrode stoichiometry of na2.7sb where the pdf indicates that a - na3xsb is the major phase in the electrode ( figure s19 ) , we estimate the stoichiometry of a - na3xsb as x = 0.4 by subtracting the contribution of sodium in the c - na3sb and within the sei , estimated to be 0.78 and 0.375 na per sb , respectively , from the sodiation level calculated from the electrochemistry . some deviation from this value is likely due to the difficulty in determining when in the electrochemical process the sodium consumed in side reactions ( sei , carbon surface reactions , etc . ) . however , further evidence of this under - sodiation comes from the second sodiation , where this amorphous a - na3xsb phase is reformed in isolation at the end of s2-c ( see section 3.2.3 ) , at an electrode stoichiometry of na2.5sb ( calculated from electrochemical measurements ) , making x = 0.5 ( figure s20 compares the pdfs of these phases ) . alloy phases are known to accommodate some degree of over- or under - stoichiometry , there is likely to be some stoichiometric flexibility in this highly amorphous phase . above a sodiation level of 2.9 na per sb no further crystalline sb is observed in the electrode ; all the sb the crystallization of c - na3sb is observed ; sharp high - r peaks corresponding to c - na3sb phase appear in the pdf , and at the end of discharge , pdf data can be modeled well by using the crystalline na3sb structure , reported by brauer and zintl ( figure s21 , table s4 ) in agreement with other studies that report this as the final sodiation product . no crystalline cubic na3sb was observed in contrast to a previous study that reported a minor amount of this high - pressure metastable phase alongside hexagonal na3sb prior to the full crystallization of hexagonal na3sb . the same large u33 values observed for the na2 site of the model compounds are also observed here . when linked with the na ssnmr , these results imply that na3sb has some degree of local disorder resulting in exchange of sodium between sites , which is not captured in the average structures of the previous reports . c - na3sb is broken down during d1-a , the high - r peaks disappearing while an additional interaction at 2.85 starts to appear . the additional interactions , again extracted from the residual of a constrained refinements against c - na3sb , are shown in figure 6(a ) ( middle ) for the end of d1-a at an electrode stoichiometry of na1.875sb . it is immediately obvious that no antimony connectivity similar to a - na3xsb is present , confirming that a different reaction pathway is taken on desodiation . instead , an additional phase is formed during d1-a , of approximate stoichiometry na1.7sb ( full details of these calculations are shown in the supporting information ) . the structure is highly amorphous , with no peaks observed above 10 , consistent with the broad peak observed in the na nmr for this phase , which indicates highly disordered sodium environments . there is a single , sharp , intense peak at 2.85 , a signature of sb sb bonding , along with weak , broad peaks centered at 4.6 , 6.6 , and 9.3 . the weak x - ray scattering power of na in comparison to sb , makes the presence of significant nasb or nana correlations in the pdf unlikely and therefore all peaks observed in the pdf are likely to come from sb sb correlations . the sharpness of the first peak compared to the other peaks in the pdf indicate that the local sb units are well - defined , probably as sb sb dumbbells . any more extended sb sb connectivity ( e.g. , chains ) would results in additional sharp peaks in the pdf at higher - r that are not observed here . dumbbells are known to be stable structural motifs in other alkali alloy phases such as li ge , li sn . certainly , the pdf in this region rules out the formation of nasb that is postulated by baggetto et al . because the weak peaks between 4 and 10 are not consistent with the structure ( figure s22 ) and the na nmr spectra a structure containing nasb - like helical chains disordered in relation to one another is also ruled out as the peak positions do not match those observed experimentally ( a calculated pdf is shown in figure s22 ) . in contrast , a simple geometrical argument suggests that dumbbells , offset with respect to each other to form a parallelogram of sb - atoms ( figure 6(b ) ) would give peak positions at the correct distances and approximately the correct intensities . it should be noted , however , that the structure has a very high degree of disorder beyond the well - defined first nearest - neighbor distance , and the atomic arrangement presented here is only a representation of the local structural motifs present ; no order on the long - range exists . the pdfs during d1-a can be reproduced well using a linear combination of the pdfs of c - na3sb and a - na1.7sb ( see supporting information , figure s25 ) , and the extracted phase fractions indicate a two - phase reaction between c - na3sb and a - na1.7sb during d1-a . at the end of d1-a , the pdf is a mixture of a - na1.7sb and c - na3sb , where approximately 14% of the c - na3sb phase remains , based on the relative scale factor obtained by least - squares refinement against pdf data . during d1-b , the remaining c - na3sb is broken down and there is a growth in intensity and change to the positions of low - r peaks , indicating both a growth of the amount of amorphous phase , and structural rearrangements within the amorphous phase . figure 6 ( top ) shows the pdf for the phase formed in this region , the na - content at this point corresponding to a stoichiometry of na1.0sb ( for the calculations of the sodiation level of this phase , see supporting information ) . the peak at 2.9 remains , a signature of the prevalence of sb sb bonding , while the intensity of the 6.6 peak increases relative to that observed for a - na1.7sb and shows no change in position . larger changes occur between these distances ; the peak at 4.6 is no longer evident , but two new peaks at 3.7 and 4.3 can be deconvoluted from the pdf ( figure s23 ) . these additional strong peaks at low - r indicate that a more highly connected antimony network results from the removal of sodium . the pdf for this structure shows a striking resemblance to a pdf reported for amorphous explosive , who propose a structural model based on a three - connected random network of antimony , where all antimony are bonded to three other antimony atoms . a large range of dihedral angles result in the broadening of peaks beyond the first nearest neighbor and no long - range order to atom atom correlations . no peak is observed at 3.3 , where the correlation between the puckered layers in c - sb is observed . this disordered antimony phase is believed to have heteroatoms terminating sb - fragments in the case of the sample of krebs , this is likely to be residual chloride . within the electrode , residual sodium or organic components from the decomposition of the organic electrolyte are likely to remain . we postulate that the lower density resulting from the lack of layering in the material compared to c - sb could accommodate the residual sodium present at this composition . the residual sodium is likely to be highly disordered , broadening out the already weak contribution to the pdf , meaning that no clear na na or na peaks at high - r appear ; these are modeled well by c - sb ( figure s26 , table s8 ) indicating that removal of further sodium from na1.0sb results in the crystallization of some areas of the electrode , consistent with previous xrd report by darwiche et al . the correlation length of this c - sb at the end of charge is limited to 20 nm . it is clear , however , that a significant amount of the a - na1.0sb remains in the electrode ; a comparison of the scale factors for the c - sb at the end of d1-c and for the pristine electrode estimates that 40% of the sb is present as c - sb , 60% of the antimony is present as the a - na1.0sb network . the ratio of the first peaks in the pdf assumed to be as a result of each sb bonding to 3 other sb atoms for the amorphous component ( extracted from the residual of pdf refinements carried out the r - range of 2050 ) and the crystalline component is 7:3 , again estimating that 70% of the antimony remains as a - na1.0sb ( figure s1 ) . at the end of desodiation , approximately 0.64 na per sb have not been removed in addition to the 0.375 na per sb that have been assigned to sei formation during the first sodiation , either because of the loss of connection of areas of the electrode or because of sodium trapped in the electrode . we propose that loss of connection is unlikely , given the reproducibility of the effect in multiple electrodes and also in reports by other authors ( table s1 ) and , because the na nmr results indicate that very little na3sb / a - na1.7sb remains in the electrode at full desodiation . reason that the isomeric shift observed for antimony at the end of charge is shifted from crystalline antimony due to residual sodium present in the material . the increase to the c - lattice parameter of the desodiated c - sb phase compared to the pristine electrode ( 11.38(4 ) vs 11.27(1 ) , table s3 ) , seems too small for the c - sb to contain any significant amount of sodium . it is more likely that sodium is trapped in the sei and within the amorphous component of the electrode that remains in a stoichiometry of na1.0sb , the latter in turn preventing the crystallization of this part of the electrode . a total capacity of 544 mahg is observed in four distinct voltage processes indicating that several sequential sodiation reactions take place . careful analysis of the pdf data allows the structural changes associated with each electrochemical signature to be isolated : the crystalline and amorphous electrode components were again separated by refining the crystalline structures using high - r ( 2050 ) data and the information from these refinements then used to constrain the refinements using the full data set and extract the amorphous component . the changes in the pdfs for the two components are shown in figure s8 . during s2-a , there is little change to the pdf at high - r ( figure s8 , right ) , indicating that crystalline sb does not take part in sodiation reactions at this voltage . however , the pdf for the amorphous component of the electrode shows considerable change at this voltage ( figure s8 , left ) ; the peaks at distances between 2.85 and 9 lower in intensity signifying breakdown of the amorphous antimony network . the pdf of the amorphous component at the end of s2-a contains features that are similar to that of a - na1.7sb , formed during d1-a ( figure s27 compares these two pdfs ) , consistent with na nmr spectra that show the reappearance of the broad resonance at 27 ppm , ascribed in the previous section to sodium within a - na1.7sb . therefore , we propose that the s2-a process is characteristic of the breakup of the amorphous antimony network ( a - na1.0sb ) to reform a - na1.7sb . high - r peaks in the pdf disappear in this region as the nanocrystalline sb component of the electrode is broken down ( figure s8 , right ) to form amorphous phases ; the reaction does not result in any additional crystalline phases . the 2.85 sb sb correlation remains almost constant , but a defined shoulder develops on the high - r side of this peak 3.44 , accompanied by the appearance of an additional peak at around 5.5 . both peaks are characteristic of a - na3xsb indicating that this phase begins to form during s1-b . linear combinations of the pdfs of a - na1.7sb ( taken from the pdf for this phase extracted at the end of d1-a ) and a - na3xsb ( taken from the pdf at the end of s2-c ) , ( figure 7 ) , indicate that c - sb does not form the dumbbell phase na1.7sb : the scale factor for the na1.7sb remains constant during s2-b , while that for the a - na3xsb increases linearly . this would indicate that the c - sb reacts straight to form a na3sb - like phase , probably due to the structural similarity between the two phases discussed earlier . at the end of s2-b , the pdf can be modeled as a linear combination of the pdfs of a - na1.7sb and a - na3xsb with phase fractions of 0.53 and 0.47 , respectively ( figure 7 ) . at this stage , a - na1.7sb is still the dominant phase in the na nmr spectra , and the weaker resonance from a - na3xsb at 37 ppm must be buried under that of the a - na1.7sb resonance . we note that the relative amounts of a - na3xsb are likely to be closely related to the exact amount of c - sb that is reformed at the height of charge and may explain why very little is produced in some samples , and subtle differences between in situ and ex situ samples may also result in differences to the phase fractions obtained in regions where metastable phases are dominant . results of linear combination fitting of the pdfs extracted during processes s2-b and s2-c with pdfs for a - na1.7sb ( obtained from d1-a ) and a - na3xsb ( from the end of d2-c ) . top : example of the fit of the linear combination ( orange line ) with experimental data ( gray circles ) . the difference between the experimental data and the fit is shown offset in gray . the contribution of a - na1.7sb ( blue solid line ) and a - na3xsb ( green dashed line ) is shown offset below . bottom : variation of the scale factors for a - na1.7sb ( blue circles ) and a - na3xsb ( orange squares ) with sodiation level . the plateau at 450 mv , s2-c , is reminiscent of the s1-a process that dominates the first sodiation . 0.45 na per sb are inserted into the electrode and peaks are observed in the pdf to higher - r ( 20 ) , indicating that a more ordered structure is formed . the 2.85 peak in the pdf disappears during this process , and the peak at 3.1 becomes the dominant low - r peak , signifying that sb sb bonds are broken during this region to form a - na3xsb . a single - phase a - na3xsb electrode , where x 0.5 based on the stoichiometry calculated from electrochemical measurements , is formed at the end of s2-c . a starting model based on c - na3sb structure was refined against the pdf data obtained at the end of s2-c , using very large thermal parameters to capture the disorder in the material and a spherical particle envelope to capture the limited correlation length in the material . sodium occupancies were constrained to be 0.83 , based on the sodiation level calculated from the electrochemical measurements . the refinement is shown in figure s28 and structural parameters are found in table s9 . the structure that is obtained from this refinement comprises of hexagonal layers of alternating na and sb , similar on a local scale to c - na3sb , but with more closely spaced layer evidenced by the decrease in c - lattice parameter from 9.49(1 ) to 9.19(1 ) , presumably due to sodium vacancies between the layers . this is consistent with na nmr results that indicate a more sb - rich sodium environment in this phase compared to c - na3sb . thermal parameters for both the antimony and sodium atoms in the interplanar direction are very large , which indicates significant turbostratic disorder of sodium within the structure ; a large number of sodium environments are likely to exist within a - na3xsb , a result that is consistent with the broad peak observed in the na nmr spectra for this species . this approach captures most of the features in the pdf and allows us to propose that the local structure of the a - na3xsb is very similar to the c - na3sb structure , and the similarity of both c - na3sb and a - na3xsb to c - sb discussed above is likely to make this reaction pathway kinetically facile . the pdf results indicate that a - na3xsb exists in isolation at the end of s2-c , but despite several attempts to obtain an ex situ sample containing pure a - na3xsb , none were successful . the presence of small amounts of c - na3sb in all ex situ spectra where a - na3xsb is present is ascribed to the metastability of the a - na3xsb ; once removed from the battery , some of the material may react to form c - na3sb and other reaction products appearing overlapped in the sei region . the exact proportion of the two phases is likely to depend on the exact sodiation at the point where the cell is stopped . during s2-d , the correlation length of the electrode increases as c - na3sb is formed from a - na3xsb . the structural parameters after refinement against pdf data are not significantly different from the end of the first sodiation ( table s4 ) . the na nmr spectra at the end of the second sodiation confirm c - na3sb as the final product . the model for a - na3xsb obtained from refinement against data at the end of s2-c was used to model the a - na3xsb formed during the first sodiation . using the structural parameters for c - na3sb and c - sb obtained from two - phase least - squares refinements against the pdf data at high - r , refinements were performed using the full r - range ( 250 ) , using a - na3xsb as an additional phase , refining only the scale factors for the three phases . this approach improves the fit to data in the intermediate region between 0.62.8 na per sb ( figure 4 , bottom ) ; rw improve in all cases , but particularly in the intermediate region where the sodiation level is between 1.8 and 3 ( table s6 ) . the approach also improves the fit of the sodiation level expected from structural data with that obtained from the electrochemical measurements ( figure 4 , top ) . some deviation is still observed at low sodiation levels , because of surface processes ( reaction with the carbon ) and sei formation that take place early on in the sodiation process and to which the pdf is not sensitive . density - functional theory calculations were performed on structures generated by our species - swapping method ( see section si.4 for details of the method ) . the only stable structures found on a zero temperature convex hull ( figure s9 ) were nasb and na3sb having p21/c and p63/mmc symmetries , respectively . at low sodiation , only 0.009 ev / formula unit ( f.u . ) above the hull tie - line is a nasb2c2/m phase . sb bonds must be broken even at low na content , producing structures that are not simply na intercalated sb ; thus c - sb is not easy to sodiate , consistent with the experimental results . a 6 formula unit per cell na3sb phase with p63 cm symmetry was found only 0.018 ev / f.u . above the ground state this indicates that for the na3sb stoichiometry at room temperature the system will easily explore lower symmetry and larger repeat - length structures ; i.e. , there are many low - energy amorphous structures . the conversion of c - sb to c - na3sb is predicted to be at 0.57 v in good agreement with experiment . there are no other obvious thermally accessible phases in the species - swapped convex hull diagram , indicating that any additional phases are likely to be metastable phases , in agreement with the results of saubanre et al . the first sodiation of antimony is dominated by the breakdown of the crystalline antimony electrode . the overpotential required to breakdown the crystalline lattice results a single pseudo - plateau at approximately 500 mv . both nmr and pdf data indicate that amorphous na3xsb is the major product during s1-a , though some c - na3sb is formed concurrently as a result of the overpotential . however , significant formation of c - na3sb only occurs once the majority of the crystalline antimony has been broken down into na3xsb , indicating there are kinetic difficulties associated with nucleating crystalline na3sb ( c - na3sb ) while sb si and li ge , where amorphous phases are formed during the breakage of dumbbell phases , and crystalline li15si4 and li15ge4 only form when the si si or ge dft calculations indicate that lower symmetry c - na3sb - like structures with more disordered sodium sites can easily be formed , indicating a propensity of this structure to disorder , and therefore to accommodate some under / over stoichiometry . we observe no evidence that environments similar to nasb are formed , in contrast to the results of baggetto et al . however , we do note that the overpotential on the first sodiation is likely to be highly dependent on the particle size and formulation ( film thickness , additives , etc . ) and therefore the mechanism for very thin films on the first sodiation may differ from what is presented here . na nmr spectra for c - na3sb indicate high sodium mobility in this structure , and structural refinements against pdf data indicate some inherent disorder in the material . potentiostatic intermittent titration technique ( pitt ) measurements reported previously indicate the sodium diffusion coefficient is high ( 34 times larger than the lithium diffusion coefficient in cubic li3sb ) close to 0 v. on the basis of the nmr and pdf experiments reported here , we suggest that a different pathway is taken on desodiation . a 0.2 v difference between sodiation and desodiation curves determined from galvanostatic intermittent titration technique ( gitt ) experiments the formation of sb sb bonding immediately on desodiation is apparent from the peak at 2.85 , which appears in the pdf during d1-a ; no resonance corresponding to a - na3xsb is observed in the nmr , ruling out the reformation of a - na3xsb . instead , the good fit of a linear combination of c - na3sb and a - na1.7sb to the pdfs collected during d1-a indicates that a two - phase reaction between c - na3sb and a - na1.7sb , the latter a highly amorphous phase containing sb - dumbbell units and no significant ordering of sodium , takes place . these results are in contrast to those reported by baggetto et al . , who proposed the reformation of the same intermediate formed on sodiation of thin films . we ascribe the different pathway taken on desodiation to poor kinetics in the electrode ; variable temperature nmr experiments have indicated that the sodium mobility in c - na3sb is high , and therefore its removal on initial desodiation is should be facile . presumably the limited antimony mobility prevents crystallization of the thermodynamically stable nasb phase , and sb - dimers , instead of more extended sb sb phase diagram , indicating that the pathway must occur via a kinetically - favored structural motif . the formation of dimers when the na : sb ratio is close to 2:1 is unsurprising since zintl counting rules predict that the antimony should be present with a formal charge of sb ; this results in the same connectivity found in halogen molecules , i.e. , x2 , and dimers are to be expected . future experiments to probe the effect of temperature on the phases formed are planned , but it is clear from both nmr and pdf results that in room - temperature batteries , the nasb phase is not formed on either sodiation or desodiation . as more sodium is removed , the antimony connectivity increases to form a network , similar to that reported for amorphous antimony . in areas where additional sodium can be removed the electrode forms crystalline antimony , but with a significantly reduced correlation length indicating it may be present as nanoparticles . it is not clear whether complete na removal ( from the amorphous phase ) is prevented due to difficulties in na transport through the amorphous phase , or whether the ( kinetic ) formation of the amorphous phase hinders further crystallization of the sb phase again due to the difficulty of breaking and reforming sb bonds . on the basis of pdf results , the different electrochemical profile observed on the second sodiation can be attributed to two key factors both intimately connected to the complex structure of the electrode formed on desodiation . first , the amorphous and crystalline phases sodiate at different voltages , something that is clearly observed in the pdf data . antimony contained as amorphous a - na1.0sb reacts during s2-a to reform a - na1.7sb . crystalline antimony does not react until lower voltages ( s2-b ) and , significantly , it appears that it reacts straight to the a - na3xsb phase without forming the na1.7sb phase , similar to the behavior observed on the first sodiation . second , the correlation length / particle size and crystallinity of the c - sb that is present at the start of the second sodiation is reduced compared to the pristine starting material . as a result of this , the overpotential required to break down the crystalline lattice that is responsible for the single plateau observed in the first sodiation , is reduced or removed entirely on the second sodiation , and as a result of this additional plateaus ( s2-b / c ) can be resolved in the electrochemistry . on the basis of the structural studies outlined here , we can propose that several structural factors are responsible for the excellent cycling and rate performance of antimony , in addition to those factors discussed by previous authors i.e . , the good electrical conductivity and the low packing density of the antimony structure that promotes ion movement through the structure . first , we show that the two intermediates , a - na3xsb and a - na1.7sb are highly amorphous structures ; this reduces the strain associated with multiple phase transitions . second , pdf measurements show the formation of a complex electrode containing both amorphous and crystalline antimony networks at full desodiation . the different reaction voltages of these components lead to a more sequential reaction profile , where an inactive component can , to some extent , accommodate the strain resulting from reactions in other phases of the electrode , thus preventing harmful side processes that lead to capacity fade . lastly , the high sodium mobility within the final c - na3sb structure is likely to contribute to the good rate performance of antimony by increasing sodium movement in and out of the phase leading to facile structural transformations . finally , we note that examination of the second sodiation process is vital , not only for understanding the structure of the active electrode material - which in this case differs considerably from the single - phase pristine electrode and , therefore , the ( de)sodiation mechanism of antimony in real systems , but also for understanding the complex processes going on in the electrode that can be masked by the presence of an overpotential in the first sodiation . using in - depth analysis of the pdf data , constrained by structural and chemical information from known model naxsb phases and na ssnmr , we are able to demonstrate , for the first time , the separation of amorphous and crystalline phases formed in a sodium - ion battery and link structural information to all electrochemical signatures observed , allowing a comprehensive mechanism of ( de)sodiation to be assembled ( figure 8) . operando pdf analysis of sodium - ion batteries , when constrained by chemical information from na nmr spectroscopy , referenced to known model compounds nasb and na3sb , was used to separate the amorphous and crystalline structures formed in antimony anodes during ( de)sodiation . this approach has led to the identification of previously unknown amorphous intermediate phases a - na3xsb and a - na1.7sb and tracking of their interconversion within a sodium - ion battery . we propose that a - na3xsb ( 0.4 < x < 0.5 ) is locally similar to crystalline na3sb , with hexagonal layers of antimony interspersed with sodium , but with a reduced interlayer spacing resulting from significant numbers of na - vacancies between the sb - layers . the pdf for a - na1.7sb has a single sharp peak at 2.85 indicative of a sb - dumbbell motif present within the highly amorphous structure . inclusion of amorphous phases into real - space least - squares refinements results in a better match of the sodium level calculated from structural refinements with that obtained from electrochemical measurements , far extending previously known mechanistic details . pdf and nmr - derived mechanism of ( de)sodiation of antimony from the first desodiation during galvanostatic cycling at a rate of c/20 . the structural origins of the different electrochemical profiles observed on the first and second sodiations are explored ; ( de)sodiation processes during the first cycle result in a change to the active electrode from crystalline antimony to a composite electrode containing both amorphous and crystalline antimony networks at full desodiation . the different reaction voltages of the amorphous and crystalline components of the electrode are responsible for the additional processes observed in subsequent cycles . na nmr spectroscopy highlights the anomalously high sodium mobility within the na3sb final sodiation product , a likely contributing factor to the exceptional rate performance of antimony compared to other alloying anodes . we show that the sodiation of antimony is controlled to a large degree by the kinetics of the system . dft calculations show no additional thermodynamically stable phases exist on the zero temperature convex hull . formation of undersodiated na3sb ( a - na3xsb ) takes place due to difficulties in nucleating c - na3sb in regions where sb the absence of nasb in both sodiation and desodiation is also ascribed to the lack of mobility of sb within the naxsb phases , particularly after the initial sb sb bonds ( in the dumbbells ) have already formed , instead forming amorphous structure containing first sb - dumbbells and later extended , but disordered , networks of antimony . the electrode formed after desodiation is a composite of amorphous and crystalline antimony networks . we show that these connectivities react at distinct voltages via different sodiation pathways ; the sequential manner of these transformations enhances the cyclability of the electrode by having an inactive component capable of buffering of strain associated with multiple phase transitions . we highlight the role that connectivity plays in determining the sodiation pathway in kinetically controlled systems ; here , amorphous and crystalline antimony networks react through different intermediate phases that are structurally related to the starting connectivity . this result could have wider implications for understanding other kinetically limited alloying systems where the starting connectivity is likely to have a large effect on the pathway taken and the capacities observed . for example , germanium nanowire anodes for sodium - ion batteries show little capacity in their crystalline form , but high capacity and good rate performance when prelithiated and amorphised . our results have additional implications for understanding high - rate nanostructured antimony anodes , because the electrochemical processes observed in bulk antimony are also observed in nanostructured systems beyond the first sodiation , indicating that the same antimony - based phase transitions take place . study of bulk antimony as a model system helps to decouple the processes associated with surface oxide groups with those associated with naxsb phase transitions in such nanostructured electrodes .

operando pair distribution function ( pdf ) analysis and ex situ 23na magic - angle spinning solid - state nuclear magnetic resonance ( mas ssnmr ) spectroscopy are used to gain insight into the alloying mechanism of high - capacity antimony anodes for sodium - ion batteries . subtraction of the pdf of crystalline naxsb phases from the total pdf , an approach constrained by chemical phase information gained from 23na ssnmr in reference to relevant model compounds , identifies two previously uncharacterized intermediate species formed electrochemically ; a - na3xsb ( x 0.40.5 ) , a structure locally similar to crystalline na3sb ( c - na3sb ) but with significant numbers of sodium vacancies and a limited correlation length , and a - na1.7sb , a highly amorphous structure featuring some sb sb bonding . the first sodiation breaks down the crystalline antimony to form first a - na3xsb and , finally , crystalline na3sb . desodiation results in the formation of an electrode formed of a composite of crystalline and amorphous antimony networks . we link the different reactivity of these networks to a series of sequential sodiation reactions manifesting as a cascade of processes observed in the electrochemical profile of subsequent cycles . the amorphous network reacts at higher voltages reforming a - na1.7sb , then a - na3xsb , whereas lower potentials are required for the sodiation of crystalline antimony , which reacts to form a - na3xsb without the formation of a - na1.7sb . a - na3xsb is converted to crystalline na3sb at the end of the second discharge . we find no evidence of formation of nasb . variable temperature 23na nmr experiments reveal significant sodium mobility within c - na3sb ; this is a possible contributing factor to the excellent rate performance of sb anodes .

Introduction Experimental Methods Results Discussion Conclusions

this raises questions about the origin of this effect as well as what the structure of the electrode at the top of charge is , as this effectively becomes the active the intermediate phases that are formed during cycling are also unknown ; x - ray diffraction ( xrd ) studies confirm that crystalline na3sb ( c - na3sb ) is formed at the end of both the first and second sodiation , but other than the formation of this crystalline phase , the electrode is amorphous for large sections of the first sodiation and all of the subsequent ( de)sodiation processes , meaning xrd is unable to probe their structure . furthermore , while x - ray pdf is highly sensitive to the components of the electrode that are strongly scattering in this case meaning that changes to the antimony connectivity during sodium insertion are very distinct , we present a pair distribution function and high - resolution na magic - angle spinning solid - state nuclear magnetic resonance spectroscopy ( mas ssnmr ) study of antimony anodes for sodium - ion batteries . additional amorphous phases were added to the refinements for the first sodiation process using a model that was refined against data from the second sodiation ( at the end of the d2-c process ) , where the amorphous phase is assumed to be phase - pure . of note , is the large value of the u33 thermal displacement parameter obtained from the refinement for the na2 ( table s4 ) , the sodium - ions within the hexagonal layers of na3sb , corresponding to displacements in the c - direction , normal to the hexagonal layers of the material . during the second sodiation , both intermediate species and c - na3sb are reformed ; the resonance at 27 ppm is the dominant naxsb phase at the end of s2-a , and grows in intensity during s2-b . during s2-c resonances at both 37 ppm and from c - na3sb appear , and at the end of the second sodiation , these results demonstrate that sodium environments consistent with c - nasb are not formed during ( de)sodiation ; instead two other naxsb intermediates species , characterized by broad na resonances at 37 ppm and 27 ppm are formed . the positions of the low - r peaks are close to the na sb and sb sb nearest correlations in c - na3sb ( the pdf for both of these phases is shown in figure 6a ) , suggesting local environments similar to c - na3sb are formed during s1-a , but without the long - range correlations present in a crystalline material . ( a ) pdfs for the amorphous phases formed during ( de)sodiation of antimony extracted from experimental data : a - na1.0sb extracted from the amorphous component of the pdf at the end of d1-b ; a - na1.7sb extracted from the amorphous component of the pdf at the end of d1-a ; a - na3xsb is the pdf extracted from the end of s2-c . using data collected at a total electrode stoichiometry of na2.7sb where the pdf indicates that a - na3xsb is the major phase in the electrode ( figure s19 ) , we estimate the stoichiometry of a - na3xsb as x = 0.4 by subtracting the contribution of sodium in the c - na3sb and within the sei , estimated to be 0.78 and 0.375 na per sb , respectively , from the sodiation level calculated from the electrochemistry . however , further evidence of this under - sodiation comes from the second sodiation , where this amorphous a - na3xsb phase is reformed in isolation at the end of s2-c ( see section 3.2.3 ) , at an electrode stoichiometry of na2.5sb ( calculated from electrochemical measurements ) , making x = 0.5 ( figure s20 compares the pdfs of these phases ) . above a sodiation level of 2.9 na per sb no further crystalline sb is observed in the electrode ; all the sb the crystallization of c - na3sb is observed ; sharp high - r peaks corresponding to c - na3sb phase appear in the pdf , and at the end of discharge , pdf data can be modeled well by using the crystalline na3sb structure , reported by brauer and zintl ( figure s21 , table s4 ) in agreement with other studies that report this as the final sodiation product . the additional interactions , again extracted from the residual of a constrained refinements against c - na3sb , are shown in figure 6(a ) ( middle ) for the end of d1-a at an electrode stoichiometry of na1.875sb . the pdfs during d1-a can be reproduced well using a linear combination of the pdfs of c - na3sb and a - na1.7sb ( see supporting information , figure s25 ) , and the extracted phase fractions indicate a two - phase reaction between c - na3sb and a - na1.7sb during d1-a . at the end of d1-a , the pdf is a mixture of a - na1.7sb and c - na3sb , where approximately 14% of the c - na3sb phase remains , based on the relative scale factor obtained by least - squares refinement against pdf data . the peak at 2.9 remains , a signature of the prevalence of sb sb bonding , while the intensity of the 6.6 peak increases relative to that observed for a - na1.7sb and shows no change in position . the residual sodium is likely to be highly disordered , broadening out the already weak contribution to the pdf , meaning that no clear na na or na peaks at high - r appear ; these are modeled well by c - sb ( figure s26 , table s8 ) indicating that removal of further sodium from na1.0sb results in the crystallization of some areas of the electrode , consistent with previous xrd report by darwiche et al . it is clear , however , that a significant amount of the a - na1.0sb remains in the electrode ; a comparison of the scale factors for the c - sb at the end of d1-c and for the pristine electrode estimates that 40% of the sb is present as c - sb , 60% of the antimony is present as the a - na1.0sb network . the ratio of the first peaks in the pdf assumed to be as a result of each sb bonding to 3 other sb atoms for the amorphous component ( extracted from the residual of pdf refinements carried out the r - range of 2050 ) and the crystalline component is 7:3 , again estimating that 70% of the antimony remains as a - na1.0sb ( figure s1 ) . at the end of desodiation , approximately 0.64 na per sb have not been removed in addition to the 0.375 na per sb that have been assigned to sei formation during the first sodiation , either because of the loss of connection of areas of the electrode or because of sodium trapped in the electrode . careful analysis of the pdf data allows the structural changes associated with each electrochemical signature to be isolated : the crystalline and amorphous electrode components were again separated by refining the crystalline structures using high - r ( 2050 ) data and the information from these refinements then used to constrain the refinements using the full data set and extract the amorphous component . the pdf of the amorphous component at the end of s2-a contains features that are similar to that of a - na1.7sb , formed during d1-a ( figure s27 compares these two pdfs ) , consistent with na nmr spectra that show the reappearance of the broad resonance at 27 ppm , ascribed in the previous section to sodium within a - na1.7sb . linear combinations of the pdfs of a - na1.7sb ( taken from the pdf for this phase extracted at the end of d1-a ) and a - na3xsb ( taken from the pdf at the end of s2-c ) , ( figure 7 ) , indicate that c - sb does not form the dumbbell phase na1.7sb : the scale factor for the na1.7sb remains constant during s2-b , while that for the a - na3xsb increases linearly . at the end of s2-b , the pdf can be modeled as a linear combination of the pdfs of a - na1.7sb and a - na3xsb with phase fractions of 0.53 and 0.47 , respectively ( figure 7 ) . at this stage , a - na1.7sb is still the dominant phase in the na nmr spectra , and the weaker resonance from a - na3xsb at 37 ppm must be buried under that of the a - na1.7sb resonance . we note that the relative amounts of a - na3xsb are likely to be closely related to the exact amount of c - sb that is reformed at the height of charge and may explain why very little is produced in some samples , and subtle differences between in situ and ex situ samples may also result in differences to the phase fractions obtained in regions where metastable phases are dominant . results of linear combination fitting of the pdfs extracted during processes s2-b and s2-c with pdfs for a - na1.7sb ( obtained from d1-a ) and a - na3xsb ( from the end of d2-c ) . the 2.85 peak in the pdf disappears during this process , and the peak at 3.1 becomes the dominant low - r peak , signifying that sb sb bonds are broken during this region to form a - na3xsb . a starting model based on c - na3sb structure was refined against the pdf data obtained at the end of s2-c , using very large thermal parameters to capture the disorder in the material and a spherical particle envelope to capture the limited correlation length in the material . thermal parameters for both the antimony and sodium atoms in the interplanar direction are very large , which indicates significant turbostratic disorder of sodium within the structure ; a large number of sodium environments are likely to exist within a - na3xsb , a result that is consistent with the broad peak observed in the na nmr spectra for this species . this approach captures most of the features in the pdf and allows us to propose that the local structure of the a - na3xsb is very similar to the c - na3sb structure , and the similarity of both c - na3sb and a - na3xsb to c - sb discussed above is likely to make this reaction pathway kinetically facile . the pdf results indicate that a - na3xsb exists in isolation at the end of s2-c , but despite several attempts to obtain an ex situ sample containing pure a - na3xsb , none were successful . the presence of small amounts of c - na3sb in all ex situ spectra where a - na3xsb is present is ascribed to the metastability of the a - na3xsb ; once removed from the battery , some of the material may react to form c - na3sb and other reaction products appearing overlapped in the sei region . the model for a - na3xsb obtained from refinement against data at the end of s2-c was used to model the a - na3xsb formed during the first sodiation . using the structural parameters for c - na3sb and c - sb obtained from two - phase least - squares refinements against the pdf data at high - r , refinements were performed using the full r - range ( 250 ) , using a - na3xsb as an additional phase , refining only the scale factors for the three phases . however , significant formation of c - na3sb only occurs once the majority of the crystalline antimony has been broken down into na3xsb , indicating there are kinetic difficulties associated with nucleating crystalline na3sb ( c - na3sb ) while sb si and li ge , where amorphous phases are formed during the breakage of dumbbell phases , and crystalline li15si4 and li15ge4 only form when the si si or ge dft calculations indicate that lower symmetry c - na3sb - like structures with more disordered sodium sites can easily be formed , indicating a propensity of this structure to disorder , and therefore to accommodate some under / over stoichiometry . a 0.2 v difference between sodiation and desodiation curves determined from galvanostatic intermittent titration technique ( gitt ) experiments the formation of sb sb bonding immediately on desodiation is apparent from the peak at 2.85 , which appears in the pdf during d1-a ; no resonance corresponding to a - na3xsb is observed in the nmr , ruling out the reformation of a - na3xsb . instead , the good fit of a linear combination of c - na3sb and a - na1.7sb to the pdfs collected during d1-a indicates that a two - phase reaction between c - na3sb and a - na1.7sb , the latter a highly amorphous phase containing sb - dumbbell units and no significant ordering of sodium , takes place . we ascribe the different pathway taken on desodiation to poor kinetics in the electrode ; variable temperature nmr experiments have indicated that the sodium mobility in c - na3sb is high , and therefore its removal on initial desodiation is should be facile . crystalline antimony does not react until lower voltages ( s2-b ) and , significantly , it appears that it reacts straight to the a - na3xsb phase without forming the na1.7sb phase , similar to the behavior observed on the first sodiation . as a result of this , the overpotential required to break down the crystalline lattice that is responsible for the single plateau observed in the first sodiation , is reduced or removed entirely on the second sodiation , and as a result of this additional plateaus ( s2-b / c ) can be resolved in the electrochemistry . first , we show that the two intermediates , a - na3xsb and a - na1.7sb are highly amorphous structures ; this reduces the strain associated with multiple phase transitions . lastly , the high sodium mobility within the final c - na3sb structure is likely to contribute to the good rate performance of antimony by increasing sodium movement in and out of the phase leading to facile structural transformations . finally , we note that examination of the second sodiation process is vital , not only for understanding the structure of the active electrode material - which in this case differs considerably from the single - phase pristine electrode and , therefore , the ( de)sodiation mechanism of antimony in real systems , but also for understanding the complex processes going on in the electrode that can be masked by the presence of an overpotential in the first sodiation . using in - depth analysis of the pdf data , constrained by structural and chemical information from known model naxsb phases and na ssnmr , we are able to demonstrate , for the first time , the separation of amorphous and crystalline phases formed in a sodium - ion battery and link structural information to all electrochemical signatures observed , allowing a comprehensive mechanism of ( de)sodiation to be assembled ( figure 8) . operando pdf analysis of sodium - ion batteries , when constrained by chemical information from na nmr spectroscopy , referenced to known model compounds nasb and na3sb , was used to separate the amorphous and crystalline structures formed in antimony anodes during ( de)sodiation . this approach has led to the identification of previously unknown amorphous intermediate phases a - na3xsb and a - na1.7sb and tracking of their interconversion within a sodium - ion battery . we propose that a - na3xsb ( 0.4 < x < 0.5 ) is locally similar to crystalline na3sb , with hexagonal layers of antimony interspersed with sodium , but with a reduced interlayer spacing resulting from significant numbers of na - vacancies between the sb - layers . the structural origins of the different electrochemical profiles observed on the first and second sodiations are explored ; ( de)sodiation processes during the first cycle result in a change to the active electrode from crystalline antimony to a composite electrode containing both amorphous and crystalline antimony networks at full desodiation . na nmr spectroscopy highlights the anomalously high sodium mobility within the na3sb final sodiation product , a likely contributing factor to the exceptional rate performance of antimony compared to other alloying anodes . formation of undersodiated na3sb ( a - na3xsb ) takes place due to difficulties in nucleating c - na3sb in regions where sb the absence of nasb in both sodiation and desodiation is also ascribed to the lack of mobility of sb within the naxsb phases , particularly after the initial sb sb bonds ( in the dumbbells ) have already formed , instead forming amorphous structure containing first sb - dumbbells and later extended , but disordered , networks of antimony .

linagliptin ( tradjenta , boehringer ingelheim , ridgefield , ct , usa ) is a newly approved medication for the treatment of type 2 diabetes mellitus.1 this agent is a potent inhibitor of the serine protease enzyme , dipeptidyl peptidase-4 ( dpp-4 ) , which is responsible for rapid degradation of two incretin hormones , glucagon - like - peptide 1 ( glp-1 ) and glucose insulinotropic polypeptide ( gip ) . glp-1 and gip have distinct physiologic actions in the regulation of glucose that would make their augmentation attractive in the patient with type 2 diabetes due to a propensity to achieve decreased levels of both hormones.2 glp-1 is secreted from intestine endocrine l - cells in response to glucose and is responsible for stimulation of insulin release from the pancreas in a glucose - dependent manner . glp-1 inhibits the release of glucagon , thereby decreasing hepatic gluco - neogenesis and insulin inhibition . glp-1 decreases gastric emptying , delaying arrival of glucose into the vasculature , and works centrally in the brain by increasing satiety with a decrease in food intake . lastly , glp-1 can increase -cell mass by decreasing apoptosis and by increasing proliferation and neogenesis of -cells.3 however , this has only been shown in animal models , with no evidence of this noted in humans as yet.4 gip is secreted from the k - cells of the intestine wall , stimulates insulin secretion from the pancreas , and has been shown to decrease cellular death and increase regeneration of -cells.3 linagliptin has been shown to be a potent long - acting dpp-4 inhibitor . an in vitro study showed that linagliptin inhibited dpp-4 with a 50% inhibition concentration ( ic50 ) of about 1 nm , compared with sitagliptin ( 19 nm ) , alogliptin ( 24 nm ) , saxagliptin ( 50 nm ) , and vidagliptin ( 62 nm).5 linagliptin has an elimination half - life of 131 hours,6 and achieves steady - state concentrations after three doses of 5 mg daily.1 linagliptin has also been shown to inhibit dpp-4 activity by more than 80% over 24 hours.68 the presence of these characteristics allows for once - daily oral dosing.7 linagliptin undergoes primarily hepatic elimination , with approximately 85% of the drug excreted unchanged in the feces.9 despite having a predominately hepatic route of elimination , the main metabolite is pharmacologically inactive.1 the overall pharmacokinetic profile of linagliptin may avoid the need to adjust the dose in patients with renal or hepatic impairment . the recommendations provided by the package insert indicate no dose adjustments are required for renal or hepatic impairment.1 multiple therapies have now been introduced to the market that target the incretin hormone system . current guidelines recommend that these treatments be considered as part of a patient - centered approach and be used as a component of a two - drug or three - drug regimen in conjunction with metformin if a patient does not meet their individualized glycosylated hemoglobin ( hba1c ) goal.10 a medline search was performed using the keywords linagliptin , dpp-4 inhibitor , and type 2 diabetes for articles published through july 2012 . the literature search was limited by the following criteria : publication in the english language , clinical trials , randomized controlled trials , and research conducted in humans ( figure 1 ) . here we summarize the available data with a focus on the clinical utility and tolerability of linagliptin . this phase iia study conducted by forst et al followed a randomized , double - blind , within - dose level , parallel , placebo - controlled design and examined the pharmacokinetic and pharmacodynamic properties of linagliptin in patients with type 2 diabetes after 4 weeks of treatment.11 participants enrolled in this study were either treatment - nave or had received up to two oral antidiabetic therapies other than a thiazolidinedione . participants were 2170 ( median 62 ) years of age , had a body mass index of 18.535 kg / m , and had an hba1c 8.5% for treatment - nave and/or one oral antidiabetic therapy , and 8.0% for patients treated with two oral antidiabetic therapies . the hba1c for the total cohort of 77 patients was 7.0% . in participants receiving an oral antidiabetic therapy , eligible patients were randomly assigned to receive linagliptin 2.5 mg ( n = 26 ) , 5 mg ( n = 16 ) , 10 mg ( n = 19 ) , or placebo ( n = 16 ) . statistically significant decreases in mean hba1c from baseline were observed at the end of the 4-week period for all the linagliptin groups compared with placebo . the placebo - corrected mean change in hba1c was 0.31% for linagliptin 2.5 mg , 0.37% for linagliptin 5 mg , and 0.28% for linagliptin 10 mg ( p = < 0.025 ) . statistically significant decreases in fasting plasma glucose and postprandial plasma glucose were also observed from baseline to the end of the study period for all linagliptin doses ( see table 1 ) . another randomized , double - blind , parallel - group study comparing treatment with either linagliptin 5 mg or placebo for 24 weeks in patients with type 2 diabetes was conducted by del prato et al.12 patients were aged 1880 ( mean 55.7 ) years with a body mass index 40 kg / m , and were either treatment - nave or previously treated with one oral antidiabetic therapy other than a thiazolidinedione . pretreated patients underwent a 6-week washout period , with the last 2 weeks being an open - label placebo run - in . treatment - nave patients entered directly into the 2-week placebo run - in period . hba1c levels had to be between 6.5% and 9.0% in non - treatment - naive patients or between 7.0% and 10% in treatment - nave patients . eligible patients were then randomized to receive treatment with linagliptin 5 mg or placebo for 24 weeks . the adjusted mean difference in the change of hba1c comparing linagliptin and placebo was 0.69% ( p < 0.0001 ) . treatment with linagliptin also resulted in significant decreases in fasting plasma glucose and postprandial plasma glucose compared with placebo ( see table 1 ) . taskinen et al performed a randomized , double - blind , placebo - controlled , multicenter , parallel - group study in 701 patients with type 2 diabetes aged 1880 years.13 subjects included had a mean age of 56.5 years , a body mass index 40 kg / m , and a mean baseline hba1c of 8.1% . subjects eligible for inclusion needed to have received metformin at a dose 1500 mg / day ( or the maximum tolerated dose ) and not more than one other oral antidiabetic therapy . in patients who had previously been treated with metformin monotherapy , hba1c had to be 7.0%10.0% at screening ; for patients treated with an additional medication , a1c had to be 6.5%9.0% . patients taking antidiabetic therapy in addition to metformin were instructed to stop the medication and then underwent a 6-week washout period that included an open - label placebo run - in phase in the last 2 weeks . for patients taking metformin monotherapy at enrolment , all eligible patients continued their usual dose of metformin and were then randomized to treatment with either linagliptin 5 mg once daily or placebo for 24 weeks . the primary endpoint was the change from baseline hba1c , adjusted for baseline hba1c and the use of monotherapy versus combination therapy at enrolment , after 24 weeks of treatment . at the end of the study , linagliptin reduced the mean hba1c level by 0.49% , whereas hba1c in the placebo group rose by 0.15% ( p linagliptin also led to significant reductions versus placebo in both fasting plasma glucose and postprandial plasma glucose ( p < 0.0001 , see table 2 ) . haak et al conducted a 24-week , randomized , double - blind , placebo - controlled phase iii trial in 791 patients who were either treatment - nave or had been treated with one other antidiabetic therapy.15 eligible patients were 1880 years of age , had a diagnosis of type 2 diabetes , and had a body mass index of 40 kg / m . in treatment - nave participants , hba1c had to be 7.5% and < 11% , and for patients pre - treated with one antidiabetic therapy had to be 7.0% to 10.5% . patients pretreated with one antidiabetic therapy entered a 4-week washout period followed by a 2-week placebo run - in period that all patients participated in . subjects were then treated for 24 weeks with one of two free combinations of linagliptin ( linagliptin 2.5 mg twice daily + metformin 500 mg twice daily or 1000 mg twice daily ) or placebo , linagliptin 5 mg once daily , metformin 500 mg twice daily , or metformin 1000 mg twice daily monotherapy . the primary endpoint was change in hba1c from baseline to 24 weeks of treatment , adjusted for baseline hba1c and previous oral antidiabetic therapy . mean baseline hba1c values were similar for all treatment groups , with an overall mean of 8.7% . the adjusted placebo - corrected mean ( 95% confidence interval ) changes in hba1c were 1.7% ( 2.0% , 1.4% ) for linagliptin + metformin 1000 mg ; 1.3% ( 1.6 , 1.1 ) for linagliptin + metformin 500 mg ; 1.2% ( 1.5% , 0.9% ) for metformin 1000 mg ; 0.8% ( 1.0 , 0.5 ) for metformin 500 mg ; and 0.6% ( 0.9% , 0.3% ) for linagliptin monotherapy ( all p < 0.0001 ) . significant reductions in fasting plasma glucose from baseline to the end of the study period were seen with combination therapies relative to metformin monotherapy . the placebo - corrected changes in fasting plasma glucose from baseline were also statistically significant for each group . this study did not assess changes in postprandial plasma glucose ( see table 2 ) . this randomized , placebo - controlled , double - blind , parallel - group study enrolled subjects with type 2 diabetes receiving metformin 1500 mg / day ( or the maximum tolerated dose ) and the maximum tolerated dose of sulfonylurea.16 patients were 1880 ( mean 58.1 ) years of age , with a body mass index 40 kg / m and hba1c 7.0% and 10.0% ( mean 8.14% ) . following a 2-week placebo run - in , a total of 1055 participants were randomized to treatment with linagliptin 5 mg once daily or placebo , in addition to the established background therapy of metformin in combination with a sulfonylurea . the primary endpoint was the change in hba1c levels between baseline and 24 weeks , stratified by baseline hba1c value . after 24 weeks , linagliptin was superior to placebo for the adjusted mean change in hba1c from baseline . the linagliptin placebo - corrected adjusted mean change from baseline was 0.62% ( p < 0.0001 ) . linagliptin also produced greater reductions in fasting plasma glucose than placebo at week 24 ( p < 0.0001 , see table 2 ) . this randomized , double - blind , parallel - group , active - controlled , noninferiority trial was conducted by gallwitz et al in 1519 patients with type 2 diabetes , aged 1880 years , and a body mass index of 40 kg / m.17 eligible subjects were receiving metformin 1500 mg / day ( or the maximum tolerated dose ) alone with an hba1c of 6.5%10.0% or 6.0%9.0% on metformin and one other oral antidiabetic therapy . mean baseline hba1c and age were 7.7% and 59.8 years in each group , respectively . participants receiving metformin and one additional antidiabetic therapy entered a 6-week wash - out period followed by a 2-week open - label placebo run - in . those receiving metformin monotherapy entered directly into a 2-week , open - label , placebo run - in period . subjects who met the inclusion criteria were then randomly assigned to treatment with linagliptin 5 mg once daily or glimepiride at an initial dose of 1 mg daily added to the current dose of metformin . the primary endpoint was the change in hba1c from baseline to week 104 , and was stratified by baseline hba1c and previous antidiabetic therapy use . after 2 years of treatment , linagliptin was noninferior to glimepiride in reducing hba1c . the difference between the treatment groups met the noninferiority criteria and was 0.20% ( p < 0.125 ) . as add - on to metformin , both linagliptin and glimepiride caused significant reductions in fasting plasma glucose and postprandial plasma glucose . the treatment differences for reductions in fasting and postprandial plasma glucose , respectively , were 6.31 mg / dl ( p = 0.012 ) and 9.73 mg / dl ( p = 0.0918 , see table 2 ) . the 12-week , multicenter , randomized , double - blind , placebo - controlled , five parallel - group study conducted by forst et al included patients with type 2 diabetes aged 2175 ( mean 60 ) years with a body mass index of 2540 kg / m.14 patients were eligible if they were pretreated with metformin alone ( baseline hba1c levels had to be 7.5%10% ) or treated with metformin and one other oral antidiabetic therapy other than a thiazolidinedione ( baseline hba1c levels had to be 7.0%9.0% ) . eligible patients who had already received metformin monotherapy entered a 2-week open - label run - in phase . patients who received metformin plus one other antidiabetic therapy entered a 6-week washout period , with the last 2 weeks being an open - label run - in phase . three doses of linagliptin ( 1 , 5 , and 10 mg once daily ) were explored when added to metformin . there was also an open - label treatment arm where patients were randomized to receive glimepiride ( 1 , 2 , or 3 mg once daily ) as add - on therapy to metformin . the mean placebo - corrected lowering of hba1c levels was 0.39% for linagliptin 1 mg ( p = 0.005 ) , 0.75% for 5 mg ( p < 0.001 ) , and 0.73% for 10 mg the change in mean placebo - corrected hba1c from baseline was 0.90% for glimepiride . the reduction in hba1c with open - label glimepiride was numerically greater versus linagliptin , but not statistically significant . fasting plasma glucose reductions were also found to be significantly greater with all doses of linagliptin than with placebo at week 12 . postprandial plasma glucose changes were not addressed in this study ( see table 2 ) . this randomized , double - blind , placebo - controlled , multicenter , parallel - group study was conducted by gomis et al in 389 patients with type 2 diabetes and aged 1880 ( mean 57.5 ) years.18 at baseline , the patients had hba1c concentrations of 7.5%11.0% ( mean 8.6% ) and a body mass index 40 kg / m . patients pretreated with oral antidiabetic therapies underwent a 6-week washout period that included an open - label placebo run - in phase in the last 2 weeks . for treatment - nave patients , only the 2-week run - in phase eligible subjects were then randomized to receive pioglitazone 30 mg once daily and linagliptin 5 mg once daily or pioglitazone 30 mg once daily and placebo for 24 weeks . the primary endpoint was change from baseline hba1c , adjusted for baseline hba1c and baseline antidiabetic therapy , after 24 weeks of treatment . at the end of the study , the adjusted mean change in hba1c from baseline for linagliptin plus pioglitazone was 1.06% compared with 0.56% for placebo plus pioglitazone . changes in fasting plasma glucose were assessed as a secondary endpoint , showing a significantly greater reduction for linagliptin plus pioglitazone than for placebo plus pioglitazone . changes in postprandial plasma glucose were not addressed in this study ( see table 2 ) . a 78-week open - label extension conducted by gomis et al evaluated participants who had previously completed one of the four 24-week , randomized , double - blind , placebo - controlled parent trials.19 these subjects received either linagliptin monotherapy , linagliptin plus metformin , linagliptin plus metformin and sulfonylurea , or linagliptin plus pioglitazone . all patients receiving one of these treatments during a previous trial continued the same treatment for an additional 78 weeks ( n = 1532 ) . those patients previously treated with placebo were switched to linagliptin monotherapy ( n = 589 ) . overall , the cohort of patients had a mean age of 57.7 years and mean baseline hba1c of 7.5% . this extension study was conducted primarily to assess the long - term safety and tolerability of linagliptin . secondary efficacy outcomes evaluated the changes in hba1c and fasting plasma glucose from baseline to 102 weeks . in participants randomized to treatment with linagliptin in the four previous trials , the mean change from baseline hba1c during the initial 24 weeks of treatment was 0.8% . this was maintained over the 78 weeks of the extension study , with a change from baseline hba1c of 0.8% . the largest observed reduction in hba1c from baseline to week 102 was in the group receiving linagliptin plus pioglitazone at 1.5% . this was followed by those patients receiving metformin and metformin plus a sulfonylurea in combination with linagliptin ( 0.7% ) . lastly , patients receiving linagliptin monotherapy showed a reduction of 0.5% at week 102 . similarly , fasting plasma glucose values already reduced during the previous trials further decreased during the extension period . in subjects randomized to placebo in the previous trials and switched to linagliptin monotherapy in the extension phase , the change in mean hba1c was 0.90% . fasting plasma glucose values also decreased from baseline over the study period ( see table 2 ) . most adverse events with linagliptin were considered to be mild to moderate in nature . adverse reactions that occurred in 2% of patients treated with linagliptin included nasopharyngitis , diarrhea , cough , urinary tract infection , and hypertriglyceridemia ( in combination with sulfonylurea therapy ) , hyperlipidemia , and weight increase ( in combination with pioglitazone).1 weight changes were reported or addressed in each of the studies above . no significant changes with regard to body weight were found when linagliptin was given as monotherapy . with regard to sulfonylurea therapy , two of the studies revealed an increase in body weight in patients treated with glimepiride versus those receiving linagliptin.14,17 however , in a study in which all patients received metformin and sulfonylurea therapy and were then randomized to placebo or linagliptin , no significant changes in body weight were seen.16 when patients received pioglitazone and either placebo or linagliptin , both groups showed an increase in body weight from baseline . the amount of weight gain was larger in patients receiving linagliptin , but the mean weight for patients receiving linagliptin was lower than that in patients receiving placebo at baseline.18 in general , linagliptin showed a low propensity to cause hypoglycemia . when used as monotherapy , no patients experienced hypoglycemia in the two studies reviewed.11,15 one study reviewing linagliptin as monotherapy versus placebo reported hypoglycemia occurring in one patient in each group.12 when combined with metformin and sulfonylurea , a higher percentage of patients receiving linagliptin experienced hypoglycemia versus placebo . however , a smaller percentage of patients experienced severe hypoglycemia when compared with placebo.16 three studies discussed or reported the occurrence of pancreatitis . one study reported zero cases15 while another study reported one case of pancreatitis in a patient receiving linagliptin.17 a 78-week , open - label extension study , which included 2121subjects , reported four cases of pancreatitis in patients who had received linagliptin for a total of 102 weeks , with two cases being acute and two chronic . this was an incidence of 0.2% in the overall treated set.19 according to the prescribing information , pancreatitis was reported more often in patients treated with linagliptin ( 21.9 per 10,000 patient years ) versus placebo ( eight per 10,000 patient years).1 one study prospectively assessed cardiovascular safety for linagliptin versus sulfonylurea ( e.g. glimepiride ) . major cardiovascular events occurred in 2% of patients treated with linagliptin and 3% treated with glimepiride ( p = 0.0213 ) . this finding was mainly attributable to a significantly lower number of nonfatal strokes with linagliptin compared with glimepiride , without any relation to hypoglycemia.17 this phase iia study conducted by forst et al followed a randomized , double - blind , within - dose level , parallel , placebo - controlled design and examined the pharmacokinetic and pharmacodynamic properties of linagliptin in patients with type 2 diabetes after 4 weeks of treatment.11 participants enrolled in this study were either treatment - nave or had received up to two oral antidiabetic therapies other than a thiazolidinedione . participants were 2170 ( median 62 ) years of age , had a body mass index of 18.535 kg / m , and had an hba1c 8.5% for treatment - nave and/or one oral antidiabetic therapy , and 8.0% for patients treated with two oral antidiabetic therapies . the hba1c for the total cohort of 77 patients was 7.0% . in participants receiving an oral antidiabetic therapy , eligible patients were randomly assigned to receive linagliptin 2.5 mg ( n = 26 ) , 5 mg ( n = 16 ) , 10 mg ( n = 19 ) , or placebo ( n = 16 ) . statistically significant decreases in mean hba1c from baseline were observed at the end of the 4-week period for all the linagliptin groups compared with placebo . the placebo - corrected mean change in hba1c was 0.31% for linagliptin 2.5 mg , 0.37% for linagliptin 5 mg , and 0.28% for linagliptin 10 mg ( p = < 0.025 ) . statistically significant decreases in fasting plasma glucose and postprandial plasma glucose were also observed from baseline to the end of the study period for all linagliptin doses ( see table 1 ) . another randomized , double - blind , parallel - group study comparing treatment with either linagliptin 5 mg or placebo for 24 weeks in patients with type 2 diabetes was conducted by del prato et al.12 patients were aged 1880 ( mean 55.7 ) years with a body mass index 40 kg / m , and were either treatment - nave or previously treated with one oral antidiabetic therapy other than a thiazolidinedione . pretreated patients underwent a 6-week washout period , with the last 2 weeks being an open - label placebo run - in . treatment - nave patients entered directly into the 2-week placebo run - in period . hba1c levels had to be between 6.5% and 9.0% in non - treatment - naive patients or between 7.0% and 10% in treatment - nave patients . eligible patients were then randomized to receive treatment with linagliptin 5 mg or placebo for 24 weeks . the adjusted mean difference in the change of hba1c comparing linagliptin and placebo was 0.69% ( p < 0.0001 ) . treatment with linagliptin also resulted in significant decreases in fasting plasma glucose and postprandial plasma glucose compared with placebo ( see table 1 ) . taskinen et al performed a randomized , double - blind , placebo - controlled , multicenter , parallel - group study in 701 patients with type 2 diabetes aged 1880 years.13 subjects included had a mean age of 56.5 years , a body mass index 40 kg / m , and a mean baseline hba1c of 8.1% . subjects eligible for inclusion needed to have received metformin at a dose 1500 mg / day ( or the maximum tolerated dose ) and not more than one other oral antidiabetic therapy . in patients who had previously been treated with metformin monotherapy , hba1c had to be 7.0%10.0% at screening ; for patients treated with an additional medication , a1c had to be 6.5%9.0% . patients taking antidiabetic therapy in addition to metformin were instructed to stop the medication and then underwent a 6-week washout period that included an open - label placebo run - in phase in the last 2 weeks . for patients taking metformin monotherapy at enrolment , all eligible patients continued their usual dose of metformin and were then randomized to treatment with either linagliptin 5 mg once daily or placebo for 24 weeks . the primary endpoint was the change from baseline hba1c , adjusted for baseline hba1c and the use of monotherapy versus combination therapy at enrolment , after 24 weeks of treatment . at the end of the study , linagliptin reduced the mean hba1c level by 0.49% , whereas hba1c in the placebo group rose by 0.15% ( p < 0.0001 ) . linagliptin also led to significant reductions versus placebo in both fasting plasma glucose and postprandial plasma glucose ( p < 0.0001 , see table 2 ) . haak et al conducted a 24-week , randomized , double - blind , placebo - controlled phase iii trial in 791 patients who were either treatment - nave or had been treated with one other antidiabetic therapy.15 eligible patients were 1880 years of age , had a diagnosis of type 2 diabetes , and had a body mass index of 40 kg / m . in treatment - nave participants , hba1c had to be 7.5% and < 11% , and for patients pre - treated with one antidiabetic therapy had to be 7.0% to 10.5% . patients pretreated with one antidiabetic therapy entered a 4-week washout period followed by a 2-week placebo run - in period that all patients participated in . subjects were then treated for 24 weeks with one of two free combinations of linagliptin ( linagliptin 2.5 mg twice daily + metformin 500 mg twice daily or 1000 mg twice daily ) or placebo , linagliptin 5 mg once daily , metformin 500 mg twice daily , or metformin 1000 mg twice daily monotherapy . the primary endpoint was change in hba1c from baseline to 24 weeks of treatment , adjusted for baseline hba1c and previous oral antidiabetic therapy . mean baseline hba1c values were similar for all treatment groups , with an overall mean of 8.7% . the adjusted placebo - corrected mean ( 95% confidence interval ) changes in hba1c were 1.7% ( 2.0% , 1.4% ) for linagliptin + metformin 1000 mg ; 1.3% ( 1.6 , 1.1 ) for linagliptin + metformin 500 mg ; 1.2% ( 1.5% , 0.9% ) for metformin 1000 mg ; 0.8% ( 1.0 , 0.5 ) for metformin 500 mg ; and 0.6% ( 0.9% , 0.3% ) for linagliptin monotherapy ( all p < 0.0001 ) . significant reductions in fasting plasma glucose from baseline to the end of the study period were seen with combination therapies relative to metformin monotherapy . the placebo - corrected changes in fasting plasma glucose from baseline were also statistically significant for each group . this study did not assess changes in postprandial plasma glucose ( see table 2 ) . this randomized , placebo - controlled , double - blind , parallel - group study enrolled subjects with type 2 diabetes receiving metformin 1500 mg / day ( or the maximum tolerated dose ) and the maximum tolerated dose of sulfonylurea.16 patients were 1880 ( mean 58.1 ) years of age , with a body mass index 40 kg / m and hba1c 7.0% and 10.0% ( mean 8.14% ) . following a 2-week placebo run - in , a total of 1055 participants were randomized to treatment with linagliptin 5 mg once daily or placebo , in addition to the established background therapy of metformin in combination with a sulfonylurea . the primary endpoint was the change in hba1c levels between baseline and 24 weeks , stratified by baseline hba1c value . after 24 weeks , linagliptin was superior to placebo for the adjusted mean change in hba1c from baseline . the linagliptin placebo - corrected adjusted mean change from baseline was 0.62% ( p < 0.0001 ) . linagliptin also produced greater reductions in fasting plasma glucose than placebo at week 24 ( p < 0.0001 , see table 2 ) . this randomized , double - blind , parallel - group , active - controlled , noninferiority trial was conducted by gallwitz et al in 1519 patients with type 2 diabetes , aged 1880 years , and a body mass index of 40 kg / m.17 eligible subjects were receiving metformin 1500 mg / day ( or the maximum tolerated dose ) alone with an hba1c of 6.5%10.0% or 6.0%9.0% on metformin and one other oral antidiabetic therapy . mean baseline hba1c and age were 7.7% and 59.8 years in each group , respectively . participants receiving metformin and one additional antidiabetic therapy entered a 6-week wash - out period followed by a 2-week open - label placebo run - in . those receiving metformin monotherapy entered directly into a 2-week , open - label , placebo run - in period . subjects who met the inclusion criteria were then randomly assigned to treatment with linagliptin 5 mg once daily or glimepiride at an initial dose of 1 mg daily added to the current dose of metformin . the primary endpoint was the change in hba1c from baseline to week 104 , and was stratified by baseline hba1c and previous antidiabetic therapy use . after 2 years of treatment , linagliptin was noninferior to glimepiride in reducing hba1c . the difference between the treatment groups met the noninferiority criteria and was 0.20% ( p < 0.125 ) . as add - on to metformin , both linagliptin and glimepiride caused significant reductions in fasting plasma glucose and postprandial plasma glucose . the treatment differences for reductions in fasting and postprandial plasma glucose , respectively , were 6.31 mg / dl ( p = 0.012 ) and 9.73 mg / dl ( p = 0.0918 , see table 2 ) . the 12-week , multicenter , randomized , double - blind , placebo - controlled , five parallel - group study conducted by forst et al included patients with type 2 diabetes aged 2175 ( mean 60 ) years with a body mass index of 2540 kg / m.14 patients were eligible if they were pretreated with metformin alone ( baseline hba1c levels had to be 7.5%10% ) or treated with metformin and one other oral antidiabetic therapy other than a thiazolidinedione ( baseline hba1c levels had to be 7.0%9.0% ) . eligible patients who had already received metformin monotherapy entered a 2-week open - label run - in phase . patients who received metformin plus one other antidiabetic therapy entered a 6-week washout period , with the last 2 weeks being an open - label run - in phase . three doses of linagliptin ( 1 , 5 , and 10 mg once daily ) were explored when added to metformin . there was also an open - label treatment arm where patients were randomized to receive glimepiride ( 1 , 2 , or 3 mg once daily ) as add - on therapy to metformin . the mean placebo - corrected lowering of hba1c levels was 0.39% for linagliptin 1 mg ( p = 0.005 ) , 0.75% for 5 mg ( p < 0.001 ) , and 0.73% for 10 mg ( p < 0.001 ) . the change in mean placebo - corrected hba1c from baseline was 0.90% for glimepiride . the reduction in hba1c with open - label glimepiride was numerically greater versus linagliptin , but not statistically significant . fasting plasma glucose reductions were also found to be significantly greater with all doses of linagliptin than with placebo at week 12 . postprandial plasma glucose changes were not addressed in this study ( see table 2 ) . this randomized , double - blind , placebo - controlled , multicenter , parallel - group study was conducted by gomis et al in 389 patients with type 2 diabetes and aged 1880 ( mean 57.5 ) years.18 at baseline , the patients had hba1c concentrations of 7.5%11.0% ( mean 8.6% ) and a body mass index 40 kg / m . patients pretreated with oral antidiabetic therapies underwent a 6-week washout period that included an open - label placebo run - in phase in the last 2 weeks . for treatment - nave patients , only the 2-week run - in phase eligible subjects were then randomized to receive pioglitazone 30 mg once daily and linagliptin 5 mg once daily or pioglitazone 30 mg once daily and placebo for 24 weeks . the primary endpoint was change from baseline hba1c , adjusted for baseline hba1c and baseline antidiabetic therapy , after 24 weeks of treatment . at the end of the study , the adjusted mean change in hba1c from baseline for linagliptin plus pioglitazone was 1.06% compared with 0.56% for placebo plus pioglitazone . changes in fasting plasma glucose were assessed as a secondary endpoint , showing a significantly greater reduction for linagliptin plus pioglitazone than for placebo plus pioglitazone . changes in postprandial plasma glucose were not addressed in this study ( see table 2 ) . a 78-week open - label extension conducted by gomis et al evaluated participants who had previously completed one of the four 24-week , randomized , double - blind , placebo - controlled parent trials.19 these subjects received either linagliptin monotherapy , linagliptin plus metformin , linagliptin plus metformin and sulfonylurea , or linagliptin plus pioglitazone . all patients receiving one of these treatments during a previous trial continued the same treatment for an additional 78 weeks ( n = 1532 ) . those patients previously treated with placebo were switched to linagliptin monotherapy ( n = 589 ) . overall , the cohort of patients had a mean age of 57.7 years and mean baseline hba1c of 7.5% . this extension study was conducted primarily to assess the long - term safety and tolerability of linagliptin . secondary efficacy outcomes evaluated the changes in hba1c and fasting plasma glucose from baseline to 102 weeks . in participants randomized to treatment with linagliptin in the four previous trials , the mean change from baseline hba1c during the initial 24 weeks of treatment was 0.8% . this was maintained over the 78 weeks of the extension study , with a change from baseline hba1c of 0.8% . the largest observed reduction in hba1c from baseline to week 102 was in the group receiving linagliptin plus pioglitazone at 1.5% . this was followed by those patients receiving metformin and metformin plus a sulfonylurea in combination with linagliptin ( 0.7% ) . lastly , patients receiving linagliptin monotherapy showed a reduction of 0.5% at week 102 . similarly , fasting plasma glucose values already reduced during the previous trials further decreased during the extension period . in subjects randomized to placebo in the previous trials and switched to linagliptin monotherapy in the extension phase , the change in mean hba1c was 0.90% . fasting plasma glucose values also decreased from baseline over the study period ( see table 2 ) . taskinen et al performed a randomized , double - blind , placebo - controlled , multicenter , parallel - group study in 701 patients with type 2 diabetes aged 1880 years.13 subjects included had a mean age of 56.5 years , a body mass index 40 kg / m , and a mean baseline hba1c of 8.1% . subjects eligible for inclusion needed to have received metformin at a dose 1500 mg / day ( or the maximum tolerated dose ) and not more than one other oral antidiabetic therapy . in patients who had previously been treated with metformin monotherapy , hba1c had to be 7.0%10.0% at screening ; for patients treated with an additional medication , a1c had to be 6.5%9.0% . patients taking antidiabetic therapy in addition to metformin were instructed to stop the medication and then underwent a 6-week washout period that included an open - label placebo run - in phase in the last 2 weeks . for patients taking metformin monotherapy at enrolment , all eligible patients continued their usual dose of metformin and were then randomized to treatment with either linagliptin 5 mg once daily or placebo for 24 weeks . the primary endpoint was the change from baseline hba1c , adjusted for baseline hba1c and the use of monotherapy versus combination therapy at enrolment , after 24 weeks of treatment . at the end of the study , linagliptin reduced the mean hba1c level by 0.49% , whereas hba1c in the placebo group rose by 0.15% ( p < 0.0001 ) . linagliptin also led to significant reductions versus placebo in both fasting plasma glucose and postprandial plasma glucose ( p < 0.0001 , see table 2 ) . haak et al conducted a 24-week , randomized , double - blind , placebo - controlled phase iii trial in 791 patients who were either treatment - nave or had been treated with one other antidiabetic therapy.15 eligible patients were 1880 years of age , had a diagnosis of type 2 diabetes , and had a body mass index of 40 kg / m . in treatment - nave participants , hba1c had to be 7.5% and < 11% , and for patients pre - treated with one antidiabetic therapy had to be 7.0% to 10.5% . patients pretreated with one antidiabetic therapy entered a 4-week washout period followed by a 2-week placebo run - in period that all patients participated in . subjects were then treated for 24 weeks with one of two free combinations of linagliptin ( linagliptin 2.5 mg twice daily + metformin 500 mg twice daily or 1000 mg twice daily ) or placebo , linagliptin 5 mg once daily , metformin 500 mg twice daily , or metformin 1000 mg twice daily monotherapy . the primary endpoint was change in hba1c from baseline to 24 weeks of treatment , adjusted for baseline hba1c and previous oral antidiabetic therapy . mean baseline hba1c values were similar for all treatment groups , with an overall mean of 8.7% . the adjusted placebo - corrected mean ( 95% confidence interval ) changes in hba1c were 1.7% ( 2.0% , 1.4% ) for linagliptin + metformin 1000 mg ; 1.3% ( 1.6 , 1.1 ) for linagliptin + metformin 500 mg ; 1.2% ( 1.5% , 0.9% ) for metformin 1000 mg ; 0.8% ( 1.0 , 0.5 ) for metformin 500 mg ; and 0.6% ( 0.9% , 0.3% ) for linagliptin monotherapy ( all p < 0.0001 ) . significant reductions in fasting plasma glucose from baseline to the end of the study period were seen with combination therapies relative to metformin monotherapy . the placebo - corrected changes in fasting plasma glucose from baseline were also statistically significant for each group . this study did not assess changes in postprandial plasma glucose ( see table 2 ) . this randomized , placebo - controlled , double - blind , parallel - group study enrolled subjects with type 2 diabetes receiving metformin 1500 mg / day ( or the maximum tolerated dose ) and the maximum tolerated dose of sulfonylurea.16 patients were 1880 ( mean 58.1 ) years of age , with a body mass index 40 kg / m and hba1c 7.0% and 10.0% ( mean 8.14% ) . following a 2-week placebo run - in , a total of 1055 participants were randomized to treatment with linagliptin 5 mg once daily or placebo , in addition to the established background therapy of metformin in combination with a sulfonylurea . the primary endpoint was the change in hba1c levels between baseline and 24 weeks , stratified by baseline hba1c value . after 24 weeks , linagliptin was superior to placebo for the adjusted mean change in hba1c from baseline . the linagliptin placebo - corrected adjusted mean change from baseline was 0.62% ( p < 0.0001 ) . linagliptin also produced greater reductions in fasting plasma glucose than placebo at week 24 ( p < 0.0001 , see table 2 ) . this randomized , double - blind , parallel - group , active - controlled , noninferiority trial was conducted by gallwitz et al in 1519 patients with type 2 diabetes , aged 1880 years , and a body mass index of 40 kg / m.17 eligible subjects were receiving metformin 1500 mg / day ( or the maximum tolerated dose ) alone with an hba1c of 6.5%10.0% or 6.0%9.0% on metformin and one other oral antidiabetic therapy . mean baseline hba1c and age were 7.7% and 59.8 years in each group , respectively . participants receiving metformin and one additional antidiabetic therapy entered a 6-week wash - out period followed by a 2-week open - label placebo run - in . those receiving metformin monotherapy entered directly into a 2-week , open - label , placebo run - in period . subjects who met the inclusion criteria were then randomly assigned to treatment with linagliptin 5 mg once daily or glimepiride at an initial dose of 1 mg daily added to the current dose of metformin . the primary endpoint was the change in hba1c from baseline to week 104 , and was stratified by baseline hba1c and previous antidiabetic therapy use . after 2 years of treatment , linagliptin was noninferior to glimepiride in reducing hba1c . the difference between the treatment groups met the noninferiority criteria and was 0.20% ( p < 0.125 ) . as add - on to metformin , both linagliptin and glimepiride caused significant reductions in fasting plasma glucose and postprandial plasma glucose . the treatment differences for reductions in fasting and postprandial plasma glucose , respectively , were 6.31 mg / dl ( p = 0.012 ) and 9.73 mg / dl ( p = 0.0918 , see table 2 ) . the 12-week , multicenter , randomized , double - blind , placebo - controlled , five parallel - group study conducted by forst et al included patients with type 2 diabetes aged 2175 ( mean 60 ) years with a body mass index of 2540 kg / m.14 patients were eligible if they were pretreated with metformin alone ( baseline hba1c levels had to be 7.5%10% ) or treated with metformin and one other oral antidiabetic therapy other than a thiazolidinedione ( baseline hba1c levels had to be 7.0%9.0% ) . eligible patients who had already received metformin monotherapy entered a 2-week open - label run - in phase . patients who received metformin plus one other antidiabetic therapy entered a 6-week washout period , with the last 2 weeks being an open - label run - in phase . three doses of linagliptin ( 1 , 5 , and 10 mg once daily ) were explored when added to metformin . there was also an open - label treatment arm where patients were randomized to receive glimepiride ( 1 , 2 , or 3 mg once daily ) as add - on therapy to metformin . the mean placebo - corrected lowering of hba1c levels was 0.39% for linagliptin 1 mg ( p = 0.005 ) , 0.75% for 5 mg ( p < 0.001 ) , and 0.73% for 10 mg ( p < 0.001 ) . the change in mean placebo - corrected hba1c from baseline was 0.90% for glimepiride . the reduction in hba1c with open - label glimepiride was numerically greater versus linagliptin , but not statistically significant . fasting plasma glucose reductions were also found to be significantly greater with all doses of linagliptin than with placebo at week 12 . postprandial plasma glucose changes were not addressed in this study ( see table 2 ) . this randomized , double - blind , placebo - controlled , multicenter , parallel - group study was conducted by gomis et al in 389 patients with type 2 diabetes and aged 1880 ( mean 57.5 ) years.18 at baseline , the patients had hba1c concentrations of 7.5%11.0% ( mean 8.6% ) and a body mass index 40 kg / m . patients pretreated with oral antidiabetic therapies underwent a 6-week washout period that included an open - label placebo run - in phase in the last 2 weeks . for treatment - nave patients , only the 2-week run - in phase was required . eligible subjects were then randomized to receive pioglitazone 30 mg once daily and linagliptin 5 mg once daily or pioglitazone 30 mg once daily and placebo for 24 weeks . the primary endpoint was change from baseline hba1c , adjusted for baseline hba1c and baseline antidiabetic therapy , after 24 weeks of treatment . at the end of the study , the adjusted mean change in hba1c from baseline for linagliptin plus pioglitazone was 1.06% compared with 0.56% for placebo plus pioglitazone . changes in fasting plasma glucose were assessed as a secondary endpoint , showing a significantly greater reduction for linagliptin plus pioglitazone than for placebo plus pioglitazone . changes in postprandial plasma glucose were not addressed in this study ( see table 2 ) . a 78-week open - label extension conducted by gomis et al evaluated participants who had previously completed one of the four 24-week , randomized , double - blind , placebo - controlled parent trials.19 these subjects received either linagliptin monotherapy , linagliptin plus metformin , linagliptin plus metformin and sulfonylurea , or linagliptin plus pioglitazone . all patients receiving one of these treatments during a previous trial continued the same treatment for an additional 78 weeks ( n = 1532 ) . those patients previously treated with placebo were switched to linagliptin monotherapy ( n = 589 ) . overall , the cohort of patients had a mean age of 57.7 years and mean baseline hba1c of 7.5% . this extension study was conducted primarily to assess the long - term safety and tolerability of linagliptin . secondary efficacy outcomes evaluated the changes in hba1c and fasting plasma glucose from baseline to 102 weeks . in participants randomized to treatment with linagliptin in the four previous trials , the mean change from baseline hba1c during the initial 24 weeks of treatment was 0.8% . this was maintained over the 78 weeks of the extension study , with a change from baseline hba1c of 0.8% . the largest observed reduction in hba1c from baseline to week 102 was in the group receiving linagliptin plus pioglitazone at 1.5% . this was followed by those patients receiving metformin and metformin plus a sulfonylurea in combination with linagliptin ( 0.7% ) . . similarly , fasting plasma glucose values already reduced during the previous trials further decreased during the extension period . in subjects randomized to placebo in the previous trials and switched to linagliptin monotherapy in the extension phase , the change in mean hba1c was 0.90% . fasting plasma glucose values also decreased from baseline over the study period ( see table 2 ) . most adverse events with linagliptin were considered to be mild to moderate in nature . adverse reactions that occurred in 2% of patients treated with linagliptin included nasopharyngitis , diarrhea , cough , urinary tract infection , and hypertriglyceridemia ( in combination with sulfonylurea therapy ) , hyperlipidemia , and weight increase ( in combination with pioglitazone).1 weight changes were reported or addressed in each of the studies above . no significant changes with regard to body weight were found when linagliptin was given as monotherapy . with regard to sulfonylurea therapy , two of the studies revealed an increase in body weight in patients treated with glimepiride versus those receiving linagliptin.14,17 however , in a study in which all patients received metformin and sulfonylurea therapy and were then randomized to placebo or linagliptin , no significant changes in body weight were seen.16 when patients received pioglitazone and either placebo or linagliptin , both groups showed an increase in body weight from baseline . the amount of weight gain was larger in patients receiving linagliptin , but the mean weight for patients receiving linagliptin was lower than that in patients receiving placebo at baseline.18 in general , linagliptin showed a low propensity to cause hypoglycemia . when used as monotherapy , no patients experienced hypoglycemia in the two studies reviewed.11,15 one study reviewing linagliptin as monotherapy versus placebo reported hypoglycemia occurring in one patient in each group.12 when combined with metformin and sulfonylurea , a higher percentage of patients receiving linagliptin experienced hypoglycemia versus placebo . however , a smaller percentage of patients experienced severe hypoglycemia when compared with placebo.16 three studies discussed or reported the occurrence of pancreatitis . one study reported zero cases15 while another study reported one case of pancreatitis in a patient receiving linagliptin.17 a 78-week , open - label extension study , which included 2121subjects , reported four cases of pancreatitis in patients who had received linagliptin for a total of 102 weeks , with two cases being acute and two chronic . this was an incidence of 0.2% in the overall treated set.19 according to the prescribing information , pancreatitis was reported more often in patients treated with linagliptin ( 21.9 per 10,000 patient years ) versus placebo ( eight per 10,000 patient years).1 one study prospectively assessed cardiovascular safety for linagliptin versus sulfonylurea ( e.g. glimepiride ) . major cardiovascular events occurred in 2% of patients treated with linagliptin and 3% treated with glimepiride ( p = 0.0213 ) . this finding was mainly attributable to a significantly lower number of nonfatal strokes with linagliptin compared with glimepiride , without any relation to hypoglycemia.17 data from the clinical trials suggest that linagliptin administered as monotherapy or in combination with other antidiabetic therapies improves hba1c and reduces fasting plasma glucose.1119 when used as monotherapy , linagliptin resulted in a placebo - corrected change in hba1c ranging from 0.28% to 0.69%.11,12 when linagliptin was added to metformin or metformin and a sulfonylurea , similar hba1c reductions ranging from 0.39% to 0.75% were observed.13,14,16 when comparing linagliptin with glimepiride as add - on therapy to metformin , a numerically greater response was seen with glimepiride , but this was not statistically significant . however , when linagliptin was used in combination with pioglitazone , larger reductions in placebo - corrected hba1c of 1.06% were seen.18 those studies that evaluated the impact of linagliptin therapy on postprandial plasma glucose also reported an improvement.1113 when used as monotherapy , linagliptin decreased postprandial plasma glucose in the range of 27.257.7 mg / dl , and when used in combination with metformin , postprandial plasma glucose decreased by 66.7 mg / dl.1113 with this , the data suggest linagliptin used as monotherapy or in combination with other antidiabetic therapies offers improvement in glycemic control . specific populations that may particularly benefit from linagliptin therapy should also be considered . in patients experiencing renal impairment precluding the use of metformin , linagliptin may have a niche in managing glycemia because it does not require dose adjustment in renal compromise . several of the studies discussed in this review stratified the change in hba1c according to the baseline value . reduction in hba1c was greater in patients with a baseline hba1c > 9% , offering another possible niche for linagliptin therapy . dpp-4 inhibitors as a class are generally well tolerated . a minimal risk of hypoglycemia when used as monotherapy and lack of weight gain are some of the desirable characteristics of this class of medications . overall , linagliptin has been shown to be well tolerated , with adverse events similar to others within its class . it is important to note that although linagliptin offers a low risk of hypoglycemia , this risk increases when this agent is combined with secretagogue therapy . pancreatitis is also of concern and is a class effect of dpp-4 inhibitors , although the risk of the condition seems very low with this medication . a long - term safety and efficacy study evaluated linagliptin therapy in over 2000 patients for a total of 102 weeks and found an overall incidence of 0.2%.19 however , recent discussions have noted that the prevalence of pancreatitis among patients with type 2 diabetes is similar to that seen with incretin hormones . a study published in 2009 by noel et al found that patients with type 2 diabetes had an almost three - fold greater risk of pancreatitis than those patients without diabetes.20 this information suggests that there may not be an increased risk of pancreatitis with incretin therapy . it has a long half - life and undergoes less renal excretion , avoiding the need for dose adjustments in patients with renal impairment.6,9 however , to date , there are no head - to - head studies comparing the efficacy of this agent with other dpp-4 inhibitors in its class . linagliptin is a newly approved dpp-4 inhibitor for use as a once - daily oral medication in the treatment of type 2 diabetes . the use of linagliptin as monotherapy or in combination with metformin or pioglitazone led to reductions in hba1c and fasting plasma glucose after 1224 weeks of therapy . this improvement in glycemic control was shown to be maintained for up to 102 weeks . it is generally considered to be weight neutral , unless used in combination with a thiazolidinedione , and has a low risk of hypoglycemia .

background : the purpose of this paper is to review the efficacy , safety , and tolerability of linagliptin in the management of hyperglycemia in adults with type 2 diabetes mellitus.methods:a medline search was performed using the keywords linagliptin and type 2 diabetes for articles published september 2010 through july 2012 . the literature search was limited by the following criteria : articles publication in the english language , clinical trials , randomized controlled trials , and research conducted in humans.results:a review of the data for linagliptin in the treatment of type 2 diabetes as monotherapy or in combination with other antidiabetic therapies suggests clinical efficacy in terms of reductions in glycosylated hemoglobin , fasting plasma glucose , and postprandial glucose . most adverse events with linagliptin are considered to be mild to moderate in nature . although linagliptin therapy may offer a low risk of hypoglycemia , the risk increases when it is used in combination with insulin secretagogues . linagliptin can generally be considered weight neutral , but a weight increase was observed when linagliptin was used in combination with a thiazolidinedione.conclusion:linagliptin is a once - daily oral medication used for the treatment of type 2 diabetes . the use of linagliptin as monotherapy or in combination with metformin , sulfonylureas , or pioglitazone led to improvement in glycemic control and was well tolerated by most patients .

Introduction Materials and methods Results Linagliptin monotherapy Combination therapy Linagliptin versus placebo as add-on therapy to metformin Linagliptin + metformin versus linagliptin alone, metformin alone, and placebo Linagliptin versus placebo in combination with metformin and sulfonylurea Linagliptin versus glimepiride in combination with metformin Linagliptin versus placebo as add-on to pioglitazone therapy Open-label extension: linagliptin monotherapy or in combination with other oral antidiabetic therapies Safety and tolerability Discussion Conclusion

linagliptin ( tradjenta , boehringer ingelheim , ridgefield , ct , usa ) is a newly approved medication for the treatment of type 2 diabetes mellitus.1 this agent is a potent inhibitor of the serine protease enzyme , dipeptidyl peptidase-4 ( dpp-4 ) , which is responsible for rapid degradation of two incretin hormones , glucagon - like - peptide 1 ( glp-1 ) and glucose insulinotropic polypeptide ( gip ) . lastly , glp-1 can increase -cell mass by decreasing apoptosis and by increasing proliferation and neogenesis of -cells.3 however , this has only been shown in animal models , with no evidence of this noted in humans as yet.4 gip is secreted from the k - cells of the intestine wall , stimulates insulin secretion from the pancreas , and has been shown to decrease cellular death and increase regeneration of -cells.3 linagliptin has been shown to be a potent long - acting dpp-4 inhibitor . an in vitro study showed that linagliptin inhibited dpp-4 with a 50% inhibition concentration ( ic50 ) of about 1 nm , compared with sitagliptin ( 19 nm ) , alogliptin ( 24 nm ) , saxagliptin ( 50 nm ) , and vidagliptin ( 62 nm).5 linagliptin has an elimination half - life of 131 hours,6 and achieves steady - state concentrations after three doses of 5 mg daily.1 linagliptin has also been shown to inhibit dpp-4 activity by more than 80% over 24 hours.68 the presence of these characteristics allows for once - daily oral dosing.7 linagliptin undergoes primarily hepatic elimination , with approximately 85% of the drug excreted unchanged in the feces.9 despite having a predominately hepatic route of elimination , the main metabolite is pharmacologically inactive.1 the overall pharmacokinetic profile of linagliptin may avoid the need to adjust the dose in patients with renal or hepatic impairment . current guidelines recommend that these treatments be considered as part of a patient - centered approach and be used as a component of a two - drug or three - drug regimen in conjunction with metformin if a patient does not meet their individualized glycosylated hemoglobin ( hba1c ) goal.10 a medline search was performed using the keywords linagliptin , dpp-4 inhibitor , and type 2 diabetes for articles published through july 2012 . the literature search was limited by the following criteria : publication in the english language , clinical trials , randomized controlled trials , and research conducted in humans ( figure 1 ) . this phase iia study conducted by forst et al followed a randomized , double - blind , within - dose level , parallel , placebo - controlled design and examined the pharmacokinetic and pharmacodynamic properties of linagliptin in patients with type 2 diabetes after 4 weeks of treatment.11 participants enrolled in this study were either treatment - nave or had received up to two oral antidiabetic therapies other than a thiazolidinedione . statistically significant decreases in fasting plasma glucose and postprandial plasma glucose were also observed from baseline to the end of the study period for all linagliptin doses ( see table 1 ) . another randomized , double - blind , parallel - group study comparing treatment with either linagliptin 5 mg or placebo for 24 weeks in patients with type 2 diabetes was conducted by del prato et al.12 patients were aged 1880 ( mean 55.7 ) years with a body mass index 40 kg / m , and were either treatment - nave or previously treated with one oral antidiabetic therapy other than a thiazolidinedione . taskinen et al performed a randomized , double - blind , placebo - controlled , multicenter , parallel - group study in 701 patients with type 2 diabetes aged 1880 years.13 subjects included had a mean age of 56.5 years , a body mass index 40 kg / m , and a mean baseline hba1c of 8.1% . at the end of the study , linagliptin reduced the mean hba1c level by 0.49% , whereas hba1c in the placebo group rose by 0.15% ( p linagliptin also led to significant reductions versus placebo in both fasting plasma glucose and postprandial plasma glucose ( p < 0.0001 , see table 2 ) . haak et al conducted a 24-week , randomized , double - blind , placebo - controlled phase iii trial in 791 patients who were either treatment - nave or had been treated with one other antidiabetic therapy.15 eligible patients were 1880 years of age , had a diagnosis of type 2 diabetes , and had a body mass index of 40 kg / m . as add - on to metformin , both linagliptin and glimepiride caused significant reductions in fasting plasma glucose and postprandial plasma glucose . the treatment differences for reductions in fasting and postprandial plasma glucose , respectively , were 6.31 mg / dl ( p = 0.012 ) and 9.73 mg / dl ( p = 0.0918 , see table 2 ) . the 12-week , multicenter , randomized , double - blind , placebo - controlled , five parallel - group study conducted by forst et al included patients with type 2 diabetes aged 2175 ( mean 60 ) years with a body mass index of 2540 kg / m.14 patients were eligible if they were pretreated with metformin alone ( baseline hba1c levels had to be 7.5%10% ) or treated with metformin and one other oral antidiabetic therapy other than a thiazolidinedione ( baseline hba1c levels had to be 7.0%9.0% ) . a 78-week open - label extension conducted by gomis et al evaluated participants who had previously completed one of the four 24-week , randomized , double - blind , placebo - controlled parent trials.19 these subjects received either linagliptin monotherapy , linagliptin plus metformin , linagliptin plus metformin and sulfonylurea , or linagliptin plus pioglitazone . in participants randomized to treatment with linagliptin in the four previous trials , the mean change from baseline hba1c during the initial 24 weeks of treatment was 0.8% . most adverse events with linagliptin were considered to be mild to moderate in nature . adverse reactions that occurred in 2% of patients treated with linagliptin included nasopharyngitis , diarrhea , cough , urinary tract infection , and hypertriglyceridemia ( in combination with sulfonylurea therapy ) , hyperlipidemia , and weight increase ( in combination with pioglitazone).1 weight changes were reported or addressed in each of the studies above . when used as monotherapy , no patients experienced hypoglycemia in the two studies reviewed.11,15 one study reviewing linagliptin as monotherapy versus placebo reported hypoglycemia occurring in one patient in each group.12 when combined with metformin and sulfonylurea , a higher percentage of patients receiving linagliptin experienced hypoglycemia versus placebo . this finding was mainly attributable to a significantly lower number of nonfatal strokes with linagliptin compared with glimepiride , without any relation to hypoglycemia.17 this phase iia study conducted by forst et al followed a randomized , double - blind , within - dose level , parallel , placebo - controlled design and examined the pharmacokinetic and pharmacodynamic properties of linagliptin in patients with type 2 diabetes after 4 weeks of treatment.11 participants enrolled in this study were either treatment - nave or had received up to two oral antidiabetic therapies other than a thiazolidinedione . statistically significant decreases in fasting plasma glucose and postprandial plasma glucose were also observed from baseline to the end of the study period for all linagliptin doses ( see table 1 ) . another randomized , double - blind , parallel - group study comparing treatment with either linagliptin 5 mg or placebo for 24 weeks in patients with type 2 diabetes was conducted by del prato et al.12 patients were aged 1880 ( mean 55.7 ) years with a body mass index 40 kg / m , and were either treatment - nave or previously treated with one oral antidiabetic therapy other than a thiazolidinedione . treatment with linagliptin also resulted in significant decreases in fasting plasma glucose and postprandial plasma glucose compared with placebo ( see table 1 ) . taskinen et al performed a randomized , double - blind , placebo - controlled , multicenter , parallel - group study in 701 patients with type 2 diabetes aged 1880 years.13 subjects included had a mean age of 56.5 years , a body mass index 40 kg / m , and a mean baseline hba1c of 8.1% . haak et al conducted a 24-week , randomized , double - blind , placebo - controlled phase iii trial in 791 patients who were either treatment - nave or had been treated with one other antidiabetic therapy.15 eligible patients were 1880 years of age , had a diagnosis of type 2 diabetes , and had a body mass index of 40 kg / m . following a 2-week placebo run - in , a total of 1055 participants were randomized to treatment with linagliptin 5 mg once daily or placebo , in addition to the established background therapy of metformin in combination with a sulfonylurea . this randomized , double - blind , parallel - group , active - controlled , noninferiority trial was conducted by gallwitz et al in 1519 patients with type 2 diabetes , aged 1880 years , and a body mass index of 40 kg / m.17 eligible subjects were receiving metformin 1500 mg / day ( or the maximum tolerated dose ) alone with an hba1c of 6.5%10.0% or 6.0%9.0% on metformin and one other oral antidiabetic therapy . as add - on to metformin , both linagliptin and glimepiride caused significant reductions in fasting plasma glucose and postprandial plasma glucose . the treatment differences for reductions in fasting and postprandial plasma glucose , respectively , were 6.31 mg / dl ( p = 0.012 ) and 9.73 mg / dl ( p = 0.0918 , see table 2 ) . the 12-week , multicenter , randomized , double - blind , placebo - controlled , five parallel - group study conducted by forst et al included patients with type 2 diabetes aged 2175 ( mean 60 ) years with a body mass index of 2540 kg / m.14 patients were eligible if they were pretreated with metformin alone ( baseline hba1c levels had to be 7.5%10% ) or treated with metformin and one other oral antidiabetic therapy other than a thiazolidinedione ( baseline hba1c levels had to be 7.0%9.0% ) . in participants randomized to treatment with linagliptin in the four previous trials , the mean change from baseline hba1c during the initial 24 weeks of treatment was 0.8% . taskinen et al performed a randomized , double - blind , placebo - controlled , multicenter , parallel - group study in 701 patients with type 2 diabetes aged 1880 years.13 subjects included had a mean age of 56.5 years , a body mass index 40 kg / m , and a mean baseline hba1c of 8.1% . haak et al conducted a 24-week , randomized , double - blind , placebo - controlled phase iii trial in 791 patients who were either treatment - nave or had been treated with one other antidiabetic therapy.15 eligible patients were 1880 years of age , had a diagnosis of type 2 diabetes , and had a body mass index of 40 kg / m . this randomized , placebo - controlled , double - blind , parallel - group study enrolled subjects with type 2 diabetes receiving metformin 1500 mg / day ( or the maximum tolerated dose ) and the maximum tolerated dose of sulfonylurea.16 patients were 1880 ( mean 58.1 ) years of age , with a body mass index 40 kg / m and hba1c 7.0% and 10.0% ( mean 8.14% ) . as add - on to metformin , both linagliptin and glimepiride caused significant reductions in fasting plasma glucose and postprandial plasma glucose . the treatment differences for reductions in fasting and postprandial plasma glucose , respectively , were 6.31 mg / dl ( p = 0.012 ) and 9.73 mg / dl ( p = 0.0918 , see table 2 ) . the 12-week , multicenter , randomized , double - blind , placebo - controlled , five parallel - group study conducted by forst et al included patients with type 2 diabetes aged 2175 ( mean 60 ) years with a body mass index of 2540 kg / m.14 patients were eligible if they were pretreated with metformin alone ( baseline hba1c levels had to be 7.5%10% ) or treated with metformin and one other oral antidiabetic therapy other than a thiazolidinedione ( baseline hba1c levels had to be 7.0%9.0% ) . this randomized , double - blind , placebo - controlled , multicenter , parallel - group study was conducted by gomis et al in 389 patients with type 2 diabetes and aged 1880 ( mean 57.5 ) years.18 at baseline , the patients had hba1c concentrations of 7.5%11.0% ( mean 8.6% ) and a body mass index 40 kg / m . in participants randomized to treatment with linagliptin in the four previous trials , the mean change from baseline hba1c during the initial 24 weeks of treatment was 0.8% . most adverse events with linagliptin were considered to be mild to moderate in nature . adverse reactions that occurred in 2% of patients treated with linagliptin included nasopharyngitis , diarrhea , cough , urinary tract infection , and hypertriglyceridemia ( in combination with sulfonylurea therapy ) , hyperlipidemia , and weight increase ( in combination with pioglitazone).1 weight changes were reported or addressed in each of the studies above . when used as monotherapy , no patients experienced hypoglycemia in the two studies reviewed.11,15 one study reviewing linagliptin as monotherapy versus placebo reported hypoglycemia occurring in one patient in each group.12 when combined with metformin and sulfonylurea , a higher percentage of patients receiving linagliptin experienced hypoglycemia versus placebo . this finding was mainly attributable to a significantly lower number of nonfatal strokes with linagliptin compared with glimepiride , without any relation to hypoglycemia.17 data from the clinical trials suggest that linagliptin administered as monotherapy or in combination with other antidiabetic therapies improves hba1c and reduces fasting plasma glucose.1119 when used as monotherapy , linagliptin resulted in a placebo - corrected change in hba1c ranging from 0.28% to 0.69%.11,12 when linagliptin was added to metformin or metformin and a sulfonylurea , similar hba1c reductions ranging from 0.39% to 0.75% were observed.13,14,16 when comparing linagliptin with glimepiride as add - on therapy to metformin , a numerically greater response was seen with glimepiride , but this was not statistically significant . however , when linagliptin was used in combination with pioglitazone , larger reductions in placebo - corrected hba1c of 1.06% were seen.18 those studies that evaluated the impact of linagliptin therapy on postprandial plasma glucose also reported an improvement.1113 when used as monotherapy , linagliptin decreased postprandial plasma glucose in the range of 27.257.7 mg / dl , and when used in combination with metformin , postprandial plasma glucose decreased by 66.7 mg / dl.1113 with this , the data suggest linagliptin used as monotherapy or in combination with other antidiabetic therapies offers improvement in glycemic control . a minimal risk of hypoglycemia when used as monotherapy and lack of weight gain are some of the desirable characteristics of this class of medications . it is important to note that although linagliptin offers a low risk of hypoglycemia , this risk increases when this agent is combined with secretagogue therapy . a long - term safety and efficacy study evaluated linagliptin therapy in over 2000 patients for a total of 102 weeks and found an overall incidence of 0.2%.19 however , recent discussions have noted that the prevalence of pancreatitis among patients with type 2 diabetes is similar to that seen with incretin hormones . linagliptin is a newly approved dpp-4 inhibitor for use as a once - daily oral medication in the treatment of type 2 diabetes . the use of linagliptin as monotherapy or in combination with metformin or pioglitazone led to reductions in hba1c and fasting plasma glucose after 1224 weeks of therapy . this improvement in glycemic control was shown to be maintained for up to 102 weeks . it is generally considered to be weight neutral , unless used in combination with a thiazolidinedione , and has a low risk of hypoglycemia .

phospholipids containing polyunsaturated fatty acids are highly prone to modification by reactive oxygen species , thereby generating a plethora of biologically active oxidized phospholipids ( oxpls ) . a variety of ( patho- ) physiological effects ascribed to oxpls underline their relevance , e.g. , in inflammation , atherosclerosis,(6 ) or immune response.(7 ) the modes of action are diverse . first , different families of receptors were described to become activated due to oxpl binding.(1 ) second , oxpls were shown to interact with drugs , thereby influencing their pharmacokinetics.(8 ) finally , one may expect the exceptional structure of an oxpl molecule to affect its biophysical properties in the lipid membrane . in this report bioactive oxpls contain extensively modified or truncated acyl chains in the sn-2 position , typically terminated by polar carboxylic or aldehydic groups . as was shown by nuclear magnetic resonance , langmuir balance , and molecular dynamics ( md ) simulations , the truncated polar moiety can protrude into the aqueous phase , despite the energy penalty associated with the exposition of the nonpolar chain regions . as a consequence , bilayer properties such as permeability , mobility , and headgroup hydration we have recently studied the diffusional properties of a fluorescent oxpl analogue , 1-palmitoyl-2-glutaroyl - sn - glycero-3-phospho - n - alexa647-ethanolamine ( pgpe - alexa647 ) , in the live cell plasma membrane , and found exceptionally high mobility of 2 m / s,(15 ) in agreement with the expected lysolipid - like behavior . moreover , we observed transient immobilization at endocytic sites , which we attributed to a preferential partitioning within highly curved membrane regions due to the inverted cone - like shape of the pgpe - alexa647.(15 ) to better understand the behavior of oxpl - molecules in membranes we decided to further address its properties in well - defined model systems . we used pgpe - alexa647 as a representative carboxylated oxpl ; we have previously shown that this fluorescent analogue mimics closely the behavior of the nonlabeled molecule in living cells.(16 ) all data were referenced against a conventional headgroup labeled fluorescent phospholipid , dihexadecanoylphosphoethanolamine ( dhpe)-bodipy . first , we were interested whether the probe displays any preference with respect to the phase state of the membrane . the cellular plasma membrane is believed to be segregated into domains,(17 ) where a more ordered ( raft- ) phase shall coexist with a disordered phase.(18 ) generation of phase - separated model membranes has become standard in many laboratories ( for reviews see , e.g. , refs ( 1921 ) ) and provided a wealth of insights into the thermodynamics of lipid bilayers . indeed , a few reports confirmed the presence of ordered environments also in the cellular plasma membrane . in this study , we found a moderate preference of the pgpe - alexa647 for the liquid - disordered phase but also significant partitioning into the liquid ordered phase , indicating a rather promiscuous localization . second , in order to explain the high mobility of pgpe - alexa647 observed in the live cell plasma membrane , we further characterized the diffusion constant in various model membranes . experiments were performed on supported lipid bilayers ( slbs ) using line - scan fluorescence correlation spectroscopy ( fcs)(25 ) and single molecule tracking.(26 ) substantially higher oxpl mobility was observed consistently ; it could be diminished by increasing cholesterol content and intensified by introducing artificial obstacles . 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dioleoylphosphatidylcholine ; dopc ) , sphingomyelin from porcine brain ( brsm ) , n - octadecanoyl - d - erythro - sphingosine ( c18 ceramide ; cer ) , and cholesterol were purchased from avanti polar lipids ( alabaster , al , u.s.a . ) and used without further purification . two different fluorescent lipids were used as probes : n-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a - diaza - s - indacene-3-propionyl)-1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine and triethylammonium salt ( bodipy fl dhpe , invitrogen , carlsbad , ca , u.s.a . ) ; the oxidized phopholipid 1-palmitoyl-2-glutaroyl - sn - glycero-3-phospho - n - alexa647-ethanolamine ( pgpe - alexa647 ) was synthesized as described previously.(16 ) optical adhesive 88 , used to glue the mica on coverslips , was purchased from norland products inc . two different buffers were used for sample preparation : buffer a ( 150 mm nacl , 10 mm hepes , and 3 mm nan3 , ph 7.4 ) and buffer b ( pbs - buffer ; paa pasching , austria ) . buffer a was filtered through a 0.2 m filter ( nalgene , rochester , ny , u.s.a . ) prior to use . 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(cap biotinyl ) ( sodium salt ) ( 18:1 biotinyl cap pe ; dope - biotin ) was purchased from avanti polar lipids ( alabaster , al , u.s.a . ) . glass slides ( menzel , # 1 , braunschweig , germany ) were incubated in a 3:1 piranha solution of sulfuric acid ( j.t . baker , 9597% , new jersey , u.s.a . ) and hydrogen peroxide ( merck , 30% , new jersey , usa ) for 20 min , rinsed with deionized water and ethanol , and dried by nitrogen . glass slides were then glued on a measurement chamber ( lab - tek , nunc , thermo fisher scientific , rochester ) from which the glass support has been removed . a total of 10 mg of dopc was dissolved in a mixture of methanol and chloroform ( 1:3 ) . then , 10 l of a 10 mg / ml dopc solution was evaporated under nitrogen stream and diluted with 100 l buffer b. vesicle solutions were prepared by ultrasonicating for 20 min . the accrued vesicle solution was put on a glass slide or avidin - coated surface . after 20 min the bilayer had been formed and was washed with buffer b. fluorescently labeled lipids were incorporated after bilayer formation by incubation from the aqueous subphase : for this , bilayers were incubated with a 2,5 nm solution of dhpe - bodipy and with a 5 nm solution of pgpe - alexa647 for another 20 min , followed by thorough washing with buffer b. avidin - coated glass surfaces were prepared by incubating a piranha - cleaned glass - coverslip with a 10 mg / ml avidin solution for 20 min , and subsequent washing with buffer b. dopc , bsm , and cholesterol were mixed in organic solution ( 1:3/methanol : chloroform ) in a molar ratio of 2:2:1 . after solvent evaporation , the lipid film thus obtained was slowly rehydrated using buffer b at 10 mg / ml lipid concentration and resuspended through vigorous vortexing . after sonicating the suspension at 60 c , a small aliquot was diluted in buffer a and deposited in the presence of 2 mm cacl2 on a 10 m thick , freshly cleaved mica glued onto a glass coverslip . the coverslip was sealed with a home - built polypropylene chamber and incubated at 55 c for 5 min . after that , the sample was rinsed at the same temperature at least 10 times with buffer a and allowed to cool down to 25 c . at this stage , fluorescently labeled lipids could be incorporated into the bilayer by addition from the aqueous subphase . the concentration of dhpe - bodipy was varied between 0.1 to 0.01 mol % and for pgpe - alexa647 from 0.5 to 0.01 mol % . after 20 min , the sample was rinsed again to remove the fluorescent lipids from the buffer solution . single molecule experiments were performed as described previously.(27 ) briefly , a zeiss axiovert 200 microscope was equipped with a 100 na=1.46 plan - apochromat objective ( zeiss , oberkochen germany ) . samples were illuminated in objective - type total internal reflection ( tir ) configuration via the epiport using 488 nm light from an ar laser ( model 2017 - 05ar , spectra physics , mountain view , ca , u.s.a . ) with an intensity of typically 911 kw / cm and 647 nm light from a kr laser ( stabilite 2017-kr , spectra physics ) with intensities of typically 510 kw / cm . a slit aperture ( zeiss ) with a width of 7 m in the object plane was used as field stop to confine the illumination area . after appropriate filtering ( z488 647mv2 , z488 647rpc , chroma , vt , u.s.a . ) , emitted signals were split into two color channels using a custom - made dichroic wedge ( chroma ) and imaged on the same back - illuminated , liquid - nitrogen - cooled ccd camera ( micro max 1300-pb , roper scientific , trenton , nj , u.s.a . ) . for the precise control of all laser pulse trains , an acoustic - optical modulator ( 1205c , isomet , springfield , va , u.s.a . ) was used . timing protocols were generated and controlled by an in - house program package implemented in labview ( national instruments , austin , tx , u.s.a . ) . experiments were performed in a home - built incubator box equipped with a heating unit , a temperature - adjustable stage insert and an objective heater ( pecon , erbach , germany ) . signals were analyzed by fitting a two - dimensional gaussian profile , yielding the position with an accuracy of 50 nm . the sample was mounted on a high - precision xy - stage ( scan i m 120 100 , mrzhuser , germany ) . for single molecule analysis , images were analyzed using in - house algorithms implemented in matlab ( mathworks , natick , ma , u.s.a.).(27 ) individual diffraction limited signals were selected and fitted with a gaussian profile , yielding the single molecule position , the brightness b and the full width at half - maximum ( fwhm ) of the gaussian function . single molecule mobility was analyzed as described previously.(27 ) in brief , trajectories are specified by a sequence of positions x(t ) , with i ranging from 1 to the number of observations of this trajectory . the mean square displacements r were calculated as a function of the time - lag tlag = n(till + tdelay ) according to r = x(t + tlag ) x(t)) , with n denoting the difference in frame index . estimated data were analyzed by fitting with the functionyielding the lateral diffusion constant d and the single molecule localization precision . only the first two data - points were used for the linear fit.(27 ) line - scan fluorescence correlation spectroscopy ( lsfcs ) was performed at room temperature ( 25 c ) on a lsm 510 meta ( zeiss , jena , germany ) as described in ref ( 25 ) . the fiber output was coupled to a home - built fcs detection unit , consisting of an emission filter and an achromatic doublet ( linos photonics , goettingen , germany ) , to image the internal pinhole onto the optical fiber connected to an avalanche photodiode ( apd ) ( perkin - elmer , boston , ma , u.s.a . ) . correlation curves were obtained with a hardware correlator ( correlator.com , bridgewater , nj , u.s.a . ) . for confocal fluorescence microscopy , the excitation light of an argon laser at 488 nm ( or a hene laser at 543 nm ) was reflected by a dichroic mirror ( hft 488/543/633 ) and focused onto the sample by a zeiss c - apochromat 40 , na ) 1.2 , uvvisir water immersion objective . the fluorescence signal was then recollected by the same objective and , after passing through a 525/50 bandpass filter ( or a 580/60 bandpass filter ) , measured by a photomultiplier ( pmt ) . the confocal geometry was ensured by a 70 m ( 80 m ) pinhole in front of the pmt . fcs measurements were performed using the same optical path described for the fluorescence imaging , the signal from the sample being collected in this case by the avalanche photodiodes in the fcs unit . the membranes simulated in this work consisted of 2500 lipids per leaflet and contained 50 water molecules per lipid ( corresponding to 12.5 water beads in the coarse grained representation ) . the content of cholesterol was increased from 0% to 50% by substituting dopc with cholesterol . additionally , 1% of the lipid molecules was the oxidized lipid 1-palmitoyl-2-glutaroyl - sn - glycero-3-phosphocholine ( pgpc ) introduced by substituting dopc with pgpc ( figure 4a ) . the lipids were randomly placed in the plain of the bilayer and rotated using an axis normal to the membrane . atom overlaps were resolved by first expanding the bilayer in the membrane plane , followed by a size reduction in small steps to the typical area per lipid of a dopc - cholesterol mixture , involving energy minimization and geometry optimization for each deflation step.(28 ) the systems was further equilibrated by first carrying out 10 and 50 ns equilibration runs with first restraining in z the phosphate of the phospholipids and the polar oxygen of the cholesterol and then only restraining the phospholipids . after 100 ns of unconstrained equilibration we carried out a production run of 200 ns . the parameters of the oxidized tail of the pgpc lipid were extracted from all atom simulations of pgpc in 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ) using berger lipids,(31 ) missing parameters were taken from the force field and from wong - ekkabut et al.(14 ) and using the spc water model.(32 ) the truncated oxidized glycero - lipid tail was coarse grained into two beads , one containing the charged carboxy terminus , one consisting of the aliphatic carbons of the chain . bead distances and bond and dihedral angles were measured and added to the coarse grained description of pgpc . a constant temperature of 310 k was maintained using the velocity rescale ( v - rescale ) algorithm(35 ) with a coupling time of 0.3 ps , coupling independently the water and the lipids plus ions to an external bath . a pressure of 1 bar was maintained in both the membrane plane and the membrane normal , using semi - isotropic berendsen pressure coupling scheme(36 ) with a time constant of 3 ps . a shift function was applied to the van der waals interactions between 0.9 and 1.2 nm , and the electrostatic interactions were shifted over the entire range from 0 to 1.2 nm . diffusion constants were calculated by fitting the linear part of the mean square displacment curves with r = 4dtlag , after removing overall translation by fitting of the dopc molecules . it is a known limitation of the coarse grained martini force field that on an absolute scale the velocity of molecules are overestimated and scaling up to an order of magnitude has been suggested.(29 ) we applied a scaling factor of 15 to obtain accordance on the absolute scale of the experimentally observed diffusion rates . the density profiles were calculated using standard procedures after removing translational shifts of the membranes , which were determined from the mean position of all dopc headgroups . the positions of the pgpc and dopc phosphate groups along the membrane normal were extracted from the density plots by fitting the phosphate group density peak with a gaussian function ( figure 5 ) . distances between each phospholipid molecule ( represented by its phosphate group ) and the nearest cholesterol molecule ( represented by its hydroxyl group ) were calculated for each time point within a trajectory ( figure 6 ) . the resulting histograms ( bin - width 0.02 nm ) were averaged for all phospholipids in the simulation . to address the phase preference of pgpe - alexa647 , we studied its partitioning in supported lipid bilayers containing bsm : dopc : chol in a molar ratio of 2:2:1 ( figure 1 ) , which shows separation in liquid ordered ( l.o . ) and disordered ( l.d . ) phase.(39 ) pgpe - alexa647 and dhpe - bodipy were added from the aqueous phase to ensure that the probes inserted only in the upper leaflet ; flip - flop is too slow to yield probe redistribution between leaflets during the experimental time frame of less than 1 h.(40 ) while the dhpe - bodipy was largely excluded from the ordered phase ( green color channel ) , we observed only weak contrast for pgpe - alexa647 . we performed line - scan fcs to determine accurate values of the surface densities in the ordered ( lo ) versus the disordered phase ( ld ) , yielding the partition coefficients k = lo/ld for pgpe - alexa647 ( k = 0.75 ) and for dhpe - bodipy ( k = 0.32 ) ( see table 1 ) . we also calculated the diffusion constants of the probe molecules , yielding higher mobility of the oxidized phospholipid analogue versus dhpe - bodipy in both phases ( table 1 ) . partitioning and mobility of pgpe - alexa647 and dhpe - bodipy in phase - separated supported lipid bilayers . panels ac show confocal fluorescence images of a dopc / bsm / chol 2:2:1 supported bilayer doped with trace amounts dhpe - bodipy and pgpe - alexa647 : an overlay , the bodipy - channel and the alexa647-channel are shown in panels ac , respectively . we performed line - scan fcs to quantify partition coefficients and mobilities ( sketch in the inset to a : the blue cone shows the detection volume , the blue and green encircled areas correspond to the l.o . and ( d ) representative averaged autocorrelation curves measured in different lipid phases of the bilayer at room temperature . shown phase ( upper figures ) and the l.o . phase ( bottom figures ) for the two different probes dhpe - bodipy ( left ) and pgpe - alexa647 ( right ) . data were fitted for two - dimensional diffusion , yielding the diffusion constants d and the surface densities . the obtained fit values are listed in table1 . the rather high l.o .- phase partitioning of pgpe - alexa647 lead us to the hypothesis that ordered phases in general may be well accessible by the oxpl . we thus investigated a bilayer composed of bsm : dopc : chol : cer in a molar ratio of 0.64:1:1:0.36 , which yields a two phase equilibrium between a ceramide - enriched gel - like phase and a liquid disordered phase.(41 ) previous data revealed efficient exclusion of all investigated fluorescent analogues.(42 ) we indeed confirmed reduced partitioning into the ceramide - enriched phase for both probes . due the low partitioning , we estimated the surface densities directly from the confocal images : a partition coefficient of cer/ld = 0.004 was calculated for dhpe - bodipy ; yet , also in this case ceramide - phase partitioning of pgpe - alexa647 was significantly higher ( cer/ld = 0.01 ) . phase was reduced compared to the ternary system for both probes , and the mobility difference basically vanished ( table 1 ) . probe mobility in the ceramide - enriched gel - like phase was too low to be measurable . in summary , the oxidized lipid analogue shows rather weak phase preference ; in general , its mobility is higher than the mobility of a conventional phospholipid . we continued by putting our focus on the mobility difference between pgpe - alexa647 and dhpe - bodipy . glass - supported lipid bilayers of dopc were prepared and studied at 25 or 37 c . after bilayer formation , pgpe - alexa647 and dhpe - bodipy were added from the aqueous phase . we used single molecule tracking to determine the diffusion constant of both lipid analogues in the same bilayer regions . to eliminate potential influences due to the immobilization of molecules at surface defects we started each recording sequence by a photobleaching pulse , which irreversibly destroyed all fluorophores in the field of view ( figure 2a ) . after a recovery time of 0.52 s , unbleached fluorescent lipid molecules have entered the field of view and could be tracked . by this measurement mode , we could restrict the analysis to a clean fraction of freely diffusing probe molecules . for our experiments , we used stroboscopic illumination with a time delay of tdel = 2 ms between two consecutive illumination pulses and an illumination time till = 1 or 3 ms . from the single molecule trajectories , the distribution of step sizes was determined and further analyzed to assess potential heterogeneity in the sample mobility , which would yield deviations from a monoexponential behavior . however , we found here for all bilayers and probe molecules purely monoexponential functions ( exemplified in figure 2b ) . we also tested for anomalous subdiffusion behavior , being indicative for confinements of the tracer or nonequilibrated systems:(27 ) when plotting the mean square displacement ( r ) as function of the time - lag ( tlag ) , anomalous subdiffusion would yield a sublinear increase . however , for all data sets we found perfectly linear relationships ( exemplified in figure 2c ) . we found substantially higher mobility for pgpe - alexa647 compared to dhpe - bodipy ( figure 3 ) : at 25 c the oxpl showed a 1.3-fold higher diffusion constant than the conventional phospholipid , at 37 c we found even a ratio of 2.2 . interestingly , this difference was reduced and finally disappeared for dopc bilayers containing increasing amounts of cholesterol ( figure 3 ) . ( a ) the upper row sketches the applied laser timing protocol , the lower row an example of an image sequence . after recording a prebleach image the according area was totally photobleached by applying a laser pulse for tbleach250 ms . the photobleaching efficiency was controlled by recording an image immediately after the bleach pulse . after a recovery time of trec2000 ms , single fluorescently labeled lipids entering the field of view can be resolved as diffraction limited signals . by recording an image sequence with high time resolution ( till = 3 ms , tdelay = 2 ms ) panel b shows the cumulative density function cdf of square displacements between consecutive images at tlag = 10 ms for pgpe - alexa647 . ( c ) exact values for diffusion constants d were calculated by plotting r versus tlag , and fitting with eq 1 . a clear difference for d is observed by comparing pgpe - alexa647 ( dark gray line , d = 7.2 0.22 m / s ) and dhpe - bodipy ( gray line , d = 3.1 0.09 m / s ) . diffusion constant of dhpe and pgpe in a dopc / cholesterol bilayer as a function of cholesterol content , determined by single molecule tracking . dopc bilayers containing up to 50% cholesterol were labeled with pgpe - alexa647 ( full circles ) and dhpe - bodipy ( open circles ) . experiments were performed at 37 c ( a ) or 25 c ( b ) . to understand this behavior , we performed coarse - grained md simulations of a dopc matrix containing low concentrations of pgpc and varying concentrations of cholesterol . figure 4a shows a snapshot of the membrane : consistent with previous all - atom simulations on similar compounds , we observed the reversal of the truncated acyl - chain in the sn-2 position of pgpc , which frequently protruded out of the membrane into the aqueous subphase . no aggregation or alignments of the analyzed lipids was observable on length scales > 5 nm , indicating that influences of potential membrane undulations can be neglected.(46 ) we determined the diffusion coefficient for both dopc and pgpc by calculating r and fitting to the linear region according to r = 4dtlag . at zero cholesterol we observed a 1.4-fold higher mobility of the oxpl pgpc compared to the matrix lipid dopc , similar to our experimental data recorded at 25 c . since pgpc carries essentially only one chain that is inserted into the hydrophobic core of the membrane , the consequentially small area of the lipid appears to be responsible for its high mobility . dopc is show in gray , pgpc is show in blue ( phosphate group in light blue ) , and cholesterol is show in orange . ( b ) effects of increasing cholesterol on the lateral diffusion of pgpc ( full circles ) and dopc ( open circles ) . strikingly , the simulations also revealed a convergence of the mobility ratio to 1 with increasing cholesterol concentrations ( figure 4b ) , in perfect agreement to our experimental data . electron density profiles gave a first hint on the origin of the effect ( figure 5 ) . at zero cholesterol content , the pgpc headgroup region ( indicated by the phosphate group ) is shifted away from the bilayer center by 1.5 compared to the dopc headgroup region . this effect diminished with increasing cholesterol concentration down to a shift of 0.8 at 40% cholesterol . the mean interaction energy between pgpc and the host matrix increased linearly with increasing cholesterol concentration ( figure 5c ) , indicating an improved accommodation of the oxpl in the membrane . taken together , the simulations indicate that increasing cholesterol content leads to a shift of pgpc toward the bilayer center , which is accompanied by facilitated contacts and stronger interactions with the host lipids and consequentially an adaption of the mobility values between probe and matrix molecules . ( a ) total electron density profiles of dopc and cholesterol are shown along with the electron density of the phosphate groups of pgpc and dopc , which are scaled for better visualization . ( b ) we calculated the peak - shift in the maxima of the phosphate groups for dopc versus pgpc , which is shown as function of cholesterol concentration . error bars were determined by comparing peak position of the first half of the trajectory with the second half and by differences in peak to peak distances between upper and lower leaflets . ( c ) the cumulative potential energy of interaction with all membrane components per pgpc molecule is shown relative to the value obtained at 0% cholesterol content . addition of cholesterol to the membrane increases the interaction strength while no changes where observed for interaction with the aqueous phase ( not shown ) . we also analyzed the average distance of dopc or pgpc to their nearest cholesterol molecule ( figure 6 ) . two peaks are clearly visible : the first one representing probe molecules with directly bound cholesterol and the second one representing probe molecules that are shielded by dopc from the nearest cholesterol . for dopc , two effects can be observed when increasing the cholesterol concentration : the first peak gets more pronounced , as the likelihood for direct contact with cholesterol increases , and the distance to the second peaks gets smaller , reflecting the condensing effect of cholesterol . first , in contrast to dopc , the first peak is substantially lower than the second peak at low cholesterol concentrations ; the ratios approach the dopc - case at high cholesterol content . this means that pgpc has a smaller preference for being associated with cholesterol than with dopc , most visible at low cholesterol content . second , the location of the second peak does not shift as strongly as for dopc , indicating that the condensing effect in the immediate proximity of pgpc is less pronounced . distribution of distances r between each phospholipid molecule ( represented by its phosphate group ) and the nearest cholesterol molecule ( represented by its hydroxyl group ) . plots represent averages over the entire trajectory and over all phospholipids of ( a ) pgpc and ( b ) dopc . high cholesterol concentrations level out the differences in the mobility of oxpl and conventional lipids . it is thus difficult to reconcile the exceptionally high mobility of pgpe - alexa647 in the plasma membrane ( d = 1.32.4 m / s(15 ) ) with the high native cholesterol concentrations of 3040% . in comparison , standard lipids or lipid - anchored proteins show a mobility which is 5- to 10-fold lower . we suspected that the diffusion matrix in the plasma membrane may be highly structured , providing essentially a percolation matrix . to enforce this effect in our model system , we artificially introduced obstacles in the proximal leaflet by immobilizing a substantial fraction of lipids . interleaflet coupling should transmit the effect to the distal leaflet.(53 ) for this , a dopc bilayer containing 4.2 mol % biotin - dope was prepared on a glass surface coated with avidin . while pgpe - alexa647 mobility was reduced only approximately 2-fold , dhpe - bodipy became basically immobile ( figure 7a , c ) . consistently , addition of 40 mol % cholesterol substantially decreased the mobility of pgpe - alexa647 ( figure 7d ) . the presence of dope - biotin did not influence the mobility of pgpe - alexa647 or dhpe - bodipy in a dopc bilayer on pure glass ( figure 7b ) . bilayers were prepared on glass supports ( a and b ) or glass supports coated with avidin ( c and d ) . diffusion constants were determined for pgpe - alexa647 ( light gray bars ) and dhpe - bodipy ( dark gray bars ) . experiments were performed on bilayers of dopc ( a ) , dopc containing 4.2 mol % biotin - dope ( b and c ) , or bilayers containing 4.2% biotin - dope and 40% cholesterol ( d ) . we studied partitioning and mobility of the oxidized phospholipid pgpe - alexa647 with reference to the conventional phospholipid dhpe - bodipy in different lipid membranes . in summary , we made the following observations : pgpe - alexa647 shows only marginal preference for fluid versus ordered phases . in general , conventional phospholipids show altered phase partitioning compared to one chain lipids ( compare refs ( 54 ) and ( 55 ) ) . it is tempting to follow an argument of the vaz group , who ascribed the rate - limiting step for association of a lyso - lipid with a bilayer to the formation of free area with appropriate size in the membrane surface.(55 ) for the oxpl , single chain insertion is sufficient for thermodynamic equilibrium,(11 ) therefore the formation of a marginal free area will be sufficient for insertion of pgpe - alexa647 . phase show similar susceptibility for the formation of free area on the length scale of a single acyl chain cross sectional area . md simulations revealed that pgpc slightly sticks out of the headgroup region , thereby essentially reducing its effective area , which most likely causes the enhanced mobility.(56 ) the experimental data show that the effect was more pronounced at elevated temperature , indicating that the shift of pgpc out of the bilayer plane is entropically favored . increased cholesterol concentration md simulations of a dopc matrix containing varying cholesterol content and low amounts of pgpc allowed us to further elucidate the effect of cholesterol on pgpc mobility . at low cholesterol concentrations , pgpc is preferentially surrounded by dopc , and its headgroup protrudes out of the headgroup region of the dopc matrix . with increasing cholesterol content , a higher rate of association of pgpc with cholesterol could be observed . thereby the pgpc headgroup is pulled into the bilayer , with the effect of increasing its interactions with the host lipids and decreasing its mobility . moreover , in a four component mixture of bsm : dopc : chol : cer ( 0.64:1:1:0.36 ) , we found hardly any difference in the l.d . we attribute the disappearance of mobility differences to a higher cholesterol concentration in the l.d . phase ; indeed , ceramide is known to displace cholesterol from the l.gel phase,(57 ) thereby increasing the cholesterol content in the fluid phase . the mobility of pgpe - alexa647 is substantially higher than the mobility of dhpe - bodipy in ordered or obstructed matrices . in the liquid ordered phase of a ternary system , a mobility ratio of r = 2.3 was obtained . moreover , when we artificially increased the obstacle density in a fluid supported lipid bilayer by immobilizing biotin - dope in the proximal bilayer leaflet on avidin , the ratio dramatically increased to r = 51.1 . deverall et al . described the effect of random obstacles on lipid mobility by extending the free area theory(56 ) and reported nonlinear dependence on the tracer size.(58 ) essentially , large tracers are more easily stuck between obstacles than small tracers . this effect appears directly relatable to the plasma membrane , where immobilized lipids or acylated proteins on the cytosolic leaflet provide a similar obstructed matrix for tracers located at the exoplasmic leaflet . in summary , we have found that the oxidized phospholipid pgpc and its fluorescent analogue pgpe - alexa647 move with increased mobility compared to conventional phospholipids . the lysolipid - like character enables the probe to enter ordered environments , thereby providing a means for spreading rapidly and homogeneously over complex matrices like the cellular plasma membrane . mobility is not only determined by the fluidity of the matrix , but also by immobile obstacles or by the localization of the probe along the bilayer normal , which can be influenced by the cholesterol concentration .

we investigated the mobility and phase - partitioning of the fluorescent oxidized phospholipid analogue 1-palmitoyl-2-glutaroyl - sn - glycero-3-phospho - n - alexa647-ethanolamine ( pgpe - alexa647 ) in supported lipid bilayers . compared to the conventional phospholipid dihexadecanoylphosphoethanolamine ( dhpe)-bodipy we found consistently higher diffusion constants . the effect became dramatic when immobile obstacles were inserted into the bilayer , which essentially blocked the diffusion of dhpe - bodipy but hardly influenced the movements of pgpe - alexa647 . in a supported lipid bilayer made of 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc ) , the differences in probe mobility leveled off with increasing cholesterol content . using coarse - grained molecular dynamics simulations , we could ascribe this effect to increased interactions between the oxidized phospholipid and the membrane matrix , concomitant with a translation in the headgroup position of the oxidized phospholipid : at zero cholesterol content , its headgroup is shifted to the outside of the dopc headgroup region , whereas increasing cholesterol concentrations pulls the headgroup into the bilayer plane .

Introduction Materials and Methods Results Discussion Conclusions

as was shown by nuclear magnetic resonance , langmuir balance , and molecular dynamics ( md ) simulations , the truncated polar moiety can protrude into the aqueous phase , despite the energy penalty associated with the exposition of the nonpolar chain regions . as a consequence , bilayer properties such as permeability , mobility , and headgroup hydration we have recently studied the diffusional properties of a fluorescent oxpl analogue , 1-palmitoyl-2-glutaroyl - sn - glycero-3-phospho - n - alexa647-ethanolamine ( pgpe - alexa647 ) , in the live cell plasma membrane , and found exceptionally high mobility of 2 m / s,(15 ) in agreement with the expected lysolipid - like behavior . moreover , we observed transient immobilization at endocytic sites , which we attributed to a preferential partitioning within highly curved membrane regions due to the inverted cone - like shape of the pgpe - alexa647. we used pgpe - alexa647 as a representative carboxylated oxpl ; we have previously shown that this fluorescent analogue mimics closely the behavior of the nonlabeled molecule in living cells. first , we were interested whether the probe displays any preference with respect to the phase state of the membrane . in this study , we found a moderate preference of the pgpe - alexa647 for the liquid - disordered phase but also significant partitioning into the liquid ordered phase , indicating a rather promiscuous localization . second , in order to explain the high mobility of pgpe - alexa647 observed in the live cell plasma membrane , we further characterized the diffusion constant in various model membranes . experiments were performed on supported lipid bilayers ( slbs ) using line - scan fluorescence correlation spectroscopy ( fcs)(25 ) and single molecule tracking. (26 ) substantially higher oxpl mobility was observed consistently ; it could be diminished by increasing cholesterol content and intensified by introducing artificial obstacles . 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dioleoylphosphatidylcholine ; dopc ) , sphingomyelin from porcine brain ( brsm ) , n - octadecanoyl - d - erythro - sphingosine ( c18 ceramide ; cer ) , and cholesterol were purchased from avanti polar lipids ( alabaster , al , u.s.a . ) two different fluorescent lipids were used as probes : n-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a - diaza - s - indacene-3-propionyl)-1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine and triethylammonium salt ( bodipy fl dhpe , invitrogen , carlsbad , ca , u.s.a . ) ; the oxidized phopholipid 1-palmitoyl-2-glutaroyl - sn - glycero-3-phospho - n - alexa647-ethanolamine ( pgpe - alexa647 ) was synthesized as described previously. 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(cap biotinyl ) ( sodium salt ) ( 18:1 biotinyl cap pe ; dope - biotin ) was purchased from avanti polar lipids ( alabaster , al , u.s.a . ) after 20 min the bilayer had been formed and was washed with buffer b. fluorescently labeled lipids were incorporated after bilayer formation by incubation from the aqueous subphase : for this , bilayers were incubated with a 2,5 nm solution of dhpe - bodipy and with a 5 nm solution of pgpe - alexa647 for another 20 min , followed by thorough washing with buffer b. avidin - coated glass surfaces were prepared by incubating a piranha - cleaned glass - coverslip with a 10 mg / ml avidin solution for 20 min , and subsequent washing with buffer b. dopc , bsm , and cholesterol were mixed in organic solution ( 1:3/methanol : chloroform ) in a molar ratio of 2:2:1 . at this stage , fluorescently labeled lipids could be incorporated into the bilayer by addition from the aqueous subphase . the concentration of dhpe - bodipy was varied between 0.1 to 0.01 mol % and for pgpe - alexa647 from 0.5 to 0.01 mol % . a slit aperture ( zeiss ) with a width of 7 m in the object plane was used as field stop to confine the illumination area . experiments were performed in a home - built incubator box equipped with a heating unit , a temperature - adjustable stage insert and an objective heater ( pecon , erbach , germany ) . (27 ) individual diffraction limited signals were selected and fitted with a gaussian profile , yielding the single molecule position , the brightness b and the full width at half - maximum ( fwhm ) of the gaussian function . (27 ) in brief , trajectories are specified by a sequence of positions x(t ) , with i ranging from 1 to the number of observations of this trajectory . fcs measurements were performed using the same optical path described for the fluorescence imaging , the signal from the sample being collected in this case by the avalanche photodiodes in the fcs unit . additionally , 1% of the lipid molecules was the oxidized lipid 1-palmitoyl-2-glutaroyl - sn - glycero-3-phosphocholine ( pgpc ) introduced by substituting dopc with pgpc ( figure 4a ) . the lipids were randomly placed in the plain of the bilayer and rotated using an axis normal to the membrane . atom overlaps were resolved by first expanding the bilayer in the membrane plane , followed by a size reduction in small steps to the typical area per lipid of a dopc - cholesterol mixture , involving energy minimization and geometry optimization for each deflation step. the parameters of the oxidized tail of the pgpc lipid were extracted from all atom simulations of pgpc in 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ) using berger lipids,(31 ) missing parameters were taken from the force field and from wong - ekkabut et al. a pressure of 1 bar was maintained in both the membrane plane and the membrane normal , using semi - isotropic berendsen pressure coupling scheme(36 ) with a time constant of 3 ps . a shift function was applied to the van der waals interactions between 0.9 and 1.2 nm , and the electrostatic interactions were shifted over the entire range from 0 to 1.2 nm . diffusion constants were calculated by fitting the linear part of the mean square displacment curves with r = 4dtlag , after removing overall translation by fitting of the dopc molecules . the density profiles were calculated using standard procedures after removing translational shifts of the membranes , which were determined from the mean position of all dopc headgroups . the positions of the pgpc and dopc phosphate groups along the membrane normal were extracted from the density plots by fitting the phosphate group density peak with a gaussian function ( figure 5 ) . to address the phase preference of pgpe - alexa647 , we studied its partitioning in supported lipid bilayers containing bsm : dopc : chol in a molar ratio of 2:2:1 ( figure 1 ) , which shows separation in liquid ordered ( l.o . ) (39 ) pgpe - alexa647 and dhpe - bodipy were added from the aqueous phase to ensure that the probes inserted only in the upper leaflet ; flip - flop is too slow to yield probe redistribution between leaflets during the experimental time frame of less than 1 h.(40 ) while the dhpe - bodipy was largely excluded from the ordered phase ( green color channel ) , we observed only weak contrast for pgpe - alexa647 . we performed line - scan fcs to determine accurate values of the surface densities in the ordered ( lo ) versus the disordered phase ( ld ) , yielding the partition coefficients k = lo/ld for pgpe - alexa647 ( k = 0.75 ) and for dhpe - bodipy ( k = 0.32 ) ( see table 1 ) . we also calculated the diffusion constants of the probe molecules , yielding higher mobility of the oxidized phospholipid analogue versus dhpe - bodipy in both phases ( table 1 ) . partitioning and mobility of pgpe - alexa647 and dhpe - bodipy in phase - separated supported lipid bilayers . panels ac show confocal fluorescence images of a dopc / bsm / chol 2:2:1 supported bilayer doped with trace amounts dhpe - bodipy and pgpe - alexa647 : an overlay , the bodipy - channel and the alexa647-channel are shown in panels ac , respectively . we performed line - scan fcs to quantify partition coefficients and mobilities ( sketch in the inset to a : the blue cone shows the detection volume , the blue and green encircled areas correspond to the l.o . phase ( bottom figures ) for the two different probes dhpe - bodipy ( left ) and pgpe - alexa647 ( right ) . data were fitted for two - dimensional diffusion , yielding the diffusion constants d and the surface densities . the rather high l.o .- phase partitioning of pgpe - alexa647 lead us to the hypothesis that ordered phases in general may be well accessible by the oxpl . due the low partitioning , we estimated the surface densities directly from the confocal images : a partition coefficient of cer/ld = 0.004 was calculated for dhpe - bodipy ; yet , also in this case ceramide - phase partitioning of pgpe - alexa647 was significantly higher ( cer/ld = 0.01 ) . phase was reduced compared to the ternary system for both probes , and the mobility difference basically vanished ( table 1 ) . probe mobility in the ceramide - enriched gel - like phase was too low to be measurable . in summary , the oxidized lipid analogue shows rather weak phase preference ; in general , its mobility is higher than the mobility of a conventional phospholipid . we continued by putting our focus on the mobility difference between pgpe - alexa647 and dhpe - bodipy . after bilayer formation , pgpe - alexa647 and dhpe - bodipy were added from the aqueous phase . to eliminate potential influences due to the immobilization of molecules at surface defects we started each recording sequence by a photobleaching pulse , which irreversibly destroyed all fluorophores in the field of view ( figure 2a ) . by this measurement mode , we could restrict the analysis to a clean fraction of freely diffusing probe molecules . from the single molecule trajectories , the distribution of step sizes was determined and further analyzed to assess potential heterogeneity in the sample mobility , which would yield deviations from a monoexponential behavior . however , we found here for all bilayers and probe molecules purely monoexponential functions ( exemplified in figure 2b ) . we found substantially higher mobility for pgpe - alexa647 compared to dhpe - bodipy ( figure 3 ) : at 25 c the oxpl showed a 1.3-fold higher diffusion constant than the conventional phospholipid , at 37 c we found even a ratio of 2.2 . a clear difference for d is observed by comparing pgpe - alexa647 ( dark gray line , d = 7.2 0.22 m / s ) and dhpe - bodipy ( gray line , d = 3.1 0.09 m / s ) . diffusion constant of dhpe and pgpe in a dopc / cholesterol bilayer as a function of cholesterol content , determined by single molecule tracking . dopc bilayers containing up to 50% cholesterol were labeled with pgpe - alexa647 ( full circles ) and dhpe - bodipy ( open circles ) . to understand this behavior , we performed coarse - grained md simulations of a dopc matrix containing low concentrations of pgpc and varying concentrations of cholesterol . figure 4a shows a snapshot of the membrane : consistent with previous all - atom simulations on similar compounds , we observed the reversal of the truncated acyl - chain in the sn-2 position of pgpc , which frequently protruded out of the membrane into the aqueous subphase . at zero cholesterol we observed a 1.4-fold higher mobility of the oxpl pgpc compared to the matrix lipid dopc , similar to our experimental data recorded at 25 c . since pgpc carries essentially only one chain that is inserted into the hydrophobic core of the membrane , the consequentially small area of the lipid appears to be responsible for its high mobility . ( b ) effects of increasing cholesterol on the lateral diffusion of pgpc ( full circles ) and dopc ( open circles ) . strikingly , the simulations also revealed a convergence of the mobility ratio to 1 with increasing cholesterol concentrations ( figure 4b ) , in perfect agreement to our experimental data . at zero cholesterol content , the pgpc headgroup region ( indicated by the phosphate group ) is shifted away from the bilayer center by 1.5 compared to the dopc headgroup region . this effect diminished with increasing cholesterol concentration down to a shift of 0.8 at 40% cholesterol . the mean interaction energy between pgpc and the host matrix increased linearly with increasing cholesterol concentration ( figure 5c ) , indicating an improved accommodation of the oxpl in the membrane . taken together , the simulations indicate that increasing cholesterol content leads to a shift of pgpc toward the bilayer center , which is accompanied by facilitated contacts and stronger interactions with the host lipids and consequentially an adaption of the mobility values between probe and matrix molecules . ( a ) total electron density profiles of dopc and cholesterol are shown along with the electron density of the phosphate groups of pgpc and dopc , which are scaled for better visualization . ( b ) we calculated the peak - shift in the maxima of the phosphate groups for dopc versus pgpc , which is shown as function of cholesterol concentration . error bars were determined by comparing peak position of the first half of the trajectory with the second half and by differences in peak to peak distances between upper and lower leaflets . ( c ) the cumulative potential energy of interaction with all membrane components per pgpc molecule is shown relative to the value obtained at 0% cholesterol content . addition of cholesterol to the membrane increases the interaction strength while no changes where observed for interaction with the aqueous phase ( not shown ) . for dopc , two effects can be observed when increasing the cholesterol concentration : the first peak gets more pronounced , as the likelihood for direct contact with cholesterol increases , and the distance to the second peaks gets smaller , reflecting the condensing effect of cholesterol . first , in contrast to dopc , the first peak is substantially lower than the second peak at low cholesterol concentrations ; the ratios approach the dopc - case at high cholesterol content . second , the location of the second peak does not shift as strongly as for dopc , indicating that the condensing effect in the immediate proximity of pgpc is less pronounced . high cholesterol concentrations level out the differences in the mobility of oxpl and conventional lipids . it is thus difficult to reconcile the exceptionally high mobility of pgpe - alexa647 in the plasma membrane ( d = 1.32.4 m / s(15 ) ) with the high native cholesterol concentrations of 3040% . we suspected that the diffusion matrix in the plasma membrane may be highly structured , providing essentially a percolation matrix . to enforce this effect in our model system , we artificially introduced obstacles in the proximal leaflet by immobilizing a substantial fraction of lipids . interleaflet coupling should transmit the effect to the distal leaflet. while pgpe - alexa647 mobility was reduced only approximately 2-fold , dhpe - bodipy became basically immobile ( figure 7a , c ) . consistently , addition of 40 mol % cholesterol substantially decreased the mobility of pgpe - alexa647 ( figure 7d ) . the presence of dope - biotin did not influence the mobility of pgpe - alexa647 or dhpe - bodipy in a dopc bilayer on pure glass ( figure 7b ) . diffusion constants were determined for pgpe - alexa647 ( light gray bars ) and dhpe - bodipy ( dark gray bars ) . we studied partitioning and mobility of the oxidized phospholipid pgpe - alexa647 with reference to the conventional phospholipid dhpe - bodipy in different lipid membranes . in summary , we made the following observations : pgpe - alexa647 shows only marginal preference for fluid versus ordered phases . it is tempting to follow an argument of the vaz group , who ascribed the rate - limiting step for association of a lyso - lipid with a bilayer to the formation of free area with appropriate size in the membrane surface. (55 ) for the oxpl , single chain insertion is sufficient for thermodynamic equilibrium,(11 ) therefore the formation of a marginal free area will be sufficient for insertion of pgpe - alexa647 . md simulations revealed that pgpc slightly sticks out of the headgroup region , thereby essentially reducing its effective area , which most likely causes the enhanced mobility. (56 ) the experimental data show that the effect was more pronounced at elevated temperature , indicating that the shift of pgpc out of the bilayer plane is entropically favored . at low cholesterol concentrations , pgpc is preferentially surrounded by dopc , and its headgroup protrudes out of the headgroup region of the dopc matrix . with increasing cholesterol content , a higher rate of association of pgpc with cholesterol could be observed . thereby the pgpc headgroup is pulled into the bilayer , with the effect of increasing its interactions with the host lipids and decreasing its mobility . moreover , in a four component mixture of bsm : dopc : chol : cer ( 0.64:1:1:0.36 ) , we found hardly any difference in the l.d . phase ; indeed , ceramide is known to displace cholesterol from the l.gel phase,(57 ) thereby increasing the cholesterol content in the fluid phase . the mobility of pgpe - alexa647 is substantially higher than the mobility of dhpe - bodipy in ordered or obstructed matrices . moreover , when we artificially increased the obstacle density in a fluid supported lipid bilayer by immobilizing biotin - dope in the proximal bilayer leaflet on avidin , the ratio dramatically increased to r = 51.1 . in summary , we have found that the oxidized phospholipid pgpc and its fluorescent analogue pgpe - alexa647 move with increased mobility compared to conventional phospholipids . mobility is not only determined by the fluidity of the matrix , but also by immobile obstacles or by the localization of the probe along the bilayer normal , which can be influenced by the cholesterol concentration .

percutaneous coronary intervention ( pci ) has become the first - line treatment for patients suffering from obstructive coronary artery disease ( cad ) . drug - eluting stents ( des ) have significantly reduced the risk of restenosis and the need for repeat revascularization when compared to bare metal stent ( bms).1 in the ravel and sirius trials,2,3 when compared to bms , des improved restenosis rates and late lumen loss , and decreased target lesion revascularization ( tlr ) from 16.6% to 4.1% ( p<0.01 ) . although first - generation des ( 1st gen des ) were introduced to disrupt neointimal growth by the use of antiproliferative drugs , this benefit was acquired at the expense of a substantial delay in vascular healing and the clinical consequences of late and very late stent thrombosis ( lst / vlst).4 second - generation des ( 2nd gen des ) were developed with newer alloys , biocompatible polymers , thinner struts , and different drugs kinetics , resulting in a reduction of lst / vlst . small vessel cad accounts for up to 30% of all pci5 and remains an independent predictor of angiographic restenosis and tlr , even after the introduction of des.6 the cobalt chromium everolimus - eluting stent ( cocr - ees ) ( xience v ; abbott vascular , santa clara , california , usa ) has been reported as one of the most frequently used 2nd gen des with better event - free survival rates in small vessels.7 this review article discusses the preclinical , clinical , and pathological performance of cocr - ees in small vessel cad . the antiproliferative drug used is everolimus , a hydroxyethyl derivative of sirolimus which acts as an immunosuppressant . it induces cell cycle arrest in the g1 phase by inhibiting the mammalian target of rapamycin , a serine / threonine protein kinase that regulates cell growth , proliferation , motility , protein synthesis , and transcription , among others.8,9 the polymer is a thin ( 7.8 m ) bio - inert , non - erodible , and ultra - pure fluorinated copolymer ( poly - n - butyl methacrylate [ pbma ] and poly - vinylidene fluoride and hexafluoropropylene [ pvdf - hfp ] ) that provides both elasticity and stability . pbma serves as a base coat for the stent and facilitates anchorage of pvdf - hfp , which serves as a matrix layer containing the drug at a ratio of 83%/17% for polymer / everolimus , respectively , and no top - coat layer is applied . the polymer composition provides mechanical integrity after stent deployment , followed by the controlled release of everolimus at a total dose of 100 g / cm , delivering up to 80% of the drug after 4 weeks.10 the platform is the multilink vision l-605 cobalt chromium alloy with a strut thickness of 81 m mounted on a compliant tapered vision balloon,11 structurally designed to improve deliverability and conformability , and at the same time , increasing its radiopacity , radial strength , and fracture resistance.12 the xience v and xience nano share the same platform design , delivery system , drug , and coating materials . the differentiating features of the xience nano pertain to a balloon diameter of 2.25 mm with a nominal inner stent diameter of 2.25 mm , as compared to xience v , which is available at diameters of 2.5 mm , 2.75 mm , 3.0 mm , 3.5 mm , and 4.0 mm . there are several stent - related factors that have been associated with lst / vlst , such as strut thickness , polymer characteristics , coating integrity , and drug dose , among others.13,14 it is known that strut thickness affects the angiographic and clinical outcome after pci.15 in this regard , des with thinner struts have been reported to provide improvement in outcomes with respect to target vessel revascularization when compared to thicker strut des in calcified lesions.16 kolandaivelu et al17 evaluated the impact of strut thickness on thrombogenicity in a chandler loop model . two main factors were reported to determine acute thrombogenicity : 1 ) strut thickness ; and 2 ) polymer coating . thrombogenicity within the various bms designs correlated with strut thickness ; stents with thicker struts were 49% more thrombogenic than stents with thinner struts ( 0.880.38 for struts < 100 m versus 1.440.65 for struts > 100 m ; p=0.036 ) . after 3 days of implantation in porcine coronary arteries , stents with thicker struts demonstrated significantly more thrombus and 62% more clots compared to their thinner strut counterparts ( 0.210.041 mm versus 0.130.019 mm ; p=0.004 ) ; also , neointimal fibrin accumulated to a greater extent around the thicker struts compared to the thinner struts ( 1.560.40 versus 0.830.41 ; p=0.016 ) . the authors also evaluated overlapping stents , which were more thrombogenic than single length - matched controls , more so for thicker than thinner struts stents ( 2.320.96 and 3.250.11 versus 1.000.17 ; p<0.001 ) . moreover , overlapping thinner strut des ( 0.510.019 ) were less thrombogenic than overlapping bms ( p<0.001 ) and even the single bms controls ( p<0.001).17 in this landmark study , kolandaivelu et al17 also found that coated stents were less thrombogenic than corresponding bms ( 0.760.02 versus 1.000.15 ; p<0.002 ) , and clot mass was also significantly lower for des when compared with bms ( 0.670.35 versus 1.030.54 ; p=0.011 ) . hypersensitivity reactions induced by the durable polymers used in 1st gen des were suggested to contribute to the occurrence of lst.1820 recent advances in stent technology , with the introduction of more biocompatible polymers , have reduced the risk of this complication.21,22 chin - quee et al23 evaluated two different polymers currently available for des in rabbit iliac arteries where cocr stents were coated with pvdf - hfp or phosphorylcholine polymer ( without drug ) and assessed for endothelialization at 14 days by confocal and scanning electron microscopy ( sem ) . endothelialization was equivalent and near complete for pvdf - hfp versus phosphorylcholine polymer - coated stents ( > 80% by sem ) . also , acute thrombogenicity was assessed in a chandler loop model using porcine blood ; thrombus adherence was similar for both polymers ( 0.940.23 versus 0.990.20 ) . these results suggest that the polymers examined here did not impede endothelization.23 polymer coating defects can potentially change the drug elution kinetics of des , or they can lead to chronic inflammatory reactions.17 furthermore , the nonuniform coating of stent struts may impact platelet adhesion and endothelialization . finally , coating fragments may embolize downstream , resulting in myocardial ischemia . yazdani et al24 evaluated 48 des for coating integrity in cocr - ees , zotarolimus - eluting stent ( zes ) , paclitaxel - eluting stent ( pes ) , and biolimus a-9-eluting stent ( bes ) in a rabbit iliofemoral stent model for durations of 7 days , 28 days , 90 days , and 180 days . the cocr - ees and zes had the least amount of coating defects as compared to the pes and bes . however , coating defects were shown to increase over time within the zes , whereas in the cocr - ees , the amount of irregularity remained constant over time . newer generation des were designed to outperform first - generation devices in this regard . in our laboratory,25 we compared 1st gen des and 2nd gen des in new zealand white rabbits and reported at 14 days that re - endothelialization above struts was variable among stents with significantly greater coverage in cocr - ees ( 64.0%27.5% ) , followed by zes ( 30.2%14.2% ) , pes ( 26.8%15.8% ) , and sirolimus - eluting stent ( ses ) ( 6.4%4.2% ) , with a statistically significant difference versus cocr - ees ( p<0.003 ) and bms ( p<0.0001 ) as a control stent ( figure 1 ) . at 28 days , all evaluated stents had more than 60% endothelial cell coverage above struts in favor of cocr - ees , but without statistically significant differences among groups . furthermore , cocr - ees had the least percentage of struts lacking endothelial coverage compared to other des.25 based on morphometry , the greatest frequency of uncovered struts was observed in the middle stented segment , while proximal and distal segments showed overall greater coverage . we also evaluated endothelium integrity using the platelet endothelial cell adhesion molecule , pecam-1 , as a surrogate , and found that cocr - ees had significantly greater cell - to - cell contact sites above the struts , which demonstrates a functionally and biologically active endothelium.25 the antiproliferative drug used is everolimus , a hydroxyethyl derivative of sirolimus which acts as an immunosuppressant . it induces cell cycle arrest in the g1 phase by inhibiting the mammalian target of rapamycin , a serine / threonine protein kinase that regulates cell growth , proliferation , motility , protein synthesis , and transcription , among others.8,9 the polymer is a thin ( 7.8 m ) bio - inert , non - erodible , and ultra - pure fluorinated copolymer ( poly - n - butyl methacrylate [ pbma ] and poly - vinylidene fluoride and hexafluoropropylene [ pvdf - hfp ] ) that provides both elasticity and stability . pbma serves as a base coat for the stent and facilitates anchorage of pvdf - hfp , which serves as a matrix layer containing the drug at a ratio of 83%/17% for polymer / everolimus , respectively , and no top - coat layer is applied . the polymer composition provides mechanical integrity after stent deployment , followed by the controlled release of everolimus at a total dose of 100 g / cm , delivering up to 80% of the drug after 4 weeks.10 the platform is the multilink vision l-605 cobalt chromium alloy with a strut thickness of 81 m mounted on a compliant tapered vision balloon,11 structurally designed to improve deliverability and conformability , and at the same time , increasing its radiopacity , radial strength , and fracture resistance.12 the xience v and xience nano share the same platform design , delivery system , drug , and coating materials . the differentiating features of the xience nano pertain to a balloon diameter of 2.25 mm with a nominal inner stent diameter of 2.25 mm , as compared to xience v , which is available at diameters of 2.5 mm , 2.75 mm , 3.0 mm , 3.5 mm , and 4.0 mm . there are several stent - related factors that have been associated with lst / vlst , such as strut thickness , polymer characteristics , coating integrity , and drug dose , among others.13,14 it is known that strut thickness affects the angiographic and clinical outcome after pci.15 in this regard , des with thinner struts have been reported to provide improvement in outcomes with respect to target vessel revascularization when compared to thicker strut des in calcified lesions.16 kolandaivelu et al17 evaluated the impact of strut thickness on thrombogenicity in a chandler loop model . two main factors were reported to determine acute thrombogenicity : 1 ) strut thickness ; and 2 ) polymer coating . thrombogenicity within the various bms designs correlated with strut thickness ; stents with thicker struts were 49% more thrombogenic than stents with thinner struts ( 0.880.38 for struts < 100 m versus 1.440.65 for struts > 100 m ; p=0.036 ) . after 3 days of implantation in porcine coronary arteries , stents with thicker struts demonstrated significantly more thrombus and 62% more clots compared to their thinner strut counterparts ( 0.210.041 mm versus 0.130.019 mm ; p=0.004 ) ; also , neointimal fibrin accumulated to a greater extent around the thicker struts compared to the thinner struts ( 1.560.40 versus 0.830.41 ; p=0.016 ) . the authors also evaluated overlapping stents , which were more thrombogenic than single length - matched controls , more so for thicker than thinner struts stents ( 2.320.96 and 3.250.11 versus 1.000.17 ; p<0.001 ) . moreover , overlapping thinner strut des ( 0.510.019 ) were less thrombogenic than overlapping bms ( p<0.001 ) and even the single bms controls ( p<0.001).17 in this landmark study , kolandaivelu et al17 also found that coated stents were less thrombogenic than corresponding bms ( 0.760.02 versus 1.000.15 ; p<0.002 ) , and clot mass was also significantly lower for des when compared with bms ( 0.670.35 versus 1.030.54 ; p=0.011 ) . hypersensitivity reactions induced by the durable polymers used in 1st gen des were suggested to contribute to the occurrence of lst.1820 recent advances in stent technology , with the introduction of more biocompatible polymers , have reduced the risk of this complication.21,22 chin - quee et al23 evaluated two different polymers currently available for des in rabbit iliac arteries where cocr stents were coated with pvdf - hfp or phosphorylcholine polymer ( without drug ) and assessed for endothelialization at 14 days by confocal and scanning electron microscopy ( sem ) . endothelialization was equivalent and near complete for pvdf - hfp versus phosphorylcholine polymer - coated stents ( > 80% by sem ) . also , acute thrombogenicity was assessed in a chandler loop model using porcine blood ; thrombus adherence was similar for both polymers ( 0.940.23 versus 0.990.20 ) . these results suggest that the polymers examined here did not impede endothelization.23 polymer coating defects can potentially change the drug elution kinetics of des , or they can lead to chronic inflammatory reactions.17 furthermore , the nonuniform coating of stent struts may impact platelet adhesion and endothelialization . finally , coating fragments may embolize downstream , resulting in myocardial ischemia . yazdani et al24 evaluated 48 des for coating integrity in cocr - ees , zotarolimus - eluting stent ( zes ) , paclitaxel - eluting stent ( pes ) , and biolimus a-9-eluting stent ( bes ) in a rabbit iliofemoral stent model for durations of 7 days , 28 days , 90 days , and 180 days . the cocr - ees and zes had the least amount of coating defects as compared to the pes and bes . however , coating defects were shown to increase over time within the zes , whereas in the cocr - ees , the amount of irregularity remained constant over time . newer generation des were designed to outperform first - generation devices in this regard . in our laboratory,25 we compared 1st gen des and 2nd gen des in new zealand white rabbits and reported at 14 days that re - endothelialization above struts was variable among stents with significantly greater coverage in cocr - ees ( 64.0%27.5% ) , followed by zes ( 30.2%14.2% ) , pes ( 26.8%15.8% ) , and sirolimus - eluting stent ( ses ) ( 6.4%4.2% ) , with a statistically significant difference versus cocr - ees ( p<0.003 ) and bms ( p<0.0001 ) as a control stent ( figure 1 ) . at 28 days , all evaluated stents had more than 60% endothelial cell coverage above struts in favor of cocr - ees , but without statistically significant differences among groups . furthermore , cocr - ees had the least percentage of struts lacking endothelial coverage compared to other des.25 based on morphometry , the greatest frequency of uncovered struts was observed in the middle stented segment , while proximal and distal segments showed overall greater coverage . we also evaluated endothelium integrity using the platelet endothelial cell adhesion molecule , pecam-1 , as a surrogate , and found that cocr - ees had significantly greater cell - to - cell contact sites above the struts , which demonstrates a functionally and biologically active endothelium.25 randomized controlled clinical trials have established differential outcomes in the safety and efficacy of des used in distinct clinical settings , and stent - related factors may play an important role in the scenery of small vessel pci.13,14,26 cannon et al27 evaluated the safety of xience nano in vessels > 2.25 mm but < 2.5 mm at 1-year follow - up . the authors established a performance goal ( pg ) at 20.4% for target lesion failure ( tlf ) based on clinical trials and registries , which evaluated 2.25 mm diameter des.14,28,29 the 1-year tlf rate was 8.1% , with an upper one - sided limit ( 95% confidence interval ) of 13.0% , meeting the pg of 20.4% ( p<0.0001 ) . the 1-year tlf rate was mainly driven by low cardiac death and myocardial infarction ( mi ) rates . the most important difference noted in this study was a higher tlf rate for the reference vessel diameter ( rvd ) 2.12 mm ( number [ n ] = 72 ) , which reached 13.89% , compared to 1.56% for a rvd > 2.12 mm ( n=64 ) . in a post clinically , no statistically significant differences were found for tlf ( 5.7% versus 4.9% ; p=1.0 ) and major adverse cardiovascular event rates ( mace)/tlf rates ( 7.5% versus 8.5% ; p=1.0 ) in diabetics versus nondiabetics , respectively . also , angiographically , in - stent and in - segment late loss showed no difference between diabetics and nondiabetics ( 0.220.47 mm versus 0.190.36 mm , p=0.83 ; and 0.140.48 mm versus 0.170.38 mm , respectively , p=0.76).27 small vessel cad represents a challenge for interventional cardiologists , with higher restenosis and stent thrombosis ( st ) rates.30,31 hermiller et al32 evaluated the safety of cocr - ees in small and nonsmall vessels in a real - world scenario , applying a 2.5 mm diameter cut - off . the mean rvd for small vessels was 2.550.36 mm and 3.250.46 mm for the nonsmall vessel group ( p<0.001 ) . definite or probable st rates were low and not significantly different between the groups at 0.37% versus 0.40% ( p=0.88 ) for the small and nonsmall vessel groups , respectively . the composite rate of cardiac death or mi was comparable for the small and nonsmall vessel group ( 4.5% versus 5.1% , respectively ; p=0.57 ) . the 1-year tlr rate was also comparable in the small vessel group ( small group 3.8% versus nonsmall group 3.0% ; p=0.35 ) . this study demonstrated the safety of cocr - ees in small vessels despite the fact that this group consisted of more females , those with a higher rate of diabetes , and those with more complex lesion characteristics.32 in a separate study , ito et al33 compared cocr - ees and pes for small vessel revascularization by pooling the data from the spirit iii and iv trials.34,35 from 4,689 patients , two groups were analyzed : the small vessel group ( rvd : 2.250.19 mm ; n=1,019 ) and the large vessel group ( rvd : 2.990.35 mm ; n=2,586 ) . after 1-year follow - up , in patients with small vessels disease , the tlf ( cocr - ees 4.4% versus pes 7.9% ; p=0.03 ) and mace ( cocr - ees 4.5% versus pes 7.9% ; the clinical endpoint , tlf , was composed of cardiac death , target vessel mi , and ischemia driven - tlr ( id - tlr ) . amid the others , only id - tlr showed a significant reduction at 1 year ( everolimus - eluting stents [ ees ] 2.4% versus pes 5.5% ; p=0.02 ) . although , st showed higher rates in small vessel revascularization , the authors found that st was significantly lower in patients with small vessels treated with cocr - ees than in those treated with pes ( 0.2% versus 1.2% , respectively ; p=0.04).33 currently , the factors predictive of in - stent restenosis can be divided into patient - related , procedure - related , and lesion - related factors . patient - related factors such as diabetes , a history of restenosis , and genetic factors have been reported as risk factors of in - stent restenosis.33 procedure - related factors include the number of stents implanted , the total stent length , and stent overlap . lesion - related characteristics , which impact the rate of restenosis , include small vessel size , long lesion length , and the severity of pretreatment as well as posttreatment lesion stenosis , among others.36 claessen et al37 collected data from spirit ii , iii , and iv,34,38,39 and combined three groups : short lesions in large vessels ( group a ) ; long lesions in large vessels or short lesions in small vessels ( group b ) ; and long lesions in small vessels ( group c ) to evaluate the safety and efficacy of cocr - ees versus pes . the mace rate after 2 years of follow - up was lower in group a , intermediate in group b , and highest in group c ( 5.6% versus 8.2% versus 10.4% , respectively ; p<0.0001 ) . also , a similar trend was observed for mi ( 3.3% versus 3.0% versus 4.5% , respectively ; p=0.02 ) and id - tlr ( 2.9% versus 5.0% versus 6.3% , respectively ; p=0.0002 ) . the authors also evaluated mace rates by stent type ( pes and cocr - ees ) , and found that the higher the lesion complexity , the greater the mace incidence ( 7.0% , 11.2% , and 12.8% for pes , respectively , p=0.007 ; versus 4.8% versus 6.6% versus 9.1% for cocr - ees , respectively , p=0.001 ) . on the other hand , the 2-year rate of definite or probable st ( academic research consortium , arc definition ) also increased with greater lesion complexity after pes implantation ( group a 0.7% versus group b 1.9% versus group c 2.8% ; p=0.03 ) , but that relationship was not present after cocr - ees implantation ( 0.9% versus 0.6% versus 0.6% ; p=0.65 ) . cocr - ees were associated with significantly lower rates of mace , mi , id - tlr , and st in groups b and c , but no statistical significance was found in the less complex group ( group a ) . multivariate analysis found that the use of cocr - ees rather than pes was an independent predictor of freedom from mace in group b ( p<0.0001 ) and group c ( p=0.004 ) , but not in group a ( p=0.19).37 recently , we reported40 the pathologic findings of 2nd gen des and compared these to 1st gen des . a total of 204 lesions ( ses = 73 ; pes = 85 ; cocr - ees = 46 ) from 149 autopsy cases with implant duration > 30 days and 3 years were pathologically analyzed to determine differences . the observed frequency of lst and vlst was less for cocr - ees ( 4% ) compared with ses ( 21% ; p=0.029 ) and pes ( 26% ; p=0.008 ) . the prevalence of restenosis for cocr - ees ( 17% ) did not differ significantly from that observed in ses ( 14% ) and pes ( 12% ) . the frequency of uncovered struts was markedly lower for cocr - ees ( 2.6% ) as compared to ses ( 18.0% ; p<0.0005 ) and pes ( 18.7% ; p<0.0005 ) . the prevalence of des with > 30% uncovered struts was also significantly lower in cocr - ees ( 20% ) than in ses ( 60% ; p<0.0005 ) and pes ( 67% ; p<0.0005 ) . in terms of inflammation , the overall prevalence of neoatherosclerosis after cocr - ees implantation in native coronary arteries was 29% , which did not differ significantly from ses ( 35% ; p=0.62 ) and pes ( 19% ; p=0.47).40 complex lesion characteristics and unstable plaques are associated with a greater delay in arterial healing when compared to pci of a simple and stable plaque by pathology.41 therefore , we evaluated the prevalence of > 30% uncovered struts in the setting of off - label versus on - label clinical indications . cocr - ees compared with ses and pes showed greater strut coverage for both on - label ( 14% versus 50% versus 57% , respectively ) and off - label ( 25% versus 68% versus 75% , respectively ) indications . when analyzing the cvpath stent database composed of 865 cases , 68 had cocr - ees implanted and 12 cases were found with a stent diameter of 2.5 mm or less ( table 1 ) . from those 12 cases , the mean stent length was 35.624.9 mm and the mean stent diameter was 2.30.27 mm . case 1 : a 45-year - old woman with obesity , hypertension , and diabetes who presented with mi secondary to involvement of the left circumflex artery , which was revascularized ; 5 days after the procedure , she died suddenly with st . case 2 : a 58-year - old male with a history of obesity and cad presented with non - st segment elevation mi , and left anterior descending artery occlusion in the region of the left diagonal branch ; bifurcation stenting was performed . the patient died suddenly 7 days after the procedure ; at autopsy , there was a large infarction and mild to moderate thrombus in the stented region . the other three patients had stable cad and only mild inflammation was observed with moderate peristrut fibrin and platelet deposition ( figure 2 ) . one of the three cases had thrombosis and stent fracture ( 20% ) ; the other two were nonstent - related deaths . the remaining seven cases ( table 1 ) had a duration > 30 days ( 242.9222.6 days ) . acute coronary syndrome was the indication in one case ( 14.3% ) , and the cause of death was stent - related a 72-year - old woman with obesity , hypertension , diabetes , atrial fibrillation , and a history of multiple revascularizations . she died from st 210 days later with underlying restenosis . for the rest of the cases ( n=6 ) with duration > 30 days , there were no differences in the principal histopathological findings among those presenting with acute coronary syndrome versus stable cad . overall , the histopathological analysis showed mild to moderate chronic peristrut inflammation consisting of monocytes , t - lymphocytes , and macrophages ( figure 3 ) without any significant eosinophils . restenosis was observed in one of the six cases ; however , the xience case was sandwiched between two vision stents , both of which had total occlusion . nevertheless , we must recognize the limited number of cases analyzed at autopsy . a greater number of cases and matched control groups will be required to understand the full scope of histopathological findings of cocr - ees in small vessel disease . des have progressively improved clinical outcomes , but the potential risk of st is still a concern and limits the use of des , especially in small vessel cad . overall , lst and vlst have been reported with an incidence of 0.2% and 0.4% per year , respectively.42 however , considering the large amount of stents implanted worldwide , those numbers are still high . several reports of st have been associated with 1st gen des , especially after dual anti - platelet therapy termination.43,44 over time , great effort has been made to improve the technology , thus reducing strut thickness from 140 m to approximately 7080 m , resulting in a dramatic reduction of thrombogenicity in bench studies.17,45 advances in polymer technology have been enormous ; new biocompatible polymers ( pbma or pvdf - hfp ) result in less inflammation after stent implantation , with the consequence of more complete and functional endothelization.46,47 also , cocr - ees show fewer coating defects after implantation when compared to different des , and this result was maintained over time and may improve vascular biocompatibility.25 clinical data confirmed the outstanding performance of cocr - ees , with lower rates of definitive / probable st , tlr , and mace . the pg for tlf was overperformed with cocr - ees when compared with a competitor des in small vessel disease . mace and tlf rates were similar among diabetics and nondiabetics.27 at pathology , cocr - ees revealed less inflammation and greater strut coverage when compared to 1st gen des , while maintaining similar efficacy in reducing neointimal growth . specifically , in small vessel disease , cocr - ees have been shown to be less thrombogenic compared to 1st gen des ; however , inflammation and restenosis remain a problem in this setting , and further technological and procedural progress is needed to improve patient outcomes . in the des era , small vessel cad remains a great challenge for interventional cardiologists . stent design and the material combination may provide better outcomes , especially in small vessel disease . cocr - ees with thinner struts , biocompatible polymers , reduced drug load , and better radiopacity and trackability have shown excellent results from preclinical , clinical , and pathological studies in small vessel cad .

coronary artery disease ( cad ) is the leading cause of morbidity and mortality worldwide . the pathogenesis of cad relates to the presence of atherosclerotic plaques in the coronary arteries , which are most frequently treated today by percutaneous coronary intervention . small vessel disease treatment represents one - third of all percutaneous coronary interventions with higher rates of restenosis and major adverse cardiac events . initially , drug - eluting stents ( des ) were developed to reduce in - stent restenosis , improving clinical outcomes and reducing the need for target vessel revascularization . however , late and very late stent thrombosis emerged as a new problem compromising des s long - term results . the cobalt chromium everolimus - eluting stent ( cocr - ees ) represents the results of an evolutionary process in des technology aimed at improving the shortcomings of first - generation des . small vessel cad has historically been an obstacle to long - term patency following implantation of des . antirestenotic efficacy has been shown to be of high relevance in small vessels . therefore , stent selection may play an important role in determining outcomes in this subgroup of patients . this article will review the performance of cocr - ees in the treatment of small vessel cad from preclinical , clinical , and pathology perspectives , and it will highlight the most important findings in this regard .

Introduction Device characteristics Xience V Preclinical findings Clinical findings Pathology findings Summary Conclusion

percutaneous coronary intervention ( pci ) has become the first - line treatment for patients suffering from obstructive coronary artery disease ( cad ) . drug - eluting stents ( des ) have significantly reduced the risk of restenosis and the need for repeat revascularization when compared to bare metal stent ( bms).1 in the ravel and sirius trials,2,3 when compared to bms , des improved restenosis rates and late lumen loss , and decreased target lesion revascularization ( tlr ) from 16.6% to 4.1% ( p<0.01 ) . although first - generation des ( 1st gen des ) were introduced to disrupt neointimal growth by the use of antiproliferative drugs , this benefit was acquired at the expense of a substantial delay in vascular healing and the clinical consequences of late and very late stent thrombosis ( lst / vlst).4 second - generation des ( 2nd gen des ) were developed with newer alloys , biocompatible polymers , thinner struts , and different drugs kinetics , resulting in a reduction of lst / vlst . small vessel cad accounts for up to 30% of all pci5 and remains an independent predictor of angiographic restenosis and tlr , even after the introduction of des.6 the cobalt chromium everolimus - eluting stent ( cocr - ees ) ( xience v ; abbott vascular , santa clara , california , usa ) has been reported as one of the most frequently used 2nd gen des with better event - free survival rates in small vessels.7 this review article discusses the preclinical , clinical , and pathological performance of cocr - ees in small vessel cad . pbma serves as a base coat for the stent and facilitates anchorage of pvdf - hfp , which serves as a matrix layer containing the drug at a ratio of 83%/17% for polymer / everolimus , respectively , and no top - coat layer is applied . the polymer composition provides mechanical integrity after stent deployment , followed by the controlled release of everolimus at a total dose of 100 g / cm , delivering up to 80% of the drug after 4 weeks.10 the platform is the multilink vision l-605 cobalt chromium alloy with a strut thickness of 81 m mounted on a compliant tapered vision balloon,11 structurally designed to improve deliverability and conformability , and at the same time , increasing its radiopacity , radial strength , and fracture resistance.12 the xience v and xience nano share the same platform design , delivery system , drug , and coating materials . there are several stent - related factors that have been associated with lst / vlst , such as strut thickness , polymer characteristics , coating integrity , and drug dose , among others.13,14 it is known that strut thickness affects the angiographic and clinical outcome after pci.15 in this regard , des with thinner struts have been reported to provide improvement in outcomes with respect to target vessel revascularization when compared to thicker strut des in calcified lesions.16 kolandaivelu et al17 evaluated the impact of strut thickness on thrombogenicity in a chandler loop model . after 3 days of implantation in porcine coronary arteries , stents with thicker struts demonstrated significantly more thrombus and 62% more clots compared to their thinner strut counterparts ( 0.210.041 mm versus 0.130.019 mm ; p=0.004 ) ; also , neointimal fibrin accumulated to a greater extent around the thicker struts compared to the thinner struts ( 1.560.40 versus 0.830.41 ; p=0.016 ) . moreover , overlapping thinner strut des ( 0.510.019 ) were less thrombogenic than overlapping bms ( p<0.001 ) and even the single bms controls ( p<0.001).17 in this landmark study , kolandaivelu et al17 also found that coated stents were less thrombogenic than corresponding bms ( 0.760.02 versus 1.000.15 ; p<0.002 ) , and clot mass was also significantly lower for des when compared with bms ( 0.670.35 versus 1.030.54 ; p=0.011 ) . yazdani et al24 evaluated 48 des for coating integrity in cocr - ees , zotarolimus - eluting stent ( zes ) , paclitaxel - eluting stent ( pes ) , and biolimus a-9-eluting stent ( bes ) in a rabbit iliofemoral stent model for durations of 7 days , 28 days , 90 days , and 180 days . the cocr - ees and zes had the least amount of coating defects as compared to the pes and bes . however , coating defects were shown to increase over time within the zes , whereas in the cocr - ees , the amount of irregularity remained constant over time . newer generation des were designed to outperform first - generation devices in this regard . in our laboratory,25 we compared 1st gen des and 2nd gen des in new zealand white rabbits and reported at 14 days that re - endothelialization above struts was variable among stents with significantly greater coverage in cocr - ees ( 64.0%27.5% ) , followed by zes ( 30.2%14.2% ) , pes ( 26.8%15.8% ) , and sirolimus - eluting stent ( ses ) ( 6.4%4.2% ) , with a statistically significant difference versus cocr - ees ( p<0.003 ) and bms ( p<0.0001 ) as a control stent ( figure 1 ) . at 28 days , all evaluated stents had more than 60% endothelial cell coverage above struts in favor of cocr - ees , but without statistically significant differences among groups . furthermore , cocr - ees had the least percentage of struts lacking endothelial coverage compared to other des.25 based on morphometry , the greatest frequency of uncovered struts was observed in the middle stented segment , while proximal and distal segments showed overall greater coverage . we also evaluated endothelium integrity using the platelet endothelial cell adhesion molecule , pecam-1 , as a surrogate , and found that cocr - ees had significantly greater cell - to - cell contact sites above the struts , which demonstrates a functionally and biologically active endothelium.25 the antiproliferative drug used is everolimus , a hydroxyethyl derivative of sirolimus which acts as an immunosuppressant . pbma serves as a base coat for the stent and facilitates anchorage of pvdf - hfp , which serves as a matrix layer containing the drug at a ratio of 83%/17% for polymer / everolimus , respectively , and no top - coat layer is applied . the polymer composition provides mechanical integrity after stent deployment , followed by the controlled release of everolimus at a total dose of 100 g / cm , delivering up to 80% of the drug after 4 weeks.10 the platform is the multilink vision l-605 cobalt chromium alloy with a strut thickness of 81 m mounted on a compliant tapered vision balloon,11 structurally designed to improve deliverability and conformability , and at the same time , increasing its radiopacity , radial strength , and fracture resistance.12 the xience v and xience nano share the same platform design , delivery system , drug , and coating materials . there are several stent - related factors that have been associated with lst / vlst , such as strut thickness , polymer characteristics , coating integrity , and drug dose , among others.13,14 it is known that strut thickness affects the angiographic and clinical outcome after pci.15 in this regard , des with thinner struts have been reported to provide improvement in outcomes with respect to target vessel revascularization when compared to thicker strut des in calcified lesions.16 kolandaivelu et al17 evaluated the impact of strut thickness on thrombogenicity in a chandler loop model . after 3 days of implantation in porcine coronary arteries , stents with thicker struts demonstrated significantly more thrombus and 62% more clots compared to their thinner strut counterparts ( 0.210.041 mm versus 0.130.019 mm ; p=0.004 ) ; also , neointimal fibrin accumulated to a greater extent around the thicker struts compared to the thinner struts ( 1.560.40 versus 0.830.41 ; p=0.016 ) . yazdani et al24 evaluated 48 des for coating integrity in cocr - ees , zotarolimus - eluting stent ( zes ) , paclitaxel - eluting stent ( pes ) , and biolimus a-9-eluting stent ( bes ) in a rabbit iliofemoral stent model for durations of 7 days , 28 days , 90 days , and 180 days . the cocr - ees and zes had the least amount of coating defects as compared to the pes and bes . however , coating defects were shown to increase over time within the zes , whereas in the cocr - ees , the amount of irregularity remained constant over time . newer generation des were designed to outperform first - generation devices in this regard . in our laboratory,25 we compared 1st gen des and 2nd gen des in new zealand white rabbits and reported at 14 days that re - endothelialization above struts was variable among stents with significantly greater coverage in cocr - ees ( 64.0%27.5% ) , followed by zes ( 30.2%14.2% ) , pes ( 26.8%15.8% ) , and sirolimus - eluting stent ( ses ) ( 6.4%4.2% ) , with a statistically significant difference versus cocr - ees ( p<0.003 ) and bms ( p<0.0001 ) as a control stent ( figure 1 ) . at 28 days , all evaluated stents had more than 60% endothelial cell coverage above struts in favor of cocr - ees , but without statistically significant differences among groups . furthermore , cocr - ees had the least percentage of struts lacking endothelial coverage compared to other des.25 based on morphometry , the greatest frequency of uncovered struts was observed in the middle stented segment , while proximal and distal segments showed overall greater coverage . we also evaluated endothelium integrity using the platelet endothelial cell adhesion molecule , pecam-1 , as a surrogate , and found that cocr - ees had significantly greater cell - to - cell contact sites above the struts , which demonstrates a functionally and biologically active endothelium.25 randomized controlled clinical trials have established differential outcomes in the safety and efficacy of des used in distinct clinical settings , and stent - related factors may play an important role in the scenery of small vessel pci.13,14,26 cannon et al27 evaluated the safety of xience nano in vessels > 2.25 mm but < 2.5 mm at 1-year follow - up . the authors established a performance goal ( pg ) at 20.4% for target lesion failure ( tlf ) based on clinical trials and registries , which evaluated 2.25 mm diameter des.14,28,29 the 1-year tlf rate was 8.1% , with an upper one - sided limit ( 95% confidence interval ) of 13.0% , meeting the pg of 20.4% ( p<0.0001 ) . the most important difference noted in this study was a higher tlf rate for the reference vessel diameter ( rvd ) 2.12 mm ( number [ n ] = 72 ) , which reached 13.89% , compared to 1.56% for a rvd > 2.12 mm ( n=64 ) . also , angiographically , in - stent and in - segment late loss showed no difference between diabetics and nondiabetics ( 0.220.47 mm versus 0.190.36 mm , p=0.83 ; and 0.140.48 mm versus 0.170.38 mm , respectively , p=0.76).27 small vessel cad represents a challenge for interventional cardiologists , with higher restenosis and stent thrombosis ( st ) rates.30,31 hermiller et al32 evaluated the safety of cocr - ees in small and nonsmall vessels in a real - world scenario , applying a 2.5 mm diameter cut - off . this study demonstrated the safety of cocr - ees in small vessels despite the fact that this group consisted of more females , those with a higher rate of diabetes , and those with more complex lesion characteristics.32 in a separate study , ito et al33 compared cocr - ees and pes for small vessel revascularization by pooling the data from the spirit iii and iv trials.34,35 from 4,689 patients , two groups were analyzed : the small vessel group ( rvd : 2.250.19 mm ; n=1,019 ) and the large vessel group ( rvd : 2.990.35 mm ; n=2,586 ) . after 1-year follow - up , in patients with small vessels disease , the tlf ( cocr - ees 4.4% versus pes 7.9% ; p=0.03 ) and mace ( cocr - ees 4.5% versus pes 7.9% ; the clinical endpoint , tlf , was composed of cardiac death , target vessel mi , and ischemia driven - tlr ( id - tlr ) . amid the others , only id - tlr showed a significant reduction at 1 year ( everolimus - eluting stents [ ees ] 2.4% versus pes 5.5% ; p=0.02 ) . although , st showed higher rates in small vessel revascularization , the authors found that st was significantly lower in patients with small vessels treated with cocr - ees than in those treated with pes ( 0.2% versus 1.2% , respectively ; p=0.04).33 currently , the factors predictive of in - stent restenosis can be divided into patient - related , procedure - related , and lesion - related factors . patient - related factors such as diabetes , a history of restenosis , and genetic factors have been reported as risk factors of in - stent restenosis.33 procedure - related factors include the number of stents implanted , the total stent length , and stent overlap . lesion - related characteristics , which impact the rate of restenosis , include small vessel size , long lesion length , and the severity of pretreatment as well as posttreatment lesion stenosis , among others.36 claessen et al37 collected data from spirit ii , iii , and iv,34,38,39 and combined three groups : short lesions in large vessels ( group a ) ; long lesions in large vessels or short lesions in small vessels ( group b ) ; and long lesions in small vessels ( group c ) to evaluate the safety and efficacy of cocr - ees versus pes . the authors also evaluated mace rates by stent type ( pes and cocr - ees ) , and found that the higher the lesion complexity , the greater the mace incidence ( 7.0% , 11.2% , and 12.8% for pes , respectively , p=0.007 ; versus 4.8% versus 6.6% versus 9.1% for cocr - ees , respectively , p=0.001 ) . cocr - ees were associated with significantly lower rates of mace , mi , id - tlr , and st in groups b and c , but no statistical significance was found in the less complex group ( group a ) . multivariate analysis found that the use of cocr - ees rather than pes was an independent predictor of freedom from mace in group b ( p<0.0001 ) and group c ( p=0.004 ) , but not in group a ( p=0.19).37 recently , we reported40 the pathologic findings of 2nd gen des and compared these to 1st gen des . the prevalence of restenosis for cocr - ees ( 17% ) did not differ significantly from that observed in ses ( 14% ) and pes ( 12% ) . the prevalence of des with > 30% uncovered struts was also significantly lower in cocr - ees ( 20% ) than in ses ( 60% ; p<0.0005 ) and pes ( 67% ; p<0.0005 ) . in terms of inflammation , the overall prevalence of neoatherosclerosis after cocr - ees implantation in native coronary arteries was 29% , which did not differ significantly from ses ( 35% ; p=0.62 ) and pes ( 19% ; p=0.47).40 complex lesion characteristics and unstable plaques are associated with a greater delay in arterial healing when compared to pci of a simple and stable plaque by pathology.41 therefore , we evaluated the prevalence of > 30% uncovered struts in the setting of off - label versus on - label clinical indications . a greater number of cases and matched control groups will be required to understand the full scope of histopathological findings of cocr - ees in small vessel disease . des have progressively improved clinical outcomes , but the potential risk of st is still a concern and limits the use of des , especially in small vessel cad . several reports of st have been associated with 1st gen des , especially after dual anti - platelet therapy termination.43,44 over time , great effort has been made to improve the technology , thus reducing strut thickness from 140 m to approximately 7080 m , resulting in a dramatic reduction of thrombogenicity in bench studies.17,45 advances in polymer technology have been enormous ; new biocompatible polymers ( pbma or pvdf - hfp ) result in less inflammation after stent implantation , with the consequence of more complete and functional endothelization.46,47 also , cocr - ees show fewer coating defects after implantation when compared to different des , and this result was maintained over time and may improve vascular biocompatibility.25 clinical data confirmed the outstanding performance of cocr - ees , with lower rates of definitive / probable st , tlr , and mace . the pg for tlf was overperformed with cocr - ees when compared with a competitor des in small vessel disease . specifically , in small vessel disease , cocr - ees have been shown to be less thrombogenic compared to 1st gen des ; however , inflammation and restenosis remain a problem in this setting , and further technological and procedural progress is needed to improve patient outcomes . in the des era , small vessel cad remains a great challenge for interventional cardiologists . stent design and the material combination may provide better outcomes , especially in small vessel disease . cocr - ees with thinner struts , biocompatible polymers , reduced drug load , and better radiopacity and trackability have shown excellent results from preclinical , clinical , and pathological studies in small vessel cad .

the reconstruction of multiple missing teeth with dental implants is a predictable and proven treatment technique for edentulous patients in both anterior and posterior regions.12 however , the molar teeth are often shown to be a troublesome area for implant restorations from mechanical and biological aspects,3 which is due to complicated and complex factors of implant prosthetic components and the load - related bone contact area.4 the strong occlusal forces exert harmful effects on an implant prosthesis and alveolar bone in the posterior area,56 which results in marginal bone loss and decreased implant stability , and can lead to complications in an implant fixture and its suprastructure.78 natural teeth are traditionally splinted in order to decrease the stress and increase the stability of prosthesis . this can also result in a smaller horizontal load being transferred to the supporting teeth , and can compensate for the crown - root ratio increasing in various alveolar bone - loss regions and periodontally compromised patients.39 several studies have recommended that adjacent implants should be splinted with the fixed retained prosthesis.3 when off - axis forces are applied to an implant , they induce an adverse loading that can cause mechanical failure of a restored implant and biological failure of the surrounding bone that could lead to implant failure.101112 the aim of a splinted implant restoration is to favorably distribute the stress between the implants in order to minimize the transmission of horizontal forces to the bone - implant contact area.31314 in particular , two - implant splinting ( 2-is ) in the posterior region can promote the stability in the mesiodistal direction and relieve the stress in the buccolingual direction.9 2-is can also be considered as an important treatment option in patients without anterior guidance or with parafunctional oral habits.15 nevertheless , several procedures of multiple - implant restoration splinting are highly technique - sensitive , and the accuracy of the final prosthesis is mainly limited.15 alveolar bone loss is common in patients with periodontitis , which will lead to an unfavorable crown - implant ratio ( c / i ratio ) . an off - axis force acting on an implant restoration with an increasing c / i ratio and crown height space ( chs ) of the implant - defined as the distance from the alveolar bone crest to the occlusion plane - can induce a detrimental load at the implant restoration neck area , and result in surrounding bone loss and eventual prosthetic failure.1617 according to grossmann et al.,3 the splinting technique can be an appropriate treatment option for periodontitis patients who have an impaired occlusal relationship due to the loss of alveolar bone and multiple teeth . in spite of splinted implant restoration being beneficial for periodontitis patients with severe alveolar bone loss and excessive occlusal forces in the posterior region , most of the studies have been theoretical , with insufficient clinical analyses and negative long - term results.3 several studies of splinted prostheses have involved short - term investigations and shown limited efficacies and controversial results , and so further investigations and long - term studies are required.151819 the aim of this retrospective study was to determine the efficacies of 2-is , and compare them with those of single implant restoration ( 1-ir ) in the first and second molar regions , which has been demonstrated to produce good results in previous studies . this study has also identified the appropriate clinical considerations for splinted implant restoration of the molar region . this study was approved by the institutional review board of the ilsan hospital , national health insurance service ( nhis ) ( approval no . all surgical treatment procedures were performed by periodontists at the department of periodontology , ilsan hospital , nhis . the study was limited to the posterior region , including patients with missing first and second molars only , in order to minimize the effects of position and occlusal force . the internal connection implant fixtures comprising a sand - blasted , large - grit , acid - etched surface ( implantium , dentium , seoul , korea ; straumann , institut straumann , basel , switzerland ) were placed using a one- or two - stage surgical procedure as the manufacturer 's protocol . the prosthesis type [ i.e. , occlusal screw ( os ) , lateral screw ( ls ) , cementation ( cm ) , and screw - cement - retained prosthesis ( scrp ) ] was selected depending on the condition of the patient and the preferences of the prosthodontist , and occlusal adjustment was carried out to obtain the optimal centric and eccentric contact forces . maintenance care that emphasized scaling and oral hygiene instruction was provided every 3 - 6 months , and intraoral periapical or panorama radiographs were obtained every 12 months . patients who had undergone implant surgery at the department of periodontology , ilsan hospital , nhis during 2005 - 2014 were reviewed over a mean functional loading period ( flp ) of 40 months . the following inclusion criteria were applied : sex ratio of 1 : 1 , aged 20 - 80 years ( mean age 58.5 years ) , and good systemic health condition ( including well - controlled systemic diseases ) . the implant prosthesis had been functioning for 1.1 - 102.8 months , with a mean loading period of 41.4 months . single - implant restorations in the first or second molar region were classified into the 1-ir group , while restorations involving two splinted implants in the first and second molar regions were classified into the 2-is group . patients with severe systemic disease , advanced or aggressive periodontitis , or parafunctional oral habits ( e.g. , excessive occlusal force , heavy clenching , or bruxism ) were excluded from this research . in total , 408 implants in 234 patients who conformed to the inclusion and exclusion criteria were investigated . all data related to these patients with implant treatments were based on the clinical treatment records , clinical photographs , and radiographs of the patients . the following clinical factors of the patients were considered : sex , mean age , implant location , flp , bone grafting , clinical c / i ratio , chs , horizontal distance ( hd ) between the two implants ( hdi , the first and second molar positions ) , and hd between the natural tooth in the mesial position and the implant in the distal position ( hdni , the first or second molar position).2021 based on previous studies , the clinical c / i ratio was defined as the distance ratio measured from the clinical crown to the implant fixture ( standard fulcrum located at the marginal bone ) , and chs was measured as the distance from the alveolar bone crest to the occlusion plane.22 hdi and hdni were measured as the distance at the marginal bone level on the day of implant placement . a pacs workstation ( centricity ge healthcare , waukesha , wi , usa ) was used to calculate the clinical c / i ratio , chs , hdi , and hdni on the radiographs , and distortion caused by magnification was corrected using a calibration based on the known interthread pitch of the implant ( implantium , dentium : 0.6 mm ; straumann , institut straumann : 1.25 mm ) as a reference . mechanical complications [ i.e. , sl , screw fracture ( sf ) , cf , and repeated sl ] and biological complications [ i.e. , peri - implant mucositis ( pm ) and periimplantitis ( pi ) ] were evaluated for each patient . the biological complications were examined based on mobility , suppuration , probing depth , bleeding on probing , and alveolar bone loss . a reversible inflammation of peri - implant mucosa was diagnosed as pm , and loss of alveolar bone was diagnosed as pi.23 all measurements were performed using a unc periodontal probe ( hu - friedy , chicago , il , usa ) . in comparisons and analyses of two groups , the chi - square test and student 's t - test ( two - tailed with independent samples ) were used to identify the relationships between the clinical factors ( i.e. , sex , mean age , implant location , flp , clinical c / i ratio , chs , hdi , and hdni ) and the complication rates ( i.e. , mechanical and biological complications ) . the chi - square test was applied to noncontinuous variables and student 's t - test ( two - tailed with independent samples ) was applied to continuous variables . the optimal cutoff value for flp related to the complications was evaluated using receiver operating characteristics ( roc ) analysis . the results obtained in all of the investigations were analyzed using spss software ( version 19.0 , spss , chicago , il , usa ) . the cutoff for statistical significance was set at p the subjects of this study comprised the 1-ir group , which contained 124 patients ( 69 males , 55 females ) with a mean age of 56.26 years ( range , 23 - 77 years ) , and the 2-is group , which contained 110 patients ( 53 males , 57 females ) with a mean age of 59.01 years ( range , 34 - 91 years ) . the implants in the 1-ir group were distributed in the posterior region as follows : maxillary first molar , n = 22 ( 15.9% ) ; maxillary second molar , n = 10 ( 7.3% ) ; mandibular first molar , n = 43 ( 31.2% ) ; and mandibular second molar , n = 63 ( 45.6% ) . totals of 32 ( 23.2% ) and 106 ( 76.8% ) implants were positioned in the maxilla and mandible , respectively . in the 2-is group , 134 ( 67 pairs , 49.6% ) and 136 ( 68 pairs , 50.4% ) implants were positioned in the maxilla and mandible , respectively . the mean flp was 42.87 months ( range , 1.84 - 101.25 months ) for the 1-ir group and 39.86 months ( range , 1.08 - 102.75 months ) for the 2-is group . in addition , bone grafting was performed for 32 ( 23.3% ) implants in the 1-ir group and for 108 ( 54 pairs , 40% ) implants in the 2-is group , showing an intergroup difference of about threefold . for the 1-ir group , the clinical c / i ratio was 1.11 0.47 , chs was 9.65 1.98 mm , and hdni was 2.80 1.17 mm ; the corresponding values for the 2-is group were 1.07 0.21 , 9.62 1.75 mm , and 3.26 1.30 mm , respectively . these results are consistent with a previous study finding that in order to minimize bone loss , hdi ( i.e. , interimplant distance ) should be longer than hdni ( i.e. , distance from the adjacent natural tooth to the implant).24 most of the data represents a mean value of normal distribution curve ; the deviation is observed in individual clinical situation of a patient . since periodontist performed the treatment in the controlled clinical setting , it can be said that the position of implant is appropriate in this study ( table 1 ) . mechanical complications were found in 31 ( 22.6% ) of the 138 implants in the 1-ir group , with sl ( n = 23 , 16.7% ) being the most common complication . this was followed by sf and cf ( n = 3 , 2.2% ) , and then repeated sl ( n = 2 , 1.5% ) . mechanical complications were found in 30 ( 11.1% ) of the 270 implants in the 2-is group , corresponding to approximately half the rate in the 1-ir group . cf ( n = 14 , 5.2% ) was the most common complication , followed by sl ( n = 10 , 3.7% ) and then sf ( n = 6 , 2.2% ) , while repeated sl did not occur in any of the implants . the rate of mechanical complications differed significantly between the two groups ( p = .020 ) , with only sl showing a clearly significant increase in the 1-ir group ( p < .001 ) . the rate of biological complications was markedly higher in the 2-is group than in the 1-ir group . only pi ( n = 5 , 3.6% ) was found in the 1-ir group . in contrast , out of the 44 ( 16.3% ) implants with biological complications in the 2-is group , pi was found in 26 ( 9.6% ) implants , followed by pm , which was found in 18 ( 6.7% ) implants . the rate of biological complication differed significantly between the two groups ( p < .001 ) , with significantly elevated incidence rates of pm ( p = .002 ) and pi ( p = .046 ) in the 2-is group . in 1-ir group , sl , sf , and cf occurred simultaneously on one implant . also , sl and cf occurred simultaneously on another implant , and sl and pi occurred simultaneously on the other implant . in 2-is group , sl and sf occurred simultaneously on one implant , and sl , cf , and pi occurred simultaneously on another implant ( table 2 ) . patients in the 2-is group who did or did not experience complications at least once were classified into the complication and success groups , respectively . mechanical and biological complications showed no statistically significant associations with sex , age , implant location in the jaw , and bone grafting . the flp was the only clinical factor to show a statistically significant difference with complications and success in the 2-is group ( p = .049 ) . 2-is remained relatively successful up to a mean of 38 months , while biological and mechanical complications arose after mean time periods of approximately 49 and 56 months , respectively . implant - supported fixed dental prostheses ( isfdps ) located in the posterior region can induce stress in the implant and marginal bone when there is an unfavorable c / i ratio ( anatomical and/or clinical c / i ratio of 2).25 in the present study , the overall clinical c / i ratio in the 2-is group was 1.07 0.21 ( first molar region = 1.06 0.18 , second molar region = 1.09 0.23 ) , and the maximum value was 1.84 ( table 1 ) , confirming that a favorable c / i ratio had been achieved . moreover , the clinical c / i ratio was close to 1 : 1 in both the mechanical complication and success groups ( 1.05 0.14 and 1.08 0.21 ) and the biological complication and success groups ( 1.05 0.23 and 1.08 0.20 ) , demonstrating favorable clinical c / i ratios.25 correspondingly , there were no statistically significant differences in the clinical c / i ratios . recent studies262728 have found that chs values exceeding 15 mm indicate an increased risk of implant prosthesis failure due to a vertical cantilever effect . in this study , the overall chs in the 2-is group was 9.62 1.75 mm ( first molar region = 9.82 1.65 mm , second molar region = 9.43 1.84 mm ) , with a maximum value of 14.28 mm ( table 1 ) . given that this is within the acceptable chs range ( 8 - 12 mm ) and below 15 mm , chs is not expected to have a negative effect on the prognosis of the implant prosthesis . chs did not differ significantly among the mechanical complication and success groups ( 9.67 1.31 and 9.62 1.80 mm , respectively ) and the biological complication and success groups ( 10.04 1.94 and 9.53 1.70 mm , respectively ) . hdi also did not differ significantly among the mechanical and biological complication groups ( 3.36 1.27 and 3.51 1.51 mm , respectively ) and the mechanical and biological success groups ( 3.25 1.30 and 3.21 1.25 mm , respectively ) ( table 3 ) . ls was the most commonly used type of prosthesis in the 1-ir group ( n = 84 , 60.9% ) , and was associated with the following rates of mechanical complications : sl , n = 14 ( 16.7% ) ; repeated sl , n = 1 ( 1.2% ) ; sf , n = 2 ( 2.4% ) ; and cf , n = 2 ( 2.4% ) . the next most common type of prosthesis was cm ( n = 23 , 16.7% ; sl , n = 5 , 21.7% ; repeated sl , n = 0 , 0% ; sf , n = 1 , 4.4% ; cf , n = 1 , 4.4% ) , followed by scrp ( n = 19 , 13.8% ; sl , n = 2 , 10.5% ; repeated sl , n = 0 , 0% ; sf , n = 0 , 0% ; cf , n = 0 , 0% ) and os ( n = 12 , 8.7% ; sl , n = 2 , 16.7% ; repeated sl , n = 1 , 8.3% ; sf , n = 0 , 0% ; cf , n = 0 , 0% ) . sl was the most frequent mechanical complication , and was associated with the prosthesis types as follows : os , n = 2 ( 16.7% ) ; ls , n = 14 ( 16.7% ) ; cm , n = 5 ( 21.7% ) ; and scrp , n = 2 ( 10.5% ) . although sl occurred proportionally the most often in cm , in terms of absolute numbers it occurred the most often in ls . however , there were no statistically significant associations between prosthesis types and mechanical complication rates ( p = .304 ) . ls was the most commonly used type of prosthesis in the 2-is group ( n = 150 , 55.6% ) , and was associated with the following rates of mechanical complications : sl , n = 8 ( 5.3% ) ; repeated sl , n = 0 ( 0% ) ; sf , n = 4 ( 2.7% ) ; and cf , n = 6 ( 4.0% ) . the next most common type of prosthesis was cm ( n = 35 , 25.9% ; sl , n = 2 , 2.9% ; repeated sl , n = 0 , 0% ; sf , n = 2 , 2.9% ; cf , n = 4 , 11.4% ) , followed by scrp ( n = 17 , 12.6% ; no complications ) and os ( n = 8 , 5.9% ; no complications ) . the overall mechanical complication rates were lower for all prosthesis types in the 2-is group than in the 1-ir group , with sl being particularly rare . os and scrp , which were used proportionally less , showed no complications at all . as with the 1-ir group , there were no statistically significant differences ( p = .425 ) ( table 4 ) . there was no case of pm for any of the prosthesis types in the 1-ir group . pi occurred in os ( n = 3 , 25.0% ) , ls ( n = 1 , 1.2% ) , and cm ( n = 1 , 4.4% ) , but was not observed in scrp . conversely , biological complications occurred for all prosthesis types in the 2-is group , with the following rates : os ( pm , n = 0 , 0% ; pi , n = 4 , 25.0% ) , ls ( pm , n = 4 , 2.7% ; pi , n = 18 , 12.0% ) , cm ( pm , n = 12 , 17.1% ; pi , n = 4 , 5.7% ) , and scrp ( pm , n = 2 , 5.9% ; pi , n = 0 , 0% ) . pm was most common in cm , while pi showed the highest rate in os but the highest absolute frequency in ls . in both groups , there were no statistically significant associations between prosthesis types and biological complications ( p = .385 and 0.385 , respectively ) ( table 4 ) . the results listed in table 3 indicate that flp was the only variable that had an important impact on mechanical and biological success . the roc curve for flp of mechanical and biological complications is shown in fig . the area under the roc curve ( auc ) for flp of mechanical complications is 0.725 , which indicates a reliable result since the value exceeds 0.5 . the optimal cutoff value was 46.57 months ( 95% confidence interval , 0.61 - 0.84 ) , which gave a sensitivity of 69.2% and a specificity of 69.7% . in addition , the auc for flp of biological complications was 0.615 , which is also a reliable result ( i.e. , > 0.5 ) . the optimal cutoff value was 39.80 months ( 95% confidence interval , 0.49 - 0.74 ) , which gave a sensitivity of 54.5% and a specificity of 54.9% . while 1-ir has been predictable treatment modality in the posterior edentulous region , mechanical and biological complications occur frequently.2930 these complications include sl , cf , implant fixture fracture , de - cementation , pm , and pi , and also , they often occur in multiple - implant restoration splinting.31 in addition , the implant success rate of 1-ir ( 94.3% ) was not significantly lower than that of multiple - implant restoration splinting ( 97.1%).32 therefore , these two types of the implant restoration have been reported to have similar success rates . however , there were some differences between the 1-ir and 2-is groups in the present study in the characteristics of mechanical and biological complication rates related to clinical factors . there was no significant association between the implant position ( first or second molar region ) in the 1-ir group and the occurrence of mechanical and biological complications ( p = .243 and p = .746 , respectively ) . these results were identical to those of a previous study.33 also , prosthesis types were not associated with complication rates in the 1-ir and 2-is groups , and did not affect the comparison of the two groups ( p = .276 ) . compared to 2-is sl was reported as the most common mechanical complication of implant - supported single crowns ( isscs),2934 and also in the present study this was the only major complication associated with a statistically significant increase in occurrence ( p < .001 ) . sex was the only clinical factor exerting an important influence on the occurrence of mechanical complications in 1-ir ( p = .040 ) . finally , 91.3% ( 21 implants ) of all sl cases occurred in male patients , which is possibly due to the biting force and occlusal contact area both being greater than in female patients.3536 conversely , in 2-is , there was a significant increase in the rates of biological complications ( p < .001 ) . one possible explanation is that although regular dental hospital visits following the completion of the final implant prosthesis enabled examinations during the early stages , the intervals between the dental hospital visits became longer over time , resulting in a reduced awareness about oral hygiene management . similarly , the significant increases in pm ( p = .002 ) and pi ( p = .046 ) are due to the difficulty of performing adequate oral hygiene management in splinted implant restorations . several studies have found that nonsplinted implant restoration was advantageous for oral hygiene management.3 these results emphasize the need to provide patients with specific instructions about the use of dental floss and interdental brushes , especially for 2-is . previous studies2737 found that sl induced changes in centric and eccentric contact forces and nonideal occlusion , and that this was a major cause of sf and cf . moreover , food impaction on the inferior aspect of the implant prosthesis was caused by sl , and the resulting gingival redness and swelling not only increased the occurrence rates of pm and pi but also decreased the survival and success rates of the implants.38 in the present study we also observed the simultaneous occurrence of sf , cf , and pi with sl in three implants from the 1-ir group and in two implants from the 2-is group . the superiority of 1-ir in the first and second molar regions has been reported previously.3940 2-is was advantageous over 1-ir in terms of mechanical complications but disadvantageous in terms of biological complications . the decreased incidence of mechanical complications is probably attributable to the improved stress distribution resulting from the splinting technique reducing the transfer of excessive forces to the implant fixture and surrounding bone31314 . in addition , the splinting technique is able to promote the retention and resistance of the prosthesis , and successful outcomes can also be expected under certain limiting conditions such as insufficient abutment length , abnormal loading , or long treatment period.3 the increased incidence of biological complications is probably due to structural aspects of the splinting technique . it is often difficult to ensure the formation of appropriate paths and distances when placing fixtures in patients with periodontal disease . this makes it difficult to form an appropriate contour and embrasure between the inferior aspect of the splinted implant restoration and the interproximal region , which is a limitation in oral hygiene management . given that biological complications cause inflammation and the loss of tissue and bone in the surrounding implant , and increase the risk of implant failure , these complications need to be managed carefully . conversely , the disconnection and reconnection are more convenient for 1-ir , and examining and ensuring the hygiene of the prosthesis are easier than for the splinted implant restoration.4142 the only clinical factor that strongly affected the mechanical and biological complications associated with 2-is was the flp . in 2-is , relatively successful outcomes were maintained for up to 38 months on mean after the placement of the implant prosthesis . however , biological and mechanical complications occurred after mean flp of approximately 49 and 56 months , respectively . these findings contrast with a previous study finding that mechanical complications usually occurred sooner than biological complications during follow - up periods of 1 - 2 years.43 we attribute this difference to our clinical protocol of implant treatment for preventing mechanical complications , since patients were encouraged to visit the dental hospital continuously at short intervals after completing the treatment so that their implant prostheses could be examined regularly . pm and pi were previously reported to occur in 50% and 10 - 43% of all implants , respectively.4445 however , the incidence of biological complications was considerably lower in the present study , with pm and pi occurring in 0% and 3.6% of 1-ir , respectively , and in 6.7% and 9.6% of 2-is . the dental hospital visits occurred regularly at intervals of 3 - 6 months during the 3 years after implant placement in the present study . however , after 3 years the interval between the dental hospital visits increased to 1 year , and some cases were lost to follow - up due to cancelled appointments . this led to neglect of oral hygiene management , which resulted in increased rates of biological complications due to the deposition of plaque and calculus and of mechanical complications due to the lack of regular examinations approximately 1 - 2 years later . therefore , within the limitations of this study , the optimal cutoff values for flp with significant effects on the mechanical and biological success of 2-is were calculated using roc analysis . this information can be used to determine the optimal period for follow - up by predicting the time at which complications might occur . such a strategy will help prevent complications , while making it possible to explain the importance of regular dental hospital visits to patients and also providing them with motivation . in the previous study , an excessively long hdni ( > 3.7 mm ) was a significant factor affecting the prognosis of isscs located in the first and second molar regions.33 however , in the present study , hdi was not an important cause of the complications in 2-is . this is probably due to hdi has to be longer than hdni in order to reduce the alveolar bone loss,24 and the reduction in the cantilever effect due to dispersal of the occlusal forces when the implant prosthesis is splinted . moreover , careful consideration should be given to a certain degree of hdi needing to be established for an interproximal design in order to simplify the use of oral hygiene products such as dental floss and interdental brushes . the results of this study need to be analyzed carefully because they were limited by the lack of investigations of various clinical factors that could affect implant restoration , such as the individual physiological characteristics , occlusal relationships , and parafunctional oral habits of each patient.464748 therefore , more reliable results can be expected from future investigations of restorations involving two or more splinted implants to analyze the effect of the above factors on mechanical and biological complications and from long - term evaluations . in addition , a careful prospective study of flp should be performed , since the present study found flp to be an important factor affecting mechanical and biological complications in 2-is . flp are the most significant clinical factor for the mechanical and biological complication rates of 2-is . biological complications come about in flp of 39.80 months and mechanical complications in flp of 46.57 months . therefore , clinical consideration of flp will help to prevent the mechanical and biological complications of 2-is .

purposethe aim of this study was to compare the efficacies of two - implant splinting ( 2-is ) and single - implant restoration ( 1-ir ) in the first and second molar regions over a mean functional loading period ( flp ) of 40 months , and to propose the appropriate clinical considerations for the splinting technique.materials and methodsthe following clinical factors were examined in the 1-ir and 2-is groups based on the total hospital records of the patients : sex , mean age , implant location , flp , bone grafting , clinical crown - implant ratio , crown height space , and horizontal distance . the mechanical complications [ i.e. , screw loosening ( sl ) , screw fracture , crown fracture , and repeated sl ] and biological complications [ i.e. , peri - implant mucositis ( pm ) and peri - implantitis ( pi ) ] were also evaluated for each patient . in analysis of two groups , the chi - square test and student 's t - test were used to identify the relationship between clinical factors and complication rates . the optimal cutoff value for the flp based on complications was evaluated using receiver operating characteristics analysis.resultsin total , 234 patients with 408 implants that had been placed during 2005 - 2014 were investigated . the incident rates of sl ( p<.001 ) , pm ( p=.002 ) , and pi ( p=.046 ) differed significantly between the 1-ir and 2-is groups . the flp was the only meaningful clinical factor for mechanical and biological complication rates in 2-is.conclusionthe mechanical complication rates were lower for 2-is than for 1-ir , while the biological complication rates were higher for 2-is . flp of 39.80 and 46.57 months were the reference follow - up periods for preventing biological and mechanical complications , respectively .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

the reconstruction of multiple missing teeth with dental implants is a predictable and proven treatment technique for edentulous patients in both anterior and posterior regions.12 however , the molar teeth are often shown to be a troublesome area for implant restorations from mechanical and biological aspects,3 which is due to complicated and complex factors of implant prosthetic components and the load - related bone contact area.4 the strong occlusal forces exert harmful effects on an implant prosthesis and alveolar bone in the posterior area,56 which results in marginal bone loss and decreased implant stability , and can lead to complications in an implant fixture and its suprastructure.78 natural teeth are traditionally splinted in order to decrease the stress and increase the stability of prosthesis . this can also result in a smaller horizontal load being transferred to the supporting teeth , and can compensate for the crown - root ratio increasing in various alveolar bone - loss regions and periodontally compromised patients.39 several studies have recommended that adjacent implants should be splinted with the fixed retained prosthesis.3 when off - axis forces are applied to an implant , they induce an adverse loading that can cause mechanical failure of a restored implant and biological failure of the surrounding bone that could lead to implant failure.101112 the aim of a splinted implant restoration is to favorably distribute the stress between the implants in order to minimize the transmission of horizontal forces to the bone - implant contact area.31314 in particular , two - implant splinting ( 2-is ) in the posterior region can promote the stability in the mesiodistal direction and relieve the stress in the buccolingual direction.9 2-is can also be considered as an important treatment option in patients without anterior guidance or with parafunctional oral habits.15 nevertheless , several procedures of multiple - implant restoration splinting are highly technique - sensitive , and the accuracy of the final prosthesis is mainly limited.15 alveolar bone loss is common in patients with periodontitis , which will lead to an unfavorable crown - implant ratio ( c / i ratio ) . an off - axis force acting on an implant restoration with an increasing c / i ratio and crown height space ( chs ) of the implant - defined as the distance from the alveolar bone crest to the occlusion plane - can induce a detrimental load at the implant restoration neck area , and result in surrounding bone loss and eventual prosthetic failure.1617 according to grossmann et al.,3 the splinting technique can be an appropriate treatment option for periodontitis patients who have an impaired occlusal relationship due to the loss of alveolar bone and multiple teeth . in spite of splinted implant restoration being beneficial for periodontitis patients with severe alveolar bone loss and excessive occlusal forces in the posterior region , most of the studies have been theoretical , with insufficient clinical analyses and negative long - term results.3 several studies of splinted prostheses have involved short - term investigations and shown limited efficacies and controversial results , and so further investigations and long - term studies are required.151819 the aim of this retrospective study was to determine the efficacies of 2-is , and compare them with those of single implant restoration ( 1-ir ) in the first and second molar regions , which has been demonstrated to produce good results in previous studies . this study has also identified the appropriate clinical considerations for splinted implant restoration of the molar region . , occlusal screw ( os ) , lateral screw ( ls ) , cementation ( cm ) , and screw - cement - retained prosthesis ( scrp ) ] was selected depending on the condition of the patient and the preferences of the prosthodontist , and occlusal adjustment was carried out to obtain the optimal centric and eccentric contact forces . patients who had undergone implant surgery at the department of periodontology , ilsan hospital , nhis during 2005 - 2014 were reviewed over a mean functional loading period ( flp ) of 40 months . the following inclusion criteria were applied : sex ratio of 1 : 1 , aged 20 - 80 years ( mean age 58.5 years ) , and good systemic health condition ( including well - controlled systemic diseases ) . the implant prosthesis had been functioning for 1.1 - 102.8 months , with a mean loading period of 41.4 months . single - implant restorations in the first or second molar region were classified into the 1-ir group , while restorations involving two splinted implants in the first and second molar regions were classified into the 2-is group . all data related to these patients with implant treatments were based on the clinical treatment records , clinical photographs , and radiographs of the patients . the following clinical factors of the patients were considered : sex , mean age , implant location , flp , bone grafting , clinical c / i ratio , chs , horizontal distance ( hd ) between the two implants ( hdi , the first and second molar positions ) , and hd between the natural tooth in the mesial position and the implant in the distal position ( hdni , the first or second molar position).2021 based on previous studies , the clinical c / i ratio was defined as the distance ratio measured from the clinical crown to the implant fixture ( standard fulcrum located at the marginal bone ) , and chs was measured as the distance from the alveolar bone crest to the occlusion plane.22 hdi and hdni were measured as the distance at the marginal bone level on the day of implant placement . a pacs workstation ( centricity ge healthcare , waukesha , wi , usa ) was used to calculate the clinical c / i ratio , chs , hdi , and hdni on the radiographs , and distortion caused by magnification was corrected using a calibration based on the known interthread pitch of the implant ( implantium , dentium : 0.6 mm ; straumann , institut straumann : 1.25 mm ) as a reference . mechanical complications [ i.e. , sl , screw fracture ( sf ) , cf , and repeated sl ] and biological complications [ i.e. , peri - implant mucositis ( pm ) and periimplantitis ( pi ) ] were evaluated for each patient . the biological complications were examined based on mobility , suppuration , probing depth , bleeding on probing , and alveolar bone loss . in comparisons and analyses of two groups , the chi - square test and student 's t - test ( two - tailed with independent samples ) were used to identify the relationships between the clinical factors ( i.e. , sex , mean age , implant location , flp , clinical c / i ratio , chs , hdi , and hdni ) and the complication rates ( i.e. the chi - square test was applied to noncontinuous variables and student 's t - test ( two - tailed with independent samples ) was applied to continuous variables . the optimal cutoff value for flp related to the complications was evaluated using receiver operating characteristics ( roc ) analysis . the cutoff for statistical significance was set at p the subjects of this study comprised the 1-ir group , which contained 124 patients ( 69 males , 55 females ) with a mean age of 56.26 years ( range , 23 - 77 years ) , and the 2-is group , which contained 110 patients ( 53 males , 57 females ) with a mean age of 59.01 years ( range , 34 - 91 years ) . in addition , bone grafting was performed for 32 ( 23.3% ) implants in the 1-ir group and for 108 ( 54 pairs , 40% ) implants in the 2-is group , showing an intergroup difference of about threefold . for the 1-ir group , the clinical c / i ratio was 1.11 0.47 , chs was 9.65 1.98 mm , and hdni was 2.80 1.17 mm ; the corresponding values for the 2-is group were 1.07 0.21 , 9.62 1.75 mm , and 3.26 1.30 mm , respectively . mechanical complications were found in 31 ( 22.6% ) of the 138 implants in the 1-ir group , with sl ( n = 23 , 16.7% ) being the most common complication . this was followed by sf and cf ( n = 3 , 2.2% ) , and then repeated sl ( n = 2 , 1.5% ) . mechanical complications were found in 30 ( 11.1% ) of the 270 implants in the 2-is group , corresponding to approximately half the rate in the 1-ir group . cf ( n = 14 , 5.2% ) was the most common complication , followed by sl ( n = 10 , 3.7% ) and then sf ( n = 6 , 2.2% ) , while repeated sl did not occur in any of the implants . the rate of mechanical complications differed significantly between the two groups ( p = .020 ) , with only sl showing a clearly significant increase in the 1-ir group ( p < .001 ) . the rate of biological complications was markedly higher in the 2-is group than in the 1-ir group . the rate of biological complication differed significantly between the two groups ( p < .001 ) , with significantly elevated incidence rates of pm ( p = .002 ) and pi ( p = .046 ) in the 2-is group . mechanical and biological complications showed no statistically significant associations with sex , age , implant location in the jaw , and bone grafting . the flp was the only clinical factor to show a statistically significant difference with complications and success in the 2-is group ( p = .049 ) . 2-is remained relatively successful up to a mean of 38 months , while biological and mechanical complications arose after mean time periods of approximately 49 and 56 months , respectively . implant - supported fixed dental prostheses ( isfdps ) located in the posterior region can induce stress in the implant and marginal bone when there is an unfavorable c / i ratio ( anatomical and/or clinical c / i ratio of 2).25 in the present study , the overall clinical c / i ratio in the 2-is group was 1.07 0.21 ( first molar region = 1.06 0.18 , second molar region = 1.09 0.23 ) , and the maximum value was 1.84 ( table 1 ) , confirming that a favorable c / i ratio had been achieved . moreover , the clinical c / i ratio was close to 1 : 1 in both the mechanical complication and success groups ( 1.05 0.14 and 1.08 0.21 ) and the biological complication and success groups ( 1.05 0.23 and 1.08 0.20 ) , demonstrating favorable clinical c / i ratios.25 correspondingly , there were no statistically significant differences in the clinical c / i ratios . in this study , the overall chs in the 2-is group was 9.62 1.75 mm ( first molar region = 9.82 1.65 mm , second molar region = 9.43 1.84 mm ) , with a maximum value of 14.28 mm ( table 1 ) . chs did not differ significantly among the mechanical complication and success groups ( 9.67 1.31 and 9.62 1.80 mm , respectively ) and the biological complication and success groups ( 10.04 1.94 and 9.53 1.70 mm , respectively ) . hdi also did not differ significantly among the mechanical and biological complication groups ( 3.36 1.27 and 3.51 1.51 mm , respectively ) and the mechanical and biological success groups ( 3.25 1.30 and 3.21 1.25 mm , respectively ) ( table 3 ) . ls was the most commonly used type of prosthesis in the 1-ir group ( n = 84 , 60.9% ) , and was associated with the following rates of mechanical complications : sl , n = 14 ( 16.7% ) ; repeated sl , n = 1 ( 1.2% ) ; sf , n = 2 ( 2.4% ) ; and cf , n = 2 ( 2.4% ) . ls was the most commonly used type of prosthesis in the 2-is group ( n = 150 , 55.6% ) , and was associated with the following rates of mechanical complications : sl , n = 8 ( 5.3% ) ; repeated sl , n = 0 ( 0% ) ; sf , n = 4 ( 2.7% ) ; and cf , n = 6 ( 4.0% ) . the overall mechanical complication rates were lower for all prosthesis types in the 2-is group than in the 1-ir group , with sl being particularly rare . conversely , biological complications occurred for all prosthesis types in the 2-is group , with the following rates : os ( pm , n = 0 , 0% ; pi , n = 4 , 25.0% ) , ls ( pm , n = 4 , 2.7% ; pi , n = 18 , 12.0% ) , cm ( pm , n = 12 , 17.1% ; pi , n = 4 , 5.7% ) , and scrp ( pm , n = 2 , 5.9% ; pi , n = 0 , 0% ) . in both groups , there were no statistically significant associations between prosthesis types and biological complications ( p = .385 and 0.385 , respectively ) ( table 4 ) . the results listed in table 3 indicate that flp was the only variable that had an important impact on mechanical and biological success . the optimal cutoff value was 46.57 months ( 95% confidence interval , 0.61 - 0.84 ) , which gave a sensitivity of 69.2% and a specificity of 69.7% . in addition , the auc for flp of biological complications was 0.615 , which is also a reliable result ( i.e. the optimal cutoff value was 39.80 months ( 95% confidence interval , 0.49 - 0.74 ) , which gave a sensitivity of 54.5% and a specificity of 54.9% . while 1-ir has been predictable treatment modality in the posterior edentulous region , mechanical and biological complications occur frequently.2930 these complications include sl , cf , implant fixture fracture , de - cementation , pm , and pi , and also , they often occur in multiple - implant restoration splinting.31 in addition , the implant success rate of 1-ir ( 94.3% ) was not significantly lower than that of multiple - implant restoration splinting ( 97.1%).32 therefore , these two types of the implant restoration have been reported to have similar success rates . however , there were some differences between the 1-ir and 2-is groups in the present study in the characteristics of mechanical and biological complication rates related to clinical factors . there was no significant association between the implant position ( first or second molar region ) in the 1-ir group and the occurrence of mechanical and biological complications ( p = .243 and p = .746 , respectively ) . these results were identical to those of a previous study.33 also , prosthesis types were not associated with complication rates in the 1-ir and 2-is groups , and did not affect the comparison of the two groups ( p = .276 ) . compared to 2-is sl was reported as the most common mechanical complication of implant - supported single crowns ( isscs),2934 and also in the present study this was the only major complication associated with a statistically significant increase in occurrence ( p < .001 ) . sex was the only clinical factor exerting an important influence on the occurrence of mechanical complications in 1-ir ( p = .040 ) . finally , 91.3% ( 21 implants ) of all sl cases occurred in male patients , which is possibly due to the biting force and occlusal contact area both being greater than in female patients.3536 conversely , in 2-is , there was a significant increase in the rates of biological complications ( p < .001 ) . similarly , the significant increases in pm ( p = .002 ) and pi ( p = .046 ) are due to the difficulty of performing adequate oral hygiene management in splinted implant restorations . moreover , food impaction on the inferior aspect of the implant prosthesis was caused by sl , and the resulting gingival redness and swelling not only increased the occurrence rates of pm and pi but also decreased the survival and success rates of the implants.38 in the present study we also observed the simultaneous occurrence of sf , cf , and pi with sl in three implants from the 1-ir group and in two implants from the 2-is group . the superiority of 1-ir in the first and second molar regions has been reported previously.3940 2-is was advantageous over 1-ir in terms of mechanical complications but disadvantageous in terms of biological complications . in addition , the splinting technique is able to promote the retention and resistance of the prosthesis , and successful outcomes can also be expected under certain limiting conditions such as insufficient abutment length , abnormal loading , or long treatment period.3 the increased incidence of biological complications is probably due to structural aspects of the splinting technique . conversely , the disconnection and reconnection are more convenient for 1-ir , and examining and ensuring the hygiene of the prosthesis are easier than for the splinted implant restoration.4142 the only clinical factor that strongly affected the mechanical and biological complications associated with 2-is was the flp . however , biological and mechanical complications occurred after mean flp of approximately 49 and 56 months , respectively . these findings contrast with a previous study finding that mechanical complications usually occurred sooner than biological complications during follow - up periods of 1 - 2 years.43 we attribute this difference to our clinical protocol of implant treatment for preventing mechanical complications , since patients were encouraged to visit the dental hospital continuously at short intervals after completing the treatment so that their implant prostheses could be examined regularly . pm and pi were previously reported to occur in 50% and 10 - 43% of all implants , respectively.4445 however , the incidence of biological complications was considerably lower in the present study , with pm and pi occurring in 0% and 3.6% of 1-ir , respectively , and in 6.7% and 9.6% of 2-is . therefore , within the limitations of this study , the optimal cutoff values for flp with significant effects on the mechanical and biological success of 2-is were calculated using roc analysis . in the previous study , an excessively long hdni ( > 3.7 mm ) was a significant factor affecting the prognosis of isscs located in the first and second molar regions.33 however , in the present study , hdi was not an important cause of the complications in 2-is . the results of this study need to be analyzed carefully because they were limited by the lack of investigations of various clinical factors that could affect implant restoration , such as the individual physiological characteristics , occlusal relationships , and parafunctional oral habits of each patient.464748 therefore , more reliable results can be expected from future investigations of restorations involving two or more splinted implants to analyze the effect of the above factors on mechanical and biological complications and from long - term evaluations . flp are the most significant clinical factor for the mechanical and biological complication rates of 2-is . biological complications come about in flp of 39.80 months and mechanical complications in flp of 46.57 months . therefore , clinical consideration of flp will help to prevent the mechanical and biological complications of 2-is .

worldwide , approximately 1.38 million women are diagnosed with breast cancer each year . in developed countries , 80 % of women with breast cancer will survive at least 60 months due to early detection techniques and effective anti - cancer treatments . in the uk , there are currently around 550,000 breast cancer survivors . breast cancer treatments can cause chronic side effects such as oestrogen deprivation symptoms , athralgias , fatigue , lymphoedema , peripheral neuropathy , reduced bone health , upper extremity functional impairments and overall functional decline . a considerable number of breast cancer survivors experience some of these side effects although there is currently no accurate quantitative data on the incidence of these symptoms . the evidence that exercise is effective in treating many of these chronic or late appearing side effects is compelling : a recent systematic review and meta - analysis supported the use of exercise to prevent or treat fatigue and lymphoedema and to improve functional status and upper body range of movement . in addition , prospective observational studies suggest that around 3 h of aerobic activity per week can significantly reduce the risk of cancer recurrence and breast cancer mortality . there is now a need for randomised controlled trials ( rcts ) to examine the long - term effects of exercise interventions for improving outcomes such as quality of life , symptom management and ultimately cancer recurrence and mortality . to date , the longest follow - up with cancer survivors after an exercise intervention is 2 years , with most rcts only following participants up to 6 months post intervention . the aim of this study was to follow - up participants from a rct during adjuvant treatment 18 and 60 months after the exercise intervention . the original study s aim was to determine if there were functional and psychological benefits of a 12-week supervised group exercise programme during treatment for early stage breast cancer , including a 6-month follow - up . the study was designed as a pragmatic randomised controlled prospective open trial and was set in three oncology clinics in scotland for recruitment and in community facilities for the exercise intervention . the participants were 203 women with breast cancer with 177 completing the 6-month follow - up . the intervention incorporated a variety of safe cardiovascular , muscular strength and flexibility exercises and group discussion of exercise behaviour change techniques , in addition to usual care . the control group received usual care until the 6-month follow - up when they had a one to one discussion about how to incorporate physical activity into their lifestyle . the main outcome measures were : quality of life ( fact ) questionnaire , beck depression inventory ( bdi ) , positive and negative affect scale ( panas ) , body mass index ( bmi ) , 7-day recall of physical activity from the scottish physical activity questionnaire-2 ( spaq ) , 12-min walk test and assessment of shoulder mobility . the results showed significant intervention effects at 12 weeks and 6 months follow - up for metres walked in 12 min , minutes of moderate intensity activity reported in a week , shoulder mobility , breast cancer specific subscale of quality of life and for positive mood . it was concluded that a supervised group exercise programme provided functional and psychological benefit after a 12-week intervention and six months later . to determine if intervention effects continued after the 6-month follow - upto determine if women who had higher levels of activity after diagnosis and treatment had a different functional or psychological profile than women who had lower levels of activity and to elicit views from the women concerning their experience of physical activity post intervention to determine if intervention effects continued after the 6-month follow - up to determine if women who had higher levels of activity after diagnosis and treatment had a different functional or psychological profile than women who had lower levels of activity and to elicit views from the women concerning their experience of physical activity post intervention however , in this paper , we will not report on the qualitative analysis of the women s views . all women who had participated in the original study and who had agreed to being contacted again ( n = 148 ) were contacted at 18 months after the intervention and invited to participate in the follow - up study . sixty months after the intervention , all women ( regardless of 18-month participation ) were contacted again and invited to participate in a further follow - up . each woman s general practitioner ( gp ) was contacted to ensure it was appropriate to write to the participant . women who agreed to take part were then contacted by telephone to arrange for reassessment at the local sports facility where the original assessments had been carried out . all procedures and outcome measures were identical to the original study . each appointment lasted approximately 2 h. all procedures were approved by the local nhs research ethics committee and informed consent obtained . in the original study , the participants were seen at the beginning and end of the exercise intervention period ( i.e. baseline and 3 months ) and at the 6-month follow - up ( i.e. 6 months from the end of the intervention period ) . in this study , we add 18 and 60-month follow - up time points . at each time point , the participants self - reported the total number of minutes of leisure time activity undertaken in the previous week , using a validated questionnaire ( spaq ) . at each time point , they were classified as more or less active if they were above or below , respectively , the sample median leisure time activity for their age group . baseline demographics were summarised and compared for those who had and had not dropped out of the study at each follow - up time point . the effect of the intervention on change from baseline in each outcome was modelled over time using a linear mixed effects model with a random intercept for subject , adjusting for study site , therapy received at baseline and age . the mean difference in change from baseline between the intervention groups at each time point was estimated with a 95 % confidence interval ( ci ) and p value . the model implicitly accounts for missing data by considering the individual trends over time as well as the observed group means at each time point to give a more accurate estimate ( than the observed group mean alone ) of the population group means over time . for example , if women with lower scores at earlier time points tend to be more likely to drop out later , then the estimated mean at later time points will be adjusted downwards slightly to account for this . the differences in the outcomes at each time point between the more and less active group were estimated using similar models , additionally adjusting for intervention group . note that it was not possible to consider change from baseline for this comparison because women were not necessarily in the same activity groups at different time points and so the outcomes were the actual scores rather than change from baseline . all women who had participated in the original study and who had agreed to being contacted again ( n = 148 ) were contacted at 18 months after the intervention and invited to participate in the follow - up study . sixty months after the intervention , all women ( regardless of 18-month participation ) were contacted again and invited to participate in a further follow - up . each woman s general practitioner ( gp ) was contacted to ensure it was appropriate to write to the participant . women who agreed to take part were then contacted by telephone to arrange for reassessment at the local sports facility where the original assessments had been carried out . all procedures and outcome measures were identical to the original study . each appointment lasted approximately 2 h. all procedures were approved by the local nhs research ethics committee and informed consent obtained . in the original study , the participants were seen at the beginning and end of the exercise intervention period ( i.e. baseline and 3 months ) and at the 6-month follow - up ( i.e. 6 months from the end of the intervention period ) . in this study , we add 18 and 60-month follow - up time points . at each time point , the participants self - reported the total number of minutes of leisure time activity undertaken in the previous week , using a validated questionnaire ( spaq ) . at each time point , they were classified as more or less active if they were above or below , respectively , the sample median leisure time activity for their age group . baseline demographics were summarised and compared for those who had and had not dropped out of the study at each follow - up time point . the effect of the intervention on change from baseline in each outcome was modelled over time using a linear mixed effects model with a random intercept for subject , adjusting for study site , therapy received at baseline and age . the mean difference in change from baseline between the intervention groups at each time point was estimated with a 95 % confidence interval ( ci ) and p value . the model implicitly accounts for missing data by considering the individual trends over time as well as the observed group means at each time point to give a more accurate estimate ( than the observed group mean alone ) of the population group means over time . for example , if women with lower scores at earlier time points tend to be more likely to drop out later , then the estimated mean at later time points will be adjusted downwards slightly to account for this . the differences in the outcomes at each time point between the more and less active group were estimated using similar models , additionally adjusting for intervention group . note that it was not possible to consider change from baseline for this comparison because women were not necessarily in the same activity groups at different time points and so the outcomes were the actual scores rather than change from baseline . one hundred and fourteen women attended follow - up at 18 months and 87 women attended at 60 months . the flow of participants through this follow - up study is shown in fig . 1 ( see reference 8 for the flow diagram for the original study ) . baseline demographic characteristics of those that took part in the study at the 18 and 60-month follow - up versus those that did not are shown in table 1 . those who participated in the follow - up at 60 months were , at baseline , 3 years older and 5 kg lighter on average and were faster walkers ( i.e. probably fitter ) ; and may have been slightly less depressed and with less negative mood than those who did not participate at 60 months . women in work prior to diagnosis and those that were less deprived were more likely to participate than those who were housewives or more deprived . there were no differences in the proportions of control and exercise group women that responded at either 18 or 60 months.fig . 1participant flow through follow - up studytable 1demographics at baseline for women that took part in the follow - up study ( responders ) and women that did not take part in the follow - up study ( non - responders ) at each subsequent time point : summary statistics and p values for differences between responders and non - responders ( wilcoxon / fisher s test)18 months5 yearsresponderpresponderpnoyesnoyesage ( years)n87114<0.01114870.03mean ( sd)49.0 ( 9.2)53.5 ( 9.3)50.3 ( 9.5)53.2 ( 9.3)baseline weight ( kg)n85114<0.0111287<0.01mean ( sd)74.2 ( 15.5)68.3 ( 13.3)73.2 ( 15.2)67.8 ( 13.2)height ( cm)n871120.20114850.14mean ( sd)161.0 ( 6.3)159.9 ( 6.0)160.9 ( 6.3)159.7 ( 5.9)bdi scoren851120.06112850.06mean ( sd)13.3 ( 7.2)11.4 ( 7.1)13.1 ( 7.5)11.1 ( 6.6)panas positiven871120.10113860.13mean ( sd)26.5 ( 8.4)28.9 ( 9.0)26.8 ( 8.6)29.2 ( 8.9)panas negativen871120.05113860.09mean ( sd)19.5 ( 7.9)17.2 ( 6.8)19.1 ( 7.8)17.0 ( 6.5)12-min walk ( m)n851140.18112870.03mean ( sd)973.4 ( 220.8)996.0 ( 224.8)958.1 ( 242.4)1,022.7 ( 190.0)spaq leisure time activity ( min)n851100.32110850.71mean ( sd)367.0 ( 330.3)365.1 ( 267.9)375.1 ( 323.2)354.1 ( 257.7)srm total scoren871140.80114870.98mean ( sd)30.7 ( 5.8)30.8 ( 5.3)30.6 ( 5.7)30.9 ( 5.4)bmi ( kg / m)n851120.01112850.02mean ( sd)28.6 ( 6.0)26.8 ( 5.3)28.3 ( 5.9)26.6 ( 5.3)exercise groupcontrol46/102 ( 45.1)56/102 ( 54.9)0.6759/102 ( 57.8)43/102 ( 42.2)0.78exercise41/99 ( 41.4)58/99 ( 58.6)55/99 ( 55.6)44/99 ( 44.4)study centregri16/33 ( 48.5)17/33 ( 51.5)0.5521/33 ( 63.6)12/33 ( 36.4)0.57boc62/151 ( 41.1)89/151 ( 58.9)85/151 ( 56.3)66/151 ( 43.7)other9/17 ( 52.9)8/17 ( 47.1)8/17 ( 47.1)9/17 ( 52.9)therapychemotherapy7/15 ( 46.7)8/15 ( 53.3)0.527/15 ( 46.7)8/15 ( 53.3)0.67radiotherapy21/57 ( 36.8)36/57 ( 63.2)32/57 ( 56.1)25/57 ( 43.9)combination59/129 ( 45.7)70/129 ( 54.3)75/129 ( 58.1)54/129 ( 41.9)surgery typemast only31/57 ( 54.4)26/57 ( 45.6)0.1838/57 ( 66.7)19/57 ( 33.3)0.20lump only48/116 ( 41.4)68/116 ( 58.6)65/116 ( 56.0)51/116 ( 44.0)lump and mast1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)lump and recon0/1 ( 0.0)1/1 ( 100.0)0/1 ( 0.0)1/1 ( 100.0)mast and recon6/22 ( 27.3)16/22 ( 72.7)9/22 ( 40.9)13/22 ( 59.1)other1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)tamoxifen usedno57/117 ( 48.7)60/117 ( 51.3)0.0872/117 ( 61.5)45/117 ( 38.5)0.15yes30/83 ( 36.1)53/83 ( 63.9)42/83 ( 50.6)41/83 ( 49.4)highest education levelschool40/92 ( 43.5)52/92 ( 56.5)1.0054/92 ( 58.7)38/92 ( 41.3)0.77other43/99 ( 43.4)56/99 ( 56.6)55/99 ( 55.6)44/99 ( 44.4)employment status ( prior to diagnosis)ft / pt10/29 ( 34.5)19/29 ( 65.5)0.0111/29 ( 37.9)18/29 ( 62.1)0.02sick52/111 ( 46.8)59/111 ( 53.2)66/111 ( 59.5)45/111 ( 40.5)housewife17/26 ( 65.4)9/26 ( 34.6)20/26 ( 76.9)6/26 ( 23.1)retired8/35 ( 22.9)27/35 ( 77.1)17/35 ( 48.6)18/35 ( 51.4)occupation ( prior to diagnosis)professional17/48 ( 35.4)31/48 ( 64.6)0.7221/48 ( 43.8)27/48 ( 56.2)0.39managerial14/35 ( 40.0)21/35 ( 60.0)18/35 ( 51.4)17/35 ( 48.6)clerical25/55 ( 45.5)30/55 ( 54.5)32/55 ( 58.2)23/55 ( 41.8)manual15/33 ( 45.5)18/33 ( 54.5)20/33 ( 60.6)13/33 ( 39.4)carstairs deprivation1217/58 ( 29.3)41/58 ( 70.7)0.0423/58 ( 39.7)35/58 ( 60.3)0.013542/86 ( 48.8)44/86 ( 51.2)53/86 ( 61.6)33/86 ( 38.4)6726/53 ( 49.1)27/53 ( 50.9)35/53 ( 66.0)18/53 ( 34.0)periodsno70/169 ( 41.4)99/169 ( 58.6)0.3695/169 ( 56.2)74/169 ( 43.8)0.74irregular8/17 ( 47.1)9/17 ( 52.9)9/17 ( 52.9)8/17 ( 47.1)regular9/15 ( 60.0)6/15 ( 40.0)10/15 ( 66.7)5/15 ( 33.3)hysterectomyno80/179 ( 44.7)99/179 ( 55.3)0.36101/179 ( 56.4)78/179 ( 43.6)1.00yes7/22 ( 31.8)15/22 ( 68.2)13/22 ( 59.1)9/22 ( 40.9)hrtnever58/124 ( 46.8)66/124 ( 53.2)0.3173/124 ( 58.9)51/124 ( 41.1)0.69former24/67 ( 35.8)43/67 ( 64.2)35/67 ( 52.2)32/67 ( 47.8)current5/10 ( 50.0)5/10 ( 50.0)6/10 ( 60.0)4/10 ( 40.0 ) participant flow through follow - up study demographics at baseline for women that took part in the follow - up study ( responders ) and women that did not take part in the follow - up study ( non - responders ) at each subsequent time point : summary statistics and p values for differences between responders and non - responders ( wilcoxon / fisher s test ) to determine if there were any lasting effects of the intervention , comparisons were made between the original treatment and control groups . table 2 shows descriptive statistics of the outcome data at 18 and 60 months with corresponding treatment effect estimates . there were significant differences between the intervention and control groups at 60 months for spaq leisure time activity over the previous week and panas positive mood score with the intervention group reporting higher activity and more positive mood . even for outcomes for which there was no significant difference at 60 months , the intervention group was consistently observed to do better than the control group throughout the entire 5-year follow - up period . the treatment effect estimates at 18 and 60 months are also displayed in units of 1 standard deviation in fig . 2 for all outcomes measured and are of similar magnitude at both time points . at 5 years , the intervention group achieved on average around 200 min of activity each week more than the control group ( see table 2 for related data ) . in general , our analyses suggested that 5 years subsequent to taking part in such an exercise intervention similar patients would be likely to achieve on average 50 to 350 min of extra physical activity per week than patients treated as usual . this is a substantial difference which could lead to considerable health benefit.table 2main outcomes : summary statistics at 18 and 60 months for the control and exercise intervention groups and model effect estimates of treatment effect differences for change from baselinesummaries at each time pointeffect estimate ( exercise control)baseline18 months5 years18 m baseline5y baselinefact - galln20111487mean74.480.787.1(sd)(14.3)(14.6)(11.1)controln10256432.20.9mean72.779.685.7(1.8 , 6.2)(3.4 , 5.2)(sd)(15.6)(14.7)(11.4)0.2860.683exercisen995844mean76.181.788.5(sd)(12.6)(14.6)(10.7)bdi scorealln19711487mean12.29.37.00.80.2(sd)(7.2)(7.7)(6.7)(3.1 , 1.5)(2.8 , 2.4)controln9856430.4950.857mean12.99.77.5(sd)(7.5)(7.7)(6.7)exercisen995844mean11.58.96.6(sd)(6.9)(7.8)(6.7)panas positivealln19911487mean27.831.034.2(sd)(8.8)(9.8)(8.3)controln10056431.53.4mean28.030.633.1(1.4 , 4.3)(0.2 , 6.7)(sd)(9.2)(10.1)(8.7)0.3120.040exercisen995844mean27.731.335.2(sd)(8.4)(9.6)(7.8)panas negativealln199114870.80.5mean18.216.715.4(2.9 , 1.4)(2.9 , 1.8)(sd)(7.4)(7.5)(5.3)0.4870.655controln1005643mean19.117.416.3(sd)(7.7)(8.1)(5.6)exercisen995844mean17.316.014.5(sd)(6.9)(6.9)(5.0)12-min walkalln1999583mean9861,0851,065(sd)(223)(192)(158)controln10047402040mean9751,0661,031(33 , 74)(16 , 97)(sd)(235)(169)(163)0.4630.164exercisen994843mean9971,1041,096(sd)(211)(213)(147)spaq leisure time activity ( min)alln1951118479204mean366533557(48 , 206)(54 , 354)(sd)(296)(355)(321)0.2220.008controln995541mean365500462(sd)(288)(334)(263)exercisen965643mean367565648(sd)(306)(373)(347)srm total scorealln20111086mean30.732.432.8(sd)(5.5)(5.3)(5.1)controln10253430.31.2mean30.331.731.8(1.1 , 1.7)(0.3 , 2.7)(sd)(5.7)(5.6)(5.9)0.6520.109exercisen995743mean31.233.133.8(sd)(5.4)(5.0)(3.9)bmialln197111850.30.6mean27.627.527.3(1.2 , 0.7)(1.6 , 0.4)(sd)(5.7)(5.1)(5.4)0.5460.222controln1005643mean27.728.028.0(sd)(6.1)(6.4)(6.7)exercisen975542mean27.426.926.7(sd)(5.3)(3.3)(3.7)18 m 18 months follow - up , 5y 60 months follow - upeffects estimates are displayed as the mean estimate , 95 % confidence interval and p value . 2exercise treatment effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side main outcomes : summary statistics at 18 and 60 months for the control and exercise intervention groups and model effect estimates of treatment effect differences for change from baseline 18 m 18 months follow - up , 5y 60 months follow - up effects estimates are displayed as the mean estimate , 95 % confidence interval and p value . model adjusted for study site , baseline therapy and age exercise treatment effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side adjusting for other baseline variables ( such as deprivation category , occupation prior to diagnosis , hysterectomy status , work status ) had negligible effect on the group differences for any of the outcomes , despite some of these baseline variables showing strong relationships with the outcomes and/or differing between women that were followed up at 18 and 60 months and those that were not . to determine if there were differences between those women who self - reported themselves as being less active at each follow - up point , comparisons were made between these two categories of women , adjusting for original treatment group ( as well as baseline study site , therapy and age ) . the model - estimated trends for the main outcomes over all time points , with confidence intervals , are given in figs . figure 3 illustrates that the more active group was observed to walk a slightly longer distance in 12 min at every follow - up time point , though the differences were not significant.fig . 3model - estimated mean 12-min walk distance , beck depression inventory score , bmi and shoulder range of motion score over time for the more and less active groups , adjusted for original study site , therapy received at baseline and baseline age , with p values for tests of differences between the groups at each time pointfig . 4model - estimated mean fact - g , fact - b subscale , panas positive and panas negative scores over time for the more and less active groups , adjusted for original study site , therapy received at baseline and baseline age , with p values for tests of differences between the groups at each time point model - estimated mean 12-min walk distance , beck depression inventory score , bmi and shoulder range of motion score over time for the more and less active groups , adjusted for original study site , therapy received at baseline and baseline age , with p values for tests of differences between the groups at each time point model - estimated mean fact - g , fact - b subscale , panas positive and panas negative scores over time for the more and less active groups , adjusted for original study site , therapy received at baseline and baseline age , with p values for tests of differences between the groups at each time point the bdi score was marginally significantly different between the groups at baseline and decreased for both groups over time . a larger decrease in depression levels for the group identifying as active was associated with significant differences at all follow - up points . bmi scores were on average slightly lower for the active group throughout the study , though the difference was statistically significant only at baseline and 12 weeks . there were statistically significant differences between the activity groups for total shoulder range of motion at baseline and 6 months follow - up only , and the observed difference was not consistent over time . figure 4 shows similar increases in fact - g average scores ( and therefore quality of life improvements ) for both activity groups over time . in general , by 60 months follow - up there were no statistically significant differences in any of the quality of life scales , despite the consistency of the observed difference over time . panas negative was significantly lower in the more active group at the end of the original study period and this persisted out to 6 months follow - up , despite there being no difference at baseline . this difference was not , however , statistically significant at 18 or 60 months , though the observed difference remained similar over time . similarly , the more active group had significantly higher panas positive at baseline and at the end of the original study period and though the magnitude of this difference was similar at 60 months , it was marginally non - significant ( 0.078 ) . the physical activity effects estimates , in units of 1 standard deviation , are displayed in fig . 5physical activity effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side physical activity effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side one hundred and fourteen women attended follow - up at 18 months and 87 women attended at 60 months . the flow of participants through this follow - up study is shown in fig . 1 ( see reference 8 for the flow diagram for the original study ) . baseline demographic characteristics of those that took part in the study at the 18 and 60-month follow - up versus those that did not are shown in table 1 . those who participated in the follow - up at 60 months were , at baseline , 3 years older and 5 kg lighter on average and were faster walkers ( i.e. probably fitter ) ; and may have been slightly less depressed and with less negative mood than those who did not participate at 60 months . women in work prior to diagnosis and those that were less deprived were more likely to participate than those who were housewives or more deprived . there were no differences in the proportions of control and exercise group women that responded at either 18 or 60 months.fig . 1participant flow through follow - up studytable 1demographics at baseline for women that took part in the follow - up study ( responders ) and women that did not take part in the follow - up study ( non - responders ) at each subsequent time point : summary statistics and p values for differences between responders and non - responders ( wilcoxon / fisher s test)18 months5 yearsresponderpresponderpnoyesnoyesage ( years)n87114<0.01114870.03mean ( sd)49.0 ( 9.2)53.5 ( 9.3)50.3 ( 9.5)53.2 ( 9.3)baseline weight ( kg)n85114<0.0111287<0.01mean ( sd)74.2 ( 15.5)68.3 ( 13.3)73.2 ( 15.2)67.8 ( 13.2)height ( cm)n871120.20114850.14mean ( sd)161.0 ( 6.3)159.9 ( 6.0)160.9 ( 6.3)159.7 ( 5.9)bdi scoren851120.06112850.06mean ( sd)13.3 ( 7.2)11.4 ( 7.1)13.1 ( 7.5)11.1 ( 6.6)panas positiven871120.10113860.13mean ( sd)26.5 ( 8.4)28.9 ( 9.0)26.8 ( 8.6)29.2 ( 8.9)panas negativen871120.05113860.09mean ( sd)19.5 ( 7.9)17.2 ( 6.8)19.1 ( 7.8)17.0 ( 6.5)12-min walk ( m)n851140.18112870.03mean ( sd)973.4 ( 220.8)996.0 ( 224.8)958.1 ( 242.4)1,022.7 ( 190.0)spaq leisure time activity ( min)n851100.32110850.71mean ( sd)367.0 ( 330.3)365.1 ( 267.9)375.1 ( 323.2)354.1 ( 257.7)srm total scoren871140.80114870.98mean ( sd)30.7 ( 5.8)30.8 ( 5.3)30.6 ( 5.7)30.9 ( 5.4)bmi ( kg / m)n851120.01112850.02mean ( sd)28.6 ( 6.0)26.8 ( 5.3)28.3 ( 5.9)26.6 ( 5.3)exercise groupcontrol46/102 ( 45.1)56/102 ( 54.9)0.6759/102 ( 57.8)43/102 ( 42.2)0.78exercise41/99 ( 41.4)58/99 ( 58.6)55/99 ( 55.6)44/99 ( 44.4)study centregri16/33 ( 48.5)17/33 ( 51.5)0.5521/33 ( 63.6)12/33 ( 36.4)0.57boc62/151 ( 41.1)89/151 ( 58.9)85/151 ( 56.3)66/151 ( 43.7)other9/17 ( 52.9)8/17 ( 47.1)8/17 ( 47.1)9/17 ( 52.9)therapychemotherapy7/15 ( 46.7)8/15 ( 53.3)0.527/15 ( 46.7)8/15 ( 53.3)0.67radiotherapy21/57 ( 36.8)36/57 ( 63.2)32/57 ( 56.1)25/57 ( 43.9)combination59/129 ( 45.7)70/129 ( 54.3)75/129 ( 58.1)54/129 ( 41.9)surgery typemast only31/57 ( 54.4)26/57 ( 45.6)0.1838/57 ( 66.7)19/57 ( 33.3)0.20lump only48/116 ( 41.4)68/116 ( 58.6)65/116 ( 56.0)51/116 ( 44.0)lump and mast1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)lump and recon0/1 ( 0.0)1/1 ( 100.0)0/1 ( 0.0)1/1 ( 100.0)mast and recon6/22 ( 27.3)16/22 ( 72.7)9/22 ( 40.9)13/22 ( 59.1)other1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)tamoxifen usedno57/117 ( 48.7)60/117 ( 51.3)0.0872/117 ( 61.5)45/117 ( 38.5)0.15yes30/83 ( 36.1)53/83 ( 63.9)42/83 ( 50.6)41/83 ( 49.4)highest education levelschool40/92 ( 43.5)52/92 ( 56.5)1.0054/92 ( 58.7)38/92 ( 41.3)0.77other43/99 ( 43.4)56/99 ( 56.6)55/99 ( 55.6)44/99 ( 44.4)employment status ( prior to diagnosis)ft / pt10/29 ( 34.5)19/29 ( 65.5)0.0111/29 ( 37.9)18/29 ( 62.1)0.02sick52/111 ( 46.8)59/111 ( 53.2)66/111 ( 59.5)45/111 ( 40.5)housewife17/26 ( 65.4)9/26 ( 34.6)20/26 ( 76.9)6/26 ( 23.1)retired8/35 ( 22.9)27/35 ( 77.1)17/35 ( 48.6)18/35 ( 51.4)occupation ( prior to diagnosis)professional17/48 ( 35.4)31/48 ( 64.6)0.7221/48 ( 43.8)27/48 ( 56.2)0.39managerial14/35 ( 40.0)21/35 ( 60.0)18/35 ( 51.4)17/35 ( 48.6)clerical25/55 ( 45.5)30/55 ( 54.5)32/55 ( 58.2)23/55 ( 41.8)manual15/33 ( 45.5)18/33 ( 54.5)20/33 ( 60.6)13/33 ( 39.4)carstairs deprivation1217/58 ( 29.3)41/58 ( 70.7)0.0423/58 ( 39.7)35/58 ( 60.3)0.013542/86 ( 48.8)44/86 ( 51.2)53/86 ( 61.6)33/86 ( 38.4)6726/53 ( 49.1)27/53 ( 50.9)35/53 ( 66.0)18/53 ( 34.0)periodsno70/169 ( 41.4)99/169 ( 58.6)0.3695/169 ( 56.2)74/169 ( 43.8)0.74irregular8/17 ( 47.1)9/17 ( 52.9)9/17 ( 52.9)8/17 ( 47.1)regular9/15 ( 60.0)6/15 ( 40.0)10/15 ( 66.7)5/15 ( 33.3)hysterectomyno80/179 ( 44.7)99/179 ( 55.3)0.36101/179 ( 56.4)78/179 ( 43.6)1.00yes7/22 ( 31.8)15/22 ( 68.2)13/22 ( 59.1)9/22 ( 40.9)hrtnever58/124 ( 46.8)66/124 ( 53.2)0.3173/124 ( 58.9)51/124 ( 41.1)0.69former24/67 ( 35.8)43/67 ( 64.2)35/67 ( 52.2)32/67 ( 47.8)current5/10 ( 50.0)5/10 ( 50.0)6/10 ( 60.0)4/10 ( 40.0 ) participant flow through follow - up study demographics at baseline for women that took part in the follow - up study ( responders ) and women that did not take part in the follow - up study ( non - responders ) at each subsequent time point : summary statistics and p values for differences between responders and non - responders ( wilcoxon / fisher s test ) to determine if there were any lasting effects of the intervention , comparisons were made between the original treatment and control groups . table 2 shows descriptive statistics of the outcome data at 18 and 60 months with corresponding treatment effect estimates . there were significant differences between the intervention and control groups at 60 months for spaq leisure time activity over the previous week and panas positive mood score with the intervention group reporting higher activity and more positive mood . even for outcomes for which there was no significant difference at 60 months , the intervention group was consistently observed to do better than the control group throughout the entire 5-year follow - up period . the treatment effect estimates at 18 and 60 months are also displayed in units of 1 standard deviation in fig . 2 for all outcomes measured and are of similar magnitude at both time points . at 5 years , the intervention group achieved on average around 200 min of activity each week more than the control group ( see table 2 for related data ) . in general , our analyses suggested that 5 years subsequent to taking part in such an exercise intervention similar patients would be likely to achieve on average 50 to 350 min of extra physical activity per week than patients treated as usual . this is a substantial difference which could lead to considerable health benefit.table 2main outcomes : summary statistics at 18 and 60 months for the control and exercise intervention groups and model effect estimates of treatment effect differences for change from baselinesummaries at each time pointeffect estimate ( exercise control)baseline18 months5 years18 m baseline5y baselinefact - galln20111487mean74.480.787.1(sd)(14.3)(14.6)(11.1)controln10256432.20.9mean72.779.685.7(1.8 , 6.2)(3.4 , 5.2)(sd)(15.6)(14.7)(11.4)0.2860.683exercisen995844mean76.181.788.5(sd)(12.6)(14.6)(10.7)bdi scorealln19711487mean12.29.37.00.80.2(sd)(7.2)(7.7)(6.7)(3.1 , 1.5)(2.8 , 2.4)controln9856430.4950.857mean12.99.77.5(sd)(7.5)(7.7)(6.7)exercisen995844mean11.58.96.6(sd)(6.9)(7.8)(6.7)panas positivealln19911487mean27.831.034.2(sd)(8.8)(9.8)(8.3)controln10056431.53.4mean28.030.633.1(1.4 , 4.3)(0.2 , 6.7)(sd)(9.2)(10.1)(8.7)0.3120.040exercisen995844mean27.731.335.2(sd)(8.4)(9.6)(7.8)panas negativealln199114870.80.5mean18.216.715.4(2.9 , 1.4)(2.9 , 1.8)(sd)(7.4)(7.5)(5.3)0.4870.655controln1005643mean19.117.416.3(sd)(7.7)(8.1)(5.6)exercisen995844mean17.316.014.5(sd)(6.9)(6.9)(5.0)12-min walkalln1999583mean9861,0851,065(sd)(223)(192)(158)controln10047402040mean9751,0661,031(33 , 74)(16 , 97)(sd)(235)(169)(163)0.4630.164exercisen994843mean9971,1041,096(sd)(211)(213)(147)spaq leisure time activity ( min)alln1951118479204mean366533557(48 , 206)(54 , 354)(sd)(296)(355)(321)0.2220.008controln995541mean365500462(sd)(288)(334)(263)exercisen965643mean367565648(sd)(306)(373)(347)srm total scorealln20111086mean30.732.432.8(sd)(5.5)(5.3)(5.1)controln10253430.31.2mean30.331.731.8(1.1 , 1.7)(0.3 , 2.7)(sd)(5.7)(5.6)(5.9)0.6520.109exercisen995743mean31.233.133.8(sd)(5.4)(5.0)(3.9)bmialln197111850.30.6mean27.627.527.3(1.2 , 0.7)(1.6 , 0.4)(sd)(5.7)(5.1)(5.4)0.5460.222controln1005643mean27.728.028.0(sd)(6.1)(6.4)(6.7)exercisen975542mean27.426.926.7(sd)(5.3)(3.3)(3.7)18 m 18 months follow - up , 5y 60 months follow - upeffects estimates are displayed as the mean estimate , 95 % confidence interval and p value . 2exercise treatment effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side main outcomes : summary statistics at 18 and 60 months for the control and exercise intervention groups and model effect estimates of treatment effect differences for change from baseline 18 m 18 months follow - up , 5y 60 months follow - up effects estimates are displayed as the mean estimate , 95 % confidence interval and p value . model adjusted for study site , baseline therapy and age exercise treatment effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side adjusting for other baseline variables ( such as deprivation category , occupation prior to diagnosis , hysterectomy status , work status ) had negligible effect on the group differences for any of the outcomes , despite some of these baseline variables showing strong relationships with the outcomes and/or differing between women that were followed up at 18 and 60 months and those that were not . to determine if there were differences between those women who self - reported themselves as being less active at each follow - up point , comparisons were made between these two categories of women , adjusting for original treatment group ( as well as baseline study site , therapy and age ) . the model - estimated trends for the main outcomes over all time points , with confidence intervals , are given in figs . figure 3 illustrates that the more active group was observed to walk a slightly longer distance in 12 min at every follow - up time point , though the differences were not significant.fig . 3model - estimated mean 12-min walk distance , beck depression inventory score , bmi and shoulder range of motion score over time for the more and less active groups , adjusted for original study site , therapy received at baseline and baseline age , with p values for tests of differences between the groups at each time pointfig . 4model - estimated mean fact - g , fact - b subscale , panas positive and panas negative scores over time for the more and less active groups , adjusted for original study site , therapy received at baseline and baseline age , with p values for tests of differences between the groups at each time point model - estimated mean 12-min walk distance , beck depression inventory score , bmi and shoulder range of motion score over time for the more and less active groups , adjusted for original study site , therapy received at baseline and baseline age , with p values for tests of differences between the groups at each time point model - estimated mean fact - g , fact - b subscale , panas positive and panas negative scores over time for the more and less active groups , adjusted for original study site , therapy received at baseline and baseline age , with p values for tests of differences between the groups at each time point the bdi score was marginally significantly different between the groups at baseline and decreased for both groups over time . a larger decrease in depression levels for the group identifying as active was associated with significant differences at all follow - up points . bmi scores were on average slightly lower for the active group throughout the study , though the difference was statistically significant only at baseline and 12 weeks . there were statistically significant differences between the activity groups for total shoulder range of motion at baseline and 6 months follow - up only , and the observed difference was not consistent over time . figure 4 shows similar increases in fact - g average scores ( and therefore quality of life improvements ) for both activity groups over time . in general , by 60 months follow - up there were no statistically significant differences in any of the quality of life scales , despite the consistency of the observed difference over time . panas negative was significantly lower in the more active group at the end of the original study period and this persisted out to 6 months follow - up , despite there being no difference at baseline . this difference was not , however , statistically significant at 18 or 60 months , though the observed difference remained similar over time . similarly , the more active group had significantly higher panas positive at baseline and at the end of the original study period and though the magnitude of this difference was similar at 60 months , it was marginally non - significant ( 0.078 ) . the physical activity effects estimates , in units of 1 standard deviation , are displayed in fig . 5 for all of the outcomes at 18 and 60 months.fig . 5physical activity effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side physical activity effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side this is the first study to examine the long - term effects of an exercise intervention in a rct with cancer survivors . the number of women lost to follow - up at 18 ( 44 % ) and 60 months ( 58 % ) is higher than that observed in a 2-year follow - up of a 6-month rehabilitation programme to reduce lymphoedema after breast cancer surgery ( 27 % ) but similar to that found in a longitudinal study of older breast cancer survivors in a 6-year follow - up ( 50 % ) . five years after taking part in the study , women who were assigned to the original intervention group , who had received the opportunity to attend a 12-week programme of supervised group exercise and group discussion of behaviour change issues , self - reported more leisure time activity and more positive mood than those women originally assigned to the control group condition . this is very encouraging as our trial was designed to promote long - term exercise behaviour change . both the intervention and control groups self - reported high levels of physical activity at 5 years exceeding current public health recommendations of achieving 150 min of moderate activity in a week . thus even the control group has benefitted from being involved in this project 5 years after diagnosis . however , as the data from table 2 shows , the intervention group reported around 200 min of activity more than the control group at 5 years , and although there is also high variability in these data , this is a statistically significant difference . the possibility of over reporting physical activity because of the self - report nature of the data must be acknowledged but the difference between the two groups in terms of physical activity is substantial . this suggests that the experience of attending the group exercise sessions had influenced the ability to sustain physical activity at a high level for the intervention group . this increase in physical activity could have important additional physical and mental well - being effects such as improved mood which we have observed , and reduced risk of recurrence of breast cancer , improved bone health and biomarker levels ( e.g. insulin pathway and inflammation ) which were not measured in the original study . an important element of the exercise programme was the group discussions that happened at the end of each class . each week , for 6 weeks , a specific theme was covered in group discussion after the exercise ( for example , the health benefits of exercise , enhancing self - efficacy and setting goals ) and supported with specifically constructed materials . these themes were guided by a model of behaviour change and were designed to promote independent exercise after the intervention . the six week block was repeated on a rolling basis , allowing all participants to hear the same themes . at the end of the 12-week intervention the control group received a personal consultation after the 6-month follow - up about how to increase physical activity levels . after the final data collection , women from both groups who expressed an interest in a local exercise referral scheme were given information on how to attend . the results show that the original intervention had a long lasting effect on helping the intervention group maintain a more physically active life . the difference in physical activity level that we see at 60 months between intervention and control group can be attributed to the experience of the class and group discussion of behaviour change challenges and solutions . in a study of cancer survivors diagnosed more than 5 years ago , over 53 % reported difficulties in crouching , standing for 2 h , carrying 10 pounds and walking quarter of a mile compared to 21 % of a matched sample with no cancer history . this demonstrates the importance of helping cancer survivors maintain basic levels of physical performance for simple activities of daily living . positive mood is an indication of psychological well - being and may also be linked to increased activity levels . williams et al . found that an acute positive affective response to a single bout of moderate intensity exercise predicted physical activity participation levels 6 and 12 months later . this is consistent with other follow - up studies and recent meta - analysis which suggest positive effects of exercise on psychosocial parameters . overall the pattern of results suggests a range of benefits of participating in the supervised exercise programme providing that the programme includes discussion of behaviour change challenges and solutions . the results therefore support the implementation of exercise opportunities into cancer rehabilitation in the same way that exercise is now a mainstream component of cardiac rehabilitation . irrespective of original group allocation , those who self - reported as engaging in higher levels physical activity recorded benefits on many of the quality of life and mood variables in comparison to those who self - reported that they were less active . this suggests that being active , regardless of original group allocation to intervention or control conditions , was associated with quality of life and mood benefits . a 13-year follow - up of 374 women diagnosed with breast cancer at a young age ( < 40 ) showed that the women whose exercise activity increased following diagnosis scored significantly higher ( p = 0.005 ) on the sf-36 physical health quality of life scale . likewise a prospective study investigating physical activity and quality of life in 545 breast cancer survivors showed that greater physical activity levels 3 years post diagnosis were related to less fatigue and better physical functioning . in general , statistically significant differences are more apparent at 18-month follow - up than at 60 months , though it is important to note that the number of women responding at 60 months was lower and the magnitude of the effect for several outcomes is similar to the corresponding 18-month effect . this is first study to follow an intervention group for 60 months after an exercise intervention for women with early stage breast cancer and our response rate is similar to other studies of this length . a limitation is that there were some differences in baseline demographics and outcome scores between those that did and did not return for follow - up and a reasonably high rate of dropout at 60 months . however , we used statistical modelling methods that appropriately accounted for such missing data to give reliable estimates of the population group means and corresponding differences over time , and we adjusted the models for baseline demographics . physical activity measures in this study were self - reported and future studies should attempt objective monitoring of physical activity patterns including sedentary time . this is first study to follow an intervention group for 60 months after an exercise intervention for women with early stage breast cancer and our response rate is similar to other studies of this length . a limitation is that there were some differences in baseline demographics and outcome scores between those that did and did not return for follow - up and a reasonably high rate of dropout at 60 months . however , we used statistical modelling methods that appropriately accounted for such missing data to give reliable estimates of the population group means and corresponding differences over time , and we adjusted the models for baseline demographics . physical activity measures in this study were self - reported and future studies should attempt objective monitoring of physical activity patterns including sedentary time . some of the benefits of a supervised exercise programme that incorporated discussion of behaviour change techniques , which were reported 6 months following the original intervention , have remained 60 months after the original study ended . these include higher levels of self - reported leisure time activity and more positive mood for the intervention group in comparison to the original control group . categorising the women by self - reported activity status , rather than by original allocation to intervention and control conditions , also shows benefits over time in terms of lower levels of depression and higher levels of mood and quality of life for those who report being more active . cancer survivors should be encouraged to engage in regular physical activity and to work towards achieving the public health recommendations for sufficient physical activity during and after treatment for early stage breast cancer [ 15 , 16 ] . services to support regular physical activity might include supervised exercise sessions in early stages , similar to that provided for cardiac rehabilitation , and encouragement to make use of local physical activity opportunities . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

purposein an earlier randomised controlled trial , we showed that early stage breast cancer patients who received a supervised exercise programme , with discussion of behaviour change techniques , had psychological and functional benefits 6 months after the intervention . the purpose of this study was to determine if benefits observed at 6 months persisted 18 and 60 months later.methodswomen who were in the original trial were contacted at 18 and 60 months after intervention . original measures were repeated.resultsof the 148 women from the original study who agreed to be contacted again , 114 attended for follow - up at 18 months and 87 at 60 months . women in the original intervention group reported more leisure time physical activity and more positive moods at 60 months than women in the original control group . irrespective of original group allocation , women who were more active consistently reported lower levels of depression and increased quality of life compared to those who were less active.conclusionswe have shown that there are lasting benefits to an exercise intervention delivered during treatment to breast cancer survivors . regular activity should be encouraged for women with early stage breast cancer as this can have lasting implications for physical and psychological functioning .

Introduction and background The aim of the this follow-up study was Method Participants Procedures Data analysis Results Demographics Effects of the intervention Associations with levels of self-reported activity Discussion Strengths and limitations Conclusions Conflict of interest Open Access

the aim of this study was to follow - up participants from a rct during adjuvant treatment 18 and 60 months after the exercise intervention . the original study s aim was to determine if there were functional and psychological benefits of a 12-week supervised group exercise programme during treatment for early stage breast cancer , including a 6-month follow - up . to determine if intervention effects continued after the 6-month follow - upto determine if women who had higher levels of activity after diagnosis and treatment had a different functional or psychological profile than women who had lower levels of activity and to elicit views from the women concerning their experience of physical activity post intervention to determine if intervention effects continued after the 6-month follow - up to determine if women who had higher levels of activity after diagnosis and treatment had a different functional or psychological profile than women who had lower levels of activity and to elicit views from the women concerning their experience of physical activity post intervention however , in this paper , we will not report on the qualitative analysis of the women s views . all women who had participated in the original study and who had agreed to being contacted again ( n = 148 ) were contacted at 18 months after the intervention and invited to participate in the follow - up study . all women who had participated in the original study and who had agreed to being contacted again ( n = 148 ) were contacted at 18 months after the intervention and invited to participate in the follow - up study . 1participant flow through follow - up studytable 1demographics at baseline for women that took part in the follow - up study ( responders ) and women that did not take part in the follow - up study ( non - responders ) at each subsequent time point : summary statistics and p values for differences between responders and non - responders ( wilcoxon / fisher s test)18 months5 yearsresponderpresponderpnoyesnoyesage ( years)n87114<0.01114870.03mean ( sd)49.0 ( 9.2)53.5 ( 9.3)50.3 ( 9.5)53.2 ( 9.3)baseline weight ( kg)n85114<0.0111287<0.01mean ( sd)74.2 ( 15.5)68.3 ( 13.3)73.2 ( 15.2)67.8 ( 13.2)height ( cm)n871120.20114850.14mean ( sd)161.0 ( 6.3)159.9 ( 6.0)160.9 ( 6.3)159.7 ( 5.9)bdi scoren851120.06112850.06mean ( sd)13.3 ( 7.2)11.4 ( 7.1)13.1 ( 7.5)11.1 ( 6.6)panas positiven871120.10113860.13mean ( sd)26.5 ( 8.4)28.9 ( 9.0)26.8 ( 8.6)29.2 ( 8.9)panas negativen871120.05113860.09mean ( sd)19.5 ( 7.9)17.2 ( 6.8)19.1 ( 7.8)17.0 ( 6.5)12-min walk ( m)n851140.18112870.03mean ( sd)973.4 ( 220.8)996.0 ( 224.8)958.1 ( 242.4)1,022.7 ( 190.0)spaq leisure time activity ( min)n851100.32110850.71mean ( sd)367.0 ( 330.3)365.1 ( 267.9)375.1 ( 323.2)354.1 ( 257.7)srm total scoren871140.80114870.98mean ( sd)30.7 ( 5.8)30.8 ( 5.3)30.6 ( 5.7)30.9 ( 5.4)bmi ( kg / m)n851120.01112850.02mean ( sd)28.6 ( 6.0)26.8 ( 5.3)28.3 ( 5.9)26.6 ( 5.3)exercise groupcontrol46/102 ( 45.1)56/102 ( 54.9)0.6759/102 ( 57.8)43/102 ( 42.2)0.78exercise41/99 ( 41.4)58/99 ( 58.6)55/99 ( 55.6)44/99 ( 44.4)study centregri16/33 ( 48.5)17/33 ( 51.5)0.5521/33 ( 63.6)12/33 ( 36.4)0.57boc62/151 ( 41.1)89/151 ( 58.9)85/151 ( 56.3)66/151 ( 43.7)other9/17 ( 52.9)8/17 ( 47.1)8/17 ( 47.1)9/17 ( 52.9)therapychemotherapy7/15 ( 46.7)8/15 ( 53.3)0.527/15 ( 46.7)8/15 ( 53.3)0.67radiotherapy21/57 ( 36.8)36/57 ( 63.2)32/57 ( 56.1)25/57 ( 43.9)combination59/129 ( 45.7)70/129 ( 54.3)75/129 ( 58.1)54/129 ( 41.9)surgery typemast only31/57 ( 54.4)26/57 ( 45.6)0.1838/57 ( 66.7)19/57 ( 33.3)0.20lump only48/116 ( 41.4)68/116 ( 58.6)65/116 ( 56.0)51/116 ( 44.0)lump and mast1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)lump and recon0/1 ( 0.0)1/1 ( 100.0)0/1 ( 0.0)1/1 ( 100.0)mast and recon6/22 ( 27.3)16/22 ( 72.7)9/22 ( 40.9)13/22 ( 59.1)other1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)tamoxifen usedno57/117 ( 48.7)60/117 ( 51.3)0.0872/117 ( 61.5)45/117 ( 38.5)0.15yes30/83 ( 36.1)53/83 ( 63.9)42/83 ( 50.6)41/83 ( 49.4)highest education levelschool40/92 ( 43.5)52/92 ( 56.5)1.0054/92 ( 58.7)38/92 ( 41.3)0.77other43/99 ( 43.4)56/99 ( 56.6)55/99 ( 55.6)44/99 ( 44.4)employment status ( prior to diagnosis)ft / pt10/29 ( 34.5)19/29 ( 65.5)0.0111/29 ( 37.9)18/29 ( 62.1)0.02sick52/111 ( 46.8)59/111 ( 53.2)66/111 ( 59.5)45/111 ( 40.5)housewife17/26 ( 65.4)9/26 ( 34.6)20/26 ( 76.9)6/26 ( 23.1)retired8/35 ( 22.9)27/35 ( 77.1)17/35 ( 48.6)18/35 ( 51.4)occupation ( prior to diagnosis)professional17/48 ( 35.4)31/48 ( 64.6)0.7221/48 ( 43.8)27/48 ( 56.2)0.39managerial14/35 ( 40.0)21/35 ( 60.0)18/35 ( 51.4)17/35 ( 48.6)clerical25/55 ( 45.5)30/55 ( 54.5)32/55 ( 58.2)23/55 ( 41.8)manual15/33 ( 45.5)18/33 ( 54.5)20/33 ( 60.6)13/33 ( 39.4)carstairs deprivation1217/58 ( 29.3)41/58 ( 70.7)0.0423/58 ( 39.7)35/58 ( 60.3)0.013542/86 ( 48.8)44/86 ( 51.2)53/86 ( 61.6)33/86 ( 38.4)6726/53 ( 49.1)27/53 ( 50.9)35/53 ( 66.0)18/53 ( 34.0)periodsno70/169 ( 41.4)99/169 ( 58.6)0.3695/169 ( 56.2)74/169 ( 43.8)0.74irregular8/17 ( 47.1)9/17 ( 52.9)9/17 ( 52.9)8/17 ( 47.1)regular9/15 ( 60.0)6/15 ( 40.0)10/15 ( 66.7)5/15 ( 33.3)hysterectomyno80/179 ( 44.7)99/179 ( 55.3)0.36101/179 ( 56.4)78/179 ( 43.6)1.00yes7/22 ( 31.8)15/22 ( 68.2)13/22 ( 59.1)9/22 ( 40.9)hrtnever58/124 ( 46.8)66/124 ( 53.2)0.3173/124 ( 58.9)51/124 ( 41.1)0.69former24/67 ( 35.8)43/67 ( 64.2)35/67 ( 52.2)32/67 ( 47.8)current5/10 ( 50.0)5/10 ( 50.0)6/10 ( 60.0)4/10 ( 40.0 ) participant flow through follow - up study demographics at baseline for women that took part in the follow - up study ( responders ) and women that did not take part in the follow - up study ( non - responders ) at each subsequent time point : summary statistics and p values for differences between responders and non - responders ( wilcoxon / fisher s test ) to determine if there were any lasting effects of the intervention , comparisons were made between the original treatment and control groups . 2exercise treatment effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side main outcomes : summary statistics at 18 and 60 months for the control and exercise intervention groups and model effect estimates of treatment effect differences for change from baseline 18 m 18 months follow - up , 5y 60 months follow - up effects estimates are displayed as the mean estimate , 95 % confidence interval and p value . 5physical activity effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side physical activity effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side one hundred and fourteen women attended follow - up at 18 months and 87 women attended at 60 months . 1participant flow through follow - up studytable 1demographics at baseline for women that took part in the follow - up study ( responders ) and women that did not take part in the follow - up study ( non - responders ) at each subsequent time point : summary statistics and p values for differences between responders and non - responders ( wilcoxon / fisher s test)18 months5 yearsresponderpresponderpnoyesnoyesage ( years)n87114<0.01114870.03mean ( sd)49.0 ( 9.2)53.5 ( 9.3)50.3 ( 9.5)53.2 ( 9.3)baseline weight ( kg)n85114<0.0111287<0.01mean ( sd)74.2 ( 15.5)68.3 ( 13.3)73.2 ( 15.2)67.8 ( 13.2)height ( cm)n871120.20114850.14mean ( sd)161.0 ( 6.3)159.9 ( 6.0)160.9 ( 6.3)159.7 ( 5.9)bdi scoren851120.06112850.06mean ( sd)13.3 ( 7.2)11.4 ( 7.1)13.1 ( 7.5)11.1 ( 6.6)panas positiven871120.10113860.13mean ( sd)26.5 ( 8.4)28.9 ( 9.0)26.8 ( 8.6)29.2 ( 8.9)panas negativen871120.05113860.09mean ( sd)19.5 ( 7.9)17.2 ( 6.8)19.1 ( 7.8)17.0 ( 6.5)12-min walk ( m)n851140.18112870.03mean ( sd)973.4 ( 220.8)996.0 ( 224.8)958.1 ( 242.4)1,022.7 ( 190.0)spaq leisure time activity ( min)n851100.32110850.71mean ( sd)367.0 ( 330.3)365.1 ( 267.9)375.1 ( 323.2)354.1 ( 257.7)srm total scoren871140.80114870.98mean ( sd)30.7 ( 5.8)30.8 ( 5.3)30.6 ( 5.7)30.9 ( 5.4)bmi ( kg / m)n851120.01112850.02mean ( sd)28.6 ( 6.0)26.8 ( 5.3)28.3 ( 5.9)26.6 ( 5.3)exercise groupcontrol46/102 ( 45.1)56/102 ( 54.9)0.6759/102 ( 57.8)43/102 ( 42.2)0.78exercise41/99 ( 41.4)58/99 ( 58.6)55/99 ( 55.6)44/99 ( 44.4)study centregri16/33 ( 48.5)17/33 ( 51.5)0.5521/33 ( 63.6)12/33 ( 36.4)0.57boc62/151 ( 41.1)89/151 ( 58.9)85/151 ( 56.3)66/151 ( 43.7)other9/17 ( 52.9)8/17 ( 47.1)8/17 ( 47.1)9/17 ( 52.9)therapychemotherapy7/15 ( 46.7)8/15 ( 53.3)0.527/15 ( 46.7)8/15 ( 53.3)0.67radiotherapy21/57 ( 36.8)36/57 ( 63.2)32/57 ( 56.1)25/57 ( 43.9)combination59/129 ( 45.7)70/129 ( 54.3)75/129 ( 58.1)54/129 ( 41.9)surgery typemast only31/57 ( 54.4)26/57 ( 45.6)0.1838/57 ( 66.7)19/57 ( 33.3)0.20lump only48/116 ( 41.4)68/116 ( 58.6)65/116 ( 56.0)51/116 ( 44.0)lump and mast1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)lump and recon0/1 ( 0.0)1/1 ( 100.0)0/1 ( 0.0)1/1 ( 100.0)mast and recon6/22 ( 27.3)16/22 ( 72.7)9/22 ( 40.9)13/22 ( 59.1)other1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)1/2 ( 50.0)tamoxifen usedno57/117 ( 48.7)60/117 ( 51.3)0.0872/117 ( 61.5)45/117 ( 38.5)0.15yes30/83 ( 36.1)53/83 ( 63.9)42/83 ( 50.6)41/83 ( 49.4)highest education levelschool40/92 ( 43.5)52/92 ( 56.5)1.0054/92 ( 58.7)38/92 ( 41.3)0.77other43/99 ( 43.4)56/99 ( 56.6)55/99 ( 55.6)44/99 ( 44.4)employment status ( prior to diagnosis)ft / pt10/29 ( 34.5)19/29 ( 65.5)0.0111/29 ( 37.9)18/29 ( 62.1)0.02sick52/111 ( 46.8)59/111 ( 53.2)66/111 ( 59.5)45/111 ( 40.5)housewife17/26 ( 65.4)9/26 ( 34.6)20/26 ( 76.9)6/26 ( 23.1)retired8/35 ( 22.9)27/35 ( 77.1)17/35 ( 48.6)18/35 ( 51.4)occupation ( prior to diagnosis)professional17/48 ( 35.4)31/48 ( 64.6)0.7221/48 ( 43.8)27/48 ( 56.2)0.39managerial14/35 ( 40.0)21/35 ( 60.0)18/35 ( 51.4)17/35 ( 48.6)clerical25/55 ( 45.5)30/55 ( 54.5)32/55 ( 58.2)23/55 ( 41.8)manual15/33 ( 45.5)18/33 ( 54.5)20/33 ( 60.6)13/33 ( 39.4)carstairs deprivation1217/58 ( 29.3)41/58 ( 70.7)0.0423/58 ( 39.7)35/58 ( 60.3)0.013542/86 ( 48.8)44/86 ( 51.2)53/86 ( 61.6)33/86 ( 38.4)6726/53 ( 49.1)27/53 ( 50.9)35/53 ( 66.0)18/53 ( 34.0)periodsno70/169 ( 41.4)99/169 ( 58.6)0.3695/169 ( 56.2)74/169 ( 43.8)0.74irregular8/17 ( 47.1)9/17 ( 52.9)9/17 ( 52.9)8/17 ( 47.1)regular9/15 ( 60.0)6/15 ( 40.0)10/15 ( 66.7)5/15 ( 33.3)hysterectomyno80/179 ( 44.7)99/179 ( 55.3)0.36101/179 ( 56.4)78/179 ( 43.6)1.00yes7/22 ( 31.8)15/22 ( 68.2)13/22 ( 59.1)9/22 ( 40.9)hrtnever58/124 ( 46.8)66/124 ( 53.2)0.3173/124 ( 58.9)51/124 ( 41.1)0.69former24/67 ( 35.8)43/67 ( 64.2)35/67 ( 52.2)32/67 ( 47.8)current5/10 ( 50.0)5/10 ( 50.0)6/10 ( 60.0)4/10 ( 40.0 ) participant flow through follow - up study demographics at baseline for women that took part in the follow - up study ( responders ) and women that did not take part in the follow - up study ( non - responders ) at each subsequent time point : summary statistics and p values for differences between responders and non - responders ( wilcoxon / fisher s test ) to determine if there were any lasting effects of the intervention , comparisons were made between the original treatment and control groups . 2exercise treatment effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side main outcomes : summary statistics at 18 and 60 months for the control and exercise intervention groups and model effect estimates of treatment effect differences for change from baseline 18 m 18 months follow - up , 5y 60 months follow - up effects estimates are displayed as the mean estimate , 95 % confidence interval and p value . 5physical activity effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side physical activity effect estimates for all outcomes at 18 and 60 months , adjusted for original study site , therapy received at baseline and baseline age , with 95 % confidence intervals ( cis ) and corresponding p values at the right hand side this is the first study to examine the long - term effects of an exercise intervention in a rct with cancer survivors . five years after taking part in the study , women who were assigned to the original intervention group , who had received the opportunity to attend a 12-week programme of supervised group exercise and group discussion of behaviour change issues , self - reported more leisure time activity and more positive mood than those women originally assigned to the control group condition . irrespective of original group allocation , those who self - reported as engaging in higher levels physical activity recorded benefits on many of the quality of life and mood variables in comparison to those who self - reported that they were less active . this is first study to follow an intervention group for 60 months after an exercise intervention for women with early stage breast cancer and our response rate is similar to other studies of this length . this is first study to follow an intervention group for 60 months after an exercise intervention for women with early stage breast cancer and our response rate is similar to other studies of this length . some of the benefits of a supervised exercise programme that incorporated discussion of behaviour change techniques , which were reported 6 months following the original intervention , have remained 60 months after the original study ended . these include higher levels of self - reported leisure time activity and more positive mood for the intervention group in comparison to the original control group .

a couple s intentions to have a number of descendants can be thwarted in a number of ways . the most difficult of factors are those that are restrictive and outside of a couple s sphere of influence or resources . the need to have a child can be reflected in motives such as happiness , well - being ( family relationships ) , identity , parenthood ( life - fulfilment ) , social control and continuity ( dyer et al . , 2008 ) . parenthood - motives may vary according to gender , societies and cultures , but neither the level of education nor wealth can substitute the inherent need for a biological child . in developing countries particularly in the sub - saharan african region , human immunodeficiency virus ( hiv ) infection together with limited resources adds to the barriers in becoming a parent . although the south african s bill of rights ( constitution of the republic of south africa act no 108 of 1996 ) decree that south africans can make decisions concerning reproduction ; access to and the use of assisted reproduction technology ( art ) are viewed in general as prohibitively expensive and only accessible to the privileged few . resources available in the public / tertiary art units and to private practices and its clients , affect reproductive healthcare screening and concurrent diagnostic and therapeutic decisions which include ; scheduling for intra - uterine inseminations ( iui ) , in vitro fertilization ( ivf ) and intra - cytoplasmic sperm injection ( icsi ) , combined with semen decontamination for hiv - seropositive males . several factors can impact on the financial health of an art programme . cost - drivers with emphasis on art laboratory set - up and procedures in south africa will be discussed in this review , structured on a previous examination of cost - drivers in south africa ( huyser and boyd , 2012 ) , i.e. ( 1 ) art procedures , ( 2 ) detection and prevention of infections , ( 3 ) sperm preparations , and ( 4 ) laboratory facilities , including supplies needed to perform procedures ( fig . the first two tertiary art institutions in south africa were established in pretoria and cape town in 1982 ( fourie et al . , 1988 ; kruger et al . , 1985 ) , with the first test tube different forms of art services are provided in the country , i.e. public service academic - centred art units or private ventures with independent specialists utilizing office - based or corporate pathology laboratories , and larger established art associates consisting of clinical and laboratory art specialists . it is questionable if the current ( approximately ) 28 national art service providers ( cross - referenced with known providers in provinces and www.ivf-worldwide.com ) are providing an adequate reproductive health service within a nation of 52 million people with a variety of cultures and languages ( http://www.southafrica.info/about/people/population ) . the speciality , repertoire and type of art structure , i.e. private vs. public / tertiary , will impact on capacity , services offered , revenue generated and patient population of the unit . south africa has four national tertiary art units , situated in cape town ( groote schuur and tygerberg / vincent palotti , www.aevitas.co.za ) , bloemfontein ( femspes group - www.femspes.co.za ) and in pretoria ( steve biko academic hospital formerly known as pretoria academic hospital , and previously as the hf verwoerd hospital ) ( http://www.pah.org.za/departments/endocrine.html ) . two out of the four national tertiary art units , situated in cape town and pretoria are entirely dependent on public funding . the reproductive and endocrine unit , as part of the department of obstetrics and gynaecology at the university of pretoria provides both diagnostic and therapeutic art procedures , including semen decontamination for hiv+ patients , and general cryopreservation ( huyser and fourie , 2010 ) , and is an accredited training unit for clinical technologists and medical biological scientists in reproductive biology . the majority of patients presently attending the art program at the unit for diagnostic purposes are from the lower to middle income groups with an average gross household income of 1,405 967 ( 1 ) per month . patients that participate in an art attempt have a slightly higher average gross household income of 2,260 1,730 per month ( random sampling of 100 non - overlapping patients from 2012 in the diagnostic and therapeutic groups , respectively ) . two different art cost groupings , i.e. state subsidized and private patient structured categories are available at our unit , with a third low cost option that can be accessed by both categories : [ a ] a subsidized category through state funding , for couples without medical aid and an annual income of less than 4,205 per household ; with patients contributing towards registration fees ( 4.00 per consultation / visit ) , medications with partial media costs . the actual procedures will cost 97.00 for an iui and 1,269.00 for ivf / icsi in this category . [ b ] a private category ( including medication , clinical- , pathology- and laboratory fees ) for couples with a medical aid and/or a household income above 8,410 p.a . a fee of up to 435 ( case - dependent ) is payable for an iui attempt and a maximum of 1,830 for ivf / icsi procedures . [ c ] an affordable low cost option for ivf / icsi is also available to patient categories [ a ] & [ b ] , and is based on initiatives for accessible ivf ( ombelet and campo , 2007 ; ombelet et al . , 2008 ; ombelet , 2009 ) ; this includes basic medication , minimal clinical- , pathology- and laboratory fees . approximate costs range from 401 to 962 ( category [ a ] & [ b ] dependent ) for an ivf or icsi cycle . patients can select the low cost option , but need to comply with criteria based on aetiology , age and case history . access to the subsidized category is subjected to budget allocation , and only a small number of patients qualify for a subsidy in this category per year . the patient ratio of 1:6:1 for the cost categories a to b to c , respectively in 2011 - 2012 ( huyser and boyd , 2012 ) , vs. a patient ratio of 1:4:2 for 2012- early 2013 could indicate a personal budget decline in the present time . a retrospective cost analysis in 1986 for the set - up of our art unit indicated that the cost for an ivf cycle amounted to 135.50 ( fourie et al . , 1988 ) . the average cost for procedures from 20 private south african art units ( mostly from gauteng ) , were obtained telephonically ( in april 12 with a follow up in april 13 ) ( fig . total cost estimations including medications , ultrasound scans and laboratory fees were obtained for a standard iui , ivf and icsi procedure . in the private sector , ivf procedures increased in 2012 - 13 on average from 2,930 to 3,255 with a similar trend found for the icsi procedures . the average costs ( standard deviation ) per procedure in the private sector are : ( i ) iui : 542 159 , ( ii ) ivf : 3,255 576 and ( iii ) icsi : 3,302 625 . the cost for an iui procedure can vary depending on the number of inseminations . dyer and kruger ( 2012 ) referred to general out - of - pocket costs for a standard ivf cycle of 841 ( subsidized in the public sector ) and 2,944 ( within the private sector ) in south africa , which reflects the previous mentioned fee structure in 2012 . the average percentage of the major cost - drivers of an ivf cycle at an active private practice in south africa in 2012 were the following : 8% of costs are allocated for clinic fees , 28% to medication , 29% to clinicians fees & consultations , and 35% for laboratory fees ( for use of equipment and the laboratory , disposables , culture media and staff expenditures ) ( huyser and boyd , 2012 ) . interestingly , laboratory expenses accounted for 39.2% of the total ivf cycle cost at our unit in 1986 ( fourie et al . , 1988 ) . converting from an ivf procedure to an icsi procedure items used in the laboratory can amount to nearly 48% of all costs per icsi cycle . approximately 70% ( 14 out of 20 practices ) of the south african art units that were contacted during the present study ( fig . iui on the other hand can be viewed as the most cost - effective art procedure ( garceau et al . , 2002 ) , and is revealed in the low comparative costs for medications , 8% of the total expenses , vs. 23 - 28% for an ivf or icsi cycle ( proportional data communicated by one of the largest art units in south africa ) ( huyser and boyd , 2012 ) . chambers and co - workers ( 2009 ) indicated that the cost of ( art ) treatment reflects the costliness of the underlying healthcare system rather than the regulatory or funding environment . within south africa , access to art is restrictive due to limited health insurance coverage of art procedures , restrained access to a few art units within the public sector ( dyer and kruger , 2012 ) , and limited funding to public sector art providers . funding of art centers and treatment of infertility competes with a range of health priorities . similar to the debate on prevention vs. treatment of infertility ( dyer and pennings , 2010 ) , the detection and thus prevention of pathogen transmission will be less expensive and more beneficial to a large number of people than treatment alone . screening of the couple should however , be directed by the incidence of disease(s ) in the specific patient population , medical history and physical examination of the couple ( elder et al . , 2005 ) . with the prevalence of hiv in sub - saharan africa , the question arises should all art participants be screened / re - screened for blood borne viruses ( bbv ) ? correspondingly , since a variety of bacteria species are present in approximately 50% of all semen samples obtained for art procedures , with gram - negative species present in only a fraction of samples ( fourie et al . , 2012 ) , should all semen samples be submitted for bacteriological culture and sensitivity ? an answer could be prophylactic or empiric anti - microbial treatment options , with concurrent costs ( huyser and boyd , 2012 ) especially in a rural setting in the absence of pathology services or due to logistical reasons . without a south african technical directive regarding the screening and treatment of art patients for bbv / pathogens it is doubtful if requirements of the european union tissue and cells directives ( www.eur-lex.europa.eu ) for art units in the eu , whereby biological screening must be carried out at the time of donation i.e. of sperm or oocytes , can be used as a blue print for south african art units . the repeated screening ( for hiv , hbv , hcv ) of patients was probed by wingfield and cotell ( 2010 ) , who suggested an initial baseline screening with appropriate risk reduction measures to prevent cross - contaminations during an art procedure . with rapid screening technology available at affordable costs ( approximately 1.30 per hiv rapid test ) in south africa , all art patients currently the cost for a single hiv rapid test ( 2 ) is approximately 1.5% of a rt - pcr quantitative ( hiv-1 rna ) and 8% of an elisa hiv-1 test . rapid tests are inexpensive , simple to perform , individually packaged with a shelf - life of approximately 12 months ( world health organization , 2004 ) and are well suited for resource constrained settings . preliminary experiences in our laboratory on using rapid tests ( hiv , hcv , hbv ) as a first - line screening indicates that the tests are easy to execute , and takes approximately 20 minutes per test to perform . all hiv positive results were confirmed with a secondary more extensive rapid test and patients are counselled to undergo a confirmatory viral validation ( preferably with cd4 and viral load analysis ) . no false positive or false negative results were encountered for the rapid tests up to date . eight percent of patients that have never undergone an hiv - test previously , tested positive with the rapid tests ( n = 100 individuals ) , with a 3:1 ratio for females to males ( stander , 2013 , personnel communication ) . a layered risk - reduction approach is practised at our unit when dealing with contaminants in semen prior to an art cycle , i.e. provide guidelines on sample collection in native languages to male patients to reduce skin contaminants ; prescribe suitable treatment based on susceptible testing of semen prior to an assisted reproduction procedure ( opposed to prophylactic antibiotic treatment ) ; use semen washing / decontamination procedures combined with a physical device ( e.g. the proinsert , [ nidacon , sweden ] ) together with discontinuous density gradients to diminish microbe re - contamination ( huyser and fourie , 2010 ; fourie et al . , 2012 ) . semen quality is taken as a surrogate measure of male fecundity in clinical andrology , male fertility , reproductive toxicology , epidemiology and pregnancy risk assessments ( cooper et al . , 2010 ) . the appropriateness of sperm preparation techniques and costs to obtain purified sperm should be considered , since the post - processed sperm sample s quality greatly governs the choice of art procedure to follow . also , if further washing steps after density gradient centrifugation ( dgc ) will be sufficient to wash sperm free of hiv . a cochrane based review by boomsma et al . ( 2011 ) showed that no specific semen preparation technique ( i.e. dgc ; swim - up ; as well as wash and centrifugation techniques ) improved clinical outcome with reference to iui procedures . the reason being that a minimum threshold of > 1 million motile spermatozoa is needed for successful conception through iui , irrespective of the type of sperm preparation method used ( ombelet et al . , 2003 ) . one such option is an office - based semen preparation device called sep - d ( surelife media technologies ( cat no : sl 001 ) ) with a current price tag of 23.00/device , consisting of a semen preparation kit containing a syringe pre - filled with a buffered culture media . motile sperm is separated from seminal plasma through a direct swim - up technique , where after the motile sperm fraction is retained in approximately 300 medium within the device , which is then connected to an iui catheter ( 7.00/catheter when purchased separately , cat no : sl 002 - 12 ) , and used for insemination ( www.surelifeivf.com ) . within the south african market a couple who qualifies for an iui procedure and resides in a rural town , could travel to a local general practitioner or clinic for repeated inseminations . gentis and co - workers ( 2012 ) reported a good clinical outcome for iui patients in a randomized controlled study at a south african art unit while using the sep - d semen processing device . the risks associated with sperm preparation techniques should be discussed with patients ( who , 2010 ; eke and oragwu , 2011 ) . data on sperm washing for hiv - seropositive patients are merely observational in nature according to a cochrane review by eke and oragwu ( 2011 ) . sperm washing refers to a sequential three phase procedure , i.e. dgc , washing of the sperm pellet and swim - up step ; as was initiated by semprini and co - workers ( 1992 ) to prepare semen samples for art from hiv - positive males . the bold undertaking was prior to the initiation of highly active antiretroviral treatment or validation of viral particles in the washed sperm sample ( semprini et al . this safety record is backed by published data from centers worldwide using iui , ivf and icsi procedures for hiv - positive males . various factors may contribute to the lack of randomized trials in this area of art , including hiv - regulations , inequalities in art treatment modalities , as well as costs of sperm washing and art procedures ( with particular reference to resource - poor countries in africa ) ( eke and oragwu , 2011 ) . the choice of a sperm preparation technique is however vital when processing sub - optimal semen samples for ivf or icsi , with discontinuous dgc being the preferred method to optimize samples . quotes for seven known brands of gradients and wash solutions were received from south african agencies in april 2013 . five of the seven brands are included in the listed culture media products ( see section : laboratory facilities , supplies and environmental aspects ) , together with purespermmedia ( nidacon , sweden ; www.nidacon.com ) and sil - select ( fertipro n.v . , belgium ; www.fertipro.com ) . a single processing ( 2 ml semen sample ) will cost on average 16.82 3.34 ( ranging from 10.11 to 19.33 ) . the term sperm decontamination was coined at our laboratory to distinguish sperm washing from the decontamination procedure used for samples possibly containing various infectious microbes e.g. bacteria , hiv , hcv and cmv ( huyser and fourie , 2010 ) . this involves the layering of density gradients and the semen using a proinsert kit ( www.tekevent.com/nidacon/proinsert ) , at a cost of 10.10 for the device ( cat no : ni - p115 - 5 , nidacon ) . the kit consists of two conical tubes with two elongated pipettes and a proinsert device . the purified motile sperm pellet ( after centrifugation ) is retrieved using the elongated pipette without re - contaminating the pellet with infectious micro - organisms . a final washing step follows to get rid of density particles and a portion of the purified sperm sample can be submitted for testing ( hiv-1 proviral dna and rna using a sensitive molecular based technique such as reverse transcription polymerase chain reaction ( rt - pcr ) ) . more than a decade of research in the treatment of hiv+ semen samples culminated in the clinical application of procedures during art treatment of patients at our unit . a 100% and 98.1% success rate in the removal of hiv-1 rna and dna respectively , is maintained for the decontamination procedure at our laboratory ( n = 100 semen samples , post - processing pcr validations , 2011 - 2012 ) . since the costs of rt - pcr viral validations on purified sperm samples are expensive especially in a developing country , ( 86.20 per test for dna or rna at a national pathology laboratory ) the question arises if all purified samples should be tested ? due to restricted access to pathology laboratories and cost implications , only qualitative viral validations are available in most developing countries . within south africa , molecular viral testing is more accessible , and in our experiences up to 32% of patients with an undetectable hiv blood load can have a positive hiv-1 rna seminal viral load ( n = 100 patients ) . patients are informed of the procedure failure rate , hiv - related health screening ( cd4 + cell levels ) and additional infectious disease tests , general risk - reduction methods , extra costs for viral validations and cryopreservation of semen samples prior to the initiation of an art attempt ( huyser and boyd , 2012 ) . financial pressures can result in procedural shortcuts and the demand to maximize patient throughput in some private practices and laboratories offering sperm processing procedures . in the absence of national directives on the art treatment of hiv+ patients within a developing country , best practice frameworks and directives from developed countries could be adapted and used as guidelines for assisted reproduction laboratories in the developing world . all art related laboratory items except for general pharmacy articles are imported from various parts of the world to south africa . equipment , sperm processing solutions , embryo culture media and disposables have to be couriered with concomitant imported taxes . setting - up an art laboratory in any part of the world depends on economics , availability and optimal maintenance of items . procedures should be best - practice - based with reliable equipment , disposables , and techniques within a risk - reduction environment . this section will discuss the costs applicable to selected art equipment and mainstream embryo culture media that is commercially available and currently in use within south africa . the price tag for six different benchtop incubators and five media brands in south africa is compared . the costs to purchase six different benchtop incubators ( listed alphabetically ) : i.e. bt37(origio / planer scanlab equipment a / s , lynge , denmark ; www.origio.com ) ; g85 and g185 standard ( k - systems kivex biotec ltd , birkerd , denmark ; www.k-systems.dk ) ; k - minc-1000 ( cookmedical , brisbane , australia ; www.cookmedical.com ) , labo c - top ( labor - technik - gttingen , germany ; www.labotect.com ) ; miri multi - room incubator for ivf ( esco medical , singapore ; www.medical.escoglobal . table i demonstrates the south african price range from 3,769.80 to 22,763.57 for benchtop incubators which can accommodate 8 to 48 petri dishes ( 35 mm ) , respectively . the costs are vat inclusive and may consist of installation , a start - up kit or humidification container where applicable . for more details see the individual websites . when the same incubators are purchased in belgium , three out of the six incubators are currently between 6.59 - 28.69% less expensive , and three incubators are 1.44 - 25.98% more expensive compared to purchase prices in south africa . . a similar cost extrapolation should be applicable to all art equipment and disposables imported into south africa . a cost analysis for the set - up of the art programme at our unit in the eighties , indicated that the total cost for laboratory equipment amounted to 11,606 . a single inverted microscope , two water jacketed upright incubators , a stereo as well as a light microscope , a single laminar flow and biological safety cabinet , hygrometer , dry - oven , centrifuge , refrigerator , osmometer , ph - meter and electronic balance constituted the laboratory to initiate the art programme ( fourie et al . , 1988 ) . more than 90% of this equipment was still fully functional twenty years on ( 2005/6 ) , when the laboratory moved to new purpose built laboratories within steve biko academic hospital ( pretoria , south africa ) . similar equipment to date will cost between 67,283 and 92,515 , depending on the model size and/or brand type ( huyser and boyd , 2012 ) . the previously mentioned historical cost analysis article by fourie and co - workers ( 1988 ) did not refer to a micro - manipulator ( for icsi procedures ) , or any cryopreservation equipment during the initiation phase of the laboratory . five different brands of culture media are currently used in south africa : i.e. globalmedia ( lifeglobal one - step protocol ; ivfonline , usa , www.lifeglobal.com ) , medicult media ( embryoassist & blastassist two - step protocol ; origio , denmark , www.origio.com ) , quinns advantage media ( sage media products two - step protocol ; coopersurgical , ( usa , www.coopersurgical.com ) ; sydneyivf ( k - sicm & k - sibm two - step protocol ; cookmedical ireland , www.cookmedical.com ) ; and vitrolife - g - series media ( g-1 & g-2 , two - step protocol ; scandinavia ivf science , gteborg , sweden ; www.vitrolife.com ) . quotes for the media brands were obtained in april / may in 2013 from south african distributers . total costs per media brand for a full art cycle including dgc , ivf , and icsi are indicated in fig . costs were calculated per ml of medium used per patient , per cycle attempt according to laboratory protocol , with embryo culture in micro - drops under oil . a similar cost analyses were performed in 2012 ( huyser and boyd , 2012 ) . culture media for an ivf procedure for six oocytes amounts to 40.26 8.76 ( ranging from 27.25 to 51.79 ) and costs approximately15% less than media used in an icsi procedure ( mean cost of 47.14 11.04 ( ranging from 31.22 to 60.32 ) ) . cryopreservation solutions for non - vitrification procedures cost on average 19.96 9.20 ( ranging from 10.18 to 28.74 ) , with a 37% difference in cost for a vitrification attempt ( mean cost of 31.85 10.37 ( ranging from 14.55 to 41.38 ) ) . an average annual increase of 7% for vitrification cryopreservation , whereas an annual average decrease of 3% for non - vitrification solutions was noted . these costs exclude the cryopreservation carriers / devices available as open or closed single - straw systems , cryo - storage tanks , or liquid nitrogen . courier services and/or company representatives , which manage the transport and delivery of art culture media timeously , play a vital role in failures or successes in the art laboratory . durable packaging and protection of temperature sensitive culture media while in transit through developing countries are also extremely important during ( occasional ) protracted customs clearance , especially in summertime . the first two tertiary art institutions in south africa were established in pretoria and cape town in 1982 ( fourie et al . , 1988 ; kruger et al . , 1985 ) , with the first test tube different forms of art services are provided in the country , i.e. public service academic - centred art units or private ventures with independent specialists utilizing office - based or corporate pathology laboratories , and larger established art associates consisting of clinical and laboratory art specialists . it is questionable if the current ( approximately ) 28 national art service providers ( cross - referenced with known providers in provinces and www.ivf-worldwide.com ) are providing an adequate reproductive health service within a nation of 52 million people with a variety of cultures and languages ( http://www.southafrica.info/about/people/population ) . the speciality , repertoire and type of art structure , i.e. private vs. public / tertiary , will impact on capacity , services offered , revenue generated and patient population of the unit . south africa has four national tertiary art units , situated in cape town ( groote schuur and tygerberg / vincent palotti , www.aevitas.co.za ) , bloemfontein ( femspes group - www.femspes.co.za ) and in pretoria ( steve biko academic hospital formerly known as pretoria academic hospital , and previously as the hf verwoerd hospital ) ( http://www.pah.org.za/departments/endocrine.html ) . two out of the four national tertiary art units , situated in cape town and pretoria are entirely dependent on public funding . the reproductive and endocrine unit , as part of the department of obstetrics and gynaecology at the university of pretoria provides both diagnostic and therapeutic art procedures , including semen decontamination for hiv+ patients , and general cryopreservation ( huyser and fourie , 2010 ) , and is an accredited training unit for clinical technologists and medical biological scientists in reproductive biology . the majority of patients presently attending the art program at the unit for diagnostic purposes are from the lower to middle income groups with an average gross household income of 1,405 967 ( 1 ) per month . patients that participate in an art attempt have a slightly higher average gross household income of 2,260 1,730 per month ( random sampling of 100 non - overlapping patients from 2012 in the diagnostic and therapeutic groups , respectively ) . two different art cost groupings , i.e. state subsidized and private patient structured categories are available at our unit , with a third low cost option that can be accessed by both categories : [ a ] a subsidized category through state funding , for couples without medical aid and an annual income of less than 4,205 per household ; with patients contributing towards registration fees ( 4.00 per consultation / visit ) , medications with partial media costs . the actual procedures will cost 97.00 for an iui and 1,269.00 for ivf / icsi in this category . [ b ] a private category ( including medication , clinical- , pathology- and laboratory fees ) for couples with a medical aid and/or a household income above 8,410 p.a . . a fee of up to 435 ( case - dependent ) is payable for an iui attempt and a maximum of 1,830 for ivf / icsi procedures . [ c ] an affordable low cost option for ivf / icsi is also available to patient categories [ a ] & [ b ] , and is based on initiatives for accessible ivf ( ombelet and campo , 2007 ; ombelet et al . , 2008 ; ombelet , 2009 ) ; this includes basic medication , minimal clinical- , pathology- and laboratory fees . approximate costs range from 401 to 962 ( category [ a ] & [ b ] dependent ) for an ivf or icsi cycle . patients can select the low cost option , but need to comply with criteria based on aetiology , age and case history . access to the subsidized category is subjected to budget allocation , and only a small number of patients qualify for a subsidy in this category per year . the patient ratio of 1:6:1 for the cost categories a to b to c , respectively in 2011 - 2012 ( huyser and boyd , 2012 ) , vs. a patient ratio of 1:4:2 for 2012- early 2013 could indicate a personal budget decline in the present time . a retrospective cost analysis in 1986 for the set - up of our art unit indicated that the cost for an ivf cycle amounted to 135.50 ( fourie et al . the average cost for procedures from 20 private south african art units ( mostly from gauteng ) , were obtained telephonically ( in april 12 with a follow up in april 13 ) ( fig . total cost estimations including medications , ultrasound scans and laboratory fees were obtained for a standard iui , ivf and icsi procedure . in the private sector , ivf procedures increased in 2012 - 13 on average from 2,930 to 3,255 with a similar trend found for the icsi procedures . the average costs ( standard deviation ) per procedure in the private sector are : ( i ) iui : 542 159 , ( ii ) ivf : 3,255 576 and ( iii ) dyer and kruger ( 2012 ) referred to general out - of - pocket costs for a standard ivf cycle of 841 ( subsidized in the public sector ) and 2,944 ( within the private sector ) in south africa , which reflects the previous mentioned fee structure in 2012 . the average percentage of the major cost - drivers of an ivf cycle at an active private practice in south africa in 2012 were the following : 8% of costs are allocated for clinic fees , 28% to medication , 29% to clinicians fees & consultations , and 35% for laboratory fees ( for use of equipment and the laboratory , disposables , culture media and staff expenditures ) ( huyser and boyd , 2012 ) . interestingly , laboratory expenses accounted for 39.2% of the total ivf cycle cost at our unit in 1986 ( fourie et al . , 1988 ) . converting from an ivf procedure to an icsi procedure items used in the laboratory can amount to nearly 48% of all costs per icsi cycle . approximately 70% ( 14 out of 20 practices ) of the south african art units that were contacted during the present study ( fig . iui on the other hand can be viewed as the most cost - effective art procedure ( garceau et al . , 2002 ) , and is revealed in the low comparative costs for medications , 8% of the total expenses , vs. 23 - 28% for an ivf or icsi cycle ( proportional data communicated by one of the largest art units in south africa ) ( huyser and boyd , 2012 ) . chambers and co - workers ( 2009 ) indicated that the cost of ( art ) treatment reflects the costliness of the underlying healthcare system rather than the regulatory or funding environment . within south africa , access to art is restrictive due to limited health insurance coverage of art procedures , restrained access to a few art units within the public sector ( dyer and kruger , 2012 ) , and limited funding to public sector art providers . funding of art centers and treatment of infertility competes with a range of health priorities . similar to the debate on prevention vs. treatment of infertility ( dyer and pennings , 2010 ) , the detection and thus prevention of pathogen transmission will be less expensive and more beneficial to a large number of people than treatment alone . screening of the couple should however , be directed by the incidence of disease(s ) in the specific patient population , medical history and physical examination of the couple ( elder et al . , 2005 ) . with the prevalence of hiv in sub - saharan africa , the question arises should all art participants be screened / re - screened for blood borne viruses ( bbv ) ? correspondingly , since a variety of bacteria species are present in approximately 50% of all semen samples obtained for art procedures , with gram - negative species present in only a fraction of samples ( fourie et al . , 2012 ) , should all semen samples be submitted for bacteriological culture and sensitivity ? an answer could be prophylactic or empiric anti - microbial treatment options , with concurrent costs ( huyser and boyd , 2012 ) especially in a rural setting in the absence of pathology services or due to logistical reasons . without a south african technical directive regarding the screening and treatment of art patients for bbv / pathogens it is doubtful if requirements of the european union tissue and cells directives ( www.eur-lex.europa.eu ) for art units in the eu , whereby biological screening must be carried out at the time of donation i.e. of sperm or oocytes , can be used as a blue print for south african art units . the repeated screening ( for hiv , hbv , hcv ) of patients was probed by wingfield and cotell ( 2010 ) , who suggested an initial baseline screening with appropriate risk reduction measures to prevent cross - contaminations during an art procedure . with rapid screening technology available at affordable costs ( approximately 1.30 per hiv rapid test ) in south africa , all art patients currently the cost for a single hiv rapid test ( 2 ) is approximately 1.5% of a rt - pcr quantitative ( hiv-1 rna ) and 8% of an elisa hiv-1 test . rapid tests are inexpensive , simple to perform , individually packaged with a shelf - life of approximately 12 months ( world health organization , 2004 ) and are well suited for resource constrained settings . preliminary experiences in our laboratory on using rapid tests ( hiv , hcv , hbv ) as a first - line screening indicates that the tests are easy to execute , and takes approximately 20 minutes per test to perform . all hiv positive results were confirmed with a secondary more extensive rapid test and patients are counselled to undergo a confirmatory viral validation ( preferably with cd4 and viral load analysis ) . no false positive or false negative results were encountered for the rapid tests up to date . eight percent of patients that have never undergone an hiv - test previously , tested positive with the rapid tests ( n = 100 individuals ) , with a 3:1 ratio for females to males ( stander , 2013 , personnel communication ) . a layered risk - reduction approach is practised at our unit when dealing with contaminants in semen prior to an art cycle , i.e. provide guidelines on sample collection in native languages to male patients to reduce skin contaminants ; prescribe suitable treatment based on susceptible testing of semen prior to an assisted reproduction procedure ( opposed to prophylactic antibiotic treatment ) ; use semen washing / decontamination procedures combined with a physical device ( e.g. the proinsert , [ nidacon , sweden ] ) together with discontinuous density gradients to diminish microbe re - contamination ( huyser and fourie , 2010 ; fourie et al . , 2012 ) . semen quality is taken as a surrogate measure of male fecundity in clinical andrology , male fertility , reproductive toxicology , epidemiology and pregnancy risk assessments ( cooper et al . , 2010 ) . the appropriateness of sperm preparation techniques and costs to obtain purified sperm should be considered , since the post - processed sperm sample s quality greatly governs the choice of art procedure to follow . also , if further washing steps after density gradient centrifugation ( dgc ) will be sufficient to wash sperm free of hiv . a cochrane based review by boomsma et al . ( 2011 ) showed that no specific semen preparation technique ( i.e. dgc ; swim - up ; as well as wash and centrifugation techniques ) improved clinical outcome with reference to iui procedures . the reason being that a minimum threshold of > 1 million motile spermatozoa is needed for successful conception through iui , irrespective of the type of sperm preparation method used ( ombelet et al . , 2003 ) . one such option is an office - based semen preparation device called sep - d ( surelife media technologies ( cat no : sl 001 ) ) with a current price tag of 23.00/device , consisting of a semen preparation kit containing a syringe pre - filled with a buffered culture media . motile sperm is separated from seminal plasma through a direct swim - up technique , where after the motile sperm fraction is retained in approximately 300 medium within the device , which is then connected to an iui catheter ( 7.00/catheter when purchased separately , cat no : sl 002 - 12 ) , and used for insemination ( www.surelifeivf.com ) . within the south african market a couple who qualifies for an iui procedure and resides in a rural town , could travel to a local general practitioner or clinic for repeated inseminations . gentis and co - workers ( 2012 ) reported a good clinical outcome for iui patients in a randomized controlled study at a south african art unit while using the sep - d semen processing device . the risks associated with sperm preparation techniques should be discussed with patients ( who , 2010 ; eke and oragwu , 2011 ) . data on sperm washing for hiv - seropositive patients are merely observational in nature according to a cochrane review by eke and oragwu ( 2011 ) . sperm washing refers to a sequential three phase procedure , i.e. dgc , washing of the sperm pellet and swim - up step ; as was initiated by semprini and co - workers ( 1992 ) to prepare semen samples for art from hiv - positive males . the bold undertaking was prior to the initiation of highly active antiretroviral treatment or validation of viral particles in the washed sperm sample ( semprini et al . this safety record is backed by published data from centers worldwide using iui , ivf and icsi procedures for hiv - positive males . various factors may contribute to the lack of randomized trials in this area of art , including hiv - regulations , inequalities in art treatment modalities , as well as costs of sperm washing and art procedures ( with particular reference to resource - poor countries in africa ) ( eke and oragwu , 2011 ) . the choice of a sperm preparation technique is however vital when processing sub - optimal semen samples for ivf or icsi , with discontinuous dgc being the preferred method to optimize samples . quotes for seven known brands of gradients and wash solutions were received from south african agencies in april 2013 . five of the seven brands are included in the listed culture media products ( see section : laboratory facilities , supplies and environmental aspects ) , together with purespermmedia ( nidacon , sweden ; www.nidacon.com ) and sil - select ( fertipro n.v . a single processing ( 2 ml semen sample ) will cost on average 16.82 3.34 ( ranging from 10.11 to 19.33 ) . the term sperm decontamination was coined at our laboratory to distinguish sperm washing from the decontamination procedure used for samples possibly containing various infectious microbes e.g. bacteria , hiv , hcv and cmv ( huyser and fourie , 2010 ) . this involves the layering of density gradients and the semen using a proinsert kit ( www.tekevent.com/nidacon/proinsert ) , at a cost of 10.10 for the device ( cat no : ni - p115 - 5 , nidacon ) . the kit consists of two conical tubes with two elongated pipettes and a proinsert device . the purified motile sperm pellet ( after centrifugation ) is retrieved using the elongated pipette without re - contaminating the pellet with infectious micro - organisms . a final washing step follows to get rid of density particles and a portion of the purified sperm sample can be submitted for testing ( hiv-1 proviral dna and rna using a sensitive molecular based technique such as reverse transcription polymerase chain reaction ( rt - pcr ) ) . more than a decade of research in the treatment of hiv+ semen samples culminated in the clinical application of procedures during art treatment of patients at our unit . a 100% and 98.1% success rate in the removal of hiv-1 rna and dna respectively , is maintained for the decontamination procedure at our laboratory ( n = 100 semen samples , post - processing pcr validations , 2011 - 2012 ) . since the costs of rt - pcr viral validations on purified sperm samples are expensive especially in a developing country , ( 86.20 per test for dna or rna at a national pathology laboratory ) the question arises if all purified samples should be tested ? due to restricted access to pathology laboratories and cost implications , only qualitative viral validations are available in most developing countries . within south africa , molecular viral testing is more accessible , and in our experiences up to 32% of patients with an undetectable hiv blood load can have a positive hiv-1 rna seminal viral load ( n = 100 patients ) . patients are informed of the procedure failure rate , hiv - related health screening ( cd4 + cell levels ) and additional infectious disease tests , general risk - reduction methods , extra costs for viral validations and cryopreservation of semen samples prior to the initiation of an art attempt ( huyser and boyd , 2012 ) . financial pressures can result in procedural shortcuts and the demand to maximize patient throughput in some private practices and laboratories offering sperm processing procedures . in the absence of national directives on the art treatment of hiv+ patients within a developing country , best practice frameworks and directives from developed countries could be adapted and used as guidelines for assisted reproduction laboratories in the developing world . all art related laboratory items except for general pharmacy articles are imported from various parts of the world to south africa . equipment , sperm processing solutions , embryo culture media and disposables have to be couriered with concomitant imported taxes . setting - up an art laboratory in any part of the world depends on economics , availability and optimal maintenance of items . procedures should be best - practice - based with reliable equipment , disposables , and techniques within a risk - reduction environment . this section will discuss the costs applicable to selected art equipment and mainstream embryo culture media that is commercially available and currently in use within south africa . the price tag for six different benchtop incubators and five media brands in south africa is compared . the costs to purchase six different benchtop incubators ( listed alphabetically ) : i.e. bt37(origio / planer scanlab equipment a / s , lynge , denmark ; www.origio.com ) ; g85 and g185 standard ( k - systems kivex biotec ltd , birkerd , denmark ; www.k-systems.dk ) ; k - minc-1000 ( cookmedical , brisbane , australia ; www.cookmedical.com ) , labo c - top ( labor - technik - gttingen , germany ; www.labotect.com ) ; miri multi - room incubator for ivf ( esco medical , singapore ; www.medical.escoglobal . table i demonstrates the south african price range from 3,769.80 to 22,763.57 for benchtop incubators which can accommodate 8 to 48 petri dishes ( 35 mm ) , respectively . the costs are vat inclusive and may consist of installation , a start - up kit or humidification container where applicable . for more details see the individual websites . when the same incubators are purchased in belgium , three out of the six incubators are currently between 6.59 - 28.69% less expensive , and three incubators are 1.44 - 25.98% more expensive compared to purchase prices in south africa . . a similar cost extrapolation should be applicable to all art equipment and disposables imported into south africa . a cost analysis for the set - up of the art programme at our unit in the eighties , indicated that the total cost for laboratory equipment amounted to 11,606 . a single inverted microscope , two water jacketed upright incubators , a stereo as well as a light microscope , a single laminar flow and biological safety cabinet , hygrometer , dry - oven , centrifuge , refrigerator , osmometer , ph - meter and electronic balance constituted the laboratory to initiate the art programme ( fourie et al . , 1988 ) . more than 90% of this equipment was still fully functional twenty years on ( 2005/6 ) , when the laboratory moved to new purpose built laboratories within steve biko academic hospital ( pretoria , south africa ) . similar equipment to date will cost between 67,283 and 92,515 , depending on the model size and/or brand type ( huyser and boyd , 2012 ) . the previously mentioned historical cost analysis article by fourie and co - workers ( 1988 ) did not refer to a micro - manipulator ( for icsi procedures ) , or any cryopreservation equipment during the initiation phase of the laboratory . five different brands of culture media are currently used in south africa : i.e. globalmedia ( lifeglobal one - step protocol ; ivfonline , usa , www.lifeglobal.com ) , medicult media ( embryoassist & blastassist two - step protocol ; origio , denmark , www.origio.com ) , quinns advantage media ( sage media products two - step protocol ; coopersurgical , ( usa , www.coopersurgical.com ) ; sydneyivf ( k - sicm & k - sibm two - step protocol ; cookmedical ireland , www.cookmedical.com ) ; and vitrolife - g - series media ( g-1 & g-2 , two - step protocol ; scandinavia ivf science , gteborg , sweden ; www.vitrolife.com ) . quotes for the media brands were obtained in april / may in 2013 from south african distributers . total costs per media brand for a full art cycle including dgc , ivf , and icsi are indicated in fig . costs were calculated per ml of medium used per patient , per cycle attempt according to laboratory protocol , with embryo culture in micro - drops under oil . a similar cost analyses were performed in 2012 ( huyser and boyd , 2012 ) . culture media for an ivf procedure for six oocytes amounts to 40.26 8.76 ( ranging from 27.25 to 51.79 ) and costs approximately15% less than media used in an icsi procedure ( mean cost of 47.14 11.04 ( ranging from 31.22 to 60.32 ) ) . cryopreservation solutions for non - vitrification procedures cost on average 19.96 9.20 ( ranging from 10.18 to 28.74 ) , with a 37% difference in cost for a vitrification attempt ( mean cost of 31.85 10.37 ( ranging from 14.55 to 41.38 ) ) . an average annual increase of 7% for vitrification cryopreservation , whereas an annual average decrease of 3% for non - vitrification solutions was noted . these costs exclude the cryopreservation carriers / devices available as open or closed single - straw systems , cryo - storage tanks , or liquid nitrogen . courier services and/or company representatives , which manage the transport and delivery of art culture media timeously , play a vital role in failures or successes in the art laboratory . durable packaging and protection of temperature sensitive culture media while in transit through developing countries are also extremely important during ( occasional ) protracted customs clearance , especially in summertime . the dual demand for art in south africa is mirrored in the provision of low cost accessible art services to lower - income nationals including middle - income private patients in the public service . the country s foreign - exchange rate can be two sides of the same coin ; i.e. equipment is more expensive to import than the purchase price within a confederacy , however comparative cut - rate art costs can lure non - nationals to south africa as a choice destination for reproductive tourism . the downturn in the global economy on the other hand has a sobering effect transnationally and should influence manufacturers of art products to develop products that can stand the test of time . art procedures need not be propelled towards the must - have and cannot - do - without approach , but providers should also reflect on the validity of the techniques and equipment , without compromising treatment virtue . art treatments should be globally within the reach of a much larger part of the population .

in developing countries especially in sub - saharan africa , human immunodeficiency virus ( hiv ) infection together with limited resources adds to the hindrances in becoming a parent . although the south african s bill of rights proclaim that south africans can make decisions concerning reproduction ; access to and the use of assisted reproduction technology ( art ) are viewed in general as excessively expensive , accessible only to the privileged few . a dissection of cost - drivers within an art laboratory , such as procedures ; sperm preparations ; laboratory supplies including embryo culture media and cryopreservation are discussed in the current overview . subject to the nature of an art practice , i.e. private vs. public / tertiary , the structure of a unit will vary with regards to patient demographics , costs and services offered . the average fees per procedure for 20 practices in the private sector in south africa are : ( i ) iui : 542 159 , ( ii ) ivf : 3,255 576 and ( iii ) icsi : 3,302 625 . laboratory costs can contribute between 35 and 48% of art fees payable in the private sector . low - cost public art services are available to citizens of the country at a few tertiary academic units . some private practices also cater specifically for middle - income citizens . art procedures need not be propelled towards the must - have and cannot - do without approach , but providers should also reflect on the validity of the techniques and equipment , without compromising treatment virtue .

Background ART procedures Screening of patients for pathogens: detection and prevention Sperm preparations Laboratory facilities, supplies and environmental aspects Conclusions

in developing countries particularly in the sub - saharan african region , human immunodeficiency virus ( hiv ) infection together with limited resources adds to the barriers in becoming a parent . although the south african s bill of rights ( constitution of the republic of south africa act no 108 of 1996 ) decree that south africans can make decisions concerning reproduction ; access to and the use of assisted reproduction technology ( art ) are viewed in general as prohibitively expensive and only accessible to the privileged few . resources available in the public / tertiary art units and to private practices and its clients , affect reproductive healthcare screening and concurrent diagnostic and therapeutic decisions which include ; scheduling for intra - uterine inseminations ( iui ) , in vitro fertilization ( ivf ) and intra - cytoplasmic sperm injection ( icsi ) , combined with semen decontamination for hiv - seropositive males . several factors can impact on the financial health of an art programme . cost - drivers with emphasis on art laboratory set - up and procedures in south africa will be discussed in this review , structured on a previous examination of cost - drivers in south africa ( huyser and boyd , 2012 ) , i.e. ( 1 ) art procedures , ( 2 ) detection and prevention of infections , ( 3 ) sperm preparations , and ( 4 ) laboratory facilities , including supplies needed to perform procedures ( fig . , 1985 ) , with the first test tube different forms of art services are provided in the country , i.e. the speciality , repertoire and type of art structure , i.e. private vs. public / tertiary , will impact on capacity , services offered , revenue generated and patient population of the unit . [ c ] an affordable low cost option for ivf / icsi is also available to patient categories [ a ] & [ b ] , and is based on initiatives for accessible ivf ( ombelet and campo , 2007 ; ombelet et al . access to the subsidized category is subjected to budget allocation , and only a small number of patients qualify for a subsidy in this category per year . in the private sector , ivf procedures increased in 2012 - 13 on average from 2,930 to 3,255 with a similar trend found for the icsi procedures . the average costs ( standard deviation ) per procedure in the private sector are : ( i ) iui : 542 159 , ( ii ) ivf : 3,255 576 and ( iii ) icsi : 3,302 625 . dyer and kruger ( 2012 ) referred to general out - of - pocket costs for a standard ivf cycle of 841 ( subsidized in the public sector ) and 2,944 ( within the private sector ) in south africa , which reflects the previous mentioned fee structure in 2012 . the average percentage of the major cost - drivers of an ivf cycle at an active private practice in south africa in 2012 were the following : 8% of costs are allocated for clinic fees , 28% to medication , 29% to clinicians fees & consultations , and 35% for laboratory fees ( for use of equipment and the laboratory , disposables , culture media and staff expenditures ) ( huyser and boyd , 2012 ) . converting from an ivf procedure to an icsi procedure items used in the laboratory can amount to nearly 48% of all costs per icsi cycle . approximately 70% ( 14 out of 20 practices ) of the south african art units that were contacted during the present study ( fig . , 2002 ) , and is revealed in the low comparative costs for medications , 8% of the total expenses , vs. 23 - 28% for an ivf or icsi cycle ( proportional data communicated by one of the largest art units in south africa ) ( huyser and boyd , 2012 ) . chambers and co - workers ( 2009 ) indicated that the cost of ( art ) treatment reflects the costliness of the underlying healthcare system rather than the regulatory or funding environment . within south africa , access to art is restrictive due to limited health insurance coverage of art procedures , restrained access to a few art units within the public sector ( dyer and kruger , 2012 ) , and limited funding to public sector art providers . similar to the debate on prevention vs. treatment of infertility ( dyer and pennings , 2010 ) , the detection and thus prevention of pathogen transmission will be less expensive and more beneficial to a large number of people than treatment alone . with the prevalence of hiv in sub - saharan africa , the question arises should all art participants be screened / re - screened for blood borne viruses ( bbv ) ? without a south african technical directive regarding the screening and treatment of art patients for bbv / pathogens it is doubtful if requirements of the european union tissue and cells directives ( www.eur-lex.europa.eu ) for art units in the eu , whereby biological screening must be carried out at the time of donation i.e. with rapid screening technology available at affordable costs ( approximately 1.30 per hiv rapid test ) in south africa , all art patients currently the cost for a single hiv rapid test ( 2 ) is approximately 1.5% of a rt - pcr quantitative ( hiv-1 rna ) and 8% of an elisa hiv-1 test . a layered risk - reduction approach is practised at our unit when dealing with contaminants in semen prior to an art cycle , i.e. ( 2011 ) showed that no specific semen preparation technique ( i.e. one such option is an office - based semen preparation device called sep - d ( surelife media technologies ( cat no : sl 001 ) ) with a current price tag of 23.00/device , consisting of a semen preparation kit containing a syringe pre - filled with a buffered culture media . sperm washing refers to a sequential three phase procedure , i.e. the bold undertaking was prior to the initiation of highly active antiretroviral treatment or validation of viral particles in the washed sperm sample ( semprini et al . various factors may contribute to the lack of randomized trials in this area of art , including hiv - regulations , inequalities in art treatment modalities , as well as costs of sperm washing and art procedures ( with particular reference to resource - poor countries in africa ) ( eke and oragwu , 2011 ) . five of the seven brands are included in the listed culture media products ( see section : laboratory facilities , supplies and environmental aspects ) , together with purespermmedia ( nidacon , sweden ; www.nidacon.com ) and sil - select ( fertipro n.v . this involves the layering of density gradients and the semen using a proinsert kit ( www.tekevent.com/nidacon/proinsert ) , at a cost of 10.10 for the device ( cat no : ni - p115 - 5 , nidacon ) . since the costs of rt - pcr viral validations on purified sperm samples are expensive especially in a developing country , ( 86.20 per test for dna or rna at a national pathology laboratory ) the question arises if all purified samples should be tested ? due to restricted access to pathology laboratories and cost implications , only qualitative viral validations are available in most developing countries . within south africa , molecular viral testing is more accessible , and in our experiences up to 32% of patients with an undetectable hiv blood load can have a positive hiv-1 rna seminal viral load ( n = 100 patients ) . patients are informed of the procedure failure rate , hiv - related health screening ( cd4 + cell levels ) and additional infectious disease tests , general risk - reduction methods , extra costs for viral validations and cryopreservation of semen samples prior to the initiation of an art attempt ( huyser and boyd , 2012 ) . financial pressures can result in procedural shortcuts and the demand to maximize patient throughput in some private practices and laboratories offering sperm processing procedures . in the absence of national directives on the art treatment of hiv+ patients within a developing country , best practice frameworks and directives from developed countries could be adapted and used as guidelines for assisted reproduction laboratories in the developing world . all art related laboratory items except for general pharmacy articles are imported from various parts of the world to south africa . equipment , sperm processing solutions , embryo culture media and disposables have to be couriered with concomitant imported taxes . setting - up an art laboratory in any part of the world depends on economics , availability and optimal maintenance of items . this section will discuss the costs applicable to selected art equipment and mainstream embryo culture media that is commercially available and currently in use within south africa . table i demonstrates the south african price range from 3,769.80 to 22,763.57 for benchtop incubators which can accommodate 8 to 48 petri dishes ( 35 mm ) , respectively . when the same incubators are purchased in belgium , three out of the six incubators are currently between 6.59 - 28.69% less expensive , and three incubators are 1.44 - 25.98% more expensive compared to purchase prices in south africa . a cost analysis for the set - up of the art programme at our unit in the eighties , indicated that the total cost for laboratory equipment amounted to 11,606 . five different brands of culture media are currently used in south africa : i.e. courier services and/or company representatives , which manage the transport and delivery of art culture media timeously , play a vital role in failures or successes in the art laboratory . durable packaging and protection of temperature sensitive culture media while in transit through developing countries are also extremely important during ( occasional ) protracted customs clearance , especially in summertime . , 1985 ) , with the first test tube different forms of art services are provided in the country , i.e. it is questionable if the current ( approximately ) 28 national art service providers ( cross - referenced with known providers in provinces and www.ivf-worldwide.com ) are providing an adequate reproductive health service within a nation of 52 million people with a variety of cultures and languages ( http://www.southafrica.info/about/people/population ) . the speciality , repertoire and type of art structure , i.e. private vs. public / tertiary , will impact on capacity , services offered , revenue generated and patient population of the unit . the reproductive and endocrine unit , as part of the department of obstetrics and gynaecology at the university of pretoria provides both diagnostic and therapeutic art procedures , including semen decontamination for hiv+ patients , and general cryopreservation ( huyser and fourie , 2010 ) , and is an accredited training unit for clinical technologists and medical biological scientists in reproductive biology . the average cost for procedures from 20 private south african art units ( mostly from gauteng ) , were obtained telephonically ( in april 12 with a follow up in april 13 ) ( fig . in the private sector , ivf procedures increased in 2012 - 13 on average from 2,930 to 3,255 with a similar trend found for the icsi procedures . the average costs ( standard deviation ) per procedure in the private sector are : ( i ) iui : 542 159 , ( ii ) ivf : 3,255 576 and ( iii ) dyer and kruger ( 2012 ) referred to general out - of - pocket costs for a standard ivf cycle of 841 ( subsidized in the public sector ) and 2,944 ( within the private sector ) in south africa , which reflects the previous mentioned fee structure in 2012 . the average percentage of the major cost - drivers of an ivf cycle at an active private practice in south africa in 2012 were the following : 8% of costs are allocated for clinic fees , 28% to medication , 29% to clinicians fees & consultations , and 35% for laboratory fees ( for use of equipment and the laboratory , disposables , culture media and staff expenditures ) ( huyser and boyd , 2012 ) . approximately 70% ( 14 out of 20 practices ) of the south african art units that were contacted during the present study ( fig . , 2002 ) , and is revealed in the low comparative costs for medications , 8% of the total expenses , vs. 23 - 28% for an ivf or icsi cycle ( proportional data communicated by one of the largest art units in south africa ) ( huyser and boyd , 2012 ) . chambers and co - workers ( 2009 ) indicated that the cost of ( art ) treatment reflects the costliness of the underlying healthcare system rather than the regulatory or funding environment . within south africa , access to art is restrictive due to limited health insurance coverage of art procedures , restrained access to a few art units within the public sector ( dyer and kruger , 2012 ) , and limited funding to public sector art providers . screening of the couple should however , be directed by the incidence of disease(s ) in the specific patient population , medical history and physical examination of the couple ( elder et al . with the prevalence of hiv in sub - saharan africa , the question arises should all art participants be screened / re - screened for blood borne viruses ( bbv ) ? without a south african technical directive regarding the screening and treatment of art patients for bbv / pathogens it is doubtful if requirements of the european union tissue and cells directives ( www.eur-lex.europa.eu ) for art units in the eu , whereby biological screening must be carried out at the time of donation i.e. with rapid screening technology available at affordable costs ( approximately 1.30 per hiv rapid test ) in south africa , all art patients currently the cost for a single hiv rapid test ( 2 ) is approximately 1.5% of a rt - pcr quantitative ( hiv-1 rna ) and 8% of an elisa hiv-1 test . a layered risk - reduction approach is practised at our unit when dealing with contaminants in semen prior to an art cycle , i.e. one such option is an office - based semen preparation device called sep - d ( surelife media technologies ( cat no : sl 001 ) ) with a current price tag of 23.00/device , consisting of a semen preparation kit containing a syringe pre - filled with a buffered culture media . within the south african market a couple who qualifies for an iui procedure and resides in a rural town , could travel to a local general practitioner or clinic for repeated inseminations . sperm washing refers to a sequential three phase procedure , i.e. various factors may contribute to the lack of randomized trials in this area of art , including hiv - regulations , inequalities in art treatment modalities , as well as costs of sperm washing and art procedures ( with particular reference to resource - poor countries in africa ) ( eke and oragwu , 2011 ) . five of the seven brands are included in the listed culture media products ( see section : laboratory facilities , supplies and environmental aspects ) , together with purespermmedia ( nidacon , sweden ; www.nidacon.com ) and sil - select ( fertipro n.v . this involves the layering of density gradients and the semen using a proinsert kit ( www.tekevent.com/nidacon/proinsert ) , at a cost of 10.10 for the device ( cat no : ni - p115 - 5 , nidacon ) . a final washing step follows to get rid of density particles and a portion of the purified sperm sample can be submitted for testing ( hiv-1 proviral dna and rna using a sensitive molecular based technique such as reverse transcription polymerase chain reaction ( rt - pcr ) ) . since the costs of rt - pcr viral validations on purified sperm samples are expensive especially in a developing country , ( 86.20 per test for dna or rna at a national pathology laboratory ) the question arises if all purified samples should be tested ? due to restricted access to pathology laboratories and cost implications , only qualitative viral validations are available in most developing countries . patients are informed of the procedure failure rate , hiv - related health screening ( cd4 + cell levels ) and additional infectious disease tests , general risk - reduction methods , extra costs for viral validations and cryopreservation of semen samples prior to the initiation of an art attempt ( huyser and boyd , 2012 ) . financial pressures can result in procedural shortcuts and the demand to maximize patient throughput in some private practices and laboratories offering sperm processing procedures . in the absence of national directives on the art treatment of hiv+ patients within a developing country , best practice frameworks and directives from developed countries could be adapted and used as guidelines for assisted reproduction laboratories in the developing world . all art related laboratory items except for general pharmacy articles are imported from various parts of the world to south africa . equipment , sperm processing solutions , embryo culture media and disposables have to be couriered with concomitant imported taxes . setting - up an art laboratory in any part of the world depends on economics , availability and optimal maintenance of items . this section will discuss the costs applicable to selected art equipment and mainstream embryo culture media that is commercially available and currently in use within south africa . when the same incubators are purchased in belgium , three out of the six incubators are currently between 6.59 - 28.69% less expensive , and three incubators are 1.44 - 25.98% more expensive compared to purchase prices in south africa . a cost analysis for the set - up of the art programme at our unit in the eighties , indicated that the total cost for laboratory equipment amounted to 11,606 . five different brands of culture media are currently used in south africa : i.e. courier services and/or company representatives , which manage the transport and delivery of art culture media timeously , play a vital role in failures or successes in the art laboratory . durable packaging and protection of temperature sensitive culture media while in transit through developing countries are also extremely important during ( occasional ) protracted customs clearance , especially in summertime . the dual demand for art in south africa is mirrored in the provision of low cost accessible art services to lower - income nationals including middle - income private patients in the public service . the country s foreign - exchange rate can be two sides of the same coin ; i.e. equipment is more expensive to import than the purchase price within a confederacy , however comparative cut - rate art costs can lure non - nationals to south africa as a choice destination for reproductive tourism . the downturn in the global economy on the other hand has a sobering effect transnationally and should influence manufacturers of art products to develop products that can stand the test of time . art procedures need not be propelled towards the must - have and cannot - do - without approach , but providers should also reflect on the validity of the techniques and equipment , without compromising treatment virtue . art treatments should be globally within the reach of a much larger part of the population .

native hawaiians are descendents of the aboriginal peoples inhabiting the hawaiian archipelago prior to western contact in 1778 and exercising sovereign governance prior to the 1892 overthrown of the hawaiian kingdom by the united states ( usa ) [ 1 , 2 ] . the 2000 us census enumerated 401,000 americans ( 0.1% of the total population ) of full or part - hawaiian ethnicity , about 60% of whom reside in the state of hawaii . native hawaiians comprise about 24.3% of the state 's current population . as in other states , the population of hawaii is aging , with an increasing number of residents living into old age . although life expectancy in hawaii exceeds that of other us states , studies conducted within the state reveal continuing ethnic differences in life expectancy . as depicted in figure 1 , over six decades ( 19502000 ) native hawaiians have consistently had the lowest life expectancy when compared to the state 's three other largest groups , namely , caucasians , filipinos ( americans ) , and japanese ( americans ) . notably , the magnitude of this disparity about 10 years lower than the longest lived group has not changed over time . about 16% of deaths among native hawaiians in 2005 occurred before 45 years , which is at least two times higher than for any other ethnic group living in the state . mortality disparities are particularly significant when comparing native hawaiians with japanese ; in 2005 , 60% of deaths among japanese occurred at age 80 + years , compared to only 25% of native hawaiians . as a result , native hawaiians are underrepresented in the older age groups . in 2008 , 21.4% of the state 's overall population was 60 + years of age but only 11.1% of native hawaiians are in this age group . to look at it another way , native hawaiians comprised 24.3% of the total state population in 2008 , but only 12.6% of residents age 60 + are native hawaiians . table 1 displays population totals and age distributions of the state 's four largest ethnic groups . other investigators have examined data for the native hawaiian population in general and have found that native hawaiians have a higher prevalence of obesity , numerous chronic conditions , and greater impoverishment than other ethnic groups living in the state [ 57 ] . the relatively poor health status of the native hawaiian population overall has been of significant interest since the publication of the seminal e ola mau : the native hawaiian health needs study report . historic difficulty in native hawaiians ' use of western mainstream healthcare services due to socioeconomic disadvantage , discrimination , and cultural misunderstanding are highlighted . subsequently , social welfare researchers in gerontology and native hawaiian health have documented a number of socioeconomic disparities ( e.g. , higher rates of poverty , lower educational levels , homelessness , and incarceration ) as well as health disparities ( e.g. , higher rates of diabetes and certain types of cancer , lower utilization of health services ) . to improve native hawaiian health and well - being , these researchers have underscored the need to consider more distal factors such as historical trauma or the cumulative effect of negative physical , sociocultural , political , and economic changes on current health disparities [ 712 ] . concomitantly , they articulate the strengths of traditional native hawaiian culture , including those cultural values and practices on health , care giving , and social support that might be integrated into interventions which offer the prospect of increased longevity and enhanced quality of life [ 8 , 10 , 11 ] . importance of this research notwithstanding , no recent studies have compared health indicators by ethnicity for older adults in hawaii with a specific focus on native hawaiian elders . research described here attempts to address this gap in the current knowledge and was conducted by researchers associated with h kpuna : national resource center for native hawaiian elders . funded by the us administration on aging ( aoa ) , h kpuna is one of three resource centers for native elders in the usa . the name derives from the native hawaiian cultural belief that one 's life essence ( spiritual energy , ancestral knowledge ) is transmitted to others through sharing of the h ( breath of life ) . it is believed that such sharing allows kpuna ( elders ) to pass on vital knowledge and wisdom to subsequent generations , thus perpetuating valued cultural traditions and a positive sense of identity . grounded in these and other traditional native hawaiian cultural values , h kpuna seeks to assure the transmission of h from kpuna ( elders ) to younger generations by achieving parity in life expectancy and good health among native hawaiian older adults comparable to that of other older americans . as a center dedicated to the health of native hawaiian elders , h kpuna develops and disseminates knowledge to inform policy and service innovations . in this paper , we examine proximal influences to the health and longevity of native hawaiian elders , specifically describe the causes of premature mortality among native hawaiians , and identify the ways in which sociodemographic and behavioral factors of hawaii 's elders vary by ethnicity . our research was guided by these questions : ( 1 ) what are causes of premature mortality ? ( 2 ) how does this vary by ethnicity ? and ( 3 ) how do sociodemographic and health behavioral indicators vary by ethnicity ? to address these questions we reviewed relevant statistical data collected by two hawaii department of health surveillance programs . written requests for data were submitted to two surveillance programs of the hawaii department of health ( doh ) : vital records and the hawaii behavioral risk factor surveillance system ( hbrfss ) . data sources are briefly described , as well as methods used to arrive at population - based statistics relevant for examining the underrepresentation of native hawaiian elders in the state 's older ( 60 years ) adult population . we requested data on the state 's largest ethnic groups from the vital records program , which routinely gathers information on births , deaths , and marriages that take place in the state . based on death record data , we calculated the years of productive life lost ( ypll ) for the state 's largest ethnic groups . ypll is an index that measures the extent of premature mortality by giving a weight to each premature death from the predetermined cutoff age , with proportionally higher weights for younger deaths . because ypll is especially sensitive to the age distribution of the population , it can not be used in cross - ethnic - group comparisons . however , ypll can be converted to a rate that is independent of sample size and population distribution following a method proposed by lee . these rates , herein called total population life lost per person in a lifetime ( tpll ) , can be compared across ethnic groups . to construct tpll , we obtained resident death data from hawaii for the 3-year period , 19992001 , for each age group by ethnicity , sex , and underlying cause of death of the deceased . averaging 3 years of death data was done to smooth annual fluctuations in death . cause of death is coded following the international classification of disease 10th revision ( icd-10 ) . we analyzed six causes of death common among older adults cancer , heart disease , cerebrovascular disease , unintentional injuries , suicide , and diabetes in addition to total death . both population and death data were classified into age groups , < 1 , 14 years , and then in 5-year intervals . all deaths in a given age group are assumed to have occurred at the midpoint age of the interval . we set age 70 as the cutoff for premature mortality in conformance with recommendations of the national center for health statistics . with the upper limit for the premature death at 70 , the ypll owing to premature mortality is given by ( 1)ypll=(70ci)di , where , ci is the midpoint of the ith age interval and di is the number of deaths in the ith age interval . thus ( 70-ci ) is the weight given to the deaths in the ith age group . the summation runs from age 0 to 70 . for the cause - specific ypll , di is the number of deaths from that particular cause of death . ypll , as a function of di , is to a large extent determined by the size and age distribution of the population as well as premature mortality . to convert ypll to a rate , we applied the index proposed by lee , which is independent of population size and age distribution . in lee 's method , the age - specific weight of ypll is multiplied by the age - specific death rate of the population , that is , ( 2)(70cj)djpj , where pj is the population of age j. the resultant is the annual number of ypll expected to occur per person in age j. originally , lee named this measure as the cumulative rate of potential life lost ( crpll ) . but here we call it the total potential life lost per person in lifetime ( tpll ) , since it also presents the number of premature deaths . when the age is grouped , tpll is expressed by ( 3)tpll=ni(70ci)dipi , where ni is the length of the interval of the age group . this is the total number of ypll expected per person before age 70 in the study population . to establish 95% confidence intervals ( cis ) , we estimated the standard deviation of tpll in each ethnic group ( 4)[ni(70ci)]2pidipipidipi . further , as the 3-year average of death was used for di , the variance of our tpll is the above quantity divided by 3 . we requested special data runs from the hawaii behavioral risk factor surveillance system ( hbrfss ) to examine ethnic variation in sociodemographic , clinical , and behavioral factors in the state 's elders . part of the behavioral risk factor surveillance system ( brfss ) of the centers for disease control and prevention ( cdc ) , the hbrfss gathers data by telephone from about 6,000 randomly selected adults ( 18 years of age ) . respondents are queried about behaviors that directly or indirectly affect health and health - related topics . for example , brfss solicits information on height and weight to calculate body mass index ( bmi ) , health behaviors ( e.g. , smoking ) , health screening ( e.g. , use of breast , cervical and colorectal cancer early detection screening ) , and chronic disease ( e.g. , diabetes , hypertension ) . sample data are adjusted and weighted based on ethnicity distributions , and estimates are produced for native hawaiians , caucasians , filipinos , and japanese . we requested a special tabulation that averaged responses from three years of data ( 2005 , 2006 , and 2007 ) relevant to older adults ( 60 years of age ) residing in hawaii . after years of data were combined , hbrfss provided data tables for older adults as a whole , and for the four largest ethnic groups . the sample size of older adults over three years included 658 native hawaiians , 2,652 caucasians , 561 filipinos , and 1,840 japanese , for a total of 6,346 elders . for hawaii , the overall tpll before age 70 per person from premature mortality was estimated at 3.3 years in 2000 , but tpll varied by ethnic group ( table 2 ) . among the groups we analyzed , the smallest number of years lost was observed for japanese at 2.6 years and , as expected , the largest was for native hawaiians , at 5.3 years . the difference in tpll between japanese and native hawaiians is twofold , meaning that , on average , native hawaiians are losing twice as many years of potential life as japanese . among the six causes of death , cancer caused the largest number of potential years lost before age 70 , with 0.74 years per person , accounting for more than 22% of overall tpll . heart disease , ranking second ( 0.59 years ) , accounted for 18% of overall tpll . unintentional injuries ranked third ( 0.40 years ) , accounting for 12% of overall tpll . death from suicide ( 0.21 years ) , stroke ( 0.13 years ) , and diabetes ( 0.06 years ) ranked fourth , fifth , and sixth , respectively . as illustrated in figure 2 , the importance of cause of death varied considerably by ethnic group . cancer was the most important cause of tpll in all populations , except native hawaiians , while heart disease was top - ranked for native hawaiians . while cancer and heart disease were the top - leading causes of tpll in most ethnic groups , accidents ranked second for caucasians , while accidents ranked third for other groups . suicide , cerebrovascular disease , and diabetes ranked fourth , fifth , and sixth in each ethnic group . native hawaiians had the highest tpll for each of the six causes of death , almost twofold higher for cancer than the other groups , two - to - four times higher for heart disease , and two - to - three times higher for diabetes . sociodemographic data from hbrfss indicate that native hawaiian older adults have the largest proportion of females to males ; women account for 61.5% of hawaiian elders compared to caucasian females who account for 50.9% of caucasian elders . this suggests that a disproportionate amount of native hawaiian men are dying before the age of 60 years . about 38.2% of native hawaiian elders attended at least one year of college . this percent is similar to that of filipino elders ( 39.7% ) , but much lower than for japanese ( 56.1% ) and caucasians ( 72.7% ) . further , about 24% of native hawaiian elders are employed ( perhaps indicating that they have had to delay retirement due to higher impoverishment rates ) , compared to only 18% of japanese elders . according to hbrfss , the percentage of elders with health insurance was relatively high overall ( 97% ) although the percentage of native hawaiian elders reporting access to health insurance was 95% . approximately 7.2% of native hawaiian elders reported not being able to see a physician in the past year due to cost , compared to less than 1% of japanese elders . native hawaiian elders reported the highest prevalence of asthma ( 20% versus 11% overall ) and diabetes ( 25% versus 17% overall ) . however , native hawaiians reported prevalence of heart attached , angina / chd , and stroke similar to caucasians . hbrfss data indicate a relatively high prevalence of behaviors associated with increased disease risk among native hawaiian elders . for example , native hawaiian elders were most likely of the four ethnic groups to smoke every day or occasionally , about 14.6% compared to only 6.9% of japanese elders ( although native hawaiian elders with a history of smoking were most likely to state that they were trying to quit or have stopped smoking at least for one day in the past year ) . native hawaiian and caucasian elders were most likely to report drinking more than four or five alcoholic beverages on one occasion ( about 9% in each group ) . about one - third of all ethnic groups reported that they were overweight , however 36% of native hawaiian elders were obese compared to only 8% of japanese elders . data from hbrfss indicate that 28% of filipinos , 26% of native hawaiians , 23% of japanese , and 19% of caucasian elders rated their personal health as fair or poor . about 18% of hawaiian elders responded that they were most likely to need special equipment , in comparison to 13% of all elders . about 80% of female elders reported having a mammogram within the last two years , including 78% of native hawaiian female elders . among male elders , native hawaiians and filipinos were the least likely to have had a prostate - specific antigen ( psa ) test for prostate cancer in the past 12 months ( all male elders = 54% , native hawaiians = 37.7% , filipinos = 33.7% ) . about 47% of filipino elders and about 53% of hawaiian elders have ever had a sigmoidoscopy or colonscopy to check for colorectal cancer , compared to 71% of caucasians and 73% of japanese . data from two state surveillance programs highlight ethnic differences in cause of death , health , and behavioral indicators that may help to explain some of the ethnic differences in life expectancy . for example , native hawaiian elders are least likely to have attended college and most likely to have incomes below 200% of poverty . they are least likely to have access to a primary care physician , have routine checkups , or to participate in early detection cancer screening . further , they are most likely to smoke and be obese . in comparison to other major ethnic groups , native hawaiian elders have the highest or second highest prevalence of a number of chronic diseases , including asthma and diabetes . heart disease is the leading cause of premature death for native hawaiians , and native hawaiians lose significantly more years of productive life to heart disease , cancer , injuries , suicide , stroke , and diabetes than other ethnic groups . these findings for older adults are consistent with earlier research on the general hawaiian population that documents serious health and social disparities [ 36 , 9 ] . there were four major limitations to our research ; these limitations point to a number of remaining and critical gaps in knowledge about native hawaiian elders . first , the study describes , but does not explain , the relationship of ethnicity and health indicators . thus , caution must be exercised in using findings to directly inform policy or practice innovations . continued research is needed to explain the relationship of native hawaiian ethnicity and proximal health indicators . second , the study captured ethnic variation in health outcomes ; the role of gender as it interacts with ethnicity was not explored . research across the life course consistently exposes disparities by gender , and this is especially so in later life [ 19 , 20 ] . for example , poverty rates for older women are nearly twice as high as for men . our understanding of the lives of native hawaiian elders will be strengthened by the inclusion of gender in analyses of social and health disparities . third , this study used surveillance data collected from hawaii residents only ; thus , findings can not be generalized to populations outside the state , including native hawaiians living on the north american continent . at present , our knowledge of hawaiian elders residing in the continental usa is very limited . this is an important omission since nearly 40% of native hawaiians live in the contiguous states , primarily california , washington , and oregon . extending life expectancy and improving quality of life requires that we understand life expectancies , health status , health care needs , preferences for care , and utilization patterns of all native hawaiian elders . future research is needed to determine if native hawaiians on the continent experience similar disparities , such as shorter life expectancies than other ethnic groups in their new communities . fourth and finally , the current study relied on surveillance data that did not address distal factors , such as the influence of historical trauma and systemic discrimination on current health disparities . native hawaiian and other indigenous health researchers consistently emphasize the influence of historical trauma and intergenerational marginalization on current health disparities [ 1 , 5 , 812 , 2226 ] . sotero 's conceptual model of historical trauma posits that subjugation of a population by a dominant group has a cumulative effect on the physical , sociocultural , political , and economic well - being of the oppressed group . the trauma is felt by first - generation survivors ( i.e. , those who directly experienced the traumatic events ) , as well as by successive generations of their descendents . across succeeding generations , the traumatic impact may be mitigated to some degree by cultural resiliency and other group protective factors . however , the result is an excess of social and physical ills that ultimately lead to population - specific health disparities . in the case of native hawaiians , historical records document the precipitous decline in population numbers and health status of native hawaiians following contact with the west . at the earliest known point of contact in 1778 , members of the cooke exploratory expedition describe natives of the islands as hardy , robust , and capable of great physical activity . however , during the first 150 years of western contact , native hawaiians suffered disability and premature death from foreign diseases such as influenza , measles , small pox , syphilis , and mumps [ 1 , 23 ] . depopulation was severe ; in the first century of contact the native population declined from an estimated 800,000 to 50,000 . depopulation was accompanied by cultural degradation with the native language and many traditional practices outlawed and/or subordinated as inferior to the english language and western practices , respectively [ 23 , 24 ] . among the most devastating changes were those related to shifts in the land tenure system from one of collective stewardship to one of private ownership and monopoly capitalism . this shift assured the rise of westerner - owned plantations and eventually , led to the overthrow of the hawaiian kingdom in 1892 and us annexation in 1898 . the exponential growth of the plantation economy and subsequent loss of native hawaiian sovereignty coupled with severe depopulation caused a collective grief among native hawaiian survivors [ 9 , 23 , 24 ] . na makaainana ( those who tend the land , common people ) were alienated from their aina ( land ) , the source of their spiritual , social , and economic well - being . as natives lost access to their land , there was a mass exodus to port cities where they became wage laborers and in some cases , debtors , paupers , and na paahao ( prisoners , convicts ) [ 12 , 24 ] . in line with sotero , native hawaiian health researchers are linking the poor health of native hawaiians in contemporary times to this cascade of adverse historic events and intergenerational social marginalization [ 9 , 10 , 12 ] . despite the relative success of native hawaiian organizations and groups to build cultural pride , positive identity , and holistic health in communities , the social marginalization of native hawaiians for example , native hawaiian children are overrepresented in the state 's child welfare population , native hawaiian youth are disproportionately represented in the state 's juvenile justice system , and native hawaiian adult men and women are overrepresented at every stage of hawaii 's criminal justice system [ 2 , 27 ] . the overall picture remains one of a population with more social and health disparities in comparison to the other large ethnic groups in the state . in the last few decades some social policies have been enacted to redress past wrongs and support programs aimed at reducing the disparate health outcomes affecting native hawaiians ( e.g. , public law 100579 , the native hawaiian health care act of 1988 ) . however , it is clear that more must be done to increase the longevity of native hawaiians and enhance the quality of life among native hawaiian elders . our findings underscore the ongoing need to target behavioral risks affecting native hawaiian longevity across the life span . health promotions grounded in evidence - based strategies and tailored on native hawaiian cultural preferences , as well as socio - economic circumstances have been recommended to reduce smoking and sedentary lifestyle , improve dietary practices , and increase participation in early detection screening [ 913 ] . in the past , health promotions that disregard relevant cultural , socio - economic factors , and systemic barriers have been received with distrust and even resentment by native hawaiian consumers who have experienced such efforts as cultural impositions [ 1 , 12 ] . thus , in targeting behavioral risks affecting longevity , it is crucial to develop interventions that adhere to principles of community - based participatory research and meaningfully involve native hawaiian communities in identification of barriers and assets salient to intervention development and delivery . based on results from the current study , we conclude with three service and policy implications for native hawaiian elders . first , all hawaii residents need primary health care that includes health education to promote optimal health practices , as well as chronic disease prevention and control . the inclusion of health promotion and disease prevention and control in primary care is especially important for native hawaiians who as a group have shorter life expectancies than other ethnic groups and who enter their senior years with more chronic conditions and poorer health habits than most other ethnic groups . health promotion and disease management programs are needed to both inform native hawaiians of their risk for heart disease , and other chronic conditions , as well as to reduce the adverse impact of these conditions on their overall well - being . contributors to heart disease , such as hypertension and high cholesterol , can be controlled by diet , exercise , and medications . although many cancers can not be prevented , they can be cured and/or controlled if diagnosed and treated early . thus , enrollment in evidence - based health promotion and disease management programs should be encouraged financially and programmatically . second , all hawaii residents need health care to be affordable and this seems especially important for native hawaiian elders . unfortunately , the high costs of deductibles , copayments , and noninsured treatments lead to delays in seeking necessary health care and may discourage elders from completing treatment or taking medications as recommended . compounding the problem is the fragmented system of long - term care , both in the provision and funding of services . this issue has critical implications for native hawaiians , who may require long - term care services early in life due to earlier onset of disability . nationally , long - term care services and financing are undergoing major programmatic changes because of the demand for cost - effective and efficient practices for improving quality of life of individuals in need of long - term care . improvements to the long - term care system include aging and disability resource centers ( adrcs ) , which offer one - stop shopping for individuals in need of long - term care services , cash , and counseling programs ( through which elders and caregivers are provided vouchers to pay for long - term care services and providers of their choice ) , expansion of community residential care models ( such as assisted living , small group homes , and geriatric foster care ) , and the culture change movement ( i.e. , to make nursing homes more homelike and less institutional ) . the adaptation of these or other new models that aim to streamline and humanize long - term care , while reducing costs , should be balanced and sensitive to the health profile and needs of each kpuna or native hawaiian elder . third , services need to reflect older adult preferences . for native hawaiian elders this may include preferences for culturally based programs and services . as all professionals will find themselves working with increasing numbers and proportions of diverse older adults with chronic disease , this becomes an increasingly important care component . similar to all older adults , quality care for native hawaiian elders acknowledges their desire to remain in their own homes with an array of assistance from families , friends , and home and community - based services that honor and reflect their culture . not surprisingly , quality of life is influenced by the education and training of professionals and other service workers who provide the care . successful interventions for native hawaiian elders are predicated on practitioners having an understanding of cultural values and traditions that influence elder 's health practices , with cultural preferences that may include involvement of the elder 's extended family and use of health promotion approaches reflecting holistic wellness [ 911 , 13 ] . continued effort is needed to develop an affordable and culturally responsive health care system that supports acute care , as well as health education and promotion , disease prevention , early disease management and treatment , and community - based long - term care . further , services must be offered to people across the life span , thereby offering the opportunity for parity in life expectancy and the hopeful prospect of good health to native hawaiian elders comparable to that of other older adults . the information in this paper does not reflect the option of the us administration on aging .

native hawaiians comprise 24.3% of hawaii 's population , but only 12.6% of the state 's older adults . few published studies have compared health indicators across ethnicities for the state 's older adult population or focused on disparities of native hawaiian elders . the current study examines data from two state surveillance programs , with attention to cause of death and social - behavioral factors relevant to elders . findings reveal that native hawaiians have the largest years of productive life lost and the lowest life expectancy , when compared to the state 's other major ethnic groups . heart disease and cancer are leading causes of premature mortality . native hawaiian elders are more likely to report behavioral health risks such as smoking and obesity , live within / below 100199% of the poverty level , and find cost a barrier to seeking care . indicated is the need for affordable care across the lifespan and health services continuum . future research might explain behavioral factors as influenced by social determinants , including historical trauma on native hawaiian longevity .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Discussion 5. Conclusions Disclosure

native hawaiians are descendents of the aboriginal peoples inhabiting the hawaiian archipelago prior to western contact in 1778 and exercising sovereign governance prior to the 1892 overthrown of the hawaiian kingdom by the united states ( usa ) [ 1 , 2 ] . the 2000 us census enumerated 401,000 americans ( 0.1% of the total population ) of full or part - hawaiian ethnicity , about 60% of whom reside in the state of hawaii . native hawaiians comprise about 24.3% of the state 's current population . as in other states , the population of hawaii is aging , with an increasing number of residents living into old age . although life expectancy in hawaii exceeds that of other us states , studies conducted within the state reveal continuing ethnic differences in life expectancy . as depicted in figure 1 , over six decades ( 19502000 ) native hawaiians have consistently had the lowest life expectancy when compared to the state 's three other largest groups , namely , caucasians , filipinos ( americans ) , and japanese ( americans ) . about 16% of deaths among native hawaiians in 2005 occurred before 45 years , which is at least two times higher than for any other ethnic group living in the state . mortality disparities are particularly significant when comparing native hawaiians with japanese ; in 2005 , 60% of deaths among japanese occurred at age 80 + years , compared to only 25% of native hawaiians . in 2008 , 21.4% of the state 's overall population was 60 + years of age but only 11.1% of native hawaiians are in this age group . to look at it another way , native hawaiians comprised 24.3% of the total state population in 2008 , but only 12.6% of residents age 60 + are native hawaiians . table 1 displays population totals and age distributions of the state 's four largest ethnic groups . other investigators have examined data for the native hawaiian population in general and have found that native hawaiians have a higher prevalence of obesity , numerous chronic conditions , and greater impoverishment than other ethnic groups living in the state [ 57 ] . the relatively poor health status of the native hawaiian population overall has been of significant interest since the publication of the seminal e ola mau : the native hawaiian health needs study report . historic difficulty in native hawaiians ' use of western mainstream healthcare services due to socioeconomic disadvantage , discrimination , and cultural misunderstanding are highlighted . to improve native hawaiian health and well - being , these researchers have underscored the need to consider more distal factors such as historical trauma or the cumulative effect of negative physical , sociocultural , political , and economic changes on current health disparities [ 712 ] . concomitantly , they articulate the strengths of traditional native hawaiian culture , including those cultural values and practices on health , care giving , and social support that might be integrated into interventions which offer the prospect of increased longevity and enhanced quality of life [ 8 , 10 , 11 ] . importance of this research notwithstanding , no recent studies have compared health indicators by ethnicity for older adults in hawaii with a specific focus on native hawaiian elders . research described here attempts to address this gap in the current knowledge and was conducted by researchers associated with h kpuna : national resource center for native hawaiian elders . grounded in these and other traditional native hawaiian cultural values , h kpuna seeks to assure the transmission of h from kpuna ( elders ) to younger generations by achieving parity in life expectancy and good health among native hawaiian older adults comparable to that of other older americans . as a center dedicated to the health of native hawaiian elders , h kpuna develops and disseminates knowledge to inform policy and service innovations . in this paper , we examine proximal influences to the health and longevity of native hawaiian elders , specifically describe the causes of premature mortality among native hawaiians , and identify the ways in which sociodemographic and behavioral factors of hawaii 's elders vary by ethnicity . our research was guided by these questions : ( 1 ) what are causes of premature mortality ? written requests for data were submitted to two surveillance programs of the hawaii department of health ( doh ) : vital records and the hawaii behavioral risk factor surveillance system ( hbrfss ) . data sources are briefly described , as well as methods used to arrive at population - based statistics relevant for examining the underrepresentation of native hawaiian elders in the state 's older ( 60 years ) adult population . we requested data on the state 's largest ethnic groups from the vital records program , which routinely gathers information on births , deaths , and marriages that take place in the state . based on death record data , we calculated the years of productive life lost ( ypll ) for the state 's largest ethnic groups . ypll is an index that measures the extent of premature mortality by giving a weight to each premature death from the predetermined cutoff age , with proportionally higher weights for younger deaths . because ypll is especially sensitive to the age distribution of the population , it can not be used in cross - ethnic - group comparisons . to construct tpll , we obtained resident death data from hawaii for the 3-year period , 19992001 , for each age group by ethnicity , sex , and underlying cause of death of the deceased . cause of death is coded following the international classification of disease 10th revision ( icd-10 ) . we analyzed six causes of death common among older adults cancer , heart disease , cerebrovascular disease , unintentional injuries , suicide , and diabetes in addition to total death . with the upper limit for the premature death at 70 , the ypll owing to premature mortality is given by ( 1)ypll=(70ci)di , where , ci is the midpoint of the ith age interval and di is the number of deaths in the ith age interval . thus ( 70-ci ) is the weight given to the deaths in the ith age group . for the cause - specific ypll , di is the number of deaths from that particular cause of death . ypll , as a function of di , is to a large extent determined by the size and age distribution of the population as well as premature mortality . in lee 's method , the age - specific weight of ypll is multiplied by the age - specific death rate of the population , that is , ( 2)(70cj)djpj , where pj is the population of age j. the resultant is the annual number of ypll expected to occur per person in age j. originally , lee named this measure as the cumulative rate of potential life lost ( crpll ) . but here we call it the total potential life lost per person in lifetime ( tpll ) , since it also presents the number of premature deaths . further , as the 3-year average of death was used for di , the variance of our tpll is the above quantity divided by 3 . we requested special data runs from the hawaii behavioral risk factor surveillance system ( hbrfss ) to examine ethnic variation in sociodemographic , clinical , and behavioral factors in the state 's elders . sample data are adjusted and weighted based on ethnicity distributions , and estimates are produced for native hawaiians , caucasians , filipinos , and japanese . we requested a special tabulation that averaged responses from three years of data ( 2005 , 2006 , and 2007 ) relevant to older adults ( 60 years of age ) residing in hawaii . after years of data were combined , hbrfss provided data tables for older adults as a whole , and for the four largest ethnic groups . the sample size of older adults over three years included 658 native hawaiians , 2,652 caucasians , 561 filipinos , and 1,840 japanese , for a total of 6,346 elders . for hawaii , the overall tpll before age 70 per person from premature mortality was estimated at 3.3 years in 2000 , but tpll varied by ethnic group ( table 2 ) . among the groups we analyzed , the smallest number of years lost was observed for japanese at 2.6 years and , as expected , the largest was for native hawaiians , at 5.3 years . among the six causes of death , cancer caused the largest number of potential years lost before age 70 , with 0.74 years per person , accounting for more than 22% of overall tpll . as illustrated in figure 2 , the importance of cause of death varied considerably by ethnic group . cancer was the most important cause of tpll in all populations , except native hawaiians , while heart disease was top - ranked for native hawaiians . while cancer and heart disease were the top - leading causes of tpll in most ethnic groups , accidents ranked second for caucasians , while accidents ranked third for other groups . native hawaiians had the highest tpll for each of the six causes of death , almost twofold higher for cancer than the other groups , two - to - four times higher for heart disease , and two - to - three times higher for diabetes . sociodemographic data from hbrfss indicate that native hawaiian older adults have the largest proportion of females to males ; women account for 61.5% of hawaiian elders compared to caucasian females who account for 50.9% of caucasian elders . this suggests that a disproportionate amount of native hawaiian men are dying before the age of 60 years . about 38.2% of native hawaiian elders attended at least one year of college . further , about 24% of native hawaiian elders are employed ( perhaps indicating that they have had to delay retirement due to higher impoverishment rates ) , compared to only 18% of japanese elders . according to hbrfss , the percentage of elders with health insurance was relatively high overall ( 97% ) although the percentage of native hawaiian elders reporting access to health insurance was 95% . approximately 7.2% of native hawaiian elders reported not being able to see a physician in the past year due to cost , compared to less than 1% of japanese elders . native hawaiian elders reported the highest prevalence of asthma ( 20% versus 11% overall ) and diabetes ( 25% versus 17% overall ) . hbrfss data indicate a relatively high prevalence of behaviors associated with increased disease risk among native hawaiian elders . for example , native hawaiian elders were most likely of the four ethnic groups to smoke every day or occasionally , about 14.6% compared to only 6.9% of japanese elders ( although native hawaiian elders with a history of smoking were most likely to state that they were trying to quit or have stopped smoking at least for one day in the past year ) . native hawaiian and caucasian elders were most likely to report drinking more than four or five alcoholic beverages on one occasion ( about 9% in each group ) . about one - third of all ethnic groups reported that they were overweight , however 36% of native hawaiian elders were obese compared to only 8% of japanese elders . data from hbrfss indicate that 28% of filipinos , 26% of native hawaiians , 23% of japanese , and 19% of caucasian elders rated their personal health as fair or poor . about 18% of hawaiian elders responded that they were most likely to need special equipment , in comparison to 13% of all elders . about 80% of female elders reported having a mammogram within the last two years , including 78% of native hawaiian female elders . among male elders , native hawaiians and filipinos were the least likely to have had a prostate - specific antigen ( psa ) test for prostate cancer in the past 12 months ( all male elders = 54% , native hawaiians = 37.7% , filipinos = 33.7% ) . about 47% of filipino elders and about 53% of hawaiian elders have ever had a sigmoidoscopy or colonscopy to check for colorectal cancer , compared to 71% of caucasians and 73% of japanese . data from two state surveillance programs highlight ethnic differences in cause of death , health , and behavioral indicators that may help to explain some of the ethnic differences in life expectancy . for example , native hawaiian elders are least likely to have attended college and most likely to have incomes below 200% of poverty . in comparison to other major ethnic groups , native hawaiian elders have the highest or second highest prevalence of a number of chronic diseases , including asthma and diabetes . heart disease is the leading cause of premature death for native hawaiians , and native hawaiians lose significantly more years of productive life to heart disease , cancer , injuries , suicide , stroke , and diabetes than other ethnic groups . these findings for older adults are consistent with earlier research on the general hawaiian population that documents serious health and social disparities [ 36 , 9 ] . there were four major limitations to our research ; these limitations point to a number of remaining and critical gaps in knowledge about native hawaiian elders . first , the study describes , but does not explain , the relationship of ethnicity and health indicators . continued research is needed to explain the relationship of native hawaiian ethnicity and proximal health indicators . our understanding of the lives of native hawaiian elders will be strengthened by the inclusion of gender in analyses of social and health disparities . third , this study used surveillance data collected from hawaii residents only ; thus , findings can not be generalized to populations outside the state , including native hawaiians living on the north american continent . this is an important omission since nearly 40% of native hawaiians live in the contiguous states , primarily california , washington , and oregon . extending life expectancy and improving quality of life requires that we understand life expectancies , health status , health care needs , preferences for care , and utilization patterns of all native hawaiian elders . future research is needed to determine if native hawaiians on the continent experience similar disparities , such as shorter life expectancies than other ethnic groups in their new communities . fourth and finally , the current study relied on surveillance data that did not address distal factors , such as the influence of historical trauma and systemic discrimination on current health disparities . sotero 's conceptual model of historical trauma posits that subjugation of a population by a dominant group has a cumulative effect on the physical , sociocultural , political , and economic well - being of the oppressed group . in the case of native hawaiians , historical records document the precipitous decline in population numbers and health status of native hawaiians following contact with the west . at the earliest known point of contact in 1778 , members of the cooke exploratory expedition describe natives of the islands as hardy , robust , and capable of great physical activity . however , during the first 150 years of western contact , native hawaiians suffered disability and premature death from foreign diseases such as influenza , measles , small pox , syphilis , and mumps [ 1 , 23 ] . this shift assured the rise of westerner - owned plantations and eventually , led to the overthrow of the hawaiian kingdom in 1892 and us annexation in 1898 . the exponential growth of the plantation economy and subsequent loss of native hawaiian sovereignty coupled with severe depopulation caused a collective grief among native hawaiian survivors [ 9 , 23 , 24 ] . in line with sotero , native hawaiian health researchers are linking the poor health of native hawaiians in contemporary times to this cascade of adverse historic events and intergenerational social marginalization [ 9 , 10 , 12 ] . despite the relative success of native hawaiian organizations and groups to build cultural pride , positive identity , and holistic health in communities , the social marginalization of native hawaiians for example , native hawaiian children are overrepresented in the state 's child welfare population , native hawaiian youth are disproportionately represented in the state 's juvenile justice system , and native hawaiian adult men and women are overrepresented at every stage of hawaii 's criminal justice system [ 2 , 27 ] . the overall picture remains one of a population with more social and health disparities in comparison to the other large ethnic groups in the state . however , it is clear that more must be done to increase the longevity of native hawaiians and enhance the quality of life among native hawaiian elders . our findings underscore the ongoing need to target behavioral risks affecting native hawaiian longevity across the life span . health promotions grounded in evidence - based strategies and tailored on native hawaiian cultural preferences , as well as socio - economic circumstances have been recommended to reduce smoking and sedentary lifestyle , improve dietary practices , and increase participation in early detection screening [ 913 ] . in the past , health promotions that disregard relevant cultural , socio - economic factors , and systemic barriers have been received with distrust and even resentment by native hawaiian consumers who have experienced such efforts as cultural impositions [ 1 , 12 ] . based on results from the current study , we conclude with three service and policy implications for native hawaiian elders . the inclusion of health promotion and disease prevention and control in primary care is especially important for native hawaiians who as a group have shorter life expectancies than other ethnic groups and who enter their senior years with more chronic conditions and poorer health habits than most other ethnic groups . health promotion and disease management programs are needed to both inform native hawaiians of their risk for heart disease , and other chronic conditions , as well as to reduce the adverse impact of these conditions on their overall well - being . contributors to heart disease , such as hypertension and high cholesterol , can be controlled by diet , exercise , and medications . second , all hawaii residents need health care to be affordable and this seems especially important for native hawaiian elders . improvements to the long - term care system include aging and disability resource centers ( adrcs ) , which offer one - stop shopping for individuals in need of long - term care services , cash , and counseling programs ( through which elders and caregivers are provided vouchers to pay for long - term care services and providers of their choice ) , expansion of community residential care models ( such as assisted living , small group homes , and geriatric foster care ) , and the culture change movement ( i.e. the adaptation of these or other new models that aim to streamline and humanize long - term care , while reducing costs , should be balanced and sensitive to the health profile and needs of each kpuna or native hawaiian elder . for native hawaiian elders this may include preferences for culturally based programs and services . similar to all older adults , quality care for native hawaiian elders acknowledges their desire to remain in their own homes with an array of assistance from families , friends , and home and community - based services that honor and reflect their culture . successful interventions for native hawaiian elders are predicated on practitioners having an understanding of cultural values and traditions that influence elder 's health practices , with cultural preferences that may include involvement of the elder 's extended family and use of health promotion approaches reflecting holistic wellness [ 911 , 13 ] . further , services must be offered to people across the life span , thereby offering the opportunity for parity in life expectancy and the hopeful prospect of good health to native hawaiian elders comparable to that of other older adults .

while the past 20 or 30 years of development in chemoinformatics has created a plethora of published software systems and algorithms for solving chemical problems , little effort has been spent in providing the community with open components and data , to be reused and improved by communal efforts . bioinformatics , with its much younger history , adopted the principles taught by success stories of the open source movement in general , and linux in particular , from the very beginning . recent years , however , have seen the emergence of open tools and databases also in chemical informatics . these draw on the existing ideas of independent peer review and scientific collaboration , mixed with open source software development paradigms . community involvement , including assessments , suggestions , critiques , and rapid evolution , is a core component of these efforts . the benefits of open source software have been discussed in great detail by eric raymond in his seminal work the cathedral and the bazaar and following works . the open source initiative ( osi ) summarizes : open source promotes software reliability and quality by supporting independent peer review and rapid evolution of source code . to be osi certified , the software must be distributed under a license that guarantees the right to read , redistribute , modify , and use the software freely . in the beginning , most scientific software was free . but the 1980s saw the value of chemical informatics and the need to productize it . much of this was meritorious , as it brought informatics into the classroom and the research lab and helped pay for some chemistry research , but it also had hidden costs , which we are now facing today . now , several open chemistry and chemoinformatics projects ( table 1 ) have pooled forces to enhance interoperability between these tools in a movement we call the blue obelisk the name originates from an informal meeting place in san diego , california , during the american chemical society 2005 spring national meeting ( see figure 1 ) and was coined by one of the authors . because contributors to the component projects live around the world , few had met in person instead collaborating and meeting via the internet.figure 1where it all began . the blue obelisk in san diego , california , at the 2005 american chemical society meeting.table 1current blue obelisk projectsprojecturlprincipal authors cml , jumbohttp://cml.sf.net / p.m .- r . , e.l.w.nmrshiftdbhttp://www.nmrshiftdb.org/c.s.joelibhttp://joelib.sf.net/j.w.kalziumhttp://edu.kde.org/kalzium/carsten niehausoctethttp://octet.sf.net / rich apodacaopen babelhttp://openbabel.sf.net / g.r.h.qsarhttp://qsar.sf.net / e.l.w . , c.s . , j.w.the chemistry development kithttp://cdk.sf.net / e.l.w . the blue obelisk in san diego , california , at the 2005 american chemical society meeting . current blue obelisk projects we identify three core areas for the blue obelisk movement : one can use other people 's code without further permission , including changing it for one 's own use and distributing it again . the mechanisms for creating and maintaining these standards cover a wide spectrum of human organizations , including various degrees of consent . we have been heavily influenced by the mantra of the internet engineering task force : rough consensus and running code . one can obtain all data in the public domain when wanted and reuse it for whatever purpose . open access and has relevance to closed access as well . as outlined above , these areas are independent of the concept of open access to read publications freely . instead , the three points focus on access to the scientific data , algorithms , and implementations themselves , rather than the formatted manuscript . in particular , we believe that these concepts strongly continue the spirit of communal peer review and reproducibility at the heart of modern scientific research . it is well - known in software development that 80% of the costs are caused by maintaining software and not by the initial implementation . this holds both for the in - house development in pharmaceutical companies and the development for commercial chemoinformatics suppliers . besides judging software by its standardized functional quality , it can also be compared on the basis of its long - term stability and interoperability . openly standardized algorithms and chemical information can help to reduce the maintenance costs , because developers can reuse available modules or test their tools against open source software and open data . this reduces the risk for both the buy and build strategies for software implementation . we agree with de lano that the try - before - buy paradigm for open source software does not necessarily require open standards . open specifications for standard algorithms such as kekulization , chirality coding , and atom typing , however , are indispensable in academic chemoinformatics research to build better , more stable , and more reproducible chemical information systems . in this contribution , we outline several examples for how the blue obelisk projects address this need : a shared dictionary of algorithms and implementations in chemoinformatics algorithms drawing from our various software projects and a shared repository of chemoinformatics data including elemental properties , atomic radii , isotopes , atom typing rules , a set of web - based chemoinformatics services , and the process of providing open algorithms and data . all of these projects were developed with continual community involvement , an open standardization process , and provide open data to key chemoinformatics processes . anyone can take part ; we welcome those in commercial organizations , academia , government , and so forth , and contributions come as code , compilations of data and molecules , testing , and more . the world wide web as it is used today is a collection of linked html pages and other data formats . whenever there is chemical or other scientific knowledge or data published via this mechanism , it is often difficult or impossible to discover , because it lacks the semantics that would help machines the only practical way to harvest information from the internetto identify and classify it . recognizing this lack , tim berners - lee introduced the concept he termed the semantic web . the semantic web is a mesh of information linked up in such a way as to be easily processable by machines , on a global scale . one can think of it as being an efficient way of representing data on the world wide web , or as a globally linked database . an analogy of the semantic web , projected onto the currently heavily researched idea of creating global networks of computational resources , so - called grids , are the semantic grids . a semantic web , and even more a semantic grid , is predicated on the supply of information and services without requiring the user to know the details of how the resource was obtained . the users , who may be humans or robots , request precise services but should be unconcerned exactly how or where they originate . for example , the calculation of a molecular property might depend on a precise method but should not , in principle , depend on the actual program used , its version , the operating system , and the machine involved . we note that many chemical calculations are described in an imprecise manner . for example , molecular weight is an imprecise term , and the result of an algorithm returning this can not be regarded as precise . the iupac gold book describes relative molecular mass , mr : ratio of the mass of a molecule to the unified atomic mass unit . relative molar mass : molar mass divided by 1 g mol ( the latter is sometimes called the standard molar mass ) . unified atomic mass unit : non - si unit of mass ( equal to the atomic mass constant ) , defined as /12 of the mass of a carbon-12 atom in its ground state and used to express masses of atomic particles , u 1.660 540 2(10 ) 10 kg . these appear to refer to the mass of a single molecule , not to the properties of a bulk sample . however , atomic masses include the concept of average as in relative atomic mass ( atomic weight ) , ar : the ratio of the average mass of the atom to the unified atomic mass unit . there are at least two algorithms that could be used to obtain the molecular mass : sum the average masses of all the atoms in the molecule ( the normal molecular weight ) sum the precise masses of the most frequent isotopes in the molecule ( giving the high - resolution molecular mass ) . even this latter method is imprecise because , in mass spectroscopy , it relates to ions , and presumably , the mass of the ionizing electron(s ) should be accounted for . moreover , the actual values of atomic weights vary between program systems . we have frequently observed variations in molecular weights between different authorities often at the second decimal place . many chemoinformatics and computational chemistry papers use data resources which are not available to reviewers and readers and algorithms which are not portable or distributed . it is a matter of trust rather than verification whether such work is accepted by the community . we believe it is essential that computational chemistry is able to provide the basic scientific tenet of reproducibility if a scientist repeats the work in an article they should be able to duplicate the result . this is simple , in principle : computers should run reliably , and if the same data are given to the same algorithm , identical results should be obtained . however , it is surprisingly difficult to assert that the same method is being used . we can amend this to known validated data resources + known validated algorithms = validated web resources . there is relatively little practice of public validation of data resources and certification of algorithms in the field of chemistry , but without this , a global chemical semantic web is difficult to implement . this article explores the basis for such interoperability and outlines a working proof of concept . we hope that , in the long - term , appropriate bodies such as iupac and other learned societies might come to oversee this practice ; until then , the blue obelisk can be seen as an informal , neutral mechanism to which those interested in open semantics can contribute . an interoperable chemical approach requires at least the following communally agreed upon components in its architecture ( in no particular order ) : terminology , data typing , extensible data structures , conformance specification and tools , links and references , namespaces , and metadata for provenance and discoverability . syntactic support for all of these is provided by chemical markup language ( cml ) and other xml namespaces ( xhtml , mathml , etc . ) . this article is largely concerned with how the semantic containers for terminology , data , and algorithms are populated . there is also an important need for machine - enforceable behavior , which may also benefit from inheritance mechanisms but is not discussed in this work . our design and practice is heavily influenced by the practice and specifications from the international union of crystallography ( iucr ) . for the past three decades , the iucr , through its data commission and other bodies , has actively developed communal practice for the interchange of data . one of us ( p.m .- r . ) has been associated with the committee for the maintenance of the cif standard ( comcifs ) project for a decade . the crystallographic information file ( cif ) is the latest design of the iucr 's semantically rich data structures and is fully described in this journal and the recently published volume g of the int the primary approach is through dictionaries , each of which can describe a subdomain ( e.g. , core , macromolecules , powder diffraction , publications , etc . ) . the dictionaries are similarly constrained by a dictionary definition language ( ddl ) which is also recursively conformant . the groundbreaking ddl and cif specifications are the major vehicle for publications of crystallographic information , both textual and numeric . the community has developed software for validation and processing ; though , the full power of the ddl is only recently becoming realized . ddl and cif predated xml by a decade and are almost isomorphic to xml schema ( xsd ) and xml in their architecture . cif dictionaries traditionally describe the human - readable meaning of a term , together with its structure and constraints ( cardinality , lexical form , numeric range , enumerations , etc . ) . this architecture can reasonably be considered an ontology for the hard sciences . because the semantics of crystallography have been well - understood for many decades , much of the ontology , including the algorithms , can be hard - coded . more recently , through the drel specification , the iucr has started to add machine - enforceable semantics into their dictionaries . chart 1 shows a typical cif dictionary entry using the starddl approach ( courtesy of prof . this specification is being actively considered by the iucr 's comcifs committee.chart 1example of a cif dictionary entry example of a cif dictionary entry much of this example is self - explanatory . description.text ( within ; ... ;) is the human - readable meaning , where there are references to other dictionary items . the enumeration.range term describes a non - negative integer ( e.g. , xsd : nonnegativeinteger in xml schema ) . * loop_. in this loop , a piece of code , based on python and extended in the drel language , describes the precise algorithm for the evaluation of the atomic mass of the cell . it defines a mass , initially zero , and a list of atom_types in the data object ( the cif ) . the atom_types have subfields number_in_cell ( provided by the author ) and atomic_mass ( from a lookup table provided by iucr ) . the sum of the atomic masses of all of the atoms is returned as _ cell.atomic_mass , the identification of the dictionary entry . these dictionaries are now compilable and executable in a proof - of - concept system . they are powerful enough to allow the complete calculation of many crystallographic quantities ( e.g. , structure factors from atomic sites and form factors ) . the code can be run directly as python , in java through jython , and compiled into other languages through the jjtree compiler . many of the bo algorithms ( e.g. , hundreds of jumbo methods ) are sufficiently simple to be documented as machine - enforceable semantics . the dictionary approach enforces communal semantics for objects ( e.g. , through octet ) ; for example , a molecule contains atoms and bonds which can provide drel - like iterators . there may be concerns about using a procedural language rather than a functional one ( e.g. , scheme or lisp ) . we believe that the approach above is easily implemented and can run in a wide range of environments . it has the benefit of synergy with the code and systems developed in crystallography . note that the approach also contains a precise identification of , and therefore retrieval of , algorithms . the bo approach is informed by this architecture ; though , the precise syntax and semantics use xml - based approaches rather than cif . the blue obelisk chemoinformatics dictionary is our effort of defining a standard set of chemoinformatics algorithms . if a software project implements one of these algorithms , they can refer to this dictionary . by using unique identifiers , the dictionary allows using web search engines , like google.com , to find implementations for an algorithm in the dictionary . the dictionary uses the following technologies : scientific , technical , and medical markup language ( stmml ; http://www.xml-cml.org/stmml/ ) is used as a general container , and mathematical markup language ( mathml ) is used to contain mathematical formulas . likewise , scalable vector graphics ( svg ) could be used to add graphics to the dictionary ; though , this is currently not used . the full source of the latest xml source for the dictionary can be retrieved from ref ( 50400bb00021 ) . the xml document is accompanied by an xml schema document that encompasses the used xml languages . this allows xml - aware editors to syntactically validate the document and filter out syntax errors in either of the three xml languages . each entry in the dictionary has an associated identifier ( i d ) , which is unique throughout the xml document . when xml namespace technologies are used , a worldwide unique identifier can be composed that uniquely points to the entry in the dictionary . for example , by defining a namespace http://qsar.sourceforge.net/dicts/blue-obelisk with a related prefix blue - obelisk , one can uniquely point to an entry describing a kabsch algorithm to align two molecules ( id = alignmentkabsch ) within this namespace by referring to blue - obelisk : alignmentkabsch . chart 2 is an example of an entry currently used in the blue obelisk dictionaries . in this example , an entry is defined for an algorithm that finds the smallest set of smallest rings , given a molecular graph . bibtexml is used using the bibtex namespace prefix to cite the article in which the algorithm was described . the entry has a bit of meta content using the dublin core standard , for which the namespace uses the prefix dc . additionally , a classification is made ( into the area of graph theory ) , and a related entry is mentioned.chart 2example of an xml dictionary entry example of an xml dictionary entry extensible stylesheet language transformation ( xslt ) is used to transform the xml source code into an xhtml document which can be displayed by a mathml - aware web browser , like mozilla firefox . the blue obelisk movement agreed on using the same namespace prefix , that is , blue - obelisk , allowing web pages for specific software projects to cite entries in the dictionary . links from those pages currently must be made explicitly , but having the citations on those pages allows a web search engine to easily find software projects that implement a specific algorithm . the xhtml web page generated from the xml source of the dictionary contains , for each entry , a link to google.com that shows available implementations of that algorithm ( see figure 2 ) . this setup provides a powerful tool to find software that implements published algorithms.figure 2screen shot of the xhtml output of the blue obelisk chemoinformatics dictionary showing the search implementations on google.com feature . screen shot of the xhtml output of the blue obelisk chemoinformatics dictionary showing the search implementations on google.com feature . at the time of writing , cdk and jmol each provide a web page that cites and links to individual blue obelisk chemoinformatics dictionary entries . the open babel project has also included links to the dictionary in its developer documentation and is in the process of producing a complete index of entries as a separate web page . all of the projects are continuing to add entries to the dictionary for common algorithms . because many chemoinformatics projects rely on accurate atomic and molecular data such as atomic masses , isotopes , electronegativities , van der waals radii , covalent radii , and so on , we have initiated a repository of a standard set of chemoinformatics data , building on the processes involved in the dictionary mentioned above . conventional standards bodies , such as iupac , have established a variety of published data , particularly on isotopes , atomic masses , elemental abundances , element symbols and names , and so on . many chemoinformatics algorithms , however , rely on other data which may not have a clear - cut definition . for example , there is no obvious way to specify a van der waals radius not all elements are perfectly spherical , and multiple definitions exist including those taken from crystal structures , gas - phase measurements , and molecular mechanics force fields . to address these issues , software can use and refer to this repository when it needs standardized data for a wide range of chemical properties and other facts , of which an overview is given in table 2 . it is anticipated that , over the next year , the repository will considerably increase in the amount of available data.table 2current content of the data repository , with a few of the used sources.property typepropertysourcesphysical propertiesisotope abundances isotope masses31 atomic masses32 ionization energies chemical propertiesaffinities radii33 electronegativities element densities discoveryyear of discovery name and etymology otheratom type definitions 2d and 3d coloring schemes current content of the data repository , with a few of the used sources . the repository uses cml and dictionaries to allow the explicit markup of data types , units , and the experimental errors , as well as metadata like bibliographic sources , creation dates , and indications of authority . an example entry in the blue obelisk data repository for example , it states that the ionization energy is 13.5984 ev and that the mass is 1.007 94 amu . it does not explicitly state which mass is meant but refers , for the definition , to the blue obelisk dictionary ( see section 3).chart 3example of a blue obelisk data repository entry example of a blue obelisk data repository entry the preceding material has described how chemoinformatics data can be managed and accessed in a collaborative manner . another aspect of collaboration is the use of distributed functionality , that is , the use of function implementations that are not necessarily on the local machine . an example of this type of approach is the use of web services . though web - based applications are ubiquitous , they are generally full - fledged applications that are monolithic in nature . the term web services refers to functionality that can be accessed over the internet in a programmatic manner . in the context of chemoinformatics , this means that a programmer can access functions , which , for example , calculate binary fingerprints , over the internet without having to understand what language the underlying function is written in or whether the function is up - to - date . of course , this implies that the calling mechanism for the given function is well - defined and that the maintainer has kept it up - to - date . this approach is useful on a smaller scale , say , at the organizational level . the advantage of having web - based services implies that updates and modifications can be made on a single server , rather than requiring updates on individual machines . we have used the cdk to provide web services for molecular similarity and descriptor calculations , available at http://blue.chem.psu.edu/rajarshi/code/java/cdkws.html . access to these services can be programmatic ( using the soap protocol ) or by a web - based interface which simply calls the service and presents the results . since the algorithms are well - documented and the calling mechanism is well - defined , the service provides a relatively transparent method to obtain chemoinformatics functionality in a distributed manner . the downside of web service functionality is that the user does not have control . this can be a problem if the service is not documented , but at the same time , it can be an advantage in that it relieves the user of the maintenance of yet another library . furthermore , with the advent of open source and open data , a user is free to investigate the inner workings of a web service if he or she so wishes . this would allow the user to ensure that the web service does indeed do what it advertises . once again , this depends on the fact that the maintainer of the web service actually assigns an open license to the web service ( in terms of access as well as code ) . clearly , increased usage of web services is dependent on the transparency of the service . that is , a user must be able to ensure that a web service does indeed do what it says and should be able to rely on the provider of the service . we believe that the open principles underlying the blue obelisk movement are conducive to the development of transparent web services which provide easy access to a variety of functionalities in a distributed manner . it has been mentioned previously that the blue obelisk movement is a communal effort . given the three goals of the movement , it is obvious why such an endeavor must be a community effort rather than that of an individual . in this sense , the blue obelisk movement characterizes the nature of open source development in general and serves as an example of how this mode of development can be applied to problems in the field of chemical algorithms , standards , and data . a striking feature of the blue obelisk movement is the wide variety of contributors to the individual projects that make up the movement . the contributions themselves range from things as large as entire programs or frameworks to things as small as small amounts of data ( e.g. , to the data repository ) or bug reports . however , it should be understood that , though a bug report may appear to be a minor contribution compared to a whole framework , each contribution plays a vital role in the communal development and peer review of these projects . at the same time , it is important to realize that open source efforts represented by the blue obelisk movement do not always involve renumeration . thus , in many cases , the contributors work on the respective projects in their spare time . this leads to the situation where some areas in a project do not get as much attention as others , simply because it has not caught the attention of a contributor or because of a lack of expertise among the contributors . in many cases , contributions to these projects are the result of a developer having an itch that needed to be scratched . thus , compared to commercial projects , it may appear that the projects represented by the blue obelisk movement lack in certain areas . given the open nature of these projects , it is a simple matter for anybody with the interest and expertise to contribute to such an area , thus filling the gap . the above discussion paints a picture of many people contributing whatever they feel like . this is an important question because the contributors to the blue obelisk projects are located all over the world . furthermore , most projects are large enough that a single person can not always manage the contributions from a large user community . the fundamental mechanism for distributed communal development is mailing lists , that is , via e - mail . mailing lists are the mode by which the majority of decisions are made by the community for a given project , both in terms of use and development . decisions are made by consensus ; although , sometimes , the benevolent dictator model of development is followed . mailing lists also serve as archives of discussion , in addition to the use of traditional web pages and collaborative web pages ( wiki ) for the development of documentation . a more real - time mode of communication is the use of internet relay chat , which allows multiple people to convene in a virtual room and communicate in real time . in general , this is restricted to text , but current instant messaging services allow for the use of both audio- and video - based communication . this type of interaction is very fruitful , because contributors can discuss current problems and decisions in real time as they are working on the projects themselves . but how are the contributions ( such as code or documents ) themselves managed ? once again , this is a very important question because multiple people will be working on a program or document , and manually managing individual contributions does not scale for projects of even moderate size . the workhorses for managing actual contributions are version control systems such as cvs or subversion . these allow multiple contributors to submit changes to a program file or a document to a centrally located repository . if multiple contributors make changes to the same document , the system allows them to intelligently merge the resultant conflicts . these systems also allow developers to track changes and essentially view the history of a project . workflows and web services can also be used in the development process , and the utility of such types of applications has been mentioned previously . many of the blue obelisk projects make use of services provided by sourceforge.net , which is a community effort to provide open source projects with a set of tools and functionality for efficient code maintenance and communication . the site supports a number of features such as cvs , mailing lists , bug trackers , and so on , all of which are freely available to open source projects . clearly , current internet - based technology allows for easy and efficient management of contributions to the various blue obelisk projects from contributors located all over the world . in a sentence , the blue obelisk movement is an example of the use of open source technology and methods to customize tools and social practices for the development of chemical information services . we have described a communal effort to realize interoperability in chemical informatics , which we call the blue obelisk movement , named after the first meeting place of our community . the bo movement currently consists of more than 10 open source and open data projects all related to chemoinformatics . we identify concepts and algorithms , codify them in a collaborative dictionary , and link them to concrete implementations in blue obelisk projects and beyond to make them machine - searchable . we have started a public repository of chemical data of general interest , including data for chemical elements and isotopes ( boiling points , colors , electron affinities , masses , covalent radii , etc . ) , definitions of atom types , and more . all of the data is augmented with documentation , citations of origin , and a bibliography . we are working on a system of web services to provide access to chemoinformatics functionality without the knowledge of the details of the individual implementation and without the need to master the installation and programming interface of yet another chemoinformatics library . we emphasize that this work in progress , because of its emphasis on interoperability , has a value beyond that of open source and open data efforts . while standardization efforts in chemistry have a long history , modern computing and data processing , the internet , and the world wide web have , for the first time , created the possibility of effortlessly searchable and reusable data and computer programs . thus , this article addresses the old guard of developers and asks them to contribute their wisdom and their work . the result can be the survival of a work of a lifetime which otherwise might not survive the emeritation or the next sale of the company . this article is also addressed to newcomers asking them to adopt the ideas of open data and software from the very beginning . what is prized is contributions that help support the communal vision ( e.g. , raymond ) . our approach is not incompatible with commercial systems ; though , the preservation of authorship moral rights is taken very seriously .

the blue obelisk movement ( http://www.blueobelisk.org/ ) is the name used by a diverse internet group promoting reusable chemistry via open source software development , consistent and complimentary chemoinformatics research , open data , and open standards . we outline recent examples of cooperation in the blue obelisk group : a shared dictionary of algorithms and implementations in chemoinformatics algorithms drawing from our various software projects ; a shared repository of chemoinformatics data including elemental properties , atomic radii , isotopes , atom typing rules , and so forth ; and web services for the platform - independent use of chemoinformatics programs .

1. Introduction 2. The Importance of Open Specifications for Algorithms and Data 3. The Blue Obelisk Dictionary 4. The Blue Obelisk Repository 5. Web Services 6. Social Aspects 7. Conclusion

while the past 20 or 30 years of development in chemoinformatics has created a plethora of published software systems and algorithms for solving chemical problems , little effort has been spent in providing the community with open components and data , to be reused and improved by communal efforts . bioinformatics , with its much younger history , adopted the principles taught by success stories of the open source movement in general , and linux in particular , from the very beginning . these draw on the existing ideas of independent peer review and scientific collaboration , mixed with open source software development paradigms . community involvement , including assessments , suggestions , critiques , and rapid evolution , is a core component of these efforts . the benefits of open source software have been discussed in great detail by eric raymond in his seminal work the cathedral and the bazaar and following works . the open source initiative ( osi ) summarizes : open source promotes software reliability and quality by supporting independent peer review and rapid evolution of source code . to be osi certified , the software must be distributed under a license that guarantees the right to read , redistribute , modify , and use the software freely . in the beginning , most scientific software was free . much of this was meritorious , as it brought informatics into the classroom and the research lab and helped pay for some chemistry research , but it also had hidden costs , which we are now facing today . now , several open chemistry and chemoinformatics projects ( table 1 ) have pooled forces to enhance interoperability between these tools in a movement we call the blue obelisk the name originates from an informal meeting place in san diego , california , during the american chemical society 2005 spring national meeting ( see figure 1 ) and was coined by one of the authors . the blue obelisk in san diego , california , at the 2005 american chemical society meeting.table 1current blue obelisk projectsprojecturlprincipal authors cml , jumbohttp://cml.sf.net / p.m .- r . the blue obelisk in san diego , california , at the 2005 american chemical society meeting . current blue obelisk projects we identify three core areas for the blue obelisk movement : one can use other people 's code without further permission , including changing it for one 's own use and distributing it again . one can obtain all data in the public domain when wanted and reuse it for whatever purpose . instead , the three points focus on access to the scientific data , algorithms , and implementations themselves , rather than the formatted manuscript . it is well - known in software development that 80% of the costs are caused by maintaining software and not by the initial implementation . this holds both for the in - house development in pharmaceutical companies and the development for commercial chemoinformatics suppliers . openly standardized algorithms and chemical information can help to reduce the maintenance costs , because developers can reuse available modules or test their tools against open source software and open data . we agree with de lano that the try - before - buy paradigm for open source software does not necessarily require open standards . open specifications for standard algorithms such as kekulization , chirality coding , and atom typing , however , are indispensable in academic chemoinformatics research to build better , more stable , and more reproducible chemical information systems . in this contribution , we outline several examples for how the blue obelisk projects address this need : a shared dictionary of algorithms and implementations in chemoinformatics algorithms drawing from our various software projects and a shared repository of chemoinformatics data including elemental properties , atomic radii , isotopes , atom typing rules , a set of web - based chemoinformatics services , and the process of providing open algorithms and data . all of these projects were developed with continual community involvement , an open standardization process , and provide open data to key chemoinformatics processes . anyone can take part ; we welcome those in commercial organizations , academia , government , and so forth , and contributions come as code , compilations of data and molecules , testing , and more . a semantic web , and even more a semantic grid , is predicated on the supply of information and services without requiring the user to know the details of how the resource was obtained . for example , the calculation of a molecular property might depend on a precise method but should not , in principle , depend on the actual program used , its version , the operating system , and the machine involved . for example , molecular weight is an imprecise term , and the result of an algorithm returning this can not be regarded as precise . however , atomic masses include the concept of average as in relative atomic mass ( atomic weight ) , ar : the ratio of the average mass of the atom to the unified atomic mass unit . there are at least two algorithms that could be used to obtain the molecular mass : sum the average masses of all the atoms in the molecule ( the normal molecular weight ) sum the precise masses of the most frequent isotopes in the molecule ( giving the high - resolution molecular mass ) . this is simple , in principle : computers should run reliably , and if the same data are given to the same algorithm , identical results should be obtained . there is relatively little practice of public validation of data resources and certification of algorithms in the field of chemistry , but without this , a global chemical semantic web is difficult to implement . we hope that , in the long - term , appropriate bodies such as iupac and other learned societies might come to oversee this practice ; until then , the blue obelisk can be seen as an informal , neutral mechanism to which those interested in open semantics can contribute . an interoperable chemical approach requires at least the following communally agreed upon components in its architecture ( in no particular order ) : terminology , data typing , extensible data structures , conformance specification and tools , links and references , namespaces , and metadata for provenance and discoverability . this article is largely concerned with how the semantic containers for terminology , data , and algorithms are populated . for the past three decades , the iucr , through its data commission and other bodies , has actively developed communal practice for the interchange of data . has been associated with the committee for the maintenance of the cif standard ( comcifs ) project for a decade . the crystallographic information file ( cif ) is the latest design of the iucr 's semantically rich data structures and is fully described in this journal and the recently published volume g of the int the primary approach is through dictionaries , each of which can describe a subdomain ( e.g. the dictionaries are similarly constrained by a dictionary definition language ( ddl ) which is also recursively conformant . ddl and cif predated xml by a decade and are almost isomorphic to xml schema ( xsd ) and xml in their architecture . this architecture can reasonably be considered an ontology for the hard sciences . description.text ( within ; ... ;) is the human - readable meaning , where there are references to other dictionary items . in this loop , a piece of code , based on python and extended in the drel language , describes the precise algorithm for the evaluation of the atomic mass of the cell . it defines a mass , initially zero , and a list of atom_types in the data object ( the cif ) . the code can be run directly as python , in java through jython , and compiled into other languages through the jjtree compiler . note that the approach also contains a precise identification of , and therefore retrieval of , algorithms . the blue obelisk chemoinformatics dictionary is our effort of defining a standard set of chemoinformatics algorithms . by using unique identifiers , the dictionary allows using web search engines , like google.com , to find implementations for an algorithm in the dictionary . the dictionary uses the following technologies : scientific , technical , and medical markup language ( stmml ; http://www.xml-cml.org/stmml/ ) is used as a general container , and mathematical markup language ( mathml ) is used to contain mathematical formulas . the full source of the latest xml source for the dictionary can be retrieved from ref ( 50400bb00021 ) . each entry in the dictionary has an associated identifier ( i d ) , which is unique throughout the xml document . when xml namespace technologies are used , a worldwide unique identifier can be composed that uniquely points to the entry in the dictionary . chart 2 is an example of an entry currently used in the blue obelisk dictionaries . additionally , a classification is made ( into the area of graph theory ) , and a related entry is mentioned.chart 2example of an xml dictionary entry example of an xml dictionary entry extensible stylesheet language transformation ( xslt ) is used to transform the xml source code into an xhtml document which can be displayed by a mathml - aware web browser , like mozilla firefox . the blue obelisk movement agreed on using the same namespace prefix , that is , blue - obelisk , allowing web pages for specific software projects to cite entries in the dictionary . links from those pages currently must be made explicitly , but having the citations on those pages allows a web search engine to easily find software projects that implement a specific algorithm . this setup provides a powerful tool to find software that implements published algorithms.figure 2screen shot of the xhtml output of the blue obelisk chemoinformatics dictionary showing the search implementations on google.com feature . screen shot of the xhtml output of the blue obelisk chemoinformatics dictionary showing the search implementations on google.com feature . at the time of writing , cdk and jmol each provide a web page that cites and links to individual blue obelisk chemoinformatics dictionary entries . the open babel project has also included links to the dictionary in its developer documentation and is in the process of producing a complete index of entries as a separate web page . because many chemoinformatics projects rely on accurate atomic and molecular data such as atomic masses , isotopes , electronegativities , van der waals radii , covalent radii , and so on , we have initiated a repository of a standard set of chemoinformatics data , building on the processes involved in the dictionary mentioned above . conventional standards bodies , such as iupac , have established a variety of published data , particularly on isotopes , atomic masses , elemental abundances , element symbols and names , and so on . many chemoinformatics algorithms , however , rely on other data which may not have a clear - cut definition . for example , there is no obvious way to specify a van der waals radius not all elements are perfectly spherical , and multiple definitions exist including those taken from crystal structures , gas - phase measurements , and molecular mechanics force fields . it is anticipated that , over the next year , the repository will considerably increase in the amount of available data.table 2current content of the data repository , with a few of the used sources.property typepropertysourcesphysical propertiesisotope abundances isotope masses31 atomic masses32 ionization energies chemical propertiesaffinities radii33 electronegativities element densities discoveryyear of discovery name and etymology otheratom type definitions 2d and 3d coloring schemes current content of the data repository , with a few of the used sources . the repository uses cml and dictionaries to allow the explicit markup of data types , units , and the experimental errors , as well as metadata like bibliographic sources , creation dates , and indications of authority . an example entry in the blue obelisk data repository for example , it states that the ionization energy is 13.5984 ev and that the mass is 1.007 94 amu . it does not explicitly state which mass is meant but refers , for the definition , to the blue obelisk dictionary ( see section 3).chart 3example of a blue obelisk data repository entry example of a blue obelisk data repository entry the preceding material has described how chemoinformatics data can be managed and accessed in a collaborative manner . another aspect of collaboration is the use of distributed functionality , that is , the use of function implementations that are not necessarily on the local machine . an example of this type of approach is the use of web services . in the context of chemoinformatics , this means that a programmer can access functions , which , for example , calculate binary fingerprints , over the internet without having to understand what language the underlying function is written in or whether the function is up - to - date . of course , this implies that the calling mechanism for the given function is well - defined and that the maintainer has kept it up - to - date . we have used the cdk to provide web services for molecular similarity and descriptor calculations , available at http://blue.chem.psu.edu/rajarshi/code/java/cdkws.html . access to these services can be programmatic ( using the soap protocol ) or by a web - based interface which simply calls the service and presents the results . furthermore , with the advent of open source and open data , a user is free to investigate the inner workings of a web service if he or she so wishes . we believe that the open principles underlying the blue obelisk movement are conducive to the development of transparent web services which provide easy access to a variety of functionalities in a distributed manner . it has been mentioned previously that the blue obelisk movement is a communal effort . in this sense , the blue obelisk movement characterizes the nature of open source development in general and serves as an example of how this mode of development can be applied to problems in the field of chemical algorithms , standards , and data . a striking feature of the blue obelisk movement is the wide variety of contributors to the individual projects that make up the movement . however , it should be understood that , though a bug report may appear to be a minor contribution compared to a whole framework , each contribution plays a vital role in the communal development and peer review of these projects . at the same time , it is important to realize that open source efforts represented by the blue obelisk movement do not always involve renumeration . thus , compared to commercial projects , it may appear that the projects represented by the blue obelisk movement lack in certain areas . this is an important question because the contributors to the blue obelisk projects are located all over the world . mailing lists also serve as archives of discussion , in addition to the use of traditional web pages and collaborative web pages ( wiki ) for the development of documentation . a more real - time mode of communication is the use of internet relay chat , which allows multiple people to convene in a virtual room and communicate in real time . in general , this is restricted to text , but current instant messaging services allow for the use of both audio- and video - based communication . once again , this is a very important question because multiple people will be working on a program or document , and manually managing individual contributions does not scale for projects of even moderate size . workflows and web services can also be used in the development process , and the utility of such types of applications has been mentioned previously . many of the blue obelisk projects make use of services provided by sourceforge.net , which is a community effort to provide open source projects with a set of tools and functionality for efficient code maintenance and communication . the site supports a number of features such as cvs , mailing lists , bug trackers , and so on , all of which are freely available to open source projects . clearly , current internet - based technology allows for easy and efficient management of contributions to the various blue obelisk projects from contributors located all over the world . in a sentence , the blue obelisk movement is an example of the use of open source technology and methods to customize tools and social practices for the development of chemical information services . we have described a communal effort to realize interoperability in chemical informatics , which we call the blue obelisk movement , named after the first meeting place of our community . the bo movement currently consists of more than 10 open source and open data projects all related to chemoinformatics . we identify concepts and algorithms , codify them in a collaborative dictionary , and link them to concrete implementations in blue obelisk projects and beyond to make them machine - searchable . we have started a public repository of chemical data of general interest , including data for chemical elements and isotopes ( boiling points , colors , electron affinities , masses , covalent radii , etc . ) , definitions of atom types , and more . all of the data is augmented with documentation , citations of origin , and a bibliography . we are working on a system of web services to provide access to chemoinformatics functionality without the knowledge of the details of the individual implementation and without the need to master the installation and programming interface of yet another chemoinformatics library . we emphasize that this work in progress , because of its emphasis on interoperability , has a value beyond that of open source and open data efforts . while standardization efforts in chemistry have a long history , modern computing and data processing , the internet , and the world wide web have , for the first time , created the possibility of effortlessly searchable and reusable data and computer programs . this article is also addressed to newcomers asking them to adopt the ideas of open data and software from the very beginning .

the aryl hydrocarbon receptor ( ahr ) is an evolutionarily ancient protein , which first appeared within the vertebrate class 450 million years ago . although historically most frequently studied in the context of regulating responsiveness to environmental toxicants , both the existence of ahr prior to industrialization and the presence of naturally occurring ligands provide evidence for its integral importance to animal physiology.1,2 ahr can be activated through binding an array of diverse ligands that can be endogenous , naturally occurring , and/or anthropogenic.35 ahr activation by environmental pollutants , leading to its dimerization with aryl hydrocarbon nuclear transporter ( arnt ) and subsequent activation of xenobiotic metabolizing enzymes such as cytochrome p4501a1 ( cyp1a1 ) , has been a primary focus of toxicology studies for decades . industrially produced ahr ligands include halogenated aromatic hydrocarbons , which are toxic chemical contaminants of the global ecosystem produced as byproducts of pesticide production , bleaching , and combustion processes . dioxins are members of the polyhalogenated aromatic hydrocarbon family , consisting of two benzene rings connected by two oxygen atoms , with four to eight chlorine atoms added . contamination with dioxin and dioxin - like compounds is widespread throughout the biosphere , including air , water , fish , and mammals.6 the entire human population , especially those living in industrialized countries , is exposed to these toxicants that act as potent , long - acting agonists for ahr.7 although the most toxic of these man - made compounds can enter through the skin or lungs , food is the most common source , and , once internalized , these fat - soluble compounds bioaccumulate in the food chain.810 ahr is highly expressed in tissues that are exposed to the environment , including the gut , lungs , and skin . as a biological sensor , ahr is structured to respond to chemical changes in the environment . the ubiquitous presence of ligands suggests a long - standing role in responding to the components of foreign materials , including substances that are inhaled , ingested , or in contact with skin . thus , it seems a logical candidate not only to regulate responses to toxic substances but also to act as an integrator of energy metabolism . other exogenous ahr ligands are naturally occurring components of food and herbal medicines , including indole metabolites , stilbenes , carotenoids , and flavonoids.1113 the indole derivatives indole-3-carbinol ( i3c ) and it metabolite 3,3-diindolylmethane ( dim ) are components of cruciferous vegetables , including broccoli and cabbage . carotenoids , the yellow , orange , and red pigments produced by plants , are present in a variety of vegetables . although considered less toxic , these lower affinity ligands are capable of activating ahr - dependent signaling . the array of potential dietary ligands suggests that ahr activation may be commonplace and physiological . germline ahr knockouts produced by three separate laboratories have demonstrated the importance of ahr for the physiological regulation of growth , fertility , and liver and cardiovascular development , as well as the expected decreased sensitivity to dioxin exposure.14 more recently , tissue - specific deletion models indicate that ahr is an important regulator of hepatic energy homeostasis , adipocyte and dendritic cell function , and cerebellar granule cell development.1517 although knockout studies are frequently cited as evidence for the activation of ahr in the absence of exogenous ligands , the range of molecules derived from food sources outlined above calls this idea into question . nevertheless , knockout studies also prompted the search for endogenous ligands , and several have been identified . for example , ultraviolet light exposure produces tryptophan derivatives in the skin and liver in rodents and humans that bind ahr with high affinity.18,19 activation of ahr by these compounds , although transient due to their rapid metabolism , potentially links ahr activity to daily oscillations in environmental illumination and to regulation of the circadian clock.2022 a cocktail of tryptophan photo - products or the specific photoproduct 6-formylindolo[3,2-b ] carbazole ( ficz ) activates ahr and alters expression patterns of circadian clock genes in vivo in the liver , as well as in a cell line derived from the rat suprachiasmatic nucleus ( scn ) , the brain s master clock.14 these data highlight the potential physiological functions of endogenous agonists , and demonstrate that ahr can interact with the circadian clock . in the absence of a ligand , the inactive ahr is bound by a group of chaperone proteins that include heat shock protein 90 ( hsp-90 ) and the ahr - interacting protein comprised pas - a and prostaglandin e synthase ( p23 ) , and localizes within the cellular cytoplasm . pas - b regions act as interactive surfaces for heterodimer and homodimer formation and function as the ligand - binding surface . lipophilic ahr agonists enter the cell and bind specifically to the pas - b region of the cytoplasmic ahr . ligand binding causes a conformational change that exposes a nuclear localization sequence , and after phosphorylation by protein kinase c , the ligand - bound ahr complex subsequently translocates into the nucleus.2325 in the nucleus , the pas - a domain of ahr dimerizes with the aryl hydrocarbon receptor nuclear translocator ( arnt ) through its pas domain , and this heterodimer binds specific dna sequences , identified as xenobiotic response elements within the promoters of target genes . classical ahr target genes include phase i metabolizing enzymes , cytochrome p450 , family 1 , member 1a ( cyp1a1 ) , cytochrome p450 , family 1 , member 2a ( cyp1a2 ) , cytochrome p450 , family 1 , sub family b ( cyp1b1 ) , phase ii metabolizing enzymes , and ahr repressor , although many other genes may also be regulated by ahr / arnt.26 classical ahr signaling does not , however , account for all the cellular effects attributed to the activated ahr . ahr signaling may be influenced by the affinity of the ligand for the ahr , as well as by cell - type - specific properties and other environmental factors . non - canonical signaling through ahr includes crosstalk with other nuclear receptors , regulation of cell cycle and map kinase cascades , modulation of the immune system , activation of immediate early genes , and interaction with the molecular circadian clock . crosstalk between ahr and estrogen receptor ( er ) signaling is perhaps the most well studied among the nuclear receptor interactions . proposed mechanisms include direct binding of liganded ahr to er , competition between ahr and er for the same transcriptional co - activators and co - repressors , enhanced estrogen metabolism resulting from inductions of phase i metabolizing enzymes , and proteosomal targeting of er for degradation ( reviewed in the studies by shanle and xu27 and swedenborg and pongratz28 ) . although ahr activation is most commonly anti - estrogenic , some ahr ligands are weakly estrogenic and the effects are context - dependent.27 ahr crosstalk with signaling pathways critical to cell - cycle progression highlights the involvement of ahr in tumorigenesis . the complex interface with the cell cycle is not completely delineated , but is dependent on protein protein interactions with the retinoblastoma tumor suppressor protein ( rb ) . this heterodimer acts transcriptionally to regulate both co - activation and co - repression during the g1 phase of the cell cycle . ahr also induces the p27 kip1 cyclin / cdk inhibitor and the cyclin - dependent kinase inhibitor p21 . similarly , ahr is also an important regulator of immune cell compartments , especially in inflammation and autoimmunity.29 clearly , ahr is a versatile receptor with a unique ability to sense chemical change in the environment . how it relates that chemical change into physiological adaptation remains a major topic for discovery . the perpetual existence of daily oscillations of the 24-hour light / dark cycle has led to an integration of environmental diurnality with physiological function . the endogenous circadian clock that drives internal timing synchronizes with the cyclic changes of the external environment to regulate processes such as the sleep wake cycle , locomotion , feeding , and temperature such that they coordinately function at the appropriate time of day.30,31 in mammals , the central oscillator resides in a region of the basal hypothalamus called scn and receives and relays information from the external environment.30 circadian rhythms are generated by core clock genes that form transcriptional and translational feedback loops , controlling their own mrna and protein levels . specifically , the pas - containing proteins clock and bmal1 form a heterodimer and bind to enhancer - box ( ebox ) regions upstream of the pas domain containing clock genes , most notably the period and cryptochrome ( cry ) families.32,33 increased levels of per and cry form heterodimers that feed back to inhibit clock and bmal1 directed transcription . posttranslational mechanisms of casein kinase 1 delta and 1 epsilon phosphorylate per proteins and target them for polyubiquitination and degradation , releasing the inhibition of clock and bmal1 , thereby maintaining the 24-hour cycle of clock genes.34 although the circadian clock is self - regulating , it is capable of sensing and adapting to change . similar to ahr - dependent mechanisms that sense xenobiotics through pas regions , the circadian clock uses clock , bmal1 , and per , pas domain - containing proteins to regulate the circadian period . pas domains , comprised of two 70 amino acid repeats identified as pas a and pas b , allow these proteins to function as environmental sensors and communicate downstream signals through gene transcription.3,35 ahr and arnt are members of the same protein family , both containing basic helix loop helix ( bhlh ) domains , composed of two helices with an inner loop sequence , and pas structural motifs . the bhlh - pas proteins are a subfamily of the bhlh family.3 the pas domain facilitates heterodimer and homodimer formation among family members.35 pas - domain - containing proteins are promiscuous in both their ligand binding and the formation of heterodimers , which allows for varied combinations of protein protein interactions and crosstalk among intracellular signaling pathways . this promiscuity suggests that ahr can interact with other pas - containing proteins outside its established canonical pathway , enhancing the possibility of activation of myriad signaling pathways.36 following agonist - induced activation , ahr forms a heterodimer with the protein arnt , which has a sequence homology similar to the clock protein bmal1 , including similar intron / exon splice patterns and conservation of five exons that compose the pas domain.37 after ligand binding , the activated ahr enters the nucleus , where it can also form a heterodimer with bmal1 in cultured hepatoma cells as well as in the ovary.38,39 this ahr / bmal1 heterodimer disrupts the normal clock / bmal1 activation of per1 on the per1 promoter , resulting in the inhibition of per1 transcription and dampened per1 rhythm in liver ( fig . 1).39,40 furthermore , per1 and bmal1 rhythms are altered in the scn of mice exposed to 2,3,7,8-tetrachlorodibenzodioxin ( tcdd).41 ahr is widely expressed in the central nervous system , including the hypo - thalamic scn , the central circadian clock.42 following intravenous injection of tcdd in rats , low levels of the dioxin are distributed in the brain , and the ahr target genes are upregulated.43,44 ahr activation in mice and hamsters alters rhythms of feeding and activity , gene expression , and the hormones prolactin , corticosterone , and melatonin.4551 human exposure to pesticides , which contain potent ahr agonists , increases the risk for idiopathic rapid eye movement sleep behavior disorder , further establishing a link between ahr , sleep , and circadian rhythms.52 since ahr and arnt are expressed within hypothalamic nuclei that regulate circadian rhythmicity , feeding behavior , and hormone secretion , and ahr activation alters gene expression in the hypothalamus , ahr - dependent mechanisms should be further explored.41,42,48,50 activation of ahr alters the expression patterns of circadian clock genes and suppresses circadian rhythms . reciprocally , genetic alteration of the circadian clock influences ahr signaling and sensitivity to agonist - induced activation of ahr and ahr target genes.40,5355 rhythmic expression of ahr mrna and protein levels is regulated by an ebox dna - binding region within the ahr promoter , influencing rhythmic control of ahr expression through clock / bmal1-induced transcription.5659 ahr is rhythmically expressed in the scn and peripheral tissues.41 ahr target gene expression of cyp1a1 , cyp1b1 , and arnt , although expressed at low levels under basal conditions , has a diurnal expression pattern with a peak that occurs during the day.41,57,58,60 in response to agonist exposure , cyp1a1 , a key target gene and indicator of ahr activation , has a rhythmic sensitivity that peaks during the night . however , in per1/2 mutant mice , rhythmic sensitivity to agonist - induced cyp1a1 expression is abolished , and overall cyp1a1 levels are enhanced.5355 additionally , rhythmic oscillation of ahr mrna and time - dependent sensitivity of target gene upregulation are absent in clock mutant mice.61 rhythmic expression of ahr and ahr target genes under basal conditions and time - dependent variations in sensitivity of ahr activation imply that the circadian system influences both physiological and exogenous ahr mechanisms . overall , the circadian clock acts as a check on ahr sensitivity to ligands . without an intact circadian clock , rhythmic expression of ahr is abolished , and , not surprisingly , the rhythm in sensitivity of the ahr to ligand - induced activation is also suppressed . however , without per1 , overall levels of cyp1a1 are enhanced in response to agonist treatment . an examination of rhythmic expression patterns suggests that highest levels of ahr , with likely highest levels of sensitivity to agonist activation , occur when per levels are near their trough . thus , per may act to suppress ahr protein , likely through an influence on clock : bmal1 transcriptional activity ( fig . conversely , ahr also influences circadian integrity . under control conditions , mice exposed to a 30-minute light pulse early during the lights - off period perceive this nocturnal light exposure as an extension of the lights - on period . in response , the circadian clock is delayed on subsequent days , which can be observed as an activity onset that occurs later than predicted on the following days . however , this light - induced phase delay in activity onset is severely attenuated in mice treated with either tcdd or -napthoflavone ( bnf ) , both ahr agonists.41 this effect holds when ahr agonists are applied to scn - containing brain slices in vitro . application of the neurotransmitter glutamate to scn - containing brain slices mimics the effects of light , causing the same phase delay described above . pretreatment of scn slices with the ahr agonist ficz blocks glutamate - induced phase resetting of the scn electrical activity rhythm.22 these data suggest that ahr activation directly within the scn impacts the ability of the clock to respond to phase - resetting stimuli . in addition to behavioral and electrical activity changes , circadian clock genes per1 and bmal1 in liver and scn are altered in response to ahr agonists.40,41,62 ahr activation with bnf attenuates light - induced induction of per1 in the scn , providing an essential mechanism for the effects of ahr activation on light - induced changes in behavioral rhythmicity . furthermore , activation of the ahr by ficz in the scn cell line scn2.2 alters the expression of the clock genes per1 , cry1 and cry2.22 collectively , the data suggest that ahr expression , even under basal conditions , influences circadian rhythm strength . overall , ahr expression and activation dampens the response of the circadian clock to perturbations in light and dampens the rhythmicity of clock gene rhythms . in contrast , when ahr is deleted , there is a tendency for the clock to have enhanced responsiveness to light at night and for increased amplitude of the per1 rhythm.39,41,63 further experimentation in ahr - null mice is needed for confirmation . generally , the aggregate data suggest that ahr may act as a gain control on the circadian clock ; activation of ahr suppresses rhythm amplitude , whereas inhibition of ahr strengthens rhythm amplitude ( fig . obesity and type 2 diabetes continue to burden society as a result of lifestyle choices , chemical exposures , genetics , and other contributing factors . in 2011 , the world health organization predicted that 347 million people suffer from type 2 diabetes.64 both circadian disruption and dioxin exposure promote metabolic dysfunction through parallel and overlapping mechanisms . epidemiological evidence links circadian disruption , by shift work , to higher body mass index , increased triglycerides , glucose dysregulation , obesity , and higher incidence of metabolic syndrome , defined by the national cholesterol education program as the presence of three or more of the following criteria : high waist circumference , blood pressure , fasting triglyceride levels , fasting high - density lipoprotein , or high fasting glucose levels.6568 mouse models of circadian disruption produce altered rhythms in core circadian genes as well as clock - controlled genes to include numerous rate - limiting metabolic genes , disrupted rhythms in lipid and glucose metabolism , and hepatic ste - atosis.69,70 ahr status influences circadian rhythm strength , even under controlled conditions . rhythms of the clock genes per1 and bmal1 are significantly enhanced in the liver of ahr mice compared to wild - type mice.41 genetic manipulation to reduce ahr expression in mice enhances the ability of mice to adjust their behavior in response to an alteration in the timing of the light / dark cycle ( jaeger and tischkau , unpublished results , april 2016 ) . together , these data suggest that the circadian timing system is more robust , both in terms of amplitude of oscillation and in adaptability to environmental change , when the ahr is inhibited or reduced . toxic responses to dioxins are mediated through the ligand - activated transcription factor ahr and upregulation of target genes that regulate xenobiotic metabolism.60,71 chronic low - level exposure to dioxins is linked to insulin resistance and development of type 2 diabetes . activation of ahr alters glucose metabolism , glucose tolerance , and insulin levels , and increases the risk of diabetes mellitus.7276 ahr activation specifically within adipose tissue promotes inflammation and impairs glucose and insulin tolerance.16,7779 additionally , signaling downstream of tumor necrosis factor nuclear factor - k , which decreases glucose transporter type 4 expression and contributes to insulin resistance , is increased in adipose tissue of vietnam veterans exposed to dioxins.80,81 ahr activation alters hepatic genes involved in fatty acid , lipid , and cholesterol metabolism pathways , including the peroxisome proliferator - activated receptor ( ppar ) pathway , all of which mediate glucose and lipid homeostasis.62,82,83 additionally , ahr activation induces an inflammatory response , which is implicated in the pathogenesis of metabolic disorders.78,84 this evidence points to a clear interaction between activated ahr and systemic energy metabolism . it seems likely that ahr plays a role in maintaining physiological balance in energy metabolism and that constant pathological activation of ahr upsets this balance , thereby contributing to metabolic disease . perhaps most importantly , mechanisms that underlie physiological regulation of metabolism by ahr provide an opportunity to increase our understanding of how the body reacts to environmental stimulation . around 90% of human exposure to dioxins occurs through our diet , primarily through animal fat consumption.8 besides tcdd and toxic pollutants , many compounds that influence ahr activity exist as natural components of fruits and vegetables.11,85,86 additionally , the arachidonic acid metabolite , lipoxin a4 , prostaglandins , specifically prostaglandin g2 , and an arachidonic acid metabolite produced in inflammatory disease conditions , namely , 12(r)-hydroxy-5(z),8(z),10e , 14(z)-dicosatetraenoic acid ( 12(r)-hete ) , can act as ahr agonists.8789 lipid and lipid derivatives , such as oxidized low - density lipoproteins ( oxldl ) , have been identified as ahr agonists.90 oxldl and saturated fatty acids contained in a western diet activate ahr and contribute to obesity and inflammation in c57bl/6 j mice.91 ahr activation also occurs through exposure to flavonoids and metabolites from fruits and vegetables , indole-3-carbinol , and herbal supplements including ginseng.12,92,93 additionally , the consumption of fish and shrimp , which bioaccumulate organohalogens , increases potential dietary exposure.94 with a wide variety of ahr agonists present within food , or present due to secondary effects of diets that increase inflammatory mediators , exposure to ahr activation and its consequences are prevalent . rather than focusing on the contribution of a single ahr ligand to ahr activation and downstream sequelae , it may be more important to consider the overall activation of ahr in response to the constellation of ligands , in terms of the overall metabolic consequences . mice lacking ahr expression have enhanced insulin sensitivity and improved glucose tolerance , although improved metabolism may differ depending on age , sex , housing , diet , and possible genetic drift.63,9597 mice with a low - affinity ahr allele fed a high - fat diet ( hfd ) are less susceptible to obesity , exhibiting differences in fat mass , liver physiology , and liver gene expression compared to mice with high - affinity ahr.98 ahr and ahr mice are resistant to the harmful effects of diet - induced obesity through protection against hepatic steatosis , insulin resistance , and inflammation . ahr - deficient mice fed an hfd have enhanced energy expenditure resulting from increased brown adipose tissue activity compared to ahr mice fed an hfd.99 hfd consumption disrupts circadian rhythms by altering circadian regulation of gene expression through inhibition of clock / bmal1 recruitment to chromatin , resulting in lost oscillation or phase advances of genes . a large number of metabolites in carbohydrate , lipid , metabolic , and xenobiotic pathways are regulated by clock / bmal1 binding to their promoter regions.100,101 genes that lose rhythmic expression following hfd consumption often peak during the time period when clock / bmal1 is recruited to chromatin , which also coincides with peak the expression of ahr.41,102,103 persistent activation of ahr alters circadian rhythm , primarily through inhibition of clock / bmal1-mediated target genes.3941 therefore , ahr - induced disruption of clock / bmal1 activity may play a role in hfd - induced disruption of metabolic rhythms . furthermore , even deletion of a single ahr allele ( ahr ) protects mice against diet - induced changes in transcriptional oscillations and also against glucose and metabolic gene rhythm disruption ( jaeger and tischkau , unpublished results , april 2016 ) . these studies highlight the involvement of ahr and the clock in the regulation of energy metabolism and provide interesting opportunities to explore novel therapies to combat poor metabolic health . ahr is widely expressed throughout the body.104,105 peripheral ahr activation and interaction with circadian signaling in metabolic tissues such as liver , adipose , and muscle has the potential to alter metabolism , but further studies are required to elucidate their detailed mechanisms . to study ahr signaling in vitro , use of the mouse hepa-1c1c7 cell line ( high ahr expression ) and the hepa-1c1c7-derived hepa-1c1c12 ( low ahr expression ) allows comparison between varied levels of ahr expression.40 in addition to liver - derived hepa-1c1c7 cells , cell lines derived from lung epithelia , keratinocytes , and fibroblasts can be used to study ahr signaling.106 in dispersed cells , however , the circadian clock frequently becomes desynchronized because of the lack of synchronizing signals provided in vivo by the scn , the endocrine and autonomic nervous systems , and diet . short exposure to high concentrations of serum ( serum shock ) can provide a synchronizing stimulus for circadian gene expression in mammalian tissue cultures . the mechanism of serum shock is hypothesized to mimic light - induced immediate early genes and synchronize circadian cycles , inherent within individual cells , to the population of cells in the dish.107 thus , knowledge of the circadian system and its behavior in cultured cells , together with cell lines that differentially express the ahr , provide unique , yet underutilized opportunities to study the interaction between circadian rhythms and ahr signaling . in addition to sharing pas domain structure with circadian proteins , ahr demonstrates a rhythmic expression of transcription and sensitivity to activation by the ahr agonist tcdd.41,57,58 reciprocally , ahr activation influences rhythmic strength of circadian clock genes , hormones , and behavioral responses to light - induced phase shifts.41,47,50 correlations between ahr variants in mouse models and humans allow us to study how altered ahr affinity affects downstream signaling pathways.108 epidemiological studies that explore the link between ahr polymorphisms and obesity may provide important information regarding the influence of ahr on physiological metabolism . decreasing ahr levels in mice by either knocking it out completely ( ahr ) or knocking it down by at least 50% ( ahr ) seems to enhance rhythms and bolster metabolism . in addition , the broad ligand - binding capacity of ahr suggests that manipulation of the receptor by an antagonist may promote healthier metabolism . the dietary component curcumin , a natural phenol found in spices , and resveratrol , a natural phenol found in red wine , along with synthetic compounds ch-223191 , 6,2,4-trimethoxyflavone , and gnf351 , have been reported as ahr antagonists.109114 given its importance in xenobiotic metabolism , complete blockade of ahr may not be desirable . thus , partial inhibition of ahr , or specific inhibition of ahr - induced effects on clock / bmal1 and nuclear receptors ppar and ppar and their downstream target genes that regulate glucose metabolism , may provide benefits while minimizing undesirable effects . it is becoming more evident that multiple mechanisms of ahr activation and downstream interaction with circadian rhythms and metabolism exist , but not all ahr - dependent consequences can be explained through dioxin response element ( dre)-dependent mechanisms . understanding the variety of mechanisms by which ahr exerts its effects on metabolism may reveal new targets of interest for the conservation of homeostasis and reinforces the significance of ahr in clock disruption and metabolic disorder .

the prevalence of metabolic syndrome , a clustering of three or more risk factors that include abdominal obesity , increased blood pressure , and high levels of glucose , triglycerides , and high - density lipoproteins , has reached dangerous and costly levels worldwide . increases in morbidity and mortality result from a combination of factors that promote altered glucose metabolism , insulin resistance , and metabolic dysfunction . although diet and exercise are commonly touted as important determinants in the development of metabolic dysfunction , other environmental factors , including circadian clock disruption and activation of the aryl hydrocarbon receptor ( ahr ) by dietary or other environmental sources , must also be considered . ahr binds a range of ligands , which prompts protein protein interactions with other per - arnt - sim ( pas)-domain - containing proteins and subsequent transcriptional activity . this review focuses on the reciprocal crosstalk between the activated ahr and the molecular circadian clock . ahr exhibits a rhythmic expression and time - dependent sensitivity to activation by ahr agonists . conversely , ahr activation influences the amplitude and phase of expression of circadian clock genes , hormones , and the behavioral responses of the clock system to changes in environmental illumination . both the clock and ahr status and activation play significant and underappreciated roles in metabolic homeostasis . this review highlights the state of knowledge regarding how ahr may act together with the circadian clock to influence energy metabolism . understanding the variety of ahr - dependent mechanisms , including its interactions with the circadian timing system that promote metabolic dysfunction , reveals new targets of interest for maintenance of healthy metabolism .

AhR Ligands AhR Activation and Signaling AhR and the Circadian Clock AhR and the Circadian Clock in Metabolic Disruption Summary and Future Studies

the aryl hydrocarbon receptor ( ahr ) is an evolutionarily ancient protein , which first appeared within the vertebrate class 450 million years ago . although historically most frequently studied in the context of regulating responsiveness to environmental toxicants , both the existence of ahr prior to industrialization and the presence of naturally occurring ligands provide evidence for its integral importance to animal physiology.1,2 ahr can be activated through binding an array of diverse ligands that can be endogenous , naturally occurring , and/or anthropogenic.35 ahr activation by environmental pollutants , leading to its dimerization with aryl hydrocarbon nuclear transporter ( arnt ) and subsequent activation of xenobiotic metabolizing enzymes such as cytochrome p4501a1 ( cyp1a1 ) , has been a primary focus of toxicology studies for decades . germline ahr knockouts produced by three separate laboratories have demonstrated the importance of ahr for the physiological regulation of growth , fertility , and liver and cardiovascular development , as well as the expected decreased sensitivity to dioxin exposure.14 more recently , tissue - specific deletion models indicate that ahr is an important regulator of hepatic energy homeostasis , adipocyte and dendritic cell function , and cerebellar granule cell development.1517 although knockout studies are frequently cited as evidence for the activation of ahr in the absence of exogenous ligands , the range of molecules derived from food sources outlined above calls this idea into question . for example , ultraviolet light exposure produces tryptophan derivatives in the skin and liver in rodents and humans that bind ahr with high affinity.18,19 activation of ahr by these compounds , although transient due to their rapid metabolism , potentially links ahr activity to daily oscillations in environmental illumination and to regulation of the circadian clock.2022 a cocktail of tryptophan photo - products or the specific photoproduct 6-formylindolo[3,2-b ] carbazole ( ficz ) activates ahr and alters expression patterns of circadian clock genes in vivo in the liver , as well as in a cell line derived from the rat suprachiasmatic nucleus ( scn ) , the brain s master clock.14 these data highlight the potential physiological functions of endogenous agonists , and demonstrate that ahr can interact with the circadian clock . in the absence of a ligand , the inactive ahr is bound by a group of chaperone proteins that include heat shock protein 90 ( hsp-90 ) and the ahr - interacting protein comprised pas - a and prostaglandin e synthase ( p23 ) , and localizes within the cellular cytoplasm . ligand binding causes a conformational change that exposes a nuclear localization sequence , and after phosphorylation by protein kinase c , the ligand - bound ahr complex subsequently translocates into the nucleus.2325 in the nucleus , the pas - a domain of ahr dimerizes with the aryl hydrocarbon receptor nuclear translocator ( arnt ) through its pas domain , and this heterodimer binds specific dna sequences , identified as xenobiotic response elements within the promoters of target genes . classical ahr target genes include phase i metabolizing enzymes , cytochrome p450 , family 1 , member 1a ( cyp1a1 ) , cytochrome p450 , family 1 , member 2a ( cyp1a2 ) , cytochrome p450 , family 1 , sub family b ( cyp1b1 ) , phase ii metabolizing enzymes , and ahr repressor , although many other genes may also be regulated by ahr / arnt.26 classical ahr signaling does not , however , account for all the cellular effects attributed to the activated ahr . non - canonical signaling through ahr includes crosstalk with other nuclear receptors , regulation of cell cycle and map kinase cascades , modulation of the immune system , activation of immediate early genes , and interaction with the molecular circadian clock . although ahr activation is most commonly anti - estrogenic , some ahr ligands are weakly estrogenic and the effects are context - dependent.27 ahr crosstalk with signaling pathways critical to cell - cycle progression highlights the involvement of ahr in tumorigenesis . the complex interface with the cell cycle is not completely delineated , but is dependent on protein protein interactions with the retinoblastoma tumor suppressor protein ( rb ) . the endogenous circadian clock that drives internal timing synchronizes with the cyclic changes of the external environment to regulate processes such as the sleep wake cycle , locomotion , feeding , and temperature such that they coordinately function at the appropriate time of day.30,31 in mammals , the central oscillator resides in a region of the basal hypothalamus called scn and receives and relays information from the external environment.30 circadian rhythms are generated by core clock genes that form transcriptional and translational feedback loops , controlling their own mrna and protein levels . specifically , the pas - containing proteins clock and bmal1 form a heterodimer and bind to enhancer - box ( ebox ) regions upstream of the pas domain containing clock genes , most notably the period and cryptochrome ( cry ) families.32,33 increased levels of per and cry form heterodimers that feed back to inhibit clock and bmal1 directed transcription . posttranslational mechanisms of casein kinase 1 delta and 1 epsilon phosphorylate per proteins and target them for polyubiquitination and degradation , releasing the inhibition of clock and bmal1 , thereby maintaining the 24-hour cycle of clock genes.34 although the circadian clock is self - regulating , it is capable of sensing and adapting to change . similar to ahr - dependent mechanisms that sense xenobiotics through pas regions , the circadian clock uses clock , bmal1 , and per , pas domain - containing proteins to regulate the circadian period . the bhlh - pas proteins are a subfamily of the bhlh family.3 the pas domain facilitates heterodimer and homodimer formation among family members.35 pas - domain - containing proteins are promiscuous in both their ligand binding and the formation of heterodimers , which allows for varied combinations of protein protein interactions and crosstalk among intracellular signaling pathways . this promiscuity suggests that ahr can interact with other pas - containing proteins outside its established canonical pathway , enhancing the possibility of activation of myriad signaling pathways.36 following agonist - induced activation , ahr forms a heterodimer with the protein arnt , which has a sequence homology similar to the clock protein bmal1 , including similar intron / exon splice patterns and conservation of five exons that compose the pas domain.37 after ligand binding , the activated ahr enters the nucleus , where it can also form a heterodimer with bmal1 in cultured hepatoma cells as well as in the ovary.38,39 this ahr / bmal1 heterodimer disrupts the normal clock / bmal1 activation of per1 on the per1 promoter , resulting in the inhibition of per1 transcription and dampened per1 rhythm in liver ( fig . 1).39,40 furthermore , per1 and bmal1 rhythms are altered in the scn of mice exposed to 2,3,7,8-tetrachlorodibenzodioxin ( tcdd).41 ahr is widely expressed in the central nervous system , including the hypo - thalamic scn , the central circadian clock.42 following intravenous injection of tcdd in rats , low levels of the dioxin are distributed in the brain , and the ahr target genes are upregulated.43,44 ahr activation in mice and hamsters alters rhythms of feeding and activity , gene expression , and the hormones prolactin , corticosterone , and melatonin.4551 human exposure to pesticides , which contain potent ahr agonists , increases the risk for idiopathic rapid eye movement sleep behavior disorder , further establishing a link between ahr , sleep , and circadian rhythms.52 since ahr and arnt are expressed within hypothalamic nuclei that regulate circadian rhythmicity , feeding behavior , and hormone secretion , and ahr activation alters gene expression in the hypothalamus , ahr - dependent mechanisms should be further explored.41,42,48,50 activation of ahr alters the expression patterns of circadian clock genes and suppresses circadian rhythms . reciprocally , genetic alteration of the circadian clock influences ahr signaling and sensitivity to agonist - induced activation of ahr and ahr target genes.40,5355 rhythmic expression of ahr mrna and protein levels is regulated by an ebox dna - binding region within the ahr promoter , influencing rhythmic control of ahr expression through clock / bmal1-induced transcription.5659 ahr is rhythmically expressed in the scn and peripheral tissues.41 ahr target gene expression of cyp1a1 , cyp1b1 , and arnt , although expressed at low levels under basal conditions , has a diurnal expression pattern with a peak that occurs during the day.41,57,58,60 in response to agonist exposure , cyp1a1 , a key target gene and indicator of ahr activation , has a rhythmic sensitivity that peaks during the night . however , in per1/2 mutant mice , rhythmic sensitivity to agonist - induced cyp1a1 expression is abolished , and overall cyp1a1 levels are enhanced.5355 additionally , rhythmic oscillation of ahr mrna and time - dependent sensitivity of target gene upregulation are absent in clock mutant mice.61 rhythmic expression of ahr and ahr target genes under basal conditions and time - dependent variations in sensitivity of ahr activation imply that the circadian system influences both physiological and exogenous ahr mechanisms . without an intact circadian clock , rhythmic expression of ahr is abolished , and , not surprisingly , the rhythm in sensitivity of the ahr to ligand - induced activation is also suppressed . an examination of rhythmic expression patterns suggests that highest levels of ahr , with likely highest levels of sensitivity to agonist activation , occur when per levels are near their trough . in response , the circadian clock is delayed on subsequent days , which can be observed as an activity onset that occurs later than predicted on the following days . pretreatment of scn slices with the ahr agonist ficz blocks glutamate - induced phase resetting of the scn electrical activity rhythm.22 these data suggest that ahr activation directly within the scn impacts the ability of the clock to respond to phase - resetting stimuli . in addition to behavioral and electrical activity changes , circadian clock genes per1 and bmal1 in liver and scn are altered in response to ahr agonists.40,41,62 ahr activation with bnf attenuates light - induced induction of per1 in the scn , providing an essential mechanism for the effects of ahr activation on light - induced changes in behavioral rhythmicity . furthermore , activation of the ahr by ficz in the scn cell line scn2.2 alters the expression of the clock genes per1 , cry1 and cry2.22 collectively , the data suggest that ahr expression , even under basal conditions , influences circadian rhythm strength . overall , ahr expression and activation dampens the response of the circadian clock to perturbations in light and dampens the rhythmicity of clock gene rhythms . in contrast , when ahr is deleted , there is a tendency for the clock to have enhanced responsiveness to light at night and for increased amplitude of the per1 rhythm.39,41,63 further experimentation in ahr - null mice is needed for confirmation . generally , the aggregate data suggest that ahr may act as a gain control on the circadian clock ; activation of ahr suppresses rhythm amplitude , whereas inhibition of ahr strengthens rhythm amplitude ( fig . epidemiological evidence links circadian disruption , by shift work , to higher body mass index , increased triglycerides , glucose dysregulation , obesity , and higher incidence of metabolic syndrome , defined by the national cholesterol education program as the presence of three or more of the following criteria : high waist circumference , blood pressure , fasting triglyceride levels , fasting high - density lipoprotein , or high fasting glucose levels.6568 mouse models of circadian disruption produce altered rhythms in core circadian genes as well as clock - controlled genes to include numerous rate - limiting metabolic genes , disrupted rhythms in lipid and glucose metabolism , and hepatic ste - atosis.69,70 ahr status influences circadian rhythm strength , even under controlled conditions . rhythms of the clock genes per1 and bmal1 are significantly enhanced in the liver of ahr mice compared to wild - type mice.41 genetic manipulation to reduce ahr expression in mice enhances the ability of mice to adjust their behavior in response to an alteration in the timing of the light / dark cycle ( jaeger and tischkau , unpublished results , april 2016 ) . activation of ahr alters glucose metabolism , glucose tolerance , and insulin levels , and increases the risk of diabetes mellitus.7276 ahr activation specifically within adipose tissue promotes inflammation and impairs glucose and insulin tolerance.16,7779 additionally , signaling downstream of tumor necrosis factor nuclear factor - k , which decreases glucose transporter type 4 expression and contributes to insulin resistance , is increased in adipose tissue of vietnam veterans exposed to dioxins.80,81 ahr activation alters hepatic genes involved in fatty acid , lipid , and cholesterol metabolism pathways , including the peroxisome proliferator - activated receptor ( ppar ) pathway , all of which mediate glucose and lipid homeostasis.62,82,83 additionally , ahr activation induces an inflammatory response , which is implicated in the pathogenesis of metabolic disorders.78,84 this evidence points to a clear interaction between activated ahr and systemic energy metabolism . around 90% of human exposure to dioxins occurs through our diet , primarily through animal fat consumption.8 besides tcdd and toxic pollutants , many compounds that influence ahr activity exist as natural components of fruits and vegetables.11,85,86 additionally , the arachidonic acid metabolite , lipoxin a4 , prostaglandins , specifically prostaglandin g2 , and an arachidonic acid metabolite produced in inflammatory disease conditions , namely , 12(r)-hydroxy-5(z),8(z),10e , 14(z)-dicosatetraenoic acid ( 12(r)-hete ) , can act as ahr agonists.8789 lipid and lipid derivatives , such as oxidized low - density lipoproteins ( oxldl ) , have been identified as ahr agonists.90 oxldl and saturated fatty acids contained in a western diet activate ahr and contribute to obesity and inflammation in c57bl/6 j mice.91 ahr activation also occurs through exposure to flavonoids and metabolites from fruits and vegetables , indole-3-carbinol , and herbal supplements including ginseng.12,92,93 additionally , the consumption of fish and shrimp , which bioaccumulate organohalogens , increases potential dietary exposure.94 with a wide variety of ahr agonists present within food , or present due to secondary effects of diets that increase inflammatory mediators , exposure to ahr activation and its consequences are prevalent . rather than focusing on the contribution of a single ahr ligand to ahr activation and downstream sequelae , it may be more important to consider the overall activation of ahr in response to the constellation of ligands , in terms of the overall metabolic consequences . mice lacking ahr expression have enhanced insulin sensitivity and improved glucose tolerance , although improved metabolism may differ depending on age , sex , housing , diet , and possible genetic drift.63,9597 mice with a low - affinity ahr allele fed a high - fat diet ( hfd ) are less susceptible to obesity , exhibiting differences in fat mass , liver physiology , and liver gene expression compared to mice with high - affinity ahr.98 ahr and ahr mice are resistant to the harmful effects of diet - induced obesity through protection against hepatic steatosis , insulin resistance , and inflammation . a large number of metabolites in carbohydrate , lipid , metabolic , and xenobiotic pathways are regulated by clock / bmal1 binding to their promoter regions.100,101 genes that lose rhythmic expression following hfd consumption often peak during the time period when clock / bmal1 is recruited to chromatin , which also coincides with peak the expression of ahr.41,102,103 persistent activation of ahr alters circadian rhythm , primarily through inhibition of clock / bmal1-mediated target genes.3941 therefore , ahr - induced disruption of clock / bmal1 activity may play a role in hfd - induced disruption of metabolic rhythms . furthermore , even deletion of a single ahr allele ( ahr ) protects mice against diet - induced changes in transcriptional oscillations and also against glucose and metabolic gene rhythm disruption ( jaeger and tischkau , unpublished results , april 2016 ) . these studies highlight the involvement of ahr and the clock in the regulation of energy metabolism and provide interesting opportunities to explore novel therapies to combat poor metabolic health . to study ahr signaling in vitro , use of the mouse hepa-1c1c7 cell line ( high ahr expression ) and the hepa-1c1c7-derived hepa-1c1c12 ( low ahr expression ) allows comparison between varied levels of ahr expression.40 in addition to liver - derived hepa-1c1c7 cells , cell lines derived from lung epithelia , keratinocytes , and fibroblasts can be used to study ahr signaling.106 in dispersed cells , however , the circadian clock frequently becomes desynchronized because of the lack of synchronizing signals provided in vivo by the scn , the endocrine and autonomic nervous systems , and diet . the mechanism of serum shock is hypothesized to mimic light - induced immediate early genes and synchronize circadian cycles , inherent within individual cells , to the population of cells in the dish.107 thus , knowledge of the circadian system and its behavior in cultured cells , together with cell lines that differentially express the ahr , provide unique , yet underutilized opportunities to study the interaction between circadian rhythms and ahr signaling . in addition to sharing pas domain structure with circadian proteins , ahr demonstrates a rhythmic expression of transcription and sensitivity to activation by the ahr agonist tcdd.41,57,58 reciprocally , ahr activation influences rhythmic strength of circadian clock genes , hormones , and behavioral responses to light - induced phase shifts.41,47,50 correlations between ahr variants in mouse models and humans allow us to study how altered ahr affinity affects downstream signaling pathways.108 epidemiological studies that explore the link between ahr polymorphisms and obesity may provide important information regarding the influence of ahr on physiological metabolism . the dietary component curcumin , a natural phenol found in spices , and resveratrol , a natural phenol found in red wine , along with synthetic compounds ch-223191 , 6,2,4-trimethoxyflavone , and gnf351 , have been reported as ahr antagonists.109114 given its importance in xenobiotic metabolism , complete blockade of ahr may not be desirable . thus , partial inhibition of ahr , or specific inhibition of ahr - induced effects on clock / bmal1 and nuclear receptors ppar and ppar and their downstream target genes that regulate glucose metabolism , may provide benefits while minimizing undesirable effects . understanding the variety of mechanisms by which ahr exerts its effects on metabolism may reveal new targets of interest for the conservation of homeostasis and reinforces the significance of ahr in clock disruption and metabolic disorder .

pharmacies assume an imperative part in the provision of health care and well - being at the community level . generally , pharmacies have extended working hours , are commonly visited , and conveniently placed within the heart of community dwellings . the pharmacist 's contribution is significant in health promotion , health maintenance , and health improvement of the communities in which they serve . additionally , pharmacists are in a unique position to understand the needs of community members through daily interaction with patients and customers . in a few areas , pharmacists are often the patient 's first point of contact , and for some their only contact with the health care professional . pharmacists are capable of providing oral health information based on request by the patient , and a few did it proactively . most of the pharmacists believed that providing oral health advice is within the realm of their profession . additionally , individuals with lack of access to dental services and those from lower socioeconomic conditions are more likely to seek oral health - related advice from pharmacists . at present , a range of products effective in curing several of the oral health ailments and meeting the patient 's expectations are available in the market , especially in pharmacies . there are products for managing dental decay , tartar buildup , gingivitis , dental hypersensitivity , teeth staining , and dental erosion , etc . self - medication with over - the - counter drugs has for quite some time been a typical practice in communities . presently , people have easy access to certain dental treatments because of the availability of numerous over - the - counter dental remedies , which were available only through the dental professionals in the past . due to changes in conveyance of primary health care the role played by pharmacists in the provision of overall and oral health cares has been acknowledged as an important and relevant issue by the government of the united kingdom . recently , the pharmacist 's function evolved from dispenser of drugs to the approved member of the health care team . a study of pharmacists found that the pharmacist has to face at least one inquiry each week on some mouth - related issue ; almost 50% of these inquiries were identified with oral sores or ulcers . pharmacists provided some oral health advice , and in the coming years the demand for such advice is going to increase for which pharmacists have expressed their readiness to expand their knowledge related to oral health . data from saudi arabia suggested that almost 34% of the pharmacists reported 10 daily requests for oral health advice mainly related to toothache , mouth ulcers , and mouth malodor . these complaints were mainly managed by medications and only very small percentages of the dental patients were referred to the dentist . most of the recommendations of oral health products made by pharmacists in riyadh , riyadh province , saudi arabia were mainly based on personal experience and patient inputs rather than scientific information highlighting deficiencies in oral health knowledge . there are many ways in which the pharmacist can come forward to provide services such as encouraging the use of fluoridated tooth pastes and soft - bristle toothbrushes , advising on a healthy diet and eating habits , encouraging the use of preventive and therapeutic oral care services , and providing the needed information , skills , and motivation to individuals and caregivers about the prevention of oral diseases . by doing all these , pharmacists can actively participate in oral disease identification , assessment , management , referral , and prevention . hence , there is a need for the pharmacist to be included as a member of a multidisciplinary oral health squad . identifying and addressing gaps in oral health knowledge , attitudes , and practices of pharmacists is of utmost importance before they can be considered as a member of oral health promotion team . hence , the aim of the study was to determine the prevailing oral health knowledge , attitude , and self - care practices among a sample of pharmacists from riyadh , riyadh province , saudi arabia . a cross - sectional study was conducted among a sample of pharmacists working in community- and hospital - based pharmacies in riyadh , riyadh province , saudi arabia . a list of pharmacies and hospitals in riyadh was obtained using online business and health insurance directories . from a total of 379 pharmacies , 100 pharmacies and 200 pharmacists were selected in the study by employing a convenience sampling approach based on the ease of accessibility . a sample size of 200 was determined by considering a value of 0.05 , required power of 0.82 , and effect size of 0.18 for one - tailed test for pearson product coefficient , which is computationally similar to spearman 's rank correlation coefficient . ( franz faul , universitat kiel , germany ) a self - administered questionnaire was distributed to the study participants by trained dental interns and the data were collected . in addition , dental interns provided the needed explanation requested by the respondents . the study questionnaire was designed by a team of dental professionals after a thorough literature review . after an initial draft of the questionnaire was prepared in english , it was validated in two steps . first , the questionnaire was sent to dental public health professionals to obtain their expert opinion with regard to its simplicity , relativity , and importance . second , a pilot study was conducted by selecting a small sample ( n = 20 ) of pharmacists , and content authenticity was pretested to determine practicability , cogency , and rendition of responses . moreover , all the amendments were incorporated into the questionnaire while ensuring its consistency with the published literature . after an in - depth discussion , questionnaire was finalized and subsequently distributed to the pharmacists for their response . the first part consisted of demographic information ( gender , nationality , qualification , experience , and type of pharmacy ) of the respondents . the second part elicited the basic knowledge of oral health , by asking 15 questions about dental plaque , causes , symptoms , consequences , prevention , and treatment of common oral diseases . the third part determined the attitude of the pharmacists toward oral health by asking five questions , in which their responses were assessed through a 5-point likert scale ( strongly agree , agree , indifferent , disagree , and strongly disagree ) of agreement . the fourth part assessed the oral self - care behavior by considering three questions on toothbrushing frequency , timing , and material / method . every correct response in the knowledge section was scored 1 , creating a scale in range of 015 . the overall knowledge scores were categorized into poor ( 05 ) , average ( 5.110 ) , and good ( 10.115 ) . similarly , for the attitude section strongly agree and agree responses were scored 1 while indifferent , disagree , and strongly disagree responses were scored 0 . a mean score of less than 4 was considered as negative attitude while a score of 45 was taken as positive attitude . in the oral self - care practices section , more appropriate responses of twice daily brushing , the morning and night after food and toothbrushing with paste were scored 1 with all other responses being scored 0 . a mean practice score of less than 2.4 was considered as inadequate and a score of 2.4 and above was deemed to be adequate for oral self - care practice . the higher the score , the better the respondent 's oral health knowledge , attitude , and self - care practices . the study was approved by the research center of riyadh colleges of dentistry and pharmacy . additionally , written informed consent was obtained from the respondents prior to participation in the study . data collected through the questionnaire were entered and statistical analysis was performed by using ibm spss statistics for windows , version 21.0 . the mean and standard deviations for knowledge , attitude , and practice scores were calculated . normality distribution of the data was assessed by applying kolmogorov smirnov and shapiro wilk 's tests , which showed significant p value ( p < 0.05 ) indicating nonnormal distribution of the data . whitney u and kruskal wallis tests were applied as a part of inferential statistics . additionally , spearman 's rank correlation coefficient was used to assess the association between knowledge attitude , knowledge practice , and attitude practice . for all statistical purposes , p < a cross - sectional study was conducted among a sample of pharmacists working in community- and hospital - based pharmacies in riyadh , riyadh province , saudi arabia . a list of pharmacies and hospitals in riyadh was obtained using online business and health insurance directories . from a total of 379 pharmacies , 100 pharmacies and 200 pharmacists were selected in the study by employing a convenience sampling approach based on the ease of accessibility . a sample size of 200 was determined by considering a value of 0.05 , required power of 0.82 , and effect size of 0.18 for one - tailed test for pearson product coefficient , which is computationally similar to spearman 's rank correlation coefficient . a self - administered questionnaire was distributed to the study participants by trained dental interns and the data were collected . the study questionnaire was designed by a team of dental professionals after a thorough literature review . after an initial draft of the questionnaire was prepared in english , it was validated in two steps . first , the questionnaire was sent to dental public health professionals to obtain their expert opinion with regard to its simplicity , relativity , and importance . second , a pilot study was conducted by selecting a small sample ( n = 20 ) of pharmacists , and content authenticity was pretested to determine practicability , cogency , and rendition of responses . moreover , the pharmacists ' opinions were sought to make the questionnaire simpler and shorter . all the amendments were incorporated into the questionnaire while ensuring its consistency with the published literature . data from the pilot study were not utilized in the final analysis . after an in - depth discussion , questionnaire was finalized and subsequently distributed to the pharmacists for their response . the first part consisted of demographic information ( gender , nationality , qualification , experience , and type of pharmacy ) of the respondents . the second part elicited the basic knowledge of oral health , by asking 15 questions about dental plaque , causes , symptoms , consequences , prevention , and treatment of common oral diseases . the third part determined the attitude of the pharmacists toward oral health by asking five questions , in which their responses were assessed through a 5-point likert scale ( strongly agree , agree , indifferent , disagree , and strongly disagree ) of agreement . the fourth part assessed the oral self - care behavior by considering three questions on toothbrushing frequency , timing , and material / method . every correct response in the knowledge section the overall knowledge scores were categorized into poor ( 05 ) , average ( 5.110 ) , and good ( 10.115 ) . similarly , for the attitude section strongly agree and agree responses were scored 1 while indifferent , disagree , and strongly disagree responses were scored 0 . a mean score of less than 4 was considered as negative attitude while a score of 45 was taken as positive attitude . in the oral self - care practices section , more appropriate responses of twice daily brushing , the morning and night after food and toothbrushing with paste were scored 1 with all other responses being scored 0 . a mean practice score of less than 2.4 was considered as inadequate and a score of 2.4 and above was deemed to be adequate for oral self - care practice . the higher the score , the better the respondent 's oral health knowledge , attitude , and self - care practices . the study was approved by the research center of riyadh colleges of dentistry and pharmacy . additionally , written informed consent was obtained from the respondents prior to participation in the study . data collected through the questionnaire were entered and statistical analysis was performed by using ibm spss statistics for windows , version 21.0 . the mean and standard deviations for knowledge , attitude , and practice scores were calculated . normality distribution of the data was assessed by applying kolmogorov smirnov and shapiro wilk 's tests , which showed significant p value ( p < 0.05 ) indicating nonnormal distribution of the data . hence , nonparametric mann whitney u and kruskal wallis tests were applied as a part of inferential statistics . additionally , spearman 's rank correlation coefficient was used to assess the association between knowledge attitude , knowledge practice , and attitude practice . for all statistical purposes , p < a total of 200 pharmacists responded to the questionnaire , making the response rate 100% . a mjority of them were males ( 67.5% ) of non - saudi nationality ( 65.5% ) . most of the respondents had ( 90.0% ) a bachelor of pharmacy qualification with 40% having 610 years of experience . moreover , a majority of the pharmacists ( 65% ) were working with chains of pharmacies , as mentioned in table 1 . characteristics of the study participan more than 90% of the study participants had knowledge about the purpose of toothbrushing , prevention of tooth decay and gum disease , that bleeding gum indicated gum disease , that the effect of sweet retention on dentition led to tooth decay , methods of prevention of tooth decay , impact of oral health on general health , and consequences teeth loss interfering with speech . around 83% of the study participants knew about tobacco chewing or smoking as the cause of oral cancer and 87.5% agreed about the possibility of moving irregularly placed teeth into the correct position . nearly 77.5% of the participants said that improper toothbrushing was the reason behind gum diseases and 72.5% said regular toothbrushing and flossing could prevent gum diseases . about 72.5% of the participants mentioned 13 months interval as the duration for changing the toothbrush . less than half of the study participants knew about that tooth decay and gum disease due to dental plaque and 47% pointed out that the bacteria in dental plaque was the reason for tooth decay . only 12.5% of the study participants could recognize dental plaque as soft deposit on the teeth and 30% reported that gum disease was the common reason for tooth loss in old age as shown table 2 . correct responses to the knowledge questions by pharmacists ( n=200 ) when enquired about the attitude of study participants toward a regular visit to the dentist and replacing missing teeth by artificial teeth , 62.5% and 42.5% responded positively . a very high percentage ( 94.5% ) of the participants strongly agreed that the smoking was a bad habit and 27.5% of the study participants showed a strong disagreement with the statement that dentists care only about treatment and not prevention . similarly , 88.5% of the study participants showed a strong agreement with the statement that treatment of toothache was similar to the treatment of any other organ in the body , as mentioned in table 3 . attitude toward oral health oral self - care practices among the study participants varied with less than half brushing their teeth twice daily and just 23% brushing in the morning and at night . additionally , 92.5% of them brushed their teeth by using toothpaste and toothbrush , as shown in figure 1 . oral self - care practices among pharmacists ( % ) the association of demographic characteristics and mean knowledge , attitude , and practices questions are expressed in table 4 . male participants showed significantly more knowledge ( 5.44 vs 4.92 , p = 0.001 ) and practice scores ( 2.18 vs 1.94 , p = 0.008 ) toward oral health compared to their female counterparts . similarly , non - saudis and those working in chain pharmacies showed significantly higher knowledge ( 5.42 vs 4.99 , p = 0.003 and 5.42 vs 5 , p = 0.005 ) and practice scores ( 2.18 vs 1.94 , p = 0.007 and 2.19 vs 1.93 , p = 0.003 ) compared to their saudi counterparts and those working in hospital - based pharmacies . mean and standard deviations of knowledge , attitude , and practice scores pharmacists with master 's degree qualification showed significantly higher mean knowledge scores ( 6.43 vs 5.83 vs 5.16 , p = 0.001 ) compared to those with diploma and bachelor 's qualifications . on the contrary , the mean attitude score was significantly higher among ( 3.99 vs 3 vs 2.93 , p = 0.001 ) pharmacists with bachelor 's degree qualification as compared to those with diploma and master 's degree qualifications . but the mean practice score was found to be significantly higher among ( 3.00 vs 2.64 vs 2.03 , p = 0.001 ) pharmacists with diploma compared to those with master 's or bachelor 's degree qualifications . similarly , pharmacists with 1115 years of experience showed a significantly higher mean knowledge score as compared to those with 1620 years , 610 years , and 05 years ' experience ( 5.96 vs 5.20 vs 5.19 vs 5.14 , p = 0.008 ) . the mean practice score was significantly higher among pharmacists with 05 years of experience ( 2.27 vs 2.04 vs 2.03 vs 1.92 , p = 0.027 ) as compared to those with 1620 years , 610 years , and 1115 years ' experience as shown in table 4 . the overall mean scores of oral health knowledge , attitude , and self - care practices were reported to be 5.27 1.05 , 3.89 0.83 , and 2.1 0.61 , respectively , suggesting average oral health knowledge , negative attitude , and inadequate oral self - care practices as shown in the figure 2 . mean and standard deviations of knowledge , attitudes , and practice scores among pharmacists the spearman correlation test revealed a significant positive correlation between knowledge and practice ( r = 0.262 , p < 0.01 ) , whereas knowledge and attitude ( r = -0.149 , p < 0.05 ) as well as attitudes and practices ( r = -0.196 , p < 0.01 ) were negatively correlated as shown in the table 5 . spearman 's correlation coefficients between knowledge - attitude ( k - a ) , knowledge - practice ( k - p ) , and attitude - practice ( a - p ) practice scor riyadh is rapidly growing capital city with many extension areas built to accommodate the increasing population . pharmacies were the first to open and provide 24-h service to cater to the health needs of the population before hospitals or polyclinics were opened in such areas . many of the residents staying in such localities receive some health- and oral health - related advices from the pharmacists . there are a variety of reasons as to why people seek health and oral health advices from the pharmacist in their vicinity . these include financial limitations , nonavailability of hospitals or polyclinics nearby , busy schedule , and difficulty in making appointment with the dentist or physician . the pharmacist 's own knowledge , attitude , and oral self - care practices are major determinants for his / her role as an oral health promoter in a community setting . hence , this study disclosed the knowledge , attitude , and self - care practices toward oral health among a sample of pharmacists from riyadh , riyadh province , saudi arabia . the resent study revealed average oral health knowledge among a sample of pharmacists from riyadh , riyadh province , saudi arabia . this could be due to several factors such as the lack of the pharmacist 's education and training in oral health , lesser degree of motivation by pharmacists to know about oral health , lack of time , limited interaction with dental professionals , and lack of opportunities for professional development . additionally , it has been reported that 50% of the pharmacists never met the dental professionals practicing close to their pharmacies though 90% of the pharmacies were located near dental clinics . knowledge of dental plaque was limited and this finding was lower than that reported among other health professionals . a similar finding of poor oral health knowledge has been reported among bachelor of pharmacy students from malaysia . in general , the possible explanation for such a finding could be that most of the male pharmacists work in community - based pharmacies in which many people seek oral health advice and information about oral health care products . on the contrary , female pharmacists predominantly work in hospital - based pharmacies dispensing mainly doctor - prescribed medications to a limited number of patients . this could have resulted in less exposure to oral health information by female pharmacists unlike community - based pharmacists . moreover , frequent interaction between oral health professionals and male pharmacists might be the reason for increased oral health knowledge among male pharmacists . another possible factor could be frequent contact between medical representatives and male pharmacists with the former explaining about the oral health care products . the present study result is a contrast to other reported studies in which females demonstrated higher oral health literacy . in saudi arabia , community pharmacies are either owned by a single person or attached to a network of pharmacies scattered across a city , province , or the country . most of the community pharmacies are managed by non - saudi pharmacists while saudi pharmacists prefer to work in the government sector . in the present study , non - saudi pharmacists showed significantly higher oral health knowledge as compared to saudi pharmacists . the probable reason could have been a higher content of oral health information in pharmacy education in the institutes from which non - saudis graduated . attitude refers to the inclination to react in a certain way to a certain situation , and to see and interpret events according to certain predispositions . in the present study , it can be speculated that the average oral health knowledge among pharmacists could be the main reason for such a finding . in the present study , a significant positive correlation between the pharmacist 's knowledge and practice was observed . on the contrary , a significantly negative correlation between knowledge attitude and attitude practice was observed . from this finding , it can be interpreted that lack of knowledge adversely influences the attitude leading to poor oral self - care practices of the pharmacists . however , higher oral health knowledge has a direct impact on oral self - care practices by improving the individual 's self - awareness , self - protection , and personal hygiene performances . the strengths of the present study was a sufficient sample size and the varied oral health - related questions designed to disclose an existing level of oral health knowledge , attitude , and self - care practices . the limitations of the present study include the lack of a standard questionnaire for measuring the oral health knowledge and the nonavailability of any reported comparable study instrument . therefore , the results of the present study were compared to the oral health knowledge of other health professionals . moreover , the results of present study rely on self - reported data ; the oral health information may have been biased through over- and underreporting due to social desirability . dental publications offer conflicting results on the impact of the oral health knowledge , attitude , and self - care practices , and oral diseases . however , gathering such data has been useful in planning an oral health education program targeted toward pharmacists . there is scarce data available regarding oral health knowledge of pharmacists toward their own oral health knowledge , attitudes , and practices . to develop a sound strategy for improving the oral health of pharmacists , for this , additional studies are needed to be conducted in a wide geographic area , preferably by considering a nationally representative sample of pharmacists from saudi arabia by using reliable and indigenously developed measures . with enhanced oral health knowledge and practices , pharmacists can serve as oral health promoters in community pharmacy setups by catering to the oral health needs of the society . pharmacists who were considered in the present study demonstrated an average knowledge , negative attitude , and inadequate self - care practices toward oral health . however , increasing oral health knowledge can have a profound improvement in oral self - care practices . hence , there is a need to increase the basic oral health knowledge of the pharmacist to improve his / her own oral self - care practices and empower him / her to be utilized as a member of the oral health care team , thereby enabling him / her to provide evidence - based advice and guidance to clients . to achieve this , professional organizations such as the saudi dental society and saudi pharmaceutical society should take proactive steps to provide regular oral health educational programs to pharmacists .

aim : identifying and addressing gaps in the oral health knowledge , attitude , and practices of pharmacists is important before they can be considered as a member of the oral health promotion team . the aim of this study was to determine the prevailing oral health knowledge , attitude , and self - care practices among a sample of pharmacists from riyadh , riyadh province , saudi arabia.materials and methods : a cross - sectional study involving 200 pharmacists working in community- and hospital - based pharmacies was conducted using a structured , self - administered , close - ended questionnaire . the responses were collected and descriptive statistics of the mean scores of knowledge , attitude , and self - care practices were calculated . mann whitney u and kruskal wallis tests were performed to compare the different groups . spearman 's rank correlation coefficient was used to assess the association among knowledge attitude , knowledge practice , and attitudepractice.results:overall , the mean scores of oral health knowledge , attitude , and self - care practices were found to be 5.27 1.05 , 3.89 0.83 , and 2.1 0.61 , respectively . male non - saudi pharmacists working in chain pharmacies , having 1115 years of experience with a master 's degree qualification showed significantly higher mean knowledge and practices scores as compared to their counterparts . spearman 's correlation tests revealed a significant positive correlation of knowledge practice ( r = 0.262 , p < 0.01 ) , whereas knowledge attitude ( r = -0.149 , p < 0.05 ) as well as attitudes practices ( r = -0.196 , p < 0.01 ) were negatively correlated.conclusion:pharmacists exhibited an average knowledge , negative attitude , and inadequate self - care practices toward oral health . however , increasing oral health knowledge can have profound improvement in oral self - care practices .

INTRODUCTION MATERIALS AND METHODS Study design, site and participants Determination of sample size Questionnaire design Ethical approval Statistical analysis RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

the pharmacist 's contribution is significant in health promotion , health maintenance , and health improvement of the communities in which they serve . in a few areas , pharmacists are often the patient 's first point of contact , and for some their only contact with the health care professional . pharmacists are capable of providing oral health information based on request by the patient , and a few did it proactively . most of the pharmacists believed that providing oral health advice is within the realm of their profession . additionally , individuals with lack of access to dental services and those from lower socioeconomic conditions are more likely to seek oral health - related advice from pharmacists . at present , a range of products effective in curing several of the oral health ailments and meeting the patient 's expectations are available in the market , especially in pharmacies . self - medication with over - the - counter drugs has for quite some time been a typical practice in communities . presently , people have easy access to certain dental treatments because of the availability of numerous over - the - counter dental remedies , which were available only through the dental professionals in the past . due to changes in conveyance of primary health care the role played by pharmacists in the provision of overall and oral health cares has been acknowledged as an important and relevant issue by the government of the united kingdom . recently , the pharmacist 's function evolved from dispenser of drugs to the approved member of the health care team . a study of pharmacists found that the pharmacist has to face at least one inquiry each week on some mouth - related issue ; almost 50% of these inquiries were identified with oral sores or ulcers . pharmacists provided some oral health advice , and in the coming years the demand for such advice is going to increase for which pharmacists have expressed their readiness to expand their knowledge related to oral health . data from saudi arabia suggested that almost 34% of the pharmacists reported 10 daily requests for oral health advice mainly related to toothache , mouth ulcers , and mouth malodor . these complaints were mainly managed by medications and only very small percentages of the dental patients were referred to the dentist . most of the recommendations of oral health products made by pharmacists in riyadh , riyadh province , saudi arabia were mainly based on personal experience and patient inputs rather than scientific information highlighting deficiencies in oral health knowledge . there are many ways in which the pharmacist can come forward to provide services such as encouraging the use of fluoridated tooth pastes and soft - bristle toothbrushes , advising on a healthy diet and eating habits , encouraging the use of preventive and therapeutic oral care services , and providing the needed information , skills , and motivation to individuals and caregivers about the prevention of oral diseases . by doing all these , pharmacists can actively participate in oral disease identification , assessment , management , referral , and prevention . hence , there is a need for the pharmacist to be included as a member of a multidisciplinary oral health squad . identifying and addressing gaps in oral health knowledge , attitudes , and practices of pharmacists is of utmost importance before they can be considered as a member of oral health promotion team . hence , the aim of the study was to determine the prevailing oral health knowledge , attitude , and self - care practices among a sample of pharmacists from riyadh , riyadh province , saudi arabia . a cross - sectional study was conducted among a sample of pharmacists working in community- and hospital - based pharmacies in riyadh , riyadh province , saudi arabia . from a total of 379 pharmacies , 100 pharmacies and 200 pharmacists were selected in the study by employing a convenience sampling approach based on the ease of accessibility . a sample size of 200 was determined by considering a value of 0.05 , required power of 0.82 , and effect size of 0.18 for one - tailed test for pearson product coefficient , which is computationally similar to spearman 's rank correlation coefficient . ( franz faul , universitat kiel , germany ) a self - administered questionnaire was distributed to the study participants by trained dental interns and the data were collected . first , the questionnaire was sent to dental public health professionals to obtain their expert opinion with regard to its simplicity , relativity , and importance . second , a pilot study was conducted by selecting a small sample ( n = 20 ) of pharmacists , and content authenticity was pretested to determine practicability , cogency , and rendition of responses . the first part consisted of demographic information ( gender , nationality , qualification , experience , and type of pharmacy ) of the respondents . the second part elicited the basic knowledge of oral health , by asking 15 questions about dental plaque , causes , symptoms , consequences , prevention , and treatment of common oral diseases . the third part determined the attitude of the pharmacists toward oral health by asking five questions , in which their responses were assessed through a 5-point likert scale ( strongly agree , agree , indifferent , disagree , and strongly disagree ) of agreement . the fourth part assessed the oral self - care behavior by considering three questions on toothbrushing frequency , timing , and material / method . the overall knowledge scores were categorized into poor ( 05 ) , average ( 5.110 ) , and good ( 10.115 ) . similarly , for the attitude section strongly agree and agree responses were scored 1 while indifferent , disagree , and strongly disagree responses were scored 0 . a mean score of less than 4 was considered as negative attitude while a score of 45 was taken as positive attitude . in the oral self - care practices section , more appropriate responses of twice daily brushing , the morning and night after food and toothbrushing with paste were scored 1 with all other responses being scored 0 . a mean practice score of less than 2.4 was considered as inadequate and a score of 2.4 and above was deemed to be adequate for oral self - care practice . the higher the score , the better the respondent 's oral health knowledge , attitude , and self - care practices . the mean and standard deviations for knowledge , attitude , and practice scores were calculated . normality distribution of the data was assessed by applying kolmogorov smirnov and shapiro wilk 's tests , which showed significant p value ( p < 0.05 ) indicating nonnormal distribution of the data . whitney u and kruskal wallis tests were applied as a part of inferential statistics . additionally , spearman 's rank correlation coefficient was used to assess the association between knowledge attitude , knowledge practice , and attitude practice . for all statistical purposes , p < a cross - sectional study was conducted among a sample of pharmacists working in community- and hospital - based pharmacies in riyadh , riyadh province , saudi arabia . from a total of 379 pharmacies , 100 pharmacies and 200 pharmacists were selected in the study by employing a convenience sampling approach based on the ease of accessibility . a sample size of 200 was determined by considering a value of 0.05 , required power of 0.82 , and effect size of 0.18 for one - tailed test for pearson product coefficient , which is computationally similar to spearman 's rank correlation coefficient . a self - administered questionnaire was distributed to the study participants by trained dental interns and the data were collected . first , the questionnaire was sent to dental public health professionals to obtain their expert opinion with regard to its simplicity , relativity , and importance . second , a pilot study was conducted by selecting a small sample ( n = 20 ) of pharmacists , and content authenticity was pretested to determine practicability , cogency , and rendition of responses . the first part consisted of demographic information ( gender , nationality , qualification , experience , and type of pharmacy ) of the respondents . the second part elicited the basic knowledge of oral health , by asking 15 questions about dental plaque , causes , symptoms , consequences , prevention , and treatment of common oral diseases . the third part determined the attitude of the pharmacists toward oral health by asking five questions , in which their responses were assessed through a 5-point likert scale ( strongly agree , agree , indifferent , disagree , and strongly disagree ) of agreement . the fourth part assessed the oral self - care behavior by considering three questions on toothbrushing frequency , timing , and material / method . every correct response in the knowledge section the overall knowledge scores were categorized into poor ( 05 ) , average ( 5.110 ) , and good ( 10.115 ) . similarly , for the attitude section strongly agree and agree responses were scored 1 while indifferent , disagree , and strongly disagree responses were scored 0 . a mean score of less than 4 was considered as negative attitude while a score of 45 was taken as positive attitude . in the oral self - care practices section , more appropriate responses of twice daily brushing , the morning and night after food and toothbrushing with paste were scored 1 with all other responses being scored 0 . a mean practice score of less than 2.4 was considered as inadequate and a score of 2.4 and above was deemed to be adequate for oral self - care practice . the higher the score , the better the respondent 's oral health knowledge , attitude , and self - care practices . additionally , written informed consent was obtained from the respondents prior to participation in the study . the mean and standard deviations for knowledge , attitude , and practice scores were calculated . normality distribution of the data was assessed by applying kolmogorov smirnov and shapiro wilk 's tests , which showed significant p value ( p < 0.05 ) indicating nonnormal distribution of the data . hence , nonparametric mann whitney u and kruskal wallis tests were applied as a part of inferential statistics . additionally , spearman 's rank correlation coefficient was used to assess the association between knowledge attitude , knowledge practice , and attitude practice . for all statistical purposes , p < a total of 200 pharmacists responded to the questionnaire , making the response rate 100% . a mjority of them were males ( 67.5% ) of non - saudi nationality ( 65.5% ) . most of the respondents had ( 90.0% ) a bachelor of pharmacy qualification with 40% having 610 years of experience . moreover , a majority of the pharmacists ( 65% ) were working with chains of pharmacies , as mentioned in table 1 . characteristics of the study participan more than 90% of the study participants had knowledge about the purpose of toothbrushing , prevention of tooth decay and gum disease , that bleeding gum indicated gum disease , that the effect of sweet retention on dentition led to tooth decay , methods of prevention of tooth decay , impact of oral health on general health , and consequences teeth loss interfering with speech . around 83% of the study participants knew about tobacco chewing or smoking as the cause of oral cancer and 87.5% agreed about the possibility of moving irregularly placed teeth into the correct position . only 12.5% of the study participants could recognize dental plaque as soft deposit on the teeth and 30% reported that gum disease was the common reason for tooth loss in old age as shown table 2 . similarly , 88.5% of the study participants showed a strong agreement with the statement that treatment of toothache was similar to the treatment of any other organ in the body , as mentioned in table 3 . attitude toward oral health oral self - care practices among the study participants varied with less than half brushing their teeth twice daily and just 23% brushing in the morning and at night . oral self - care practices among pharmacists ( % ) the association of demographic characteristics and mean knowledge , attitude , and practices questions are expressed in table 4 . male participants showed significantly more knowledge ( 5.44 vs 4.92 , p = 0.001 ) and practice scores ( 2.18 vs 1.94 , p = 0.008 ) toward oral health compared to their female counterparts . similarly , non - saudis and those working in chain pharmacies showed significantly higher knowledge ( 5.42 vs 4.99 , p = 0.003 and 5.42 vs 5 , p = 0.005 ) and practice scores ( 2.18 vs 1.94 , p = 0.007 and 2.19 vs 1.93 , p = 0.003 ) compared to their saudi counterparts and those working in hospital - based pharmacies . mean and standard deviations of knowledge , attitude , and practice scores pharmacists with master 's degree qualification showed significantly higher mean knowledge scores ( 6.43 vs 5.83 vs 5.16 , p = 0.001 ) compared to those with diploma and bachelor 's qualifications . on the contrary , the mean attitude score was significantly higher among ( 3.99 vs 3 vs 2.93 , p = 0.001 ) pharmacists with bachelor 's degree qualification as compared to those with diploma and master 's degree qualifications . but the mean practice score was found to be significantly higher among ( 3.00 vs 2.64 vs 2.03 , p = 0.001 ) pharmacists with diploma compared to those with master 's or bachelor 's degree qualifications . similarly , pharmacists with 1115 years of experience showed a significantly higher mean knowledge score as compared to those with 1620 years , 610 years , and 05 years ' experience ( 5.96 vs 5.20 vs 5.19 vs 5.14 , p = 0.008 ) . the mean practice score was significantly higher among pharmacists with 05 years of experience ( 2.27 vs 2.04 vs 2.03 vs 1.92 , p = 0.027 ) as compared to those with 1620 years , 610 years , and 1115 years ' experience as shown in table 4 . the overall mean scores of oral health knowledge , attitude , and self - care practices were reported to be 5.27 1.05 , 3.89 0.83 , and 2.1 0.61 , respectively , suggesting average oral health knowledge , negative attitude , and inadequate oral self - care practices as shown in the figure 2 . mean and standard deviations of knowledge , attitudes , and practice scores among pharmacists the spearman correlation test revealed a significant positive correlation between knowledge and practice ( r = 0.262 , p < 0.01 ) , whereas knowledge and attitude ( r = -0.149 , p < 0.05 ) as well as attitudes and practices ( r = -0.196 , p < 0.01 ) were negatively correlated as shown in the table 5 . spearman 's correlation coefficients between knowledge - attitude ( k - a ) , knowledge - practice ( k - p ) , and attitude - practice ( a - p ) practice scor riyadh is rapidly growing capital city with many extension areas built to accommodate the increasing population . many of the residents staying in such localities receive some health- and oral health - related advices from the pharmacists . these include financial limitations , nonavailability of hospitals or polyclinics nearby , busy schedule , and difficulty in making appointment with the dentist or physician . the pharmacist 's own knowledge , attitude , and oral self - care practices are major determinants for his / her role as an oral health promoter in a community setting . hence , this study disclosed the knowledge , attitude , and self - care practices toward oral health among a sample of pharmacists from riyadh , riyadh province , saudi arabia . the resent study revealed average oral health knowledge among a sample of pharmacists from riyadh , riyadh province , saudi arabia . this could be due to several factors such as the lack of the pharmacist 's education and training in oral health , lesser degree of motivation by pharmacists to know about oral health , lack of time , limited interaction with dental professionals , and lack of opportunities for professional development . additionally , it has been reported that 50% of the pharmacists never met the dental professionals practicing close to their pharmacies though 90% of the pharmacies were located near dental clinics . a similar finding of poor oral health knowledge has been reported among bachelor of pharmacy students from malaysia . in general , the possible explanation for such a finding could be that most of the male pharmacists work in community - based pharmacies in which many people seek oral health advice and information about oral health care products . on the contrary , female pharmacists predominantly work in hospital - based pharmacies dispensing mainly doctor - prescribed medications to a limited number of patients . this could have resulted in less exposure to oral health information by female pharmacists unlike community - based pharmacists . moreover , frequent interaction between oral health professionals and male pharmacists might be the reason for increased oral health knowledge among male pharmacists . another possible factor could be frequent contact between medical representatives and male pharmacists with the former explaining about the oral health care products . in saudi arabia , community pharmacies are either owned by a single person or attached to a network of pharmacies scattered across a city , province , or the country . most of the community pharmacies are managed by non - saudi pharmacists while saudi pharmacists prefer to work in the government sector . in the present study , non - saudi pharmacists showed significantly higher oral health knowledge as compared to saudi pharmacists . the probable reason could have been a higher content of oral health information in pharmacy education in the institutes from which non - saudis graduated . in the present study , it can be speculated that the average oral health knowledge among pharmacists could be the main reason for such a finding . in the present study , a significant positive correlation between the pharmacist 's knowledge and practice was observed . from this finding , it can be interpreted that lack of knowledge adversely influences the attitude leading to poor oral self - care practices of the pharmacists . however , higher oral health knowledge has a direct impact on oral self - care practices by improving the individual 's self - awareness , self - protection , and personal hygiene performances . the strengths of the present study was a sufficient sample size and the varied oral health - related questions designed to disclose an existing level of oral health knowledge , attitude , and self - care practices . the limitations of the present study include the lack of a standard questionnaire for measuring the oral health knowledge and the nonavailability of any reported comparable study instrument . therefore , the results of the present study were compared to the oral health knowledge of other health professionals . moreover , the results of present study rely on self - reported data ; the oral health information may have been biased through over- and underreporting due to social desirability . dental publications offer conflicting results on the impact of the oral health knowledge , attitude , and self - care practices , and oral diseases . however , gathering such data has been useful in planning an oral health education program targeted toward pharmacists . there is scarce data available regarding oral health knowledge of pharmacists toward their own oral health knowledge , attitudes , and practices . to develop a sound strategy for improving the oral health of pharmacists , for this , additional studies are needed to be conducted in a wide geographic area , preferably by considering a nationally representative sample of pharmacists from saudi arabia by using reliable and indigenously developed measures . with enhanced oral health knowledge and practices , pharmacists can serve as oral health promoters in community pharmacy setups by catering to the oral health needs of the society . pharmacists who were considered in the present study demonstrated an average knowledge , negative attitude , and inadequate self - care practices toward oral health . however , increasing oral health knowledge can have a profound improvement in oral self - care practices . hence , there is a need to increase the basic oral health knowledge of the pharmacist to improve his / her own oral self - care practices and empower him / her to be utilized as a member of the oral health care team , thereby enabling him / her to provide evidence - based advice and guidance to clients .

this randomized , examinerblind , paralleldesign , controlled , singlecentre study was conducted in the usa between 1 november and 2 december 2011 . the primary objective was to compare the efficacy in reducing gingivitis and plaque of an experimental mouthrinse containing 0.15% lae with that of a mouthrinse containing 5% hydroalcohol ( negative control ) after 4 weeks of use as adjuncts to tooth brushing . the secondary objective was to compare the efficacy of these mouthrinses in reducing gingivitis and plaque after 2 weeks use and their ability to reduce gingival bleeding after 2 and 4 weeks use . the study was conducted in accordance with the protocol , the abbreviated investigational device exemption regulations ( 21 cfr part 812 ) , international conference on harmonisation harmonised tripartite guideline for good clinical practice ( 1996 ) , the declaration of helsinki ( 2000 ) and applicable local regulatory requirements and laws . , subjects made three visits to the clinic : on day 1 ( screening / baseline visit 1 ) , day 15 1 day ( visit 2 ) and day 29 1 day ( visit 3 ) . subjects were required to refrain from oral hygiene practices for 1218 h and from eating , drinking or smoking for 4 h before study visits . subjects were to refrain from using unassigned oral care products ( including interdental cleaning devices except for the removal of impacted food ) or having any dental work done ( except for emergency procedures ) throughout the study . subjects received test materials in blinded packaging , although taste differences were perceivable upon product use . the sponsor provided blinded test materials ; personnel dispensing the test products or supervising their use did not participate in the examination of subjects . at the screening visit , qualifying subjects provided written informed consent before their participation in the study . subjects completed a medical / dental history questionnaire and underwent an oral examination and evaluation for plaque , gingivitis and bleeding . subjects then received dental prophylaxis to remove plaque ( confirmed by disclosure ) , stains and calculus . the subjects were randomly assigned to one of two treatment groups to receive either an experimental 0.15% laecontaining mouthrinse or a 5% hydroalcohol negativecontrol mouthrinse ( both manufactured by johnson & johnson healthcare products division of mcneilppc inc . , skillman , nj , usa ) . subjects were assigned a unique randomization number , allocated sequentially by site staff , based on a randomization schedule provided by the study sponsor . all subjects received a standard fluoride toothpaste ( colgate cavity protection toothpaste ; manufactured by colgate palmolive company , new york , ny , usa ) and a softbristled toothbrush ( reach advanced design toothbrush ; distributed by johnson & johnson healthcare products division of mcneilppc , inc . ) , instruction on oral hygiene , and diary cards . subjects were instructed to brush their teeth twice daily in their usual manner and to rinse for 30 s after each brushing with 20 ml of their assigned mouthrinse . use of the mouthrinse was supervised at visits 1 and 2 . at visits 2 and 3 subjects subjects compliance with studyproduct usage instructions was assessed by review of their completed diary cards and by collecting and weighing mouthrinse bottles at visits 2 and 3 . three indices were used to assess clinical efficacy : the turesky modification of the quigleyhein plaque index ( pi ; turesky et al . 1982 ) , the modified gingival index ( mgi ; lobene et al . 1986 ) and the bleeding index ( bi ; ainamo & bay 1975 , saxton & van der ouderaa 1989 ) . the coprimary endpoints of the study were wholemouth mean pi score at visit 3 ( week 4 ) and wholemouth mean mgi score at visit 3 . secondary endpoints included wholemouth mean pi and mgi scores at visit 2 ( week 2 ) ; wholemouth mean bi scores at visits 2 and 3 ; and microbiological absolute and log counts for oral microbes derived from plaque samples collected at visit 1 and visit 3 . men and women aged 18 years and in good general health with signs of adequate oral hygiene ( i.e. daily tooth brushing and no signs of oral neglect ) were eligible for inclusion in the study . all subjects had to have : 20 natural teeth with scorable surfaces ; a mean mgi score 1.95 ; a baseline mean pi score 1.95 for overnight plaque accumulation ; and an absence of significant oral soft tissue pathology , periodontitis or extensive subgingival calculus . exclusion criteria included : a history of significant adverse events following use of oral hygiene products ; conditions requiring prophylactic use of antibiotics before dental surgery according to united states clinical practice ; use of antibiotics , antiinflammatory or anticoagulant therapy or any medication that might interfere with efficacy evaluations in the 4 weeks before the study ; regular use of antiplaque/gingivitis dental products within 2 weeks of the study ; and any severe acute or chronic medical condition or laboratory abnormality that might pose a risk to the participant or interfere with interpretation of the results of the study . subjects were assessed for adverse events , gingivitis , gingival bleeding and plaque , in that order . plaque accumulation was scored at all visits using the pi ( turesky et al . 1982 ) for six surfaces ( distobuccal , midbuccal , mesiobuccal , distolingual , midlingual and mesiolingual ) of all scorable teeth after disclosing ( 0 : no plaque ; 1 : separate flecks or discontinuous band of plaque at the gingival margin ; 2 : thin [ up to 1 mm ] continuous band of plaque at the gingival margin ; 3 : band of plaque wider than 1 mm but less than onethird of surface ; 4 : plaque covering more than onethird but less than twothirds of surface ; 5 : plaque covering more than twothirds of surface ) . gingivitis was assessed at all visits on the buccal and lingual marginal gingivae and interdental papillae of all scorable teeth using the mgi ( 0 : normal ; 1 : mild inflammation of any point of the gingival unit ; 2 : mild inflammation of the entire gingival unit ; 3 : moderate inflammation of the gingival unit ; 4 : severe inflammation of the gingival unit ; lobene et al . gingival bleeding was assessed at all visits . a periodontal probe ( 0.5 mm diameter tip ) was inserted into the gingival crevice and swept distal to mesial at a 60 angle while maintaining contact with the sulcular epithelium . four areas around each tooth were assessed ( distobuccal , midbuccal , midlingual and mesiolingual ) . bleeding was recorded using the gingival bi ( 0 : absence after 30 s ; 1 : bleeding after 30 s ; 2 : immediate bleeding ; ainamo & bay 1975 , saxton & van der ouderaa 1989 ) 30 s after an entire surface ( e.g. , buccal ) in each quadrant was probed . plaque samples were collected , using a sterile curette , from the buccal surfaces of teeth 28 in the upper right quadrant by a trained dental professional at visits 1 and 3 following the clinical assessments . microbiological assessment of the plaque samples using dna : dna hybridization was conducted to assess shifts in the oral microflora . dna isolated from the bacteria in the plaque samples was fixed in lanes to nylon membranes using a minislot 60 device ( immunetics , cambridge , ma , usa ) and hybridized by checkerboard hybridization ( socransky et al . 2004 ) in a miniblotter 45 ( immunetics ) to digoxigeninlabelled whole genomic dna probes for 41 species . the bacterial species recognized by the probes were categorized as shown in table 1 . dna probes were detected using an antibody to digoxigenin conjugated with alkaline phosphatase , and chemifluorescence detection . signals were detected using attophos substrate ( amersham life sciences , arlington heights , il , usa ) and read with a storm fluoroimager ( molecular dynamics , sunnyvale , ca , usa ) . standards for each species were included at a concentration of 10 and 10 cells on the membrane as controls . the assay sensitivity was adjusted to permit detection of 10 cells of a given species by adjusting the concentration of the dna probe . signals were converted to absolute counts by comparison with the standards on the same membrane . bacterial species detected by genomic dna probes in dna : dna hybridization analysis of dental plaque samples oral examinations of the buccal and sublingual mucosa , lips / labial mucosa , mucobuccal fold , gingiva , tongue , hard and soft palate , uvula , oropharynx , teeth and dental restorations were conducted at each visit to monitor oral tolerability . emergent or worsening adverse events were recorded at each recall visit , after a review of medical history and a thorough examination of the oral cavity . abnormalities , such as lip bites , food burns and traumatic ulcers , that were not considered clinically significant by the investigator were not recorded as adverse events . the safety analysis was conducted on all randomized subjects who used at least one dose of the study products . the number and proportion of subjects experiencing adverse events throughout the study period were summarized according to the medical dictionary for regulatory activities system organ class and preferred term version 14.1 . the planned sample size of 40 subjects in each treatment group was based on estimates of standard deviations and means from a 4week pilot study , and provides 95% power to detect a difference in means of 0.102 ( assuming a standard deviation of 0.125 ) for wholemouth mean pi and wholemouth mean mgi at the 0.05 level of significance ( twosided ) . demographics and baseline characteristics were compared betweenv treatment groups using analysis of variance or a chisquared test . fisher 's exact test was used instead of a chisquared test if the number of subjects was sufficiently small . the primary and secondary efficacy analyses were based on the full analysis set ( i.e. randomized subjects who used at least one dose of study product and had at least one postbaseline efficacy assessment ) using an analysis of covariance with treatment as a factor and corresponding baseline value as the covariate . microbiological counts for each category of oral microbe were reported using summary statistics . for this study , data imputations were not performed as the number of missing data was expected to be negligible . this randomized , examinerblind , paralleldesign , controlled , singlecentre study was conducted in the usa between 1 november and 2 december 2011 . the primary objective was to compare the efficacy in reducing gingivitis and plaque of an experimental mouthrinse containing 0.15% lae with that of a mouthrinse containing 5% hydroalcohol ( negative control ) after 4 weeks of use as adjuncts to tooth brushing . the secondary objective was to compare the efficacy of these mouthrinses in reducing gingivitis and plaque after 2 weeks use and their ability to reduce gingival bleeding after 2 and 4 weeks use . the study was conducted in accordance with the protocol , the abbreviated investigational device exemption regulations ( 21 cfr part 812 ) , international conference on harmonisation harmonised tripartite guideline for good clinical practice ( 1996 ) , the declaration of helsinki ( 2000 ) and applicable local regulatory requirements and laws . , subjects made three visits to the clinic : on day 1 ( screening / baseline visit 1 ) , day 15 1 day ( visit 2 ) and day 29 1 day ( visit 3 ) . subjects were required to refrain from oral hygiene practices for 1218 h and from eating , drinking or smoking for 4 h before study visits . subjects were to refrain from using unassigned oral care products ( including interdental cleaning devices except for the removal of impacted food ) or having any dental work done ( except for emergency procedures ) throughout the study . subjects received test materials in blinded packaging , although taste differences were perceivable upon product use . the sponsor provided blinded test materials ; personnel dispensing the test products or supervising their use did not participate in the examination of subjects . at the screening visit , qualifying subjects provided written informed consent before their participation in the study . subjects completed a medical / dental history questionnaire and underwent an oral examination and evaluation for plaque , gingivitis and bleeding . subjects then received dental prophylaxis to remove plaque ( confirmed by disclosure ) , stains and calculus . the subjects were randomly assigned to one of two treatment groups to receive either an experimental 0.15% laecontaining mouthrinse or a 5% hydroalcohol negativecontrol mouthrinse ( both manufactured by johnson & johnson healthcare products division of mcneilppc inc . , skillman , nj , usa ) . subjects were assigned a unique randomization number , allocated sequentially by site staff , based on a randomization schedule provided by the study sponsor . all subjects received a standard fluoride toothpaste ( colgate cavity protection toothpaste ; manufactured by colgate palmolive company , new york , ny , usa ) and a softbristled toothbrush ( reach advanced design toothbrush ; distributed by johnson & johnson healthcare products division of mcneilppc , inc . ) , instruction on oral hygiene , and diary cards . subjects were instructed to brush their teeth twice daily in their usual manner and to rinse for 30 s after each brushing with 20 ml of their assigned mouthrinse . use of the mouthrinse was supervised at visits 1 and 2 . at visits 2 and 3 subjects subjects compliance with studyproduct usage instructions was assessed by review of their completed diary cards and by collecting and weighing mouthrinse bottles at visits 2 and 3 . three indices were used to assess clinical efficacy : the turesky modification of the quigleyhein plaque index ( pi ; turesky et al . 1982 ) , the modified gingival index ( mgi ; lobene et al . 1986 ) and the bleeding index ( bi ; ainamo & bay 1975 , saxton & van der ouderaa 1989 ) . the coprimary endpoints of the study were wholemouth mean pi score at visit 3 ( week 4 ) and wholemouth mean mgi score at visit 3 . secondary endpoints included wholemouth mean pi and mgi scores at visit 2 ( week 2 ) ; wholemouth mean bi scores at visits 2 and 3 ; and microbiological absolute and log counts for oral microbes derived from plaque samples collected at visit 1 and visit 3 . men and women aged 18 years and in good general health with signs of adequate oral hygiene ( i.e. daily tooth brushing and no signs of oral neglect ) were eligible for inclusion in the study . all subjects had to have : 20 natural teeth with scorable surfaces ; a mean mgi score 1.95 ; a baseline mean pi score 1.95 for overnight plaque accumulation ; and an absence of significant oral soft tissue pathology , periodontitis or extensive subgingival calculus . exclusion criteria included : a history of significant adverse events following use of oral hygiene products ; conditions requiring prophylactic use of antibiotics before dental surgery according to united states clinical practice ; use of antibiotics , antiinflammatory or anticoagulant therapy or any medication that might interfere with efficacy evaluations in the 4 weeks before the study ; regular use of antiplaque/gingivitis dental products within 2 weeks of the study ; and any severe acute or chronic medical condition or laboratory abnormality that might pose a risk to the participant or interfere with interpretation of the results of the study . subjects were assessed for adverse events , gingivitis , gingival bleeding and plaque , in that order . plaque accumulation was scored at all visits using the pi ( turesky et al . 1982 ) for six surfaces ( distobuccal , midbuccal , mesiobuccal , distolingual , midlingual and mesiolingual ) of all scorable teeth after disclosing ( 0 : no plaque ; 1 : separate flecks or discontinuous band of plaque at the gingival margin ; 2 : thin [ up to 1 mm ] continuous band of plaque at the gingival margin ; 3 : band of plaque wider than 1 mm but less than onethird of surface ; 4 : plaque covering more than onethird but less than twothirds of surface ; 5 : plaque covering more than twothirds of surface ) . gingivitis was assessed at all visits on the buccal and lingual marginal gingivae and interdental papillae of all scorable teeth using the mgi ( 0 : normal ; 1 : mild inflammation of any point of the gingival unit ; 2 : mild inflammation of the entire gingival unit ; 3 : moderate inflammation of the gingival unit ; 4 : severe inflammation of the gingival unit ; lobene et al . gingival bleeding was assessed at all visits . a periodontal probe ( 0.5 mm diameter tip ) was inserted into the gingival crevice and swept distal to mesial at a 60 angle while maintaining contact with the sulcular epithelium . four areas around each tooth were assessed ( distobuccal , midbuccal , midlingual and mesiolingual ) . bleeding was recorded using the gingival bi ( 0 : absence after 30 s ; 1 : bleeding after 30 s ; 2 : immediate bleeding ; ainamo & bay 1975 , saxton & van der ouderaa 1989 ) 30 s after an entire surface ( e.g. , buccal ) in each quadrant was probed . plaque samples were collected , using a sterile curette , from the buccal surfaces of teeth 28 in the upper right quadrant by a trained dental professional at visits 1 and 3 following the clinical assessments . microbiological assessment of the plaque samples using dna : dna hybridization was conducted to assess shifts in the oral microflora . dna isolated from the bacteria in the plaque samples was fixed in lanes to nylon membranes using a minislot 60 device ( immunetics , cambridge , ma , usa ) and hybridized by checkerboard hybridization ( socransky et al . 2004 ) in a miniblotter 45 ( immunetics ) to digoxigeninlabelled whole genomic dna probes for 41 species . the bacterial species recognized by the probes were categorized as shown in table 1 . dna probes were detected using an antibody to digoxigenin conjugated with alkaline phosphatase , and chemifluorescence detection . signals were detected using attophos substrate ( amersham life sciences , arlington heights , il , usa ) and read with a storm fluoroimager ( molecular dynamics , sunnyvale , ca , usa ) . standards for each species were included at a concentration of 10 and 10 cells on the membrane as controls . the assay sensitivity was adjusted to permit detection of 10 cells of a given species by adjusting the concentration of the dna probe . signals were converted to absolute counts by comparison with the standards on the same membrane . bacterial species detected by genomic dna probes in dna : dna hybridization analysis of dental plaque samples oral examinations of the buccal and sublingual mucosa , lips / labial mucosa , mucobuccal fold , gingiva , tongue , hard and soft palate , uvula , oropharynx , teeth and dental restorations were conducted at each visit to monitor oral tolerability . emergent or worsening adverse events were recorded at each recall visit , after a review of medical history and a thorough examination of the oral cavity . abnormalities , such as lip bites , food burns and traumatic ulcers , that were not considered clinically significant by the investigator were not recorded as adverse events . the safety analysis was conducted on all randomized subjects who used at least one dose of the study products . the number and proportion of subjects experiencing adverse events throughout the study period were summarized according to the medical dictionary for regulatory activities system organ class and preferred term version 14.1 . the planned sample size of 40 subjects in each treatment group was based on estimates of standard deviations and means from a 4week pilot study , and provides 95% power to detect a difference in means of 0.102 ( assuming a standard deviation of 0.125 ) for wholemouth mean pi and wholemouth mean mgi at the 0.05 level of significance ( twosided ) . demographics and baseline characteristics were compared betweenv treatment groups using analysis of variance or a chisquared test . fisher 's exact test was used instead of a chisquared test if the number of subjects was sufficiently small . the primary and secondary efficacy analyses were based on the full analysis set ( i.e. randomized subjects who used at least one dose of study product and had at least one postbaseline efficacy assessment ) using an analysis of covariance with treatment as a factor and corresponding baseline value as the covariate . microbiological counts for each category of oral microbe were reported using summary statistics . for this study , data imputations were not performed as the number of missing data was expected to be negligible . eightyseven subjects were randomized to the two study treatments : 43 to receive the 0.15% lae mouthrinse and 44 to receive the negative control . one subject in the control group experienced a serious adverse event ( testicular swelling , unrelated to the mouthrinse ) and discontinued study treatment . the demographic and baseline variables of the two study groups are shown in table 2 ; no statistically significant differences were noted between the groups . demographics and baseline characteristics ( all randomized subjects ) bi , bleeding index ; lae , ethyl lauroyl arginate ; mgi , modified gingival index ; pi , plaque index ; sd , standard deviation . the 0.15% laecontaining mouthrinse was associated with a statistically significantly greater reduction from baseline in the wholemouth mean pi score compared with the control mouthrinse , with a betweentreatment difference in the least squares means of 1.23 ( 95% confidence interval [ 95% ci ] : 1.07 , 1.39 ) , equating to a 42.6% greater reduction versus control ( p < 0.001 ; table 3 ) . the reduction in the wholemouth mean mgi score was also statistically significantly greater with the lae mouthrinse ( difference : 0.23 [ 95% ci : 0.19 , 0.28 ] ; 10.7% reduction compared with the control ; p < 0.001 ) . statistically significant differences in wholemouth mean pi and mgi scores between the 0.15% lae mouthrinse and control mouthrinse , in favour of the lae mouthrinse , were also evident after 2 weeks of treatment ( table 3 ) . wholemouth mean pi , mgi and bi scores ( full analysis set ) p < 0.001 versus control ( based on analysis of covariance model ) . bi , bleeding index ; ci , confidence interval ; lae , ethyl lauroyl arginate ; mgi , modified gingival index ; pi , plaque index ; sd , standard deviation ; se , standard error . in the gingival bleeding assessment ( table 3 ) , the 0.15% laecontaining mouthrinse was associated with statistically significantly greater reductions from baseline in wholemouth mean bi score ( p < 0.001 ) at both weeks 2 and 4 compared with the control . at week 2 , a 36.3% reduction in the proportion of bleeding sites was observed compared with the control group ( 0.077 versus 0.121 ; difference 0.04 [ 95% ci : 0.03 , 0.06 ] ) . at week 4 , a 50.9% reduction in the proportion of bleeding sites was observed compared with the control group ( 0.058 versus 0.119 ; difference 0.06 [ 95% ci : 0.04 , 0.08 ] ) . all bleeding scores were either 0 or 1 in both treatment groups across all study visits . therefore , in this study , the mean scores were equivalent to proportions of bleeding sites . microbiological analysis of the plaque samples collected at baseline and week 4 ( visit 3 ) revealed no significant compositional changes in the oral microflora . total microbiological counts ( log10 ) of complexes at baseline and week 4 were similar for both treatment groups , with most differences within 0.5 log ( fig . proportions of each complex were also similar at baseline and week 4 ( fig . 2b ) , with the exception of the purple complex , which was slightly less common at week 4 than at baseline following treatment with both the laecontaining mouthrinse ( 4.19% versus 5.43% respectively ) and the control mouthrinse ( 5.12% versus 7.86% respectively ) ; and the orange complex , which was slightly more common at week 4 than at baseline ( 23.82% versus 16.87% respectively ) in the laecontaining mouthrinse group . microbiological analysis of supragingival plaque collected at baseline and after 4 weeks of treatment with 0.15% laecontaining mouthrinse or 0.5% hydroalcohol control mouthrinse . ( a ) total microbiological counts ( log10 ) for each complex of bacterial species ; ( b ) summary of percentage of counts represented by each complex . for details of bacterial species detected by each category of probe , see table 1 . no adverse events related to oral soft tissue were recorded during the study in either treatment group . one subject who received the control mouthrinse experienced a serious adverse event of testicular swelling requiring hospitalization and discontinued from the study . this was related to testicular cancer and not to the use of the control mouthrinse . no incidences of tooth staining were recorded as adverse events . in both groups there were minor protocol deviations , but no violations associated with subject compliance . the findings of this 4week randomized controlled study show that twicedaily use of an experimental 0.15% laecontaining mouthrinse as an adjunct to tooth brushing resulted in statistically significant reductions from baseline in plaque accumulation , gingivitis and bleeding after 2 and 4 weeks of use in subjects with mildtomoderate gingivitis . plaque was reduced by 42.6% ( difference in least squares means of scores : 1.23 [ 95% ci : 1.07 , 1.39 ] relative to the negative control by week 4 , and gingivitis reduced by 10.7% ( difference 0.23 [ 95% ci : 0.19 , 0.28 ] ) . notably , a reduction in bleeding of 50.9% versus the negative control was evident after 4 weeks of use ( difference 0.06 [ 95% ci : 0.04 , 0.08 ] ) . the 0.15% laecontaining mouthrinse was well tolerated , with no adverse events affecting the oral soft tissue . microbiological analysis of plaque samples obtained throughout the study indicated no microbial shift in the oral microflora tested , suggesting that there are no safety concerns with use of the 0.15% laecontaining mouthrinse when used over a 4week period . a longer study ( e.g. 6 months ) would provide more information on the efficacy and safety of 0.15% laecontaining mouthrinse when used over the long term . in addition , no formal assessment of tooth staining or calculus was included in this study . the effects of the mouthrinse are consistent with its proposed action of reducing the adhesion of dental plaque to the dental pellicle ( giertsen et al . 2007 ) . in a previous in situ study , in which subjects wore acrylic appliances containing proteincoated discs on the buccal surface of their teeth , use of a 0.5% laecontaining mouthrinse threetimes daily significantly reduced the total number of bacteria and most of the taxa tested in plaque collected from the discs ( giertsen et al . 2007 ) . the control of plaque , and the subsequent reduction in gingivitis , is a key goal in the maintenance of gingival and oral health ( american academy of periodontology 2005 ) . many factors influence oral health ; some are controllable , e.g. toothbrushing time , frequency and duration , while others are difficult to control or change , e.g. host response , motivation and dexterity . for example , of those individuals who use dental floss , less than half utilize it correctly ( lang et al . the efforts of dentists and hygienists to improve their patients oral hygiene habits could be significantly assisted by the use of adjunctive oral care products , which are easy to use and help to mitigate the compliance / technique issues associated with interdental cleaning ( van der ouderaa 1991 , warren & chater 1996 ) . ideally , an oral antiplaque agent should prevent biofilm formation yet have no adverse effects on the oral microflora . in this study , the negligible changes in plaque and gingivitis levels in the control group effectively confirmed the lack of change in the subjects mechanical plaquecontrol practices . any meaningful change in these levels ( worsening or improvement ) would suggest a change from their prestudy habits ; worsening in the control group would suggest that study instructions inhibited subjects usual oral hygiene practices , while improvement in the control group would likely suggest greater compliance with the mechanical regimen the fact that the control group started and completed the study with significant ( and nearly identical ) levels of plaque and gingivitis indicates that , in this population , brushing twice daily in their usual manner was not sufficient . the results of this study thus demonstrate that mechanical oral hygiene alone does not adequately improve the gingival health of individuals with gingivitis . it is well recognized that mechanical cleaning is the most important component of oral hygiene ; however , it only appears to maintain control at a certain level of plaque and gingivitis . therefore , to improve upon an individual 's baseline level , a mouthrinse is a useful adjunct to mechanical home care . however , as with any therapeutic intervention , adequate compliance with recommended product usage is of paramount importance . it is recognized that compliance with rinsing twice a day , to achieve the added benefit , may represent a challenge to some individuals . therapeutic components of oral care rinses have been shown to be effective in reducing gingivitis in numerous clinical trials and systematic reviews ( wu & savitt 2002 , zimmer et al . more generalized use of chlorhexidinecontaining mouthrinses for plaque and gingivitis control is limited by the potential for poor compliance due to tooth staining and calculus formation with daily use ( charles et al . there is therefore a need for dailyuse products with comparable efficacy but which lack these side effects . the results of this study suggest that the laecontaining mouthrinse may be a suitable alternative adjunctive treatment for the control of mildtomoderate gingivitis in adults . the mechanism of action of lae differs from chlorhexidine in that lae has a physical effect by preventing attachment of bacteria to the pellicle ( giertsen et al . 2007 ) rather than bactericidal or bacteriostatic effects . the surfaceactive compound delmopinol , which is available as an antimicrobial mouthrinse , also binds to the pellicle and prevents adherence of dental plaque ( vassilakos et al . this study show that a mouthrinse containing 0.15% lae used as an adjunct to tooth brushing was effective in the reduction of plaque , gingivitis and bleeding at both 2 and 4 weeks of use in subjects with mildtomoderate gingivitis , and was well tolerated .

abstractaimthis 4week , singlecentre , randomized , examinerblind , controlled study investigated the efficacy and safety of 0.15% ethyl lauroyl arginate ( lae)containing mouthrinse in adults with mildtomoderate gingivitis.material and methodssubjects were randomized to use 0.15% laecontaining mouthrinse or 5% hydroalcoholnegative control twice daily after brushing with standard fluoride toothpaste . plaque , gingivitis and bleeding were assessed at baseline and weeks 2 and 4 . the oral microflora was analysed at baseline and week 4.resultseightyseven subjects were randomized to treatment . the 0.15% laecontaining mouthrinse was associated with statistically significantly ( p < 0.001 ) greater reductions in mean plaque and gingivitis scores versus the negative control at week 2 ( difference [ 95% confidence interval ] : plaque 0.83 [ 0.64 , 1.02 ] , 29.1% ; gingivitis 0.11 [ 0.07 , 0.14 ] , 4.8% ) and week 4 ( coprimary endpoints : plaque 1.23 [ 1.07 , 1.39 ] , 42.6% ; gingivitis 0.23 [ 0.19 , 0.28 ] , 10.7% ) . bleedingindex scores were significantly ( p < 0.001 ) reduced versus the control at weeks 2 ( by 0.04 [ 0.03 , 0.06 ] , 36.3% ) and 4 ( by 0.06 [ 0.04 , 0.08 ] , 50.9% ) . no shifts were detected in the oral microflora . there were no treatmentrelated adverse events.conclusionsthe 0.15% laecontaining mouthrinse was well tolerated and significantly reduced plaque , gingivitis and bleeding when used as an adjunct to tooth brushing for 4 weeks .

Materials and methods Study design Subjects Assessments Safety Statistical analyses Results Discussion

this randomized , examinerblind , paralleldesign , controlled , singlecentre study was conducted in the usa between 1 november and 2 december 2011 . the primary objective was to compare the efficacy in reducing gingivitis and plaque of an experimental mouthrinse containing 0.15% lae with that of a mouthrinse containing 5% hydroalcohol ( negative control ) after 4 weeks of use as adjuncts to tooth brushing . the secondary objective was to compare the efficacy of these mouthrinses in reducing gingivitis and plaque after 2 weeks use and their ability to reduce gingival bleeding after 2 and 4 weeks use . the study was conducted in accordance with the protocol , the abbreviated investigational device exemption regulations ( 21 cfr part 812 ) , international conference on harmonisation harmonised tripartite guideline for good clinical practice ( 1996 ) , the declaration of helsinki ( 2000 ) and applicable local regulatory requirements and laws . , subjects made three visits to the clinic : on day 1 ( screening / baseline visit 1 ) , day 15 1 day ( visit 2 ) and day 29 1 day ( visit 3 ) . subjects were required to refrain from oral hygiene practices for 1218 h and from eating , drinking or smoking for 4 h before study visits . subjects were to refrain from using unassigned oral care products ( including interdental cleaning devices except for the removal of impacted food ) or having any dental work done ( except for emergency procedures ) throughout the study . subjects completed a medical / dental history questionnaire and underwent an oral examination and evaluation for plaque , gingivitis and bleeding . the subjects were randomly assigned to one of two treatment groups to receive either an experimental 0.15% laecontaining mouthrinse or a 5% hydroalcohol negativecontrol mouthrinse ( both manufactured by johnson & johnson healthcare products division of mcneilppc inc . all subjects received a standard fluoride toothpaste ( colgate cavity protection toothpaste ; manufactured by colgate palmolive company , new york , ny , usa ) and a softbristled toothbrush ( reach advanced design toothbrush ; distributed by johnson & johnson healthcare products division of mcneilppc , inc . ) subjects were instructed to brush their teeth twice daily in their usual manner and to rinse for 30 s after each brushing with 20 ml of their assigned mouthrinse . use of the mouthrinse was supervised at visits 1 and 2 . 1986 ) and the bleeding index ( bi ; ainamo & bay 1975 , saxton & van der ouderaa 1989 ) . the coprimary endpoints of the study were wholemouth mean pi score at visit 3 ( week 4 ) and wholemouth mean mgi score at visit 3 . secondary endpoints included wholemouth mean pi and mgi scores at visit 2 ( week 2 ) ; wholemouth mean bi scores at visits 2 and 3 ; and microbiological absolute and log counts for oral microbes derived from plaque samples collected at visit 1 and visit 3 . daily tooth brushing and no signs of oral neglect ) were eligible for inclusion in the study . exclusion criteria included : a history of significant adverse events following use of oral hygiene products ; conditions requiring prophylactic use of antibiotics before dental surgery according to united states clinical practice ; use of antibiotics , antiinflammatory or anticoagulant therapy or any medication that might interfere with efficacy evaluations in the 4 weeks before the study ; regular use of antiplaque/gingivitis dental products within 2 weeks of the study ; and any severe acute or chronic medical condition or laboratory abnormality that might pose a risk to the participant or interfere with interpretation of the results of the study . subjects were assessed for adverse events , gingivitis , gingival bleeding and plaque , in that order . 1982 ) for six surfaces ( distobuccal , midbuccal , mesiobuccal , distolingual , midlingual and mesiolingual ) of all scorable teeth after disclosing ( 0 : no plaque ; 1 : separate flecks or discontinuous band of plaque at the gingival margin ; 2 : thin [ up to 1 mm ] continuous band of plaque at the gingival margin ; 3 : band of plaque wider than 1 mm but less than onethird of surface ; 4 : plaque covering more than onethird but less than twothirds of surface ; 5 : plaque covering more than twothirds of surface ) . gingival bleeding was assessed at all visits . four areas around each tooth were assessed ( distobuccal , midbuccal , midlingual and mesiolingual ) . microbiological assessment of the plaque samples using dna : dna hybridization was conducted to assess shifts in the oral microflora . dna isolated from the bacteria in the plaque samples was fixed in lanes to nylon membranes using a minislot 60 device ( immunetics , cambridge , ma , usa ) and hybridized by checkerboard hybridization ( socransky et al . dna probes were detected using an antibody to digoxigenin conjugated with alkaline phosphatase , and chemifluorescence detection . signals were detected using attophos substrate ( amersham life sciences , arlington heights , il , usa ) and read with a storm fluoroimager ( molecular dynamics , sunnyvale , ca , usa ) . emergent or worsening adverse events were recorded at each recall visit , after a review of medical history and a thorough examination of the oral cavity . the number and proportion of subjects experiencing adverse events throughout the study period were summarized according to the medical dictionary for regulatory activities system organ class and preferred term version 14.1 . the planned sample size of 40 subjects in each treatment group was based on estimates of standard deviations and means from a 4week pilot study , and provides 95% power to detect a difference in means of 0.102 ( assuming a standard deviation of 0.125 ) for wholemouth mean pi and wholemouth mean mgi at the 0.05 level of significance ( twosided ) . this randomized , examinerblind , paralleldesign , controlled , singlecentre study was conducted in the usa between 1 november and 2 december 2011 . the primary objective was to compare the efficacy in reducing gingivitis and plaque of an experimental mouthrinse containing 0.15% lae with that of a mouthrinse containing 5% hydroalcohol ( negative control ) after 4 weeks of use as adjuncts to tooth brushing . the secondary objective was to compare the efficacy of these mouthrinses in reducing gingivitis and plaque after 2 weeks use and their ability to reduce gingival bleeding after 2 and 4 weeks use . , subjects made three visits to the clinic : on day 1 ( screening / baseline visit 1 ) , day 15 1 day ( visit 2 ) and day 29 1 day ( visit 3 ) . subjects were required to refrain from oral hygiene practices for 1218 h and from eating , drinking or smoking for 4 h before study visits . subjects completed a medical / dental history questionnaire and underwent an oral examination and evaluation for plaque , gingivitis and bleeding . the subjects were randomly assigned to one of two treatment groups to receive either an experimental 0.15% laecontaining mouthrinse or a 5% hydroalcohol negativecontrol mouthrinse ( both manufactured by johnson & johnson healthcare products division of mcneilppc inc . subjects were assigned a unique randomization number , allocated sequentially by site staff , based on a randomization schedule provided by the study sponsor . all subjects received a standard fluoride toothpaste ( colgate cavity protection toothpaste ; manufactured by colgate palmolive company , new york , ny , usa ) and a softbristled toothbrush ( reach advanced design toothbrush ; distributed by johnson & johnson healthcare products division of mcneilppc , inc . ) subjects were instructed to brush their teeth twice daily in their usual manner and to rinse for 30 s after each brushing with 20 ml of their assigned mouthrinse . use of the mouthrinse was supervised at visits 1 and 2 . 1986 ) and the bleeding index ( bi ; ainamo & bay 1975 , saxton & van der ouderaa 1989 ) . the coprimary endpoints of the study were wholemouth mean pi score at visit 3 ( week 4 ) and wholemouth mean mgi score at visit 3 . secondary endpoints included wholemouth mean pi and mgi scores at visit 2 ( week 2 ) ; wholemouth mean bi scores at visits 2 and 3 ; and microbiological absolute and log counts for oral microbes derived from plaque samples collected at visit 1 and visit 3 . daily tooth brushing and no signs of oral neglect ) were eligible for inclusion in the study . exclusion criteria included : a history of significant adverse events following use of oral hygiene products ; conditions requiring prophylactic use of antibiotics before dental surgery according to united states clinical practice ; use of antibiotics , antiinflammatory or anticoagulant therapy or any medication that might interfere with efficacy evaluations in the 4 weeks before the study ; regular use of antiplaque/gingivitis dental products within 2 weeks of the study ; and any severe acute or chronic medical condition or laboratory abnormality that might pose a risk to the participant or interfere with interpretation of the results of the study . subjects were assessed for adverse events , gingivitis , gingival bleeding and plaque , in that order . four areas around each tooth were assessed ( distobuccal , midbuccal , midlingual and mesiolingual ) . plaque samples were collected , using a sterile curette , from the buccal surfaces of teeth 28 in the upper right quadrant by a trained dental professional at visits 1 and 3 following the clinical assessments . microbiological assessment of the plaque samples using dna : dna hybridization was conducted to assess shifts in the oral microflora . dna isolated from the bacteria in the plaque samples was fixed in lanes to nylon membranes using a minislot 60 device ( immunetics , cambridge , ma , usa ) and hybridized by checkerboard hybridization ( socransky et al . signals were detected using attophos substrate ( amersham life sciences , arlington heights , il , usa ) and read with a storm fluoroimager ( molecular dynamics , sunnyvale , ca , usa ) . emergent or worsening adverse events were recorded at each recall visit , after a review of medical history and a thorough examination of the oral cavity . abnormalities , such as lip bites , food burns and traumatic ulcers , that were not considered clinically significant by the investigator were not recorded as adverse events . the planned sample size of 40 subjects in each treatment group was based on estimates of standard deviations and means from a 4week pilot study , and provides 95% power to detect a difference in means of 0.102 ( assuming a standard deviation of 0.125 ) for wholemouth mean pi and wholemouth mean mgi at the 0.05 level of significance ( twosided ) . eightyseven subjects were randomized to the two study treatments : 43 to receive the 0.15% lae mouthrinse and 44 to receive the negative control . one subject in the control group experienced a serious adverse event ( testicular swelling , unrelated to the mouthrinse ) and discontinued study treatment . demographics and baseline characteristics ( all randomized subjects ) bi , bleeding index ; lae , ethyl lauroyl arginate ; mgi , modified gingival index ; pi , plaque index ; sd , standard deviation . the 0.15% laecontaining mouthrinse was associated with a statistically significantly greater reduction from baseline in the wholemouth mean pi score compared with the control mouthrinse , with a betweentreatment difference in the least squares means of 1.23 ( 95% confidence interval [ 95% ci ] : 1.07 , 1.39 ) , equating to a 42.6% greater reduction versus control ( p < 0.001 ; table 3 ) . the reduction in the wholemouth mean mgi score was also statistically significantly greater with the lae mouthrinse ( difference : 0.23 [ 95% ci : 0.19 , 0.28 ] ; 10.7% reduction compared with the control ; p < 0.001 ) . statistically significant differences in wholemouth mean pi and mgi scores between the 0.15% lae mouthrinse and control mouthrinse , in favour of the lae mouthrinse , were also evident after 2 weeks of treatment ( table 3 ) . wholemouth mean pi , mgi and bi scores ( full analysis set ) p < 0.001 versus control ( based on analysis of covariance model ) . bi , bleeding index ; ci , confidence interval ; lae , ethyl lauroyl arginate ; mgi , modified gingival index ; pi , plaque index ; sd , standard deviation ; se , standard error . in the gingival bleeding assessment ( table 3 ) , the 0.15% laecontaining mouthrinse was associated with statistically significantly greater reductions from baseline in wholemouth mean bi score ( p < 0.001 ) at both weeks 2 and 4 compared with the control . at week 2 , a 36.3% reduction in the proportion of bleeding sites was observed compared with the control group ( 0.077 versus 0.121 ; difference 0.04 [ 95% ci : 0.03 , 0.06 ] ) . at week 4 , a 50.9% reduction in the proportion of bleeding sites was observed compared with the control group ( 0.058 versus 0.119 ; difference 0.06 [ 95% ci : 0.04 , 0.08 ] ) . therefore , in this study , the mean scores were equivalent to proportions of bleeding sites . microbiological analysis of the plaque samples collected at baseline and week 4 ( visit 3 ) revealed no significant compositional changes in the oral microflora . total microbiological counts ( log10 ) of complexes at baseline and week 4 were similar for both treatment groups , with most differences within 0.5 log ( fig . proportions of each complex were also similar at baseline and week 4 ( fig . 2b ) , with the exception of the purple complex , which was slightly less common at week 4 than at baseline following treatment with both the laecontaining mouthrinse ( 4.19% versus 5.43% respectively ) and the control mouthrinse ( 5.12% versus 7.86% respectively ) ; and the orange complex , which was slightly more common at week 4 than at baseline ( 23.82% versus 16.87% respectively ) in the laecontaining mouthrinse group . microbiological analysis of supragingival plaque collected at baseline and after 4 weeks of treatment with 0.15% laecontaining mouthrinse or 0.5% hydroalcohol control mouthrinse . no adverse events related to oral soft tissue were recorded during the study in either treatment group . one subject who received the control mouthrinse experienced a serious adverse event of testicular swelling requiring hospitalization and discontinued from the study . this was related to testicular cancer and not to the use of the control mouthrinse . no incidences of tooth staining were recorded as adverse events . in both groups there were minor protocol deviations , but no violations associated with subject compliance . the findings of this 4week randomized controlled study show that twicedaily use of an experimental 0.15% laecontaining mouthrinse as an adjunct to tooth brushing resulted in statistically significant reductions from baseline in plaque accumulation , gingivitis and bleeding after 2 and 4 weeks of use in subjects with mildtomoderate gingivitis . plaque was reduced by 42.6% ( difference in least squares means of scores : 1.23 [ 95% ci : 1.07 , 1.39 ] relative to the negative control by week 4 , and gingivitis reduced by 10.7% ( difference 0.23 [ 95% ci : 0.19 , 0.28 ] ) . notably , a reduction in bleeding of 50.9% versus the negative control was evident after 4 weeks of use ( difference 0.06 [ 95% ci : 0.04 , 0.08 ] ) . the 0.15% laecontaining mouthrinse was well tolerated , with no adverse events affecting the oral soft tissue . microbiological analysis of plaque samples obtained throughout the study indicated no microbial shift in the oral microflora tested , suggesting that there are no safety concerns with use of the 0.15% laecontaining mouthrinse when used over a 4week period . 6 months ) would provide more information on the efficacy and safety of 0.15% laecontaining mouthrinse when used over the long term . in a previous in situ study , in which subjects wore acrylic appliances containing proteincoated discs on the buccal surface of their teeth , use of a 0.5% laecontaining mouthrinse threetimes daily significantly reduced the total number of bacteria and most of the taxa tested in plaque collected from the discs ( giertsen et al . the control of plaque , and the subsequent reduction in gingivitis , is a key goal in the maintenance of gingival and oral health ( american academy of periodontology 2005 ) . the efforts of dentists and hygienists to improve their patients oral hygiene habits could be significantly assisted by the use of adjunctive oral care products , which are easy to use and help to mitigate the compliance / technique issues associated with interdental cleaning ( van der ouderaa 1991 , warren & chater 1996 ) . ideally , an oral antiplaque agent should prevent biofilm formation yet have no adverse effects on the oral microflora . in this study , the negligible changes in plaque and gingivitis levels in the control group effectively confirmed the lack of change in the subjects mechanical plaquecontrol practices . any meaningful change in these levels ( worsening or improvement ) would suggest a change from their prestudy habits ; worsening in the control group would suggest that study instructions inhibited subjects usual oral hygiene practices , while improvement in the control group would likely suggest greater compliance with the mechanical regimen the fact that the control group started and completed the study with significant ( and nearly identical ) levels of plaque and gingivitis indicates that , in this population , brushing twice daily in their usual manner was not sufficient . it is well recognized that mechanical cleaning is the most important component of oral hygiene ; however , it only appears to maintain control at a certain level of plaque and gingivitis . therefore , to improve upon an individual 's baseline level , a mouthrinse is a useful adjunct to mechanical home care . more generalized use of chlorhexidinecontaining mouthrinses for plaque and gingivitis control is limited by the potential for poor compliance due to tooth staining and calculus formation with daily use ( charles et al . the results of this study suggest that the laecontaining mouthrinse may be a suitable alternative adjunctive treatment for the control of mildtomoderate gingivitis in adults . this study show that a mouthrinse containing 0.15% lae used as an adjunct to tooth brushing was effective in the reduction of plaque , gingivitis and bleeding at both 2 and 4 weeks of use in subjects with mildtomoderate gingivitis , and was well tolerated .

physical stimuli can significantly alter cellular behavior , giving rise to biochemical signals involved in molecular response.1 this process is called mechanotransduction , and the responsible structures sensitive to mechanical forces are most probably cytoskeleton elements.2 a number of studies have demonstrated that cells are sensitive to several kinds of physical cues ( shear stress , topography , mechanical deformation , etc ) , influencing cell migration,3 differentiation,4 and proliferation.5 among these stimuli , gravity is required for the correct development of land - based organisms , and in particular for the skeleton and for the muscle and nervous systems.6 an increasing amount of research is focused on the effects of gravity alterations on the physiological processes , but also on the possibility to exploit this stimulus as a potential therapeutic cue.79 as an example , improved regeneration of infarcted myocardium has been achieved after injection of stem cells differentiated following a 2 g hypergravity treatment , which enhanced the activities of cardiac marker mef-2 by promoting the nuclear export of histone deacetylase 5.10 chang et al investigated altered gravity effects on human lung adenocarcinoma , demonstrating the ability of simulated microgravity to decrease the metastatic potential of this tumor cell line.11 other researchers used microgravity stimulation as an approach for the development of a large amount of -cell spheroids , which once transplanted in mice are able to improve the symptoms of diabetes.12 among different tissues , bone is particularly affected by altered gravity conditions : evidence regarding bone regeneration suggests that hypergravity exposure conversely to microgravity , which negatively affects osteogenesis may enhance the osteogenic potential of osteoblast precursors.13 the ability of mesenchymal stem cells ( mscs ) to differentiate into osteoblasts is well known , but the osteogenic potential of mscs decreases with the prolonged culture duration necessary to obtain an appropriate number of cells for clinical applications.14 some countermeasures to this issue could come from nanotechnology , which proposes many different typologies of nanoparticles ( nps ) for stem cell labeling , tracking , delivery , and stimulation,15 including several examples of nanomaterials able to foster osteogenesis in mscs.1618 our group , as an example , successfully exploited barium titanate nps ( btnps ; figure 1a ) as a possible agent for the improvement of osteogenic differentiation of mscs.19 btnps belong to a class of ferroelectric materials showing high piezoelectricity,20 and with regard to biomedical applications , they demonstrate high cytocompatibility,21 excellent properties as nonlinear imaging probes,22 and the ability to deliver doxorubicin in cancer cells by improving drug uptake.23 moreover , as previously mentioned , btnps were proven to enhance the osteogenesis of mscs , as demonstrated by an increment of hydroxyapatite deposition . starting from these findings , we decided to develop a protocol for msc stimulation by combining incubation with btnps with treatment in hypergravity , with the goal of improving the differentiation process toward osteoblasts , and thus to obtain stem cells with enhanced osteogenic differentiation potential . 2013 campaign at the european space agency ( esa ; noordwijk , the netherlands ) , taking advantage of the large - diameter centrifuge for the hypergravity treatment . with the proposed experimental protocol , we had the aims of 1 ) investigating cell morphology and differentiation ( at gene , protein , and phenotype level ) following the combination of hypergravity treatment and btnp administration , and 2 ) evaluating np uptake by stem cells in altered gravity conditions , thus investigating the possibility of enhancing cellular internalization by simply exploiting an increased gravitational force . rat mscs ( scr027 ; millipore ) were used at the second passage in all the experiments . for the maintenance of mscs , the medium was composed of dulbecco s modified eagle s medium supplemented with 10% fetal bovine serum , 100 u / ml penicillin , 100 mg / ml streptomycin , and 200 mm glutamine ( all these reagents from gibco ) . cultures were maintained in an incubator at standard culture conditions ( 37c , 5% co2 , and 100% humidity ) . for all the experiments , mscs were trypsinized and seeded on glass slides ( diameter 13 mm ) 48 hours before hypergravity treatment at 10,000/cm for tests in proliferation conditions and at 30,000/cm for tests under osteogenic differentiation . osteogenesis was induced in the differentiation samples immediately before the hypergravity treatment by supplementing the medium with 100 nm dexamethasone , 200 m ascorbic acid 2-phosphate , and 10 mm glycerol 2-phosphate ( all these reagents from sigma - aldrich ) . some samples ( both proliferating and differentiating ) were moreover provided with btnps ( 20 g / ml ) , also in this case immediately before the hypergravity treatment . this dose was selected based on our previous results of an analysis of btnp effects on mscs , where 20 g / ml was found to be the optimal concentration at which nps did not negatively affect cellular functions.19 the nps , obtained from nanostructured and amorphous materials ( houston , tx , usa ) , were about 150 nm in radius , and were administered to the cell culture upon stabilization in gum arabic ( sigma - aldrich ) . images of the final dispersion of btnps were acquired after gold - sputtering by scanning electron microscopy through a dual - beam system ( fei helios 600 ; figure 1a ) . further details on np characterization have been previously reported.19 glass slides supporting the cell cultures were transferred in cylindrical vials filled with biocompatible polydimethylsiloxane ( 10:1 base : cross - linking agent ratio , curing temperature 60c ) , and fully covered with 800 l of the appropriate cell - culture medium to exclude shear - stress effects ( figure 1b ) . for the positioning of the samples , we used a delrin structure designed to support 30 cylindrical vials ( figure 1c ) . it is composed of four large rotating arms with a swing gondola at each extremity ( figure 1d ) , and can support hypergravity levels between 1 g and 20 g. we performed analyses on proliferating and differentiating rat mscs provided with and without 20 g / ml of btnps immediately before the altered gravity treatment , as previously described . the sample support was put in an incubator inside a gondola and accelerated at 20 g for 3 hours at 32c . at the end of the hypergravity stimulation , the proliferating samples were immediately processed for subsequent analysis , while differentiating samples were incubated for further 48 hours in differentiating conditions ( in incubator , 37c , 5% co2 ) before the assessment of osteogenesis . analogous experiments , as normal - gravity controls , were performed , maintaining cells at the same conditions of atmosphere and temperature for 3 hours at 1 g. all the procedures were performed in duplicate . in summary , we examined the following experimental classes : 1 ) proliferating cells at 1 g without btnps , 2 ) proliferating cells at 1 g with btnps , 3 ) proliferating cells at 20 g without btnps , 4 ) proliferating cells at 20 g with btnps , 5 ) differentiating cells at 1 g without btnps , 6 ) differentiating cells at 1 g with btnps , 7 ) differentiating cells at 20 g without btnps , and 8) differentiating cells at 20 g with btnps . immediately after the treatment , some proliferative samples were processed in order to evaluate the effects of hypergravity and btnps on msc adhesion and shape . to assess changes in morphology , cytoskeleton conformation , and adhesion , immunofluorescence with the cytoskeleton / focal cells were incubated for 45 minutes with a vinculin primary monoclonal antibody ( mouse antirat , diluted 1:100 in 10% goat serum in phosphate - buffered saline [ pbs ] ) after fixation with 4% paraformaldehyde in pbs for 20 minutes at 4c , permeabilization for 15 minutes with 0.1% triton x-100 ( sigma - aldrich ) , and treatment with a blocking solution ( 10% goat serum in pbs ) for 1 hour . thereafter , a staining solution composed of a green fluorescent labeled secondary antibody ( goat antimouse , diluted 1:50 in 10% goat serum ) , 100 m tetramethylrhodamine isothiocyanate phalloidin for f - actin labeling , and 1 m 4,6-diamidino-2-phenylindole ( dapi ) for nucleus counterstaining were added . samples were finally washed several times with pbs before observation under a confocal laser - scanning microscope ( c2s ; nikon ) . cell shape was analyzed on proliferative samples stained with coomassie brilliant blue ( 0.2% for 5 minutes ) . cell area and different shape descriptors were calculated with imagej software ( http://rsb.info.nih.gov/ij ) analyzing at least 50 well - distinct cells , acquired with an inverted optical microscope ( nikon eclipse ti ) . in particular , the solidity ( s ) , circularity ( c ) , and roundness ( r ) of cells were investigated according to the following equations:24 roundness = r = ab(1)where a and b are the width and length of the minimum bounding ( the smallest rectangle enclosing the selection ) , respectively , circularity = c=4ap2(2)where p is the perimeter and a is the cell area , and solidity = s = aconvexa(3)where convexa is the area enclosed by the smallest shell that borders all the points of the cell . np internalization has been investigated by a multimodal microscope with an in - plane resolution of approximately 300 nm and a resolution of 1 m along the optical axis . coherent anti - stokes raman scattering ( cars ) has been exploited to obtain images of cells , based on a degenerate pump - and - probe beam ( papb ) created by a ti sa pulsed laser ( chameleon vision ii ; coherent ) and a supercontinuum generator ( photonic crystal fiber scg-800 ; newport ) that produces a broadband stokes beam . the beams were chirped through their transmission by two sf6 glass blocks a 10 cm - long one for the papb , and a 15 cm - long one for the stokes radiation in order to optimize the spectral resolution . for the excitation of ch2 bonds at a raman shift of 2,850 cm and thus for the cell imaging , we adjusted the delay between the papb and the stokes beam . for the localization of btnps , an 806 nm papb was combined with stokes photons producing a sum - frequency generation ( sfg ) signal from btnps at approximately 452 nm . the images were subject to analysis with imagej software , and the amount of btnp internalization was calculated as the ratio of the area occupied by the nps inside the cells and the total cell area , on at least 50 cells for each experimental treatment . quantitative real - time reverse - transcription polymerase chain reaction ( qpcr ) was used to investigate the messenger ribonucleic acid ( mrna ) transcription of osteogenesis - marker genes ( runt related transcription factor 2 [ runx2 ] , collagen type i alpha-1 [ col1a1 ] , and alkaline phosphatase [ alpl ] ) and of rhoa ( ras homolog gene family , member a ) , which regulates osteogenesis - activating runx2 following mechanical stimulation.25 after the experimental procedures , cells were trypsinized and centrifuged , and the total rna was extracted from the samples with the high pure rna isolation kit ( roche ) according to the manufacturer s instructions . the quantity and purity of rna was verified through spectrophotometric analysis ( nanodrop ; thermo scientific ) . thereafter , complementary deoxyribonucleic acid was obtained from the reverse transcription of 100 ng of rna through iscript reverse transcription supermix ( bio - rad ) . the following protocol was used for the retrotranscription : 25c for 5 minutes , 42c for 45 minutes , 48c for 15 minutes , and finally 85c for 5 minutes . the amplification was carried out on the cfx connect real - time pcr detection system ( bio - rad ) thermocycler with the ssoadvanced sybr green supermix ( bio - rad ) . the temperature steps for the amplification reaction were : one cycle at 98c for 30 seconds , 40 cycles at 98c for 3 seconds , and 60c for 7 seconds , a temperature ramp from 65c to 95c , with 0.5c / second increments ( for melting curve generation ) . the housekeeping genes adopted were gusb and rpl19 ; the cycle threshold ( ct ) value relative to the control sample ( cultures performed at 1 g , without np treatment ) was considered as the reference for the calculation of ct ( difference between ct values deriving from difference between ct of target and housekeeping genes ) for the subsequent samples.26 primer sequences ( forward and reverse ) of the investigated genes are reported in table 1 . the analysis was focused on the main proteins involved in the early stage of osteogenesis ( alpl and col1 ; glyceraldehyde-3-phosphate dehydrogenase [ gapdh ] was adopted as the reference ) . for protein extraction , after trypsinization and centrifugation , cell pellets were lysed in 50 mm tris - hcl ( ph 7.6 ) , 2 mm ethylenediaminetetraacetic acid , 100 mm nacl , 1% nonidet p40 , and antiproteases 1 ( all these reagents from sigma ) . protein quantification was performed through the pierce bca protein assay kit ( thermo scientific ) following the manufacturer s protocol . twenty micrograms of protein for each sample was separated on a 4%15% mini - protean tgx stain - free gel ( bio - rad ) under reducing conditions , and then transferred to nitrocellulose membrane . the membranes were saturated for 45 minutes with 4% nonfat dry milk in pbs for blocking nonspecific binding sites , and then probed with primary antibodies against alpl ( rabbit antirat , diluted 1:8,000 , abcam biotechnology ) , col1 ( rabbit antirat , diluted 1:1,000 , abcam biotechnology ) , or gapdh ( mouse antirat , diluted 1:2,000 , abcam biotechnology ) overnight at 4c . finally , specific secondary horseradish peroxidase - conjugated anti - rabbit or antimouse antibodies ( kpl , final concentration 0.2 g / ml ) were used , and the immunocomplexes were detected by chemiluminescence ( ecl clarity ; bio - rad ) using the chemi - doc xrs+ system ( bio - rad ) . the intensity of the bands was quantified through the chemi - doc xrs+ software by adopting the 1 g sample values as reference for the relative expression and by normalizing to the gapdh values . alizarin red solution ( millipore ) was used to stain in orange red the calcium deposits of the msc cultures induced to differentiate . following paraformaldehyde fixation , cells were incubated with 500 l of alizarin red solution at room temperature for 30 minutes . after extensive washing steps with deionized water , cells were visualized in bright field under the optical microscope for image acquisition . imagej was used to detect and automatically quantify the size of alizarin red - positive areas for each experimental group , analyzing at least ten random fields per sample . all the described experiments were carried out at least in triplicate , and all procedures were replicated twice . with regard to image analysis , data were tested with the nonparametric kruskal each box plot is composed of whiskers indicating the minimum and maximum of the data , while the top and the bottom of the box are the first and third quartiles , respectively ; the band inside the box is the median . qpcr data were analyzed with bio - rad cfx manager software . in all cases , rat mscs ( scr027 ; millipore ) were used at the second passage in all the experiments . for the maintenance of mscs , the medium was composed of dulbecco s modified eagle s medium supplemented with 10% fetal bovine serum , 100 u / ml penicillin , 100 mg / ml streptomycin , and 200 mm glutamine ( all these reagents from gibco ) . cultures were maintained in an incubator at standard culture conditions ( 37c , 5% co2 , and 100% humidity ) . for all the experiments , mscs were trypsinized and seeded on glass slides ( diameter 13 mm ) 48 hours before hypergravity treatment at 10,000/cm for tests in proliferation conditions and at 30,000/cm for tests under osteogenic differentiation . osteogenesis was induced in the differentiation samples immediately before the hypergravity treatment by supplementing the medium with 100 nm dexamethasone , 200 m ascorbic acid 2-phosphate , and 10 mm glycerol 2-phosphate ( all these reagents from sigma - aldrich ) . some samples ( both proliferating and differentiating ) were moreover provided with btnps ( 20 g / ml ) , also in this case immediately before the hypergravity treatment . this dose was selected based on our previous results of an analysis of btnp effects on mscs , where 20 g / ml was found to be the optimal concentration at which nps did not negatively affect cellular functions.19 the nps , obtained from nanostructured and amorphous materials ( houston , tx , usa ) , were about 150 nm in radius , and were administered to the cell culture upon stabilization in gum arabic ( sigma - aldrich ) . images of the final dispersion of btnps were acquired after gold - sputtering by scanning electron microscopy through a dual - beam system ( fei helios 600 ; figure 1a ) . further details on np characterization have been previously reported.19 glass slides supporting the cell cultures were transferred in cylindrical vials filled with biocompatible polydimethylsiloxane ( 10:1 base : cross - linking agent ratio , curing temperature 60c ) , and fully covered with 800 l of the appropriate cell - culture medium to exclude shear - stress effects ( figure 1b ) . for the positioning of the samples , we used a delrin structure designed to support 30 cylindrical vials ( figure 1c ) . it is composed of four large rotating arms with a swing gondola at each extremity ( figure 1d ) , and can support hypergravity levels between 1 g and 20 g. we performed analyses on proliferating and differentiating rat mscs provided with and without 20 g / ml of btnps immediately before the altered gravity treatment , as previously described . the sample support was put in an incubator inside a gondola and accelerated at 20 g for 3 hours at 32c . at the end of the hypergravity stimulation , the proliferating samples were immediately processed for subsequent analysis , while differentiating samples were incubated for further 48 hours in differentiating conditions ( in incubator , 37c , 5% co2 ) before the assessment of osteogenesis . analogous experiments , as normal - gravity controls , were performed , maintaining cells at the same conditions of atmosphere and temperature for 3 hours at 1 g. all the procedures were performed in duplicate . in summary , we examined the following experimental classes : 1 ) proliferating cells at 1 g without btnps , 2 ) proliferating cells at 1 g with btnps , 3 ) proliferating cells at 20 g without btnps , 4 ) proliferating cells at 20 g with btnps , 5 ) differentiating cells at 1 g without btnps , 6 ) differentiating cells at 1 g with btnps , 7 ) differentiating cells at 20 g without btnps , and 8) differentiating cells at 20 g with btnps . immediately after the treatment , some proliferative samples were processed in order to evaluate the effects of hypergravity and btnps on msc adhesion and shape . to assess changes in morphology , cytoskeleton conformation , and adhesion , immunofluorescence with the cytoskeleton / focal adhesion staining kit ( millipore ) was performed . cells were incubated for 45 minutes with a vinculin primary monoclonal antibody ( mouse antirat , diluted 1:100 in 10% goat serum in phosphate - buffered saline [ pbs ] ) after fixation with 4% paraformaldehyde in pbs for 20 minutes at 4c , permeabilization for 15 minutes with 0.1% triton x-100 ( sigma - aldrich ) , and treatment with a blocking solution ( 10% goat serum in pbs ) for 1 hour . thereafter , a staining solution composed of a green fluorescent labeled secondary antibody ( goat antimouse , diluted 1:50 in 10% goat serum ) , 100 m tetramethylrhodamine isothiocyanate phalloidin for f - actin labeling , and 1 m 4,6-diamidino-2-phenylindole ( dapi ) for nucleus counterstaining were added . samples were finally washed several times with pbs before observation under a confocal laser - scanning microscope ( c2s ; nikon ) . cell shape was analyzed on proliferative samples stained with coomassie brilliant blue ( 0.2% for 5 minutes ) . cell area and different shape descriptors were calculated with imagej software ( http://rsb.info.nih.gov/ij ) analyzing at least 50 well - distinct cells , acquired with an inverted optical microscope ( nikon eclipse ti ) . in particular , the solidity ( s ) , circularity ( c ) , and roundness ( r ) of cells were investigated according to the following equations:24 roundness = r = ab(1)where a and b are the width and length of the minimum bounding ( the smallest rectangle enclosing the selection ) , respectively , circularity = c=4ap2(2)where p is the perimeter and a is the cell area , and solidity = s = aconvexa(3)where convexa is the area enclosed by the smallest shell that borders all the points of the cell . np internalization has been investigated by a multimodal microscope with an in - plane resolution of approximately 300 nm and a resolution of 1 m along the optical axis . coherent anti - stokes raman scattering ( cars ) has been exploited to obtain images of cells , based on a degenerate pump - and - probe beam ( papb ) created by a ti sa pulsed laser ( chameleon vision ii ; coherent ) and a supercontinuum generator ( photonic crystal fiber scg-800 ; newport ) that produces a broadband stokes beam . the beams were chirped through their transmission by two sf6 glass blocks a 10 cm - long one for the papb , and a 15 cm - long one for the stokes radiation in order to optimize the spectral resolution . for the excitation of ch2 bonds at a raman shift of 2,850 cm and thus for the cell imaging , we adjusted the delay between the papb and the stokes beam . for the localization of btnps , an 806 nm papb was combined with stokes photons producing a sum - frequency generation ( sfg ) signal from btnps at approximately 452 nm . the images were subject to analysis with imagej software , and the amount of btnp internalization was calculated as the ratio of the area occupied by the nps inside the cells and the total cell area , on at least 50 cells for each experimental treatment . quantitative real - time reverse - transcription polymerase chain reaction ( qpcr ) was used to investigate the messenger ribonucleic acid ( mrna ) transcription of osteogenesis - marker genes ( runt related transcription factor 2 [ runx2 ] , collagen type i alpha-1 [ col1a1 ] , and alkaline phosphatase [ alpl ] ) and of rhoa ( ras homolog gene family , member a ) , which regulates osteogenesis - activating runx2 following mechanical stimulation.25 after the experimental procedures , cells were trypsinized and centrifuged , and the total rna was extracted from the samples with the high pure rna isolation kit ( roche ) according to the manufacturer s instructions . the quantity and purity of rna was verified through spectrophotometric analysis ( nanodrop ; thermo scientific ) . thereafter , complementary deoxyribonucleic acid was obtained from the reverse transcription of 100 ng of rna through iscript reverse transcription supermix ( bio - rad ) . the following protocol was used for the retrotranscription : 25c for 5 minutes , 42c for 45 minutes , 48c for 15 minutes , and finally 85c for 5 minutes . the amplification was carried out on the cfx connect real - time pcr detection system ( bio - rad ) thermocycler with the ssoadvanced sybr green supermix ( bio - rad ) . the temperature steps for the amplification reaction were : one cycle at 98c for 30 seconds , 40 cycles at 98c for 3 seconds , and 60c for 7 seconds , a temperature ramp from 65c to 95c , with 0.5c / second increments ( for melting curve generation ) . the housekeeping genes adopted were gusb and rpl19 ; the cycle threshold ( ct ) value relative to the control sample ( cultures performed at 1 g , without np treatment ) was considered as the reference for the calculation of ct ( difference between ct values deriving from difference between ct of target and housekeeping genes ) for the subsequent samples.26 primer sequences ( forward and reverse ) of the investigated genes are reported in table 1 . the analysis was focused on the main proteins involved in the early stage of osteogenesis ( alpl and col1 ; glyceraldehyde-3-phosphate dehydrogenase [ gapdh ] was adopted as the reference ) . for protein extraction , after trypsinization and centrifugation , cell pellets were lysed in 50 mm tris - hcl ( ph 7.6 ) , 2 mm ethylenediaminetetraacetic acid , 100 mm nacl , 1% nonidet p40 , and antiproteases 1 ( all these reagents from sigma ) . protein quantification was performed through the pierce bca protein assay kit ( thermo scientific ) following the manufacturer s protocol . twenty micrograms of protein for each sample was separated on a 4%15% mini - protean tgx stain - free gel ( bio - rad ) under reducing conditions , and then transferred to nitrocellulose membrane . the membranes were saturated for 45 minutes with 4% nonfat dry milk in pbs for blocking nonspecific binding sites , and then probed with primary antibodies against alpl ( rabbit antirat , diluted 1:8,000 , abcam biotechnology ) , col1 ( rabbit antirat , diluted 1:1,000 , abcam biotechnology ) , or gapdh ( mouse antirat , diluted 1:2,000 , abcam biotechnology ) overnight at 4c . finally , specific secondary horseradish peroxidase - conjugated anti - rabbit or antimouse antibodies ( kpl , final concentration 0.2 g / ml ) were used , and the immunocomplexes were detected by chemiluminescence ( ecl clarity ; bio - rad ) using the chemi - doc xrs+ system ( bio - rad ) . the intensity of the bands was quantified through the chemi - doc xrs+ software by adopting the 1 g sample values as reference for the relative expression and by normalizing to the gapdh values . alizarin red solution ( millipore ) was used to stain in orange red the calcium deposits of the msc cultures induced to differentiate . following paraformaldehyde fixation , cells were incubated with 500 l of alizarin red solution at room temperature for 30 minutes . after extensive washing steps with deionized water , cells were visualized in bright field under the optical microscope for image acquisition . imagej was used to detect and automatically quantify the size of alizarin red - positive areas for each experimental group , analyzing at least ten random fields per sample . all the described experiments were carried out at least in triplicate , and all procedures were replicated twice . with regard to image analysis , data were tested with the nonparametric kruskal wallis analysis followed by the nemenyi damico wolfe each box plot is composed of whiskers indicating the minimum and maximum of the data , while the top and the bottom of the box are the first and third quartiles , respectively ; the band inside the box is the median . qpcr data were analyzed with bio - rad cfx manager software . in all cases , immunofluorescent staining of cytoskeleton ( f - actin in red , vinculin in green ) of proliferating mscs qualitatively showed evident effects following hypergravity treatment ( figure 2 ) . in particular , 20 g - treated cells appeared more stretched and with f - actin organized in parallel fibers with respect to the 1 g control cultures . in order to gain quantitative data about the influence of hypergravity and nps on cell morphology , we calculated cell area and cell - shape descriptors ( solidity , circularity , roundness ) on coomassie blue - stained cultures in proliferative conditions immediately after the hypergravity treatment ( figure 3a ) . concerning area evaluation ( figure 3b ) , we observed a significant increase when cells were treated with btnps ( 8,6482,419 m ) , hypergravity ( 9,7352,795 m ) , or with the combination of the two stimuli ( 9,7332,739 m ) , compared to the control at 1 g not treated with the nps ( 6,1551,602 m ) ( p<0.05 in all the three treatments with respect to the control ) . the solidity of the cells ( figure 3c ) after hypergravity stimulation , independently of the presence of btnps ( 0.830.05 and 0.810.04 for 20 g and 20 g + btnps , respectively ) was significantly lower than that of the cells grown at 1 g ( 0.930.02 and 0.980.02 for 1 g and 1 g + btnps , respectively ; p<0.05 in the 20 g treatments with respect to the 1 g treatments ) . a similar trend was observed for circularity ( figure 3d ) , the distributions of which denoted a significant decrease of the values of proliferating mscs subjected to 20 g acceleration ( 0.440.10 and 0.460.06 for 20 g and 20 g + btnps , respectively ) with respect to 1 g conditions ( 0.720.07 and 0.770.08 for 1 g and 1 g + btnps , respectively ) ( p<0.05 ) . finally , we found an analogous trend in the reduction of the roundness ( figure 3e ) for the cells that underwent hypergravity treatment ( 0.420.13 and 0.540.10 for 20 g and 20 g + btnps , respectively ) when compared to the controls at 1 g ( 0.710.11 and 0.670.08 for 1 g and 1 g + btnps , respectively ) . nevertheless , only the roundness reduction at 20 g was statistically significant with respect to 1 g ( p<0.05 ) . results of nonlinear microscopy on proliferating and differentiating cells internalizing btnps at 1 g and 20 g are reported in figure 4 . figure 4a depicts images representative of the four experimental conditions , highlighting a perinuclear cytoplasmic accumulation of btnps . the cars signal from cells is represented in green , the sfg signal from nps in red : we can appreciate the emission spectrum from a bundle of nps when irradiated with the papb and the stokes beam in figure 4b . four individual bands are visible , originating from : a second harmonic generation shg signal from the 806 nm pump beam , sfg from the combination of the 806 nm pump beam and the portion of the stokes beam temporarily overlapping to it ( ie , corresponding to approximately 1,045 nm ) , broadband second harmonic generation from the stokes beam , and nonresonant cars background . a quantitative evaluation of np uptake ( figure 4c ) performed in terms of percentage of the cytoplasmic area occupied by the btnps revealed a strong enhancement of btnp internalization in cells that underwent hypergravity stimulation ( p<0.05 ) , both in differentiation ( 6.80.8% at 20 g , 3.60.3% at 1 g ) and in proliferation ( 2.70.3% at 20 g , 1.40.1% at 1 g ) conditions . the generally higher internalization in differentiation conditions was simply due to a longer btnp incubation time ( 3 + 48 hours for differentiation samples versus 3 hours for proliferation samples ) . the evaluation of the gene transcription through qpcr of samples under differentiation conditions is shown in the plots of figure 5a . runx2 was significantly upregulated in 20 g - treated samples ( 1.5-fold at 20 g , 1.8-fold at 20 g + btnps ) with respect to the 1 g and 1 g + btnp cultures . col1a1 transcription was significantly enhanced ( 1.5-fold ) only in the double - stimulation 20 g + btnps , while down - regulation ( 0.6-fold ) was noticed in cultures performed at 1 g in the presence of the nps . finally , once again alpl was significantly upregulated ( 1.6-fold ) when cells were synergistically stimulated with hypergravity and nps with respect to all the other treatments . to evaluate how hypergravity affected the osteogenic pathway , we focused also on the transcription of rhoa in proliferating cells ( thus without any osteoinductive chemical cue ) immediately after the hypergravity treatment , since rhoa codes for a small guanosine triphosphatase protein known to play a key role in osteogenesis following mechanical stimulation.27 figure 5b shows a significant upregulation ( p<0.05 ) of rhoa mrna transcription in all treatments ( 1 g + btnps 2.0-fold , 20 g 2.5-fold , 20 g + btnps 2.8-fold ) with respect to the 1 g control not treated with btnps . moreover , this is interesting to highlight , as the 20 g + btnp group revealed a significant upregulation of rhoa with respect to the 1 g + btnp group ( 1.4-fold , p<0.05 ) , thus suggesting a role of both nps and hypergravity in the enhancement of msc differentiation . in order to assess the effects of our stimulation procedures at the protein - expression level also , western blotting of collagen type i and alkaline phosphatase was performed , and the results are reported in figure 6a . quantitative values obtained evaluating band intensities ( figure 6b ) proved an enhancement of collagen type i expression in all the experimental groups with respect to the control at 1 g not treated with btnps , but only hypergravity conditions were statistically significant ( 20 g 1.3-fold , 20 g + btnps 1.5-fold ; p<0.05 ) . moreover , the increasing expression of collagen type i in the synergic stimulation hypergravity + nps is significant ( p<0.05 ) when compared to the 1 g + btnp treatment ( 1.3-fold , figure 6b ) . with regard to alkaline phosphatase , no significant expression differences were detected among any of the treatments . the alizarin red assay ( figure 7a ) revealed an increase of the calcium deposition ( in terms of size of calcium nodules ) in samples treated with btnps both at 20 g ( 11,9091,691 m , 45% higher with respect to 20 g without btnps 8,1921,988 m ) and at 1 g ( 8,8132,615 m , 74% higher with respect to 1 g without btnps 5,0712,265 m ) . interestingly , there was also around a 35% increment in 20 g + btnp cultures with respect to the 1 g + btnp ones ; however , statistically significant differences were only detected in the samples stimulated at 20 g + btnps with respect both to 1 g ( 134% , p<0.005 ) and 20 g ( 45% , p<0.005 ; figure 7b ) . immunofluorescent staining of cytoskeleton ( f - actin in red , vinculin in green ) of proliferating mscs qualitatively showed evident effects following hypergravity treatment ( figure 2 ) . in particular , 20 g - treated cells appeared more stretched and with f - actin organized in parallel fibers with respect to the 1 g control cultures . in order to gain quantitative data about the influence of hypergravity and nps on cell morphology , we calculated cell area and cell - shape descriptors ( solidity , circularity , roundness ) on coomassie blue - stained cultures in proliferative conditions immediately after the hypergravity treatment ( figure 3a ) . concerning area evaluation ( figure 3b ) , we observed a significant increase when cells were treated with btnps ( 8,6482,419 m ) , hypergravity ( 9,7352,795 m ) , or with the combination of the two stimuli ( 9,7332,739 m ) , compared to the control at 1 g not treated with the nps ( 6,1551,602 m ) ( p<0.05 in all the three treatments with respect to the control ) . the solidity of the cells ( figure 3c ) after hypergravity stimulation , independently of the presence of btnps ( 0.830.05 and 0.810.04 for 20 g and 20 g + btnps , respectively ) was significantly lower than that of the cells grown at 1 g ( 0.930.02 and 0.980.02 for 1 g and 1 g + btnps , respectively ; p<0.05 in the 20 g treatments with respect to the 1 g treatments ) . a similar trend was observed for circularity ( figure 3d ) , the distributions of which denoted a significant decrease of the values of proliferating mscs subjected to 20 g acceleration ( 0.440.10 and 0.460.06 for 20 g and 20 g + btnps , respectively ) with respect to 1 g conditions ( 0.720.07 and 0.770.08 for 1 g and 1 g + btnps , respectively ) ( p<0.05 ) . finally , we found an analogous trend in the reduction of the roundness ( figure 3e ) for the cells that underwent hypergravity treatment ( 0.420.13 and 0.540.10 for 20 g and 20 g + btnps , respectively ) when compared to the controls at 1 g ( 0.710.11 and 0.670.08 for 1 g and 1 g + btnps , respectively ) . nevertheless , only the roundness reduction at 20 g was statistically significant with respect to 1 g ( p<0.05 ) . results of nonlinear microscopy on proliferating and differentiating cells internalizing btnps at 1 g and 20 g are reported in figure 4 . figure 4a depicts images representative of the four experimental conditions , highlighting a perinuclear cytoplasmic accumulation of btnps . the cars signal from cells is represented in green , the sfg signal from nps in red : we can appreciate the emission spectrum from a bundle of nps when irradiated with the papb and the stokes beam in figure 4b . four individual bands are visible , originating from : a second harmonic generation shg signal from the 806 nm pump beam , sfg from the combination of the 806 nm pump beam and the portion of the stokes beam temporarily overlapping to it ( ie , corresponding to approximately 1,045 nm ) , broadband second harmonic generation from the stokes beam , and nonresonant cars background . a quantitative evaluation of np uptake ( figure 4c ) performed in terms of percentage of the cytoplasmic area occupied by the btnps revealed a strong enhancement of btnp internalization in cells that underwent hypergravity stimulation ( p<0.05 ) , both in differentiation ( 6.80.8% at 20 g , 3.60.3% at 1 g ) and in proliferation ( 2.70.3% at 20 g , 1.40.1% at 1 g ) conditions . the generally higher internalization in differentiation conditions was simply due to a longer btnp incubation time ( 3 + 48 hours for differentiation samples versus 3 hours for proliferation samples ) . the evaluation of the gene transcription through qpcr of samples under differentiation conditions is shown in the plots of figure 5a . runx2 was significantly upregulated in 20 g - treated samples ( 1.5-fold at 20 g , 1.8-fold at 20 g + btnps ) with respect to the 1 g and 1 g + btnp cultures . col1a1 transcription was significantly enhanced ( 1.5-fold ) only in the double - stimulation 20 g + btnps , while down - regulation ( 0.6-fold ) was noticed in cultures performed at 1 g in the presence of the nps . finally , once again alpl was significantly upregulated ( 1.6-fold ) when cells were synergistically stimulated with hypergravity and nps with respect to all the other treatments . to evaluate how hypergravity affected the osteogenic pathway , we focused also on the transcription of rhoa in proliferating cells ( thus without any osteoinductive chemical cue ) immediately after the hypergravity treatment , since rhoa codes for a small guanosine triphosphatase protein known to play a key role in osteogenesis following mechanical stimulation.27 figure 5b shows a significant upregulation ( p<0.05 ) of rhoa mrna transcription in all treatments ( 1 g + btnps 2.0-fold , 20 g 2.5-fold , 20 g + btnps 2.8-fold ) with respect to the 1 g control not treated with btnps . moreover , this is interesting to highlight , as the 20 g + btnp group revealed a significant upregulation of rhoa with respect to the 1 g + btnp group ( 1.4-fold , p<0.05 ) , thus suggesting a role of both nps and hypergravity in the enhancement of msc differentiation . in order to assess the effects of our stimulation procedures at the protein - expression level also , western blotting of collagen type i and alkaline phosphatase was performed , and the results are reported in figure 6a . quantitative values obtained evaluating band intensities ( figure 6b ) proved an enhancement of collagen type i expression in all the experimental groups with respect to the control at 1 g not treated with btnps , but only hypergravity conditions were statistically significant ( 20 g 1.3-fold , 20 g + btnps 1.5-fold ; p<0.05 ) . moreover , the increasing expression of collagen type i in the synergic stimulation hypergravity + nps is significant ( p<0.05 ) when compared to the 1 g + btnp treatment ( 1.3-fold , figure 6b ) . with regard to alkaline phosphatase the alizarin red assay ( figure 7a ) revealed an increase of the calcium deposition ( in terms of size of calcium nodules ) in samples treated with btnps both at 20 g ( 11,9091,691 m , 45% higher with respect to 20 g without btnps 8,1921,988 m ) and at 1 g ( 8,8132,615 m , 74% higher with respect to 1 g without btnps 5,0712,265 m ) . interestingly , there was also around a 35% increment in 20 g + btnp cultures with respect to the 1 g + btnp ones ; however , statistically significant differences were only detected in the samples stimulated at 20 g + btnps with respect both to 1 g ( 134% , p<0.005 ) and 20 g ( 45% , p<0.005 ; figure 7b ) . it is widely recognized that several kinds of cells , including endothelial cells,28 osteoblasts,29 myoblasts,30 and stem cells,31 are sensitive to a change of gravity - force intensity . in particular , it was found that hypergravity treatments could enhance osteogenesis in osteoblast - like cells.32 in this paper , we have reported on the hypothesis that upon hypergravity stimulation and btnp administration , mscs enhance their commitment toward osteogenesis . mechanical forces are well known to induce cytoskeleton rearrangements , and these conformation changes deeply affect stem cell behavior.33 to analyze these phenomena in our experimental conditions , we performed a quantitative evaluation of several cell - shape descriptors . the morphometric analysis demonstrated that following a hypergravity treatment , cells were less circular , more spread , and cover a larger area when compared to the 1 g control . overall , the characterization of cell shape and morphology showed that an increment of gravitational force , both in the presence or not of btnps , provided as a consequence a more irregular and spread morphology . this effect , consistent with other results available in the literature,34 could contribute to the maturation toward osteoblasts , given that spread cells are committed to osteogenesis , and conversely , a rounded shape promotes adipogenesis.35 furthermore , the f - actin organization in parallel and well - defined stress fibers qualitatively observed after hypergravity stimulation is a further hint of cytoskeleton - tension enhancement , and consequently of the mechanotransduction leading to osteogenesis ( mediated by rhoa and runx2).36 among the impressive variety of inorganic nanomaterials investigated in nanomedicine , btnps are still relatively unexplored yet most promising , owing to their good biocompatibility,21 osteoinductive properties,19 and nonlinear optical properties.22 nonlinear microscopy performed thanks to these particular features allowed the enhancement of np uptake following hypergravity treatment to be assessed . our results proved that hypergravity enhances np internalization , both in proliferation and differentiation cultures . a number of studies in the literature have aimed to enhance nanocarrier internalization by using physical approaches , such as ultrasounds37 or magnetic fields.38 however , these methods suffer some disadvantages related to cell damage and/or undesired effects on metabolism and functions . hypergravity could therefore represent a valid alternative for enhanced drug and gene delivery mediated by nps for plenty of in vitro applications . since osteogenic differentiation was induced for just 2 days ( because of technical constrains at the esa facilities ) , the mrna - expression analysis was focused on genes involved in the early stage of osteogenesis ( ie , runx2 , col1a1 , alpl ) . the obtained results , which indicated general gene upregulation following hypergravity and btnp treatments , are particularly interesting , since runx2 encodes for the key transcription factor that induces osteogenic differentiation,39 col1a1 product is the major component of the bone organic matrix,40 and alpl is involved in the mineralization process.41 corroborating our findings , similar results were obtained following msc stimulation at 2 g and at 10 g.42,43 our results highlight that the performed stimulations ( hypergravity and incubation with btnps ) can synergistically act in improving the early stage maturation of mscs toward osteoblasts . western blotting showed an increment of col1 expression in hypergravity - stimulated cultures , these being data in line with other findings on osteoblast - like cells treated for 24 hours at 13 g , which demonstrated a significant increment in collagen expression.44 in our case , moreover , the combination of hypergravity with btnp incubation further increased collagen production . therefore , increased col1 mrna expression was also translated to enhanced protein production ; instead , concerning alpl , we observed upregulation of the mrna , but not enhancement of the protein expression , most probably because of a too - early stage of differentiation.45 obviously , gene activation does not always result in an increase in protein levels , because of the translation - regulation and protein - degradation mechanisms ; however , even though this point is interesting , it is out of the scope of this work to deeply analyze the molecular mechanisms of transduction regulation involved in this process . finally , we investigated the osteoblast phenotype through the alizarin red assay , which revealed an enhancement of calcium deposits in the presence of btnps and hypergravity treatment with respect to the other experimental groups . indeed , the size of calcium nodules in samples under hypergravity conditions and btnp treatment was found to be significantly higher when compared to the hypergravity stimulation alone , thus further suggesting a synergic effect of the double stimulation . these data are consistent with the results obtained by prodanov et al who demonstrated an increase of calcium content in mscs cultured on nanotextured substrate and subjected to a 10 g treatment.43 taken together , all the obtained results suggest two hypotheses about the mechanism of msc osteogenesis enhancement following hypergravity treatment and btnp incubation . the first hypothesis is based on the already proven osteoinduction effectiveness of btnps:46 as np uptake is increased following hypergravity treatment , osteogenic maturation is enhanced because of a higher number of internalized btnps . however , we have to consider also improvements of msc maturation when hypergravity is applied without the presence of nps . in order to understand whether hypergravity could itself act as a mechanical cue able to induce osteogenesis , we performed an analysis of the transcription of rhoa , as the activity of this gene is altered following cell - shape changes , and more in general following mechanical stimulation of cells.47 our results provide the evidence that a 3-hour 20 g stimulation makes cells more spread and elongated , reducing their circularity . several studies have reported that cell shape affects rhoa regulation:4850 spread cells cause rhoa activation that responds to mechanical stimuli enhancing cell tension and stiffness , and upregulating biochemical factors for the induction of the osteogenic pathway , while adipogenesis is inhibited . we therefore analyzed rhoa transcription in the absence of any osteoinductive chemical stimulation and immediately after the treatments . a significant increase of rhoa mrna was observed in both 1 g + btnp and in 20 g treatments , thus demonstrating that both stimuli are involved in the activation of runx2 transcription ( triggered by rhoa product ) , and thus that both stimuli are osteoinductive . in a recent work,19 it was indeed demonstrated that btnps are able to mechanically stimulate mscs by remodeling their cytoskeleton and by increasing their stiffness . therefore , the rhoa upregulation we observed after btnp treatment at 1 g is consistent with the reported mechanical stimulation induced by the nps . interestingly , the combination of btnps and hypergravity had an even more pronounced effect on the upregulation of rhoa with respect to the single treatments , thus indicating that their combination intensifies osteogenesis . we can thus deduce that hypergravity acts as an osteoinductuive stimulus per se , and at the same time , by enhancing np internalization , further increases the osteoinductive potential of the btnps , thus achieving a double - level and synergic effect following the applied treatments . the goal of this research was to propose new strategies to overcome difficulties in the osteogenic differentiation of mscs . collected data demonstrated a short treatment ( 3 hours ) at 20 g combined with incubation with 20 g / ml of btnps synergistically promoted the osteogenesis of mscs , evaluated both at the gene and phenotype levels . hypergravity , in addition to providing per se osteogenic stimulation , is able to promote np uptake , thus further enhancing np effects at low doses . all the collected results , even if preliminary , are promising for the elaboration of new approaches in several biomedical fields , including drug delivery , tissue engineering , and regenerative medicine .

backgroundenhancement of the osteogenic potential of mesenchymal stem cells ( mscs ) is highly desirable in the field of bone regeneration . this paper proposes a new approach for the improvement of osteogenesis combining hypergravity with osteoinductive nanoparticles ( nps).materials and methodsin this study , we aimed to investigate the combined effects of hypergravity and barium titanate nps ( btnps ) on the osteogenic differentiation of rat mscs , and the hypergravity effects on np internalization . to obtain the hypergravity condition , we used a large - diameter centrifuge in the presence of a btnp - doped culture medium . we analyzed cell morphology and np internalization with immunofluorescent staining and coherent anti - stokes raman scattering , respectively . moreover , cell differentiation was evaluated both at the gene level with quantitative real - time reverse - transcription polymerase chain reaction and at the protein level with western blotting.resultsfollowing a 20 g treatment , we found alterations in cytoskeleton conformation , cellular shape and morphology , as well as a significant increment of expression of osteoblastic markers both at the gene and protein levels , jointly pointing to a substantial increment of np uptake . taken together , our findings suggest a synergistic effect of hypergravity and btnps in the enhancement of the osteogenic differentiation of mscs.conclusionthe obtained results could become useful in the design of new approaches in bone - tissue engineering , as well as for in vitro drug - delivery strategies where an increment of nanocarrier internalization could result in a higher drug uptake by cell and/or tissue constructs .

Introduction Materials and methods Cell culture and experimental procedures in the large-diameter centrifuge Cytoskeleton/focal adhesion staining and cell shape-descriptor analysis Nanoparticle-internalization analysis Quantitative real-time reverse-transcription polymerase chain reaction Western blotting Alizarin red staining Statistical analysis Results Cell morphology and nanoparticle internalization Cell-differentiation assessment: gene and protein analysis and alizarin red staining Discussion Conclusion

physical stimuli can significantly alter cellular behavior , giving rise to biochemical signals involved in molecular response.1 this process is called mechanotransduction , and the responsible structures sensitive to mechanical forces are most probably cytoskeleton elements.2 a number of studies have demonstrated that cells are sensitive to several kinds of physical cues ( shear stress , topography , mechanical deformation , etc ) , influencing cell migration,3 differentiation,4 and proliferation.5 among these stimuli , gravity is required for the correct development of land - based organisms , and in particular for the skeleton and for the muscle and nervous systems.6 an increasing amount of research is focused on the effects of gravity alterations on the physiological processes , but also on the possibility to exploit this stimulus as a potential therapeutic cue.79 as an example , improved regeneration of infarcted myocardium has been achieved after injection of stem cells differentiated following a 2 g hypergravity treatment , which enhanced the activities of cardiac marker mef-2 by promoting the nuclear export of histone deacetylase 5.10 chang et al investigated altered gravity effects on human lung adenocarcinoma , demonstrating the ability of simulated microgravity to decrease the metastatic potential of this tumor cell line.11 other researchers used microgravity stimulation as an approach for the development of a large amount of -cell spheroids , which once transplanted in mice are able to improve the symptoms of diabetes.12 among different tissues , bone is particularly affected by altered gravity conditions : evidence regarding bone regeneration suggests that hypergravity exposure conversely to microgravity , which negatively affects osteogenesis may enhance the osteogenic potential of osteoblast precursors.13 the ability of mesenchymal stem cells ( mscs ) to differentiate into osteoblasts is well known , but the osteogenic potential of mscs decreases with the prolonged culture duration necessary to obtain an appropriate number of cells for clinical applications.14 some countermeasures to this issue could come from nanotechnology , which proposes many different typologies of nanoparticles ( nps ) for stem cell labeling , tracking , delivery , and stimulation,15 including several examples of nanomaterials able to foster osteogenesis in mscs.1618 our group , as an example , successfully exploited barium titanate nps ( btnps ; figure 1a ) as a possible agent for the improvement of osteogenic differentiation of mscs.19 btnps belong to a class of ferroelectric materials showing high piezoelectricity,20 and with regard to biomedical applications , they demonstrate high cytocompatibility,21 excellent properties as nonlinear imaging probes,22 and the ability to deliver doxorubicin in cancer cells by improving drug uptake.23 moreover , as previously mentioned , btnps were proven to enhance the osteogenesis of mscs , as demonstrated by an increment of hydroxyapatite deposition . starting from these findings , we decided to develop a protocol for msc stimulation by combining incubation with btnps with treatment in hypergravity , with the goal of improving the differentiation process toward osteoblasts , and thus to obtain stem cells with enhanced osteogenic differentiation potential . 2013 campaign at the european space agency ( esa ; noordwijk , the netherlands ) , taking advantage of the large - diameter centrifuge for the hypergravity treatment . with the proposed experimental protocol , we had the aims of 1 ) investigating cell morphology and differentiation ( at gene , protein , and phenotype level ) following the combination of hypergravity treatment and btnp administration , and 2 ) evaluating np uptake by stem cells in altered gravity conditions , thus investigating the possibility of enhancing cellular internalization by simply exploiting an increased gravitational force . for the maintenance of mscs , the medium was composed of dulbecco s modified eagle s medium supplemented with 10% fetal bovine serum , 100 u / ml penicillin , 100 mg / ml streptomycin , and 200 mm glutamine ( all these reagents from gibco ) . for the positioning of the samples , we used a delrin structure designed to support 30 cylindrical vials ( figure 1c ) . it is composed of four large rotating arms with a swing gondola at each extremity ( figure 1d ) , and can support hypergravity levels between 1 g and 20 g. we performed analyses on proliferating and differentiating rat mscs provided with and without 20 g / ml of btnps immediately before the altered gravity treatment , as previously described . immediately after the treatment , some proliferative samples were processed in order to evaluate the effects of hypergravity and btnps on msc adhesion and shape . coherent anti - stokes raman scattering ( cars ) has been exploited to obtain images of cells , based on a degenerate pump - and - probe beam ( papb ) created by a ti sa pulsed laser ( chameleon vision ii ; coherent ) and a supercontinuum generator ( photonic crystal fiber scg-800 ; newport ) that produces a broadband stokes beam . quantitative real - time reverse - transcription polymerase chain reaction ( qpcr ) was used to investigate the messenger ribonucleic acid ( mrna ) transcription of osteogenesis - marker genes ( runt related transcription factor 2 [ runx2 ] , collagen type i alpha-1 [ col1a1 ] , and alkaline phosphatase [ alpl ] ) and of rhoa ( ras homolog gene family , member a ) , which regulates osteogenesis - activating runx2 following mechanical stimulation.25 after the experimental procedures , cells were trypsinized and centrifuged , and the total rna was extracted from the samples with the high pure rna isolation kit ( roche ) according to the manufacturer s instructions . for the positioning of the samples , we used a delrin structure designed to support 30 cylindrical vials ( figure 1c ) . it is composed of four large rotating arms with a swing gondola at each extremity ( figure 1d ) , and can support hypergravity levels between 1 g and 20 g. we performed analyses on proliferating and differentiating rat mscs provided with and without 20 g / ml of btnps immediately before the altered gravity treatment , as previously described . immediately after the treatment , some proliferative samples were processed in order to evaluate the effects of hypergravity and btnps on msc adhesion and shape . coherent anti - stokes raman scattering ( cars ) has been exploited to obtain images of cells , based on a degenerate pump - and - probe beam ( papb ) created by a ti sa pulsed laser ( chameleon vision ii ; coherent ) and a supercontinuum generator ( photonic crystal fiber scg-800 ; newport ) that produces a broadband stokes beam . quantitative real - time reverse - transcription polymerase chain reaction ( qpcr ) was used to investigate the messenger ribonucleic acid ( mrna ) transcription of osteogenesis - marker genes ( runt related transcription factor 2 [ runx2 ] , collagen type i alpha-1 [ col1a1 ] , and alkaline phosphatase [ alpl ] ) and of rhoa ( ras homolog gene family , member a ) , which regulates osteogenesis - activating runx2 following mechanical stimulation.25 after the experimental procedures , cells were trypsinized and centrifuged , and the total rna was extracted from the samples with the high pure rna isolation kit ( roche ) according to the manufacturer s instructions . in order to gain quantitative data about the influence of hypergravity and nps on cell morphology , we calculated cell area and cell - shape descriptors ( solidity , circularity , roundness ) on coomassie blue - stained cultures in proliferative conditions immediately after the hypergravity treatment ( figure 3a ) . concerning area evaluation ( figure 3b ) , we observed a significant increase when cells were treated with btnps ( 8,6482,419 m ) , hypergravity ( 9,7352,795 m ) , or with the combination of the two stimuli ( 9,7332,739 m ) , compared to the control at 1 g not treated with the nps ( 6,1551,602 m ) ( p<0.05 in all the three treatments with respect to the control ) . the solidity of the cells ( figure 3c ) after hypergravity stimulation , independently of the presence of btnps ( 0.830.05 and 0.810.04 for 20 g and 20 g + btnps , respectively ) was significantly lower than that of the cells grown at 1 g ( 0.930.02 and 0.980.02 for 1 g and 1 g + btnps , respectively ; p<0.05 in the 20 g treatments with respect to the 1 g treatments ) . finally , we found an analogous trend in the reduction of the roundness ( figure 3e ) for the cells that underwent hypergravity treatment ( 0.420.13 and 0.540.10 for 20 g and 20 g + btnps , respectively ) when compared to the controls at 1 g ( 0.710.11 and 0.670.08 for 1 g and 1 g + btnps , respectively ) . a quantitative evaluation of np uptake ( figure 4c ) performed in terms of percentage of the cytoplasmic area occupied by the btnps revealed a strong enhancement of btnp internalization in cells that underwent hypergravity stimulation ( p<0.05 ) , both in differentiation ( 6.80.8% at 20 g , 3.60.3% at 1 g ) and in proliferation ( 2.70.3% at 20 g , 1.40.1% at 1 g ) conditions . col1a1 transcription was significantly enhanced ( 1.5-fold ) only in the double - stimulation 20 g + btnps , while down - regulation ( 0.6-fold ) was noticed in cultures performed at 1 g in the presence of the nps . to evaluate how hypergravity affected the osteogenic pathway , we focused also on the transcription of rhoa in proliferating cells ( thus without any osteoinductive chemical cue ) immediately after the hypergravity treatment , since rhoa codes for a small guanosine triphosphatase protein known to play a key role in osteogenesis following mechanical stimulation.27 figure 5b shows a significant upregulation ( p<0.05 ) of rhoa mrna transcription in all treatments ( 1 g + btnps 2.0-fold , 20 g 2.5-fold , 20 g + btnps 2.8-fold ) with respect to the 1 g control not treated with btnps . moreover , this is interesting to highlight , as the 20 g + btnp group revealed a significant upregulation of rhoa with respect to the 1 g + btnp group ( 1.4-fold , p<0.05 ) , thus suggesting a role of both nps and hypergravity in the enhancement of msc differentiation . in order to assess the effects of our stimulation procedures at the protein - expression level also , western blotting of collagen type i and alkaline phosphatase was performed , and the results are reported in figure 6a . in order to gain quantitative data about the influence of hypergravity and nps on cell morphology , we calculated cell area and cell - shape descriptors ( solidity , circularity , roundness ) on coomassie blue - stained cultures in proliferative conditions immediately after the hypergravity treatment ( figure 3a ) . the solidity of the cells ( figure 3c ) after hypergravity stimulation , independently of the presence of btnps ( 0.830.05 and 0.810.04 for 20 g and 20 g + btnps , respectively ) was significantly lower than that of the cells grown at 1 g ( 0.930.02 and 0.980.02 for 1 g and 1 g + btnps , respectively ; p<0.05 in the 20 g treatments with respect to the 1 g treatments ) . finally , we found an analogous trend in the reduction of the roundness ( figure 3e ) for the cells that underwent hypergravity treatment ( 0.420.13 and 0.540.10 for 20 g and 20 g + btnps , respectively ) when compared to the controls at 1 g ( 0.710.11 and 0.670.08 for 1 g and 1 g + btnps , respectively ) . a quantitative evaluation of np uptake ( figure 4c ) performed in terms of percentage of the cytoplasmic area occupied by the btnps revealed a strong enhancement of btnp internalization in cells that underwent hypergravity stimulation ( p<0.05 ) , both in differentiation ( 6.80.8% at 20 g , 3.60.3% at 1 g ) and in proliferation ( 2.70.3% at 20 g , 1.40.1% at 1 g ) conditions . col1a1 transcription was significantly enhanced ( 1.5-fold ) only in the double - stimulation 20 g + btnps , while down - regulation ( 0.6-fold ) was noticed in cultures performed at 1 g in the presence of the nps . to evaluate how hypergravity affected the osteogenic pathway , we focused also on the transcription of rhoa in proliferating cells ( thus without any osteoinductive chemical cue ) immediately after the hypergravity treatment , since rhoa codes for a small guanosine triphosphatase protein known to play a key role in osteogenesis following mechanical stimulation.27 figure 5b shows a significant upregulation ( p<0.05 ) of rhoa mrna transcription in all treatments ( 1 g + btnps 2.0-fold , 20 g 2.5-fold , 20 g + btnps 2.8-fold ) with respect to the 1 g control not treated with btnps . moreover , this is interesting to highlight , as the 20 g + btnp group revealed a significant upregulation of rhoa with respect to the 1 g + btnp group ( 1.4-fold , p<0.05 ) , thus suggesting a role of both nps and hypergravity in the enhancement of msc differentiation . in order to assess the effects of our stimulation procedures at the protein - expression level also , western blotting of collagen type i and alkaline phosphatase was performed , and the results are reported in figure 6a . with regard to alkaline phosphatase the alizarin red assay ( figure 7a ) revealed an increase of the calcium deposition ( in terms of size of calcium nodules ) in samples treated with btnps both at 20 g ( 11,9091,691 m , 45% higher with respect to 20 g without btnps 8,1921,988 m ) and at 1 g ( 8,8132,615 m , 74% higher with respect to 1 g without btnps 5,0712,265 m ) . overall , the characterization of cell shape and morphology showed that an increment of gravitational force , both in the presence or not of btnps , provided as a consequence a more irregular and spread morphology . this effect , consistent with other results available in the literature,34 could contribute to the maturation toward osteoblasts , given that spread cells are committed to osteogenesis , and conversely , a rounded shape promotes adipogenesis.35 furthermore , the f - actin organization in parallel and well - defined stress fibers qualitatively observed after hypergravity stimulation is a further hint of cytoskeleton - tension enhancement , and consequently of the mechanotransduction leading to osteogenesis ( mediated by rhoa and runx2).36 among the impressive variety of inorganic nanomaterials investigated in nanomedicine , btnps are still relatively unexplored yet most promising , owing to their good biocompatibility,21 osteoinductive properties,19 and nonlinear optical properties.22 nonlinear microscopy performed thanks to these particular features allowed the enhancement of np uptake following hypergravity treatment to be assessed . since osteogenic differentiation was induced for just 2 days ( because of technical constrains at the esa facilities ) , the mrna - expression analysis was focused on genes involved in the early stage of osteogenesis ( ie , runx2 , col1a1 , alpl ) . the obtained results , which indicated general gene upregulation following hypergravity and btnp treatments , are particularly interesting , since runx2 encodes for the key transcription factor that induces osteogenic differentiation,39 col1a1 product is the major component of the bone organic matrix,40 and alpl is involved in the mineralization process.41 corroborating our findings , similar results were obtained following msc stimulation at 2 g and at 10 g.42,43 our results highlight that the performed stimulations ( hypergravity and incubation with btnps ) can synergistically act in improving the early stage maturation of mscs toward osteoblasts . western blotting showed an increment of col1 expression in hypergravity - stimulated cultures , these being data in line with other findings on osteoblast - like cells treated for 24 hours at 13 g , which demonstrated a significant increment in collagen expression.44 in our case , moreover , the combination of hypergravity with btnp incubation further increased collagen production . therefore , increased col1 mrna expression was also translated to enhanced protein production ; instead , concerning alpl , we observed upregulation of the mrna , but not enhancement of the protein expression , most probably because of a too - early stage of differentiation.45 obviously , gene activation does not always result in an increase in protein levels , because of the translation - regulation and protein - degradation mechanisms ; however , even though this point is interesting , it is out of the scope of this work to deeply analyze the molecular mechanisms of transduction regulation involved in this process . finally , we investigated the osteoblast phenotype through the alizarin red assay , which revealed an enhancement of calcium deposits in the presence of btnps and hypergravity treatment with respect to the other experimental groups . these data are consistent with the results obtained by prodanov et al who demonstrated an increase of calcium content in mscs cultured on nanotextured substrate and subjected to a 10 g treatment.43 taken together , all the obtained results suggest two hypotheses about the mechanism of msc osteogenesis enhancement following hypergravity treatment and btnp incubation . the first hypothesis is based on the already proven osteoinduction effectiveness of btnps:46 as np uptake is increased following hypergravity treatment , osteogenic maturation is enhanced because of a higher number of internalized btnps . in order to understand whether hypergravity could itself act as a mechanical cue able to induce osteogenesis , we performed an analysis of the transcription of rhoa , as the activity of this gene is altered following cell - shape changes , and more in general following mechanical stimulation of cells.47 our results provide the evidence that a 3-hour 20 g stimulation makes cells more spread and elongated , reducing their circularity . we can thus deduce that hypergravity acts as an osteoinductuive stimulus per se , and at the same time , by enhancing np internalization , further increases the osteoinductive potential of the btnps , thus achieving a double - level and synergic effect following the applied treatments . the goal of this research was to propose new strategies to overcome difficulties in the osteogenic differentiation of mscs . collected data demonstrated a short treatment ( 3 hours ) at 20 g combined with incubation with 20 g / ml of btnps synergistically promoted the osteogenesis of mscs , evaluated both at the gene and phenotype levels . all the collected results , even if preliminary , are promising for the elaboration of new approaches in several biomedical fields , including drug delivery , tissue engineering , and regenerative medicine .

the malaria parasite , plasmodium , is a major public health burden in the developing world , and despite the existence of antimalarial treatment active in blood stages of infection , there is a continual need for novel drug design as the parasite develops resistance to current treatments . additionally , treatment for the hypnozoite - causing species , plasmodium vivax , requires primaquine , which has severe side effects and causes hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency . discovery of novel compounds in antimalarial drug development is essential for future intervention strategies . atg8 is the ubiquitin - like ( ubl ) protein necessary for formation and maturation of autophagosomes in autophagy in eukarya . in yeast and mammals , atg8 is lipidated to the autophagosome membrane , but in plasmodium , atg8 is partially conjugated to the membrane of the apicoplast under nonstarvation conditions . the apicoplast is a nonphotosynthetic chloroplast - like organelle present in apicomplexans and is essential for isoprenoid synthesis . under starvation conditions , atg8 relocates to acidic vesicles with rab7 in and near the food vacuole . atg8 is essential to the plasmodium parasite and has been proposed as a target for antimalarial drug design . in most eukaryotes , lipidation of atg8 to phosphatidylethanolamine ( pe ) in membranes normally requires proteolytic processing of the c - terminus of atg8 by atg4 and activation via adenosine 5-triphosphate ( atp ) followed by intermediate thioester bond formation with the e1-activating enzyme atg7 . atg8 is then transferred to its e2-like conjugating enzyme atg3 , forming a second thioester intermediate before being conjugated to the nitrogen of pe ( figure 1 ) . this process also requires noncovalent interaction between atg8 and atg3 through a well - characterized atg8-interacting motif ( aim ) in atg3 and two hydrophobic pockets , termed the w and l - site , in atg8 . notably , in plasmodium , atg8 is synthesized with a c - terminal glycine and therefore does not require activation by atg4 . recently published studies showed a drastic growth defect in plasmodium falciparum when levels of pfatg7 were reduced . this along with the essentiality of plasmodium atg8 suggests that targeting pfatg8 lipidation is a good strategy for drug intervention . we previously elucidated the protein crystal structure of p. falciparum atg8 bound to a peptide corresponding to pfatg3 s aim ( pdb code 4eoy ) . regions of diversity exist between the human and plasmodium system that may be exploitable through small molecule inhibition . our mutational and interaction studies suggest that the plasmodium atg8-atg3 interaction requires atg8 s w / l site as well as the apicomplexan loop on atg8 ( residues 6776 ) , termed the a - loop . here , we report the identification of a class of compounds that inhibit the plasmodium atg8-atg3 interaction and that inhibit in vitro growth of p. falciparum in blood- and liver - stage assays , presumably through prevention of pfatg8 lipidation . previously , we developed a surface plasmon resonance ( spr)-based competition assay to identify compounds that disrupt the pfatg8-pfatg3 noncovalent interaction.pfatg3 is immobilized onto an spr chip , and pfatg8 is injected in the presence of dimethyl sulfoxide ( dmso , control ) or a compound ( dissolved in dmso ) , and binding is measured by the spr response . the medicines for malaria venture ( mmv ) malaria box of 200 druglike and 200 probelike molecules was screened at 5 m in a primary spr screen ( figure 2a ) . six compounds met the cutoff for at least 25% inhibition of the pfatg8-pfatg3 interaction : ( n-(4-methylphenyl)-4-pyridin-2-yl-1,3-thiazol-2-amine ) ( 1 ) , ( 2-methylsulfanyl - n-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide ) ( 2 ) , ( 2-bromo - n-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide ) ( 3 ) , n-[2-chloro-5-(trifluoromethyl)phenyl]-2-[2-(4-methylphenyl)pyrazolo[1,5-a]pyrazin-4-yl]sulfanylacetamide ( 4 ) , 1-[4-(dimethylamino)phenyl]-6,6-dimethyl-1,3,5-triazine-2,4-diamine ( 5 ) , and 2-n,3-n - bis(4-bromophenyl)quinoxaline-2,3-diamine ( 6 ) ( figure 2a,2b , table 1 ) . in subsequent dose - dependent studies , 46 demonstrated a constant level of inhibition independent of concentration of the small molecule and were not further investigated . compounds 13 led to dose - dependent inhibition with an spr inhibitory concentration ( ic50 spr ) ranging from 6 to 18 m ( figure 2c ) . interestingly , these compounds shared a common scaffold : 4-pyridin-2-yl-1,3-thiazol-2-amine ( pta ) incorporated as the n - substituent in various anilines or benzamides . 13 were tested for their effect on the stability of pfatg8 using fluorescence - based thermal shift assays ( tsas ) . none of the compounds significantly affected the melting temperature ( tm ) indicating that they most likely did not disturb the tertiary structure of pfatg8 ( figure 2d ) . pfatg8 was immobilized onto an spr ni - nitrilotriacetic acid ( nta ) chip via a 12 histidine n - terminal tag . 1 , chosen for its better solubility , was injected over the chip , and binding was measured . compound 1 led to a dose - dependent increase in spr response , indicating binding to pfatg8 ( figure 3a ) . we next sought to determine the binding site for the pta compounds with in silico docking . the openeye software package ( www.eyesopen.com ) was used to dock conformers of the compounds against the x - ray structure of pfatg8 ( pdb code 4eoy ) . all three compounds docked to the w - site of pfatg8 ( figure 3b ) . 2 and 3 bound with the pyridine ring in the w - site , whereas 1 was predicted to bind with the pyridine ring in the l - site , the thiazole ring positioned between the pockets , and the methylbenzene group in the w - site . an alternate pose was enriched within the top 10 poses output by the docking study in which 1 binds solely within the w - site in a more compact conformation . 1 is missing a donor oxygen compared to 2 and 3 and , therefore , may adopt a different mode of binding to the w- and l - sites of pfatg8 . 2 docked with the pyridine ring in the w - site , and the methylsulfanylbenzene group bound to the l - site of pfatg8 . docking was repeated using a version of the receptor for which the carbonyl of lys47 was input as a hydrogen bond acceptor constraint ( figure 3b inset ) . 1 had a similar binding pose to the highest ranked pose from the unconstrained docking , while 2 was slightly different with the pyridine ring rotated 90 in the w - site and the benzene ring positioned just below and to the left of the l - site pocket with the methylsulfanyl group reaching into the mostly hydrophobic l - site . the half maximal inhibitory concentrations ( ic50 ) for these compounds in p. falciparum 3d7 blood stages were previously reported and are located on the ncbi pubchem database ( http://pubchem.ncbi.nlm.nih.gov ) . 1 has a reported ic50 of 350400 nm ( pubchem bioassay i d ( aid ) : 660866 and 449703 ) . the reported ic50 for 2 ranged from 0.20 to 6.8 m , while 3 ranged from 1.36 to 4.52 m ( pubchem aid : 660866 and 449707 ) . we focused on compound 1 for further studies because the reported cytoxicity in human cell lines is much lower than that of compounds 2 or 3 ( pubchem aid : 660872 , 685525 , and 449705 ) . pfatg8 is expressed and lipidated during the liver stage where it partially localizes to the apicoplast . treatment of early liver stage parasites with the autophagy inhibitor 3-methyladenine is reported to delay conversion of the parasite into its trophozoite form . 1 was previously tested in plasmodium yoelii liver stage cultures and did not display > 50% inhibition at the screening concentration of 10 m ; an ic50 was not reported ( pubchem aid : 602118 and 602156).p . yoelii and plasmodium berghei are often used to test drugs for liver stage inhibition as they are easier to culture . however , these are rodent malaria models and may not be indicative of activity in p. falciparum . analysis of the w / l - site in these three species revealed differences in the amino acid composition that could affect drugs predicted to bind in that region ( figure 4 ) . we utilized a recently established p. falciparum liver stage in vitro model in which sporozoites isolated from infected mosquitos salivary glands invade hc-04 hepatocytes . hc-04 is a unique immortalized cell line that exhibits the expression of biochemical markers characteristic for normal hepatocytes and allows for the full development of the human malaria parasite , p. falciparum.(2022 ) using this system , we assessed the effect of 1 on the development of p. falciparum 3d7-green fluorescent protein ( gfp ) parasites in human hepatocytes in vitro . no change in the viability of hc-04 cells was detected in response to treatment with 3 m or 30 m of 1 for 96 h ( figure 5a ) . using flow cytometry , we observed about a 50% decrease in the proportion of hepatocytes infected with p. falciparum 3d7-gfp sporozoites ( gfp+/propidium iodide ( pi)- cells ) in response to treatment with 30 m , but not with 3 m of 1 ( figure 5b , c ) . additionally , there was a dose - dependent reduction in the intensity of gfp fluorescence at both concentrations of 1 , indicating inhibition of parasite development within hepatocytes , at least in vitro ( figure 5d ) . because 1 did not affect cell survival or cell growth of hc-04 cells ( figure 5a ) , the compound s effect on the parasite is unlikely to result from host cell cytotoxicity . we next sought to determine whether 1 had an effect on pfatg8 in p. falciparum blood stage cultures . in immunoblot assays , very low levels of endogenous pfatg8 were detected in dmso - treated control cells . incubation of cells in minimal media lacking human serum for 5 h led to a very slight increase in pfatg8 . in contrast , treatment with 50 m cytocidal levels of compound 1 led to a drastic increase in pfatg8 protein levels as well as to a shift in mobility , likely corresponding to the unlipidated form of pfatg8 . overall protein levels were unchanged , indicating an up - regulation or accumulation of pfatg8 in the presence of 1 under conditions that are likely leading to cell death ( figure s1 of the supporting information ) . after treating the parasites for 6 hours , a dose - dependent increase in delipidation of pfatg8 is already observed at 12.5 m of 1 , and at 25 m , unlipidated pfatg8 is the predominant species ( figure 6a , figures s2s4 of the supporting information ) . when investigating the soluble versus insoluble membrane fraction , pfatg8 could only be detected in the soluble fraction with the number of parasites used per lane . we attribute this to the low amount of pfatg8 present in the untreated control and to reaching the detection limit of our assay ( data not shown ) . at the treatment concentration and duration used in the study , our studies indicated that the pta scaffold is a good platform for hit - optimization . we synthesized a pta - benzaldehyde derivative , 4-formyl - n-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide ( 7 ) , with a functional handle extending off the common hydrophobic ring system ( table 1 , scheme 1 ) . 7 was prepared from commercially available pta and 4-formyl benzoic acid through a dicyclohexylcarbodiimide ( dcc)-promoted amide coupling . compound 7 can be tethered through a dialkoxyamine linker to a library of aldehydes and can be screened using our primary spr competition assay against the pfatg8-pfatg3 interaction . in docking studies , 7 bound the w- and l - site of pfatg8 in a fashion similar to compound 2 with the functional handle positioned toward the a - loop pocket ( figure 7a ) . a gain in parasite selectivity for such bifunctional analogues is expected as the a - loop is missing in the human atg8 homologues . to confirm binding to pfatg8 , we tethered 7 to ( + ) -biotinamidohexanoic acid hydrazide ( bach ) through its reactive aldehyde group and tested binding with spr . his12-pfatg8 injected at various concentrations led to a dose - dependent increase in spr response indicating pfatg8 directly binds 8 with a kd of 540 nm . in contrast , the human atg8 homologue , microtubule - associated protein light chain 3 ( hlc3 ) showed much lower affinity for 8 with a kd of 18 m , indicating specificity of the pta scaffold for p. falciparum ( figure 7b ) . additionally , recombinant pfatg3 did not bind immobilized 8 ( data not shown ) in agreement with our docking studies suggesting binding to the w - site of pfatg8 . compound 7 was converted to the hydrochloride salt and was subjected to acid - catalyzed acetal formation with methanol and trimethyl orthoformate under microwave irradiation to provide the dimethyl acetal compound 9 , an unreactive derivative , to confirm the inhibitory activity of the starting platform ( table 1 , scheme 1 ) . 4-(dimethoxymethyl)-n-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide 9 has a similar shape and distribution of the acceptor and donor pairs as the original pta compounds ( figure 7c ) and docked onto pfatg8 in a fashion similar to 7 ( figure 7a ) . spr studies confirmed that 9 inhibited the pfatg8-pfatg3 interaction with an ic50 of 2.86 m in spr ( figure 8a ) . we next measured growth inhibition of p. falciparum 3d7 by 1 using the sybr green i assay . this assay exploits the absence of nuclei in erythrocytes with a fluorescent dye that is unquenched upon binding to nucleic acids , preferentially double - stranded dna . in two of three independent experiments , the ic50 of 1 was 768 nm , similar to previously published results , while in a third experiment , the ic50 was 3.3 m , resulting in an average ic50 of 1.61 1.47 m . using this assay , 9 had a potency similar to that of compound 1 against the blood stage of p. falciparum with an average ic50 of 1.48 0.6 m ( figure 8b ) . we identified compound 1 through a screen for inhibitors against the plasmodium atg8-atg3 protein protein interaction ( ppi ) . targeting protein protein interactions has long been overlooked in the drug development field . it was thought to be difficult because of the shallower nature of many pockets and the more extensive network of residues involved in the interaction compared to an enzymatic site . however , ppis also offer the opportunity for more selectivity , an important concept when targeting a eukaryotic parasitic protein within a eukaryotic host . great methodological and technological advances have been made recently using this approach with promising leads in cancer drug development . in the case of our inhibitor , in addition to its potential as a therapeutic drug , any 1-derived inhibitor could serve as a tool to elucidate the function of pfatg8 during different stages of the malaria life cycle as its function is currently unclear . treatment of p. falciparum with high levels of 1 led to a drastic increase in pfatg8 protein levels , presumably the unlipidated form as judged by its migration in sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) . this increase could be due to an up - regulation of pfatg8 synthesis to compensate for inhibition or due to a buildup of existing protein levels because of a blockade in autophagic degradation . in yeast , nitrogen starvation leads to induction of atg8 expression while inhibition of later stages of autophagy leads to accumulation and even greater protein levels of atg8 . further studies are necessary to determine if pfatg8 is up - regulated at the transcriptional , translational , or degradation level in response to treatment with 1 . this could be due to an accumulation of the drug inside the parasite or could be because even slight inhibition of pfatg8 lipidation has drastic effects on parasite growth , similar to reaching the tipping point on a balance . an alternative explanation is the result of off - target effects ; however , the observed delipidation of pfatg8 could not be attributed to an off - target effect . 1 was previously reported to have low cytotoxicity with an ld50 ( lethal dose ) of 18.2 m in hepg2 cells and half maximal cytotoxicity concentration ( cc50 ) and ic50 of 32 m in huh7 cells ( pubchem aid : 685525 , 660872 , 449705 ) . in our study together , this indicates 1 may be a good starting scaffold for antimalarial drug design . compound 1 had lower activity in the liver stage than in the blood stage , which could either indicate that pfatg8 is less important in the liver stage or that less compound is delivered to the parasite in liver cells . there is precedence for this variability in the efficacy of antimalarials between the liver and blood stages . compound 1 showed 50% inhibition of liver stage parasites at 30 m , which is within the range for cytotoxicity in human cells . we suggest the use of 1 and the pta scaffold for further probe development to increase specificity and potency in both the liver and blood stages . all pta - containing mmv compounds had a ligand efficiency by atom number ( lean ) score greater than 0.3 in the blood stage assay , indicative of good ligand efficiency and good potential for future drug optimization . this combined with the higher affinity of 8 for pfatg8 compared to human lc3 makes the pta scaffold a promising starting point for expansion . we are currently pursuing optimization through a combinatorial oxime library approach using 7 with the goal to extend the inhibitor into the a - loop pocket and to gain selectivity toward pfatg8 . our spr data confirm that 1 directly binds to pfatg8 , while our docking studies suggest that the pta scaffolds of compounds 13 , 7 , and 9 bind the w - site . the w - sites of atg8 homologues in mammals and yeast participate in numerous protein protein interactions through binding to the aromatic residue of an aim , including nonautophagic proteins . therefore , our inhibitor and its derivatives could be used to identify novel plasmodium atg8 interactions as well as to confirm paralogous interactions known to occur in yeast and mammalian cells . taken together , our bioinformatics analysis of the pta - binding site and the spr binding studies strongly suggest that the amino acid differences near the l- and w - site may have contributed to the failure of 1 in the p. yoelii liver stage drug - screening assay . two residue differences ( i8v , p9s ) adjacent to the w - site may lead to a widening of the w - site in p. yoelli because of their shorter side chains , and one residue ( v62i ) participating directly in the l - site results in an extended side chain and therefore may decrease the volume of the l - site pocket . additionally , l115 m just below the l - site binding pocket may also contribute to a decreased volume as a secondary shell residue . care and emphasis on choice of model system should be taken into account when studying protein ligand interactions , as single amino acid substitutions may have drastic effects on binding . his12-pfatg8 variants were expressed and purified with cobalt - nta affinity columns similar to his6-pfatg8 , as previously published with the exception that proteins were eluted from cobalt - charged talon resin ( clonetech ) in buffer containing 50 mm ethylenediaminetetraacetic acid ( edta ) rather than imidazole . spr runs were conducted on a biacore 3000 instrument ( ge healthcare ) at 25 c with a flow rate of 50 l / min , unless otherwise specified . running buffer ( rb ) consisted of 1 phosphate - buffered saline ( pbs ) ( 1 mm kh2po4 , 5.6 mm na2hpo4 , 154.5 mm nacl , ph 7.4 ) , 0.01% v / v p20 , and varying amounts of dmso ( quality biologicals ) . a double referencing method was applied to correct for nonspecific binding to the chip with interspersed blank injections correcting for baseline drifts . changes in refractive index because of dmso were accounted for with a dmso calibration curve . mbp - pfatg3 was immobilized onto a cm5 chip ( biacore ) as described previously with mbp immobilized on a reference flowcell . compounds were added to 300 nm his6-pfatg8 in rb at a final concentration of 5 m , and 40 l was injected , followed by a 12.5 l injection of 2 m mgcl2 for dissociation and regeneration of the spr chip surface . thirty microliters of pfatg8 at 200 nm was injected in the presence of a 2-fold dilution series of compound ( highest concentration of 50 m ) or equivalent volume of dmso ( final dmso concentration was 1% ) . his12-pfatg8 was injected over an nta - chip ( ge healthcare ) preconditioned with nickel , leading to capture of 3000 response units ( rus ) . running a 2-fold - dilution series of compounds , highest concentration of 75 m , was injected over pfatg8 variants at 40 l / min . 8 was injected over a neutravidin - coated spr chip ( ge healthcare ) on one flowcell , with 130 rus immobilized , while bach was injected over a reference flowcell ( 150 rus immobilized ) . his6-pfatg8 , human lc3 , or his6-pfatg3 was injected in duplicate in running buffer containing 10 mm hepes ph 7.5 , 150 mm nacl , 0.05% p20 . protein was dissociated from the chip after each cycle with 10 l injection of 20 mm hepes ph 7.4 , 1% w / v sds regeneration solution . all reagents were obtained from commercial suppliers and were used without further purification . acetonitrile was distilled after drying on cah2 and then was stored over 3 molecular sieves . dynamic adsorbents 3263 m silica gel was used for flash column chromatography , and 250 m f254 plates were used for thin layer chromatography ( tlc ) . microwave - assisted reactions were carried out using a biotage initiator microwave synthesizer ( 300 w ) . h and c nmr spectra were acquired on a bruker avance iii 500 spectrometer operating at 500 mhz for h and 125 mhz for c. chemical shift values are reported as ( ppm ) relative to chcl3 at 7.27 ppm and dmso at 2.50 ppm for h nmr and chcl3 at 77.0 ppm and dmso at 39.51 ppm for c nmr . mass spectrometry analysis was carried out at university of illinois at urbana - champagne , school of chemical sciences , mass spectrometry laboratory . the purity of synthesized compounds was 95% as analyzed by high - performance liquid chromatography ( hplc , beckman gold nouveau system gold ) on a c18 column ( grace alltima 3 m c18 analytical rocket column , 53 mm 7 mm ) using triethylammonium acetate buffer ( 50 mm , ph 7 ) and acetonitrile ( acn ) as eluent , flow rate 3 ml / min , and detection at 300 nm . to a solution of 4-(pyridin-2-yl)-1,3-thiazol-2-amine ( 0.059 g , 0.33 mmol ) in acetonitrile ( 2.0 ml ) was added sequentially dicyclohexylcarbodiimide ( 0.076 g , 0.37 mmol ) , 4-formylbenzoic acid ( 0.050 g , 0.33 mmol ) , and n , n - dimethylamino pyridine ( 0.012 g , 0.10 mmol ) . the mixture was heated at 50 c for 17 h and then was allowed to cool to ambient temperature . solids were removed by vacuum filtration , and the resulting filtrate was condensed under reduced pressure . the resulting yellow solid was redissolved in chcl3 ( 5 ml ) , and 1 m hcl ( 5 ml ) was added to give a yellow - tan emulsion at the liquid this solid was collected by centrifugation at 4000 rpm for 5 min followed by manual collection of the resulting cake ( this acid precipitation was necessary to remove closely eluting impurities ) . the solid was then purified by silica flash column chromatography ( dichloromethane(dcm):meoh : triethylamine 94:5:1 ) rf = 0.32 . the product was obtained as a yellow powder ( 23 mg , 22% yield ) . h nmr ( 500 mhz , dmso - d6 ) ( ppm ) = 12.95 ( br . s. , 1h ) , 10.13 ( s , 1h ) , 8.63 ( d , j = 3.93 hz , 1h ) , 8.31 ( d , j = 8.17 hz , 2h ) , 8.07 ( d , j = 8.33 hz , 2h ) , 8.03 ( d , j = 7.86 hz , 1h ) , 7.92 ( s , 1h ) 7.91 ( td , j = 2.00 hz , 8.75 hz , 1h ) , 7.35 ( ddd , j = 1.10 , 4.79 , 7.47 hz , 1h ) c nmr ( 500 mhz , dmso - d6 ) ( ppm ) = 192.90 , 164.74 , 158.91 , 152.03 , 149.54 , 149.38 , 138.50 , 137.30 , 137.15 , 129.41 , 128.94 , 122.88 , 120.06 , 112.30 . high - resolution mass spectrometry ( hrms , electrospray ionization , esi ) m / z : calcd 310.0650 ( m h ) ; found 310.0651 ( m h ) . twenty - five microliters 50 mm 7 dissolved in dmso was incubated with 20 l 50 mm bach ( 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a - hexahydrothieno[3,4-d]imidazol-4-yl]-n-(6-hydrazinyl-6-oxohexyl)pentanamide ) ( sigma - aldrich ) dissolved in dmso and 10 l sodium acetate ( ph 4.5 ) with 0.02% sodium azide at 37 c overnight . 7 dissolved in chcl3 was treated with 1 m hcl as described above to form the hydrochloride salt . 7 hcl ( 0.040 g , 0.12 mmol ) was suspended in meoh ( 0.5 ml ) , and trimethylorthoformate ( 0.010 ml , 0.91 mmol ) was added followed by p - toulene sulfonic acid monohydrate ( 0.003 g , 0.012 mmol ) . this solution was heated by microwave irradiation at 130 c in a sealed vial for 5 min and then was stirred at ambient temperature for 36 h at which time a precipitate formed . the solvent was removed under reduced pressure , and the residue was dissolved in dcm ( 10 ml ) and was washed with saturated nahco3 ( 10 ml ) and brine ( 10 ml ) and was dried with na2so4 . condensation under reduced pressure yielded the product as a yellow powder ( 26 mg , 61% yield ) . h nmr ( 500 mhz , cdcl3 ) ( ppm ) = 9.81 ( br s , 1h ) , 8.64 ( d , j = 4.24 hz , 1h ) , 7.96 ( d , j = 8.17 hz , 2h ) , 7.91 ( d , j = 7.86 hz , 1h ) , 7.74 ( s , 1h ) , 7.74 ( td , j = 1.73 , 7.70 hz , 1h ) , 7.62 ( d , j = 8.17 hz , 2h ) , 7.22 ( dd , j = 4.95 , 6.84 hz , 1h ) , 5.47 ( s , 1h ) , 3.35 ( s , 6h ) c nmr ( 500 mhz , cdcl3 ) ( ppm ) = 164.24 , 158.12 , 152.24 , 149.85 , 149.62 , 143.32 , 136.84 , 131.80 , 127.51 , 127.29 , 122.69 , 120.50 , 112.21 , 102.08 , 52.71 . hrms ( esi ) m / z : calcd 356.1069 ( m h ) ; found 356.1070 ( m h ) . assays were conducted in 1 pbs with 1:1800 final dilution of sypro orange dye ( invitrogen ) . fluorescence was measured from 20 to 80 c in a biorad c1000 thermal cycler . one hundred microliters of pta compounds or equivalent volume of dmso was added to his6-pfatg8 . the hc-04 cell line ( atcc , manassas , va , u.s . ) was maintained in complete medium ( imdm containing 2.5% fcs , 100 units / ml penicillin , 100 g / ml streptomycin , and 2 mm l - glutamine , all from gibco , life technologies , grand island , ny ) . p. falciparum 3d7-gfp parasite strain was propagated in the parasitology core facility , the johns hopkins malaria research institute . in vitro infection of human hepatocytes briefly , salivary glands were sequestered from infected anopheles gambiae mosquitoes at day 17 after exposure to infective blood meal , and homogenates were separated on an optiprep density gradient ( sigma - aldrich , st . sporozoites were collected from the gradient interface , were washed in complete medium , were counted using a hemocytometer , and were incubated with hc-04 cells at 3:1 sporozoite to hepatocyte ratio for 2 h at 37 c . infected cultures were further propagated in complete medium alone or in medium supplemented with 3 m or 30 m of 1 . flow cytometry based detection of infected cells was done 72 h post infection using facscalibur flow cytometer ( bd biosciences ) and was analyzed using flowjo software ( tree star , inc . , effect of 1 on the viability of in vitro propagated hc-04 cells was monitored as follows : 0.3 10 cells per well were seeded into the 24-well plate and were treated with 3 m or 30 m of 1 in complete medium for 96 h , and a relevant amount of dmso was used as a vehicle control . detection of annexin - v positive and pi - positive cells in hepatocyte cultures was done by flow cytometry according to the manufacturer s instruction ( invitrogen , life technologies , grand island , ny , u.s . ) . p. falciparum 3d7 and fcr3 cultures were maintained using modified , previously published methods at 37 c , 2% hematocrit of human red blood cells . complete culture media consisted of sterile rpmi 1640 media ( life technologies ) supplemented with 10% human serum and 0.005% hypoxanthine and buffered with final concentrations of 0.6% hepes and 0.26% nahco3 . the fcr3 strain was maintained at 3% co2 and 5% o2 , 92% n2 atmosphere , while the 3d7 strain was maintained at 5% co2 , 5% o2 , and 90% n2 atmosphere . j. smith , seattle biomed ) asynchronous culture , 25% parasitemia , was washed in starvation media lacking human serum and was resuspended in complete media with 50 m compound 1 or equivalent dmso or in starvation media with equivalent dmso for 5 h. rbcs were harvested with centrifugation and were lysed with 0.2% saponin , and rbc lysate was removed through three 1 pbs washes . parasites were harvested by centrifugation and were washed in 1 pbs with complete edta - free protease inhibitors ( roche ) and were lysed by repeated vortexing and boiling in sds reducing sample buffer . lysates were separated with sds - page on a 420% polyacrylamide gel and were subjected to western blotting with 1:400 -tgatg8 , demonstrated to be cross - reactive with pfatg8 ( generously provided by dr . hrp - conjugated secondary antibodies ( southern biotech ) were detected by supersignal west femto ( thermo scientific ) or amersham ecl prime ( ge healthcare ) chemiluminescent substrate . ap - conjugated secondary antibodies ( emd millipore ) were detected using nbt / bcip ( promega ) colorimetric stain . total protein levels were visualized with proact membrane stain ( amresco ) and were quantified with imagej . parasite morphology at time of harvesting was visualized with light microscopy at 100 magnification on olympus bx53 system microscope ( olympus america , inc . ) . ten microliters of 10 compound diluted in rpmi 1640 media ( gibco ) with a constant concentration of 1% dmso was added to a 96 well plate ( costar ) , 90 l of 1.5% ring stage , synchronized with 5% w / v sorbitol p. falciparum 3d7 parasites , 1% hematocrit , in culture media with 10% v / v human serum with 10 g / ml gentamycin . each compound concentration and 1% v / v dmso controls were run in triplicate . plates were incubated at 37 c in 5% o2 , 5% co2 , and 90% n2 for 72 h. plates were frozen , thawed , and incubated with 100 l 2 sybr green in lysis buffer ( 20 mm tris ph 7.5 , 5 mm edta , 0.008% saponin , 0.08% tritonx-100 ) in the dark for at least 1 h. fluorescence was measured with a plate reader ( hts 7000 , perkinelmer ) at excitation / emission wavelengths of 485/535 nm . docking was conducted using the openeye software package using standard parameters if not specified otherwise ( www.eyesopen.com ) . a receptor for pfatg8 was made with make_receptor from oedocking toolkit without constraints covering the whole molecule to detect potential binding pockets on the surface . three main pockets ( w - site , l - site , a - site ) were detected with 377 , 271 , and 513 volumes , used in first docking studies . a second receptor was generated with specific constraints to the carbonyl of lys47 as hydrogen bond donor . a maximum of 2000 conformers for each compound from the mmv malaria box was prepared with omega2 . fred was used to dock these conformers onto both the constrained and unconstrained receptor . docking results were visualized using the openeye visualization software , vida . using itasser , models of p. yoelii and p. berghei atg8 were generated with pdb code 4eoy as the parent molecule . default values as suggested by the web server were used to generate these models . sequences for p. yoelii ( pyym_0504500 ) and p. berghei ( pbanka_050410 ) were obtained from plasmodb . atg8 , represented by the rectangle , is essential to the elongation of the autophagosomal membrane . ( b ) generic conjugation pathway shown for yeast system . in plasmodium , atg8 is synthesized with a c - terminal glycine that does not require proteolytic processing for activation . identification of a common scaffold that inhibits atg8-atg3 from the mmv malaria box screen . ( b ) bar graph showing inhibition of hits in primary screen , denoted by compound number . inhibition was measured with increasing amount of compound in spr competition screen . mean and standard deviation ( sd ) of three injections are shown . error bars show sd of three measurements . identification of 1 binding site on pfatg8 . 1 was injected over immobilized pfatg8-variants in two separate runs at four concentrations . ( b ) in silico docking of pta compounds to pfatg8 ( pdb code 4eoy ) . overall structure of pfatg8 with w- and l - site and a - loop demarcated . docking was also performed with a hydrogen bond constraint to the carbonyl of lys47 , located between the w- and l - site . predicted pose for constrained docking is shown in green . for 1 , an alternate pose , it is highly ranked in the unconstrained docking and is enriched in the top 10 poses as shown in magenta . amino acid changes between the species are shown in red with p. falciparum letter and numbering followed by p. yoelii . w - site , l - site , and a - loop pockets are shown in mesh in cyan , purple , and green , respectively . falciparum atg8 pocket sizes were calculated with openeye vida visualization software ( www.eyesopen.com ) . effect of 1 treatment on the development of p. falciparum 3d7 gfp parasite in hc-04 cells in vitro . ( a ) flow cytometry based detection of annexin - v positive and pi - positive cells in hepatocyte cultures treated with 1 ( as described in experimental section ) . dot plot graphs demonstrate representative pattern of staining , and bar graphs show summary ( mean sd ) of viable cell detection obtained in three independent hepatocyte cultures . ( b ) viable infected hepatocytes ( gfp+/pi ) were detected by flow cytometry in hc-04 cultures 72 h post infection with p. falciparum . dot plot graphs demonstrate representative pattern of staining , and numbers reflect percentages of gfp positive cells in total pi negative cell populations . ( c ) summary ( mean sd ) of viable infected cell detection obtained in three independent hepatocyte cultures . ( d ) gfp - specific fluorescence was assessed in infected cultures exposed to 1 or dmso for 72 h. histograms demonstrate one representative staining pattern , and numbers reflect mean fluorescence intensity ( mfi ) in gfp - positive populations . bar graphs reflect gfp - specific mfi ( mean sd ) in viable cell population detected in three independent hepatocyte cultures . ( a ) dose - dependent immunoblot analysis of p. falciparum treated with dmso or 3.375 , 6.75 , 12.5 , or 25 m 1 for 6 h. chloroquine ( cq ) at 50 nm was used as a positive control of autophagy inhibition . the blot was probed with antibody against tgatg8 , demonstrated to be cross reactive against pfatg8 . ( b ) blood smears of p. falciparum after treatment with dmso or 50 m 1 for 5 h , observed at 100 magnification . representative images for different stages are shown , progressing from ring stage on the left to late schizont on the right . 7 ( yellow ) and 9 ( green ) were docked onto the constrained receptor of pfatg8 ( pdb code 4eoy ) with openeye docking suite . pfatg8 and hlc3 were injected over immobilized 8 , and binding was measured with spr . ( c ) superposition of small molecule hits derived from mmv malaria box with 9 . the individual molecular surfaces with their corresponding electrostatic potential are depicted in side and top view . all four molecules share the pta moiety and were superimposed using rocs via shape complementarity and tanimoto color scoring function . the figure was prepared with vida and was rendered in povray ( www.povray.org ) . ( a ) inhibition of pfatg8-pfatg3 interaction by 9 . spr response of pfatg8 injected over immobilized pfatg3 was measured in the presence of increasing concentration of 9 . sybr green i assays were used to measure inhibition by chloroquine ( cq ) , 1 , and 9 . growth inhibition curves are shown for one experiment with ic50 values from two to three experiments in table inset . pta - containing compounds used in studies listed with pubchem compound identification ( cid ) , mmv i d , chemical structure , molecular weight ( g / mol ) , ic50 in spr and blood stage assays , and lean score , calculated as log ( ic50)/number of heavy atoms . the organisms are maintained in licensed bsl2 facilities , and approvals are obtained annually for all consortia laboratories . human erythrocytes are obtained either commercially or from healthy volunteers under johns hopkins irb - approved protocols . because these cells are provided to the lab without identifiers

atg8 is a ubiquitin - like autophagy protein in eukaryotes that is covalently attached ( lipidated ) to the elongating autophagosomal membrane . autophagy is increasingly appreciated as a target in diverse diseases from cancer to eukaryotic parasitic infections . some of the autophagy machinery is conserved in the malaria parasite , plasmodium . although atg8 s function in the parasite is not well understood , it is essential for plasmodium growth and survival and partially localizes to the apicoplast , an indispensable organelle in apicomplexans . here , we describe the identification of inhibitors from the malaria medicine venture malaria box against the interaction of pfatg8 with its e2-conjugating enzyme , pfatg3 , by surface plasmon resonance . inhibition of this protein protein interaction prevents pfatg8 lipidation with phosphatidylethanolamine . these small molecule inhibitors share a common scaffold and have activity against both blood and liver stages of infection by plasmodium falciparum . we have derivatized this scaffold into a functional platform for further optimization .

Introduction Results Discussion Experimental Section

the malaria parasite , plasmodium , is a major public health burden in the developing world , and despite the existence of antimalarial treatment active in blood stages of infection , there is a continual need for novel drug design as the parasite develops resistance to current treatments . discovery of novel compounds in antimalarial drug development is essential for future intervention strategies . atg8 is the ubiquitin - like ( ubl ) protein necessary for formation and maturation of autophagosomes in autophagy in eukarya . in yeast and mammals , atg8 is lipidated to the autophagosome membrane , but in plasmodium , atg8 is partially conjugated to the membrane of the apicoplast under nonstarvation conditions . the apicoplast is a nonphotosynthetic chloroplast - like organelle present in apicomplexans and is essential for isoprenoid synthesis . atg8 is essential to the plasmodium parasite and has been proposed as a target for antimalarial drug design . atg8 is then transferred to its e2-like conjugating enzyme atg3 , forming a second thioester intermediate before being conjugated to the nitrogen of pe ( figure 1 ) . notably , in plasmodium , atg8 is synthesized with a c - terminal glycine and therefore does not require activation by atg4 . recently published studies showed a drastic growth defect in plasmodium falciparum when levels of pfatg7 were reduced . this along with the essentiality of plasmodium atg8 suggests that targeting pfatg8 lipidation is a good strategy for drug intervention . regions of diversity exist between the human and plasmodium system that may be exploitable through small molecule inhibition . here , we report the identification of a class of compounds that inhibit the plasmodium atg8-atg3 interaction and that inhibit in vitro growth of p. falciparum in blood- and liver - stage assays , presumably through prevention of pfatg8 lipidation . previously , we developed a surface plasmon resonance ( spr)-based competition assay to identify compounds that disrupt the pfatg8-pfatg3 noncovalent interaction.pfatg3 is immobilized onto an spr chip , and pfatg8 is injected in the presence of dimethyl sulfoxide ( dmso , control ) or a compound ( dissolved in dmso ) , and binding is measured by the spr response . the medicines for malaria venture ( mmv ) malaria box of 200 druglike and 200 probelike molecules was screened at 5 m in a primary spr screen ( figure 2a ) . six compounds met the cutoff for at least 25% inhibition of the pfatg8-pfatg3 interaction : ( n-(4-methylphenyl)-4-pyridin-2-yl-1,3-thiazol-2-amine ) ( 1 ) , ( 2-methylsulfanyl - n-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide ) ( 2 ) , ( 2-bromo - n-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide ) ( 3 ) , n-[2-chloro-5-(trifluoromethyl)phenyl]-2-[2-(4-methylphenyl)pyrazolo[1,5-a]pyrazin-4-yl]sulfanylacetamide ( 4 ) , 1-[4-(dimethylamino)phenyl]-6,6-dimethyl-1,3,5-triazine-2,4-diamine ( 5 ) , and 2-n,3-n - bis(4-bromophenyl)quinoxaline-2,3-diamine ( 6 ) ( figure 2a,2b , table 1 ) . in subsequent dose - dependent studies , 46 demonstrated a constant level of inhibition independent of concentration of the small molecule and were not further investigated . interestingly , these compounds shared a common scaffold : 4-pyridin-2-yl-1,3-thiazol-2-amine ( pta ) incorporated as the n - substituent in various anilines or benzamides . 13 were tested for their effect on the stability of pfatg8 using fluorescence - based thermal shift assays ( tsas ) . none of the compounds significantly affected the melting temperature ( tm ) indicating that they most likely did not disturb the tertiary structure of pfatg8 ( figure 2d ) . the openeye software package ( www.eyesopen.com ) was used to dock conformers of the compounds against the x - ray structure of pfatg8 ( pdb code 4eoy ) . all three compounds docked to the w - site of pfatg8 ( figure 3b ) . 1 is missing a donor oxygen compared to 2 and 3 and , therefore , may adopt a different mode of binding to the w- and l - sites of pfatg8 . 2 docked with the pyridine ring in the w - site , and the methylsulfanylbenzene group bound to the l - site of pfatg8 . docking was repeated using a version of the receptor for which the carbonyl of lys47 was input as a hydrogen bond acceptor constraint ( figure 3b inset ) . 1 had a similar binding pose to the highest ranked pose from the unconstrained docking , while 2 was slightly different with the pyridine ring rotated 90 in the w - site and the benzene ring positioned just below and to the left of the l - site pocket with the methylsulfanyl group reaching into the mostly hydrophobic l - site . pfatg8 is expressed and lipidated during the liver stage where it partially localizes to the apicoplast . treatment of early liver stage parasites with the autophagy inhibitor 3-methyladenine is reported to delay conversion of the parasite into its trophozoite form . analysis of the w / l - site in these three species revealed differences in the amino acid composition that could affect drugs predicted to bind in that region ( figure 4 ) . hc-04 is a unique immortalized cell line that exhibits the expression of biochemical markers characteristic for normal hepatocytes and allows for the full development of the human malaria parasite , p. (2022 ) using this system , we assessed the effect of 1 on the development of p. falciparum 3d7-green fluorescent protein ( gfp ) parasites in human hepatocytes in vitro . no change in the viability of hc-04 cells was detected in response to treatment with 3 m or 30 m of 1 for 96 h ( figure 5a ) . using flow cytometry , we observed about a 50% decrease in the proportion of hepatocytes infected with p. falciparum 3d7-gfp sporozoites ( gfp+/propidium iodide ( pi)- cells ) in response to treatment with 30 m , but not with 3 m of 1 ( figure 5b , c ) . additionally , there was a dose - dependent reduction in the intensity of gfp fluorescence at both concentrations of 1 , indicating inhibition of parasite development within hepatocytes , at least in vitro ( figure 5d ) . because 1 did not affect cell survival or cell growth of hc-04 cells ( figure 5a ) , the compound s effect on the parasite is unlikely to result from host cell cytotoxicity . in contrast , treatment with 50 m cytocidal levels of compound 1 led to a drastic increase in pfatg8 protein levels as well as to a shift in mobility , likely corresponding to the unlipidated form of pfatg8 . overall protein levels were unchanged , indicating an up - regulation or accumulation of pfatg8 in the presence of 1 under conditions that are likely leading to cell death ( figure s1 of the supporting information ) . after treating the parasites for 6 hours , a dose - dependent increase in delipidation of pfatg8 is already observed at 12.5 m of 1 , and at 25 m , unlipidated pfatg8 is the predominant species ( figure 6a , figures s2s4 of the supporting information ) . when investigating the soluble versus insoluble membrane fraction , pfatg8 could only be detected in the soluble fraction with the number of parasites used per lane . we attribute this to the low amount of pfatg8 present in the untreated control and to reaching the detection limit of our assay ( data not shown ) . at the treatment concentration and duration used in the study , our studies indicated that the pta scaffold is a good platform for hit - optimization . we synthesized a pta - benzaldehyde derivative , 4-formyl - n-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide ( 7 ) , with a functional handle extending off the common hydrophobic ring system ( table 1 , scheme 1 ) . compound 7 can be tethered through a dialkoxyamine linker to a library of aldehydes and can be screened using our primary spr competition assay against the pfatg8-pfatg3 interaction . in docking studies , 7 bound the w- and l - site of pfatg8 in a fashion similar to compound 2 with the functional handle positioned toward the a - loop pocket ( figure 7a ) . a gain in parasite selectivity for such bifunctional analogues is expected as the a - loop is missing in the human atg8 homologues . additionally , recombinant pfatg3 did not bind immobilized 8 ( data not shown ) in agreement with our docking studies suggesting binding to the w - site of pfatg8 . compound 7 was converted to the hydrochloride salt and was subjected to acid - catalyzed acetal formation with methanol and trimethyl orthoformate under microwave irradiation to provide the dimethyl acetal compound 9 , an unreactive derivative , to confirm the inhibitory activity of the starting platform ( table 1 , scheme 1 ) . we next measured growth inhibition of p. falciparum 3d7 by 1 using the sybr green i assay . this assay exploits the absence of nuclei in erythrocytes with a fluorescent dye that is unquenched upon binding to nucleic acids , preferentially double - stranded dna . using this assay , 9 had a potency similar to that of compound 1 against the blood stage of p. falciparum with an average ic50 of 1.48 0.6 m ( figure 8b ) . we identified compound 1 through a screen for inhibitors against the plasmodium atg8-atg3 protein protein interaction ( ppi ) . targeting protein protein interactions has long been overlooked in the drug development field . it was thought to be difficult because of the shallower nature of many pockets and the more extensive network of residues involved in the interaction compared to an enzymatic site . however , ppis also offer the opportunity for more selectivity , an important concept when targeting a eukaryotic parasitic protein within a eukaryotic host . in the case of our inhibitor , in addition to its potential as a therapeutic drug , any 1-derived inhibitor could serve as a tool to elucidate the function of pfatg8 during different stages of the malaria life cycle as its function is currently unclear . this increase could be due to an up - regulation of pfatg8 synthesis to compensate for inhibition or due to a buildup of existing protein levels because of a blockade in autophagic degradation . in yeast , nitrogen starvation leads to induction of atg8 expression while inhibition of later stages of autophagy leads to accumulation and even greater protein levels of atg8 . this could be due to an accumulation of the drug inside the parasite or could be because even slight inhibition of pfatg8 lipidation has drastic effects on parasite growth , similar to reaching the tipping point on a balance . an alternative explanation is the result of off - target effects ; however , the observed delipidation of pfatg8 could not be attributed to an off - target effect . compound 1 had lower activity in the liver stage than in the blood stage , which could either indicate that pfatg8 is less important in the liver stage or that less compound is delivered to the parasite in liver cells . there is precedence for this variability in the efficacy of antimalarials between the liver and blood stages . compound 1 showed 50% inhibition of liver stage parasites at 30 m , which is within the range for cytotoxicity in human cells . we suggest the use of 1 and the pta scaffold for further probe development to increase specificity and potency in both the liver and blood stages . the w - sites of atg8 homologues in mammals and yeast participate in numerous protein protein interactions through binding to the aromatic residue of an aim , including nonautophagic proteins . taken together , our bioinformatics analysis of the pta - binding site and the spr binding studies strongly suggest that the amino acid differences near the l- and w - site may have contributed to the failure of 1 in the p. yoelii liver stage drug - screening assay . two residue differences ( i8v , p9s ) adjacent to the w - site may lead to a widening of the w - site in p. yoelli because of their shorter side chains , and one residue ( v62i ) participating directly in the l - site results in an extended side chain and therefore may decrease the volume of the l - site pocket . a double referencing method was applied to correct for nonspecific binding to the chip with interspersed blank injections correcting for baseline drifts . compounds were added to 300 nm his6-pfatg8 in rb at a final concentration of 5 m , and 40 l was injected , followed by a 12.5 l injection of 2 m mgcl2 for dissociation and regeneration of the spr chip surface . thirty microliters of pfatg8 at 200 nm was injected in the presence of a 2-fold dilution series of compound ( highest concentration of 50 m ) or equivalent volume of dmso ( final dmso concentration was 1% ) . protein was dissociated from the chip after each cycle with 10 l injection of 20 mm hepes ph 7.4 , 1% w / v sds regeneration solution . the resulting yellow solid was redissolved in chcl3 ( 5 ml ) , and 1 m hcl ( 5 ml ) was added to give a yellow - tan emulsion at the liquid this solid was collected by centrifugation at 4000 rpm for 5 min followed by manual collection of the resulting cake ( this acid precipitation was necessary to remove closely eluting impurities ) . p. falciparum 3d7-gfp parasite strain was propagated in the parasitology core facility , the johns hopkins malaria research institute . , effect of 1 on the viability of in vitro propagated hc-04 cells was monitored as follows : 0.3 10 cells per well were seeded into the 24-well plate and were treated with 3 m or 30 m of 1 in complete medium for 96 h , and a relevant amount of dmso was used as a vehicle control . detection of annexin - v positive and pi - positive cells in hepatocyte cultures was done by flow cytometry according to the manufacturer s instruction ( invitrogen , life technologies , grand island , ny , u.s . ) plates were incubated at 37 c in 5% o2 , 5% co2 , and 90% n2 for 72 h. plates were frozen , thawed , and incubated with 100 l 2 sybr green in lysis buffer ( 20 mm tris ph 7.5 , 5 mm edta , 0.008% saponin , 0.08% tritonx-100 ) in the dark for at least 1 h. fluorescence was measured with a plate reader ( hts 7000 , perkinelmer ) at excitation / emission wavelengths of 485/535 nm . a second receptor was generated with specific constraints to the carbonyl of lys47 as hydrogen bond donor . a maximum of 2000 conformers for each compound from the mmv malaria box was prepared with omega2 . atg8 , represented by the rectangle , is essential to the elongation of the autophagosomal membrane . in plasmodium , atg8 is synthesized with a c - terminal glycine that does not require proteolytic processing for activation . identification of a common scaffold that inhibits atg8-atg3 from the mmv malaria box screen . ( b ) bar graph showing inhibition of hits in primary screen , denoted by compound number . identification of 1 binding site on pfatg8 . overall structure of pfatg8 with w- and l - site and a - loop demarcated . for 1 , an alternate pose , it is highly ranked in the unconstrained docking and is enriched in the top 10 poses as shown in magenta . 7 ( yellow ) and 9 ( green ) were docked onto the constrained receptor of pfatg8 ( pdb code 4eoy ) with openeye docking suite . ( c ) superposition of small molecule hits derived from mmv malaria box with 9 . ( a ) inhibition of pfatg8-pfatg3 interaction by 9 . spr response of pfatg8 injected over immobilized pfatg3 was measured in the presence of increasing concentration of 9 . because these cells are provided to the lab without identifiers

retinitis pigmentosa ( rp ) is the term used for a group of retinal diseases that are characterized by inherited , progressive degeneration of retinal tissue , mainly rod and secondarily cone photoreceptors . the clinical features are night blindness and visual field ( vf ) loss due to loss of rod photo - receptors . many patients fall into a classical pattern of difficulties with dark adaptation and night blindness and loss of midperipheral vision field . as the disease advances , they lose peripheral vision , eventually developing tunnel vision with the remaining cone photoreceptors , and finally lose central vision and visual acuities ( vas ) as these cones secondarily degenerate in the macular region.1,2 typically , it takes several years until the patients lose their central vision ; therefore , it is important to protect the cone photoreceptors in the macular area for rp patients . because rp is caused by various mutations in any of > 45 responsible genes , the processes of degeneration are considered to be not uniform , and no effective treatment other than nutritional supplementation of vitamin a currently exists . recently , noorwez et al3 reported that valproic acid ( vpa ) can increase the yield of properly folded rp mutant rhodopsins ; by using their high - throughput screening method involving in silico , cell - based , and in vitro assays , the authors were able to identify pharmacological chaperones of misfolded rhodopsin . based on the data , clemson et al4 reported in their retrospective study that treatment with vpa in patients with rp improved vas and vfs . however , there were controversies over that study,57 and another group , bhalla et al,8 not only claimed no improvement in va and vf in their study but also stated that vpa may facilitate some adverse side effects . to date , various clinical studies have been performed,9,10 but no conclusion has been reached regarding the efficacy of using vpa in patients with rp . the pharmacological basis of the antiepileptic action of vpa has been related to the regulation of the glutamate excitatory neurotransmission and/or gamma - aminobutyric acid ( gaba ) inhibitory neurotransmission.11 recent studies showed that vpa is an effective inhibitor of histone deacetylases , the key enzymes for the control of histone acetylation state and hence for the epigenetic regulation of gene expression . mainly through inhibition of histone deacetylases , vpa induces apoptosis of microglia cells12 and activates bdnf promoter.13 moreover , vpa induces neuronal differentiation but suppresses astrocytic and oligodendrocytic differentiation of neural stem cells14 and promotes neurite outgrowth.15 in this prospective study , we examined the efficacy and safety of vpa use in japanese patients with rp . this study is registered with the clinical trials registry of the japan medical association center for clinical trials , number jma - iia00053 . in this prospective , interventional , noncomparative case study ethics committee approval was granted by the ethics committee at the institute of biomedical research and innovation . all the patients were seen at the institute of biomedical research and innovation hospital ( kobe , japan ) from december 2010 to january 2013 . the diagnosis of rp was based on the patients symptoms of night blindness , ring scotoma or concentric constriction of vfs , markedly reduced or nonrecordable a- and b - wave amplitudes on electroretinogram ( erg ) testing , and ophthalmoscopic findings ( ie , characteristic fundus changes in attenuated retinal vessels and bone - spicule - like pigment clumping ) . the inclusion criteria were the following : 20 years old or older , best - corrected visual acuities ( bcvas ) of > 20/200 and < 20/30 , and vfs of 10 measured using goldmann perimeter with isopter i4 . the exclusion criteria were the following : patients with retinal diseases other than rp , including retinal degeneration secondary to inflammation or infection and retinal vascular or macular diseases ; cataractous lens gradings of more than n1 , c2 , or p1 according to lens opacities classification system iii grading ; previous intraocular surgery except for uncomplicated cataract extraction ; women of childbearing potential who were pregnant , nursing , or planning a pregnancy ; presentation of liver disease or a urea cycle disorder ; patients who had drug hypersensitivity ; patients who had attempted suicide or had suicidal thoughts with manic depressive illness ; and patients who were using contraindication medicine . prior to treatment at the initial study visit , each patient underwent ophthalmic examinations , including bcva measured using a landolt chart , intraocular pressure measurements , vf measured with the humphrey field analyzer ( hfa ; zeiss - humphrey systems , dublin , ca , usa ) 10 - 2 program , slit - lamp biomicroscopy , and dilated indirect fundus ophthalmoscopy . after baseline measurements were obtained , all the patients were instructed to receive oral supplementation of 400 mg ( the lowest dosage used for anticonvulsant therapy ) of vpa ( depakene - r ; kyowa hakko kirin , tokyo , japan ) daily for 6 months . the patients returned to our clinic for follow - up visits and were asked to report on the development of any subjective visual changes as well as any systemic adverse events . bcvas , vfs , and ophthalmic findings were collected throughout the entire study at months 1 , 3 , 6 ( end of supplementation ) , 9 , and 12 . blood samples were collected to check blood counts , clinical biochemistry , and the blood concentration of vpa at months 1 , 2 , 3 , 4 , and 6 ( end of supplementation ) . bcva was converted to the value of logarithm of the minimum angle of resolution ( logmar ) for all analyses . vf test results were summarized using the mean deviation ( md ) value calculated by the hfa provided software . the calculation of the md value involved averaging the differences between the measured sensitivities and the age - adjusted normal sensitivities ( total deviations ) at each test point . although not included as routine tests in our study design , microperimetry-1 ( mp-1 ; nidek , gamagori , japan ) or multifocal erg ( veris ; electro - diagnostic imaging , inc . , redwood , ca , usa and le-4000 ; tomey , nagoya , japan ) were also recorded in some patients at pretreatment and at 6 months or at 6 months and 12 months . the primary end point of this study was improvement in bcva after the 6 months of treatment with vpa , and the secondary end points were vf and the occurrence of adverse events . the variance approximation for the estimation of sample size was obtained from previous studies that have tested a similar hypothesis.4 a sample size calculation was performed before the study , assuming a maximum dropout rate of 30% . accordingly , we assumed that at least 30 patients were going to fail power of 80% ( -1 ) to detect a logmar 0.2 difference in va between patients before and after receiving vpa . all parameters obtained prior to vpa treatment and at 1 month , 3 months , 6 months , 9 months , and 12 months after treatment initiation were compared using the wilcoxon signed - rank test with bonferroni correction . we utilized a bonferroni adjustment for multiple comparisons , after which p - values 0.01 were considered as statistically significant . all statistical analyses were performed using the statistical analysis software ( spss inc . , chicago , il , usa ) . this study is registered with the clinical trials registry of the japan medical association center for clinical trials , number jma - iia00053 . in this prospective , interventional , noncomparative case study , the study protocols adhered to the tenets of the declaration of helsinki . ethics committee approval was granted by the ethics committee at the institute of biomedical research and innovation . all the patients were seen at the institute of biomedical research and innovation hospital ( kobe , japan ) from december 2010 to january 2013 . the diagnosis of rp was based on the patients symptoms of night blindness , ring scotoma or concentric constriction of vfs , markedly reduced or nonrecordable a- and b - wave amplitudes on electroretinogram ( erg ) testing , and ophthalmoscopic findings ( ie , characteristic fundus changes in attenuated retinal vessels and bone - spicule - like pigment clumping ) . the inclusion criteria were the following : 20 years old or older , best - corrected visual acuities ( bcvas ) of > 20/200 and < 20/30 , and vfs of 10 measured using goldmann perimeter with isopter i4 . the exclusion criteria were the following : patients with retinal diseases other than rp , including retinal degeneration secondary to inflammation or infection and retinal vascular or macular diseases ; cataractous lens gradings of more than n1 , c2 , or p1 according to lens opacities classification system iii grading ; previous intraocular surgery except for uncomplicated cataract extraction ; women of childbearing potential who were pregnant , nursing , or planning a pregnancy ; presentation of liver disease or a urea cycle disorder ; patients who had drug hypersensitivity ; patients who had attempted suicide or had suicidal thoughts with manic depressive illness ; and patients who were using contraindication medicine . prior to treatment at the initial study visit , each patient underwent ophthalmic examinations , including bcva measured using a landolt chart , intraocular pressure measurements , vf measured with the humphrey field analyzer ( hfa ; zeiss - humphrey systems , dublin , ca , usa ) 10 - 2 program , slit - lamp biomicroscopy , and dilated indirect fundus ophthalmoscopy . after baseline measurements were obtained , all the patients were instructed to receive oral supplementation of 400 mg ( the lowest dosage used for anticonvulsant therapy ) of vpa ( depakene - r ; kyowa hakko kirin , tokyo , japan ) daily for 6 months . the patients returned to our clinic for follow - up visits and were asked to report on the development of any subjective visual changes as well as any systemic adverse events . bcvas , vfs , and ophthalmic findings were collected throughout the entire study at months 1 , 3 , 6 ( end of supplementation ) , 9 , and 12 . blood samples were collected to check blood counts , clinical biochemistry , and the blood concentration of vpa at months 1 , 2 , 3 , 4 , and 6 ( end of supplementation ) . bcva was converted to the value of logarithm of the minimum angle of resolution ( logmar ) for all analyses . vf test results were summarized using the mean deviation ( md ) value calculated by the hfa provided software . the calculation of the md value involved averaging the differences between the measured sensitivities and the age - adjusted normal sensitivities ( total deviations ) at each test point . although not included as routine tests in our study design , microperimetry-1 ( mp-1 ; nidek , gamagori , japan ) or multifocal erg ( veris ; electro - diagnostic imaging , inc . , redwood , ca , usa and le-4000 ; tomey , nagoya , japan ) were also recorded in some patients at pretreatment and at 6 months or at 6 months and 12 months . the primary end point of this study was improvement in bcva after the 6 months of treatment with vpa , and the secondary end points were vf and the occurrence of adverse events . the variance approximation for the estimation of sample size was obtained from previous studies that have tested a similar hypothesis.4 a sample size calculation was performed before the study , assuming a maximum dropout rate of 30% . accordingly , we assumed that at least 30 patients were going to fail power of 80% ( -1 ) to detect a logmar 0.2 difference in va between patients before and after receiving vpa . all parameters obtained prior to vpa treatment and at 1 month , 3 months , 6 months , 9 months , and 12 months after treatment initiation were compared using the wilcoxon signed - rank test with bonferroni correction . we utilized a bonferroni adjustment for multiple comparisons , after which p - values 0.01 were considered as statistically significant . all statistical analyses were performed using the statistical analysis software ( spss inc . , chicago , il , usa ) . two patients were lost to follow - up after the 6 months visit ; the data for these patients were not included in the analysis . overall , 29 patients ( 12 males and 17 females ) with rp completed the 12 months study period . the patients ages ranged from 30 years to 72 years ( mean sd : 52.511.5 years ) . mendelian inheritance studies disclosed 13 sporadic , eleven autosomal recessive , and five autosomal dominant patterns . the age , sex , mendelian inheritance , bcva , md value of the vf , and mean blood vpa concentration are shown in table 1 . first , we evaluated the changes in the visual function during the vpa administration period ( from baseline to 6 months ) and the cessation period ( from 6 months to 12 months ) . the median changes in the value of log - mar bcva per month were 0.00 ( interquartile range [ iqr ] , 0.180.00 ) during administration and 0.00 ( iqr , 0.000.20 ) during cessation , which showed statistically significant difference ( p=0.001 ; figure 1a ) . the median changes in the md value of vf per month were 0.11 db ( iqr , 0.180.03 ) during administration and 0.48 db ( iqr , 0.170.28 ) during cessation , which also showed statistically significant difference ( p=0.001 ; figure 1b ) . next , we evaluated the shift in va ( logmar ) and vf ( db ) over time ( figure 2 ) . the median logmar bcva values were 0.39 ( iqr , 0.300.69 ) at baseline , 0.39 ( iqr , 0.300.52 ; p=0.08 ) at 1 month , 0.39 ( iqr , 0.300.61 ; p=0.02 ) at 3 months , 0.39 ( iqr , 0.300.52 ; p=0.006 ) at 6 months , 0.39 ( iqr , 0.300.69 ; p=0.14 ) at 9 months , and 0.39 ( iqr , 0.300.76 ; p=0.62 ) at 12 months . compared with baseline , the logmar bcva value was significantly improved at 6 months , during the period of vpa treatment ( figure 2a ) . the median md values of vf were 28.89 db ( iqr , 20.2633.18 ) at baseline , 27.54 db ( iqr , 18.9232.52 ; p=0.001 ) at 1 month , 28.14 db ( iqr , 18.2932.60 ; p=0.004 ) at 3 months , 28.16 db ( iqr,18.5931.96 ; p=0.004 ) at 6 months , 27.97 db ( iqr , 19.5133.11 ; p=0.88 ) at 9 months , and 29.15 db ( iqr , 18.6833.06 ; p=0.97 ) at 12 months . compared with baseline , the median md values of vf were significantly improved at 1 month , 3 months , and 6 months , corresponding to the period of vpa treatment ( figure 2b ) . the mean blood concentration of vpa increased to 43.0416.96 g / ml at 1 month after intake . during the internal use period , the blood concentration of vpa was stable in each patient and the total mean blood concentration value of vpa within 6 months was 39.0012.47 g / ml . as shown in figure 3 , there were no significant relations between the mean blood vpa concentration values of each patient and the changes in bcva ( r=0.06 , p=0.73 ) and vf ( r=0.18 , p=0.33 ) at 6 months . the patients subjective visual symptoms reported during and after vpa treatment are summarized in table 2 . during intake of vpa , eleven of 29 ( 38% ) patients felt no change in their vision , eight ( 28% ) patients felt clearer color vision , four ( 14% ) patients felt legibleness , and four ( 14% ) patients felt brightness . after the cessation of vpa intake , eight ( 28% ) patients reported having blurred vision , four ( 14% ) patients reported seeing dimness , three ( 10% ) patients reported having photophobia , two ( 7% ) patients reported difficulty seeing colors , and one ( 3% ) patient experienced fatigue . although not included in the study design and statistical analysis , we performed mp-1 and multifocal erg tests on some patients , and the preliminary results are shown in the supplementary materials section . some patients gained sensitivity by mp-1 at 6 months after vpa treatment ( figure s1 ) . with multifocal erg , the amplitudes of ( p1-n1 ) in rings 3 and 4 ( perifoveal area)16 were relatively unchanged between the two periods , whereas ( p1-n1 ) amplitudes of ring 1 ( center of the fovea ) seem to increase in some patients after 6 months of treatment , whereas the changes after vpa cessation seem to be insignificant ( figure s2 ) . throughout the study period , no systemic drug - related serious adverse events were observed in the study participants ; checked blood counts from collected blood samples and clinical biochemistry were within normal lesion . during the period of vpa intake , dizziness stomatitis , alopecia , and diarrhea were each reported in one ( 3% ) patient . while several reports showed that oral vpa treatment improved vas and vfs of rp patients , the opposite results were shown in other reports . the results of existing reports in vpa treatment are summarized in table 3 . in the present study of rp patients , the bcva and md values in the hfa 10 - 2 program significantly improved on average after 6 months of vpa intake and returned to the baseline levels after the cessation of the vpa treatment ( figure 2 ) . based on a large cohort study of natural courses of rp , the mean annual rates of decline of remaining bcva and vf were estimated to be 1.0%8.6% and 2.6%13.5% , respectively.17 compared to these data , our results showed some beneficial effect of vpa on rp patients at the 12-month time point even with its confined periodic effect . we can not deny the possibility of placebo effect , but two previous studies using docosahexaenoic acid or 9-cis -carotene had a randomized placebo treatment group without provision of multivitamin , and the placebo group in both of these studies worsened in vf by 1.41.32 db with hfa between years 0 and 4 or by 0.54.5 cm with goldmann perimetry within 90 days.18,19 indeed , we can not make a direct comparison between our results and these past studies , but we think that the vpa effect observed here is worthy of further investigation with a controlled study design in the future . nevertheless , the effect of vpa on bcva observed in our study may not seem to be remarkable as one might expect ( 0.2 log units ) . a large cohort study showing the significant effect to slow the progression of rp by nutritional supplements such as vitamin and docosahexaenoic acid presented its efficacy by vf and erg amplitudes but not by bcva.20,21 altogether , these results indicate that the change in bcva may not be sufficiently sensitive to evaluate the efficacy of a treatment for rp patients . we also performed mp-1 and , more objectively , multifocal ergs on a limited number of patients . although the data collection was performed in the limited number of patients , we observed the increased p1-n1 amplitudes specifically in ring 1 central fovea in some patients after 6 months of vpa treatment . this may imply the effect of vpa on the area of remaining photo - receptors , but the overall amplitudes were very small . we need more data to objectively evaluate the effect of vpa on foveal function . in this study , there were no significant relationship between the mean blood vpa concentrations of each patient and the changes in bcva and vf . first of all , vpa dosage used in this study was relatively low in order to minimize adverse effect of vpa , and the plasma vpa levels in our patients were below the therapeutic range ( 45100 mg / l ) . with this low concentration , albumin binding sites on vpa were unsaturated , and therefore , vpa binds to albumin at variable degrees , making its pharmacological behavior difficult to estimate.2224 if we use the therapeutic range of vpa , it might be able to show clearer effect of vpa on bcva or vf . we also observed a significant improvement in md values of hfa 10 - 2 programs with vpa treatment . in the original report by clemson et al , their studies were based on an in vitro experiment that vpa acts as a molecular chaperone of rhodopsin proteins that increases the yield of properly folded mutant rhodopsins . therefore , these authors suggested a potential effect of vpa on autosomal dominant rp , targeting improperly folded mutant rhodopsins , in rod photoreceptors . our current study included rp patients with seemingly various causal genes with 13 sporadic , eleven autosomal recessive , and five autosomal dominant hereditary patterns . our hfa examination with central 10 - 2 program may also represent cone photoreceptor functions . additionally , close hearing of patient s subjective symptoms also suggested some improvement in cone photoreceptor function : 16 of 29 ( 55% ) patients felt it was easier to see during the period of vpa intake , whereas after cessation , eye discomfort was registered in 18 of 29 ( 62% ) patients and half of the patients made some description related to color vision . stasheff et al25,26 found that after degeneration started , ganglion cells exhibited hyperactivity , firing spontaneously at rates many times greater than normal in rd1 and rd10 mice , strains with closely related rp . because the pharmacological basis of the antiepileptic action of vpa has been related to reduction in neuronal excitability by the increase in gabaergic activity , it is possible that vpa reduced hyperactivity of ganglion cells . kimura et al27 also reported that vpa reduced retinal ganglion cell death in a mouse model of normal tension glaucoma . in this report , they indicated that vpa exerts neuroprotective effects through suppression of oxidative stress and stimulation of cell survival signaling . therefore , patients may have felt that it was easier to see with reduced visual noise from spontaneous firing of ganglion cells or with neuroprotective effects . this phenomenon may also explain why bcva and vf were improved in our study with various types of genetic patterns . however , this hypothesis was based on subjective symptoms of vpa - treated rp patients , and we need further objective evaluation of cone - related functions , including color vision or contrast sensitivity , in vpa - treated patients . vpa has been widely used as an antiepileptic drug for several decades , and the use of vpa monotherapy in the treatment of epilepsy was not associated with vf defects.28,29 however , abnormal color visions in epileptic adolescents treated with vpa were reported.30,31 sisk7 reported three cases with complications of vpa treatment , two of which had severe decrease in bcva ; the two patients were 8 years and 15 years of age and received 10 mg / kg / d of vpa for 45 months . bhalla et al8 reported that 12 ( 39% ) of 31 vpa - treated patients reported systemic side effects , of whom nine ( 29% ) discontinued vpa intake due to side effects . in this study , we did not observe a severe decline in visual functions or systemic adverse events to discontinue vpa treatment . one possible explanation is that our patients were all at the age of 30 years or older and the dosage of vpa was relatively low ( 400 mg / d ) . it is still necessary to carefully observe the patients systemic conditions during vpa treatment and to exercise caution when using vpa for young rp patients . in this prospective study , we found the following : 1 ) while in use , oral intake of vpa was suggestive of a short - term benefit to patients with rp and 2 ) regardless of the genotype , there were no systemic drug - related adverse events . it is necessary to examine the effect of a longer vpa supplementation in a controlled study design . the changes in the retinal sensitivity during the vpa administration period and the cessation period . notes : scatter plots show changes in retinal sensitivity during the vpa administration period ( from baseline to 6 months ; n=24 ) ( a ) and the cessation period ( from 6 months to 12 months ; n=24 ) ( b ) . retinal sensitivities of the central 2 the changes in the ( p1-n1 ) amplitude during the vpa administration period and the cessation period . notes : scatter plots showing changes in retinal sensitivities during the vpa administration period ( from baseline to 6 months ; n=21 ) ( a ) and the cessation period ( from 6 months to 12 months ; n=9 ) ( b ) . multifocal ergs of the central 20 were measured with veris ( electro - diagnostics , inc . , ) and le-4000 ( tomey ) . abbreviations : p1-n1 , first positive wave minus first negative wave ; vpa , valproic acid ; erg , electroretinogram ; deg , degree .

purposethe purpose of this study was to examine the efficacy and safety of valproic acid ( vpa ) use in patients with retinitis pigmentosa ( rp).patients and methodsthis was a prospective , interventional , noncomparative case study . in total , 29 eyes from 29 patients with rp whose best - corrected visual acuities ( bcvas ) in logarithm of the minimum angle of resolution ( logmar ) ranged from 1.0 to 0.16 with visual fields ( vfs ) of 10 ( measured using goldmann perimeter with i4 ) were recruited . the patients received oral supplementation with 400 mg of vpa daily for 6 months and were followed for an additional 6 months . bcvas , vfs ( measured with the humphrey field analyzer central 10 - 2 program ) , and subjective questionnaires were examined before , during , and after the cessation of vpa supplementation.resultsthe changes in bcva and vf showed statistically significant differences during the internal use of vpa , compared with after cessation ( p=0.001 ) . with vpa intake , bcva in logmar significantly improved from baseline to 6 months ( p=0.006 ) . the mean deviation value of the vf significantly improved from baseline to 1 month ( p=0.001 ) , 3 months ( p=0.004 ) , and 6 months ( p=0.004 ) . these efficacies , however , were reversed to the baseline levels after the cessation of vpa intake . there were no significant relations between the mean blood vpa concentrations of each patient and the changes in bcva and vf . during the internal use of vpa , 15 of 29 patients answered easier to see , whereas blurred vision was registered in 21 of 29 patients on cessation . no systemic drug - related adverse events were observed.conclusionwhile in use , oral intake of vpa indicated a short - term benefit to patients with rp . it is necessary to examine the effect of a longer vpa supplementation in a controlled study design .

Introduction Patients and methods Trial registration Ethics Patients Study protocols Determination of sample size Statistical analysis Results Discussion Conclusion Supplementary materials

as the disease advances , they lose peripheral vision , eventually developing tunnel vision with the remaining cone photoreceptors , and finally lose central vision and visual acuities ( vas ) as these cones secondarily degenerate in the macular region.1,2 typically , it takes several years until the patients lose their central vision ; therefore , it is important to protect the cone photoreceptors in the macular area for rp patients . recently , noorwez et al3 reported that valproic acid ( vpa ) can increase the yield of properly folded rp mutant rhodopsins ; by using their high - throughput screening method involving in silico , cell - based , and in vitro assays , the authors were able to identify pharmacological chaperones of misfolded rhodopsin . based on the data , clemson et al4 reported in their retrospective study that treatment with vpa in patients with rp improved vas and vfs . however , there were controversies over that study,57 and another group , bhalla et al,8 not only claimed no improvement in va and vf in their study but also stated that vpa may facilitate some adverse side effects . to date , various clinical studies have been performed,9,10 but no conclusion has been reached regarding the efficacy of using vpa in patients with rp . the pharmacological basis of the antiepileptic action of vpa has been related to the regulation of the glutamate excitatory neurotransmission and/or gamma - aminobutyric acid ( gaba ) inhibitory neurotransmission.11 recent studies showed that vpa is an effective inhibitor of histone deacetylases , the key enzymes for the control of histone acetylation state and hence for the epigenetic regulation of gene expression . mainly through inhibition of histone deacetylases , vpa induces apoptosis of microglia cells12 and activates bdnf promoter.13 moreover , vpa induces neuronal differentiation but suppresses astrocytic and oligodendrocytic differentiation of neural stem cells14 and promotes neurite outgrowth.15 in this prospective study , we examined the efficacy and safety of vpa use in japanese patients with rp . this study is registered with the clinical trials registry of the japan medical association center for clinical trials , number jma - iia00053 . in this prospective , interventional , noncomparative case study ethics committee approval was granted by the ethics committee at the institute of biomedical research and innovation . the diagnosis of rp was based on the patients symptoms of night blindness , ring scotoma or concentric constriction of vfs , markedly reduced or nonrecordable a- and b - wave amplitudes on electroretinogram ( erg ) testing , and ophthalmoscopic findings ( ie , characteristic fundus changes in attenuated retinal vessels and bone - spicule - like pigment clumping ) . the inclusion criteria were the following : 20 years old or older , best - corrected visual acuities ( bcvas ) of > 20/200 and < 20/30 , and vfs of 10 measured using goldmann perimeter with isopter i4 . prior to treatment at the initial study visit , each patient underwent ophthalmic examinations , including bcva measured using a landolt chart , intraocular pressure measurements , vf measured with the humphrey field analyzer ( hfa ; zeiss - humphrey systems , dublin , ca , usa ) 10 - 2 program , slit - lamp biomicroscopy , and dilated indirect fundus ophthalmoscopy . after baseline measurements were obtained , all the patients were instructed to receive oral supplementation of 400 mg ( the lowest dosage used for anticonvulsant therapy ) of vpa ( depakene - r ; kyowa hakko kirin , tokyo , japan ) daily for 6 months . the patients returned to our clinic for follow - up visits and were asked to report on the development of any subjective visual changes as well as any systemic adverse events . bcvas , vfs , and ophthalmic findings were collected throughout the entire study at months 1 , 3 , 6 ( end of supplementation ) , 9 , and 12 . blood samples were collected to check blood counts , clinical biochemistry , and the blood concentration of vpa at months 1 , 2 , 3 , 4 , and 6 ( end of supplementation ) . bcva was converted to the value of logarithm of the minimum angle of resolution ( logmar ) for all analyses . the calculation of the md value involved averaging the differences between the measured sensitivities and the age - adjusted normal sensitivities ( total deviations ) at each test point . , redwood , ca , usa and le-4000 ; tomey , nagoya , japan ) were also recorded in some patients at pretreatment and at 6 months or at 6 months and 12 months . the primary end point of this study was improvement in bcva after the 6 months of treatment with vpa , and the secondary end points were vf and the occurrence of adverse events . all parameters obtained prior to vpa treatment and at 1 month , 3 months , 6 months , 9 months , and 12 months after treatment initiation were compared using the wilcoxon signed - rank test with bonferroni correction . this study is registered with the clinical trials registry of the japan medical association center for clinical trials , number jma - iia00053 . in this prospective , interventional , noncomparative case study , the study protocols adhered to the tenets of the declaration of helsinki . the diagnosis of rp was based on the patients symptoms of night blindness , ring scotoma or concentric constriction of vfs , markedly reduced or nonrecordable a- and b - wave amplitudes on electroretinogram ( erg ) testing , and ophthalmoscopic findings ( ie , characteristic fundus changes in attenuated retinal vessels and bone - spicule - like pigment clumping ) . the inclusion criteria were the following : 20 years old or older , best - corrected visual acuities ( bcvas ) of > 20/200 and < 20/30 , and vfs of 10 measured using goldmann perimeter with isopter i4 . prior to treatment at the initial study visit , each patient underwent ophthalmic examinations , including bcva measured using a landolt chart , intraocular pressure measurements , vf measured with the humphrey field analyzer ( hfa ; zeiss - humphrey systems , dublin , ca , usa ) 10 - 2 program , slit - lamp biomicroscopy , and dilated indirect fundus ophthalmoscopy . after baseline measurements were obtained , all the patients were instructed to receive oral supplementation of 400 mg ( the lowest dosage used for anticonvulsant therapy ) of vpa ( depakene - r ; kyowa hakko kirin , tokyo , japan ) daily for 6 months . the patients returned to our clinic for follow - up visits and were asked to report on the development of any subjective visual changes as well as any systemic adverse events . bcvas , vfs , and ophthalmic findings were collected throughout the entire study at months 1 , 3 , 6 ( end of supplementation ) , 9 , and 12 . blood samples were collected to check blood counts , clinical biochemistry , and the blood concentration of vpa at months 1 , 2 , 3 , 4 , and 6 ( end of supplementation ) . bcva was converted to the value of logarithm of the minimum angle of resolution ( logmar ) for all analyses . the calculation of the md value involved averaging the differences between the measured sensitivities and the age - adjusted normal sensitivities ( total deviations ) at each test point . , redwood , ca , usa and le-4000 ; tomey , nagoya , japan ) were also recorded in some patients at pretreatment and at 6 months or at 6 months and 12 months . the primary end point of this study was improvement in bcva after the 6 months of treatment with vpa , and the secondary end points were vf and the occurrence of adverse events . all parameters obtained prior to vpa treatment and at 1 month , 3 months , 6 months , 9 months , and 12 months after treatment initiation were compared using the wilcoxon signed - rank test with bonferroni correction . overall , 29 patients ( 12 males and 17 females ) with rp completed the 12 months study period . the age , sex , mendelian inheritance , bcva , md value of the vf , and mean blood vpa concentration are shown in table 1 . first , we evaluated the changes in the visual function during the vpa administration period ( from baseline to 6 months ) and the cessation period ( from 6 months to 12 months ) . the median changes in the value of log - mar bcva per month were 0.00 ( interquartile range [ iqr ] , 0.180.00 ) during administration and 0.00 ( iqr , 0.000.20 ) during cessation , which showed statistically significant difference ( p=0.001 ; figure 1a ) . the median changes in the md value of vf per month were 0.11 db ( iqr , 0.180.03 ) during administration and 0.48 db ( iqr , 0.170.28 ) during cessation , which also showed statistically significant difference ( p=0.001 ; figure 1b ) . next , we evaluated the shift in va ( logmar ) and vf ( db ) over time ( figure 2 ) . the median logmar bcva values were 0.39 ( iqr , 0.300.69 ) at baseline , 0.39 ( iqr , 0.300.52 ; p=0.08 ) at 1 month , 0.39 ( iqr , 0.300.61 ; p=0.02 ) at 3 months , 0.39 ( iqr , 0.300.52 ; p=0.006 ) at 6 months , 0.39 ( iqr , 0.300.69 ; p=0.14 ) at 9 months , and 0.39 ( iqr , 0.300.76 ; p=0.62 ) at 12 months . compared with baseline , the logmar bcva value was significantly improved at 6 months , during the period of vpa treatment ( figure 2a ) . the median md values of vf were 28.89 db ( iqr , 20.2633.18 ) at baseline , 27.54 db ( iqr , 18.9232.52 ; p=0.001 ) at 1 month , 28.14 db ( iqr , 18.2932.60 ; p=0.004 ) at 3 months , 28.16 db ( iqr,18.5931.96 ; p=0.004 ) at 6 months , 27.97 db ( iqr , 19.5133.11 ; p=0.88 ) at 9 months , and 29.15 db ( iqr , 18.6833.06 ; p=0.97 ) at 12 months . compared with baseline , the median md values of vf were significantly improved at 1 month , 3 months , and 6 months , corresponding to the period of vpa treatment ( figure 2b ) . the mean blood concentration of vpa increased to 43.0416.96 g / ml at 1 month after intake . during the internal use period , the blood concentration of vpa was stable in each patient and the total mean blood concentration value of vpa within 6 months was 39.0012.47 g / ml . as shown in figure 3 , there were no significant relations between the mean blood vpa concentration values of each patient and the changes in bcva ( r=0.06 , p=0.73 ) and vf ( r=0.18 , p=0.33 ) at 6 months . during intake of vpa , eleven of 29 ( 38% ) patients felt no change in their vision , eight ( 28% ) patients felt clearer color vision , four ( 14% ) patients felt legibleness , and four ( 14% ) patients felt brightness . after the cessation of vpa intake , eight ( 28% ) patients reported having blurred vision , four ( 14% ) patients reported seeing dimness , three ( 10% ) patients reported having photophobia , two ( 7% ) patients reported difficulty seeing colors , and one ( 3% ) patient experienced fatigue . although not included in the study design and statistical analysis , we performed mp-1 and multifocal erg tests on some patients , and the preliminary results are shown in the supplementary materials section . with multifocal erg , the amplitudes of ( p1-n1 ) in rings 3 and 4 ( perifoveal area)16 were relatively unchanged between the two periods , whereas ( p1-n1 ) amplitudes of ring 1 ( center of the fovea ) seem to increase in some patients after 6 months of treatment , whereas the changes after vpa cessation seem to be insignificant ( figure s2 ) . throughout the study period , no systemic drug - related serious adverse events were observed in the study participants ; checked blood counts from collected blood samples and clinical biochemistry were within normal lesion . during the period of vpa intake , dizziness stomatitis , alopecia , and diarrhea were each reported in one ( 3% ) patient . in the present study of rp patients , the bcva and md values in the hfa 10 - 2 program significantly improved on average after 6 months of vpa intake and returned to the baseline levels after the cessation of the vpa treatment ( figure 2 ) . based on a large cohort study of natural courses of rp , the mean annual rates of decline of remaining bcva and vf were estimated to be 1.0%8.6% and 2.6%13.5% , respectively.17 compared to these data , our results showed some beneficial effect of vpa on rp patients at the 12-month time point even with its confined periodic effect . we can not deny the possibility of placebo effect , but two previous studies using docosahexaenoic acid or 9-cis -carotene had a randomized placebo treatment group without provision of multivitamin , and the placebo group in both of these studies worsened in vf by 1.41.32 db with hfa between years 0 and 4 or by 0.54.5 cm with goldmann perimetry within 90 days.18,19 indeed , we can not make a direct comparison between our results and these past studies , but we think that the vpa effect observed here is worthy of further investigation with a controlled study design in the future . nevertheless , the effect of vpa on bcva observed in our study may not seem to be remarkable as one might expect ( 0.2 log units ) . a large cohort study showing the significant effect to slow the progression of rp by nutritional supplements such as vitamin and docosahexaenoic acid presented its efficacy by vf and erg amplitudes but not by bcva.20,21 altogether , these results indicate that the change in bcva may not be sufficiently sensitive to evaluate the efficacy of a treatment for rp patients . this may imply the effect of vpa on the area of remaining photo - receptors , but the overall amplitudes were very small . we need more data to objectively evaluate the effect of vpa on foveal function . in this study , there were no significant relationship between the mean blood vpa concentrations of each patient and the changes in bcva and vf . first of all , vpa dosage used in this study was relatively low in order to minimize adverse effect of vpa , and the plasma vpa levels in our patients were below the therapeutic range ( 45100 mg / l ) . with this low concentration , albumin binding sites on vpa were unsaturated , and therefore , vpa binds to albumin at variable degrees , making its pharmacological behavior difficult to estimate.2224 if we use the therapeutic range of vpa , it might be able to show clearer effect of vpa on bcva or vf . we also observed a significant improvement in md values of hfa 10 - 2 programs with vpa treatment . our current study included rp patients with seemingly various causal genes with 13 sporadic , eleven autosomal recessive , and five autosomal dominant hereditary patterns . our hfa examination with central 10 - 2 program may also represent cone photoreceptor functions . additionally , close hearing of patient s subjective symptoms also suggested some improvement in cone photoreceptor function : 16 of 29 ( 55% ) patients felt it was easier to see during the period of vpa intake , whereas after cessation , eye discomfort was registered in 18 of 29 ( 62% ) patients and half of the patients made some description related to color vision . because the pharmacological basis of the antiepileptic action of vpa has been related to reduction in neuronal excitability by the increase in gabaergic activity , it is possible that vpa reduced hyperactivity of ganglion cells . therefore , patients may have felt that it was easier to see with reduced visual noise from spontaneous firing of ganglion cells or with neuroprotective effects . this phenomenon may also explain why bcva and vf were improved in our study with various types of genetic patterns . however , this hypothesis was based on subjective symptoms of vpa - treated rp patients , and we need further objective evaluation of cone - related functions , including color vision or contrast sensitivity , in vpa - treated patients . vpa has been widely used as an antiepileptic drug for several decades , and the use of vpa monotherapy in the treatment of epilepsy was not associated with vf defects.28,29 however , abnormal color visions in epileptic adolescents treated with vpa were reported.30,31 sisk7 reported three cases with complications of vpa treatment , two of which had severe decrease in bcva ; the two patients were 8 years and 15 years of age and received 10 mg / kg / d of vpa for 45 months . in this study , we did not observe a severe decline in visual functions or systemic adverse events to discontinue vpa treatment . one possible explanation is that our patients were all at the age of 30 years or older and the dosage of vpa was relatively low ( 400 mg / d ) . it is still necessary to carefully observe the patients systemic conditions during vpa treatment and to exercise caution when using vpa for young rp patients . in this prospective study , we found the following : 1 ) while in use , oral intake of vpa was suggestive of a short - term benefit to patients with rp and 2 ) regardless of the genotype , there were no systemic drug - related adverse events . it is necessary to examine the effect of a longer vpa supplementation in a controlled study design . the changes in the retinal sensitivity during the vpa administration period and the cessation period . notes : scatter plots show changes in retinal sensitivity during the vpa administration period ( from baseline to 6 months ; n=24 ) ( a ) and the cessation period ( from 6 months to 12 months ; n=24 ) ( b ) . retinal sensitivities of the central 2 the changes in the ( p1-n1 ) amplitude during the vpa administration period and the cessation period . notes : scatter plots showing changes in retinal sensitivities during the vpa administration period ( from baseline to 6 months ; n=21 ) ( a ) and the cessation period ( from 6 months to 12 months ; n=9 ) ( b ) . abbreviations : p1-n1 , first positive wave minus first negative wave ; vpa , valproic acid ; erg , electroretinogram ; deg , degree .

periodontal plastic surgery procedures address these esthetic and functional demands and have become an integral part of the periodontal treatment . several therapeutic modalities such as free gingival autografts , pedicle grafts , connective tissue graft , grafts combining the two modalities , and guided tissue regeneration have been used for covering the denuded roots and to augment the width and thickness of the keratinized gingival . these procedures have resulted in reduction or elimination of root hypersensitivity , improved esthetics , and facilitation of plaque control . among various surgical techniques , subepithelial connective tissue ( sect ) grafts remain the most commonly used and most successful root coverage procedures . the esthetic and functional success of sect graft techniques is highly predictable and reliable which has been documented in several longitudinal studies . thickness and volume of the tissue to be harvested from the donor site are among the important factors in determining the appropriate treatment method . variations of size and shape of the palatal vault may also affect the dimensions of the donor tissue harvested . although an ample number of studies have evaluated the results of utilizing sect graft at the recipient site , a very few studies are focused on evaluating the wound healing and assessing patient - centered outcomes at the palatal donor area . furthermore , the oral cavity provides a unique environmental challenge for the healing wounds produced during various periodontal surgical procedures . the ideal technique for procuring a connective tissue graft should harvest an adequate graft , be user - friendly , produce minimum palatal discomfort , have minimal operative complications , and create a wound in the donor area that heals quickly with minimal postoperative complications . various techniques have been developed for harvesting soft tissue grafts from the palate such as trap door technique , parallel incision technique , and single incision technique . in the present study , a new instrument unigraft knife ( also called the free gingival graft knife from ace surgical supplies ) was used which when activated elevates a partial thickness flap beneath which the connective tissue graft is procured . the healing of the palatal wound created with this knife was compared with the wound created by langer and langer trap door technique . the purpose of the present study was to evaluate and compare the healing of the wound at the palatal donor site and root coverage results of the two different techniques . sixteen systemically healthy patients with 30 sites ( gingival recession 2 mm ) were recruited from the outpatient department of periodontology and implantology who presented with miller class i and ii recession ( 2 mm ) . noncomplaint patients , patients with root surface restoration , current smokers , or tobacco users were excluded from the study . thirty recession sites were divided into two equal groups with 15 recession sites each , that is , group i , which received the graft procured with the unigraft knife ( ace surgical supply co. , ma , figure 1c ) and group ii , which received the graft harvested by the langer and langer technique . the randomization was done by a coin flip technique as the study involved only two groups . ( k ) postoperative view of recession coverage at recipient site after 6 months all the enrolled patients underwent phase 1 periodontal therapy and were given oral hygiene instructions to ensure that they would adopt the correct brushing technique . full mouth plaque scores ( fmps ) and full mouth bleeding scores ( fmbs ) were recorded initially and after scaling and root planing . surgery was not carried out till the patients reached fmps < 20% and fmbs < 20% . parameters implied for evaluation of healing pattern at the donor site included the measurement of wound size ( ws ) , immediate bleeding ( ib ) and delayed bleeding ( db ) , complete wound epithelialization ( ce ) , sensibility disorders ( sd ) , and postoperative pain ( pp ) at baseline , 1 , 4 , and 12 week postoperatively . ws measurements were made by measuring the surface area of the palate from where the graft was procured that appeared to be granulating in or clinically did not appear to be covered by epithelium . measurements were made with a periodontal probe ( unc-15 ) to the nearest measurement of 0.5 mm . ib and db were assessed and ib was recorded as positive if the donor area presented with bleeding after 2 min application of external pressure with a sterile gauze . db was measured as positive if the patient presented with prolonged hemorrhage from the palate during the postsurgical period . ce wound was assessed clinically by means of colored photographs taken at each postsurgical visit . the scores were assigned as follows : 0 = no color match with adjacent tissues , 1 = partial color match with the adjacent tissues , and 2 = complete color match with the adjacent tissues . sd was assessed by means of a periodontal probe ( unc-15 , hu - friedy ) using a 4-point discrimination scale ( coronal , apical , mesial , and distal ) around the donor area before and after the surgical procedure and the follow - up visits . identical assessment was made at the same time in the corresponding contralateral area to collect the most reliable data possible . objective sensory loss was recorded using a rubbing movement and a pin - pressure nociception . patients were asked to give a rating of their loss of sensibility based on a 3-point verbal descriptor scale ( none , mild or moderate , severe ) . pp : at the subsequent postoperative appointments , the patients were asked to rate their discomfort level in the palate for the previous week . the numbers of pills taken for pain were also noted down for each patient . visual analog scale ( vas ) was used , and pain was assessed by asking the patients to rate the intensity of the pain perceived in the palate donor area at 1 and 4 week using 100 mm horizontal scale with the left endpoint marked worst pain imaginable as the primary efficacy parameter . the patient was asked to move a finger on the vas tip which coincided with the level of pain experienced . verbal rating scale ( vrs ) ( no pain , mild pain , moderate pain , severe pain , and very severe pain ) was used to rate the discomfort level in the palate donor area at the postoperative appointments . all the clinical measurements were made by the same examiner only , to avoid any inter examiner bias . except for the evaluation of colored photographs for ce where three examiners scored the photographs separately , on evaluation of healing pattern at the recipient site , the clinical parameters were assessed at the baseline , 3 , and 6 month . these included the clinical attachment level ( measured as the distance from cementoenamel junction to the base of the pocket ) , vertical recession ( vr measured as the distance from cementoenamel junction to the free gingival margin at the mid - buccal level ) , and width of keratinized gingiva ( kt measured as the distance from most apical position of gingival margin to the mucogingival border at the buccal tooth surface ) . initially , in both groups at the recession site , a full mucoperiosteal flap using horizontal and vertical incisions was raised according to the langer and langer technique , sparing the proximal papillae . in the apical areas , all the recipient areas were treated in a similar fashion so that the only difference in the therapy each group received was the method used to obtain the graft . further , for the donor site , one of the two techniques for harvesting the graft ( unigraft knife method or langer and langer technique ) was used to harvest the required amount of sect graft for the recession sites as per the randomization table . a palatal region from the first molar to canine was utilized for procuring the required amount of sect graft . the donor area was sounded with a periodontal probe to ensure that there was a minimum of 3 mm soft tissue thickness . unigraft knife was assembled in a conventional manner to permit cutting in a pulling motion . the knife was assembled with a cutting shoe reversed so that the cutting shoe would cut in a pushing direction . it was then used to elevate a partial thickness trap door flap by pushing the knife , under control , distally across the palate . this trap door flap was retracted mesially to permit access to the connective tissue beneath it . the knife was then assembled in a conventional manner to permit cutting in the pull motion . now , starting at the distal edge of the trap door flap , the knife was then used to elevate a connective tissue flap [ figure 1a - k ] . this secondary flap , made up of connective tissue , was incised at the mesial edge . palatal donor area was sounded with a periodontal probe to ensure 3 mm soft tissue thickness . a pair of parallel incisions ( 1.5 mm apart ) were made into the palate in the area of the first molar to canine . the incisions were made with a single 1012 mm deep pass of no 15 blade mounted on bp handle . the parallel incision was made at least 23 mm from the gingival margin in the palate . vertical incisions were placed at the mesial and distal end of the most external incision . a 4 - 0 silk suture was placed through the palatal tissue to retract the palatal tissue and to provide access to the tissue between the initial incisions . the tissue was then removed by incising the mesial , distal , and medial edges between the parallel incisions . pressure was applied with wet gauze to the donor area [ figure 2a - i ] . the palatal wound was closed with sutures in the vertical incision and also using the suture that had been used to retract the palatal tissue for access . ( i ) postoperative view of recession coverage at recipient site - after 6 months in both the groups , the procured graft from the palate was secured over the recipient site using vicryl 4 - 0 sutures [ figures 1k and 2i ] . the overlying flap was sutured as coronally as possible to cover the connective tissue graft . after closure of the vertical incisions , a mild compression with gauge soaked with sterile saline solution was done for 5 min to reduce the size of the clot . a periodontal dressing was placed over the recipient site and on donor site to protect the underlying tissue for 10 days postoperatively . postoperative instructions included the use of 0.2% chlorhexidine gluconate rinse twice daily and avoidance of trauma to the surgical areas . initially , in both groups at the recession site , a full mucoperiosteal flap using horizontal and vertical incisions was raised according to the langer and langer technique , sparing the proximal papillae . in the apical areas , all the recipient areas were treated in a similar fashion so that the only difference in the therapy each group received was the method used to obtain the graft . further , for the donor site , one of the two techniques for harvesting the graft ( unigraft knife method or langer and langer technique ) was used to harvest the required amount of sect graft for the recession sites as per the randomization table . a palatal region from the first molar to canine was utilized for procuring the required amount of sect graft . the donor area was sounded with a periodontal probe to ensure that there was a minimum of 3 mm soft tissue thickness . unigraft knife was assembled in a conventional manner to permit cutting in a pulling motion . the knife was assembled with a cutting shoe reversed so that the cutting shoe would cut in a pushing direction . it was then used to elevate a partial thickness trap door flap by pushing the knife , under control , distally across the palate . this trap door flap was retracted mesially to permit access to the connective tissue beneath it . the knife was then assembled in a conventional manner to permit cutting in the pull motion . now , starting at the distal edge of the trap door flap , the knife was then used to elevate a connective tissue flap [ figure 1a - k ] . this secondary flap , made up of connective tissue , was incised at the mesial edge . palatal donor area was sounded with a periodontal probe to ensure 3 mm soft tissue thickness . a pair of parallel incisions ( 1.5 mm apart ) were made into the palate in the area of the first molar to canine . the incisions were made with a single 1012 mm deep pass of no 15 blade mounted on bp handle . the parallel incision was made at least 23 mm from the gingival margin in the palate . vertical incisions were placed at the mesial and distal end of the most external incision . a 4 - 0 silk suture was placed through the palatal tissue to retract the palatal tissue and to provide access to the tissue between the initial incisions . the tissue was then removed by incising the mesial , distal , and medial edges between the parallel incisions . pressure was applied with wet gauze to the donor area [ figure 2a - i ] . the palatal wound was closed with sutures in the vertical incision and also using the suture that had been used to retract the palatal tissue for access . ( i ) postoperative view of recession coverage at recipient site - after 6 months in both the groups , the procured graft from the palate was secured over the recipient site using vicryl 4 - 0 sutures [ figures 1k and 2i ] . the overlying flap was sutured as coronally as possible to cover the connective tissue graft . after closure of the vertical incisions , a mild compression with gauge soaked with sterile saline solution was done for 5 min to reduce the size of the clot . a periodontal dressing was placed over the recipient site and on donor site to protect the underlying tissue for 10 days postoperatively . postoperative instructions included the use of 0.2% chlorhexidine gluconate rinse twice daily and avoidance of trauma to the surgical areas . the statistical analysis was done using spss version 15.0 statistical analysis software ( statistical package for the social sciences ( spss ) , version 15.0 , ibm , chicago , il ) . the values were represented in number ( % ) and mean standard deviation and to test the significance between two means ( group i and ii ) the student 's t - test was used . for comparison of change in parameters in two groups at different time intervals , paired t - test was used . since this study was aimed to assess the early healing of the wound at the palatal donor site by comparing langer and langer trap door technique and a unigraft knife method for obtaining the connective tissue graft for the treatment of buccal gingival recession , the recipient site was treated in a similar fashion in both the groups . all the patients completed the study period , and there was no drop - out . no complications were observed in any patient , and all patients responded well to the treatment and follow - up visits . patients in both the groups exhibited pain at 1-week follow - up which was higher in group i than group ii , but it was statistically not significant . as per vas measurements , the patients reported mild to moderate pain at the donor site [ table 1 ] . comparison of complete wound epithelisation , delayed bleeding , sensibility disorders , at different time intervals on evaluation of scores from vrs scale , the pain reported by group i patients was of moderate intensity whereas for group ii , dull pain was reported by the patients at 1-week interval . however , at subsequent follow - up visits , none of the patients in either of the groups reported pain , till the conclusion of the study . pp was also assessed as per the number of nsaid pills taken by the patients in both the groups . it was found to be similar at 1 week postoperative interval , and no analgesic pill intake was reported by any patient at 4 and 12 weeks follow - up intervals . when all the parameters of the pp , i.e. vas , vrs , and nsaids pills taken were analyzed collectively , the amount of pain reported at 1 week in both the groups was significantly higher than at subsequent time intervals [ table 2 ] . comparing the vas , vrs , pills in both the groups at different time intervals reduction in ws from baseline to all follow - up time intervals was significantly faster in both the groups , but the rate of ws reduction was much faster in group ii compared to group i [ table 3 ] . comparison of wound size ( ws ) at different time intervals complete wound epithelization was also achieved at a faster rate in group ii as compared to group i. the difference in the rate of ce was significantly higher ( p < 0.001 ) from baseline to 1 week in both the groups [ table 4 ] . db was seen at 1-week follow - up in both the cases which was higher in group i , but was not statistically significant ( p = 0.825 ) . no postoperative bleeding was observed on further follow - up visits in both the groups [ table 1 ] . comparison of mean change in wound size and complete wound epithelisation at different time intervals in group i and group ii sd was noticed in group ii at 1 week , but was not seen in group i and also no sd was observed at any of the further follow - up visits in either of the groups [ table 1 ] . on evaluation of root coverage parameters at the recipient site , results showed difference in the mean vr , mean root coverage in both the groups to be nonsignificant ( p > 0.05 ; table 5 ) . intragroup comparison of mean width of keratinized gingiva ( kt ) showed no statistically significant difference ( p = 0.419 ; table 5 ) at all - time intervals . intragroup comparison of mean kt showed that both the groups gained statistically significant amount of tissue at 3 and 6 month postsurgically as compared to baseline ( p < 0.001 ) [ table 6 ] . comparison of vertical recession , probing depth , clinical attachment level and width of keratinized gingiva in two groups at different time intervals . ( group i - unigraftknife method and group ii - langer and langer method ) comparison of mean change in width of keratinized gingiva ( kt ) in two groups at different time intervals there are several techniques available to obtain suitable sect graft . among the techniques used for sect graft harvestation , langer and langer trapdoor method langer and langer method along with most of the techniques relies on free hand dissection of the palate and , therefore , is a highly technique sensitive procedure . the aim of the present study was to compare healing at the palatal donor area using two different surgical techniques to harvest a sect graft for a root coverage procedure . the recipient sites were treated in a similar manner so that the only difference in the therapy each group received was the method used to obtain the graft . thirty sites in 16 patients were selected ( gingival recession 2 mm ) for root coverage procedures . this comparative , clinical , randomized study was designed to assess the differences in healing pattern and patient discomfort between the two groups . the connective tissue graft from the palatal site was harvested 23 mm away from the gingival margin for both the techniques . harris in 1997 also advocated that at least 3 mm of palatal mucosa ( thickness ) was needed for harvesting a connective tissue graft ; therefore in our study , sounding with a periodontal probe was done in both the groups . a uniform connective tissue graft with a thickness of 1.5 mm was obtained using unigraft knife . for langer and langer trap door technique , freehand incisions were made 1.5 mm apart , and an effort was made to keep the thickness of the graft uniform . two releasing vertical incisions were also given to facilitate the removal of connective tissue graft and to aid in wound closure . at recipient site , in both the groups , attempt was made to completely cover the connective tissue graft by overlying flap . studies have shown that total vascularization in the part of the connective tissue graft occurred when the flap at the recipient site completely covered it . as per the best of our knowledge , no data have been reported in the literature regarding the assessment of depth of wound at the palatal donor area . visual cues , such as wound bed color and wound measurements , can not be relied upon . therefore , apart from measuring the wound using unc-15 , clinical photographs were taken at each postoperative visit to access complete wound epithelization . the ws was measured with unc-15 ( to the nearest of 0.5 mm ) at 1 week postoperative visit was significantly larger in group i as compared to group ii ( p < 0.001 ; table 3 ) . this could have been the result of the high rate of sloughing of the flap seen with the technique using unigraft knife . if the knife was engaged superficially , a thin trap door flap was obtained which might have resulted in high rate of sloughing . making the incisions freehand could have permitted a deeper trap door incision or a wider base on the trap door flap which may have helped to reduce the sloughing seen . at 4 weeks too , the mean ws was larger in group i as compared to that in group ii ( p < 0.001 ; table 4 ) . a deeper incision or a wider base in the unigraft knife group might have helped to reduce the sloughing . this was not possible as there are limited no of sizes of cutting shoe available for this knife . at 4 weeks follow - up visit , although the wound area was epithelized , there was a depression seen at all the palatal sites in the group i , whereas in the group ii , wound area was fully diminished at all palatal sites at 4 weeks follow - up except for 1 patient . by 12 weeks , in both the groups , complete epithelization of the palatal wound area had occurred and clinically wound area was fully diminished . the percentage of complete palatal wound epithelization using langer and langer trap door technique cited in the relevant literature is quite similar to that seen in our study . the rate of sloughing of the primary flap using the langer and langer trap door technique was similar to those seen in previous reports . edel who performed this technique also reported that degeneration of the primary flap in most patients takes about 1 week . harris , 1997 , in a comparative study of the clinical healing in the donor area demonstrated that a high rate of sloughing occurs in the superficial flap when free gingival graft knife was employed . unigraft knife method resulted in a larger wound area at 1-week postoperative visit than the langer and langer trap door method . this could have resulted due to a variation in the ws created by the unigraft knife where the flap design was such that the distal border instead of the medial one served as the base of the reflected primary flap . the base of the primary reflected flap in the langer and langer trap door was toward the mid - palate in the region of first molar and canine and was broader than the base of the primary reflected flap in the unigraft knife method . intragroup comparison of ce showed that in group i , it was completed at 12 weeks postsurgery , whereas in group ii , it was completed at 4 weeks except for one patient where it was observed at 12 weeks period . the results could be correlated to a study by del pizzo et al . who also found complete palatal wound epithelization by 4 weeks after surgery . kahnberg and thilander in a study on palatal healing in rats , noted that epithelization progressed from the wound borders , and reduction of the wound surface preceded by contraction of the wound margins and by epithelial cell migration . at none of the time db at 1-week postoperative visit was higher in group i as compared to group ii , but the difference was not statistically significant ( p = 0.825 ; table 4 ) . according to griffin et al . , 2006 , proper care was taken in both the groups to ensure that postoperative instructions are abided by . no statistically significant differences were observed regarding the return of sensibility in the palatal donor site in both the groups . literature supports that transient - postoperative sensory dysfunction is a possible complication after harvesting the graft from the palatal region . halata et al . were able to show different nerve endings in the hard palate . in addition to free nerve endings within the epithelium and lamina propria , they also found merkel nerve endings , as well as meissner and ruffini corpuscles , inside the basal lamina and the adjacent connective tissue , all of which are sensitive to touch and pressure . although sd is not an objective measurement , in our study , both 2-point discrimination and soft - touch discrimination were used which have been reported to be reliable methods to detect the function of these mechanoreceptors . in both groups , mean vas and number of analgesic pills intake in the group at 1 week were higher as compared to that in group ii , yet the difference was not statistically significant . no pain or number of analgesic pills intake was reported by any patient in both the groups after 4 and 12 weeks follow - up intervals . lengthy surgical procedures may create extensive tissue injury , prolong vasodilation that permits more fluid to accumulate in the interstitial spaces , and results in higher level of biologic mediators released by inflammatory and resident cells . however , differences in patient perception can also influence the levels of reported pp . both the unigraft knife method and the langer and langer trap door method simplify the technique for obtaining the sect graft from the palate . undoubtedly , in certain clinician 's hands , with certain skill levels and in certain situations , one technique with or without on assessing the root coverage parameters at the recipient site , it was found that the difference in the mean vr in both the groups at baseline , 3 , and 6 months was not statistically significant . mean root coverage achieved for group i was 54% , whereas for group ii , it was 68.13% at final ( 6 months ) postoperative visit . this could be explained by the fact that recipient area in both the groups was treated in a similar fashion . harris , 1997 , also observed similar trends on comparison of root coverage at the recipient site using the unigraft knife method and langer and langer trap door technique . intergroup comparison of mean width of keratinized gingiva ( kt ) showed no statistically significant ( p = 0.419 ) difference at all time intervals . intragroup comparison of mean kt showed that both the groups gained a statistically significant amount of tissue at 3 months and 6 months postsurgically as compared to baseline . transplanted sect of subsequent size has shown the availability to result in predictable root coverage and increased width of attached gingival . wennstrm and zucchelli reported that transplanted connective tissue from palate has an ability to alter the differentiation of epithelial cells of the thin covering coronally advanced flap to become keratinized cells . granulation tissue formation derived from the periodontal ligament also contributes to the increased width of keratinized gingiva . mucogingival junction regains its genetically defined position following its coronal dislocation with the coronally repositioned flap resulting in the increased gingival dimension . the increase in width of keratinized gingiva observed between 3 and 6 months has been attributed to creeping attachment . therefore , this study suggests that in terms of operator factors , both the techniques yielded sufficient sect graft suitable for periodontal plastic procedure with minimal operative complication . however , unigraft knife technique created a slightly larger wound area at the palate as compared to the free - hand incisions made in langer and langer technique . the wound in the langer and langer trap door technique method healed slightly more quickly as compared to the unigraft knife method , thus might have accounted for less patient discomfort . during the study , it was observed that the use of unigraft knife was technique - sensitive and it was difficult to adapt the instrument in situations where the palatal vault was high and narrow . because of only a few sizes of cutting shoes available for unigraft knife , its adaptation to a variety of anatomic forms of palate is limited . the langer and langer trapdoor technique was more user - friendly as the angles of the free - hand incisions could be easily adjusted if some anatomic variations were encountered .

aim : to compare the healing pattern in palate following harvestation of connective tissue graft by two different techniques and to compare the recession coverage at the recipient sites.materials and methods:30 recession sites with miller 's class i and ii recession in 16 patients were recruited for this study . sites were randomly divided into 2 treatment groups . group i used unigraft knife to harvest the connective tissue whereas in group ii patients langer & langer techniques was used to harvest the connective tissue graft from the palate . healing was evaluated at the donor site using- wound size(ws ) , immediate bleeding ( ib ) and delayed bleeding ( db ) , complete wound epithelialization ( ce ) , sensibility disorders ( s ) and post operative pain ( pp ) at baseline , 1st , 4th , and 12th week postoperatively . recession coverage was assessed by measuring clinical attachment level ( cal ) , vertical recession ( vr ) , width of keratinized gingiva ( kt).results : on comparison between group i and ii , a statistically significant larger wound size was observed in group i. cwe was higher in group ii . a non significant difference was observed when sd , and delayed bleeding were compared at all time intervals . a non - significant difference was observed in the clinical parameters at the recipient site.conclusion:when evaluating the ws and cwe , the langer and langer technique was found to be better than the unigraft knife technique for harvesting the connective tissue graft , whereas both the techniques were found to be effective in root coverage procedure outcomes .

Introduction Materials and Methods Recipient-site preparation Procedure followed for unigraft knife group: Group I Procedure followed for Langer and Langer technique: Group II Results Discussion Conclusion Financial support and sponsorship Conflicts of interest

several therapeutic modalities such as free gingival autografts , pedicle grafts , connective tissue graft , grafts combining the two modalities , and guided tissue regeneration have been used for covering the denuded roots and to augment the width and thickness of the keratinized gingival . among various surgical techniques , subepithelial connective tissue ( sect ) grafts remain the most commonly used and most successful root coverage procedures . thickness and volume of the tissue to be harvested from the donor site are among the important factors in determining the appropriate treatment method . although an ample number of studies have evaluated the results of utilizing sect graft at the recipient site , a very few studies are focused on evaluating the wound healing and assessing patient - centered outcomes at the palatal donor area . furthermore , the oral cavity provides a unique environmental challenge for the healing wounds produced during various periodontal surgical procedures . the ideal technique for procuring a connective tissue graft should harvest an adequate graft , be user - friendly , produce minimum palatal discomfort , have minimal operative complications , and create a wound in the donor area that heals quickly with minimal postoperative complications . various techniques have been developed for harvesting soft tissue grafts from the palate such as trap door technique , parallel incision technique , and single incision technique . in the present study , a new instrument unigraft knife ( also called the free gingival graft knife from ace surgical supplies ) was used which when activated elevates a partial thickness flap beneath which the connective tissue graft is procured . the healing of the palatal wound created with this knife was compared with the wound created by langer and langer trap door technique . the purpose of the present study was to evaluate and compare the healing of the wound at the palatal donor site and root coverage results of the two different techniques . sixteen systemically healthy patients with 30 sites ( gingival recession 2 mm ) were recruited from the outpatient department of periodontology and implantology who presented with miller class i and ii recession ( 2 mm ) . thirty recession sites were divided into two equal groups with 15 recession sites each , that is , group i , which received the graft procured with the unigraft knife ( ace surgical supply co. , ma , figure 1c ) and group ii , which received the graft harvested by the langer and langer technique . ( k ) postoperative view of recession coverage at recipient site after 6 months all the enrolled patients underwent phase 1 periodontal therapy and were given oral hygiene instructions to ensure that they would adopt the correct brushing technique . parameters implied for evaluation of healing pattern at the donor site included the measurement of wound size ( ws ) , immediate bleeding ( ib ) and delayed bleeding ( db ) , complete wound epithelialization ( ce ) , sensibility disorders ( sd ) , and postoperative pain ( pp ) at baseline , 1 , 4 , and 12 week postoperatively . ws measurements were made by measuring the surface area of the palate from where the graft was procured that appeared to be granulating in or clinically did not appear to be covered by epithelium . db was measured as positive if the patient presented with prolonged hemorrhage from the palate during the postsurgical period . sd was assessed by means of a periodontal probe ( unc-15 , hu - friedy ) using a 4-point discrimination scale ( coronal , apical , mesial , and distal ) around the donor area before and after the surgical procedure and the follow - up visits . identical assessment was made at the same time in the corresponding contralateral area to collect the most reliable data possible . pp : at the subsequent postoperative appointments , the patients were asked to rate their discomfort level in the palate for the previous week . visual analog scale ( vas ) was used , and pain was assessed by asking the patients to rate the intensity of the pain perceived in the palate donor area at 1 and 4 week using 100 mm horizontal scale with the left endpoint marked worst pain imaginable as the primary efficacy parameter . verbal rating scale ( vrs ) ( no pain , mild pain , moderate pain , severe pain , and very severe pain ) was used to rate the discomfort level in the palate donor area at the postoperative appointments . except for the evaluation of colored photographs for ce where three examiners scored the photographs separately , on evaluation of healing pattern at the recipient site , the clinical parameters were assessed at the baseline , 3 , and 6 month . these included the clinical attachment level ( measured as the distance from cementoenamel junction to the base of the pocket ) , vertical recession ( vr measured as the distance from cementoenamel junction to the free gingival margin at the mid - buccal level ) , and width of keratinized gingiva ( kt measured as the distance from most apical position of gingival margin to the mucogingival border at the buccal tooth surface ) . initially , in both groups at the recession site , a full mucoperiosteal flap using horizontal and vertical incisions was raised according to the langer and langer technique , sparing the proximal papillae . in the apical areas , all the recipient areas were treated in a similar fashion so that the only difference in the therapy each group received was the method used to obtain the graft . further , for the donor site , one of the two techniques for harvesting the graft ( unigraft knife method or langer and langer technique ) was used to harvest the required amount of sect graft for the recession sites as per the randomization table . it was then used to elevate a partial thickness trap door flap by pushing the knife , under control , distally across the palate . this trap door flap was retracted mesially to permit access to the connective tissue beneath it . now , starting at the distal edge of the trap door flap , the knife was then used to elevate a connective tissue flap [ figure 1a - k ] . this secondary flap , made up of connective tissue , was incised at the mesial edge . a pair of parallel incisions ( 1.5 mm apart ) were made into the palate in the area of the first molar to canine . the parallel incision was made at least 23 mm from the gingival margin in the palate . ( i ) postoperative view of recession coverage at recipient site - after 6 months in both the groups , the procured graft from the palate was secured over the recipient site using vicryl 4 - 0 sutures [ figures 1k and 2i ] . the overlying flap was sutured as coronally as possible to cover the connective tissue graft . a periodontal dressing was placed over the recipient site and on donor site to protect the underlying tissue for 10 days postoperatively . initially , in both groups at the recession site , a full mucoperiosteal flap using horizontal and vertical incisions was raised according to the langer and langer technique , sparing the proximal papillae . in the apical areas , all the recipient areas were treated in a similar fashion so that the only difference in the therapy each group received was the method used to obtain the graft . further , for the donor site , one of the two techniques for harvesting the graft ( unigraft knife method or langer and langer technique ) was used to harvest the required amount of sect graft for the recession sites as per the randomization table . this trap door flap was retracted mesially to permit access to the connective tissue beneath it . now , starting at the distal edge of the trap door flap , the knife was then used to elevate a connective tissue flap [ figure 1a - k ] . this secondary flap , made up of connective tissue , was incised at the mesial edge . a pair of parallel incisions ( 1.5 mm apart ) were made into the palate in the area of the first molar to canine . the parallel incision was made at least 23 mm from the gingival margin in the palate . the palatal wound was closed with sutures in the vertical incision and also using the suture that had been used to retract the palatal tissue for access . ( i ) postoperative view of recession coverage at recipient site - after 6 months in both the groups , the procured graft from the palate was secured over the recipient site using vicryl 4 - 0 sutures [ figures 1k and 2i ] . the overlying flap was sutured as coronally as possible to cover the connective tissue graft . a periodontal dressing was placed over the recipient site and on donor site to protect the underlying tissue for 10 days postoperatively . the values were represented in number ( % ) and mean standard deviation and to test the significance between two means ( group i and ii ) the student 's t - test was used . for comparison of change in parameters in two groups at different time intervals , paired t - test was used . since this study was aimed to assess the early healing of the wound at the palatal donor site by comparing langer and langer trap door technique and a unigraft knife method for obtaining the connective tissue graft for the treatment of buccal gingival recession , the recipient site was treated in a similar fashion in both the groups . patients in both the groups exhibited pain at 1-week follow - up which was higher in group i than group ii , but it was statistically not significant . as per vas measurements , the patients reported mild to moderate pain at the donor site [ table 1 ] . comparison of complete wound epithelisation , delayed bleeding , sensibility disorders , at different time intervals on evaluation of scores from vrs scale , the pain reported by group i patients was of moderate intensity whereas for group ii , dull pain was reported by the patients at 1-week interval . it was found to be similar at 1 week postoperative interval , and no analgesic pill intake was reported by any patient at 4 and 12 weeks follow - up intervals . vas , vrs , and nsaids pills taken were analyzed collectively , the amount of pain reported at 1 week in both the groups was significantly higher than at subsequent time intervals [ table 2 ] . comparing the vas , vrs , pills in both the groups at different time intervals reduction in ws from baseline to all follow - up time intervals was significantly faster in both the groups , but the rate of ws reduction was much faster in group ii compared to group i [ table 3 ] . comparison of wound size ( ws ) at different time intervals complete wound epithelization was also achieved at a faster rate in group ii as compared to group i. the difference in the rate of ce was significantly higher ( p < 0.001 ) from baseline to 1 week in both the groups [ table 4 ] . db was seen at 1-week follow - up in both the cases which was higher in group i , but was not statistically significant ( p = 0.825 ) . no postoperative bleeding was observed on further follow - up visits in both the groups [ table 1 ] . comparison of mean change in wound size and complete wound epithelisation at different time intervals in group i and group ii sd was noticed in group ii at 1 week , but was not seen in group i and also no sd was observed at any of the further follow - up visits in either of the groups [ table 1 ] . on evaluation of root coverage parameters at the recipient site , results showed difference in the mean vr , mean root coverage in both the groups to be nonsignificant ( p > 0.05 ; table 5 ) . intragroup comparison of mean width of keratinized gingiva ( kt ) showed no statistically significant difference ( p = 0.419 ; table 5 ) at all - time intervals . intragroup comparison of mean kt showed that both the groups gained statistically significant amount of tissue at 3 and 6 month postsurgically as compared to baseline ( p < 0.001 ) [ table 6 ] . comparison of vertical recession , probing depth , clinical attachment level and width of keratinized gingiva in two groups at different time intervals . ( group i - unigraftknife method and group ii - langer and langer method ) comparison of mean change in width of keratinized gingiva ( kt ) in two groups at different time intervals there are several techniques available to obtain suitable sect graft . among the techniques used for sect graft harvestation , langer and langer trapdoor method langer and langer method along with most of the techniques relies on free hand dissection of the palate and , therefore , is a highly technique sensitive procedure . the aim of the present study was to compare healing at the palatal donor area using two different surgical techniques to harvest a sect graft for a root coverage procedure . the recipient sites were treated in a similar manner so that the only difference in the therapy each group received was the method used to obtain the graft . thirty sites in 16 patients were selected ( gingival recession 2 mm ) for root coverage procedures . the connective tissue graft from the palatal site was harvested 23 mm away from the gingival margin for both the techniques . harris in 1997 also advocated that at least 3 mm of palatal mucosa ( thickness ) was needed for harvesting a connective tissue graft ; therefore in our study , sounding with a periodontal probe was done in both the groups . a uniform connective tissue graft with a thickness of 1.5 mm was obtained using unigraft knife . for langer and langer trap door technique , freehand incisions were made 1.5 mm apart , and an effort was made to keep the thickness of the graft uniform . two releasing vertical incisions were also given to facilitate the removal of connective tissue graft and to aid in wound closure . at recipient site , in both the groups , attempt was made to completely cover the connective tissue graft by overlying flap . studies have shown that total vascularization in the part of the connective tissue graft occurred when the flap at the recipient site completely covered it . as per the best of our knowledge , no data have been reported in the literature regarding the assessment of depth of wound at the palatal donor area . the ws was measured with unc-15 ( to the nearest of 0.5 mm ) at 1 week postoperative visit was significantly larger in group i as compared to group ii ( p < 0.001 ; table 3 ) . at 4 weeks too , the mean ws was larger in group i as compared to that in group ii ( p < 0.001 ; table 4 ) . a deeper incision or a wider base in the unigraft knife group might have helped to reduce the sloughing . at 4 weeks follow - up visit , although the wound area was epithelized , there was a depression seen at all the palatal sites in the group i , whereas in the group ii , wound area was fully diminished at all palatal sites at 4 weeks follow - up except for 1 patient . by 12 weeks , in both the groups , complete epithelization of the palatal wound area had occurred and clinically wound area was fully diminished . the percentage of complete palatal wound epithelization using langer and langer trap door technique cited in the relevant literature is quite similar to that seen in our study . the rate of sloughing of the primary flap using the langer and langer trap door technique was similar to those seen in previous reports . harris , 1997 , in a comparative study of the clinical healing in the donor area demonstrated that a high rate of sloughing occurs in the superficial flap when free gingival graft knife was employed . unigraft knife method resulted in a larger wound area at 1-week postoperative visit than the langer and langer trap door method . this could have resulted due to a variation in the ws created by the unigraft knife where the flap design was such that the distal border instead of the medial one served as the base of the reflected primary flap . the base of the primary reflected flap in the langer and langer trap door was toward the mid - palate in the region of first molar and canine and was broader than the base of the primary reflected flap in the unigraft knife method . intragroup comparison of ce showed that in group i , it was completed at 12 weeks postsurgery , whereas in group ii , it was completed at 4 weeks except for one patient where it was observed at 12 weeks period . kahnberg and thilander in a study on palatal healing in rats , noted that epithelization progressed from the wound borders , and reduction of the wound surface preceded by contraction of the wound margins and by epithelial cell migration . at none of the time db at 1-week postoperative visit was higher in group i as compared to group ii , but the difference was not statistically significant ( p = 0.825 ; table 4 ) . no statistically significant differences were observed regarding the return of sensibility in the palatal donor site in both the groups . literature supports that transient - postoperative sensory dysfunction is a possible complication after harvesting the graft from the palatal region . in both groups , mean vas and number of analgesic pills intake in the group at 1 week were higher as compared to that in group ii , yet the difference was not statistically significant . lengthy surgical procedures may create extensive tissue injury , prolong vasodilation that permits more fluid to accumulate in the interstitial spaces , and results in higher level of biologic mediators released by inflammatory and resident cells . both the unigraft knife method and the langer and langer trap door method simplify the technique for obtaining the sect graft from the palate . undoubtedly , in certain clinician 's hands , with certain skill levels and in certain situations , one technique with or without on assessing the root coverage parameters at the recipient site , it was found that the difference in the mean vr in both the groups at baseline , 3 , and 6 months was not statistically significant . mean root coverage achieved for group i was 54% , whereas for group ii , it was 68.13% at final ( 6 months ) postoperative visit . harris , 1997 , also observed similar trends on comparison of root coverage at the recipient site using the unigraft knife method and langer and langer trap door technique . intergroup comparison of mean width of keratinized gingiva ( kt ) showed no statistically significant ( p = 0.419 ) difference at all time intervals . intragroup comparison of mean kt showed that both the groups gained a statistically significant amount of tissue at 3 months and 6 months postsurgically as compared to baseline . transplanted sect of subsequent size has shown the availability to result in predictable root coverage and increased width of attached gingival . granulation tissue formation derived from the periodontal ligament also contributes to the increased width of keratinized gingiva . the increase in width of keratinized gingiva observed between 3 and 6 months has been attributed to creeping attachment . therefore , this study suggests that in terms of operator factors , both the techniques yielded sufficient sect graft suitable for periodontal plastic procedure with minimal operative complication . however , unigraft knife technique created a slightly larger wound area at the palate as compared to the free - hand incisions made in langer and langer technique . the wound in the langer and langer trap door technique method healed slightly more quickly as compared to the unigraft knife method , thus might have accounted for less patient discomfort . during the study , it was observed that the use of unigraft knife was technique - sensitive and it was difficult to adapt the instrument in situations where the palatal vault was high and narrow . the langer and langer trapdoor technique was more user - friendly as the angles of the free - hand incisions could be easily adjusted if some anatomic variations were encountered .

the diet and lifestyle questionnaire of vip , including a semi - quantitative food frequency questionnaire ( ffq ) , has been completed by almost all participants , with an average recruitment rate for period included in this study ( 19902008 ) of 59% within the target age - groups . in support of the population - based nature of the vip cohort are the nearly identical cancer incidence rates in the vip cohort and the population of vsterbotten ( 39 ) , and the minimal socio - economic selection bias ( 40 ) . in previous studies less than 1% ( n=595 ) of the vip participants have been defined as sami , and of these the majority are non - reindeer - herding sami ( 12 ) . from a total of 113,205 health examinations within the vip cohort ( 19922008 ) , of which 26,491 were repeated measures 10 years apart , a final study population of 77,319 participants was defined , based on the following exclusion criteria : subjects with missing data for more than 10% of the items in the ffq and/or portion size ( n=6,715 ) ; subjects lacking data for body mass index ( bmi ) or with bmi<10 kg / m ( n=60 ) ; subjects with unrealistic food intake level ( fil ) values , defined as a ratio of total energy intake to estimated basal metabolic rate ( 41 ) in the lowest 5th percentile or the highest 2.5th percentile , determined separately for sex and ffq version ( n=7,977 ) ; and subjects with repeated health surveys ( n=21,134 ) , in which the most recent sampling occasion was excluded from all analyses . in the present study , 3 versions of the vip ffq were used : the original , 84-item ffq ( n=25,886 ) , an older , nearly identical , 84-item ffq ( n=4,083 ) , and the most recent 65-item version , in which most foods are unchanged from the 84-item versions , some food groups have been deleted , and some similar food groups have been merged ( n=47,350 ) . all but the oldest version of the ffq have been validated against 24-hour recalls and/or biomarkers for b vitamins fatty acids and beta - carotene ( 4244 ) . in all ffq versions , meal - time portion sizes were estimated with the aid of 4 colour photographs of a plate containing proportionally increasing amounts of food stuffs representing vegetables and main sources of carbohydrate ( for example , rice or pasta ) and protein ( for example , meat or fish ) . calculation of nutrient intakes from the ffq items and portion size estimations is described elsewhere ( 45 ) . food items were recalculated into grams / day by multiplying frequency with portion size and adjusting for validated sex- and age - specific intake levels , defined by repeated 24 hour dietary recalls ( 43 ) . for boiled coffee , the original scale , occasions / day was used , to facilitate comparison with previous studies . intakes were energy adjusted by the residual method ( 46 ) . to define low - energy reporters within the data - set , a direct comparison of fil and physical activity level ( pal ) was used , which is a method appropriate for large sample sizes ( 47 ) . pal , available for 94.5% of the subjects , was estimated from 2 questions on general , work - related activity and leisure - time physical activity in exercise clothes ( 48 ) . cut - offs for definition of low - energy reporters ( 61.5% of subjects with pal - data ) were calculated by the goldberg method , modified by black ( 47 ) , and applied to the fil / pal ratio separately for ffq version and sex . a traditional sami diet score was constructed in a similar manner to the mediterranean diet score ( 1 ) , by adding 1 point for each intake above or below the median intake of several dietary items characteristic or uncharacteristic of the traditional sami diet , respectively . the selection of these dietary items was based on historical references ( 79 ) , interviews in which elderly sami were asked how they thought that their parents would have filled out the vip ffq in the 1930s1950s ( 10 ) , and present - time descriptions , including comparisons of diet and lifestyle in present - day reindeer - herding sami , non - herding sami and non - sami in the vip cohort ( 1013 ) . the score included 1 point for each intake above the median for red meat , fatty fish , total fat , berries and boiled coffee and 1 point for each intake below the median for vegetables , bread and fibre , all calculated separately for sex and ffq version , thus creating a range from 0 to 8 points for each subject . however , the sami participants with the 84-item ffq should overlap almost completely with the dataset from our previous study containing ethnicity data at an individual level ( 10 ) . in order to provide some indication of the validity of the score , we therefore used that dataset to calculate median traditional sami diet scores for reindeer - herding sami , non - reindeer - herding sami and geographically matched non - sami . a sensitivity analysis was performed by testing models in which the selected traditional sami diet score items were excluded from the score one by one , yielding 8 models of 07 points . heterogeneity between these reduced models and the original traditional sami diet score were tested by chi - square tests . furthermore , an extended , 10-point traditional sami diet score was modelled , including intakes of blood dishes and liver / kidney , in the sub - sample of subjects with 84-item ffqs . mortality end - points up to and including 31 december 2007 , were identified by linking the vip database with the swedish national cause - of - death registry . cancer mortality was defined as underlying cause of death , icd-9 codes 140208 , or icd-10 codes c00c97 . cvd mortality was defined as the main cause of death and/or underlying cause of death , icd-9 codes 390438 , or icd-10 likely predictors of mortality , including age , body mass index ( bmi , kg / m ) , current smoking ( yes / no ) , education ( lack of post - secondary , yes / no ) , sedentary lifestyle ( no regular physical activity in exercise clothes , yes / no ) , and intake of alcohol ( g / day ) , fat ( g / day ) , saturated fat ( g / day ) , and total energy ( kcal / day ) , were examined for association with traditional sami diet score categories by kruskal wallis tests ( tables i and ii ) . baseline characteristics of vsterbotten intervention program participants according to traditional sami diet score calculated separately for ffq version and sex . physical activity level was estimated from 2 questions on general , work - related activity and leisure - time physical activity in exercise clothes ( 48 ) . low outdoor activity was defined as at most 2 regular leisure time outdoor activities among the following : walking ( 1 times / day ) , biking ( 1 times / day ) , collecting berries / mushrooms ( 1 times / week ) , gardening ( 1 times / week ) or shovelling snow ( 1 times / week ) or fishing / hunting ( 1 times / month ) . sex - specific hazard ratios ( hr ) for all - cause , cancer ( including the most common cancer sites ) , and cvd mortality were calculated by cox regression . though all lifestyle variables were significantly associated with traditional sami diet score categories in at least 1 sex , none met our criterion for a confounder : altering the hr for the traditional sami diet score by 10% or more when included in a bivariate model . to facilitate comparison with other studies , common risk factors were kept in the multivariate model 2 , which thus included : age , bmi , current smoking , education , sedentary lifestyle , and intake of alcohol and total energy . proportional hazard assumptions were confirmed by schoenfeld 's test . with age categorised into 10-year age groups , all covariates fulfilled the criteria for the proportional hazard assumption in men , but in women the exposure variable , traditional sami diet score , did not , p shoenfeld 's test=0.032 . this minor deviation from proportionality indicates an increased risk of unstable results in women , but probably does not otherwise affect our results materially . we have chosen to present results for both men and women , in order to provide as complete a description of the cohort as possible , but our interpretation of the results in women is conservative . the subgroup with low metabolic risk profile included subjects free from hypertension , diabetes , and obesity , whereas a high metabolic risk profile included hypertension and/or diabetes and/or obesity . hypertension was defined as systolic blood pressure 140 mm hg and/or diastolic blood pressure 90 mm hg and/or use of medication to lower blood pressure . for classification as non - hypertensive , mmol / l and/or post - load glucose 12.2 mmol / l ( measured in capillary plasma ) . for classification as non - diabetic , measurement of fasting or post - load glucose was required . the cohort was stratified according to pal above or below the median . in order to ensure that dietary changes due to disease did not affect the results , we did analysis with excluding all subjects with follow - up times shorter than 2 years . in addition we replaced the sedentary lifestyle variable in the multivariate model with pal or with a variable representing low physical activity outdoors , the latter encompassing questions on walking , biking , picking berries or mushrooms , gardening , shovelling snow , and hunting and fishing . all tests were 2-sided , and p - values < 0.05 were considered statistically significant . the study protocol and data handling procedures were approved by the regional ethical review board of northern sweden ( dnr 07 - 165 m ) . evaluation by a specific ethical review board representing the sami society would also have been desirable , but such a board does not exist in sweden at present . all study subjects provided written informed consent , and the study was conducted in accordance with the declaration of helsinki . our study cohort consists of participants from the vsterbotten intervention program ( vip ) , in which health risk measures and anthropometric data are collected from residents of the county of vsterbotten turning 30 ( years 19851996 ) , 40 , 50 and 60 years of age . the diet and lifestyle questionnaire of vip , including a semi - quantitative food frequency questionnaire ( ffq ) , has been completed by almost all participants , with an average recruitment rate for period included in this study ( 19902008 ) of 59% within the target age - groups . in support of the population - based nature of the vip cohort are the nearly identical cancer incidence rates in the vip cohort and the population of vsterbotten ( 39 ) , and the minimal socio - economic selection bias ( 40 ) . in previous studies less than 1% ( n=595 ) of the vip participants have been defined as sami , and of these the majority are non - reindeer - herding sami ( 12 ) . from a total of 113,205 health examinations within the vip cohort ( 19922008 ) , of which 26,491 were repeated measures 10 years apart , a final study population of 77,319 participants was defined , based on the following exclusion criteria : subjects with missing data for more than 10% of the items in the ffq and/or portion size ( n=6,715 ) ; subjects lacking data for body mass index ( bmi ) or with bmi<10 kg / m ( n=60 ) ; subjects with unrealistic food intake level ( fil ) values , defined as a ratio of total energy intake to estimated basal metabolic rate ( 41 ) in the lowest 5th percentile or the highest 2.5th percentile , determined separately for sex and ffq version ( n=7,977 ) ; and subjects with repeated health surveys ( n=21,134 ) , in which the most recent sampling occasion was excluded from all analyses . in the present study , 3 versions of the vip ffq were used : the original , 84-item ffq ( n=25,886 ) , an older , nearly identical , 84-item ffq ( n=4,083 ) , and the most recent 65-item version , in which most foods are unchanged from the 84-item versions , some food groups have been deleted , and some similar food groups have been merged ( n=47,350 ) . all but the oldest version of the ffq have been validated against 24-hour recalls and/or biomarkers for b vitamins fatty acids and beta - carotene ( 4244 ) . in all ffq versions , meal - time portion sizes were estimated with the aid of 4 colour photographs of a plate containing proportionally increasing amounts of food stuffs representing vegetables and main sources of carbohydrate ( for example , rice or pasta ) and protein ( for example , meat or fish ) . calculation of nutrient intakes from the ffq items and portion size estimations is described elsewhere ( 45 ) . food items were recalculated into grams / day by multiplying frequency with portion size and adjusting for validated sex- and age - specific intake levels , defined by repeated 24 hour dietary recalls ( 43 ) . for boiled coffee , the original scale , occasions / day was used , to facilitate comparison with previous studies . to define low - energy reporters within the data - set , a direct comparison of fil and physical activity level ( pal ) was used , which is a method appropriate for large sample sizes ( 47 ) . pal , available for 94.5% of the subjects , was estimated from 2 questions on general , work - related activity and leisure - time physical activity in exercise clothes ( 48 ) . cut - offs for definition of low - energy reporters ( 61.5% of subjects with pal - data ) were calculated by the goldberg method , modified by black ( 47 ) , and applied to the fil / pal ratio separately for ffq version and sex . a traditional sami diet score was constructed in a similar manner to the mediterranean diet score ( 1 ) , by adding 1 point for each intake above or below the median intake of several dietary items characteristic or uncharacteristic of the traditional sami diet , respectively . the selection of these dietary items was based on historical references ( 79 ) , interviews in which elderly sami were asked how they thought that their parents would have filled out the vip ffq in the 1930s1950s ( 10 ) , and present - time descriptions , including comparisons of diet and lifestyle in present - day reindeer - herding sami , non - herding sami and non - sami in the vip cohort ( 1013 ) . the score included 1 point for each intake above the median for red meat , fatty fish , total fat , berries and boiled coffee and 1 point for each intake below the median for vegetables , bread and fibre , all calculated separately for sex and ffq version , thus creating a range from 0 to 8 points for each subject . however , the sami participants with the 84-item ffq should overlap almost completely with the dataset from our previous study containing ethnicity data at an individual level ( 10 ) . in order to provide some indication of the validity of the score , we therefore used that dataset to calculate median traditional sami diet scores for reindeer - herding sami , non - reindeer - herding sami and geographically matched non - sami . a sensitivity analysis was performed by testing models in which the selected traditional sami diet score items were excluded from the score one by one , yielding 8 models of 07 points . heterogeneity between these reduced models and the original traditional sami diet score were tested by chi - square tests . furthermore , an extended , 10-point traditional sami diet score was modelled , including intakes of blood dishes and liver / kidney , in the sub - sample of subjects with 84-item ffqs . mortality end - points up to and including 31 december 2007 , were identified by linking the vip database with the swedish national cause - of - death registry . cancer mortality was defined as underlying cause of death , icd-9 codes 140208 , or icd-10 codes c00c97 . cvd mortality was defined as the main cause of death and/or underlying cause of death , icd-9 codes 390438 , or icd-10 codes i00i69 . likely predictors of mortality , including age , body mass index ( bmi , kg / m ) , current smoking ( yes / no ) , education ( lack of post - secondary , yes / no ) , sedentary lifestyle ( no regular physical activity in exercise clothes , yes / no ) , and intake of alcohol ( g / day ) , fat ( g / day ) , saturated fat ( g / day ) , and total energy ( kcal / day ) , were examined for association with traditional sami diet score categories by kruskal wallis tests ( tables i and ii ) . baseline characteristics of vsterbotten intervention program participants according to traditional sami diet score calculated separately for ffq version and sex . physical activity level was estimated from 2 questions on general , work - related activity and leisure - time physical activity in exercise clothes ( 48 ) . low outdoor activity was defined as at most 2 regular leisure time outdoor activities among the following : walking ( 1 times / day ) , biking ( 1 times / day ) , collecting berries / mushrooms ( 1 times / week ) , gardening ( 1 times / week ) or shovelling snow ( 1 times / week ) or fishing / hunting ( 1 times / month ) . sex - specific hazard ratios ( hr ) for all - cause , cancer ( including the most common cancer sites ) , and cvd mortality were calculated by cox regression . though all lifestyle variables were significantly associated with traditional sami diet score categories in at least 1 sex , none met our criterion for a confounder : altering the hr for the traditional sami diet score by 10% or more when included in a bivariate model . to facilitate comparison with other studies , common risk factors were kept in the multivariate model 2 , which thus included : age , bmi , current smoking , education , sedentary lifestyle , and intake of alcohol and total energy . categorised into 10-year age groups , all covariates fulfilled the criteria for the proportional hazard assumption in men , but in women the exposure variable , traditional sami diet score , did not , p shoenfeld 's test=0.032 . this minor deviation from proportionality indicates an increased risk of unstable results in women , but probably does not otherwise affect our results materially . we have chosen to present results for both men and women , in order to provide as complete a description of the cohort as possible , but our interpretation of the results in women is conservative . the subgroup with low metabolic risk profile included subjects free from hypertension , diabetes , and obesity , whereas a high metabolic risk profile included hypertension and/or diabetes and/or obesity . hypertension was defined as systolic blood pressure 140 mm hg and/or diastolic blood pressure 90 mm hg and/or use of medication to lower blood pressure . for classification as non - hypertensive , mmol / l and/or post - load glucose 12.2 mmol / l ( measured in capillary plasma ) . for classification as non - diabetic , measurement of fasting or post - load glucose was required . the cohort was stratified according to pal above or below the median . in order to ensure that dietary changes due to disease did not affect the results , we did analysis with excluding all subjects with follow - up times shorter than 2 years . in addition we replaced the sedentary lifestyle variable in the multivariate model with pal or with a variable representing low physical activity outdoors , the latter encompassing questions on walking , biking , picking berries or mushrooms , gardening , shovelling snow , and hunting and fishing . all tests were 2-sided , and p - values < 0.05 were considered statistically significant . the study protocol and data handling procedures were approved by the regional ethical review board of northern sweden ( dnr 07 - 165 m ) . evaluation by a specific ethical review board representing the sami society would also have been desirable , but such a board does not exist in sweden at present . all study subjects provided written informed consent , and the study was conducted in accordance with the declaration of helsinki . among the 77,319 subjects included in the study , a total of 2,383 deaths occurred , including 975 cancer related and 681 cvd related . follow - up times ranged from 1 day to 19 years , with a median of 10 years . in the study population , 20.0% of men and 19.7% of women had high traditional sami diet scores ( 68 points ) . table i shows baseline characteristics of the vsterbotten intervention program participants according to traditional sami diet score . a high traditional sami diet score was associated with lower age , education level and pal , and higher prevalence of smoking , sedentary lifestyle and low outdoor activity level . in men , table ii shows the intake levels of various food items , including those making up the traditional sami diet score , as well as total energy and ethanol intakes . the greatest variability was found for vegetables and bread , of which subjects with high traditional sami diet scores had reported intakes of at most half those of subjects with low traditional sami diet scores . intakes of red meat and fatty fish also varied considerably , with higher intakes associated with higher traditional sami diet scores . further , a high traditional sami diet score was associated with a lower total energy intake and a higher ethanol intake , especially in men . baseline dietary characteristics of vsterbotten intervention program participants according to traditional sami diet score calculated separately for ffq version and sex . in table iii , hrs for all - cause , cancer and cvd mortality per 1-point increase in traditional sami diet score are shown . in the crude model 1 , adjusted only for age , increasing traditional sami diet score was associated with an elevated all - cause mortality in both men and women [ crude hr for men 1.07 ( 95% ci 1.041.10 ) p 0.001 ; crude hr for women 1.06 ( 95% ci 1.021.11 ) p 0.001 ] . in the multivariate model 2 , adjusted for age , bmi , current smoking , education , sedentary lifestyle , and intake of alcohol and total energy , risk associations were attenuated and only statistically significant in men [ multivariate hr for men 1.04 ( 95% ci 1.011.07 ) , p=0.018 ; multivariate hr for women 1.03 ( 95% ci 0.991.07 ) , p=0.130 ] . in the subgroup analyses , a statistically significantly elevated all - cause mortality risk was found only in men with a low metabolic risk profile , that is , free from hypertension , diabetes and obesity [ multivariate hr 1.06 ( ci=1.021.11 ) p=0.008 ] , and in men with a low pal [ multivariate hr 1.05 ( ci=1.011.09 ) p=0.019 ] . hazard ratios for all - cause , cancer and cardiovascular disease mortality by traditional sami diet score in participants of the vsterbotten intervention program cohort 19902008 based on intake of red meat , fatty fish , fat , berries , boiled coffee , vegetables , bread and fibre , which are intake variables characteristic of traditional sami culture ( 1013 ) . further adjusted for bmi , sedentary lifestyle , education , current smoking , intake of alcohol and total energy . diabetes and/or hypertension and/or body mass index 30.00 . physical activity level 1.6 ( median ) . estimated from 2 questionnaire items on work - related and leisure - time physical activity ( 48 ) . estimated from 2 questionnaire items on work - related and leisure - time physical activity ( 48 ) . for cancer and cvd mortality , hrs were above 1 and statistically significant in both men and women in model 1 . however , associations were not statistically significant in the multivariate model 2 , except for cvd mortality in men with a low metabolic risk profile [ multivariate hr 1.10 ( ci=1.011.20 ) p=0.023 ] . risk associations were above or very close to 1 , but not statistically significant , for the most common cancer sites : colorectum ( 127 deaths ) , pancreas ( 93 deaths ) , breast ( 80 deaths ) , prostate ( 60 deaths ) , and stomach ( 52 deaths ) ( data not shown ) , with the possible exception of respiratory tract cancer ( 122 deaths ) , in which an increased risk with increasing traditional sami diet score of borderline statistical significance was observed [ multivariate hr 1.11 ( ci = 1.001.24 ) p = 0.053 ] . in the subsample of 29,969 subjects with the 84-item ffq ( 992 male deaths , 673 female deaths ) , for whom liver / kidney and blood dishes were added to the traditional sami diet score ( 010 points ) , results were weaker ( data not shown ) . results were stronger in subjects who reported a more adequate total energy intake [ multivariate hr for all - cause mortality in men 1.07 ( ci=1.021.13 ) p=0.009 ; and women1.08 ( ci=1.001.16 ) p=0.047 , for all - cause mortality ] . excluding subjects with < 2 years of follow - up , or replacing the sedentary lifestyle variable with pal or low outdoor physical activity in the multivariate model , had no material effect on the results for all - cause , cancer or cvd mortality , except a reduction in power due to more missing values ( data not shown ) . in table iv , hrs for all - cause mortality for the individual dietary items included in the traditional sami diet score items are presented . for most of the positively defined traditional sami diet score items ( 1 point for intake exceptions were higher intake of red meat in women [ multivariate hr 1.17 ( ci=1.021.34 ) p=0.023 ] and higher intake of fat in men [ multivariate hr 1.12 ( ci=1.011.25 ) p=0.037 ] . in negatively defined traditional sami diet score items ( 1 point for intake < median ) , statistically significant risk associations were observed for low intake of vegetables [ multivariate hr 1.14 ( ci=1.021.27 ) p=0.016 ] and bread [ multivariate hr 1.14 ( ci=1.031.27 ) p=0.014 ] in men . hazard ratios for all cause mortality according to the traditional sami dietary elements included in the traditional sami diet score hazard ratios were determined by cox regression analyses . further adjusted for bmi , sedentary lifestyle , education , current smoking , intake of alcohol and total energy . adjusted as in footnote 3 , as well as for the remaining items in the traditional sami diet score . inclusion in the traditional sami diet score tested in subjects with data on intake of blood dishes and liver / kidney ( subjects with a more extensive version of the food frequency questionnaire ) . the results of the sensitivity analysis are shown in table v. the risk association for all - cause mortality was not materially affected by the removal of any 1 dietary item included in the score . these analyses were performed in men only because of the instability of the traditional sami diet score cox regression model in women , as noted in the materials and methods section . sensitivity analysis for the traditional sami diet score in men , a comparison of all - cause mortality using 8 reduced models , in which the traditional sami diet score items were excluded from the score one by one hazard ratios were determined by cox regression analyses . adjusted for age , bmi , sedentary lifestyle , education , current smoking , intake of alcohol and total energy . tests for heterogeneity between the result presented and the result for the complete traditional sami diet score were performed by a chi - square test . applying the traditional sami diet score on the data - set used in our previous study ( 10 ) , yielded a median ( 1st3rd quartile ) score of 6.0 ( 4.07.0 ) points in reindeer - herding sami , 4.5 ( 3.06.0 ) points in non - reindeer - herding sami , and 4.0 ( 3.05.0 ) points in geographically matched non - sami ( p 0.001 ) . in this largely non - sami , population - based cohort study of 77,319 men and women in northern sweden , with up to 19 years of follow up , higher traditional sami diet scores were associated with a weak increase in all - cause mortality in men . this increased risk may be equally attributed to cvd and cancer , since hrs above 1 were the general pattern for both endpoints , but it appeared to be particularly pronounced for cvd mortality in men with a low metabolic risk profile ( free from diabetes , hypertension and obesity ) . in women , we found no stable risk associations for traditional sami diet score , though all hrs were 1 . our findings , in a largely non - sami population , are generally in line with the evidence to date for the individual components of the traditional sami diet score . although the score is high in some foods widely considered to be healthy , such as fatty fish ( 25 ) and berries ( 26 ) , it is also rich in foods associated with negative health outcomes such as red meat for colorectal and other cancers ( 19 ) , and lacking in foods associated with positive health outcomes such as vegetables for reduced cvd risk ( 20 ) . null associations were also found for fatty fish and berries . in a previous report from the same northern swedish population , no associations between reported fish consumption ( lean and fatty fish combined ) and risk of acute myocardial infarction were observed ( 49 ) . in our previous interviews with elderly sami 5070 years ago , we found that intakes of both fatty fish and berries were likely much higher than the intakes of either sami or non - sami in vsterbotten today ( 10 ) . thus , the present - day diet in the population of vsterbotten may deviate from the traditional sami diet to such a degree that potential health effects can not be revealed . former studies have suggested that the slightly reduced cancer risk in sami populations might be due to traditional sami diets ( 30 ) . however , not only does the present - day diet in vsterbotten deviate considerably from our definition of traditional sami diet , but it is also possible that our traditional sami diet score does not closely enough reflect the traditional sami diet , which is , in itself difficult to define . knowledge of the diet prior to the 1700s is limited ( 7,9 ) , and the centuries since then have been marked by considerable change for the sami population ( 10 ) . traditional sami diet , for example by distinguishing between different kinds of red meat , such as wild game and reindeer meat and commercial beef , or fish bought and fish caught in the wild , or wild berries and greens and cultivated ones . such foods may differ not only in biochemical composition ( 26,50 ) , but also in related social or behavioural elements not otherwise accounted for in the statistical analyses . thus , the vip ffq is inevitably an imprecise tool for the construction of a score representing a traditional sami dietary pattern . the traditional sami diet score was not intended to reflect the diet of the sami today . however , we have previously observed differences in the diet of present - day reindeer - herding sami , non - reindeer - herding sami and non - sami groups using the vip ffq ( 10,12 ) . furthermore , traditional sami diet scores were high in reindeer - herding sami compared to the other groups in that dataset ( 10 ) , suggesting some degree of validity for the score , even in the present day . in diet score methodology , overall effects of adherence to a certain dietary pattern are studied , allowing for a single measure encompassing all potential interactions among the dietary components included , but also preventing the detection of effects related to the specific components or interactions . dietary patterns are thus considered more relevant for public health recommendations , but less relevant for understanding underlying mechanisms ( 51 ) . diet scores inevitably provide a rough estimate of diet . to preserve power and limit complexity , they are often restricted to relative , dichotomous , rather than absolute , multiple , cut - offs , and equal weighting of a limited number of dietary components comprising the score . however , as the success of the mediterranean diet illustrates , diet scores can be an important tool in the field of nutritional epidemiology . the intervention built into the vsterbotten intervention program , in which diabetes and hypertension diagnosed through the health survey are treated , may have diluted our results . the weaker risk associations in men with metabolic risk factors at baseline may reflect such an effect . chance findings may have occurred due to multiple testing , and p - values should therefore be interpreted conservatively . there is also a substantial risk of residual confounding due to factors not , or not adequately , assessed by the vip questionnaire and health survey , such as smoking history , defined only as current smoker , yes / no , and socioeconomic status , represented by education level , in the present study . physical activity , in particular physical activity patterns of the traditional sami lifestyle , is another important example of residual confounding . much of the extensive physical activity , while not directly related to occupation , was conducted essentially by need and not as leisure - time or recreational activity ( 10 ) . furthermore , recreational physical activity in exercise clothes is a rough measure of physical activity . this question , and the work - related physical activity question , in the vip ffq are therefore probably inadequate for the assessment of physical activity as a confounder or effect modifier of the relationship between traditional sami diet score and mortality . however , replacing the sedentary lifestyle variable with a low outdoor physical activity variable did not materially affect the main findings . the main strengths of this study were the population - based cohort design , the long follow up ( up to 19 years ) , and the large sample size , as well as the rather unique aspect of examining the general population from a minority perspective . given the inherent limitations of the questionnaire used , and the difficulty defining a score reflecting traditional sami diet , this study should be considered exploratory , a first attempt to address this topic . further study of cohorts with more detailed information on dietary and lifestyle items relevant for traditional sami culture is warranted . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .

objectivesto examine the relationship between traditional sami dietary pattern and mortality in a general northern swedish population.study designpopulation - based cohort study.methodswe examined 77,319 subjects from the vsterbotten intervention program ( vip ) cohort . a traditional sami diet score was constructed by adding 1 point for intake above the median level of red meat , fatty fish , total fat , berries and boiled coffee , and 1 point for intake below the median of vegetables , bread and fibre . hazard ratios ( hr ) for mortality were calculated by cox regression.resultsincreasing traditional sami diet scores were associated with slightly elevated all - cause mortality in men [ multivariate hr per 1-point increase in score 1.04 ( 95% ci 1.011.07 ) , p=0.018 ] , but not for women [ multivariate hr 1.03 ( 95% ci 0.991.07 ) , p=0.130 ] . this increased risk was approximately equally attributable to cardiovascular disease and cancer , though somewhat more apparent for cardiovascular disease mortality in men free from diabetes , hypertension and obesity at baseline [ multivariate hr 1.10 ( 95% ci 1.011.20 ) , p=0.023].conclusionsa weak increased all - cause mortality was observed in men with higher traditional sami diet scores . however , due to the complexity in defining a traditional sami diet , and the limitations of our questionnaire for this purpose , the study should be considered exploratory , a first attempt to relate a traditional sami dietary pattern to health endpoints . further investigation of cohorts with more detailed information on dietary and lifestyle items relevant for traditional sami culture is warranted .

Material and methods Study cohort Food frequency questionnaire Under-reporting Traditional Sami diet score Identification of mortality end-points Statistical analysis Ethics Results Discussion Conflict of interest and funding

in support of the population - based nature of the vip cohort are the nearly identical cancer incidence rates in the vip cohort and the population of vsterbotten ( 39 ) , and the minimal socio - economic selection bias ( 40 ) . in the present study , 3 versions of the vip ffq were used : the original , 84-item ffq ( n=25,886 ) , an older , nearly identical , 84-item ffq ( n=4,083 ) , and the most recent 65-item version , in which most foods are unchanged from the 84-item versions , some food groups have been deleted , and some similar food groups have been merged ( n=47,350 ) . for boiled coffee , the original scale , occasions / day was used , to facilitate comparison with previous studies . cut - offs for definition of low - energy reporters ( 61.5% of subjects with pal - data ) were calculated by the goldberg method , modified by black ( 47 ) , and applied to the fil / pal ratio separately for ffq version and sex . a traditional sami diet score was constructed in a similar manner to the mediterranean diet score ( 1 ) , by adding 1 point for each intake above or below the median intake of several dietary items characteristic or uncharacteristic of the traditional sami diet , respectively . the selection of these dietary items was based on historical references ( 79 ) , interviews in which elderly sami were asked how they thought that their parents would have filled out the vip ffq in the 1930s1950s ( 10 ) , and present - time descriptions , including comparisons of diet and lifestyle in present - day reindeer - herding sami , non - herding sami and non - sami in the vip cohort ( 1013 ) . the score included 1 point for each intake above the median for red meat , fatty fish , total fat , berries and boiled coffee and 1 point for each intake below the median for vegetables , bread and fibre , all calculated separately for sex and ffq version , thus creating a range from 0 to 8 points for each subject . in order to provide some indication of the validity of the score , we therefore used that dataset to calculate median traditional sami diet scores for reindeer - herding sami , non - reindeer - herding sami and geographically matched non - sami . a sensitivity analysis was performed by testing models in which the selected traditional sami diet score items were excluded from the score one by one , yielding 8 models of 07 points . heterogeneity between these reduced models and the original traditional sami diet score were tested by chi - square tests . furthermore , an extended , 10-point traditional sami diet score was modelled , including intakes of blood dishes and liver / kidney , in the sub - sample of subjects with 84-item ffqs . cvd mortality was defined as the main cause of death and/or underlying cause of death , icd-9 codes 390438 , or icd-10 likely predictors of mortality , including age , body mass index ( bmi , kg / m ) , current smoking ( yes / no ) , education ( lack of post - secondary , yes / no ) , sedentary lifestyle ( no regular physical activity in exercise clothes , yes / no ) , and intake of alcohol ( g / day ) , fat ( g / day ) , saturated fat ( g / day ) , and total energy ( kcal / day ) , were examined for association with traditional sami diet score categories by kruskal wallis tests ( tables i and ii ) . baseline characteristics of vsterbotten intervention program participants according to traditional sami diet score calculated separately for ffq version and sex . sex - specific hazard ratios ( hr ) for all - cause , cancer ( including the most common cancer sites ) , and cvd mortality were calculated by cox regression . though all lifestyle variables were significantly associated with traditional sami diet score categories in at least 1 sex , none met our criterion for a confounder : altering the hr for the traditional sami diet score by 10% or more when included in a bivariate model . with age categorised into 10-year age groups , all covariates fulfilled the criteria for the proportional hazard assumption in men , but in women the exposure variable , traditional sami diet score , did not , p shoenfeld 's test=0.032 . the subgroup with low metabolic risk profile included subjects free from hypertension , diabetes , and obesity , whereas a high metabolic risk profile included hypertension and/or diabetes and/or obesity . all study subjects provided written informed consent , and the study was conducted in accordance with the declaration of helsinki . our study cohort consists of participants from the vsterbotten intervention program ( vip ) , in which health risk measures and anthropometric data are collected from residents of the county of vsterbotten turning 30 ( years 19851996 ) , 40 , 50 and 60 years of age . in support of the population - based nature of the vip cohort are the nearly identical cancer incidence rates in the vip cohort and the population of vsterbotten ( 39 ) , and the minimal socio - economic selection bias ( 40 ) . in the present study , 3 versions of the vip ffq were used : the original , 84-item ffq ( n=25,886 ) , an older , nearly identical , 84-item ffq ( n=4,083 ) , and the most recent 65-item version , in which most foods are unchanged from the 84-item versions , some food groups have been deleted , and some similar food groups have been merged ( n=47,350 ) . for boiled coffee , the original scale , occasions / day was used , to facilitate comparison with previous studies . cut - offs for definition of low - energy reporters ( 61.5% of subjects with pal - data ) were calculated by the goldberg method , modified by black ( 47 ) , and applied to the fil / pal ratio separately for ffq version and sex . a traditional sami diet score was constructed in a similar manner to the mediterranean diet score ( 1 ) , by adding 1 point for each intake above or below the median intake of several dietary items characteristic or uncharacteristic of the traditional sami diet , respectively . the selection of these dietary items was based on historical references ( 79 ) , interviews in which elderly sami were asked how they thought that their parents would have filled out the vip ffq in the 1930s1950s ( 10 ) , and present - time descriptions , including comparisons of diet and lifestyle in present - day reindeer - herding sami , non - herding sami and non - sami in the vip cohort ( 1013 ) . the score included 1 point for each intake above the median for red meat , fatty fish , total fat , berries and boiled coffee and 1 point for each intake below the median for vegetables , bread and fibre , all calculated separately for sex and ffq version , thus creating a range from 0 to 8 points for each subject . in order to provide some indication of the validity of the score , we therefore used that dataset to calculate median traditional sami diet scores for reindeer - herding sami , non - reindeer - herding sami and geographically matched non - sami . a sensitivity analysis was performed by testing models in which the selected traditional sami diet score items were excluded from the score one by one , yielding 8 models of 07 points . heterogeneity between these reduced models and the original traditional sami diet score were tested by chi - square tests . furthermore , an extended , 10-point traditional sami diet score was modelled , including intakes of blood dishes and liver / kidney , in the sub - sample of subjects with 84-item ffqs . likely predictors of mortality , including age , body mass index ( bmi , kg / m ) , current smoking ( yes / no ) , education ( lack of post - secondary , yes / no ) , sedentary lifestyle ( no regular physical activity in exercise clothes , yes / no ) , and intake of alcohol ( g / day ) , fat ( g / day ) , saturated fat ( g / day ) , and total energy ( kcal / day ) , were examined for association with traditional sami diet score categories by kruskal wallis tests ( tables i and ii ) . baseline characteristics of vsterbotten intervention program participants according to traditional sami diet score calculated separately for ffq version and sex . sex - specific hazard ratios ( hr ) for all - cause , cancer ( including the most common cancer sites ) , and cvd mortality were calculated by cox regression . though all lifestyle variables were significantly associated with traditional sami diet score categories in at least 1 sex , none met our criterion for a confounder : altering the hr for the traditional sami diet score by 10% or more when included in a bivariate model . categorised into 10-year age groups , all covariates fulfilled the criteria for the proportional hazard assumption in men , but in women the exposure variable , traditional sami diet score , did not , p shoenfeld 's test=0.032 . the subgroup with low metabolic risk profile included subjects free from hypertension , diabetes , and obesity , whereas a high metabolic risk profile included hypertension and/or diabetes and/or obesity . all study subjects provided written informed consent , and the study was conducted in accordance with the declaration of helsinki . among the 77,319 subjects included in the study , a total of 2,383 deaths occurred , including 975 cancer related and 681 cvd related . in the study population , 20.0% of men and 19.7% of women had high traditional sami diet scores ( 68 points ) . table i shows baseline characteristics of the vsterbotten intervention program participants according to traditional sami diet score . a high traditional sami diet score was associated with lower age , education level and pal , and higher prevalence of smoking , sedentary lifestyle and low outdoor activity level . in men , table ii shows the intake levels of various food items , including those making up the traditional sami diet score , as well as total energy and ethanol intakes . intakes of red meat and fatty fish also varied considerably , with higher intakes associated with higher traditional sami diet scores . further , a high traditional sami diet score was associated with a lower total energy intake and a higher ethanol intake , especially in men . baseline dietary characteristics of vsterbotten intervention program participants according to traditional sami diet score calculated separately for ffq version and sex . in table iii , hrs for all - cause , cancer and cvd mortality per 1-point increase in traditional sami diet score are shown . in the crude model 1 , adjusted only for age , increasing traditional sami diet score was associated with an elevated all - cause mortality in both men and women [ crude hr for men 1.07 ( 95% ci 1.041.10 ) p 0.001 ; crude hr for women 1.06 ( 95% ci 1.021.11 ) p 0.001 ] . in the multivariate model 2 , adjusted for age , bmi , current smoking , education , sedentary lifestyle , and intake of alcohol and total energy , risk associations were attenuated and only statistically significant in men [ multivariate hr for men 1.04 ( 95% ci 1.011.07 ) , p=0.018 ; multivariate hr for women 1.03 ( 95% ci 0.991.07 ) , p=0.130 ] . in the subgroup analyses , a statistically significantly elevated all - cause mortality risk was found only in men with a low metabolic risk profile , that is , free from hypertension , diabetes and obesity [ multivariate hr 1.06 ( ci=1.021.11 ) p=0.008 ] , and in men with a low pal [ multivariate hr 1.05 ( ci=1.011.09 ) p=0.019 ] . hazard ratios for all - cause , cancer and cardiovascular disease mortality by traditional sami diet score in participants of the vsterbotten intervention program cohort 19902008 based on intake of red meat , fatty fish , fat , berries , boiled coffee , vegetables , bread and fibre , which are intake variables characteristic of traditional sami culture ( 1013 ) . however , associations were not statistically significant in the multivariate model 2 , except for cvd mortality in men with a low metabolic risk profile [ multivariate hr 1.10 ( ci=1.011.20 ) p=0.023 ] . risk associations were above or very close to 1 , but not statistically significant , for the most common cancer sites : colorectum ( 127 deaths ) , pancreas ( 93 deaths ) , breast ( 80 deaths ) , prostate ( 60 deaths ) , and stomach ( 52 deaths ) ( data not shown ) , with the possible exception of respiratory tract cancer ( 122 deaths ) , in which an increased risk with increasing traditional sami diet score of borderline statistical significance was observed [ multivariate hr 1.11 ( ci = 1.001.24 ) p = 0.053 ] . in the subsample of 29,969 subjects with the 84-item ffq ( 992 male deaths , 673 female deaths ) , for whom liver / kidney and blood dishes were added to the traditional sami diet score ( 010 points ) , results were weaker ( data not shown ) . results were stronger in subjects who reported a more adequate total energy intake [ multivariate hr for all - cause mortality in men 1.07 ( ci=1.021.13 ) p=0.009 ; and women1.08 ( ci=1.001.16 ) p=0.047 , for all - cause mortality ] . in table iv , hrs for all - cause mortality for the individual dietary items included in the traditional sami diet score items are presented . for most of the positively defined traditional sami diet score items ( 1 point for intake exceptions were higher intake of red meat in women [ multivariate hr 1.17 ( ci=1.021.34 ) p=0.023 ] and higher intake of fat in men [ multivariate hr 1.12 ( ci=1.011.25 ) p=0.037 ] . in negatively defined traditional sami diet score items ( 1 point for intake < median ) , statistically significant risk associations were observed for low intake of vegetables [ multivariate hr 1.14 ( ci=1.021.27 ) p=0.016 ] and bread [ multivariate hr 1.14 ( ci=1.031.27 ) p=0.014 ] in men . hazard ratios for all cause mortality according to the traditional sami dietary elements included in the traditional sami diet score hazard ratios were determined by cox regression analyses . adjusted as in footnote 3 , as well as for the remaining items in the traditional sami diet score . the results of the sensitivity analysis are shown in table v. the risk association for all - cause mortality was not materially affected by the removal of any 1 dietary item included in the score . these analyses were performed in men only because of the instability of the traditional sami diet score cox regression model in women , as noted in the materials and methods section . sensitivity analysis for the traditional sami diet score in men , a comparison of all - cause mortality using 8 reduced models , in which the traditional sami diet score items were excluded from the score one by one hazard ratios were determined by cox regression analyses . tests for heterogeneity between the result presented and the result for the complete traditional sami diet score were performed by a chi - square test . applying the traditional sami diet score on the data - set used in our previous study ( 10 ) , yielded a median ( 1st3rd quartile ) score of 6.0 ( 4.07.0 ) points in reindeer - herding sami , 4.5 ( 3.06.0 ) points in non - reindeer - herding sami , and 4.0 ( 3.05.0 ) points in geographically matched non - sami ( p 0.001 ) . in this largely non - sami , population - based cohort study of 77,319 men and women in northern sweden , with up to 19 years of follow up , higher traditional sami diet scores were associated with a weak increase in all - cause mortality in men . this increased risk may be equally attributed to cvd and cancer , since hrs above 1 were the general pattern for both endpoints , but it appeared to be particularly pronounced for cvd mortality in men with a low metabolic risk profile ( free from diabetes , hypertension and obesity ) . in women , we found no stable risk associations for traditional sami diet score , though all hrs were 1 . our findings , in a largely non - sami population , are generally in line with the evidence to date for the individual components of the traditional sami diet score . although the score is high in some foods widely considered to be healthy , such as fatty fish ( 25 ) and berries ( 26 ) , it is also rich in foods associated with negative health outcomes such as red meat for colorectal and other cancers ( 19 ) , and lacking in foods associated with positive health outcomes such as vegetables for reduced cvd risk ( 20 ) . in a previous report from the same northern swedish population , no associations between reported fish consumption ( lean and fatty fish combined ) and risk of acute myocardial infarction were observed ( 49 ) . thus , the present - day diet in the population of vsterbotten may deviate from the traditional sami diet to such a degree that potential health effects can not be revealed . however , not only does the present - day diet in vsterbotten deviate considerably from our definition of traditional sami diet , but it is also possible that our traditional sami diet score does not closely enough reflect the traditional sami diet , which is , in itself difficult to define . knowledge of the diet prior to the 1700s is limited ( 7,9 ) , and the centuries since then have been marked by considerable change for the sami population ( 10 ) . traditional sami diet , for example by distinguishing between different kinds of red meat , such as wild game and reindeer meat and commercial beef , or fish bought and fish caught in the wild , or wild berries and greens and cultivated ones . thus , the vip ffq is inevitably an imprecise tool for the construction of a score representing a traditional sami dietary pattern . the traditional sami diet score was not intended to reflect the diet of the sami today . furthermore , traditional sami diet scores were high in reindeer - herding sami compared to the other groups in that dataset ( 10 ) , suggesting some degree of validity for the score , even in the present day . in diet score methodology , overall effects of adherence to a certain dietary pattern are studied , allowing for a single measure encompassing all potential interactions among the dietary components included , but also preventing the detection of effects related to the specific components or interactions . the intervention built into the vsterbotten intervention program , in which diabetes and hypertension diagnosed through the health survey are treated , may have diluted our results . the weaker risk associations in men with metabolic risk factors at baseline may reflect such an effect . this question , and the work - related physical activity question , in the vip ffq are therefore probably inadequate for the assessment of physical activity as a confounder or effect modifier of the relationship between traditional sami diet score and mortality . the main strengths of this study were the population - based cohort design , the long follow up ( up to 19 years ) , and the large sample size , as well as the rather unique aspect of examining the general population from a minority perspective . given the inherent limitations of the questionnaire used , and the difficulty defining a score reflecting traditional sami diet , this study should be considered exploratory , a first attempt to address this topic . further study of cohorts with more detailed information on dietary and lifestyle items relevant for traditional sami culture is warranted .

patients ' active participation in their own care is known to increase motivation and adherence to prescriptions , give better treatment results , create greater satisfaction with received care , and reduce stress and anxiety . patient participation is an important basis for nursing care and medical treatment and it is also a legal right in many western countries . studies have established that patients consider participation to be both obvious and important [ 3 , 4 ] , but there are also findings showing the opposite and patients may prefer a passive recipient role [ 6 , 7 ] . knowledge of what may influence patients ' participation is thus of great importance when it comes to meeting their expectations and demands . previous research focusing on patient participation from a patient perspective has been performed primarily in medicine and is carried out by physicians [ 8 , 9 ] . research on patient participation in nursing care has defined participation in performing clinical or daily living skills . patient participation has been explored in different situations , for example , discharge planning [ 1114 ] and bedside reporting in emergency care and has primarily focused on decision - making in treatment / care ( e.g. , [ 1720 ] ) . although nursing theories emphasise participation ( e.g. , ) and studies have explored patient participation in different contexts and situations , there have not been congruence regarding definition , elements , and processes [ 8 , 22 , 23 ] . the lack of clarity is amplified by the use of several terms : patient / client / consumer / user involvement , collaboration , partnership , and influence [ 8 , 17 ] . however , when the focus is on the patient perspective , the concept of patient participation is commonly used . . found that the patient needs to have the intellectual ability to understand and choose between alternatives and make decisions about their own nursing care and the nurse must provide adequate and correct information . tutton emphasized the significance of developing a relationship between nurse and patient and the importance of understanding the patient as well as gaining and retaining an emotional connection . according to sahlsten et al . , a nurse needs to use strategies including building close co - operation with the patient , getting to know the person , and reinforcing self - care capacity . factors restricting participation were identified by wellard et al . : limited communication between nurses and patients , task - oriented nursing labour , and environmental constraints limiting patients ' privacy . . found nonparticipation ; when patients lack an equal relationship , respect , and information . according to efraimsson et al . , nonparticipation , occurs when professionals are not attuned to the concerns of the patient and individual needs and when they literally silence or disregard the patient 's wishes . . found that a nurse can lack theoretical or practical knowledge required as well as an insight that patient participation requires deliberate and planned interaction between nurse and patient together with adjusted actions within every encounter . larsson et al . recently presented barriers for participation from a patient perspective : facing own inability , meeting lack of empathy , meeting a paternalistic attitude , and sensing structural barriers . while several studies have addressed patient participation , few accounts exist based on patients ' descriptions of decisive incidents that influenced their participation in nursing care . accordingly , there is a need to explore situations related to critical incidents that influence patient participation . the aim of this study was to identify incidents and nurses ' behaviours that influence patients ' participation in nursing care based on patients ' experiences from inpatient somatic care . this study is part of a larger project regarding patient participation in nursing care from the perspective of both patient and nurse . a qualitative approach , using the critical incident technique ( cit ) , was employed . the cit is a systematic , inductive , and flexible method where specific descriptions of human behaviour in defined situations are collected . the central concept in cit is a critical incident which is a maior event of great importance to the person involved . the incidents are mostly collected in semistructured face - to - face interviews , the most satisfactory data collection method in cit for insuring that all the necessary details are supplied . the informants are asked to provide descriptions of specific incidents , positive and/or negative , which they perceive as significant . here , these descriptions were collected within the framework of the interview method in order to generate an adequate depth of response . it is usually sufficient to collect a total of 100 incidents for a qualitative analysis . the participants ( n = 17 ) in this study were recruited from somatic inpatient care . the intention was to have a range of informants able to contribute their experience as patients . the informants were ambulatory patients from three internal medical wards with neither an explicit care philosophy emphasising patient participation , nor a focus on nurse - patient continuity . the wards were focused on ( i ) stroke , ( ii ) disorder of kidney and heart , and ( iii ) lung . all informants were able to communicate in swedish and had no physical or cognitive deficits hampering the ability to describe their experiences as patients . the interviewer assisted the patients to describe the specific incidents that have influenced their participation in nursing care . the interview guide consisted of the following questions : describe a positive significant incident which was successful for your participation in your own nursing care , and describe a negative significant incident where you felt nonparticipation . after the patient had identified an event , the following questions , earlier used by kemppainen , were asked : what were the circumstances leading to that event ? the same wording in the questions was kept throughout all interviews , as recommended by flanagan . the informants were recruited from an internal medical clinic in a central hospital in west sweden . all the nurses on the selected wards were sent written information regarding aim and procedure . the nurses were asked to approach patients the day before an interview was scheduled and ask whether they were interested in participating in the study or not . the interviews were held in the patient 's own room or adjacent to the wards in a place where there would be no interruption in order to provide a relaxed environment . each interview was conducted in an open , friendly atmosphere by the main nurse researcher and lasted between 30 and 60 minutes . each interview was audio - taped and transcribed verbatim by the main researcher ( inga e. larsson ) . the data material was read repeatedly to obtain a sense of the whole . in the data reduction process , an incident , either negative or positive , was identified as critical if it was related to the aim of the study and based on a detailed and discernible narrative of a course of events with a distinct start and end . in the data material , in line with the cit tradition , the classification started with identification and extraction of the incidents . these were analysed , without consideration whether positive or negative , to find similarities . in the second step of analysis , different kinds of nurse behaviours were next identified and classified , followed by patients ' responses of these behaviours . early in the analysis , the number of nurse behaviours increased rapidly and the last three interviews resulted in no new behaviours . to increase credibility , the classifications were discussed by the researchers ( inga e. larsson , monika j. m. sahlsten ) as no coassessor was involved in the coding . in addition , two researchers ( kerstin segesten , kaety a. e. plos ) not earlier involved in the study examined the classifications including direct quotations . this final classification system consisted of two main areas and 16 nurse behaviours allocated to six patient responses . the incidents arise in everyday situations and illuminate both positive and negative turning points ( table 1 ) . the most frequently described incidents concern situations during medical ward round where the nurse provides no support for patient input , and examples are also given of no preparation ahead of the round . other incidents concern situations during nursing ward round describing genuine interest and search for patients ' experience and views but examples are also given of distance with limited support for patient input . incidents also describe situations during information session where the nurse provides meaningful and sufficient information but there are also descriptions of missing , insufficient , or inadequate information . the incidents concerning nursing documentation include descriptions of no invitation to participate and examples are also given of no recording of the patients ' views . other incidents concern situations during drug administration where the nurse leaves it to the patient to decide about tablet dosage for pain treatment but there is also examples of when the nurse provides no tablet for sleeping problems as well as routinely interrupts pain treatment infusion with little or none consideration to the individual . the least described type of incidents is meal which include examples of opportunity to choose where and when as well as what to eat and how much . in the next step of analysis , positive incidents were identified as stimulating patient participation and the negative incidents as inhibiting . in table 2 , an overview of these two main areas along with patients ' responses to nurses ' behaviours are provided . the nurses ' behaviours are illuminated using direct quotations that illustrate the connection with the narratives . when nurses care about patients and show a genuine interest , they feel treated and accepted as a unique person . the informants emphasised the importance of not being seen solely as an illness or a bed number . nurses showed that they were accessible : the nurse was there when i needed it . the nurse confirmed the patient by showing that she cared and wanted to get to know me . the fact that the nurse listened and asked questions was considered crucial : she really listened to me and understood my situation . she asked questions to get an overall picture of my condition and find out what i like and want . it helps me to understand my thoughts and how i can process different things . when nurses provide information adapted to the patient 's needs , he / she is motivated to actively participate in own care . the nurse gave the necessary explanations : she made sure i got the information i wanted and needed . she explained what the illness meant and how it was all connected , for example , why i took this pill and was given that injection . i was given time to think and ask questions , so i know what it is all about . i was given brochures and books to read , which enabled me to form my own opinion and understand better how it is all connected . then it was easier for us to talk about my illness and what was going to happen next . it was considered important that the nurse acts as a mediator of contacts : she helped me so that i got to talk with other patients about their experiences and the treatment i was going to begin on . the nurse also took me on a guided tour to say hello on the ward where i was going to be treated and see how it all works . the informants also emphasised the importance of the nurse giving tips about self - care : i was given tips about what to do to make it easier , how to take care of the bandage , give the injections at home , and take care of myself when it comes to food and exercise . when the nurse starts with and utilizes patients ' own knowledge , they feel as an asset in their cooperation . the nurse discussed and made agreements : she always included me in discussions because she needs my knowledge , said i was an expert . the nurse also handed over responsibility : i have been allowed to decide on my pain treatment and i take the pills when i need them . that means i do not have to press the call button as soon as it hurts and then i can wait longer so that i do not get so drugged and constipated . when a nurse , who is expected to provide support , seems to view patients in an unreflected way , they feel alone , ignored , and let down . she must have thought that i could do that myself and was just trying to get out of it . you have to dare meet person to person . a nurse was non - supportive during the medical ward round : i tried to give my views during the round and did n't get any help from the nurse . she was silent and did n't dare back me up in front of the physician . they talked about me , but i was n't asked a single question ; i felt ignored and upset . it would have been better to have been backed up directly instead of her coming back afterwards and trying to put everything right . the way a nurse communicates can make patients feel depreciated . a nurse disparaged a patient with baby talk : the nurse talked to me like i was a child ; that belittles me as a person and gives an impression of insincerity . a nurse made ironic remarks about an experience : i was told to point at a ruler and got the answer : my dear , you ca n't be in that much pain . if you were , you 'd be both in a cold sweat and more affected . now , you just think about it one more time . when a nurse seems to want to exercise control and does not attach any importance to patients ' views , they feel ignored and unable to participate and exert an influence . when i said we should do like this instead , the nurse said : you do n't understand this , what are you making a fuss for . a nurse answered curtly : i am inquisitive and the nurse only answered very briefly . i was constantly being told : we 'll have to wait and see what the physician has to say . surely , it is possible to answer one of my questions reasonably . maybe she 's not allowed to tell me , but she could at least tell me that . a nurse neglected making notes in records : she did n't write what i has asked to be written in my record . when so many people are involved in my care , what is written down is important and it is often wrong ; that scares me . when i read the epicrisis of the nursing care plan , i saw that they had copied the old one . it would have been good if i had been allowed to take part in the planning and evaluated my care . this study , based on patients ' experiences from inpatient somatic care , provided a picture of incidents , nurses ' behaviours that stimulate or inhibit patients ' participation and patient reactions on nurses ' behaviours . a purposeful sample was used in order to obtain a varied picture of critical incidents of significance for patient participation . in this study , 17 inpatients provided a total of 105 critical incidents which , according to flanagan , may be sufficient for a meaningful analysis . the findings are based on these informants and their ability to describe experiences of patient participation in nursing care . although a majority of these informants were able to name some of the registered nurses on his / her ward , it is not certain that they in fact were able to distinguish nursing from experiences with other care providers . the sample included informants with different experiences , which increases the possibility of shedding light on the researched question from a variety of perspectives . various ages , diagnoses , wards , and cultural backgrounds contributed to a rich variation which , taken as a whole , can be regarded as a strength . actions were taken to enhance credibility in data collection . at the end of each interview , the main conclusions were verbally summarised by the interviewer and the informant supplemented , verified , and further developed the content . when 14 interviews had been conducted , earlier data were replicated and nothing new was added . the interviews were conducted and transcribed verbatim in their entirety by the same person , which enhanced the trustworthiness of the data material collected . credibility in the analysis was enhanced by continuously switching between the whole and the parts , and comparing and revising until a final classification emerged from the data material . rigor was ensured by systematically handling the data , repeatedly reading , identifying , and reflecting on the critical incidents . to increase credibility , two of the authors ( inga e. larsson and monika j. m. sahlsten ) discussed the classifications including direct quotations in order to reduce bias which is recommended by flanagan . finally , two researchers ( kerstin segesten and kaety a. e. plos ) not previously involved in the study reviewed and commented on the classifications , which included citations . this study is based exclusively on the patients ' experiences . to provide a more complete picture , a future study may include observations of interactions between nurses , physicians , and patients but also interviews afterward to get their perspectives on why they behaved the way they did . many factors influence each interaction , and asking why could provide more insight and knowledge . only inpatient somatic care has been highlighted and , obviously , other patients and settings need to be explored . medical ward rounds still seem to be an incident not conducted in a democratic fashion . states that the rounds serves as a central marketplace for information where the main topic for physicians and nurses is medical information . the patients are only asked in order to reach agreement on decision - making or checking outcomes of treatment . nursing documentation seems also to be an incident where patients have limited opportunities to exercise influence . the hierarchical nursing classification system carried out in detail may mainly serve organisational and administrative purposes and therefore disregard the patients . the goal has been to record work done by nurses and to provide evidence for performed interventions . accordingly , nursing documentation is regarded as a matter for nurses and the fact that patients also have views on its content seems to have been noticed earlier in only two studies of patient participation [ 4 , 28 ] . drug administration appears to be an incident where ward policies and protocols seem to be emphasised rather than an individual 's comfort needs . pain is an individual experience where patient participation is of uttermost importance for the recovering . the most basic nursing care situations such as participation in daily living skills are not described with the exception of meals , indicating that it may be obvious and/or of minor importance . the findings reveal that stimulating patients ' participation occurred when nurses treated the patient as a valuable coworker . this emphasises the importance of a person - centred care and of achieving a genuine connection and trusting companionship , in line with tutton and sahlsten et al . . each patient 's own capacity needs to be reinforced in order to optimise participation where patient and nurse share control and responsibility . to achieve this balance , a nurse ought to develop a personal , ordinary , and spontaneous approach in nursing practice . our findings highlight that if patients are to feel regarded as unique persons , it is crucial to break free from preconceptions and assumptions of what their needs are and enter into each patient 's world . patients need to feel that the nurse understands their situation and unique prerequisites , which is a starting point for being actively involved in one 's own nursing care . according to the informants , it is important to become motivated and engaged through information . it might be helpful to think of the patient as using and trying to implement evidence - based practice , as pointed to by edwards . patients need to find acceptable interpretations of what is happening to them , which is essential for participation . patients collect information and take action according to their own assessment of credibility and trustworthiness of information given . if different nurses appear to provide contradictory information or opinions , the patient could be confused as it means that the starting point for coping and action strategies keeps changing . consequently , information needs to be adequate , individually adjusted , coordinated , and univocal . to meet the patients ' needs , nurses have to use pedagogical strategies that promote learning such as focusing on the patient 's process of reflection . this implies in - depth questions to induce patients to be self - reflective in order to utilise their own full potential in line with sahlsten et al . . the findings suggest that a patient , who is acknowledged as competent , presupposes stimulation and encouragement as a successful doer and owner of knowledge , in line with hughes and tutton . patients ' desire to do as much as they can by themselves may be seen as a basic human characteristic . consequently , it is only the patient who can decide what is in his / her own best interest and nurses are then engaged in supporting . if possibilities to choose and make decisions are maximised , this may result in increased motivation to take responsibility , and exert influence and control [ 36 , 39 ] . according to the informants , this leads to a sense of independence , which increases well - being , but a nurse then needs to relinquish some control , rather than exerting it . the findings reveal that inhibiting patients ' participation occurred when nurses treated patients so they felt neglected and as a helpless object of a nurse 's actions . this seems to indicate that a person - centred approach is devalued in favour of a task - centred one . a nurse might have a limited understanding of professional nursing care and focus on tasks , which could result in the patient easily becoming a passive object . when patients perceive themselves as being abandoned without backup , this indicates that nurses may use a protective mechanism to screen off emotional or advocacy aspects of their work . this may be due to working under time pressure or an idea that connecting with the patient is risky in a professional relationship . nurses may need both practical and personal support to reduce a use of blocking behaviours to be able to work in a more responsive and effective way . in order to continuously develop self - awareness and critical monitoring skills , this may increase nurses ' ability to reflect and develop their behaviour in patient encounters . to provide sufficient support during medical ward rounds was surprisingly an expectation on nurses by all informants . in order to optimise patient participation , nurses need courage to back up patients to reach self - advocacy and also to be sufficiently confident to question procedures , which are to the patient 's disadvantage . however , nurses can see themselves as , and acts as , an intermediary with the physician . patients rarely get explanations or are encouraged to ask questions , an outdated routine that does not satisfy the demands of patients today . if medical ward rounds should continue in its present shape , patients need information regarding its actual aim , which seems to be to , as physician , get a face on the patient for whom the care planning is done . when patients feel belittled verbally , a nurse may exercise the power of language or behave as a parent figure , also pointed to by hewison . the nurse disparages the patient in order to be in charge and sets the parameters for what is acceptable . mccabe claims that professional nurses need to be aware of the impact the way they choose to communicate has on their patients . communication is a powerful tool that mediates ideas , attitudes , and information , but it can also reinforce nurses ' authority and hinder or exclude patients so they become increasingly dependent according to kettunen et al . or result in reluctance . being ignored without influence mirrors nonrespect and no recognition of patients ' requests and their right to participate . by recording the patients ' views , things they regard as important will be revealed and made visible , also pointed to by krkkinen and eriksson . this presupposes that the recorder knows the patient , which perhaps was not the case here . when a nurse neglects the importance of written documentation , the informants here felt that they were exposed to risks . records can be used as working documents for both parties which may improve the content . we recommend that a nurse provide a notebook and encourage patients to keep their own notes . this could support them to remember , prepare for meetings for example , rounds , and ask questions . it can also help patients to participate and take a higher degree of control in their own care . when nurses have a bossy or patronising attitude , this reflects a belief that it is the nurse who knows best what is in the patient 's interest . the level of control that nurses themselves have over their practice has been shown to affect the level of active patient participation . if nurses perceive themselves as diminished and not seen , they may repress patients . empowering the patient this study , based on patients ' experiences from inpatient somatic care provided a picture of incidents , nurses ' behaviours that stimulate or inhibit patients ' participation and patient reactions on nurses ' behaviours . in order to promote patient participation , nurses need to be aware of the situations where they could overstep the mark and which of their own behaviours lead to promotion or hindrance . our findings suggest that there is scope for developing nurses ' behaviours in order to activate patients in their own nursing care . the findings may increase understanding of patient participation in nursing practise , education , policymaking , and evaluation . further verification of the findings is recommended , either by means of replication or other studies in different settings .

patient participation is an important basis for nursing care and medical treatment and is a legal right in many western countries . studies have established that patients consider participation to be both obvious and important , but there are also findings showing the opposite and patients often prefer a passive recipient role . knowledge of what may influence patients ' participation is thus of great importance . the aim was to identify incidents and nurses ' behaviours that influence patients ' participation in nursing care based on patients ' experiences from inpatient somatic care . the critical incident technique ( cit ) was employed . interviews were performed with patients ( n = 17 ) , recruited from somatic inpatient care at an internal medical clinic in west sweden . this study provided a picture of incidents , nurses ' behaviours that stimulate or inhibit patients ' participation , and patient reactions on nurses ' behaviours . incidents took place during medical ward round , nursing ward round , information session , nursing documentation , drug administration , and meal .

1. Introduction 2. Method 3. Results 4. Discussion 5. Conclusions

patients ' active participation in their own care is known to increase motivation and adherence to prescriptions , give better treatment results , create greater satisfaction with received care , and reduce stress and anxiety . patient participation is an important basis for nursing care and medical treatment and it is also a legal right in many western countries . studies have established that patients consider participation to be both obvious and important [ 3 , 4 ] , but there are also findings showing the opposite and patients may prefer a passive recipient role [ 6 , 7 ] . knowledge of what may influence patients ' participation is thus of great importance when it comes to meeting their expectations and demands . previous research focusing on patient participation from a patient perspective has been performed primarily in medicine and is carried out by physicians [ 8 , 9 ] . research on patient participation in nursing care has defined participation in performing clinical or daily living skills . patient participation has been explored in different situations , for example , discharge planning [ 1114 ] and bedside reporting in emergency care and has primarily focused on decision - making in treatment / care ( e.g. , [ 1720 ] ) . , ) and studies have explored patient participation in different contexts and situations , there have not been congruence regarding definition , elements , and processes [ 8 , 22 , 23 ] . the lack of clarity is amplified by the use of several terms : patient / client / consumer / user involvement , collaboration , partnership , and influence [ 8 , 17 ] . however , when the focus is on the patient perspective , the concept of patient participation is commonly used . found that the patient needs to have the intellectual ability to understand and choose between alternatives and make decisions about their own nursing care and the nurse must provide adequate and correct information . tutton emphasized the significance of developing a relationship between nurse and patient and the importance of understanding the patient as well as gaining and retaining an emotional connection . , a nurse needs to use strategies including building close co - operation with the patient , getting to know the person , and reinforcing self - care capacity . : limited communication between nurses and patients , task - oriented nursing labour , and environmental constraints limiting patients ' privacy . found nonparticipation ; when patients lack an equal relationship , respect , and information . , nonparticipation , occurs when professionals are not attuned to the concerns of the patient and individual needs and when they literally silence or disregard the patient 's wishes . found that a nurse can lack theoretical or practical knowledge required as well as an insight that patient participation requires deliberate and planned interaction between nurse and patient together with adjusted actions within every encounter . recently presented barriers for participation from a patient perspective : facing own inability , meeting lack of empathy , meeting a paternalistic attitude , and sensing structural barriers . while several studies have addressed patient participation , few accounts exist based on patients ' descriptions of decisive incidents that influenced their participation in nursing care . accordingly , there is a need to explore situations related to critical incidents that influence patient participation . the aim of this study was to identify incidents and nurses ' behaviours that influence patients ' participation in nursing care based on patients ' experiences from inpatient somatic care . this study is part of a larger project regarding patient participation in nursing care from the perspective of both patient and nurse . a qualitative approach , using the critical incident technique ( cit ) , was employed . the cit is a systematic , inductive , and flexible method where specific descriptions of human behaviour in defined situations are collected . the central concept in cit is a critical incident which is a maior event of great importance to the person involved . the incidents are mostly collected in semistructured face - to - face interviews , the most satisfactory data collection method in cit for insuring that all the necessary details are supplied . the informants are asked to provide descriptions of specific incidents , positive and/or negative , which they perceive as significant . the participants ( n = 17 ) in this study were recruited from somatic inpatient care . the intention was to have a range of informants able to contribute their experience as patients . the informants were ambulatory patients from three internal medical wards with neither an explicit care philosophy emphasising patient participation , nor a focus on nurse - patient continuity . the wards were focused on ( i ) stroke , ( ii ) disorder of kidney and heart , and ( iii ) lung . all informants were able to communicate in swedish and had no physical or cognitive deficits hampering the ability to describe their experiences as patients . the interviewer assisted the patients to describe the specific incidents that have influenced their participation in nursing care . the interview guide consisted of the following questions : describe a positive significant incident which was successful for your participation in your own nursing care , and describe a negative significant incident where you felt nonparticipation . the same wording in the questions was kept throughout all interviews , as recommended by flanagan . the informants were recruited from an internal medical clinic in a central hospital in west sweden . all the nurses on the selected wards were sent written information regarding aim and procedure . the nurses were asked to approach patients the day before an interview was scheduled and ask whether they were interested in participating in the study or not . the interviews were held in the patient 's own room or adjacent to the wards in a place where there would be no interruption in order to provide a relaxed environment . each interview was audio - taped and transcribed verbatim by the main researcher ( inga e. larsson ) . in the data reduction process , an incident , either negative or positive , was identified as critical if it was related to the aim of the study and based on a detailed and discernible narrative of a course of events with a distinct start and end . in the data material , in line with the cit tradition , the classification started with identification and extraction of the incidents . these were analysed , without consideration whether positive or negative , to find similarities . in the second step of analysis , different kinds of nurse behaviours were next identified and classified , followed by patients ' responses of these behaviours . to increase credibility , the classifications were discussed by the researchers ( inga e. larsson , monika j. m. sahlsten ) as no coassessor was involved in the coding . in addition , two researchers ( kerstin segesten , kaety a. e. plos ) not earlier involved in the study examined the classifications including direct quotations . the most frequently described incidents concern situations during medical ward round where the nurse provides no support for patient input , and examples are also given of no preparation ahead of the round . other incidents concern situations during nursing ward round describing genuine interest and search for patients ' experience and views but examples are also given of distance with limited support for patient input . incidents also describe situations during information session where the nurse provides meaningful and sufficient information but there are also descriptions of missing , insufficient , or inadequate information . the incidents concerning nursing documentation include descriptions of no invitation to participate and examples are also given of no recording of the patients ' views . other incidents concern situations during drug administration where the nurse leaves it to the patient to decide about tablet dosage for pain treatment but there is also examples of when the nurse provides no tablet for sleeping problems as well as routinely interrupts pain treatment infusion with little or none consideration to the individual . the least described type of incidents is meal which include examples of opportunity to choose where and when as well as what to eat and how much . in the next step of analysis , positive incidents were identified as stimulating patient participation and the negative incidents as inhibiting . in table 2 , an overview of these two main areas along with patients ' responses to nurses ' behaviours are provided . the nurses ' behaviours are illuminated using direct quotations that illustrate the connection with the narratives . when nurses care about patients and show a genuine interest , they feel treated and accepted as a unique person . nurses showed that they were accessible : the nurse was there when i needed it . the nurse confirmed the patient by showing that she cared and wanted to get to know me . the fact that the nurse listened and asked questions was considered crucial : she really listened to me and understood my situation . she asked questions to get an overall picture of my condition and find out what i like and want . it helps me to understand my thoughts and how i can process different things . the nurse gave the necessary explanations : she made sure i got the information i wanted and needed . then it was easier for us to talk about my illness and what was going to happen next . the nurse also took me on a guided tour to say hello on the ward where i was going to be treated and see how it all works . the informants also emphasised the importance of the nurse giving tips about self - care : i was given tips about what to do to make it easier , how to take care of the bandage , give the injections at home , and take care of myself when it comes to food and exercise . when the nurse starts with and utilizes patients ' own knowledge , they feel as an asset in their cooperation . the nurse also handed over responsibility : i have been allowed to decide on my pain treatment and i take the pills when i need them . that means i do not have to press the call button as soon as it hurts and then i can wait longer so that i do not get so drugged and constipated . when a nurse , who is expected to provide support , seems to view patients in an unreflected way , they feel alone , ignored , and let down . she must have thought that i could do that myself and was just trying to get out of it . a nurse was non - supportive during the medical ward round : i tried to give my views during the round and did n't get any help from the nurse . she was silent and did n't dare back me up in front of the physician . they talked about me , but i was n't asked a single question ; i felt ignored and upset . it would have been better to have been backed up directly instead of her coming back afterwards and trying to put everything right . a nurse disparaged a patient with baby talk : the nurse talked to me like i was a child ; that belittles me as a person and gives an impression of insincerity . a nurse made ironic remarks about an experience : i was told to point at a ruler and got the answer : my dear , you ca n't be in that much pain . if you were , you 'd be both in a cold sweat and more affected . now , you just think about it one more time . when a nurse seems to want to exercise control and does not attach any importance to patients ' views , they feel ignored and unable to participate and exert an influence . i was constantly being told : we 'll have to wait and see what the physician has to say . maybe she 's not allowed to tell me , but she could at least tell me that . a nurse neglected making notes in records : she did n't write what i has asked to be written in my record . when i read the epicrisis of the nursing care plan , i saw that they had copied the old one . this study , based on patients ' experiences from inpatient somatic care , provided a picture of incidents , nurses ' behaviours that stimulate or inhibit patients ' participation and patient reactions on nurses ' behaviours . a purposeful sample was used in order to obtain a varied picture of critical incidents of significance for patient participation . in this study , 17 inpatients provided a total of 105 critical incidents which , according to flanagan , may be sufficient for a meaningful analysis . the findings are based on these informants and their ability to describe experiences of patient participation in nursing care . although a majority of these informants were able to name some of the registered nurses on his / her ward , it is not certain that they in fact were able to distinguish nursing from experiences with other care providers . the sample included informants with different experiences , which increases the possibility of shedding light on the researched question from a variety of perspectives . various ages , diagnoses , wards , and cultural backgrounds contributed to a rich variation which , taken as a whole , can be regarded as a strength . at the end of each interview , the main conclusions were verbally summarised by the interviewer and the informant supplemented , verified , and further developed the content . the interviews were conducted and transcribed verbatim in their entirety by the same person , which enhanced the trustworthiness of the data material collected . credibility in the analysis was enhanced by continuously switching between the whole and the parts , and comparing and revising until a final classification emerged from the data material . rigor was ensured by systematically handling the data , repeatedly reading , identifying , and reflecting on the critical incidents . to increase credibility , two of the authors ( inga e. larsson and monika j. m. sahlsten ) discussed the classifications including direct quotations in order to reduce bias which is recommended by flanagan . this study is based exclusively on the patients ' experiences . to provide a more complete picture , a future study may include observations of interactions between nurses , physicians , and patients but also interviews afterward to get their perspectives on why they behaved the way they did . many factors influence each interaction , and asking why could provide more insight and knowledge . only inpatient somatic care has been highlighted and , obviously , other patients and settings need to be explored . medical ward rounds still seem to be an incident not conducted in a democratic fashion . states that the rounds serves as a central marketplace for information where the main topic for physicians and nurses is medical information . the patients are only asked in order to reach agreement on decision - making or checking outcomes of treatment . nursing documentation seems also to be an incident where patients have limited opportunities to exercise influence . the hierarchical nursing classification system carried out in detail may mainly serve organisational and administrative purposes and therefore disregard the patients . the goal has been to record work done by nurses and to provide evidence for performed interventions . accordingly , nursing documentation is regarded as a matter for nurses and the fact that patients also have views on its content seems to have been noticed earlier in only two studies of patient participation [ 4 , 28 ] . drug administration appears to be an incident where ward policies and protocols seem to be emphasised rather than an individual 's comfort needs . pain is an individual experience where patient participation is of uttermost importance for the recovering . the most basic nursing care situations such as participation in daily living skills are not described with the exception of meals , indicating that it may be obvious and/or of minor importance . the findings reveal that stimulating patients ' participation occurred when nurses treated the patient as a valuable coworker . this emphasises the importance of a person - centred care and of achieving a genuine connection and trusting companionship , in line with tutton and sahlsten et al . . each patient 's own capacity needs to be reinforced in order to optimise participation where patient and nurse share control and responsibility . to achieve this balance , a nurse ought to develop a personal , ordinary , and spontaneous approach in nursing practice . our findings highlight that if patients are to feel regarded as unique persons , it is crucial to break free from preconceptions and assumptions of what their needs are and enter into each patient 's world . patients need to feel that the nurse understands their situation and unique prerequisites , which is a starting point for being actively involved in one 's own nursing care . according to the informants , it is important to become motivated and engaged through information . it might be helpful to think of the patient as using and trying to implement evidence - based practice , as pointed to by edwards . patients need to find acceptable interpretations of what is happening to them , which is essential for participation . patients collect information and take action according to their own assessment of credibility and trustworthiness of information given . if different nurses appear to provide contradictory information or opinions , the patient could be confused as it means that the starting point for coping and action strategies keeps changing . consequently , information needs to be adequate , individually adjusted , coordinated , and univocal . to meet the patients ' needs , nurses have to use pedagogical strategies that promote learning such as focusing on the patient 's process of reflection . this implies in - depth questions to induce patients to be self - reflective in order to utilise their own full potential in line with sahlsten et al . . patients ' desire to do as much as they can by themselves may be seen as a basic human characteristic . consequently , it is only the patient who can decide what is in his / her own best interest and nurses are then engaged in supporting . if possibilities to choose and make decisions are maximised , this may result in increased motivation to take responsibility , and exert influence and control [ 36 , 39 ] . according to the informants , this leads to a sense of independence , which increases well - being , but a nurse then needs to relinquish some control , rather than exerting it . the findings reveal that inhibiting patients ' participation occurred when nurses treated patients so they felt neglected and as a helpless object of a nurse 's actions . a nurse might have a limited understanding of professional nursing care and focus on tasks , which could result in the patient easily becoming a passive object . nurses may need both practical and personal support to reduce a use of blocking behaviours to be able to work in a more responsive and effective way . in order to continuously develop self - awareness and critical monitoring skills , this may increase nurses ' ability to reflect and develop their behaviour in patient encounters . to provide sufficient support during medical ward rounds was surprisingly an expectation on nurses by all informants . in order to optimise patient participation , nurses need courage to back up patients to reach self - advocacy and also to be sufficiently confident to question procedures , which are to the patient 's disadvantage . however , nurses can see themselves as , and acts as , an intermediary with the physician . if medical ward rounds should continue in its present shape , patients need information regarding its actual aim , which seems to be to , as physician , get a face on the patient for whom the care planning is done . when patients feel belittled verbally , a nurse may exercise the power of language or behave as a parent figure , also pointed to by hewison . the nurse disparages the patient in order to be in charge and sets the parameters for what is acceptable . mccabe claims that professional nurses need to be aware of the impact the way they choose to communicate has on their patients . communication is a powerful tool that mediates ideas , attitudes , and information , but it can also reinforce nurses ' authority and hinder or exclude patients so they become increasingly dependent according to kettunen et al . being ignored without influence mirrors nonrespect and no recognition of patients ' requests and their right to participate . by recording the patients ' views , things they regard as important will be revealed and made visible , also pointed to by krkkinen and eriksson . when a nurse neglects the importance of written documentation , the informants here felt that they were exposed to risks . records can be used as working documents for both parties which may improve the content . this could support them to remember , prepare for meetings for example , rounds , and ask questions . the level of control that nurses themselves have over their practice has been shown to affect the level of active patient participation . if nurses perceive themselves as diminished and not seen , they may repress patients . empowering the patient this study , based on patients ' experiences from inpatient somatic care provided a picture of incidents , nurses ' behaviours that stimulate or inhibit patients ' participation and patient reactions on nurses ' behaviours . in order to promote patient participation , nurses need to be aware of the situations where they could overstep the mark and which of their own behaviours lead to promotion or hindrance . our findings suggest that there is scope for developing nurses ' behaviours in order to activate patients in their own nursing care . the findings may increase understanding of patient participation in nursing practise , education , policymaking , and evaluation . further verification of the findings is recommended , either by means of replication or other studies in different settings .

the development of cancer is a complex multistep process that requires the accumulation of mutations resulting in a cell acquiring the essential hallmarks of cancer : evasion of apoptosis , self - sufficiency in growth signals , insensitivity to antigrowth signals , invasive and metastatic abilities , limitless replicative potential , and sustained angiogenesis . given that normal adult stem cells already exhibit limitless replicative potential , it is hypothesized that transformed stems cells may be the cells of origin for many cancers [ 2 , 3 ] . in addition to replicative potential , long - lived stem cells have the opportunity to accumulate oncogenic mutations over years or decades from common mutagenic sources like inflammation , radiation , chemicals , or infection , unlike shorter - lived transit amplifying ( ta ) cells that rapidly proliferate and differentiate [ 4 , 5 ] . like healthy adult stem cells , transformed stem cells these ta cells would be capable of driving tumor formation and generating the heterogeneous combination of populations commonly seen in cancer [ 6 , 7 ] . transformed stem cells have been termed cancer stem cells ( cscs ) , also known as cancer initiating cells , and are defined as the fraction of cells within a tumor that are long lived , possess the potential to proliferate indefinitely , and can generate all heterogeneous lineages of the original tumor in xenograft models [ 6 , 8 ] . cscs are expected to utilize characteristics commonly found in stem cell populations such as differential metabolic activity , specific signaling pathway activity , and regulation of cell cycling characteristics , albeit with aberrant regulation [ 7 , 9 ] ( table 1 ) . importantly , cscs that survive treatment could account for tumor recurrence as a result of reactivation of proliferation in surviving cscs . traditional chemotherapy regimens target proliferating cells , potentially missing slower dividing cscs that must be eradicated to provide long - term disease - free survival . a better understanding of cscs is essential in understanding the biological and clinical consequences of existing regimens and designing new therapies to improve patient outcome . current methods for isolation and study of cscs rely on cell surface markers found to be enriched in populations with stem cell - like properties . this technique was first used by bonnet and dick in 1997 when they demonstrated that only the cd34cd38 subset of cells were capable of initiating human acute myeloid leukemia ( aml ) in immune - compromised mouse models . since the work of these two pioneers , csc populations have been identified in multiple epithelial cancers including the breast , prostate , pancreas , colon [ 1517 ] , ovaries , and brain . one issue centers on uncertainty of the functional implications of csc markers and is best exemplified by the use of cd133 in the identification of colon cscs . shortly after ricci - vitiani et al . ( 2007 ) demonstrated the use of the cd133 to identify cscs in colon tumor , shmelkov et al . demonstrated that cd133 expression was not restricted to colon cscs , but that cd133 is expressed on differentiated colonic epithelium in both mice and humans [ 16 , 20 ] . reasons behind this contradiction of data still remain unclear , but kemper et al . methodically evaluated the cd133 antibodies used by both groups and reached the conclusion that the ac133 epitope used by ricci - vitiani et al . recognized a differentially expressed form of cd133 that is not recognized by the antibody used by shmelkov et al . . it appears that cd133 is expressed in all colon epithelium , while the ac133 epitope is specific for the csc phenotype . furthermore , kemper et al . were unable to determine the functional significance of the differentially expressed isoforms of cd133 , highlighting another drawback to the use of markers to identify cscs . very little is known about the function of many of the proposed csc markers , and even less is known about the functional implications they may have for the csc phenotype . at best , markers without functional implications must be viewed as only tools for stem cell enrichment , suggesting the need for a more functionally significant means of csc identification . given the similarities between normal adult stem cells and cscs , aberrant regulation of self - renewal and quiescence is likely central to csc pathology [ 9 , 10 ] . targeting pathways that mediate stem cell quiescence is therefore an intriguing alternate method for csc identification and use in future therapy . the primary objectives of this paper are to place quiescent label - retaining studies in the context of what is currently known about adult stem cells and then review the existing evidence for quiescence in cancer stem cells . we will examine current evidence for the role of quiescence in csc resistance to conventional cancer therapy and recurrence . finally , we will explore current knowledge of quiescence regulation and how these studies might be considered when developing csc future experiments to develop targeted therapies against cscs . adult stem cells are critical for continued normal tissue homeostasis and response to wounding for many of the epithelial tissues of the body . adult stem cells are characterized by their ability to self - renew indefinitely and produce progeny capable of differentiating and repopulating tissue specific lineages . populations of adult stem cells have been identified in tissues throughout the body , including the skin [ 2325 ] , mammary glands [ 26 , 27 ] , intestine [ 28 , 29 ] , prostate , brain , and the hematopoietic system [ 32 , 33 ] . in tissues where cells are frequently lost to the environment , like those of the intestine and skin , new cells are continuously required to replenish those that are lost . to facilitate this constant need for new cells , some epithelial tissues are arranged hierarchically with slowly proliferating stem cells that asymmetrically divide to give rise to a new stem cell and a rapidly dividing transit amplifying ( ta ) cell . transit amplifying cells proliferate quickly for a limited number of divisions , allowing for the high degree of cell turnover necessary to sustain adult tissues . infrequent division or a quiescent nature is not definitive for adult stem cell but is suggested to be important for maintenance of many adult stem cell pools . evidence suggests that quiescence may play an important role in protecting stem cells from exhausting their proliferative capacity , inhibiting differentiation , and limiting accumulation of mutations during frequent rounds of dna synthesis [ 3537 ] . initial efforts to identify and study adult stem cells took advantage of the slow - cycling nature of stem cell populations in studies employing pulse / chase methodology [ 23 , 28 ] . in these studies , tritiated thymidine ( h - tdr ) or 5-bromo-2-deoxy - uridine ( brdu ) was repeatedly administered to mice or cultured cells that were then followed by an extended period of chase time . during this chase period , rapidly proliferating ta cells divide the label between daughter cells , consequently diluting the label ( figure 1 ) . in contrast , slow - cycling stem cells undergo few divisions and retain detectable quantities of label for much longer periods of time . demonstrated that label retaining cells ( lrcs ) were exclusively present in the bulge area of the mouse hair follicle . these cells were found to be relatively stem - like : primitive in cytoplasmic contents , structurally similar to other putative stem cell populations , and could be stimulated to proliferate . we utilized human skin xenografted onto immunodeficient mice to show that lrcs were present in an analogous bulge region of human skin delineated by keratin 15 ( k15 ) expression . cells present in the bulge region have been experimentally shown to be quiescent for up to 1 year , and based on the hair growth cycle of scalp skin can likely remain quiescent for up to 5 years . using the k15 promoter to drive expression of egfp or lacz , k15 positive cells were found to differentiate into all major epithelial lineages of the mouse skin . we demonstrated that k15 + bulge cells from human skin can differentiate into epidermal , sebaceous , and hair follicle lineages in vitro . array analysis of the lrc bulge population showed increased activation of smad and inhibitors of the wnt pathway , suggesting the ability for lrcs to organize their niche and communicate with neighboring mesenchymal and epithelial cells , an important characteristic for stem cell function . the work in our lab and others supports a model in which the bulge region of hair follicles represents the stem cell niche in skin . at the onset of the growth phase ( anagen ) hair follicle stem cells are activated and produce matrix ta cells that proliferate and differentiate into the seven different lineages found within the hair follicle . as matrix ta cells exhaust their proliferative potential they enter a state of destruction ( catagen ) leading to the loss of the majority of the hair follicle excluding the bulge . catagen is followed by a period of rest ( telogen ) in which the bulge stem cells remain quiescent until activation into a new anagen stage . although likely important for the maintenance of the stem cell pool , quiescence may not be a requirement for adult stem cells . using a lacz construct under a conditional promoter for the stem cell - associated protein leucine - rich g protein - coupled receptor 5 ( lgr-5 ) , jaks et al . demonstrated a distinct nonlabel retaining subpopulation of bulge cells that overlap with the cd34/k15 at telogen but not anagen . lineage tracing techniques confirmed that lgr-5 cells actively cycled during normal homeostasis and had a multipotent phenotype . the authors of this paper suggest that the lgr-5 population of cells represents a cycling population of stem cells under normal conditions , whereas the label retaining cd34/k15 stem cells may represent a reserve population that is activated after tissue damage . as yet , a conclusive relationship between these two populations can not be firmly established . similar label - retaining methods have been used to study slow - cycling cells in other tissues , such as the small intestine and colon . work conducted by potten and colleagues identified slow - cycling lrcs at the + 4 position at the base of the colon crypt . these crypt base cells were found to be maintained in a steady state of between four and six cells that go through division approximately once a week . upon irradiation , these cells demonstrated increased antiapoptotic bcl-2 expression , decreased p53 expression and were highly activated and involved in clonogenic regeneration of the crypt . detailed biochemical analysis of this population has been limited by the absence of reliable markers and methods capable of sufficiently isolating these cells . two studies involving the putative stem cell - associated rna binding protein musashi-1 ( msi-1 ) have both demonstrated colocalization of this protein with colon lrcs , but fell short of testing for clonogenicity of this population [ 44 , 45 ] . similar to stem cell populations in the skin , 1-integrin was found to be highly expressed in the lower half of the colonic crypt . when sorted via flow cytometry , 1-integrin showed enrichment for clonogenic cells ; however , an exact colocalization pattern with lrcs was not evaluated , and therefore , the connection remains only speculative . from the evidence collected in these studies and others , a model has been suggested in which slow - cycling stem cells , found at the base of the crypt , undergo periodic division to give rise to ta cells . transit amplifying cells low in the crypt undergo rapid division and slowly progress up the crypt , losing replicitative potential and differentiating as they increase in crypt height . these cells are ultimately lost to the environment [ 6 , 43 ] . as within the hair follicle , there is convincing evidence for an lgr-5 nonlabel retaining population of colon stem cells additionally found at the base of the crypt . while the lrcs reside at the + 4 population , lgr-5 cells are observed as slender wedge - shaped cells at the + 2 position . again , the exact relationship between the lrcs and the lgr-5 cells is yet to be fully explored , and more data into the lineage potential of both of these cell populations is needed to form a cohesive model . since the early identification of colon and hair follicle slow - cycling stem cell populations , label - retaining techniques have been used to identify and validate putative stem cell populations in multiple epithelial tissues . in the mammary gland , three separate label - retaining populations have been identified and proposed as possible stem cells . in a study conducted by welm et al . , lrcs were found to comprise a subpopulation of stem cell antigen-1 positive ( sca-1 ) cells . these sca-1 cells were found to be enriched for the ability to form outgrowths , leading the authors to speculate that the lrcs might represent the stem cell population contained within the sca-1 cells . in contrast to this study , shackleton et al . identified a long - term label - retaining population enriched by the marker combination lincd29cd24 that was able to reconstitute a functional mammary gland from a single cell . the lincd29cd24 did not enrich for the sca-1 population , prompting other groups to suggest a stem cell hierarchy in which multiple layers of stem cells exist within the mammary gland . using a slightly different methodology , pece used the lipophilic fluorescent dye pkh26 to identify a population of mammary label retaining cells . the use of the pkh26 allows for live sorting of lrcs , which is not possible using the nucleotide analogue brdu and ht - tdr that both require permeabilization of the cell membrane for antibody labeling . live sorting of pkh26 lrcs demonstrated increased in vitro sphere formation efficiency and regeneration of cleared fat pads over non - lrcs . pece was also able to conduct transcriptional analysis of the lrc population , from which he created a human normal mammary gland stem cell signature ( hnmsc ) consisting of the markers cd49f / dner / dll1 . unfortunately , the exact relationship between the different populations identified by these three groups is not yet clear . in the brain , high doses of h - tdr kill all but one percent of proliferating subependymal . high dose therapeutics did not affect the capacity of quiescent cells to generate spheres in vitro or repopulate the proliferating population in vivo . the ability to survive and re - enter the cell cycle suggests a stem cell phenotype for these quiescent cells . prostate slow - cycling lrcs located in the proximal ducts demonstrated high proliferative potential and the ability to reconstitute the prostate glandular structure in vitro . this ability singles them out as stem cells over more rapidly cycling ta cells located at the distal region of the ducts . finally in the pancreas , characterization of lrcs around the acini and ducts suggested a stem cell population by demonstrating increased expression of the putative stem cell marker c - met and activation in response to damage to form duct - like structures . combined , these data indicate an important role for quiescent lrcs in maintenance and longevity of multiple adult epithelial tissues . if cscs do originate from normal adult stem cells , then it is foreseeable that key stem cell regulatory traits are retained through the oncogenic transition ; quiescence is potentially one of these traits . little research has been done to address how quiescence might play a role in csc biology , but there are some indications that quiescent stem - like populations might contribute to at least some tumors . we previously identified a subpopulation of cells in human sebaceous tumors that expressed the skin stem cell marker keratin 15 ( figure 2 ) . these cells appeared to have variable expression of the proliferation marker ki-67 , suggesting a low but higher proliferative rate than normal stem cells . in primary ovarian tumors , gao et al . demonstrated that cd24 cells expressing stem cell - associated genes like nestin , oct4 , and both notch1 and notch4 were more slowly proliferating than the bulk tumor cells suggesting a quiescent phenotype . low numbers of slowly proliferating cd24 cells were shown to produce tumors in a xenograft model where bulk cells were found to be nontumorigenic . pece also observed a link between cscs and quiescence in breast tumors . using the hnmsc signature generated with normal mammary lrcs , pece turned his attention to the analysis of primary breast tumors , finding that the hnmsc signature was more commonly found in grade 3 tumors over that of grade 1 . when grade 1 and grade 3 mammospheres were analyzed for pkh label retaining cells , both populations were found to retain label , with grade 3 tumors demonstrating a higher percentage . when evaluated for tumor genicity , breast tumor cells positive for the hnmsc signature were more efficient at forming in vitro spheres and in vivo xenograft tumors that those cells lacking the hnmsc signature . cultured cancer cell lines are often used to study signaling pathways , invasion , migration , and apoptosis , but are rarely thought of as candidates for csc studies . many of the most widely used cell lines have been in passage for years , are perceived homogeneous , lack interactions with the appropriate stromal microenvironment , and change characteristics based on alterations in culture conditions . therefore , cultured cell line studies assessing csc characteristics must be evaluated critically , with data interpreted within the context of the experimental parameters , and results confirmed under biologically relevant conditions . mcf7 , sum149 , sum159 , sum1315 , and mda.mb.231 suggests that these lines may not be as homogenous and void of stem like cells as once thought . cd44/cd24/esa cells within these lines were found to contain the ability to self - renew , reconstitute the parental line , and to be up to 90% label retaining . if lrcs are found to retain the csc phenotype in cultured cell lines , these cell lines may provide an important resource for future delineation of quiescent pathway regulators . additional transitive evidence linking quiescence to cscs can be found in the work conducted by roesch et al . in melanoma . this group found that primary melanoma cell lines contained a pkh26 label retaining population that was almost specifically identified by the h3k4 demethylase jarid1b . this population of cells was found to incorporate brdu more slowly but retain it for a longer period of time , lack ki67 staining , and have a doubling time of up to 4 weeks in vitro . when egfp was placed under the control of the jarid1b promoter , gfp cells demonstrated increased sphere forming ability in vitro . interestingly , gfp cells were able to retain brdu in vivo , but did not show increased tumor initiating ability over gfp cell during the time period analyzed . small hairpin rna ( shrna ) knockdown of jarid1b resulted in the in vitro exhaustion of proliferating cells , demonstrating the need for jarid1b cells in maintenance of proliferative capacity but not initiation of tumors . when assessed more fully , both in vitro and in vivo gfp cells gave rise to heterogeneous progeny , including jarid1b gfp+ cells . the most direct evidence to date for quiescence playing a role in cscs comes from a study conducted by dembinski and krauss . in this study vybrant dii cell - labeling solution was used to label pancreatic adenocarcinoma cells and conduct cancer stem studies on flow cytometry sorted label retaining cells . dii label retaining slow - cycling cells ( dii / sccs ) comprised ~3% of total cell number . interestingly , label retaining cells also exhibited an elongated fibroblast shape and an increase in the epithelial - mesenchymal transition markers vimentin , snail , and twist . a fibroblast - like csc is consistent with evidence demonstrating an increase in stem - like properties in cells that have undergone an epithelial - mesenchymal transition . furthermore , sorted dii / sccs demonstrated a 2.510-fold increase in soft agar colony forming ability , twofold increase in invasive potential , and more than a tenfold increase in xenograft formation over nonlabel retaining cells . combined , these data suggest that dii / sccs cells represent an enriched csc population . when assessed for common csc marker status , dii / sccs were enriched but only partially overlapped with cd24/cd44 and cd133 populations . it is curious to consider how these commonly used csc markers relate to the lrc populations and what role , if any , these markers play in the slow - cycling phenotype ? like the melanoma study by roesch et al . , dembinski and krauss 's study also indicated the ability for lrcs to produce non - lrcs and surprisingly also for non - lrcs to produce lrcs . such a dynamic suggests two possibilities ( 1 ) that the true unknown csc population is favored in the lrcs , but also found in the non - lrcs and can therefore give rise to both populations , or ( 2 ) that there exists a dynamic relationship in lrc - csc populations that is context dependent and allows for interconversion between the two states . the dembinski and krauss study argues a dynamic population of cscs that might coincide with an epithelial - mesenchymal transition ( emt ) . emt plays a central role in embryogenesis and mesoderm differentiation into multiple tissue types during development . the emergence of embryonic stem cell - associated genes like nanog , oct4 , sox2 , and c - myc in high grade undifferentiated cancers is suggestive that aberrant regulations of emt and other early development pathways might be playing a role in csc characteristics . this data is a further evidence to support a dynamic quiescent slow - cycling model for many types of cancer . future studies will be important for further development and integration of these observations into the csc model for tumor initiation and propagation . at the present time , we have no clear understanding of why some patients recur and which cancers will have resistance to conventional types of therapy . tumors from different patients in the same organ are likely to have undergone different oncogenic transitions , leading to a diversity of possible regulatory mechanism and pathway activities that might be contributing to the survival of a specific cancer . while broad patterns like the dysregulation of the wnt pathway in colon carcinomas are commonly observed , the secondary mutations that may accompany these cancers could be vastly different and contribute to survival in different ways . even within the same tumor , different cscs have the possibility to accumulate unique mutations that may provide added resistance and be passed on to daughter cells . in context with the vast differences in tumorigenesis and heterogeneity with a tumor , it is not surprising that the exact contributors to chemotherapy resistance and consequently which patients will respond optimally to chemotherapy are not well understood . it has been proposed that variations in cell cycle control , antiapoptotic proteins , increased dna damage repair proteins , upregulation of cellular pumps , and increased metabolic activity may all play important roles in chemotherapy resistance [ 6 , 5962 ] . conventional chemotherapies and radiotherapies the quiescent nature of many adult stem cell pools is therefore an inherent mechanism for resistance and cell survival to conventional therapies . in the hematopoietic system , normal hematopoietic stem cells ( hscs ) when treated with the commonly used chemotherapy agent 5-fluorouracil ( 5-fu ) , mice that were p21 deficient had a significant decrease in cobblestone area - forming stem cells ( 10.8% ) than normal p21 expressing wild - type mice ( 60.5% ) . in the brain , morshead et al . demonstrated that high doses of tritiated thymidine ( h - tdr ) killed the constitutively proliferating cells in the adult mouse forebrain , but had no effect on quiescent stem cell ability to generate spheres . this data supports a model in which quiescent mouse forebrain stem cells are able to survive and re - enter the cell cycle to allow for regeneration of the damaged tissue . a similar pattern of stem cell survival and regeneration was observed 72 hours following doxorubicin treatment in mouse intestine . in this experiment , mice intestine demonstrated increased amounts of cell death via apoptosis in the + 36 positions and a parallel disappearance of mitotic activity . this period of relatively nonexistent mitotic activity was followed by stem cell re - entry into the cell cycle and tissue regeneration in the + 4 position stem cell compartment . furthermore , colon stem cell survival during chemotherapy is aided by increased expression of bh3-only bcl-2 members that inhibit apoptosis , working in parallel with quiescence to increase the likelihood of stem cell survival . in chemotherapy - induced alopecia , the rapidly dividing ta cells in the hair matrix undergo apoptosis , while the stem cells in the bulge region survive to regenerate the follicle after chemotherapy is withdrawn . potential factors involved in regulating hair follicle stem cell survival such as caveolin-1 are emerging . similar mechanisms for survival and self - renewal for cscs are plausible in instances of tumor recurrence in human patients where cytotoxic agents kill proliferative cancer cells , leaving quiescent slow - cycling . cancer stem cells that survive chemotherapy would have the ability to re - enter the cell cycle and produce highly proliferative - rapidly dividing progenitor cells that can re - establish the tumor . it is even probable that successive cycles of chemotherapy would intensify a tumor by weakening the normal stem cell pool and creating therapy resistant cscs that give rise to resistant off - spring . slow cycling csc populations in the colon , breast , ovaries , and pancreas have been shown to demonstrate both in vivo abilities to survive therapies that kill bulk tumor cells as well as a requirement for doses of up to twice that which are required to kill rapidly proliferating cells in vitro [ 18 , 55 , 62 , 67 ] . these data demonstrate how ineffective conventional therapies can be on quiescent cell populations and help to explain why tumors that seem to fully regress during treatment can recur . while large tumor populations may appear to have totally regressed after treatment , single surviving cscs would not be detectable with current diagnostic technology . populations of cscs that are resistant to chemotherapy or radiation are able to re - enter the cell cycle or never fully undergo cell cycle arrest and are primed to re - establish tumors [ 53 , 68 ] . even more devastating to the survival of patients mouse ovarian tumors have been demonstrated to undergo accelerated clonogenic production during radiotherapy regimens , expanding the csc pool and driving development of a more aggressive secondary tumor . furthermore , these cells would be more likely to produce chemotherapy resistant offspring , rendering the tumor unaffected by later rounds of treatment . while quiescence is likely to contribute to the survival of cscs in response to chemotherapy and radiation , slow cycling is not the sole mechanism and in all likelihood works in parallel with other systems to increase survival . msi-1 colon cancer cells have been demonstrated to be less sensitive to cytotoxic drugs due to increased il-4 expression and orchestration of antiapoptotic mechanisms . the expression of other antiapoptotic proteins like c - flip and bcl-2 bh-3 only family members is frequently seen in stem cell and csc populations and has been demonstrated to contribute to cell survival during radiation and chemotherapy [ 59 , 60 ] . reduced cycling may help to limit cell damage in these cases , decreasing prodeath signals and increasing the potential for csc survival . additional mechanisms for csc survival include increased dna damage repair , upregulation of cell pumps like the multidrug resistance transporter ( mdr1 ) and the adenosine triphosphate - binding cassette ( abcb1 ) , and increased metabolic activity through aldh [ 61 , 62 ] . although the quiescence contribution to these mechanisms of resistance is unclear , it is likely that reduced proliferative rate only adds to their effectiveness . additional time in s or g2 phase of the cell cycle coupled with increased dna repair protein activity may afford a survival advantage over bulk cells that continuously accrue dna damage and ultimately are forced to undergo apoptosis . reduced cycling speed together with increased pumps would facilitate more drug being removed from cscs , limiting overall cytotoxic effects during the period of treatment . additionally , quiescence would allow for increased metabolic activity of aldh and other metabolites over that of bulk cells with a shorter cell cycle period . importantly , there is no reason why combinations or all of these resistance mechanisms could not be playing a role in csc survival . future therapies may need to address all these issues to be successful in complete tumor eradication . given the importance of quiescence in the csc contribution to tumor progression and survival , understanding the mechanisms that govern quiescence will prove important in the development of future strategies to better target these cells . much of our current understanding of the mechanisms controlling quiescence come from studies using conditional induction of quiescence in normal adult fibroblasts . the induction of quiescence in fibroblasts is generally accomplished in one of three ways : mitogen deprivation , contact inhibition , or loss of adhesion . each method of inducing quiescence in fibroblast appears to yield a different quiescent transcriptional program . the three transcription programs overlap in differential expression of 131 genes that coller et al . this signature is comprised of genes that regulated cell growth and division , suppress apoptosis and differentiation , and govern intercellular communication . downregulated elements in the quiescence signature consist of genes associated with cell cycle progression including cyclin b1 , cdc20 , cul-1 , and myc . up regulated genes included important cell cycle regulators like tp53 ( p53 ) , cyclin d2 , and mxi1 . also up regulated in this signature are regulators of key stem cell - associated pathways including the wnt pathway ( fzd2 and tcf7l2 ) , the bmp pathway ( smad1 ) , and the notch pathway ( hes1 ) . notch activation of hes1 is of particular interest as it has been shown to control reversibility of fibroblast quiescence by blocking differentiation and entry in irreversible cell cycle arrest . notch pathway activity is important in mammary gland development as well as the mammary csc response immediately following irradiation , suggesting that the notch pathway may be a potential target in cscs [ 5 , 71 ] . interestingly , there exists a fourth transcriptional program in fibroblasts induced by overexpression of cyclin - dependent kinase inhibitors ( cki ) like p21 and p16 . the cki p21 has been found to control entry into quiescence and maintenance of the quiescent state , allowing cells to activate a dna damage - like response . additionally , maintenance of fibroblast quiescence has also been shown to be highly regulated by the retinoblastoma family members rb and p107 . loss of rb and p107 did not affect the ability of fibroblasts to enter g0 , but these cells were unable to maintain the quiescent state . while rb loss is generally associated with the progression of cancer , retention of rb in cscs or contribution of other rb family members like p107 may be important in csc maintenance of quiescence . developing and studying a quiescence signature in fibroblasts may be important in understanding regulation of the cell cycle , but the exact relevance to quiescent stem cell populations is not very clear . primarily , quiescence fibroblast studies are conducted on large populations of fibroblasts under biologically stressful conditions like contact inhibition or serum starvation . in contrast , individual stem cells and cscs maintain quiescence while in contact with daughter cells and stromal layers and in the presence of normal mitogenic signals . additionally , sphere forming assays commonly used for the identification of stem cells and cscs rely specifically on proliferation under nonadherent conditions . if mitogen deprivation , loss of adhesion , and contact inhibition truly activate three different transcriptional programs in quiescent fibroblast populations , it is possible that the transcriptional program facilitated by quiescent stem cells and cscs may be very different . quiescence regulation of a stem cell population is most comprehensively understood in the hematopoietic system . when compared to differentiated or cycling hscs , quiescent hscs were found to have up - regulated genes associated with cell cycle regulation , translation and rna processing , and metabolic process . down - regulated genes were generally associated with transcription factors , signaling proteins , cell cycle proteins , and inhibitors of cell cycle progression . in line with these findings , the cki p21 was found to be necessary for quiescence and maintenance of the hsc pool . mice that are p21 null demonstrate an increase in the number of stem cells present and lose the ability to repopulate the bone marrow in serial transplant experiments , suggesting uncontrolled expansion and eventual exhaustion of the stem cell pool . this deregulation of the stem cell pool is likely due to p21 downstream effects on rb family members : rb , p107 , and p130 . triple knockout of these three family members resulted in hematopoietic progenitor g1/s transition and proliferation , leading to exhaustion of the proliferative potential , similar to that seen in p21 loss . while p21 also appears to play a role in adult neural stem cell regulation and maintenance , other factors occasional exit of neural stem cells from the quiescent state is important for proper tissue maintenance and may be controlled though notch signaling via hes1 oscillations . down - regulation of hes1 in neural progenitor cells during g1 phase reduced repression of cyclind , ngn2 , and dll1 , activating notch signaling and driving cell cycle progression and generation of neural progenitors . neural progenitors and neurons continue to retain low levels of hes1 as they proliferate and differentiate . in neural stem cells , hes1 expression and control of cyclind and notch signaling increase until subsequent g1 entry . interestingly , p21 loss does not appear to play a significant role during differentiation in the brain , suggesting the need for additional means of cell cycle regulation in differentiated senescent cells . signaling pathways with interactions to other ckis also play important roles in quiescent adult stem cell regulation . in mammary glands , the hedgehog pathways components gli2 and bmi-1 have been demonstrated to regulate stem cell self - renewal . when injected into cleared mammary fat pads , gli2 or bmi-1 over expressing mammospheres were able to produce substantially more outgrowths than control mammospheres . bmi-1 has been demonstrated to transcriptionally repress the p16 and p19 , suggesting a role for bmi-1 in mammary stem cell cycle control . additional signaling pathways have been demonstrated to play important roles in stem cell quiescence , specifically the bmp pathway in skin . bmp and calcineurin signaling up - regulate the transcription factor nfat1c that has been found to highly colocalize with cd34 cells in the hair follicle . nfat1c represses transcription of cdk4 , stalling cells in g1/s phase and maintaining quiescence . loss of nfat1c permits entry into the cell cycle , shortening telogen and prompting aberrant entry into anagen . while significant advances are being made in understanding quiescence control in normal adult stem cell populations , much less very few studies have been conducted specifically addressing control of quiescent cscs , most likely due to the difficulty of isolating and analyzing pure csc populations . if cscs are truly derived from adult stem cells , then it is possible that hes1 , p21 , p16 , rb family members , bmi-1 , and nfat1c play significant roles in csc regulation . although rare , there are clues that at least some of these regulators are important in cscs . in the colon cancer cell line hct116 , p21 null cells were found to produce tenfold smaller tumors in growth assays when compared to normal cells expressing p21 . under sphere forming conditions , p21 null cells were unable to form spheres , ceased proliferation , and eventually died . this p21 dependence was found to be associated with lack of e - cadherin expression and suppression of apoptosis signals , suggesting a more complex role for p21 in tumor cells than simply regulating cell cycle . small molecule targeting of p21 or downstream p21 targets may therefore prove to be an effective means of forcing quiescent cscs to cycle or undergo apoptosis . cancers frequently have aberrant signaling in the wnt , hedgehog ( hh ) , and notch self - renewal pathways that likely contribute to cell cycle control and differentiation . increased expression of hes1 has been observed in ovarian , breast , and nonsmall cell lung carcinomas , suggesting active regulation of notch signaling . in melanoma , the slow cycling cells identified by rosech et al . , repress notch signaling directly though jarid1b interaction with the notch ligand jagged 1 promoter , consequently reducing intracellular notch and controlling proliferation . hes1 and jagged1 may therefore be potential targets in future cancer treatments designed to target cscs . targeted reduction of hes1 would increase notch signaling , driving cscs to proliferate and exhaust their proliferative potential , and making them more susceptible to conventional therapy . in colon cancers , mutations in apc or -catenin in the presence of wnt signal , -catenin is no longer taken up by an apc - dependent degradation complex and translocates to the nucleus where it binds tcf / lef transcriptions factors to control expression of cell cycle target genes . loss of apc in crypt lrg5 cells has been demonstrated to be an important step towards initiation of intestinal adenomas . interestingly , cells expressing high wnt downstream transcription factors tcf / lef in primary sphere cultures demonstrated increased clonogenicity and the generation of both cycling and noncycling cells . in tumors , these high wnt expressing cells were located near stromal fibroblasts that provided signals to activate -catenin - dependent transcription . this data suggests that csc cell cycle control may not be entirely cell autonomous and partially regulated by microenvironmental signals . targeting wnt pathway regulators or the ability for cscs to communicate with their stromal environment may represent potential mechanism for limiting csc expansion and contribution to recurrence . there is also mounting evidence for the requirement of hedgehog signaling in proliferation and survival of both colon and breast tumors . active hh - gli signaling was found to contribute to the subpopulation of human colon cd133 cells that were able to survive and self - renew in xenograft studies . in breast tumor cd44/cd24 cells , the hh pathway proteins patch ( ptch1 ) , gli1 , gli2 , and bmi-1 all demonstrated like their adult mammary stem cell counterparts , overexpression of bmi-1 in mammary cscs suggests a potential role for p16 and p19 in cell cycle regulation and suggests a potential drug target for improved csc eradication . while p21 , p16 , notch , wnt , and hedgehog signaling may provide tempting targets for the removal of cscs , targeting of these pathways would require meticulous targeting of csc or titration of inhibitors to act on cscs but not normal stem cell populations additionally , improper application of cell cycle inhibitors like p21 may fuel tumor growth and aggressiveness . the cdk inhibitor p21 acts as a tumor suppressor in dividing cells by protecting against genome instability and working with other tumor suppressors to subdue oncogenes [ 82 , 83 ] . loss of p21 combined with chemical induction of carcinogenesis has demonstrated increased induction of tumors and increased aggressiveness in resulting tumors [ 84 , 85 ] . these data highlight the necessity to be able to selectively target cscs when using cdk inhibitors and add to the challenges ahead in developing treatments to better eradiate cscs . the limited data available on the regulation of quiescence equates to a poor understanding for the role of quiescence in tumor progression and recurrence . exactly how and to what extent quiescence plays a role in tumor recurrence is at present unclear . what little evidence there is suggests that quiescence might be an important factor in tumor cell survival after conventional therapy . mechanistically , csc quiescence suggests an inherent means of resistance that when coupled with increased dna repair or metabolic activity could explain the patterns of recurrence and acquired resistance currently observed in posttherapy cancer patients . the functionally relevant identification of quiescent cscs though label - retaining assays may prove to be an important tool in ongoing csc research . future research must focus on better understanding and targeting of quiescent csc populations , specifically identifying regulators and factors that separate cscs from normal stem cells . general targeting of p21 , bmi-1 , hes1 , and other commonly shared cell cycle regulators might prove disastrous for patients if these treatments eradicate normal stem cell populations as well as cscs . aberrant regulation of normal stem cell characteristics presents a difficult paradox in fighting cscs : how to target the cancer without harming normal stem cells . hope exists that careful study of cscs will identify new or differentially expressed targets that will specifically affect tumors , minimizing toxic side effects and leaving patients cancer free .

long - lived cancer stem cells ( cscs ) with indefinite proliferative potential have been identified in multiple epithelial cancer types . these cells are likely derived from transformed adult stem cells and are thought to share many characteristics with their parental population , including a quiescent slow - cycling phenotype . various label - retaining techniques have been used to identify normal slow cycling adult stem cell populations and offer a unique methodology to functionally identify and isolate cancer stem cells . the quiescent nature of cscs represents an inherent mechanism that at least partially explains chemotherapy resistance and recurrence in posttherapy cancer patients . isolating and understanding the cell cycle regulatory mechanisms of quiescent cancer cells will be a key component to creation of future therapies that better target cscs and totally eradicate tumors . here we review the evidence for quiescent csc populations and explore potential cell cycle regulators that may serve as future targets for elimination of these cells .

1. Cancer Induction from Adult Stem Cells 2. Adult Stem Cells and Quiescence 3. Quiescence and CSCs 4. Quiescence and Resistance to Chemotherapy 5. Regulators of Quiescence 6. Conclusions and Future Directions

given that normal adult stem cells already exhibit limitless replicative potential , it is hypothesized that transformed stems cells may be the cells of origin for many cancers [ 2 , 3 ] . in addition to replicative potential , long - lived stem cells have the opportunity to accumulate oncogenic mutations over years or decades from common mutagenic sources like inflammation , radiation , chemicals , or infection , unlike shorter - lived transit amplifying ( ta ) cells that rapidly proliferate and differentiate [ 4 , 5 ] . like healthy adult stem cells , transformed stem cells these ta cells would be capable of driving tumor formation and generating the heterogeneous combination of populations commonly seen in cancer [ 6 , 7 ] . transformed stem cells have been termed cancer stem cells ( cscs ) , also known as cancer initiating cells , and are defined as the fraction of cells within a tumor that are long lived , possess the potential to proliferate indefinitely , and can generate all heterogeneous lineages of the original tumor in xenograft models [ 6 , 8 ] . cscs are expected to utilize characteristics commonly found in stem cell populations such as differential metabolic activity , specific signaling pathway activity , and regulation of cell cycling characteristics , albeit with aberrant regulation [ 7 , 9 ] ( table 1 ) . a better understanding of cscs is essential in understanding the biological and clinical consequences of existing regimens and designing new therapies to improve patient outcome . current methods for isolation and study of cscs rely on cell surface markers found to be enriched in populations with stem cell - like properties . since the work of these two pioneers , csc populations have been identified in multiple epithelial cancers including the breast , prostate , pancreas , colon [ 1517 ] , ovaries , and brain . given the similarities between normal adult stem cells and cscs , aberrant regulation of self - renewal and quiescence is likely central to csc pathology [ 9 , 10 ] . the primary objectives of this paper are to place quiescent label - retaining studies in the context of what is currently known about adult stem cells and then review the existing evidence for quiescence in cancer stem cells . we will examine current evidence for the role of quiescence in csc resistance to conventional cancer therapy and recurrence . adult stem cells are critical for continued normal tissue homeostasis and response to wounding for many of the epithelial tissues of the body . adult stem cells are characterized by their ability to self - renew indefinitely and produce progeny capable of differentiating and repopulating tissue specific lineages . populations of adult stem cells have been identified in tissues throughout the body , including the skin [ 2325 ] , mammary glands [ 26 , 27 ] , intestine [ 28 , 29 ] , prostate , brain , and the hematopoietic system [ 32 , 33 ] . in tissues where cells are frequently lost to the environment , like those of the intestine and skin , new cells are continuously required to replenish those that are lost . to facilitate this constant need for new cells , some epithelial tissues are arranged hierarchically with slowly proliferating stem cells that asymmetrically divide to give rise to a new stem cell and a rapidly dividing transit amplifying ( ta ) cell . infrequent division or a quiescent nature is not definitive for adult stem cell but is suggested to be important for maintenance of many adult stem cell pools . evidence suggests that quiescence may play an important role in protecting stem cells from exhausting their proliferative capacity , inhibiting differentiation , and limiting accumulation of mutations during frequent rounds of dna synthesis [ 3537 ] . initial efforts to identify and study adult stem cells took advantage of the slow - cycling nature of stem cell populations in studies employing pulse / chase methodology [ 23 , 28 ] . in contrast , slow - cycling stem cells undergo few divisions and retain detectable quantities of label for much longer periods of time . these cells were found to be relatively stem - like : primitive in cytoplasmic contents , structurally similar to other putative stem cell populations , and could be stimulated to proliferate . array analysis of the lrc bulge population showed increased activation of smad and inhibitors of the wnt pathway , suggesting the ability for lrcs to organize their niche and communicate with neighboring mesenchymal and epithelial cells , an important characteristic for stem cell function . at the onset of the growth phase ( anagen ) hair follicle stem cells are activated and produce matrix ta cells that proliferate and differentiate into the seven different lineages found within the hair follicle . although likely important for the maintenance of the stem cell pool , quiescence may not be a requirement for adult stem cells . the authors of this paper suggest that the lgr-5 population of cells represents a cycling population of stem cells under normal conditions , whereas the label retaining cd34/k15 stem cells may represent a reserve population that is activated after tissue damage . similar label - retaining methods have been used to study slow - cycling cells in other tissues , such as the small intestine and colon . work conducted by potten and colleagues identified slow - cycling lrcs at the + 4 position at the base of the colon crypt . upon irradiation , these cells demonstrated increased antiapoptotic bcl-2 expression , decreased p53 expression and were highly activated and involved in clonogenic regeneration of the crypt . similar to stem cell populations in the skin , 1-integrin was found to be highly expressed in the lower half of the colonic crypt . from the evidence collected in these studies and others , a model has been suggested in which slow - cycling stem cells , found at the base of the crypt , undergo periodic division to give rise to ta cells . these cells are ultimately lost to the environment [ 6 , 43 ] . as within the hair follicle , there is convincing evidence for an lgr-5 nonlabel retaining population of colon stem cells additionally found at the base of the crypt . while the lrcs reside at the + 4 population , lgr-5 cells are observed as slender wedge - shaped cells at the + 2 position . again , the exact relationship between the lrcs and the lgr-5 cells is yet to be fully explored , and more data into the lineage potential of both of these cell populations is needed to form a cohesive model . since the early identification of colon and hair follicle slow - cycling stem cell populations , label - retaining techniques have been used to identify and validate putative stem cell populations in multiple epithelial tissues . in the mammary gland , three separate label - retaining populations have been identified and proposed as possible stem cells . identified a long - term label - retaining population enriched by the marker combination lincd29cd24 that was able to reconstitute a functional mammary gland from a single cell . the lincd29cd24 did not enrich for the sca-1 population , prompting other groups to suggest a stem cell hierarchy in which multiple layers of stem cells exist within the mammary gland . pece was also able to conduct transcriptional analysis of the lrc population , from which he created a human normal mammary gland stem cell signature ( hnmsc ) consisting of the markers cd49f / dner / dll1 . the ability to survive and re - enter the cell cycle suggests a stem cell phenotype for these quiescent cells . prostate slow - cycling lrcs located in the proximal ducts demonstrated high proliferative potential and the ability to reconstitute the prostate glandular structure in vitro . if cscs do originate from normal adult stem cells , then it is foreseeable that key stem cell regulatory traits are retained through the oncogenic transition ; quiescence is potentially one of these traits . these cells appeared to have variable expression of the proliferation marker ki-67 , suggesting a low but higher proliferative rate than normal stem cells . demonstrated that cd24 cells expressing stem cell - associated genes like nestin , oct4 , and both notch1 and notch4 were more slowly proliferating than the bulk tumor cells suggesting a quiescent phenotype . pece also observed a link between cscs and quiescence in breast tumors . many of the most widely used cell lines have been in passage for years , are perceived homogeneous , lack interactions with the appropriate stromal microenvironment , and change characteristics based on alterations in culture conditions . in this study vybrant dii cell - labeling solution was used to label pancreatic adenocarcinoma cells and conduct cancer stem studies on flow cytometry sorted label retaining cells . dii label retaining slow - cycling cells ( dii / sccs ) comprised ~3% of total cell number . it is curious to consider how these commonly used csc markers relate to the lrc populations and what role , if any , these markers play in the slow - cycling phenotype ? such a dynamic suggests two possibilities ( 1 ) that the true unknown csc population is favored in the lrcs , but also found in the non - lrcs and can therefore give rise to both populations , or ( 2 ) that there exists a dynamic relationship in lrc - csc populations that is context dependent and allows for interconversion between the two states . this data is a further evidence to support a dynamic quiescent slow - cycling model for many types of cancer . future studies will be important for further development and integration of these observations into the csc model for tumor initiation and propagation . even within the same tumor , different cscs have the possibility to accumulate unique mutations that may provide added resistance and be passed on to daughter cells . in context with the vast differences in tumorigenesis and heterogeneity with a tumor , it is not surprising that the exact contributors to chemotherapy resistance and consequently which patients will respond optimally to chemotherapy are not well understood . it has been proposed that variations in cell cycle control , antiapoptotic proteins , increased dna damage repair proteins , upregulation of cellular pumps , and increased metabolic activity may all play important roles in chemotherapy resistance [ 6 , 5962 ] . conventional chemotherapies and radiotherapies the quiescent nature of many adult stem cell pools is therefore an inherent mechanism for resistance and cell survival to conventional therapies . in the hematopoietic system , normal hematopoietic stem cells ( hscs ) when treated with the commonly used chemotherapy agent 5-fluorouracil ( 5-fu ) , mice that were p21 deficient had a significant decrease in cobblestone area - forming stem cells ( 10.8% ) than normal p21 expressing wild - type mice ( 60.5% ) . this data supports a model in which quiescent mouse forebrain stem cells are able to survive and re - enter the cell cycle to allow for regeneration of the damaged tissue . this period of relatively nonexistent mitotic activity was followed by stem cell re - entry into the cell cycle and tissue regeneration in the + 4 position stem cell compartment . furthermore , colon stem cell survival during chemotherapy is aided by increased expression of bh3-only bcl-2 members that inhibit apoptosis , working in parallel with quiescence to increase the likelihood of stem cell survival . similar mechanisms for survival and self - renewal for cscs are plausible in instances of tumor recurrence in human patients where cytotoxic agents kill proliferative cancer cells , leaving quiescent slow - cycling . cancer stem cells that survive chemotherapy would have the ability to re - enter the cell cycle and produce highly proliferative - rapidly dividing progenitor cells that can re - establish the tumor . slow cycling csc populations in the colon , breast , ovaries , and pancreas have been shown to demonstrate both in vivo abilities to survive therapies that kill bulk tumor cells as well as a requirement for doses of up to twice that which are required to kill rapidly proliferating cells in vitro [ 18 , 55 , 62 , 67 ] . these data demonstrate how ineffective conventional therapies can be on quiescent cell populations and help to explain why tumors that seem to fully regress during treatment can recur . populations of cscs that are resistant to chemotherapy or radiation are able to re - enter the cell cycle or never fully undergo cell cycle arrest and are primed to re - establish tumors [ 53 , 68 ] . furthermore , these cells would be more likely to produce chemotherapy resistant offspring , rendering the tumor unaffected by later rounds of treatment . while quiescence is likely to contribute to the survival of cscs in response to chemotherapy and radiation , slow cycling is not the sole mechanism and in all likelihood works in parallel with other systems to increase survival . msi-1 colon cancer cells have been demonstrated to be less sensitive to cytotoxic drugs due to increased il-4 expression and orchestration of antiapoptotic mechanisms . the expression of other antiapoptotic proteins like c - flip and bcl-2 bh-3 only family members is frequently seen in stem cell and csc populations and has been demonstrated to contribute to cell survival during radiation and chemotherapy [ 59 , 60 ] . although the quiescence contribution to these mechanisms of resistance is unclear , it is likely that reduced proliferative rate only adds to their effectiveness . additional time in s or g2 phase of the cell cycle coupled with increased dna repair protein activity may afford a survival advantage over bulk cells that continuously accrue dna damage and ultimately are forced to undergo apoptosis . future therapies may need to address all these issues to be successful in complete tumor eradication . given the importance of quiescence in the csc contribution to tumor progression and survival , understanding the mechanisms that govern quiescence will prove important in the development of future strategies to better target these cells . downregulated elements in the quiescence signature consist of genes associated with cell cycle progression including cyclin b1 , cdc20 , cul-1 , and myc . up regulated genes included important cell cycle regulators like tp53 ( p53 ) , cyclin d2 , and mxi1 . notch pathway activity is important in mammary gland development as well as the mammary csc response immediately following irradiation , suggesting that the notch pathway may be a potential target in cscs [ 5 , 71 ] . loss of rb and p107 did not affect the ability of fibroblasts to enter g0 , but these cells were unable to maintain the quiescent state . developing and studying a quiescence signature in fibroblasts may be important in understanding regulation of the cell cycle , but the exact relevance to quiescent stem cell populations is not very clear . in contrast , individual stem cells and cscs maintain quiescence while in contact with daughter cells and stromal layers and in the presence of normal mitogenic signals . additionally , sphere forming assays commonly used for the identification of stem cells and cscs rely specifically on proliferation under nonadherent conditions . if mitogen deprivation , loss of adhesion , and contact inhibition truly activate three different transcriptional programs in quiescent fibroblast populations , it is possible that the transcriptional program facilitated by quiescent stem cells and cscs may be very different . mice that are p21 null demonstrate an increase in the number of stem cells present and lose the ability to repopulate the bone marrow in serial transplant experiments , suggesting uncontrolled expansion and eventual exhaustion of the stem cell pool . triple knockout of these three family members resulted in hematopoietic progenitor g1/s transition and proliferation , leading to exhaustion of the proliferative potential , similar to that seen in p21 loss . while p21 also appears to play a role in adult neural stem cell regulation and maintenance , other factors occasional exit of neural stem cells from the quiescent state is important for proper tissue maintenance and may be controlled though notch signaling via hes1 oscillations . in neural stem cells , hes1 expression and control of cyclind and notch signaling increase until subsequent g1 entry . interestingly , p21 loss does not appear to play a significant role during differentiation in the brain , suggesting the need for additional means of cell cycle regulation in differentiated senescent cells . signaling pathways with interactions to other ckis also play important roles in quiescent adult stem cell regulation . in mammary glands , the hedgehog pathways components gli2 and bmi-1 have been demonstrated to regulate stem cell self - renewal . bmi-1 has been demonstrated to transcriptionally repress the p16 and p19 , suggesting a role for bmi-1 in mammary stem cell cycle control . additional signaling pathways have been demonstrated to play important roles in stem cell quiescence , specifically the bmp pathway in skin . loss of nfat1c permits entry into the cell cycle , shortening telogen and prompting aberrant entry into anagen . while significant advances are being made in understanding quiescence control in normal adult stem cell populations , much less very few studies have been conducted specifically addressing control of quiescent cscs , most likely due to the difficulty of isolating and analyzing pure csc populations . if cscs are truly derived from adult stem cells , then it is possible that hes1 , p21 , p16 , rb family members , bmi-1 , and nfat1c play significant roles in csc regulation . although rare , there are clues that at least some of these regulators are important in cscs . in breast tumor cd44/cd24 cells , the hh pathway proteins patch ( ptch1 ) , gli1 , gli2 , and bmi-1 all demonstrated like their adult mammary stem cell counterparts , overexpression of bmi-1 in mammary cscs suggests a potential role for p16 and p19 in cell cycle regulation and suggests a potential drug target for improved csc eradication . while p21 , p16 , notch , wnt , and hedgehog signaling may provide tempting targets for the removal of cscs , targeting of these pathways would require meticulous targeting of csc or titration of inhibitors to act on cscs but not normal stem cell populations additionally , improper application of cell cycle inhibitors like p21 may fuel tumor growth and aggressiveness . these data highlight the necessity to be able to selectively target cscs when using cdk inhibitors and add to the challenges ahead in developing treatments to better eradiate cscs . the limited data available on the regulation of quiescence equates to a poor understanding for the role of quiescence in tumor progression and recurrence . mechanistically , csc quiescence suggests an inherent means of resistance that when coupled with increased dna repair or metabolic activity could explain the patterns of recurrence and acquired resistance currently observed in posttherapy cancer patients . the functionally relevant identification of quiescent cscs though label - retaining assays may prove to be an important tool in ongoing csc research . future research must focus on better understanding and targeting of quiescent csc populations , specifically identifying regulators and factors that separate cscs from normal stem cells . general targeting of p21 , bmi-1 , hes1 , and other commonly shared cell cycle regulators might prove disastrous for patients if these treatments eradicate normal stem cell populations as well as cscs . aberrant regulation of normal stem cell characteristics presents a difficult paradox in fighting cscs : how to target the cancer without harming normal stem cells .

are currently classified as overweight or obese ( bmi 25 kg / m ) , and one - third of them are frankly obese ( bmi 30 kg / m ) ( 1 ) . is 26.1 million in 20052010 or 12% of the population ( 2 ) . among u.s . diabetic patients , the prevalence of overweight or obesity has increased to 80% ( 3 ) . obesity is associated with increased risks of several cardiometabolic diseases , including hypertension ( 4 ) , diabetes ( 5 ) , coronary heart disease ( chd ) ( 6,7 ) , heart failure ( 8) , and stroke ( 9 ) . cardiovascular diseases , especially chd , are the leading causes of death worldwide . in recent years , several prospective studies have assessed the association between obesity and the risks of total and cvd mortality among diabetic patients , and the results are inconsistent . to date , many studies have reported positive associations ( 10,11 ) , inverse associations ( 1214 ) , u - shaped associations ( 1517 ) , or no associations ( 18 ) between bmi and mortality among patients with diabetes . all of these studies were focused on the association between bmi and cvd mortality ; however , no studies assessed the association between bmi and the risk of incident chd among diabetic patients . in this study , we examined the association between bmi and the risk of chd among patients with type 2 diabetes in the louisiana state university hospital - based longitudinal study ( lsuhls ) . between 1997 and 2012 , the lsu health care services division ( lsuhcsd ) operated seven public hospitals and affiliated clinics in louisiana providing quality medical care to the residents of louisiana regardless of their income or insurance coverage ( 1926 ) . overall , lsuhcsd facilities have served 1.6 million patients ( 35% of the louisiana population ) since 1997 . administrative , anthropometric , laboratory , clinical diagnosis , and medication data collected at these facilities are available in electronic form for both inpatients and outpatients from 1997 . using these data , a cohort of diabetic patients was established by using the icd-9 ( code 250 ) between 1 january 1999 and 31 december 2009 . both inpatients and outpatients confirmation of diabetes diagnoses was made by applying the american diabetes association criteria : a fasting plasma glucose level 126 mg / dl ; 2-h glucose level 200 mg / dl after a 75-g 2-h oral glucose tolerance test ; and one or more classic symptoms plus a random plasma glucose level 200 mg / dl ( 27,28 ) . the first record of diabetes diagnosis was used to establish the baseline for each patient in the present analyses due to the design of the cohort study . before diagnosis with diabetes the agreement of diabetes diagnosis was 97% : 20,919 of a sample of 21,566 hospital discharge diagnoses based on icd codes also had physician - confirmed diabetes by using the american diabetes associates diabetes diagnosis criteria ( 27 ) . the current study included 30,434 newly diagnosed patients ( 10,955 men and 19,479 women ) with type 2 diabetes who were 3095 years of age without a history of chd and stroke at the time of diabetes diagnosis and with complete repeated data on all risk factor variables . we only included african americans and whites because the patient numbers of hispanics , asians , and native americans are very small in the lsuhcsd hospitals . patients were excluded if they were underweight ( bmi < 18.5 kg / m ) because of limited statistical power for this group . compared with diabetic patients excluded from the present analyses due to missing data , those included in the analyses were younger ( 51.0 vs. 57.6 years old ) , had a higher frequency of african americans ( 58.6 vs. 45.4% ) , and less males ( 37.2 vs. 47.1% ) . the study and analysis plan was approved by the pennington biomedical research center and lsu health sciences center institutional review boards , lsu system . we did not obtain informed consent from participants involved in our study because we used anonymized data compiled from electronic medical records . the patient s characteristics , including age at diabetes diagnosis , sex , race / ethnicity , family income , smoking status , types of health insurance , bmi , blood pressure , total cholesterol , hdl cholesterol , ldl cholesterol , triglycerides , glycosylated hemoglobin ( hba1c ) , estimated glomerular filtration rate ( egfr ) , and medication ( antihypertensive drug , cholesterol - lowering drug , and antidiabetes drug ) within a half year before the diabetes diagnosis ( baseline ) , during follow - up after the diabetes diagnosis ( follow - up ) , and the last visit were extracted from the computerized hospitalization records . height and weight bmi was calculated by dividing weight in kilograms by the square of height in meters . values of bmi , blood pressure , hba1c , ldl cholesterol , and egfr over time were measured firstly at baseline and secondly as an updated mean of annual measurements , calculated for each participant from baseline to each year of follow - up . for example , at 1 year , the updated mean is the average of the baseline and 1-year values , and at 3 years , it is the average of baseline , 1-year , 2-year , and 3-year values . in the case of an event during follow - up , the period for estimating updated mean values was from baseline to the year before this event occurred ( 29 ) . follow - up information was obtained from the lsuhls inpatient and outpatient database by using the unique number assigned to every patient who visits the lsuhcsd hospitals . the diagnosis of chd was the primary end point of interest of the study and defined according to the following icd-9 : chd ( icd - codes 410414 ) . follow - up of each cohort member continued until the date of the diagnosis of chd , the date of the last visit if the subject stopped use of lsuhcsd hospitals , or the date of death , determined from linking to the louisiana office of public health vital records registry , or 31 may 2012 ( 23,26 ) . the association between bmi and the risk of chd was analyzed by using cox proportional hazards models . bmi was evaluated in the following two ways : 1 ) as five weight categories ( 18.524.9 [ reference group ] , 2529.9 , 3034.9 , 3539.9 , and 40 kg / m ) and 2 ) as a continuous variable . the trend over different categories of bmi was tested in models with the median of each category as a continuous variable . all analyses were adjusted for age ( continuous variable ) and race ( african american and white ) ( model 1 ) and further for smoking ( never , past , and current ) , income ( continuous variable ) , and types of insurance ( free , self - pay , medicaid , medicare , and commercial ) ( model 2 ) , and additionally for systolic blood pressure ( continuous variable ) , hba1c ( continuous variable ) , ldl cholesterol ( continuous variable ) , hdl cholesterol ( continuous variable ) , triglycerides ( continuous variable ) , egfr ( 90 , 6089 , 3059 , 1529 , and < 15 ml / min/1.73 m ) , use of antihypertensive drugs ( no use , ace inhibitor , angiotensin ii receptor blockers , -blockers , calcium channel blocker , diuretics , and other antihypertensive drugs ) , use of diabetes medications ( no use , oral hypoglycemic agents , and insulin ) , and use of cholesterol - lowering agents ( no use , statins , and other cholesterol - lowering agents ) ( model 3 ) . we stratified the samples by sex because there was a significant interaction between sex and bmi on the risk of chd . since the interactions between race and bmi on the risk of chd were not statistically significant , data for white and african americans were combined in some analyses . all statistical analyses were performed with pasw for windows , version 20.0 ( ibm spss inc . , between 1997 and 2012 , the lsu health care services division ( lsuhcsd ) operated seven public hospitals and affiliated clinics in louisiana providing quality medical care to the residents of louisiana regardless of their income or insurance coverage ( 1926 ) . overall , lsuhcsd facilities have served 1.6 million patients ( 35% of the louisiana population ) since 1997 . administrative , anthropometric , laboratory , clinical diagnosis , and medication data collected at these facilities are available in electronic form for both inpatients and outpatients from 1997 . using these data , a cohort of diabetic patients was established by using the icd-9 ( code 250 ) between 1 january 1999 and 31 december 2009 . both inpatients and outpatients confirmation of diabetes diagnoses was made by applying the american diabetes association criteria : a fasting plasma glucose level 126 mg / dl ; 2-h glucose level 200 mg / dl after a 75-g 2-h oral glucose tolerance test ; and one or more classic symptoms plus a random plasma glucose level 200 mg / dl ( 27,28 ) . the first record of diabetes diagnosis was used to establish the baseline for each patient in the present analyses due to the design of the cohort study . before diagnosis with diabetes the agreement of diabetes diagnosis was 97% : 20,919 of a sample of 21,566 hospital discharge diagnoses based on icd codes also had physician - confirmed diabetes by using the american diabetes associates diabetes diagnosis criteria ( 27 ) . the current study included 30,434 newly diagnosed patients ( 10,955 men and 19,479 women ) with type 2 diabetes who were 3095 years of age without a history of chd and stroke at the time of diabetes diagnosis and with complete repeated data on all risk factor variables . we only included african americans and whites because the patient numbers of hispanics , asians , and native americans are very small in the lsuhcsd hospitals . patients were excluded if they were underweight ( bmi < 18.5 kg / m ) because of limited statistical power for this group . compared with diabetic patients excluded from the present analyses due to missing data , those included in the analyses were younger ( 51.0 vs. 57.6 years old ) , had a higher frequency of african americans ( 58.6 vs. 45.4% ) , and less males ( 37.2 vs. 47.1% ) . the study and analysis plan was approved by the pennington biomedical research center and lsu health sciences center institutional review boards , lsu system . we did not obtain informed consent from participants involved in our study because we used anonymized data compiled from electronic medical records . the patient s characteristics , including age at diabetes diagnosis , sex , race / ethnicity , family income , smoking status , types of health insurance , bmi , blood pressure , total cholesterol , hdl cholesterol , ldl cholesterol , triglycerides , glycosylated hemoglobin ( hba1c ) , estimated glomerular filtration rate ( egfr ) , and medication ( antihypertensive drug , cholesterol - lowering drug , and antidiabetes drug ) within a half year before the diabetes diagnosis ( baseline ) , during follow - up after the diabetes diagnosis ( follow - up ) , and the last visit were extracted from the computerized hospitalization records . height and weight were measured without shoes and with light clothing according to a standardized protocol . bmi was calculated by dividing weight in kilograms by the square of height in meters . values of bmi , blood pressure , hba1c , ldl cholesterol , and egfr over time were measured firstly at baseline and secondly as an updated mean of annual measurements , calculated for each participant from baseline to each year of follow - up . for example , at 1 year , the updated mean is the average of the baseline and 1-year values , and at 3 years , it is the average of baseline , 1-year , 2-year , and 3-year values . in the case of an event during follow - up , the period for estimating updated mean values was from baseline to the year before this event occurred ( 29 ) . follow - up information was obtained from the lsuhls inpatient and outpatient database by using the unique number assigned to every patient who visits the lsuhcsd hospitals . the diagnosis of chd was the primary end point of interest of the study and defined according to the following icd-9 : chd ( icd - codes 410414 ) . follow - up of each cohort member continued until the date of the diagnosis of chd , the date of the last visit if the subject stopped use of lsuhcsd hospitals , or the date of death , determined from linking to the louisiana office of public health vital records registry , or 31 may 2012 ( 23,26 ) . the association between bmi and the risk of chd was analyzed by using cox proportional hazards models . bmi was evaluated in the following two ways : 1 ) as five weight categories ( 18.524.9 [ reference group ] , 2529.9 , 3034.9 , 3539.9 , and 40 kg / m ) and 2 ) as a continuous variable . the trend over different categories of bmi was tested in models with the median of each category as a continuous variable . all analyses were adjusted for age ( continuous variable ) and race ( african american and white ) ( model 1 ) and further for smoking ( never , past , and current ) , income ( continuous variable ) , and types of insurance ( free , self - pay , medicaid , medicare , and commercial ) ( model 2 ) , and additionally for systolic blood pressure ( continuous variable ) , hba1c ( continuous variable ) , ldl cholesterol ( continuous variable ) , hdl cholesterol ( continuous variable ) , triglycerides ( continuous variable ) , egfr ( 90 , 6089 , 3059 , 1529 , and < 15 ml / min/1.73 m ) , use of antihypertensive drugs ( no use , ace inhibitor , angiotensin ii receptor blockers , -blockers , calcium channel blocker , diuretics , and other antihypertensive drugs ) , use of diabetes medications ( no use , oral hypoglycemic agents , and insulin ) , and use of cholesterol - lowering agents ( no use , statins , and other cholesterol - lowering agents ) ( model 3 ) . we stratified the samples by sex because there was a significant interaction between sex and bmi on the risk of chd . since the interactions between race and bmi on the risk of chd were not statistically significant , data for white and african americans were combined in some analyses . all statistical analyses were performed with pasw for windows , version 20.0 ( ibm spss inc . , chicago , il ) . baseline characteristics of patients with type 2 diabetes by the outcome during follow - up data represent means or percentages . all data except age are adjusted for age and race . during a mean follow - up period of 7.3 years , 7,414 subjects ( 2,926 men and 4,488 women ) developed chd . patients who developed chd during follow - up were older and used more glucose - lowering , lipid - lowering , and antihypertensive medication compared with those who did not develop chd . the multivariable - adjusted ( age , race , smoking , income , and types of insurance ) ( model 2 ) hazard ratios ( hrs ) for chd at different levels of bmi at baseline ( 18.524.9 [ reference group ] , 2529.9 , 3034.9 , 3539.9 , and 40 kg / m ) were 1.00 , 1.14 ( 95% ci 1.001.29 ) , 1.27 ( 1.121.45 ) , 1.54 ( 1.341.78 ) , and 1.42 ( 1.231.64 ) ( ptrend < 0.001 ) in men and 1.00 , 0.95 ( 0.851.07 ) , 0.95 ( 0.841.06 ) , 1.06 ( 0.941.20 ) , and 1.09 ( 1.001.22 ) ( ptrend < 0.001 ) in women , respectively ( table 2 ) . after further adjustment for other confounding factors ( systolic blood pressure , hba1c , ldl cholesterol , hdl cholesterol , triglycerides , egfr , use of antihypertensive drugs , use of diabetes medications , and use of cholesterol - lowering agents ) , this association remained significant among men ( ptrend < 0.001 ) and women ( ptrend = 0.006 ) . hrs of chd according to different levels of bmi at baseline , during follow - up , and at last visit among patients with type 2 diabetes adjusted for age and race . adjusted for age , race , types of insurance , income , and smoking . adjusted for age , race , types of insurance , income , smoking , systolic blood pressure , ldl cholesterol , hdl cholesterol , triglycerides , hba1c , egfr , use of antihypertensive drugs ( none , ace inhibitor , angiotensin ii receptor blockers , -blockers , calcium channel blocker , diuretics , other antihypertensive drugs , and any two or more of above treatments ) , glucose - lowering agents ( none , oral hypoglycemic agents , and insulin ) , and cholesterol - lowering agents ( none , statins , and other cholesterol - lowering agents ) . when bmi was examined as a continuous variable , the multivariable - adjusted ( model 2 ) hrs of chd for each one - unit increase in bmi at baseline were 1.015 ( 95% ci 1.0111.020 ) in men and 1.004 ( 95% ci 1.0011.008 ) in women ( table 2 ) . there was a significant interaction between sex and bmi on chd risk ( = 9.86 ; 1df , p < 0.005 ) , which indicated that this positive association was stronger in men than in women . when we did an additional analysis by using an updated mean value of bmi , we found the same positive association between bmi and chd risk among both men ( ptrend < 0.001 ) and women ( ptrend < 0.001 ) ( table 2 ) . when we did another additional analysis by using the last visit value of bmi , we found a positive association between bmi and chd risk among men ( ptrend < 0.001 ) and a u - shaped association between bmi and chd risk among women ( ptrend = 0.003 ) ( table 2 ) . women who were overweight and had class i obesity ( bmi 2534.9 kg / m ) at last visit had a lower risk of chd compared with normal - weight women ( bmi < 25 kg / m ) . after excluding subjects who were diagnosed with chd during the first 2 years of follow - up ( n = 3,207 ) , the multivariable - adjusted hrs ( model 2 ) of chd for each one - unit increase in bmi at baseline , during follow - up , and at the last visit were 1.014 ( 95% ci 1.0101.019 ) , 1.017 ( 1.0121.022 ) , and 1.015 ( 1.0091.019 ) in men and 1.005 ( 1.0011.009 ) , 1.006 ( 1.0021.010 ) , and 1.005 ( 1.0001.008 ) in women ( data not shown ) , respectively . in stratified analyses , the multivariable - adjusted positive association between bmi and chd risk was present among men with different smoking status ( tables 3 and 4 ) . when stratified by age , race , and use of antidiabetic drugs , this positive association of bmi at baseline , during follow - up , and at the last visit with chd risk was still present among men in all subgroups and among women in some of the subgroups ( tables 3 and 4 ) . hrs ( 95% cis ) of chd according to different levels of bmi at baseline among various subpopulations adjusted for age , race , types of insurance , income , and smoking , other than the variable for stratification . hrs ( 95% cis ) of chd according to different levels of bmi during follow - up and at last visit among various subpopulations adjusted for age , race , types of insurance , income , and smoking , other than the variable for stratification . our study found a positive association of bmi at baseline and during follow - up with the risk of chd among both men and women with type 2 diabetes , and this association was stronger among men than among women . in addition , we found that a positive association between bmi and the risk of chd was present in both african americans and whites with type 2 diabetes and in nonsmokers and smokers . the positive association did not change among men but changed to a u - shaped association among women with type 2 diabetes when we assessed bmi of the last visit with chd risk . only a few prospective studies have evaluated the association between obesity and total and cvd mortality among diabetic patients , and the results are controversial including inverse associations ( 1214 ) , positive associations ( 10,11 ) , u - shaped associations ( 1517 ) , or no association ( 18 ) . the current study was the first , to our knowledge , to assess the association between bmi and the risk of incident chd among diabetic patients . the results of our study indicated a positive association between bmi and the risk of chd among patients with type 2 diabetes . we found this positive association of chd risk by bmi at baseline and during follow - up . in addition , this positive association was present in different race , antidiabetes medication , and smoking groups . it is noteworthy that there was a u - shaped association between bmi at the last visit and the risk of chd among women with type 2 diabetes in the current study . our study found that diabetic women who were overweight and had class i obesity ( bmi 2534.9 kg / m ) at the last visit had a lower risk of chd compared with normal - weight women ( bmi < 25 it is well known that women with diabetes have a greater or equal relative risk of chd than men with diabetes ( 30,31 ) . the current study found a significant positive association of bmi and chd risk among both men and women with type 2 diabetes , and this association is stronger among men than among women . the finding from our study is noteworthy for us to prevent chd among patients with type 2 diabetes . in addition , more studies are needed to confirm the different effect size of bmi with chd risk among men and women with type 2 diabetes . it has been suggested that three potential methodological concerns should be considered when assessing the associations between obesity and health outcomes ( 32 ) . people with a history of cvd and several other chronic diseases frequently lose weight , and thus , people with a lower weight might increase the estimated risk of death . a recent analysis pooling five longitudinal studies has found that patients who have normal weight at the time of diabetes diagnosis have a higher mortality risk than those who are overweight or obese ( 12 ) . they suggest that diabetic individuals with metabolically obese normal - weight may reflect underlying illness that predisposes to mortality ( 33 ) . despite having a normal bmi , these diabetic individuals have hyperinsulinemia , insulin resistance , and dyslipidemia , and all of these factors predispose individuals to death ( 33 ) . in the current study , we excluded patients with a history of chd and stroke at time of diabetes diagnosis , which can minimize the influence of reverse causation . moreover , we performed another sensitivity analysis by excluding the subjects who were diagnosed with chd during the first 2 years of follow - up ( n = 3,207 ) , and the positive association of bmi at baseline and during follow - up with chd risk was still present . the second major concern is that confounding factors may distort the association between body weight and chd . smoking is a particularly important factor because smokers tend to weigh less and have much higher chd risk than nonsmokers . in the current study , smoking status was considered as a confounding factor in the multivariable model , and the positive association between bmi and the risk of chd was found in both never - smokers and smokers . the third methodological concern in some analyses between weight and chd risk is that the physiologic effects of excess fatness , such as hypertension , diabetes , and dyslipidemia , were controlled for statistically , thus artificially removing some of the effects of being overweight . obesity has been found as a strong risk factor for hypertension ( 4 ) , high levels of hba1c ( 5 ) , and high serum cholesterol among diabetic patients ( 34 ) and has also been the key or important component of the metabolic syndrome ( 35 ) . all of these factors are associated with an increased risk of chd ( 3537 ) and considered as mediating factors for the physiologic effects of obesity on the chd risk . in the current study , the adjustment for systolic blood pressure , ldl cholesterol , hdl cholesterol , triglycerides , hba1c , egfr , and treatment attenuated the association between bmi and chd risk , but bmi as a continuous variable remained a statistically significant predictor of chd in the multivariable model . there are several strengths in our study , including the large sample size , high proportion of african americans , and the use of administrative databases to avoid differential recall biases . we have used baseline bmi levels , updated mean values of bmi during follow - up , and the last visit value of bmi in the analyses , which can avoid potential bias from a single baseline measurement . in addition , participants in this study used the same public health care system that minimizes the influence of accessibility to health care , particularly in comparing men and women . one limitation of our study is that our analysis was not performed on a representative sample of the population , which limits the generalizability of this study ; however , lsuhcsd hospitals are public hospitals and cover > 1.6 million patients , most of whom are low - income persons in louisiana . the results of the current study will have wide applicability for the population with low income and without health insurance in the u.s . another limitation of our study is that we did not have data on other obesity indicators , such as waist , hip , and thigh circumferences , and did not assess abdominal height , although these adiposity predictors have been shown to be associated with cvd risk ( 6,38,39 ) . third , while body weight was measured at each clinic visit , clinically measured bmi might not be as accurate as bmi measured in carefully conducted laboratory studies ( 40 ) . fourth , even though our analyses adjusted for an extensive set of confounding factors , residual confounding due to the measurement error in the assessment of confounding factors , unmeasured factors such as heart rates , physical activity , education , and dietary factors , can not be excluded . in summary , we found a positive association between bmi at baseline and during follow - up with the risk of chd among men and women with type 2 diabetes , and this association was stronger among men than among women . we also found a positive association between bmi at the last visit and the risk of chd among men with type 2 diabetes and a u - shaped association between bmi at the last visit and the risk of chd among women with type 2 diabetes .

objectivethe association between obesity and coronary heart disease ( chd ) risk remains debatable , and no studies have assessed this association among diabetic patients . the aim of our study was to investigate the association between bmi and chd risk among patients with type 2 diabetes.research design and methodsthe sample included 30,434 diabetic patients ( 10,955 men and 19,479 women ) 3095 years of age without a history of chd or stroke in the louisiana state university hospital - based longitudinal study.resultsduring a mean follow - up period of 7.3 years , 7,414 subjects developed chd . the multivariable - adjusted hazard ratios for chd across levels of bmi at baseline ( 18.524.9 , 2529.9 , 3034.9 , 3539.9 , and 40 kg / m2 ) were 1.00 , 1.14 ( 95% ci 1.001.29 ) , 1.27 ( 1.121.45 ) , 1.54 ( 1.341.78 ) , and 1.42 ( 1.231.64 ) ( ptrend < 0.001 ) in men and 1.00 , 0.95 ( 0.851.07 ) , 0.95 ( 0.841.06 ) , 1.06 ( 0.941.20 ) , and 1.09 ( 1.001.22 ) ( ptrend < 0.001 ) in women , respectively . when we used an updated mean or last visit value of bmi , the positive association between bmi and chd risk did not change in men . however , the positive association of bmi with chd changed to a u - shaped association in women when we used the last visit value of bmi.conclusionsour study suggests that there is a positive association between bmi at baseline and during follow - up with the risk of chd among patients with type 2 diabetes . we indicate a u - shaped association between bmi at the last visit and the risk of chd among women with type 2 diabetes .

Introduction Research Design and Methods Study Population Baseline Measurements Prospective Follow-up Statistical Analyses Results Conclusions

are currently classified as overweight or obese ( bmi 25 kg / m ) , and one - third of them are frankly obese ( bmi 30 kg / m ) ( 1 ) . obesity is associated with increased risks of several cardiometabolic diseases , including hypertension ( 4 ) , diabetes ( 5 ) , coronary heart disease ( chd ) ( 6,7 ) , heart failure ( 8) , and stroke ( 9 ) . in recent years , several prospective studies have assessed the association between obesity and the risks of total and cvd mortality among diabetic patients , and the results are inconsistent . to date , many studies have reported positive associations ( 10,11 ) , inverse associations ( 1214 ) , u - shaped associations ( 1517 ) , or no associations ( 18 ) between bmi and mortality among patients with diabetes . all of these studies were focused on the association between bmi and cvd mortality ; however , no studies assessed the association between bmi and the risk of incident chd among diabetic patients . in this study , we examined the association between bmi and the risk of chd among patients with type 2 diabetes in the louisiana state university hospital - based longitudinal study ( lsuhls ) . the current study included 30,434 newly diagnosed patients ( 10,955 men and 19,479 women ) with type 2 diabetes who were 3095 years of age without a history of chd and stroke at the time of diabetes diagnosis and with complete repeated data on all risk factor variables . compared with diabetic patients excluded from the present analyses due to missing data , those included in the analyses were younger ( 51.0 vs. 57.6 years old ) , had a higher frequency of african americans ( 58.6 vs. 45.4% ) , and less males ( 37.2 vs. 47.1% ) . we did not obtain informed consent from participants involved in our study because we used anonymized data compiled from electronic medical records . the patient s characteristics , including age at diabetes diagnosis , sex , race / ethnicity , family income , smoking status , types of health insurance , bmi , blood pressure , total cholesterol , hdl cholesterol , ldl cholesterol , triglycerides , glycosylated hemoglobin ( hba1c ) , estimated glomerular filtration rate ( egfr ) , and medication ( antihypertensive drug , cholesterol - lowering drug , and antidiabetes drug ) within a half year before the diabetes diagnosis ( baseline ) , during follow - up after the diabetes diagnosis ( follow - up ) , and the last visit were extracted from the computerized hospitalization records . values of bmi , blood pressure , hba1c , ldl cholesterol , and egfr over time were measured firstly at baseline and secondly as an updated mean of annual measurements , calculated for each participant from baseline to each year of follow - up . for example , at 1 year , the updated mean is the average of the baseline and 1-year values , and at 3 years , it is the average of baseline , 1-year , 2-year , and 3-year values . in the case of an event during follow - up , the period for estimating updated mean values was from baseline to the year before this event occurred ( 29 ) . follow - up of each cohort member continued until the date of the diagnosis of chd , the date of the last visit if the subject stopped use of lsuhcsd hospitals , or the date of death , determined from linking to the louisiana office of public health vital records registry , or 31 may 2012 ( 23,26 ) . the association between bmi and the risk of chd was analyzed by using cox proportional hazards models . bmi was evaluated in the following two ways : 1 ) as five weight categories ( 18.524.9 [ reference group ] , 2529.9 , 3034.9 , 3539.9 , and 40 kg / m ) and 2 ) as a continuous variable . all analyses were adjusted for age ( continuous variable ) and race ( african american and white ) ( model 1 ) and further for smoking ( never , past , and current ) , income ( continuous variable ) , and types of insurance ( free , self - pay , medicaid , medicare , and commercial ) ( model 2 ) , and additionally for systolic blood pressure ( continuous variable ) , hba1c ( continuous variable ) , ldl cholesterol ( continuous variable ) , hdl cholesterol ( continuous variable ) , triglycerides ( continuous variable ) , egfr ( 90 , 6089 , 3059 , 1529 , and < 15 ml / min/1.73 m ) , use of antihypertensive drugs ( no use , ace inhibitor , angiotensin ii receptor blockers , -blockers , calcium channel blocker , diuretics , and other antihypertensive drugs ) , use of diabetes medications ( no use , oral hypoglycemic agents , and insulin ) , and use of cholesterol - lowering agents ( no use , statins , and other cholesterol - lowering agents ) ( model 3 ) . since the interactions between race and bmi on the risk of chd were not statistically significant , data for white and african americans were combined in some analyses . the current study included 30,434 newly diagnosed patients ( 10,955 men and 19,479 women ) with type 2 diabetes who were 3095 years of age without a history of chd and stroke at the time of diabetes diagnosis and with complete repeated data on all risk factor variables . compared with diabetic patients excluded from the present analyses due to missing data , those included in the analyses were younger ( 51.0 vs. 57.6 years old ) , had a higher frequency of african americans ( 58.6 vs. 45.4% ) , and less males ( 37.2 vs. 47.1% ) . we did not obtain informed consent from participants involved in our study because we used anonymized data compiled from electronic medical records . the patient s characteristics , including age at diabetes diagnosis , sex , race / ethnicity , family income , smoking status , types of health insurance , bmi , blood pressure , total cholesterol , hdl cholesterol , ldl cholesterol , triglycerides , glycosylated hemoglobin ( hba1c ) , estimated glomerular filtration rate ( egfr ) , and medication ( antihypertensive drug , cholesterol - lowering drug , and antidiabetes drug ) within a half year before the diabetes diagnosis ( baseline ) , during follow - up after the diabetes diagnosis ( follow - up ) , and the last visit were extracted from the computerized hospitalization records . values of bmi , blood pressure , hba1c , ldl cholesterol , and egfr over time were measured firstly at baseline and secondly as an updated mean of annual measurements , calculated for each participant from baseline to each year of follow - up . for example , at 1 year , the updated mean is the average of the baseline and 1-year values , and at 3 years , it is the average of baseline , 1-year , 2-year , and 3-year values . in the case of an event during follow - up , the period for estimating updated mean values was from baseline to the year before this event occurred ( 29 ) . follow - up of each cohort member continued until the date of the diagnosis of chd , the date of the last visit if the subject stopped use of lsuhcsd hospitals , or the date of death , determined from linking to the louisiana office of public health vital records registry , or 31 may 2012 ( 23,26 ) . the association between bmi and the risk of chd was analyzed by using cox proportional hazards models . bmi was evaluated in the following two ways : 1 ) as five weight categories ( 18.524.9 [ reference group ] , 2529.9 , 3034.9 , 3539.9 , and 40 kg / m ) and 2 ) as a continuous variable . all analyses were adjusted for age ( continuous variable ) and race ( african american and white ) ( model 1 ) and further for smoking ( never , past , and current ) , income ( continuous variable ) , and types of insurance ( free , self - pay , medicaid , medicare , and commercial ) ( model 2 ) , and additionally for systolic blood pressure ( continuous variable ) , hba1c ( continuous variable ) , ldl cholesterol ( continuous variable ) , hdl cholesterol ( continuous variable ) , triglycerides ( continuous variable ) , egfr ( 90 , 6089 , 3059 , 1529 , and < 15 ml / min/1.73 m ) , use of antihypertensive drugs ( no use , ace inhibitor , angiotensin ii receptor blockers , -blockers , calcium channel blocker , diuretics , and other antihypertensive drugs ) , use of diabetes medications ( no use , oral hypoglycemic agents , and insulin ) , and use of cholesterol - lowering agents ( no use , statins , and other cholesterol - lowering agents ) ( model 3 ) . we stratified the samples by sex because there was a significant interaction between sex and bmi on the risk of chd . since the interactions between race and bmi on the risk of chd were not statistically significant , data for white and african americans were combined in some analyses . baseline characteristics of patients with type 2 diabetes by the outcome during follow - up data represent means or percentages . during a mean follow - up period of 7.3 years , 7,414 subjects ( 2,926 men and 4,488 women ) developed chd . patients who developed chd during follow - up were older and used more glucose - lowering , lipid - lowering , and antihypertensive medication compared with those who did not develop chd . the multivariable - adjusted ( age , race , smoking , income , and types of insurance ) ( model 2 ) hazard ratios ( hrs ) for chd at different levels of bmi at baseline ( 18.524.9 [ reference group ] , 2529.9 , 3034.9 , 3539.9 , and 40 kg / m ) were 1.00 , 1.14 ( 95% ci 1.001.29 ) , 1.27 ( 1.121.45 ) , 1.54 ( 1.341.78 ) , and 1.42 ( 1.231.64 ) ( ptrend < 0.001 ) in men and 1.00 , 0.95 ( 0.851.07 ) , 0.95 ( 0.841.06 ) , 1.06 ( 0.941.20 ) , and 1.09 ( 1.001.22 ) ( ptrend < 0.001 ) in women , respectively ( table 2 ) . after further adjustment for other confounding factors ( systolic blood pressure , hba1c , ldl cholesterol , hdl cholesterol , triglycerides , egfr , use of antihypertensive drugs , use of diabetes medications , and use of cholesterol - lowering agents ) , this association remained significant among men ( ptrend < 0.001 ) and women ( ptrend = 0.006 ) . hrs of chd according to different levels of bmi at baseline , during follow - up , and at last visit among patients with type 2 diabetes adjusted for age and race . when bmi was examined as a continuous variable , the multivariable - adjusted ( model 2 ) hrs of chd for each one - unit increase in bmi at baseline were 1.015 ( 95% ci 1.0111.020 ) in men and 1.004 ( 95% ci 1.0011.008 ) in women ( table 2 ) . there was a significant interaction between sex and bmi on chd risk ( = 9.86 ; 1df , p < 0.005 ) , which indicated that this positive association was stronger in men than in women . when we did an additional analysis by using an updated mean value of bmi , we found the same positive association between bmi and chd risk among both men ( ptrend < 0.001 ) and women ( ptrend < 0.001 ) ( table 2 ) . when we did another additional analysis by using the last visit value of bmi , we found a positive association between bmi and chd risk among men ( ptrend < 0.001 ) and a u - shaped association between bmi and chd risk among women ( ptrend = 0.003 ) ( table 2 ) . women who were overweight and had class i obesity ( bmi 2534.9 kg / m ) at last visit had a lower risk of chd compared with normal - weight women ( bmi < 25 kg / m ) . after excluding subjects who were diagnosed with chd during the first 2 years of follow - up ( n = 3,207 ) , the multivariable - adjusted hrs ( model 2 ) of chd for each one - unit increase in bmi at baseline , during follow - up , and at the last visit were 1.014 ( 95% ci 1.0101.019 ) , 1.017 ( 1.0121.022 ) , and 1.015 ( 1.0091.019 ) in men and 1.005 ( 1.0011.009 ) , 1.006 ( 1.0021.010 ) , and 1.005 ( 1.0001.008 ) in women ( data not shown ) , respectively . in stratified analyses , the multivariable - adjusted positive association between bmi and chd risk was present among men with different smoking status ( tables 3 and 4 ) . when stratified by age , race , and use of antidiabetic drugs , this positive association of bmi at baseline , during follow - up , and at the last visit with chd risk was still present among men in all subgroups and among women in some of the subgroups ( tables 3 and 4 ) . hrs ( 95% cis ) of chd according to different levels of bmi at baseline among various subpopulations adjusted for age , race , types of insurance , income , and smoking , other than the variable for stratification . hrs ( 95% cis ) of chd according to different levels of bmi during follow - up and at last visit among various subpopulations adjusted for age , race , types of insurance , income , and smoking , other than the variable for stratification . our study found a positive association of bmi at baseline and during follow - up with the risk of chd among both men and women with type 2 diabetes , and this association was stronger among men than among women . in addition , we found that a positive association between bmi and the risk of chd was present in both african americans and whites with type 2 diabetes and in nonsmokers and smokers . the positive association did not change among men but changed to a u - shaped association among women with type 2 diabetes when we assessed bmi of the last visit with chd risk . only a few prospective studies have evaluated the association between obesity and total and cvd mortality among diabetic patients , and the results are controversial including inverse associations ( 1214 ) , positive associations ( 10,11 ) , u - shaped associations ( 1517 ) , or no association ( 18 ) . the current study was the first , to our knowledge , to assess the association between bmi and the risk of incident chd among diabetic patients . the results of our study indicated a positive association between bmi and the risk of chd among patients with type 2 diabetes . we found this positive association of chd risk by bmi at baseline and during follow - up . it is noteworthy that there was a u - shaped association between bmi at the last visit and the risk of chd among women with type 2 diabetes in the current study . our study found that diabetic women who were overweight and had class i obesity ( bmi 2534.9 kg / m ) at the last visit had a lower risk of chd compared with normal - weight women ( bmi < 25 it is well known that women with diabetes have a greater or equal relative risk of chd than men with diabetes ( 30,31 ) . the current study found a significant positive association of bmi and chd risk among both men and women with type 2 diabetes , and this association is stronger among men than among women . the finding from our study is noteworthy for us to prevent chd among patients with type 2 diabetes . in addition , more studies are needed to confirm the different effect size of bmi with chd risk among men and women with type 2 diabetes . people with a history of cvd and several other chronic diseases frequently lose weight , and thus , people with a lower weight might increase the estimated risk of death . in the current study , we excluded patients with a history of chd and stroke at time of diabetes diagnosis , which can minimize the influence of reverse causation . moreover , we performed another sensitivity analysis by excluding the subjects who were diagnosed with chd during the first 2 years of follow - up ( n = 3,207 ) , and the positive association of bmi at baseline and during follow - up with chd risk was still present . the second major concern is that confounding factors may distort the association between body weight and chd . in the current study , smoking status was considered as a confounding factor in the multivariable model , and the positive association between bmi and the risk of chd was found in both never - smokers and smokers . the third methodological concern in some analyses between weight and chd risk is that the physiologic effects of excess fatness , such as hypertension , diabetes , and dyslipidemia , were controlled for statistically , thus artificially removing some of the effects of being overweight . obesity has been found as a strong risk factor for hypertension ( 4 ) , high levels of hba1c ( 5 ) , and high serum cholesterol among diabetic patients ( 34 ) and has also been the key or important component of the metabolic syndrome ( 35 ) . all of these factors are associated with an increased risk of chd ( 3537 ) and considered as mediating factors for the physiologic effects of obesity on the chd risk . in the current study , the adjustment for systolic blood pressure , ldl cholesterol , hdl cholesterol , triglycerides , hba1c , egfr , and treatment attenuated the association between bmi and chd risk , but bmi as a continuous variable remained a statistically significant predictor of chd in the multivariable model . there are several strengths in our study , including the large sample size , high proportion of african americans , and the use of administrative databases to avoid differential recall biases . we have used baseline bmi levels , updated mean values of bmi during follow - up , and the last visit value of bmi in the analyses , which can avoid potential bias from a single baseline measurement . one limitation of our study is that our analysis was not performed on a representative sample of the population , which limits the generalizability of this study ; however , lsuhcsd hospitals are public hospitals and cover > 1.6 million patients , most of whom are low - income persons in louisiana . another limitation of our study is that we did not have data on other obesity indicators , such as waist , hip , and thigh circumferences , and did not assess abdominal height , although these adiposity predictors have been shown to be associated with cvd risk ( 6,38,39 ) . in summary , we found a positive association between bmi at baseline and during follow - up with the risk of chd among men and women with type 2 diabetes , and this association was stronger among men than among women . we also found a positive association between bmi at the last visit and the risk of chd among men with type 2 diabetes and a u - shaped association between bmi at the last visit and the risk of chd among women with type 2 diabetes .

diabetes mellitus has long been considered a disease of carbohydrate metabolism , and before the discovery of insulin in 1921 , low carbohydrate starvation diets were the default treatment . from the 1930s through to the 1960s , many experts continued to advise strict carbohydrate restriction , with the result that most people with diabetes adopted a high fat , low carbohydrate diet . however , some early work in the 1920s and 1930s had suggested that high carbohydrate diets improved glucose tolerance , and the dramatic increase in deaths from vascular disease in those whose lives were prolonged by insulin treatment led to a volte - face in the 1980s , with authorities now recommending low fat , high carbohydrate diets . the pendulum has again swung the other way , and there is renewed interest in very low carbohydrate diets for the treatment of diabetes , with various physicians extolling the virtues of dietary carbohydrate restriction as the first approach in diabetes management , and some authorities recognizing low carbohydrate diets as a suitable weight - loss strategy for those with type 2 diabetes [ 5 , 6 ] . interestingly , the carbohydrate debate seems to be based on strong personal opinion and those working in the area tend to cherry - pick the evidence to support their particular view , whether that of low , moderate , or high carbohydrate . debates about the issue can become very passionate , and it is worth reminding ourselves that passion in science is an infallible marker of lack of evidence . the evidence available is contradictory at best , and leaves both health professionals and people with diabetes alike wondering if low carbohydrate diets do live up to the hype surrounding them , and whether they should be recommended as a suitable treatment . recent systematic reviews and meta - analyses including people with type 2 diabetes report that although low carbohydrate diets lead to significantly greater weight loss and improvements in glycated hemoglobin ( hba1c ) and lipids over the short term [ 7 , 8 ] , there is no greater advantage over the longer term [ 9 , 10 ] . despite this evidence , low carbohydrate diets remain an area of controversy and this review aims to provide an overview of the latest evidence , and to explore the role of low carbohydrate diets for people with type 2 diabetes . this article is based on previously conducted studies , and does not involve any new studies of human or animal subjects conducted by the author . one of the issues with the term low carbohydrate is uncertainty about what this means in terms of carbohydrate intake . ketosis readily occurs at carbohydrate intakes below 50 g / day , and these very low carbohydrate , ketogenic diets ( vlckd ) appear to have more pronounced effects than other , less restricted carbohydrate diets . the taxonomy for diets containing various amounts of dietary carbohydrate has been suggested in a recent paper , see table 1 . in practice , most atkins - style diets are designed to be very low in carbohydrate ( less than 20 g / day initially ) and high in protein and fat , and other diets , e.g. , the zone and the south beach diet , promote a moderate carbohydrate restriction together with high protein and low fat intakes.table 1taxonomy of diets containing differing amounts of carbohydratedescriptionamount of carbohydrateg / day% total energy intakevery low carbohydrate ketogenic diet205010low carbohydrate<130<26moderate carbohydrate1302302645high carbohydrate>230>45adapted from feinman et al . treating type 2 diabetes is challenging , encompassing as it does management of glycemia , cardiovascular disease ( cvd ) risk factors , obesity , and other co - morbidities by a combination of lifestyle strategies ( diet and physical activity ) , behavioral and psychological interventions , pharmaceutical treatment , and bariatric surgery . medical management of type 2 diabetes has led to cynicism about the efficacy of lifestyle management , particularly dietary strategies , and at present the components of the most effective diet remain unknown . a recent systematic review and meta - analysis suggested that low carbohydrate , low glycemic index ( gi ) , mediterranean , and high protein diets all showed greater improvements in glycemic control than control diets . despite criticism of the statistical analysis due to heterogeneity of the studies included , this review supports the premise that improvements in glycemic control , cvd risk , and weight loss are achievable with different diets with varying amounts of carbohydrate , and that low carbohydrate diets are not necessarily superior in effect . treating type 2 diabetes is challenging , encompassing as it does management of glycemia , cardiovascular disease ( cvd ) risk factors , obesity , and other co - morbidities by a combination of lifestyle strategies ( diet and physical activity ) , behavioral and psychological interventions , pharmaceutical treatment , and bariatric surgery . medical management of type 2 diabetes has led to cynicism about the efficacy of lifestyle management , particularly dietary strategies , and at present the components of the most effective diet remain unknown . a recent systematic review and meta - analysis suggested that low carbohydrate , low glycemic index ( gi ) , mediterranean , and high protein diets all showed greater improvements in glycemic control than control diets . despite criticism of the statistical analysis due to heterogeneity of the studies included , this review supports the premise that improvements in glycemic control , cvd risk , and weight loss are achievable with different diets with varying amounts of carbohydrate , and that low carbohydrate diets are not necessarily superior in effect . an electronic search of english language articles was performed using medline ( 2010may 2015 ) , embase ( 2010may 2015 ) , and the cochrane central register of controlled trials ( 2010may 2015 ) using the search terms low carbohydrate diet and type 2 diabetes . the selection criteria included all randomized controlled trials ( rcts ) comparing interventions evaluating reduced carbohydrate intake with higher carbohydrate intake in people with diagnosed type 2 diabetes . the title and abstract of each record retrieved from the search were screened by the author ( pad ) , and full articles were retrieved if the information given suggested that the study met the selection criteria . data were extracted using a specially designed form and included information about authors , country , year of publication , primary and secondary outcomes , intervention , and outcomes . thirteen of these studies were excluded and eight met the inclusion criteria [ 1825 ] . the flow diagram illustrating the search and selection of studies . a descriptive summary of the included trials and main results are shown in tables 2 , 3 , and 4 . it proved impossible to combine the results of the eight selected studies using statistical methods as the studies were heterogeneous in terms of the dietary intervention ( carbohydrate intakes ranged from more than 20 g to 166 g / day ) , length of follow - up ( 624 months ) , data quality , and data reporting . 1study flow diagram showing number of studies screened , assessed for eligibility , and included in the narrative reviewtable 2descriptive summary of recent low carbohydrate trials for people with type 2 diabetesfirst author , yearduration ( months)numbersdieticiciqbal , 2010 24707430 g / day carbohydrate30% fat , 500 kcal / day energy deficitelhayany , 2010 128517435% carbohydrate , 45% fat ( 50 % of which was mufa)1 . mediterranean diet : as ada diet , but mufa fatlarsen , 2011 12534640% carbohydrate , 30% protein , 30% fat55% carbohydrate , 15% protein , 30% fatguldbrand , 2012 24303120% carbohydrate , 30% protein , 50% fat5560% carbohydrate , 1025% protein , 30% fatkrebs , 2013 2420721140% carbohydrate , 30% protein , 30% fat55% carbohydrate , 15% protein , 30% fatmayer , 2014 11.5222420 g / day carbohydrate30% fat , 5001000 kcal / day energy deficit and orlistatyamada , 2014 6121270130 g / day carbohydrate to avoid ketosis5060% carbohydrate , < 20% protein , < 25% fat , with energy restriction based on japanese recommendationstay , 2015 64647<50 g / day carbohydrate ( 14%)53% low gi carbohydrate , 17% protein , 30% fat ada american diabetes association , c comparator , gi glycemic index , i intervention , mufa monounsaturated fatty acidstable 3summary of results of recent low carbohydrate trials for people with type 2 diabetes ( body weight and glycemic control)first author , yearbody weight loss ( kg)changes in hba1c ( % ) ici c p valueici c p valueiqbal , 2010 1.70.21.20.290.10.20.1nselhayany , 2010 8.97.61.40.5572.01.60.40.88larsen , 2011 2.232.170.070.90.230.280.040.76guldbrand , 2012 2.02.90.90.330.00.20.20.76krebs , 2013 3.96.02.10.730.10.10.00.5mayer , 2014 7.58.10.60.80.70.20.80.045yamada , 2014 2.61.41.20.80.60.20.40.03tay , 2014 12.011.50.50.57nrnrnrnr c comparator , hba1c glycated hemoglobin , i intervention , nr not reported , ns no significant differencetable 4summary of results of recent low carbohydrate trials for people with type 2 diabetes ( cardiovascular risk)first author , yeartotal cholesterol ( mmol / l)hdl ( mmol / l)ldl ( mmol / l)triglycerides ( mmol / l)systolic bp ( mm / hg)diastolic bp ( mm / hg)i c p valuei c p valuei c p valuei c p valuei c p valuei c p valueiqbal , 2010 0.03ns0.0ns0.06ns0.14ns6.7ns0.5nselhayany , 2010 0.020.2040.18<0.0010.240.0360.64<0.001nrnrnrnrlarsen , 2011 0.160.320.010.840.010.30.170.344.30.050.40.7guldbrand , 2012 0.20.330.120.150.00.160.10.3520.7430.75krebs , 2013 0.070.030.010.410.030.320.030.0210.870.00.96mayer , 2014 0.230.40.030.50.250.30.280.3110.00660.013yamada , 2014 nrnr0.250.130.080.490.580.081.70.544.60.3tay , 2014 0.00.89nrnr0.00.810.40.0012.30.261.80.1 bp blood pressure , c comparator , hdl high density lipoprotein , i intervention , ldl low density lipoprotein , nr not reported , ns no significant difference study flow diagram showing number of studies screened , assessed for eligibility , and included in the narrative review descriptive summary of recent low carbohydrate trials for people with type 2 diabetes ada american diabetes association , c comparator , gi glycemic index , i intervention , mufa monounsaturated fatty acids summary of results of recent low carbohydrate trials for people with type 2 diabetes ( body weight and glycemic control ) c comparator , hba1c glycated hemoglobin , i intervention , nr not reported , ns no significant difference summary of results of recent low carbohydrate trials for people with type 2 diabetes ( cardiovascular risk ) bp blood pressure , c comparator , hdl high density lipoprotein , i intervention , ldl low density lipoprotein , nr not reported , ns no significant difference all eight studies reported weight loss in the group receiving the reduced carbohydrate intervention , with mean weight losses ranging from 1.7 kg to 12.0 kg . the greatest weight loss was reported in the shortest study lasting 6 months . there appeared to be no relationship between degree of carbohydrate restriction and weight loss . however , these studies all included a control group receiving dietary interventions that provided higher carbohydrate intakes but were designed for weight loss , consequently those in the control groups also lost weight during the course of the studies . mean weight losses in the control group were similar to those in the reduced carbohydrate group and ranged from 0.2 kg to 11.5 kg , with the result that none of the eight studies reported significantly greater weight loss in the group receiving the reduced carbohydrate intervention . despite no significant differences in weight losses , three of the studies reported significantly greater reductions in hba1c in the reduced carbohydrate intervention group [ 19 , 23 , 24 ] . one of the studies did not report hba1c despite the fact that this measurement was defined as the primary outcome , leading to the speculation that there were no differences in glycemic control between the two groups . this has now been confirmed , with recent publication of a follow - up at 12 months reporting no difference in hba1c reductions between the low and high carbohydrate intakes . changes in hba1c in the reduced carbohydrate intervention groups were variable between studies , ranging from + 0.1% to 2.0% , with the greatest reduction seen in studies of shorter duration . there appeared to be little correlation between the degree of carbohydrate restriction and changes in glycemic control . hba1c levels were also reduced in five of the seven control groups , with changes ranging from + 0.1% to 0.3% . in summary , one study failed to report hba1c , three studies showed significant reductions in hba1c in the reduced carbohydrate group [ 19 , 23 , 24 ] , and four studies showed no significant differences between the two groups [ 18 , 2022 ] . all eight studies measured lipid concentrations , and seven studies measured blood pressure [ 18 , 2025 ] . most studies reported reductions in lipid concentrations in both the reduced carbohydrate intervention and higher carbohydrate control group , with no significant differences between the two groups . however , significantly greater reductions in the reduced carbohydrate group were reported for total cholesterol concentrations in one study , low density lipoprotein ( ldl ) and high density lipoprotein ( hdl ) concentrations in one study , and triglycerides in three studies [ 19 , 22 , 25 ] . changes in blood pressure were variable and showed no significant differences in six of the seven studies reporting outcomes ; four studies reported reductions in systolic blood pressure ( sbp ) in the reduced carbohydrate group compared to the higher carbohydrate group [ 18 , 20 , 23 , 25 ] , and three reported increases [ 21 , 22 , 24 ] ; for diastolic blood pressure four reported decreases in the reduced carbohydrate intervention group [ 18 , 20 , 23 , 25 ] and three reported increases [ 21 , 22 , 24 ] . in summary , although there was no evidence of a deleterious effect of a reduced carbohydrate diet on cvd risk , equally , there was no evidence of superiority over a higher carbohydrate intake . adherence to the prescribed intervention was assessed by self - reported dietary intake using a variety of methods including 24-hour diet histories and 3- , 4- , and 7-day food diaries . in the majority of the studies , mean intake of carbohydrate in the reduced carbohydrate intervention group was higher than that prescribed ; in only two studies did the participants achieve target intakes [ 24 , 25 ] . attrition rates were reported for seven studies , and ranged from no dropouts to 60% . there were no differences in attrition rates between the intervention and control groups in any of the studies . in general , lower attrition rates were reported for shorter studies , and for those with fewer participants . all eight studies reported weight loss in the group receiving the reduced carbohydrate intervention , with mean weight losses ranging from 1.7 kg to 12.0 kg . however , these studies all included a control group receiving dietary interventions that provided higher carbohydrate intakes but were designed for weight loss , consequently those in the control groups also lost weight during the course of the studies . mean weight losses in the control group were similar to those in the reduced carbohydrate group and ranged from 0.2 kg to 11.5 kg , with the result that none of the eight studies reported significantly greater weight loss in the group receiving the reduced carbohydrate intervention . despite no significant differences in weight losses , three of the studies reported significantly greater reductions in hba1c in the reduced carbohydrate intervention group [ 19 , 23 , 24 ] . one of the studies did not report hba1c despite the fact that this measurement was defined as the primary outcome , leading to the speculation that there were no differences in glycemic control between the two groups . this has now been confirmed , with recent publication of a follow - up at 12 months reporting no difference in hba1c reductions between the low and high carbohydrate intakes . changes in hba1c in the reduced carbohydrate intervention groups were variable between studies , ranging from + 0.1% to 2.0% , with the greatest reduction seen in studies of shorter duration . there appeared to be little correlation between the degree of carbohydrate restriction and changes in glycemic control . hba1c levels were also reduced in five of the seven control groups , with changes ranging from + 0.1% to 0.3% . in summary , one study failed to report hba1c , three studies showed significant reductions in hba1c in the reduced carbohydrate group [ 19 , 23 , 24 ] , and four studies showed no significant differences between the two groups [ 18 , 2022 ] . cardiovascular risk was assessed by changes in lipid concentrations and blood pressure . all eight studies measured lipid concentrations , and seven studies measured blood pressure [ 18 , 2025 ] . most studies reported reductions in lipid concentrations in both the reduced carbohydrate intervention and higher carbohydrate control group , with no significant differences between the two groups . however , significantly greater reductions in the reduced carbohydrate group were reported for total cholesterol concentrations in one study , low density lipoprotein ( ldl ) and high density lipoprotein ( hdl ) concentrations in one study , and triglycerides in three studies [ 19 , 22 , 25 ] . changes in blood pressure were variable and showed no significant differences in six of the seven studies reporting outcomes ; four studies reported reductions in systolic blood pressure ( sbp ) in the reduced carbohydrate group compared to the higher carbohydrate group [ 18 , 20 , 23 , 25 ] , and three reported increases [ 21 , 22 , 24 ] ; for diastolic blood pressure four reported decreases in the reduced carbohydrate intervention group [ 18 , 20 , 23 , 25 ] and three reported increases [ 21 , 22 , 24 ] . in summary , although there was no evidence of a deleterious effect of a reduced carbohydrate diet on cvd risk , equally , there was no evidence of superiority over a higher carbohydrate intake . adherence to the prescribed intervention was assessed by self - reported dietary intake using a variety of methods including 24-hour diet histories and 3- , 4- , and 7-day food diaries . in the majority of the studies , mean intake of carbohydrate in the reduced carbohydrate intervention group was higher than that prescribed ; in only two studies did the participants achieve target intakes [ 24 , 25 ] . attrition rates were reported for seven studies , and ranged from no dropouts to 60% . there were no differences in attrition rates between the intervention and control groups in any of the studies . in general , lower attrition rates were reported for shorter studies , and for those with fewer participants . this review of recent studies evaluating the effects of low carbohydrate diets in people with type 2 diabetes supports previous meta - analyses showing that although there may be greater short - term improvements in glycemic control , weight loss , and cvd risk , this is not sustained over the longer - term . many studies have attempted to determine the ideal macronutrient ( protein , fat , and carbohydrate ) intake for people with type 2 diabetes , and evidence to date is inconclusive . one of the best predictors of improved outcomes in people with type 2 diabetes is energy restriction and weight loss , and there are a variety of strategies by which this may be achieved , with no clear indication of the superiority of low carbohydrate diets . this is true for both those with type 2 diabetes and those without [ 10 , 29 , 30 ] . much of the positive effect of low carbohydrate diets is due to weight loss , and the effect independent of weight change is difficult to assess . in the absence of categorical evidence supporting the use of low carbohydrate diets , one wonders why they have gained such strong support and media attention over the past few years . many proponents of low carbohydrate diets maintain that recent healthy eating guidelines promoting carbohydrate and restricting fat have been counterproductive and have led to escalating rates of obesity and type 2 diabetes . the cause of obesity is extremely complex and it is unlikely that one factor , that of carbohydrate intake , is the root cause . there is also contrary evidence indicating that diets high in fruit , vegetables , whole grains , and legumes ( all of which contain carbohydrate ) actually protect against obesity , cvd , and , to a lesser extent , type 2 diabetes . studies often fail to address the type of carbohydrate included in the diet , and this may affect outcomes . there is now accumulating evidence that unprocessed carbohydrates , including whole grains , fruit , vegetables , and legumes , have health benefits , and those from refined sources , including white bread and white rice and particularly sugar and sugar - sweetened beverages ( ssb ) , are associated with increased risk of obesity , cvd , and type 2 diabetes [ 3639 ] . it could be speculated that the benefits of low carbohydrate diets are associated with a reduction in refined carbohydrate and not total carbohydrate per se . for people with type 2 diabetes , there is evidence from a large , long - term rct suggesting that higher carbohydrate diets can improve weight loss , glycemic control , and cvd risk factors ( although not cvd mortality ) . the look ahead trial ( clinicaltrials.gov identifier , nct0017953 ) reported greater weight loss , improvements in glycemia and cvd risk factors , and reduced risk of microvascular complications , depression , sleep apnea , and urinary incontinence at 9.6-year follow - up in those allocated an intensive lifestyle education ( ile ) program compared to standard diabetes education ( des ) . those in the ile group were encouraged to increase physical activity and adopt an energy - reduced , low fat , partial meal replacement plan . at 1-year follow - up , they derived a higher proportion of energy from carbohydrate ( ile 50.8% vs. des 42.5% ) and a lower proportion from fat ( ile 34.2% vs. des 39.7% ) , demonstrating that a higher carbohydrate , lower fat diet was associated with improved outcomes . concern has been expressed about the long - term health effects of low carbohydrate diets on renal function , calcium metabolism , lack of essential nutrients , and cvd risk , and a systematic review and meta - analysis reported that low carbohydrate diets were associated with a significantly higher risk of all - cause mortality . reductions in carbohydrate intake may also be associated with an increased risk of hypoglycemia in those treated with insulin or insulin secretagogues , and to reduce this medical supervision , reductions in medication and self - monitoring of blood glucose concentrations are recommended for those adopting a low carbohydrate diet . low carbohydrate diets tend to be higher in protein , and this may have an adverse effect on renal function . there are very few studies investigating renal function and low carbohydrate diets , although a recent study suggested that improvements in renal function are related to weight loss , and that this occurs to a similar extent with low carbohydrate , mediterranean , and low fat diets . in obese people without diabetes , studies have shown that low carbohydrate diets have no harmful effects on glomerular filtration rate ( gfr ) , albuminuria , fluid or electrolyte balance when compared to a low fat diet [ 47 , 48 ] . it has been postulated that as very low carbohydrate diets cause ketosis , this induces acidosis , promoting urinary calcium loss and leading to low bone mineral density and increased risk of osteoporosis . there is very little research in this field , and none at all in people with diabetes , making it challenging to draw firm conclusions . one animal study showed that low carbohydrate diets induce low bone mineral density in rats , and two small studies in obese subjects reported deleterious effects on urinary calcium loss and markers for bone formation . conversely , another study reported no effect of a low carbohydrate diet on bone turnover markers . the long - term effects of low carbohydrate diets on calcium metabolism and bone health are unknown . other claims about the negative aspects of low carbohydrate diets include that of nutritional deficiencies , namely those commonly found in unprocessed carbohydrate foods including vitamins , minerals , dietary fiber , and phytochemicals with antioxidant properties . there is no evidence to either endorse or refute this suggestion , although a computer - generated analysis showed that low carbohydrate diets are deficient in many micronutrients , and an analysis of four popular diets from the usa ( atkins , learn , ornish , and zone ) demonstrated that all diets showed a degree of deficiency : specifically thiamine , folic acid , vitamin c , iron , and magnesium in the case of low carbohydrate diets . low carbohydrate diets may be low in dietary fiber and epidemiological evidence suggests that low intakes of dietary fiber are associated with increased risk of lower gastrointestinal disorders , including colon cancer [ 34 , 55 ] , and this may be further exacerbated by high intakes of red meat and meat products . the most controversial aspect of low carbohydrate diets is that they may increase the risk of cvd as they are associated with higher total and saturated fat intakes . there is little evidence for this in people with type 2 diabetes as there are very few studies ; as a result many commentators have extrapolated from studies in the general population . there are some issues with the quality of evidence used to define the relationship between fat intake and cvd risk as most studies are short - term rcts with surrogate end points , or observational and epidemiological studies , where associations do not prove causation . recent meta - analyses and systematic reviews have reported that there is no association between cvd and type of dietary fat , whether saturated fatty acids ( sfa ) , polyunsaturated fatty acids ( pufa ) , or monounsaturated fatty acids ( mufa ) [ 57 , 58 ] , leading to headlines stating that scientists have been wrong for decades and have mislead the public with low - fat , healthy eating recommendations . however , both these reviews have been widely criticized for omitting important cohort studies , incorrect extraction of data , incorrect interpretation , and a failure to mention the results of other , superior analyses . many experts still maintain that there is an association between sfa and cvd , and that the evidence supports substitution of sfa by unsaturated fat . the recently published cochrane review also supports this recommendation , stating that there is a small but potentially important reduction in cvd risk with the reduction of sfa . it is worth remembering that most studies examining the relationship between fat intake and cvd include fat intakes in a fairly narrow range of approximately 3040 % of total energy intake , and little is known about the relative effects of intakes above these values . this may be an issue for some individuals adopting a low carbohydrate diet where fat , often sfa , is actively promoted to induce ketosis and increase palatability . as is the case with glycemic control , weight reduction improves cvd risk factors and if weight loss is achieved , there are no significant differences between either low fat , high carbohydrate diets and low carbohydrate diets for primary prevention of cvd . on balance , there is little evidence to support changing current recommendations for fat intake in people with type 2 diabetes . there is a further consideration that is now coming to the fore , and that is the challenge of sustainable nutrition . sustainable diets , as defined by the food and agriculture organization ( fao ) , are nutritionally adequate , safe , affordable , and culturally acceptable and are sparing of natural and human resources . the carbon footprint of different foodstuffs has been investigated , and the results show that red meat is the most carbon intensive process , followed by dairy , fruit , chicken , and vegetables . low carbohydrate diets tend to include foods with the biggest carbon footprint and large - scale adoption of these diets will increase greenhouse gas emissions . in terms of cultural acceptance newly industrialized countries such as china and india are experiencing a rapid increase in the prevalence of diabetes [ 66 , 67 ] , and it is estimated that by 2030 , 551 billion people ( 10 % of the world s population ) will have diabetes . for many of these people , a low carbohydrate diet is either unacceptable for religious or cultural reasons , or simply unaffordable . concern has been expressed about the long - term health effects of low carbohydrate diets on renal function , calcium metabolism , lack of essential nutrients , and cvd risk , and a systematic review and meta - analysis reported that low carbohydrate diets were associated with a significantly higher risk of all - cause mortality . reductions in carbohydrate intake may also be associated with an increased risk of hypoglycemia in those treated with insulin or insulin secretagogues , and to reduce this medical supervision , reductions in medication and self - monitoring of blood glucose concentrations are recommended for those adopting a low carbohydrate diet . low carbohydrate diets tend to be higher in protein , and this may have an adverse effect on renal function . there are very few studies investigating renal function and low carbohydrate diets , although a recent study suggested that improvements in renal function are related to weight loss , and that this occurs to a similar extent with low carbohydrate , mediterranean , and low fat diets . in obese people without diabetes , studies have shown that low carbohydrate diets have no harmful effects on glomerular filtration rate ( gfr ) , albuminuria , fluid or electrolyte balance when compared to a low fat diet [ 47 , 48 ] . it has been postulated that as very low carbohydrate diets cause ketosis , this induces acidosis , promoting urinary calcium loss and leading to low bone mineral density and increased risk of osteoporosis . there is very little research in this field , and none at all in people with diabetes , making it challenging to draw firm conclusions . one animal study showed that low carbohydrate diets induce low bone mineral density in rats , and two small studies in obese subjects reported deleterious effects on urinary calcium loss and markers for bone formation . conversely , another study reported no effect of a low carbohydrate diet on bone turnover markers . the long - term effects of low carbohydrate diets on calcium metabolism and bone health are unknown . other claims about the negative aspects of low carbohydrate diets include that of nutritional deficiencies , namely those commonly found in unprocessed carbohydrate foods including vitamins , minerals , dietary fiber , and phytochemicals with antioxidant properties . there is no evidence to either endorse or refute this suggestion , although a computer - generated analysis showed that low carbohydrate diets are deficient in many micronutrients , and an analysis of four popular diets from the usa ( atkins , learn , ornish , and zone ) demonstrated that all diets showed a degree of deficiency : specifically thiamine , folic acid , vitamin c , iron , and magnesium in the case of low carbohydrate diets . low carbohydrate diets may be low in dietary fiber and epidemiological evidence suggests that low intakes of dietary fiber are associated with increased risk of lower gastrointestinal disorders , including colon cancer [ 34 , 55 ] , and this may be further exacerbated by high intakes of red meat and meat products . the most controversial aspect of low carbohydrate diets is that they may increase the risk of cvd as they are associated with higher total and saturated fat intakes . there is little evidence for this in people with type 2 diabetes as there are very few studies ; as a result many commentators have extrapolated from studies in the general population . there are some issues with the quality of evidence used to define the relationship between fat intake and cvd risk as most studies are short - term rcts with surrogate end points , or observational and epidemiological studies , where associations do not prove causation . recent meta - analyses and systematic reviews have reported that there is no association between cvd and type of dietary fat , whether saturated fatty acids ( sfa ) , polyunsaturated fatty acids ( pufa ) , or monounsaturated fatty acids ( mufa ) [ 57 , 58 ] , leading to headlines stating that scientists have been wrong for decades and have mislead the public with low - fat , healthy eating recommendations . however , both these reviews have been widely criticized for omitting important cohort studies , incorrect extraction of data , incorrect interpretation , and a failure to mention the results of other , superior analyses . many experts still maintain that there is an association between sfa and cvd , and that the evidence supports substitution of sfa by unsaturated fat . the recently published cochrane review also supports this recommendation , stating that there is a small but potentially important reduction in cvd risk with the reduction of sfa . it is worth remembering that most studies examining the relationship between fat intake and cvd include fat intakes in a fairly narrow range of approximately 3040 % of total energy intake , and little is known about the relative effects of intakes above these values . this may be an issue for some individuals adopting a low carbohydrate diet where fat , often sfa , is actively promoted to induce ketosis and increase palatability . as is the case with glycemic control , weight reduction improves cvd risk factors and if weight loss is achieved , there are no significant differences between either low fat , high carbohydrate diets and low carbohydrate diets for primary prevention of cvd . on balance , there is little evidence to support changing current recommendations for fat intake in people with type 2 diabetes . there is a further consideration that is now coming to the fore , and that is the challenge of sustainable nutrition . sustainable diets , as defined by the food and agriculture organization ( fao ) , are nutritionally adequate , safe , affordable , and culturally acceptable and are sparing of natural and human resources . the carbon footprint of different foodstuffs has been investigated , and the results show that red meat is the most carbon intensive process , followed by dairy , fruit , chicken , and vegetables . low carbohydrate diets tend to include foods with the biggest carbon footprint and large - scale adoption of these diets will increase greenhouse gas emissions . in terms of cultural acceptance newly industrialized countries such as china and india are experiencing a rapid increase in the prevalence of diabetes [ 66 , 67 ] , and it is estimated that by 2030 , 551 billion people ( 10 % of the world s population ) will have diabetes . for many of these people , a low carbohydrate diet is either unacceptable for religious or cultural reasons , or simply unaffordable . to date the evidence suggests that low carbohydrate diets are effective for weight loss and improvements in glycemic control and cvd risk , but that they are not superior to other dietary approaches . for this reason , low carbohydrate diets can not be recommended as the default strategy for people with type 2 diabetes . however , they are another useful tool for those who wish to adopt them , although long - term side effects of these diets remain unknown . the question remains how much carbohydrate should someone with type 2 diabetes eat ? both diabetes uk and the american diabetes association recommend an individualized approach , where health professionals work with the person with diabetes to identify an eating pattern that is based on that individual s lifestyle , culture , and preferences . both authorities identify carbohydrate management as a key strategy and address both type and amount of carbohydrate , emphasizing unprocessed carbohydrate from whole grains , fruit , and vegetable sources . perhaps it is time to abandon the macronutrient approach to nutritional advice and begin to talk about specific foods and eating patterns and encourage those associated with health . there is no ideal eating pattern that will benefit all people with diabetes , although total energy intake is an important consideration , especially in those who are overweight or obese . epidemiological and observational studies show that there are dietary patterns that are associated with better overall health outcomes and which are rich in vegetables , fruit , whole grains , seafood , legumes , and nuts , contain moderate amounts of dairy products , and are lower in red and processed meat , sugar , and refined grains . in summary , although low carbohydrate diets appear to be safe and effective in people with diabetes , there are more sustainable alternatives available and this should be fully explained to all those with type 2 diabetes . this article is based on previously conducted studies , and does not involve any new studies of human or animal subjects conducted by the author . this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license ( http://creativecommons.org/licenses/by-nc/4.0/ ) , which permits any noncommercial use , distribution , and reproduction in any medium , provided you give appropriate credit to the original author(s ) and the source , provide a link to the creative commons license , and indicate if changes were made .

introductionlow carbohydrate diets are again in the spotlight and have been identified as particularly appropriate for people with type 2 diabetes . there is confusion amongst both health professionals and people with diabetes about the suitability of these diets . this review aims to provide an overview of the latest evidence and to explore the role of low carbohydrate diets for people with type 2 diabetes.methodsan electronic search of english language articles was performed using medline ( 2010may 2015 ) , embase ( 2010may 2015 ) , and the cochrane central register of controlled trials ( 2010may 2015 ) . only randomized controlled trials comparing interventions evaluating reduced carbohydrate intake with higher carbohydrate intake in people with diagnosed type 2 diabetes were included . primary outcomes included weight , glycated hemoglobin , and lipid concentrations.resultslow carbohydrate diets in people with type 2 diabetes were effective for short - term improvements in glycemic control , weight loss , and cardiovascular risk , but this was not sustained over the longer term . overall , low carbohydrate diets failed to show superiority over higher carbohydrate intakes for any of the measures evaluated including weight loss , glycemic control , lipid concentrations , blood pressure , and compliance with treatment.conclusionrecent studies suggest that low carbohydrate diets appear to be safe and effective over the short term , but show no statistical differences from control diets with higher carbohydrate content and can not be recommended as the default treatment for people with type 2 diabetes .

Introduction Definition of Low Carbohydrate Diets The Role of Low Carbohydrate Diets in Treating Type 2 Diabetes Methods Results Weight Loss Glycemic Control Cardiovascular Risk Adherence and Attrition Discussion Disadvantages of Low Carbohydrate Diets Conclusions Disclosures Compliance with ethics guidelines Open Access

diabetes mellitus has long been considered a disease of carbohydrate metabolism , and before the discovery of insulin in 1921 , low carbohydrate starvation diets were the default treatment . the pendulum has again swung the other way , and there is renewed interest in very low carbohydrate diets for the treatment of diabetes , with various physicians extolling the virtues of dietary carbohydrate restriction as the first approach in diabetes management , and some authorities recognizing low carbohydrate diets as a suitable weight - loss strategy for those with type 2 diabetes [ 5 , 6 ] . the evidence available is contradictory at best , and leaves both health professionals and people with diabetes alike wondering if low carbohydrate diets do live up to the hype surrounding them , and whether they should be recommended as a suitable treatment . recent systematic reviews and meta - analyses including people with type 2 diabetes report that although low carbohydrate diets lead to significantly greater weight loss and improvements in glycated hemoglobin ( hba1c ) and lipids over the short term [ 7 , 8 ] , there is no greater advantage over the longer term [ 9 , 10 ] . despite this evidence , low carbohydrate diets remain an area of controversy and this review aims to provide an overview of the latest evidence , and to explore the role of low carbohydrate diets for people with type 2 diabetes . ketosis readily occurs at carbohydrate intakes below 50 g / day , and these very low carbohydrate , ketogenic diets ( vlckd ) appear to have more pronounced effects than other , less restricted carbohydrate diets . treating type 2 diabetes is challenging , encompassing as it does management of glycemia , cardiovascular disease ( cvd ) risk factors , obesity , and other co - morbidities by a combination of lifestyle strategies ( diet and physical activity ) , behavioral and psychological interventions , pharmaceutical treatment , and bariatric surgery . medical management of type 2 diabetes has led to cynicism about the efficacy of lifestyle management , particularly dietary strategies , and at present the components of the most effective diet remain unknown . a recent systematic review and meta - analysis suggested that low carbohydrate , low glycemic index ( gi ) , mediterranean , and high protein diets all showed greater improvements in glycemic control than control diets . despite criticism of the statistical analysis due to heterogeneity of the studies included , this review supports the premise that improvements in glycemic control , cvd risk , and weight loss are achievable with different diets with varying amounts of carbohydrate , and that low carbohydrate diets are not necessarily superior in effect . medical management of type 2 diabetes has led to cynicism about the efficacy of lifestyle management , particularly dietary strategies , and at present the components of the most effective diet remain unknown . a recent systematic review and meta - analysis suggested that low carbohydrate , low glycemic index ( gi ) , mediterranean , and high protein diets all showed greater improvements in glycemic control than control diets . despite criticism of the statistical analysis due to heterogeneity of the studies included , this review supports the premise that improvements in glycemic control , cvd risk , and weight loss are achievable with different diets with varying amounts of carbohydrate , and that low carbohydrate diets are not necessarily superior in effect . an electronic search of english language articles was performed using medline ( 2010may 2015 ) , embase ( 2010may 2015 ) , and the cochrane central register of controlled trials ( 2010may 2015 ) using the search terms low carbohydrate diet and type 2 diabetes . the selection criteria included all randomized controlled trials ( rcts ) comparing interventions evaluating reduced carbohydrate intake with higher carbohydrate intake in people with diagnosed type 2 diabetes . it proved impossible to combine the results of the eight selected studies using statistical methods as the studies were heterogeneous in terms of the dietary intervention ( carbohydrate intakes ranged from more than 20 g to 166 g / day ) , length of follow - up ( 624 months ) , data quality , and data reporting . 1study flow diagram showing number of studies screened , assessed for eligibility , and included in the narrative reviewtable 2descriptive summary of recent low carbohydrate trials for people with type 2 diabetesfirst author , yearduration ( months)numbersdieticiciqbal , 2010 24707430 g / day carbohydrate30% fat , 500 kcal / day energy deficitelhayany , 2010 128517435% carbohydrate , 45% fat ( 50 % of which was mufa)1 . mediterranean diet : as ada diet , but mufa fatlarsen , 2011 12534640% carbohydrate , 30% protein , 30% fat55% carbohydrate , 15% protein , 30% fatguldbrand , 2012 24303120% carbohydrate , 30% protein , 50% fat5560% carbohydrate , 1025% protein , 30% fatkrebs , 2013 2420721140% carbohydrate , 30% protein , 30% fat55% carbohydrate , 15% protein , 30% fatmayer , 2014 11.5222420 g / day carbohydrate30% fat , 5001000 kcal / day energy deficit and orlistatyamada , 2014 6121270130 g / day carbohydrate to avoid ketosis5060% carbohydrate , < 20% protein , < 25% fat , with energy restriction based on japanese recommendationstay , 2015 64647<50 g / day carbohydrate ( 14%)53% low gi carbohydrate , 17% protein , 30% fat ada american diabetes association , c comparator , gi glycemic index , i intervention , mufa monounsaturated fatty acidstable 3summary of results of recent low carbohydrate trials for people with type 2 diabetes ( body weight and glycemic control)first author , yearbody weight loss ( kg)changes in hba1c ( % ) ici c p valueici c p valueiqbal , 2010 1.70.21.20.290.10.20.1nselhayany , 2010 8.97.61.40.5572.01.60.40.88larsen , 2011 2.232.170.070.90.230.280.040.76guldbrand , 2012 2.02.90.90.330.00.20.20.76krebs , 2013 3.96.02.10.730.10.10.00.5mayer , 2014 7.58.10.60.80.70.20.80.045yamada , 2014 2.61.41.20.80.60.20.40.03tay , 2014 12.011.50.50.57nrnrnrnr c comparator , hba1c glycated hemoglobin , i intervention , nr not reported , ns no significant differencetable 4summary of results of recent low carbohydrate trials for people with type 2 diabetes ( cardiovascular risk)first author , yeartotal cholesterol ( mmol / l)hdl ( mmol / l)ldl ( mmol / l)triglycerides ( mmol / l)systolic bp ( mm / hg)diastolic bp ( mm / hg)i c p valuei c p valuei c p valuei c p valuei c p valuei c p valueiqbal , 2010 0.03ns0.0ns0.06ns0.14ns6.7ns0.5nselhayany , 2010 0.020.2040.18<0.0010.240.0360.64<0.001nrnrnrnrlarsen , 2011 0.160.320.010.840.010.30.170.344.30.050.40.7guldbrand , 2012 0.20.330.120.150.00.160.10.3520.7430.75krebs , 2013 0.070.030.010.410.030.320.030.0210.870.00.96mayer , 2014 0.230.40.030.50.250.30.280.3110.00660.013yamada , 2014 nrnr0.250.130.080.490.580.081.70.544.60.3tay , 2014 0.00.89nrnr0.00.810.40.0012.30.261.80.1 bp blood pressure , c comparator , hdl high density lipoprotein , i intervention , ldl low density lipoprotein , nr not reported , ns no significant difference study flow diagram showing number of studies screened , assessed for eligibility , and included in the narrative review descriptive summary of recent low carbohydrate trials for people with type 2 diabetes ada american diabetes association , c comparator , gi glycemic index , i intervention , mufa monounsaturated fatty acids summary of results of recent low carbohydrate trials for people with type 2 diabetes ( body weight and glycemic control ) c comparator , hba1c glycated hemoglobin , i intervention , nr not reported , ns no significant difference summary of results of recent low carbohydrate trials for people with type 2 diabetes ( cardiovascular risk ) bp blood pressure , c comparator , hdl high density lipoprotein , i intervention , ldl low density lipoprotein , nr not reported , ns no significant difference all eight studies reported weight loss in the group receiving the reduced carbohydrate intervention , with mean weight losses ranging from 1.7 kg to 12.0 kg . however , these studies all included a control group receiving dietary interventions that provided higher carbohydrate intakes but were designed for weight loss , consequently those in the control groups also lost weight during the course of the studies . all eight studies measured lipid concentrations , and seven studies measured blood pressure [ 18 , 2025 ] . however , significantly greater reductions in the reduced carbohydrate group were reported for total cholesterol concentrations in one study , low density lipoprotein ( ldl ) and high density lipoprotein ( hdl ) concentrations in one study , and triglycerides in three studies [ 19 , 22 , 25 ] . changes in blood pressure were variable and showed no significant differences in six of the seven studies reporting outcomes ; four studies reported reductions in systolic blood pressure ( sbp ) in the reduced carbohydrate group compared to the higher carbohydrate group [ 18 , 20 , 23 , 25 ] , and three reported increases [ 21 , 22 , 24 ] ; for diastolic blood pressure four reported decreases in the reduced carbohydrate intervention group [ 18 , 20 , 23 , 25 ] and three reported increases [ 21 , 22 , 24 ] . in summary , although there was no evidence of a deleterious effect of a reduced carbohydrate diet on cvd risk , equally , there was no evidence of superiority over a higher carbohydrate intake . however , these studies all included a control group receiving dietary interventions that provided higher carbohydrate intakes but were designed for weight loss , consequently those in the control groups also lost weight during the course of the studies . mean weight losses in the control group were similar to those in the reduced carbohydrate group and ranged from 0.2 kg to 11.5 kg , with the result that none of the eight studies reported significantly greater weight loss in the group receiving the reduced carbohydrate intervention . in summary , one study failed to report hba1c , three studies showed significant reductions in hba1c in the reduced carbohydrate group [ 19 , 23 , 24 ] , and four studies showed no significant differences between the two groups [ 18 , 2022 ] . all eight studies measured lipid concentrations , and seven studies measured blood pressure [ 18 , 2025 ] . however , significantly greater reductions in the reduced carbohydrate group were reported for total cholesterol concentrations in one study , low density lipoprotein ( ldl ) and high density lipoprotein ( hdl ) concentrations in one study , and triglycerides in three studies [ 19 , 22 , 25 ] . changes in blood pressure were variable and showed no significant differences in six of the seven studies reporting outcomes ; four studies reported reductions in systolic blood pressure ( sbp ) in the reduced carbohydrate group compared to the higher carbohydrate group [ 18 , 20 , 23 , 25 ] , and three reported increases [ 21 , 22 , 24 ] ; for diastolic blood pressure four reported decreases in the reduced carbohydrate intervention group [ 18 , 20 , 23 , 25 ] and three reported increases [ 21 , 22 , 24 ] . in summary , although there was no evidence of a deleterious effect of a reduced carbohydrate diet on cvd risk , equally , there was no evidence of superiority over a higher carbohydrate intake . this review of recent studies evaluating the effects of low carbohydrate diets in people with type 2 diabetes supports previous meta - analyses showing that although there may be greater short - term improvements in glycemic control , weight loss , and cvd risk , this is not sustained over the longer - term . many studies have attempted to determine the ideal macronutrient ( protein , fat , and carbohydrate ) intake for people with type 2 diabetes , and evidence to date is inconclusive . one of the best predictors of improved outcomes in people with type 2 diabetes is energy restriction and weight loss , and there are a variety of strategies by which this may be achieved , with no clear indication of the superiority of low carbohydrate diets . much of the positive effect of low carbohydrate diets is due to weight loss , and the effect independent of weight change is difficult to assess . in the absence of categorical evidence supporting the use of low carbohydrate diets , one wonders why they have gained such strong support and media attention over the past few years . many proponents of low carbohydrate diets maintain that recent healthy eating guidelines promoting carbohydrate and restricting fat have been counterproductive and have led to escalating rates of obesity and type 2 diabetes . there is now accumulating evidence that unprocessed carbohydrates , including whole grains , fruit , vegetables , and legumes , have health benefits , and those from refined sources , including white bread and white rice and particularly sugar and sugar - sweetened beverages ( ssb ) , are associated with increased risk of obesity , cvd , and type 2 diabetes [ 3639 ] . for people with type 2 diabetes , there is evidence from a large , long - term rct suggesting that higher carbohydrate diets can improve weight loss , glycemic control , and cvd risk factors ( although not cvd mortality ) . concern has been expressed about the long - term health effects of low carbohydrate diets on renal function , calcium metabolism , lack of essential nutrients , and cvd risk , and a systematic review and meta - analysis reported that low carbohydrate diets were associated with a significantly higher risk of all - cause mortality . low carbohydrate diets tend to be higher in protein , and this may have an adverse effect on renal function . there are very few studies investigating renal function and low carbohydrate diets , although a recent study suggested that improvements in renal function are related to weight loss , and that this occurs to a similar extent with low carbohydrate , mediterranean , and low fat diets . there is very little research in this field , and none at all in people with diabetes , making it challenging to draw firm conclusions . other claims about the negative aspects of low carbohydrate diets include that of nutritional deficiencies , namely those commonly found in unprocessed carbohydrate foods including vitamins , minerals , dietary fiber , and phytochemicals with antioxidant properties . there is no evidence to either endorse or refute this suggestion , although a computer - generated analysis showed that low carbohydrate diets are deficient in many micronutrients , and an analysis of four popular diets from the usa ( atkins , learn , ornish , and zone ) demonstrated that all diets showed a degree of deficiency : specifically thiamine , folic acid , vitamin c , iron , and magnesium in the case of low carbohydrate diets . there is little evidence for this in people with type 2 diabetes as there are very few studies ; as a result many commentators have extrapolated from studies in the general population . recent meta - analyses and systematic reviews have reported that there is no association between cvd and type of dietary fat , whether saturated fatty acids ( sfa ) , polyunsaturated fatty acids ( pufa ) , or monounsaturated fatty acids ( mufa ) [ 57 , 58 ] , leading to headlines stating that scientists have been wrong for decades and have mislead the public with low - fat , healthy eating recommendations . as is the case with glycemic control , weight reduction improves cvd risk factors and if weight loss is achieved , there are no significant differences between either low fat , high carbohydrate diets and low carbohydrate diets for primary prevention of cvd . on balance , there is little evidence to support changing current recommendations for fat intake in people with type 2 diabetes . concern has been expressed about the long - term health effects of low carbohydrate diets on renal function , calcium metabolism , lack of essential nutrients , and cvd risk , and a systematic review and meta - analysis reported that low carbohydrate diets were associated with a significantly higher risk of all - cause mortality . there are very few studies investigating renal function and low carbohydrate diets , although a recent study suggested that improvements in renal function are related to weight loss , and that this occurs to a similar extent with low carbohydrate , mediterranean , and low fat diets . in obese people without diabetes , studies have shown that low carbohydrate diets have no harmful effects on glomerular filtration rate ( gfr ) , albuminuria , fluid or electrolyte balance when compared to a low fat diet [ 47 , 48 ] . there is very little research in this field , and none at all in people with diabetes , making it challenging to draw firm conclusions . the long - term effects of low carbohydrate diets on calcium metabolism and bone health are unknown . other claims about the negative aspects of low carbohydrate diets include that of nutritional deficiencies , namely those commonly found in unprocessed carbohydrate foods including vitamins , minerals , dietary fiber , and phytochemicals with antioxidant properties . there is no evidence to either endorse or refute this suggestion , although a computer - generated analysis showed that low carbohydrate diets are deficient in many micronutrients , and an analysis of four popular diets from the usa ( atkins , learn , ornish , and zone ) demonstrated that all diets showed a degree of deficiency : specifically thiamine , folic acid , vitamin c , iron , and magnesium in the case of low carbohydrate diets . the most controversial aspect of low carbohydrate diets is that they may increase the risk of cvd as they are associated with higher total and saturated fat intakes . there is little evidence for this in people with type 2 diabetes as there are very few studies ; as a result many commentators have extrapolated from studies in the general population . as is the case with glycemic control , weight reduction improves cvd risk factors and if weight loss is achieved , there are no significant differences between either low fat , high carbohydrate diets and low carbohydrate diets for primary prevention of cvd . on balance , there is little evidence to support changing current recommendations for fat intake in people with type 2 diabetes . low carbohydrate diets tend to include foods with the biggest carbon footprint and large - scale adoption of these diets will increase greenhouse gas emissions . to date the evidence suggests that low carbohydrate diets are effective for weight loss and improvements in glycemic control and cvd risk , but that they are not superior to other dietary approaches . for this reason , low carbohydrate diets can not be recommended as the default strategy for people with type 2 diabetes . in summary , although low carbohydrate diets appear to be safe and effective in people with diabetes , there are more sustainable alternatives available and this should be fully explained to all those with type 2 diabetes . this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license ( http://creativecommons.org/licenses/by-nc/4.0/ ) , which permits any noncommercial use , distribution , and reproduction in any medium , provided you give appropriate credit to the original author(s ) and the source , provide a link to the creative commons license , and indicate if changes were made .

male fvb / n jcl mice ( clea japan , tokyo , japan ) were studied at 1216 weeks of age . the animals were housed individually in plastic cages at 24 1c with lights on from 0600 to 1800 h , and they were maintained with free access to a laboratory diet ( oriental yeast , tokyo , japan ) and water . mice were anesthetized by intraperitoneal injection of ketamine ( 100 mg / kg body mass ) and xylazine ( 10 mg / kg ) , and a chronic double - walled stainless steel cannula was implanted stereotaxically and unilaterally into the right side of the vmh , arc , dmh , or pvh or into the lateral ventricle according to the atlas of franklin and paxinos ( 26 ) . the stereotaxic coordinates were ap 1.3 ( 1.3 mm anterior to the bregma ) , l 0.3 ( 0.3 mm lateral to the bregma ) , and h 5.8 ( 5.8 mm below the bregma on the surface of the skull ) for the vmh ; ap 1.6 , l 0.2 , and h 6.1 for the arc ; ap 1.6 , l 0.3 , and h 5.5 for the dmh ; ap 0.75 , l 0.2 , and h 4.9 for the pvh ; and ap 0.3 , l 1.0 , and h 2.25 for the lateral ventricle . cannulas were anchored firmly to the skull . in experiments featuring administration of the mcr antagonist shu9119 , cannulas were implanted both into vmh and intracerebroventricularly . three days before determination of tissue glucose uptake , a silicone catheter was implanted into the external jugular vein . animals were handled repeatedly during the recovery period ( 2 weeks ) after cannula implantation to habituate them to the injection and blood - sampling procedures . correct placement of the cannula tips was verified microscopically in brain sections in all experiments , with > 90% of animals manifesting correct placement . tyrosine phosphorylation of signal transducer and activator of transcription 3 ( stat3 ) in each medial hypothalamic nucleus was examined after injection of leptin , as described below . all animal experiments were performed in accordance with institutional guidelines for the care and handling of experimental animals , and they were approved by the ethics committee for animal experiments of the national institute for physiological sciences . leptin ( 5 ng ) ( national hormones and pituitary program , torrance , ca ) or mt - ii ( 10 ng ) ( phoenix pharmaceuticals , burlingame , ca ) dissolved in 0.1 l physiological saline was injected with the use of a hamilton microsyringe into the right side of the vmh , dmh , pvh , or arc of freely moving mice through the unilateral cannula implanted into the corresponding nucleus . the concentrations of leptin ( 3 mol / l ) and mt - ii ( 100 mol / l ) dissolved in 0.1 l physiological saline injected into the medial hypothalamic nuclei are at least 10 times higher than those necessary for the maximum activation of leptin receptor and mcrs ( 2729 ) . alternatively , mt - ii ( 3 g ) in 0.5 l saline was injected into the lateral ventricle . shu9119 ( 1 g ) ( phoenix pharmaceuticals ) in 0.5 l saline was also injected intracerebroventricularly immediately before leptin injection . the dose of mt - ii and shu9119 injected intracerebroventricularly altered food intake significantly ( 30,31 ) . control animals received 0.1 l saline delivered into the various nuclei or 0.5 l saline delivered intracerebroventricularly , respectively . the rate constant of net tissue uptake of 2-[h]deoxy - d - glucose ( 2[h]dg ) in peripheral tissues was determined , as described previously ( 32,33 ) , by injecting a mixture of 6.25 ci 2[h]dg ( 10 ci / mmol ) and 1.25 ci [ c]sucrose ( 10 ci / mmol ) ( american radiolabeled chemicals , st . louis , mo ) through the jugular vein catheter 3 or 6 h after the microinjection of leptin or mt - ii . blood was collected 0 , 10 , 15 , and 20 min after injection of the radioactive tracers . immediately after collection of the final blood sample ( 20 min ) , an overdose of pentobarbital sodium ( 100 mg / kg ) was injected through the jugular vein catheter and the mice were rapidly decapitated . hypothalamic nuclei were then rapidly dissected as described below and frozen in liquid nitrogen for subsequent immunoblot analysis . skeletal muscle ( soleus , red and white portions of the gastrocnemius , and extensor digitorum longus [ edl ] ) as well as interscapular bat , heart , spleen , and epididymal white adipose tissue ( wat ) were rapidly dissected , weighed , and assayed for the radioactivity . plasma samples were also analyzed for glucose ( glucose cii test ; wako , osaka , japan ) and insulin ( mouse insulin elisa kit [ u - type ] ; shibayagi , gunma , japan ) concentrations . the rate constant of net tissue uptake of 2[h]dg was calculated as described previously ( 32,33 ) . the radioactivity of [ c]sucrose in tissues was used to calculate the 2[h]dg radioactivity remaining in the extracellular space ( 33 ) . the accuracy of the dissection was assessed by measurement of mrnas for neuropeptides or transcription factors such as corticotropin - releasing factor ( crf ) mrna for the pvh , pomc and npy mrnas for the arc , sf1 mrna for the vmh , and the absence of these various mrnas for the dmh . the right side of the pvh , arc , vmh , or dmh was dissected from a 1-mm - thick sagittal section prepared from the midline of the fresh brain ( online appendix supplemental fig the homogenates were centrifuged , and the resulting supernatants ( 5 g of protein ) were fractionated by sds - page . immunoblot analysis was then performed with antibodies ( 1 g / ml ) , including those to the tyr - phosphorylated or total forms of stat3 ( cell signaling technology , danvers , ma ) , to c - fos ( santa cruz biotechnology , santa cruz , ca ) , or to -actin ( cell signaling technology ) . immune complexes were visualized with horseradish peroxidase conjugated secondary antibodies ( santa cruz biotechnology ) and enhanced chemiluminescence reagents ( ge healthcare , tokyo , japan ) . protein bands were quantified using image j software ( national institutes of health , http://rsbweb.nih.gov/ij ) . total rna was isolated from hypothalamic and peripheral tissue with the use of isogen ( nippon gene , wako , japan ) , and portions of the rna ( 300 ng ) were subjected to reverse transcription with an oligo(dt ) primer and avian myeloblastosis virus reverse transcriptase ( takara , shiga , japan ) . the resulting cdna was subjected to the pcr with la taq ( takara ) and primers obtained from sigma genosys ( ishikari , japan ) . for quantitative real - time pcr , cdna was amplified using sybr green pcr master mix with an abi 7500 real - time pcr system ( applied biosystems , tokyo , japan ) . data were normalized by the amount of eukaryotic elongation factor 2 ( eef2 ) mrna . statistical analysis of stat3 phosphorylation , c - fos expression , and real - time pcr was performed by student t test , and analysis for other experiments was performed by anova followed by dunnett test . a p value of < 0.05 was considered statistically significant . leptin ( 5 ng ) ( national hormones and pituitary program , torrance , ca ) or mt - ii ( 10 ng ) ( phoenix pharmaceuticals , burlingame , ca ) dissolved in 0.1 l physiological saline was injected with the use of a hamilton microsyringe into the right side of the vmh , dmh , pvh , or arc of freely moving mice through the unilateral cannula implanted into the corresponding nucleus . the concentrations of leptin ( 3 mol / l ) and mt - ii ( 100 mol / l ) dissolved in 0.1 l physiological saline injected into the medial hypothalamic nuclei are at least 10 times higher than those necessary for the maximum activation of leptin receptor and mcrs ( 2729 ) . alternatively , mt - ii ( 3 g ) in 0.5 l saline was injected into the lateral ventricle . shu9119 ( 1 g ) ( phoenix pharmaceuticals ) in 0.5 l saline was also injected intracerebroventricularly immediately before leptin injection . the dose of mt - ii and shu9119 injected intracerebroventricularly altered food intake significantly ( 30,31 ) . control animals received 0.1 l saline delivered into the various nuclei or 0.5 l saline delivered intracerebroventricularly , respectively . the rate constant of net tissue uptake of 2-[h]deoxy - d - glucose ( 2[h]dg ) in peripheral tissues was determined , as described previously ( 32,33 ) , by injecting a mixture of 6.25 ci 2[h]dg ( 10 ci / mmol ) and 1.25 ci [ c]sucrose ( 10 ci / mmol ) ( american radiolabeled chemicals , st . louis , mo ) through the jugular vein catheter 3 or 6 h after the microinjection of leptin or mt - ii . blood was collected 0 , 10 , 15 , and 20 min after injection of the radioactive tracers . immediately after collection of the final blood sample ( 20 min ) , an overdose of pentobarbital sodium ( 100 mg / kg ) was injected through the jugular vein catheter and the mice were rapidly decapitated . hypothalamic nuclei were then rapidly dissected as described below and frozen in liquid nitrogen for subsequent immunoblot analysis . skeletal muscle ( soleus , red and white portions of the gastrocnemius , and extensor digitorum longus [ edl ] ) as well as interscapular bat , heart , spleen , and epididymal white adipose tissue ( wat ) were rapidly dissected , weighed , and assayed for the radioactivity . plasma samples were also analyzed for glucose ( glucose cii test ; wako , osaka , japan ) and insulin ( mouse insulin elisa kit [ u - type ] ; shibayagi , gunma , japan ) concentrations . the rate constant of net tissue uptake of 2[h]dg was calculated as described previously ( 32,33 ) . the radioactivity of [ c]sucrose in tissues was used to calculate the 2[h]dg radioactivity remaining in the extracellular space ( 33 ) . the accuracy of the dissection was assessed by measurement of mrnas for neuropeptides or transcription factors such as corticotropin - releasing factor ( crf ) mrna for the pvh , pomc and npy mrnas for the arc , sf1 mrna for the vmh , and the absence of these various mrnas for the dmh . the right side of the pvh , arc , vmh , or dmh was dissected from a 1-mm - thick sagittal section prepared from the midline of the fresh brain ( online appendix supplemental fig . 1 [ available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0638/dc1 ] ) . the homogenates were centrifuged , and the resulting supernatants ( 5 g of protein ) were fractionated by sds - page . immunoblot analysis was then performed with antibodies ( 1 g / ml ) , including those to the tyr - phosphorylated or total forms of stat3 ( cell signaling technology , danvers , ma ) , to c - fos ( santa cruz biotechnology , santa cruz , ca ) , or to -actin ( cell signaling technology ) . immune complexes were visualized with horseradish peroxidase conjugated secondary antibodies ( santa cruz biotechnology ) and enhanced chemiluminescence reagents ( ge healthcare , tokyo , japan ) . protein bands were quantified using image j software ( national institutes of health , http://rsbweb.nih.gov/ij ) . total rna was isolated from hypothalamic and peripheral tissue with the use of isogen ( nippon gene , wako , japan ) , and portions of the rna ( 300 ng ) were subjected to reverse transcription with an oligo(dt ) primer and avian myeloblastosis virus reverse transcriptase ( takara , shiga , japan ) . the resulting cdna was subjected to the pcr with la taq ( takara ) and primers obtained from sigma genosys ( ishikari , japan ) . for quantitative real - time pcr , cdna was amplified using sybr green pcr master mix with an abi 7500 real - time pcr system ( applied biosystems , tokyo , japan ) . data were normalized by the amount of eukaryotic elongation factor 2 ( eef2 ) mrna . c - fos expression , and real - time pcr was performed by student t test , and analysis for other experiments was performed by anova followed by dunnett test . microinjection of leptin ( 5 ng ) into the vmh induced a significant increase in the rate constant of 2[h]dg uptake in the red type of skeletal muscle ( soleus and red portion of gastrocnemius ) , mixed type of skeletal muscle ( edl ) , bat , and heart but not in spleen or epididymal wat ( fig . 1 ) . glucose uptake in red or mixed skeletal muscle was significantly increased at 6 h after leptin injection ( fig . 1a ) , whereas that in bat and heart was increased at both 3 and 6 h ( fig . glucose uptake in the white portion of the gastrocnemius showed a tendency to increase in response to leptin , but the change was not statistically significant . glucose uptake in peripheral tissues at 3 h after saline injection into the vmh did not differ from that apparent at 6 h ( data not shown ) . injection of leptin into the vmh significantly increased glut4 mrna in bat and heart but not in soleus muscle at 6 h after leptin injection ( fig . hexokinase ii mrna did not change in soleus , bat , or heart ( fig . effects of leptin injection into the vmh on glucose uptake and gene expression in peripheral tissues . the rate constant of 2[h]dg uptake was measured in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) at 3 and 6 h after injection of leptin or saline ( control ) into the vmh of mice . , saline vmh ( 6 h ) ; , leptin vmh ( 3 h ) ; , leptin vmh ( 6 h ) . e : the mrna levels of glut4 , hexokinase ii ( hkii ) , and ucp1 in soleus , bat , and heart were quantified by real - time pcr . values are normalized by the level of eef2 mrna . gastro - r , red portion of gastrocnemius ; gastro - w , white portion of gastrocnemius . p < 0.05 vs. the corresponding value for saline - injected controls . , saline vmh ( 6 h ) ; , leptin vmh ( 6 h ) . injection of maximal dose of leptin into the arc induced a small but significant increase in the rate constant of 2[h]dg uptake in bat at 6 h after injection , but it had no effect on glucose uptake in skeletal muscle , heart , spleen , or wat ( fig . injection of leptin into the dmh or pvh had no effect on glucose uptake in peripheral tissues ( fig . 2 ) . glucose uptake in peripheral tissues after saline injection into the dmh or pvh did not differ from that apparent after saline injection into the arc ( data not shown ) . plasma glucose and insulin concentrations were not affected by leptin injection into the vmh or other hypothalamic nuclei ( supplemental table 2 ) , consistent with previous observations ( 13,15 ) . effects of leptin injection into the arc , dmh , or pvh on glucose uptake in peripheral tissues . the rate constant of 2[h]dg uptake was measured in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) at 6 h after injection of leptin into the hypothalamic nuclei of mice . data are means se for six or seven mice . * p < 0.05 vs. the corresponding value for control animals injected with saline into arc . , saline arc ; , leptin arc ; leptin dmh ; , leptin pvh . to determine whether leptin activated ob - rb in the hypothalamic nuclei , we examined the tyrosine phosphorylation of stat3 in tissue samples therefrom . rt - pcr analysis confirmed that the isolated pvh , arc , and vmh specimens were enriched in crf mrna , pomc and npy mrnas , and sf1 mrna , respectively , and that the dmh was largely devoid of these mrnas ( fig . microinjection of leptin into the vmh , dmh , or pvh preferentially increased the tyrosine phosphorylation of stat3 to the similar extent in the corresponding nucleus at 6 h after injection ( fig . injection of leptin into the arc increased the tyrosine phosphorylation of stat3 in the dmh as well as in the arc ( fig . 3e ) , possibly as a result of leakage of leptin into the dmh through the surface of the injection cannula , given that the arc cannula passed through the dmh . together , these results suggested that the vmh is a key target of leptin in its regulation of glucose uptake in skeletal muscle , heart , and bat , whereas the leptin receptor in the arc mediates stimulation of glucose uptake in bat . effects of leptin injection into medial hypothalamic nuclei on tyrosine phosphorylation of stat3 and c - fos expression . a : rt - pcr analysis of crf , pomc , npy , sf1 , and eef2 ( loading control ) mrnas in the pvh , arc , vmh , and dmh . b e : immunoblot analysis of the phosphorylation of stat3 on tyr in the medial hypothalamic nuclei at 6 h after leptin injection into the vmh ( b ) , dmh ( c ) , pvh ( d ) , or arc ( e ) . leptin was injected into the right side of the hypothalamic nuclei , and the same side of the pvh ( p ) , arc ( a ) , vmh ( v ) , and dmh ( d ) was collected . representative blots of tyr - phosphorylated stat3 are shown in the upper panels , and quantitative data ( means se ) from four to six mice are shown in the lower panels . the amount of tyr -phosphorylated stat3 was normalized by that of total stat3 , and the normalized values were expressed relative to the corresponding value for the pvh of control mice . f : immunoblot analysis of c - fos expression in the medial hypothalamic nuclei after leptin injection into the vmh . the right side of the pvh , arc , vmh , and dmh was collected at 6 h after injection of leptin or saline into the same side of the vmh . a representative blot is shown in the upper panel , and quantitative data ( means se ) from four to six mice are shown in the lower panel . the amount of c - fos was normalized by that of -actin , and the normalized values were expressed relative to the corresponding value for the saline - injected control mice . * p < 0.05 . we next examined whether leptin injection into the vmh might increase neuronal activity in other hypothalamic nuclei by measuring expression of the transcription factor c - fos . leptin injection into the vmh significantly increased c - fos expression in the arc as well as in the vmh at 6 h after injection ( fig . leptin injection into the vmh increased the phosphorylation of stat3 in the nucleus preferentially at 1 h after the injection , similar to that at 6 h. however , it did not increase c - fos expression in any nucleus at 1 h ( supplemental fig . leptin injection into the vmh did not increase pomc mrna in the arc at 6 h after injection ( supplemental fig . we next examined the role of mcrs in glucose uptake in peripheral tissues induced by injection of leptin into the vmh ( fig . injection of the mcr antagonist shu9119 ( 1 g ) into the lateral ventricle ( intracerebroventricularly ) abolished the increase in 2[h]dg uptake in peripheral tissues normally apparent at 6 h after the injection of leptin into the vmh . injection of shu9119 alone did not affect glucose uptake in peripheral tissues . plasma glucose and insulin levels were also not changed in response to intracerebroventricular injection of shu9119 ( supplemental table 2 ) . effect of intracerebroventricular injection of shu9119 on glucose uptake in peripheral tissues induced by injection of leptin into the vmh . the rate constant of 2[h]dg uptake in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) was measured 6 h after injection of leptin or saline ( control ) into the vmh . we tested the effects of intracerebroventricular injection of the mcr agonist mt - ii on glucose uptake in peripheral tissues ( fig . the intracerebroventricular injection of mt - ii ( 3 g ) increased 2[h]dg uptake in bat , heart , and all types of skeletal muscle , including the white portion of the gastrocnemius , but not in spleen or epididymal wat , at 3 or 6 h after injection . glucose uptake in peripheral tissues at 3 h after saline injection did not differ from that apparent at 6 h ( data not shown ) . the intracerebroventricular injection of mt - ii increased the plasma glucose level at both 3 and 6 h after injection ( supplemental table 2 ) . these results thus suggested that intracerebroventricular injection of mt - ii promotes glucose production as well as glucose uptake in certain peripheral tissues . in contrast , plasma insulin concentration did not change after intracerebroventricular injection of mt - ii , despite the associated hyperglycemia , suggesting that mt - ii inhibits insulin secretion from pancreatic -cells . the rate constant of 2[h]dg uptake in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) was measured at 3 or 6 h after intracerebroventricular injection of mt - ii or saline ( control ) . finally , we examined the effects of direct injection of mt - ii into the individual medial hypothalamic nuclei on glucose uptake in peripheral tissues ( fig . microinjection of mt - ii ( 10 ng ) into the vmh increased 2[h]dg uptake in bat , heart , and all types of skeletal muscle but not in spleen or epididymal wat . in contrast , injection of mt - ii into the pvh increased glucose uptake only in bat , and that into the dmh or arc did not affect glucose uptake in any of the peripheral tissues examined . plasma glucose and insulin levels did not change in response to injection of mt - ii into any of the hypothalamic nuclei ( supplemental table 2 ) . effects of mt - ii injection into vmh , pvh , dmh , or arc on glucose uptake in peripheral tissues . the rate constant of 2[h]dg uptake in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) was measured at 6 h after the injection of mt - ii into the individual hypothalamic nuclei . , vmh ; , mt - ii vmh ; , mt - ii pvh ; , mt - ii dmh ; , mt - ii arc . microinjection of leptin ( 5 ng ) into the vmh induced a significant increase in the rate constant of 2[h]dg uptake in the red type of skeletal muscle ( soleus and red portion of gastrocnemius ) , mixed type of skeletal muscle ( edl ) , bat , and heart but not in spleen or epididymal wat ( fig . 1 ) . glucose uptake in red or mixed skeletal muscle was significantly increased at 6 h after leptin injection ( fig . 1a ) , whereas that in bat and heart was increased at both 3 and 6 h ( fig . glucose uptake in the white portion of the gastrocnemius showed a tendency to increase in response to leptin , but the change was not statistically significant . glucose uptake in peripheral tissues at 3 h after saline injection into the vmh did not differ from that apparent at 6 h ( data not shown ) . injection of leptin into the vmh significantly increased glut4 mrna in bat and heart but not in soleus muscle at 6 h after leptin injection ( fig . hexokinase ii mrna did not change in soleus , bat , or heart ( fig . effects of leptin injection into the vmh on glucose uptake and gene expression in peripheral tissues . the rate constant of 2[h]dg uptake was measured in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) at 3 and 6 h after injection of leptin or saline ( control ) into the vmh of mice . , saline vmh ( 6 h ) ; , leptin vmh ( 3 h ) ; , leptin vmh ( 6 h ) . e : the mrna levels of glut4 , hexokinase ii ( hkii ) , and ucp1 in soleus , bat , and heart were quantified by real - time pcr . values are normalized by the level of eef2 mrna . gastro - r , red portion of gastrocnemius ; gastro - w , white portion of gastrocnemius . p < 0.05 vs. the corresponding value for saline - injected controls . , saline vmh ( 6 h ) ; , leptin vmh ( 6 h ) . injection of maximal dose of leptin into the arc induced a small but significant increase in the rate constant of 2[h]dg uptake in bat at 6 h after injection , but it had no effect on glucose uptake in skeletal muscle , heart , spleen , or wat ( fig . injection of leptin into the dmh or pvh had no effect on glucose uptake in peripheral tissues ( fig . 2 ) . glucose uptake in peripheral tissues after saline injection into the dmh or pvh did not differ from that apparent after saline injection into the arc ( data not shown ) . plasma glucose and insulin concentrations were not affected by leptin injection into the vmh or other hypothalamic nuclei ( supplemental table 2 ) , consistent with previous observations ( 13,15 ) . effects of leptin injection into the arc , dmh , or pvh on glucose uptake in peripheral tissues . the rate constant of 2[h]dg uptake was measured in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) at 6 h after injection of leptin into the hypothalamic nuclei of mice . data are means se for six or seven mice . * p < 0.05 vs. the corresponding value for control animals injected with saline into arc . , saline arc ; , leptin arc ; leptin dmh ; , leptin pvh . to determine whether leptin activated ob - rb in the hypothalamic nuclei , we examined the tyrosine phosphorylation of stat3 in tissue samples therefrom . rt - pcr analysis confirmed that the isolated pvh , arc , and vmh specimens were enriched in crf mrna , pomc and npy mrnas , and sf1 mrna , respectively , and that the dmh was largely devoid of these mrnas ( fig . microinjection of leptin into the vmh , dmh , or pvh preferentially increased the tyrosine phosphorylation of stat3 to the similar extent in the corresponding nucleus at 6 h after injection ( fig . injection of leptin into the arc increased the tyrosine phosphorylation of stat3 in the dmh as well as in the arc ( fig . 3e ) , possibly as a result of leakage of leptin into the dmh through the surface of the injection cannula , given that the arc cannula passed through the dmh . together , these results suggested that the vmh is a key target of leptin in its regulation of glucose uptake in skeletal muscle , heart , and bat , whereas the leptin receptor in the arc mediates stimulation of glucose uptake in bat . effects of leptin injection into medial hypothalamic nuclei on tyrosine phosphorylation of stat3 and c - fos expression . a : rt - pcr analysis of crf , pomc , npy , sf1 , and eef2 ( loading control ) mrnas in the pvh , arc , vmh , and dmh . b e : immunoblot analysis of the phosphorylation of stat3 on tyr in the medial hypothalamic nuclei at 6 h after leptin injection into the vmh ( b ) , dmh ( c ) , pvh ( d ) , or arc ( e ) . leptin was injected into the right side of the hypothalamic nuclei , and the same side of the pvh ( p ) , arc ( a ) , vmh ( v ) , and dmh ( d ) was collected . representative blots of tyr - phosphorylated stat3 are shown in the upper panels , and quantitative data ( means se ) from four to six mice are shown in the lower panels . the amount of tyr -phosphorylated stat3 was normalized by that of total stat3 , and the normalized values were expressed relative to the corresponding value for the pvh of control mice . f : immunoblot analysis of c - fos expression in the medial hypothalamic nuclei after leptin injection into the vmh . the right side of the pvh , arc , vmh , and dmh was collected at 6 h after injection of leptin or saline into the same side of the vmh . a representative blot is shown in the upper panel , and quantitative data ( means se ) from four to six mice are shown in the lower panel . the amount of c - fos was normalized by that of -actin , and the normalized values were expressed relative to the corresponding value for the saline - injected control mice . * p < 0.05 . we next examined whether leptin injection into the vmh might increase neuronal activity in other hypothalamic nuclei by measuring expression of the transcription factor c - fos . leptin injection into the vmh significantly increased c - fos expression in the arc as well as in the vmh at 6 h after injection ( fig . leptin injection into the vmh increased the phosphorylation of stat3 in the nucleus preferentially at 1 h after the injection , similar to that at 6 h. however , it did not increase c - fos expression in any nucleus at 1 h ( supplemental fig . leptin injection into the vmh did not increase pomc mrna in the arc at 6 h after injection ( supplemental fig . we next examined the role of mcrs in glucose uptake in peripheral tissues induced by injection of leptin into the vmh ( fig . 4 ) . injection of the mcr antagonist shu9119 ( 1 g ) into the lateral ventricle ( intracerebroventricularly ) abolished the increase in 2[h]dg uptake in peripheral tissues normally apparent at 6 h after the injection of leptin into the vmh . injection of shu9119 alone did not affect glucose uptake in peripheral tissues . plasma glucose and insulin levels were also not changed in response to intracerebroventricular injection of shu9119 ( supplemental table 2 ) . effect of intracerebroventricular injection of shu9119 on glucose uptake in peripheral tissues induced by injection of leptin into the vmh . the rate constant of 2[h]dg uptake in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) was measured 6 h after injection of leptin or saline ( control ) into the vmh . , saline vmh ; , shu9119 i.c.v . ; , leptin vmh ; , leptin vmh + shu9119 i.c.v . we tested the effects of intracerebroventricular injection of the mcr agonist mt - ii on glucose uptake in peripheral tissues ( fig . the intracerebroventricular injection of mt - ii ( 3 g ) increased 2[h]dg uptake in bat , heart , and all types of skeletal muscle , including the white portion of the gastrocnemius , but not in spleen or epididymal wat , at 3 or 6 h after injection . glucose uptake in peripheral tissues at 3 h after saline injection did not differ from that apparent at 6 h ( data not shown ) . the intracerebroventricular injection of mt - ii increased the plasma glucose level at both 3 and 6 h after injection ( supplemental table 2 ) . these results thus suggested that intracerebroventricular injection of mt - ii promotes glucose production as well as glucose uptake in certain peripheral tissues . in contrast , plasma insulin concentration did not change after intracerebroventricular injection of mt - ii , despite the associated hyperglycemia , suggesting that mt - ii inhibits insulin secretion from pancreatic -cells . the rate constant of 2[h]dg uptake in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) was measured at 3 or 6 h after intracerebroventricular injection of mt - ii or saline ( control ) . finally , we examined the effects of direct injection of mt - ii into the individual medial hypothalamic nuclei on glucose uptake in peripheral tissues ( fig . 6 ) . microinjection of mt - ii ( 10 ng ) into the vmh increased 2[h]dg uptake in bat , heart , and all types of skeletal muscle but not in spleen or epididymal wat . in contrast , injection of mt - ii into the pvh increased glucose uptake only in bat , and that into the dmh or arc did not affect glucose uptake in any of the peripheral tissues examined . plasma glucose and insulin levels did not change in response to injection of mt - ii into any of the hypothalamic nuclei ( supplemental table 2 ) . effects of mt - ii injection into vmh , pvh , dmh , or arc on glucose uptake in peripheral tissues . the rate constant of 2[h]dg uptake in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) was measured at 6 h after the injection of mt - ii into the individual hypothalamic nuclei . data are means sem for six or seven mice . * p < 0.05 vs. the corresponding value for saline - injected control animals . , vmh ; , mt - ii vmh ; , mt - ii pvh ; , mt - ii dmh ; , mt - ii arc . leptin is a physiologically and clinically important hormone that regulates glucose metabolism in peripheral tissues . we have previously shown that microinjection of leptin into the vmh and nearby medial hypothalamic area preferentially increased glucose uptake in skeletal muscle , heart , and bat ( 1416 ) . in the present study , we found that activation of the leptin receptor specifically in the vmh , as detected by measurement of the tyrosine phosphorylation of stat3 , resulted in a marked increase in glucose uptake in those peripheral tissues , similar to the effects of intracerebroventricular or peripheral administration of leptin ( 13,36 ) . in contrast , injection of leptin into the arc increased glucose uptake only in bat . injection of leptin into the dmh or pvh had no effect on glucose uptake in any of the peripheral tissues examined . the present data thus suggest that the leptin receptor in the vmh and arc regulates glucose uptake in different peripheral tissues . our present results further indicate that mcr activation is necessary for the increase in glucose uptake in peripheral tissues induced by injection of leptin into the vmh . the intracerebroventricular injection of shu9119 thus abolished the effect of leptin injected into the vmh on peripheral glucose uptake , whereas intracerebroventricular injection of mt - ii increased glucose uptake in peripheral tissues . moreover , injection of mt - ii into vmh , but not that into the dmh or arc , increased glucose uptake in skeletal muscle , heart , and bat , whereas injection of mt - ii into the pvh increased glucose uptake preferentially in bat . both mcrs in the vmh and pvh may play an important role in the regulation of glucose uptake in peripheral tissues . pomc neurons in the arc receive strong excitatory input from the dorsomedial region of the vmh ( 37 ) . the restoration of ob - rb expression in the vmh by adeno - associated virus mediated gene transfer in koletsky rats increased the amount of pomc mrna in the arc ( 20 ) . we have now shown that injection of leptin into the vmh increased c - fos expression in the arc without an effect on stat3 phosphorylation in the arc . furthermore , the effect of leptin injected into the vmh on glucose uptake in peripheral tissues was found to be dependent on mcrs in the brain . we therefore propose that stimulation of vmh neurons by leptin results in activation of a set of pomc neurons in the arc and thereby increases glucose uptake in skeletal muscle , heart , and bat . mcrs in the vmh and pvh contribute to the upregulation of glucose uptake in peripheral tissues induced by injection of leptin into the vmh . although c - fos expression did not increase in the pvh in response to leptin injection into the vmh , this may have been due to the operation of -aminobutyric acid ( gaba)-mediated neurotransmission in the pvh ( 6 ) . the mechanism by which injection of leptin into the arc increased glucose uptake specifically in bat remains unclear . one possible explanation for this observation is that the injection of leptin into the arc activated a selective set of pomc neurons in the arc that regulate glucose uptake in bat alone , with pomc neurons being abundant in both the anterior and posterior regions of the arc ( 38 ) . it is also possible that pomc neurons in the arc that regulate glucose uptake in skeletal muscle and heart require excitatory input as well as leptin for their full activation . the intracerebroventricular injection of mt - ii increased glucose uptake in bat to a markedly greater extent than did the maximum dose of injection of mt - ii into the vmh or pvh . mc4r - expressing neurons in several regions of the brain stem as well as in the hypothalamic nuclei connect polysynaptically with interscapular bat ( 39 ) . direct injection of mt - ii into the raphe pallidus induces a thermogenic response in interscapular bat ( 40 ) . glucose uptake in bat might thus be regulated by mcrs in multiple brain regions . to date , there is little histological evidence of a connection between vmh neurons and interscapular bat , although injection of leptin into the vmh increases plasma catecholamine levels more effectively than does that into other hypothalamic regions ( 41 ) . we previously showed that injection of leptin into the medial hypothalamus increased glucose uptake in peripheral tissues through the activation of sympathetic nerves ( 14,15 ) . injection of leptin into the medial hypothalamus also increased insulin sensitivity in peripheral tissues by a -adrenergic mechanism ( 14 ) . whereas the molecular mechanism remains elusive , a direct effect of -adrenergic receptors expressed in peripheral tissues as well as a -adrenergic receptor dependent increase in blood flow appear to contribute to leptin - induced glucose uptake in these tissues . the dmh is an important hypothalamic nucleus in the regulation of thermogenesis in bat ( 42 ) . injection of a gaba typea ( gabaa ) receptor antagonist into the dmh thus increased thermogenic activity in bat ( 42 ) . moreover , injection of a gabaa receptor agonist into the dmh blocked sympathetic , thermogenic , and cardiovascular responses induced either by injection of prostaglandin e2 into the medial preoptic area ( 43 ) or by skin cooling ( 44 ) . both ob - rb and mc4r are abundant in the dmh ( 7 ) . furthermore , dmh neurons , including those expressing mc4r , connect polysynaptically with interscapular bat ( 39 ) . however , we have now shown that injection of leptin or mt - ii into the dmh did not increase glucose uptake in the peripheral tissues examined . although we can not exclude the possibility that injection of leptin or mt - ii activated only a subset of dmh neurons expressing ob - rb or mcr , our results suggest that the leptin receptor and mcr in the dmh have other roles , such as the regulation of food intake or modulation of bat thermogenesis in response to cold stimuli . our present results indicate that the vmh regulates glucose uptake in skeletal muscle as well as in heart and bat , accompanying increased glut4 mrna in bat and heart . thus , increased expression of glut4 may involve the acute increase in glucose uptake in bat and heart in response to leptin , while other mechanism might be involved in the leptin - induced glucose uptake in skeletal muscle . intracerebroventricular injection of mt - ii has been shown to increase glut4 mrna expression in skeletal muscle at 24 h after the injection ( 22 ) . gene expression in skeletal muscle in response to leptin and mt - ii may require > 24 h. the present results showed that injection of leptin or mt - ii in some medial hypothalamic nuclei increased glucose uptake in certain peripheral tissues , while it did not alter plasma glucose level . these results suggest that leptin and mt - ii stimulates hepatic glucose production as well as glucose utilization in peripheral tissues . the result was supported by the previous report ( 13 ) showing that leptin increased plasma glucose turnover in mice at 6 h after the injection . moreover , gutierrez - juarez et al . ( 45 ) has shown that intracerebroventricular infusion of mcr agonist increased hepatic glucose production in lean rats at 6 h after the start of the infusion , while it inhibited hepatic glucose production at 7 days ( 46 ) . these results suggest that the effects of leptin and melanocortin receptor agonist on hepatic glucose production alter time dependently . recent study suggests that mt - ii seems to be a promising agent to bypass leptin resistance and to improve energy homeostasis in diet - induced obese mice ( 47 ) . we have shown that intracerebroventricular injection of mt - ii but not leptin activates amp - activated protein kinase in skeletal muscle in diet - induced obese mice ( 48 ) . the pvh and vmh may be target sites of mt - ii to improve glucose metabolism in these mice . together , our results suggest that the vmh plays a key role in leptin- and mt - ii induced glucose uptake in skeletal muscle , heart , and bat , whereas the leptin receptor in the arc and mcrs in the pvh regulate glucose uptake in bat . the medial hypothalamic nuclei thus appear to play distinct roles in the regulation of glucose uptake in peripheral tissues by leptin and mt - ii .

objectivethe medial hypothalamus mediates leptin - induced glucose uptake in peripheral tissues , and brain melanocortin receptors ( mcrs ) mediate certain central effects of leptin . however , the contributions of the leptin receptor and mcrs in individual medial hypothalamic nuclei to regulation of peripheral glucose uptake have remained unclear . we examined the effects of an injection of leptin and the mcr agonist mt - ii into medial hypothalamic nuclei on glucose uptake in peripheral tissues.research design and methodsleptin or mt - ii was injected into the ventromedial ( vmh ) , dorsomedial ( dmh ) , arcuate nucleus ( arc ) , or paraventricular ( pvh ) hypothalamus or the lateral ventricle ( intracerebroventricularly ) in freely moving mice . the mcr antagonist shu9119 was injected intracerebroventricularly . glucose uptake was measured by the 2-[3h]deoxy - d - glucose method.resultsleptin injection into the vmh increased glucose uptake in skeletal muscle , brown adipose tissue ( bat ) , and heart , whereas that into the arc increased glucose uptake in bat , and that into the dmh or pvh had no effect . shu9119 abolished these effects of leptin injected into the vmh . injection of mt - ii either into the vmh or intracerebroventricularly increased glucose uptake in skeletal muscle , bat , and heart , whereas that into the pvh increased glucose uptake in bat , and that into the dmh or arc had no effect.conclusionsthe vmh mediates leptin- and mt - ii induced glucose uptake in skeletal muscle , bat , and heart . these effects of leptin are dependent on mcr activation . the leptin receptor in the arc and mcr in the pvh regulate glucose uptake in bat . medial hypothalamic nuclei thus play distinct roles in leptin- and mt - ii induced glucose uptake in peripheral tissues .

RESEARCH DESIGN AND METHODS Administration of leptin, MT-II, and SHU9119. Measurement of the rate constant of 2-[ Sampling of medial hypothalamic nuclei. Immunoblot analysis. RNA extraction, RT-PCR analysis, and quantitative real-time PCR. Statistical analysis. RESULTS Effects of leptin injection into medial hypothalamic nuclei on glucose uptake in peripheral tissues. Effect of intracerebroventricular injection of an MCR antagonist on glucose uptake in peripheral tissues induced by injection of leptin into the VMH. Effects of intracerebroventricular injection of an MCR agonist on glucose uptake in peripheral tissues. Effects of MT-II injected into medial hypothalamic nuclei on glucose uptake in peripheral tissues. DISCUSSION Supplementary Material

mice were anesthetized by intraperitoneal injection of ketamine ( 100 mg / kg body mass ) and xylazine ( 10 mg / kg ) , and a chronic double - walled stainless steel cannula was implanted stereotaxically and unilaterally into the right side of the vmh , arc , dmh , or pvh or into the lateral ventricle according to the atlas of franklin and paxinos ( 26 ) . the stereotaxic coordinates were ap 1.3 ( 1.3 mm anterior to the bregma ) , l 0.3 ( 0.3 mm lateral to the bregma ) , and h 5.8 ( 5.8 mm below the bregma on the surface of the skull ) for the vmh ; ap 1.6 , l 0.2 , and h 6.1 for the arc ; ap 1.6 , l 0.3 , and h 5.5 for the dmh ; ap 0.75 , l 0.2 , and h 4.9 for the pvh ; and ap 0.3 , l 1.0 , and h 2.25 for the lateral ventricle . leptin ( 5 ng ) ( national hormones and pituitary program , torrance , ca ) or mt - ii ( 10 ng ) ( phoenix pharmaceuticals , burlingame , ca ) dissolved in 0.1 l physiological saline was injected with the use of a hamilton microsyringe into the right side of the vmh , dmh , pvh , or arc of freely moving mice through the unilateral cannula implanted into the corresponding nucleus . the concentrations of leptin ( 3 mol / l ) and mt - ii ( 100 mol / l ) dissolved in 0.1 l physiological saline injected into the medial hypothalamic nuclei are at least 10 times higher than those necessary for the maximum activation of leptin receptor and mcrs ( 2729 ) . alternatively , mt - ii ( 3 g ) in 0.5 l saline was injected into the lateral ventricle . leptin ( 5 ng ) ( national hormones and pituitary program , torrance , ca ) or mt - ii ( 10 ng ) ( phoenix pharmaceuticals , burlingame , ca ) dissolved in 0.1 l physiological saline was injected with the use of a hamilton microsyringe into the right side of the vmh , dmh , pvh , or arc of freely moving mice through the unilateral cannula implanted into the corresponding nucleus . the concentrations of leptin ( 3 mol / l ) and mt - ii ( 100 mol / l ) dissolved in 0.1 l physiological saline injected into the medial hypothalamic nuclei are at least 10 times higher than those necessary for the maximum activation of leptin receptor and mcrs ( 2729 ) . alternatively , mt - ii ( 3 g ) in 0.5 l saline was injected into the lateral ventricle . the accuracy of the dissection was assessed by measurement of mrnas for neuropeptides or transcription factors such as corticotropin - releasing factor ( crf ) mrna for the pvh , pomc and npy mrnas for the arc , sf1 mrna for the vmh , and the absence of these various mrnas for the dmh . microinjection of leptin ( 5 ng ) into the vmh induced a significant increase in the rate constant of 2[h]dg uptake in the red type of skeletal muscle ( soleus and red portion of gastrocnemius ) , mixed type of skeletal muscle ( edl ) , bat , and heart but not in spleen or epididymal wat ( fig . glucose uptake in peripheral tissues at 3 h after saline injection into the vmh did not differ from that apparent at 6 h ( data not shown ) . effects of leptin injection into the vmh on glucose uptake and gene expression in peripheral tissues . the rate constant of 2[h]dg uptake was measured in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) at 3 and 6 h after injection of leptin or saline ( control ) into the vmh of mice . injection of maximal dose of leptin into the arc induced a small but significant increase in the rate constant of 2[h]dg uptake in bat at 6 h after injection , but it had no effect on glucose uptake in skeletal muscle , heart , spleen , or wat ( fig . injection of leptin into the dmh or pvh had no effect on glucose uptake in peripheral tissues ( fig . glucose uptake in peripheral tissues after saline injection into the dmh or pvh did not differ from that apparent after saline injection into the arc ( data not shown ) . effects of leptin injection into the arc , dmh , or pvh on glucose uptake in peripheral tissues . the rate constant of 2[h]dg uptake was measured in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) at 6 h after injection of leptin into the hypothalamic nuclei of mice . together , these results suggested that the vmh is a key target of leptin in its regulation of glucose uptake in skeletal muscle , heart , and bat , whereas the leptin receptor in the arc mediates stimulation of glucose uptake in bat . b e : immunoblot analysis of the phosphorylation of stat3 on tyr in the medial hypothalamic nuclei at 6 h after leptin injection into the vmh ( b ) , dmh ( c ) , pvh ( d ) , or arc ( e ) . leptin was injected into the right side of the hypothalamic nuclei , and the same side of the pvh ( p ) , arc ( a ) , vmh ( v ) , and dmh ( d ) was collected . the right side of the pvh , arc , vmh , and dmh was collected at 6 h after injection of leptin or saline into the same side of the vmh . we next examined the role of mcrs in glucose uptake in peripheral tissues induced by injection of leptin into the vmh ( fig . injection of the mcr antagonist shu9119 ( 1 g ) into the lateral ventricle ( intracerebroventricularly ) abolished the increase in 2[h]dg uptake in peripheral tissues normally apparent at 6 h after the injection of leptin into the vmh . effect of intracerebroventricular injection of shu9119 on glucose uptake in peripheral tissues induced by injection of leptin into the vmh . the rate constant of 2[h]dg uptake in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) was measured 6 h after injection of leptin or saline ( control ) into the vmh . we tested the effects of intracerebroventricular injection of the mcr agonist mt - ii on glucose uptake in peripheral tissues ( fig . the intracerebroventricular injection of mt - ii ( 3 g ) increased 2[h]dg uptake in bat , heart , and all types of skeletal muscle , including the white portion of the gastrocnemius , but not in spleen or epididymal wat , at 3 or 6 h after injection . these results thus suggested that intracerebroventricular injection of mt - ii promotes glucose production as well as glucose uptake in certain peripheral tissues . the rate constant of 2[h]dg uptake in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) was measured at 3 or 6 h after intracerebroventricular injection of mt - ii or saline ( control ) . finally , we examined the effects of direct injection of mt - ii into the individual medial hypothalamic nuclei on glucose uptake in peripheral tissues ( fig . microinjection of mt - ii ( 10 ng ) into the vmh increased 2[h]dg uptake in bat , heart , and all types of skeletal muscle but not in spleen or epididymal wat . in contrast , injection of mt - ii into the pvh increased glucose uptake only in bat , and that into the dmh or arc did not affect glucose uptake in any of the peripheral tissues examined . plasma glucose and insulin levels did not change in response to injection of mt - ii into any of the hypothalamic nuclei ( supplemental table 2 ) . effects of mt - ii injection into vmh , pvh , dmh , or arc on glucose uptake in peripheral tissues . the rate constant of 2[h]dg uptake in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) was measured at 6 h after the injection of mt - ii into the individual hypothalamic nuclei . microinjection of leptin ( 5 ng ) into the vmh induced a significant increase in the rate constant of 2[h]dg uptake in the red type of skeletal muscle ( soleus and red portion of gastrocnemius ) , mixed type of skeletal muscle ( edl ) , bat , and heart but not in spleen or epididymal wat ( fig . glucose uptake in peripheral tissues at 3 h after saline injection into the vmh did not differ from that apparent at 6 h ( data not shown ) . effects of leptin injection into the vmh on glucose uptake and gene expression in peripheral tissues . the rate constant of 2[h]dg uptake was measured in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) at 3 and 6 h after injection of leptin or saline ( control ) into the vmh of mice . injection of maximal dose of leptin into the arc induced a small but significant increase in the rate constant of 2[h]dg uptake in bat at 6 h after injection , but it had no effect on glucose uptake in skeletal muscle , heart , spleen , or wat ( fig . injection of leptin into the dmh or pvh had no effect on glucose uptake in peripheral tissues ( fig . glucose uptake in peripheral tissues after saline injection into the dmh or pvh did not differ from that apparent after saline injection into the arc ( data not shown ) . effects of leptin injection into the arc , dmh , or pvh on glucose uptake in peripheral tissues . the rate constant of 2[h]dg uptake was measured in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) at 6 h after injection of leptin into the hypothalamic nuclei of mice . injection of leptin into the arc increased the tyrosine phosphorylation of stat3 in the dmh as well as in the arc ( fig . together , these results suggested that the vmh is a key target of leptin in its regulation of glucose uptake in skeletal muscle , heart , and bat , whereas the leptin receptor in the arc mediates stimulation of glucose uptake in bat . b e : immunoblot analysis of the phosphorylation of stat3 on tyr in the medial hypothalamic nuclei at 6 h after leptin injection into the vmh ( b ) , dmh ( c ) , pvh ( d ) , or arc ( e ) . leptin was injected into the right side of the hypothalamic nuclei , and the same side of the pvh ( p ) , arc ( a ) , vmh ( v ) , and dmh ( d ) was collected . the right side of the pvh , arc , vmh , and dmh was collected at 6 h after injection of leptin or saline into the same side of the vmh . we next examined the role of mcrs in glucose uptake in peripheral tissues induced by injection of leptin into the vmh ( fig . injection of the mcr antagonist shu9119 ( 1 g ) into the lateral ventricle ( intracerebroventricularly ) abolished the increase in 2[h]dg uptake in peripheral tissues normally apparent at 6 h after the injection of leptin into the vmh . effect of intracerebroventricular injection of shu9119 on glucose uptake in peripheral tissues induced by injection of leptin into the vmh . the rate constant of 2[h]dg uptake in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) was measured 6 h after injection of leptin or saline ( control ) into the vmh . we tested the effects of intracerebroventricular injection of the mcr agonist mt - ii on glucose uptake in peripheral tissues ( fig . the intracerebroventricular injection of mt - ii ( 3 g ) increased 2[h]dg uptake in bat , heart , and all types of skeletal muscle , including the white portion of the gastrocnemius , but not in spleen or epididymal wat , at 3 or 6 h after injection . these results thus suggested that intracerebroventricular injection of mt - ii promotes glucose production as well as glucose uptake in certain peripheral tissues . the rate constant of 2[h]dg uptake in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) was measured at 3 or 6 h after intracerebroventricular injection of mt - ii or saline ( control ) . finally , we examined the effects of direct injection of mt - ii into the individual medial hypothalamic nuclei on glucose uptake in peripheral tissues ( fig . microinjection of mt - ii ( 10 ng ) into the vmh increased 2[h]dg uptake in bat , heart , and all types of skeletal muscle but not in spleen or epididymal wat . in contrast , injection of mt - ii into the pvh increased glucose uptake only in bat , and that into the dmh or arc did not affect glucose uptake in any of the peripheral tissues examined . plasma glucose and insulin levels did not change in response to injection of mt - ii into any of the hypothalamic nuclei ( supplemental table 2 ) . effects of mt - ii injection into vmh , pvh , dmh , or arc on glucose uptake in peripheral tissues . the rate constant of 2[h]dg uptake in skeletal muscle ( a ) , bat and heart ( b ) , spleen ( c ) , and epididymal wat ( d ) was measured at 6 h after the injection of mt - ii into the individual hypothalamic nuclei . we have previously shown that microinjection of leptin into the vmh and nearby medial hypothalamic area preferentially increased glucose uptake in skeletal muscle , heart , and bat ( 1416 ) . in the present study , we found that activation of the leptin receptor specifically in the vmh , as detected by measurement of the tyrosine phosphorylation of stat3 , resulted in a marked increase in glucose uptake in those peripheral tissues , similar to the effects of intracerebroventricular or peripheral administration of leptin ( 13,36 ) . in contrast , injection of leptin into the arc increased glucose uptake only in bat . injection of leptin into the dmh or pvh had no effect on glucose uptake in any of the peripheral tissues examined . the present data thus suggest that the leptin receptor in the vmh and arc regulates glucose uptake in different peripheral tissues . our present results further indicate that mcr activation is necessary for the increase in glucose uptake in peripheral tissues induced by injection of leptin into the vmh . the intracerebroventricular injection of shu9119 thus abolished the effect of leptin injected into the vmh on peripheral glucose uptake , whereas intracerebroventricular injection of mt - ii increased glucose uptake in peripheral tissues . moreover , injection of mt - ii into vmh , but not that into the dmh or arc , increased glucose uptake in skeletal muscle , heart , and bat , whereas injection of mt - ii into the pvh increased glucose uptake preferentially in bat . both mcrs in the vmh and pvh may play an important role in the regulation of glucose uptake in peripheral tissues . furthermore , the effect of leptin injected into the vmh on glucose uptake in peripheral tissues was found to be dependent on mcrs in the brain . we therefore propose that stimulation of vmh neurons by leptin results in activation of a set of pomc neurons in the arc and thereby increases glucose uptake in skeletal muscle , heart , and bat . mcrs in the vmh and pvh contribute to the upregulation of glucose uptake in peripheral tissues induced by injection of leptin into the vmh . the mechanism by which injection of leptin into the arc increased glucose uptake specifically in bat remains unclear . one possible explanation for this observation is that the injection of leptin into the arc activated a selective set of pomc neurons in the arc that regulate glucose uptake in bat alone , with pomc neurons being abundant in both the anterior and posterior regions of the arc ( 38 ) . it is also possible that pomc neurons in the arc that regulate glucose uptake in skeletal muscle and heart require excitatory input as well as leptin for their full activation . the intracerebroventricular injection of mt - ii increased glucose uptake in bat to a markedly greater extent than did the maximum dose of injection of mt - ii into the vmh or pvh . we previously showed that injection of leptin into the medial hypothalamus increased glucose uptake in peripheral tissues through the activation of sympathetic nerves ( 14,15 ) . whereas the molecular mechanism remains elusive , a direct effect of -adrenergic receptors expressed in peripheral tissues as well as a -adrenergic receptor dependent increase in blood flow appear to contribute to leptin - induced glucose uptake in these tissues . however , we have now shown that injection of leptin or mt - ii into the dmh did not increase glucose uptake in the peripheral tissues examined . although we can not exclude the possibility that injection of leptin or mt - ii activated only a subset of dmh neurons expressing ob - rb or mcr , our results suggest that the leptin receptor and mcr in the dmh have other roles , such as the regulation of food intake or modulation of bat thermogenesis in response to cold stimuli . our present results indicate that the vmh regulates glucose uptake in skeletal muscle as well as in heart and bat , accompanying increased glut4 mrna in bat and heart . thus , increased expression of glut4 may involve the acute increase in glucose uptake in bat and heart in response to leptin , while other mechanism might be involved in the leptin - induced glucose uptake in skeletal muscle . gene expression in skeletal muscle in response to leptin and mt - ii may require > 24 h. the present results showed that injection of leptin or mt - ii in some medial hypothalamic nuclei increased glucose uptake in certain peripheral tissues , while it did not alter plasma glucose level . we have shown that intracerebroventricular injection of mt - ii but not leptin activates amp - activated protein kinase in skeletal muscle in diet - induced obese mice ( 48 ) . together , our results suggest that the vmh plays a key role in leptin- and mt - ii induced glucose uptake in skeletal muscle , heart , and bat , whereas the leptin receptor in the arc and mcrs in the pvh regulate glucose uptake in bat . the medial hypothalamic nuclei thus appear to play distinct roles in the regulation of glucose uptake in peripheral tissues by leptin and mt - ii .

the street foods play an important socioeconomic role in meeting food and nutritional requirements of city consumers at affordable prices to the lower and middle income groups and are appreciated for their unique flavors and convenience [ 13 ] . street foods also assure food security for low income urban population and livelihood for a significant proportion of the population in many developing countries . street foods are described as wide range of ready - to - eat foods and beverages or prepared at home and consumed on the streets without further preparation . these food items are usually sold by vendors and hawkers in the streets or other similar public places . while street vended foods are appreciated for their unique flavors as well as their convenience , they are also important in contributing to the nutritional status of the population . in contrast to these potential benefits , it is also recognized that street food vendors are often poor , uneducated , and lack knowledge in safe food handling , environment , sanitation and hygiene , mode of food display , food service and hand washing , sources of raw materials , and use of potable water . consequently , street foods are perceived to be a major public health risk . foodborne illnesses of microbial origin are a major health problem associated with street foods [ 68 ] . in addition , resistance of foodborne microorganisms in multi - drug made the food safety situation more vulnerable in public health . diarrheal diseases are the most common food poisoning cases in bangladesh and in some cases , these can cause death . the diseases are caused by either toxin from the microbe or by the human body 's reactions to the microbe . the traditional processing methods that are used in the preparation , inappropriate holding temperature , and poor personal hygiene of food handlers are some of the main causes of contamination of street foods [ 11 , 12 ] . also the foods are not effectively protected from flies and dust [ 13 , 14 ] . in bangladesh , street foods are mostly prepared and processed manually and sold to the public at various lorry terminals , by the roadside or by itinerant vendors . a study of the socioeconomic conditions and determination of the hygienic and sanitary practices of street food vendors in dhaka city corporation was carried out by fao 2010 . the study result demonstrated that 25% street food vendors are illiterate and can not write their names and have no formal education . as street food business requires low investment , most of the vendors ( 88% ) were found to own the business . most of the vending shops ( 68% ) were located on the footpath irrespective of areas surveyed and 30% vending carts were placed near the municipal drain and 18% near the sewerage . microbiological study of different foods items , drinking water , and hand swab samples showed the prevalence of overwhelmingly high numbers of aerobic bacteria , coliform bacteria , and pathogens . for the sake of public health , it is important to understand the epidemiology of foodborne illnesses because it will help in prevention and control efforts , appropriately allocating resources to control foodborne illness , monitoring , and evaluation of food safety measures , development of new food safety standards , and assessment of the cost - effectiveness of interventions . the purpose of this study is to see the microbial risk of food poisoning associated with street food in order to determine the magnitude of the problem , risk factors , monitoring and surveillance , and measures of control . the street food vendors of bangladesh are not enumerated in the formal sector of country 's economy . they are identified as the informal sector where their businesses are conducted as a form of irregular , unstable , and marginal economic activities . as such there is no systematic documentation of the numbers of street food vendors , their scale of businesses , or the viability of their pursuits . after rickshaw - pulling , street vending is probably the second most important employment opportunity for the urban poor in bangladesh , and particularly important for young and middle - aged men who have migrated to dhaka in the past five to ten years . dhaka is among the world 's cities with the highest number of hawkers : in asia , only mumbai ( ~250,000 ) , delhi ( ~200,000 ) , calcutta ( ~150,000 ) , and bangkok ( ~100,000 ) have similarly large numbers of street vendors . however , benjamin conducted a survey on street food vendors in dhaka , over a period of three years ( 2007 to 2010 ) . this survey and official labour statistics demonstrated that between 90,000 and 100,000 street vendors sell prepared food items , and around 418,000 people or 2.9 percent of dhaka 's total population depend on the income generated by street food vendors . this implies that almost eight million people or 55 percent of the population of dhaka take some street food everyday . the significance of street food system of dhaka is beyond doubt and selling street food is not a marginal economic activity , but a normal yet highly visible social practice , that is , economically efficient and deeply embedded in the urban economy and in urban life [ 2 , 4 , 20 ] . a glimpse of the socioeconomic background of the vendors is presented below to help understand who the street food vendors are.both males and females and married and unmarried operate as street food vendors . their age range is between 25 and 60 years with a majority being in the age group of 3040 years.many street food vendors and their families have their origin in rural backgrounds or have moved to urban centers at a later stage or else live in rural areas and travel daily to the city for their business operations.the level of education achieved by the street food vendors is comparatively low and in the case of a majority , education levels varied between grades 5 and 8.many street food vendors are constrained by the unstable socioeconomic backgrounds in their families.employment history of the street food vendors shows their previous involvement in several urban - based , irregular , and low - paid income generating activities , which required hard manual labor , prior to their involvement in the street food business . their age range is between 25 and 60 years with a majority being in the age group of 3040 years . many street food vendors and their families have their origin in rural backgrounds or have moved to urban centers at a later stage or else live in rural areas and travel daily to the city for their business operations . the level of education achieved by the street food vendors is comparatively low and in the case of a majority , education levels varied between grades 5 and 8 . employment history of the street food vendors shows their previous involvement in several urban - based , irregular , and low - paid income generating activities , which required hard manual labor , prior to their involvement in the street food business . street food vendors are a self - employed category of small entrepreneurs who are not dependent on any institutional structures to find their livelihoods . their enterprises evolve exclusively around their own individual strengths and the support extended to them by their immediate social networks such as family members and other close associates . the earnings from their business enterprises are a means of living for the vendors themselves and their dependent family members . as such , these economic activities of the street food vendors have not only provided a source of livelihood to the vendors and their dependent family members but also have reduced the plight of their becoming an economic and social burden on the state . street food , therefore , not only meets the food requirements particularly of those of the low income categories but also the busy customers who do not have much time either to prepare their own food or to go to other eating houses where probably the food is more expensive and servicing is time consuming . street vendors face unique kinds of livelihood risks because of the legal , physical , and sociocultural environment in which they work . the most pressing and ongoing risk for many street vendors is the possibility that local government authorities will forcibly remove them from the streets or confiscate their merchandise . this risk of displacement often increases in the context of elections , mega events , or efforts to beautify historic city centers . just like formal business operators , street vendors are less productive in unstable institutional environments where rules are irregular and unpredictable [ 5 , 19 ] . many must lift and haul heavy loads of goods to and from their point of sale each day . the physical environments in which they work typically lack proper infrastructure , such as clean running water , toilets , and solid waste removal ( table 1 ) . street vendors are exposed to physical harm from the improper provision of fire safety equipment and the improper regulation of traffic in commercial areas . they are also exposed to a high concentration of air pollutants and to inclement weather . these physical risks take a particular toll on young children who must accompany their mothers to vend in the streets . given the growing numbers of the street food vendors and the customers who patronize them , the issues and problems the vendors encounter need special attention of the authorities concerned . harassment on the part of local authorities including evictions , confiscation of merchandise , and demands for bribes is a common source of income risk for street vendors . street vendors legal status can act as a bridge between their employment conditions and the range of employment risks they face ( table 1 and figure 1 ) . a vendor with a fixed structure in a designated market , for example , may be more likely to hold a license or permit , and in turn would be less exposed to certain kinds of risks . likewise , a street vendor who works as an employee selling a particular kind of product , such as newspapers , may be better protected by law and therefore less vulnerable . obtaining legal status of some kind is therefore a key demand of street trading organizations in many cities . the number of people living in the country 's capital dhaka almost doubled from 5.3 to 9.3 million . this development has led to an increase in the demand for relatively inexpensive and ready - to - eat foods as many urban residents spend most of the day outside of the house and have little time and money to spend on food . rapid urbanization also turned street - food vending into an important business ; in dhaka alone , around 200,000 people earn their living by selling street foods [ 4 , 23 ] . the low cost , accessibility , and convenience are the key factors for the growing popularity of street foods . women play a very vital role in the street food sector through their direct and/or indirect involvement in the business . additionally a significant number of street vendors are woman - headed households [ 1 , 19 ] . the diversity that exists among street food vendors is reflected in the type of food they prepare / sell , the scale of their business , the mode in which they are operating , the locations in which they prepare and sell food , the type of clients to whom they sell food , and so forth . there are so many varieties that it is impossible to provide a menu of all the different street foods consumed around the world . in bangladesh , street foods include chola boot ( chickpeas ) , bhelpuri ( puffed rice with potatoes ) , and samucha ( deep - fried dough stuffed with vegetables and/or meat ) as well as drinks like sugar - cane juice and lassi ( yoghurt and water ) . other popular snacks are ghugni ( boiled and mashed white peas with spices ) , singara ( flour wraps stuffed with vegetables , spices , and occasionally liver ) , and different types of cakes . the customer surveys undertaken by fao 2006 and other investigators revealed that the main consumers of street foods in most countries were other members of the informal sector , such as fellow hawkers and hustlers and casual wage laborers . other important categories of customer were children and students , office workers , and housewives . the studies also found that street foods were consumed across all income groups and the proportion of the daily household food budget spent on street foods was high , ranging from 25 percent in bogor to 47 percent in chonburi , thailand . the frequency and regularity of consumption were variable : in some countries , street foods were bought daily and formed an integral part of the diet ; in others , notably in bangladesh , they appeared supplementary and few customers bought them daily [ 6 , 26 ] . some categories of consumer ( students , itinerant unskilled laborers , and the homeless ) were found to buy almost all their food from vendors . the cost of street foods is usually competitive compared with that of foods purchased from larger food establishments , such as restaurants and fast food outlets . also , due to the sometimes high costs of fuel and ingredients in urban contexts , economies of scale can create a street food cheaper than the same food prepared at home . the hygienic aspects of street food vending are a major concern for food control officers . vending stands are often crude structures , and running water , washing facilities , and toilettes may not be available . improved safety of street foods can be achieved through awareness raising programmes involving several partners such as local authorities , the food vendors , government departments , consumer organizations , standard setting bodies , and some nongovernmental organizations . in some instances , the vendors are keen to participate in programmes that provide basic facilities that make it possible for them to work in clean environments . for example , in a survey of street food vendors in lusaka and harare , the vendors indicated that they would be willing to pay for basic facilities such as running water and electricity but would want the local authorities to provide the water points , refuse receptacles , and washing facilities . a viable partnership involving local authorities , vendors and policy makers is therefore encouraged as this should lead to the improvement of business conditions and allow for the improvement of the livelihoods of vendors and their families . the battle against foodborne diseases is facing new challenges due to the globalization of the food market , climate change , and changing patterns of human consumption as fresh and convenient foods are currently preferred . as food is biological in nature , it is capable of supporting the growth of microorganisms and foodborne diseases result from the ingestion of contaminated foods and food products . more than 250 different types of viruses , bacteria , parasites , toxins , metals , and prions are associated with foodborne diseases in humans . although viruses are more responsible for more than 50% of all foodborne illnesses ; generally hospitalizations and deaths associated with foodborne infections are due to bacterial agents . the infections range from mild gastroenteritis to life - threatening neurologic , hepatic , and renal syndromes caused by either toxin from the disease - causing microbe or by the human body 's reaction to the microbe itself . of the many thousands different bacterial species , more than 90% of food - poisoning illnesses are caused by species of staphylococcus , salmonella , clostridium , campylobacter , listeria , vibrio , bacillus , and enteropathogenic escherichia coli . for instance , in the us and france , in the last decade of the 20th century , salmonella was the most frequent cause of bacterial foodborne illness ( 5,70010,200 cases ) , followed by campylobacter ( 2,6003,500 cases ) and listeria ( 304 cases ) . in south africa , species of listeria , enterobacter , and aeromonas were the most prevalent bacteria in ready - to - eat foods . however , no such databases are available for bangladesh as well as other developing countries . therefore , from october 2012 to the present , our laboratory conducted a series of experiments to assess the microbial quality of street foods of bangladesh . more than 100 street foods samples of 20 kinds including singara , jhal - muri , chatpati , chetoi pitha , chola / bengal gram , jilapi , jar drinking water , pickles , amra , tehari , vegetable rolls , sugarcane juice , raw cucumber slices , milk , other juices , beverages , and bread were analyzed for major foodborne pathogens including , salmonella spp . , escherichia coli o157 , o111 , o26 , and other e. coli , other coliforms , listeria spp . , and staphylococcus spp . the presence of all the above mentioned pathogenic organisms found in the street foods was presented in table 2 . in addition , the isolated pathogenic organisms were found resistant to at least 7 antibiotics . these studies demonstrated that foods sold in the street of dhaka city constitute a potential microbial hazard to human health . in addition , mamun et al . reported that among different items of street vended foods , 54% of sliced fruits samples , 59% of jhalmuri samples , 29% of chotpotis samples , 53% of vajavuji samples , and all ( 100% ) sharbat samples were found unsatisfactory microbial quality . this finding also reflected poor microbiological quality of the school - based street vended foods indicating a health threat to the school children of dhaka city [ 1 , 8 ] . nonfood grade chemical additives , such as colorants and preservatives , and contaminants , such as pesticide residues , have also been found in street foods . a chemical analysis of street foods in bogor found unpermitted coloring agents such as textile dyes and also pesticide residues . proper use of salt , spices , nitrates , and sugar is an important means of preventing food spoilage , but the drive to keep prices low may lead to the purchase of cheap ingredients containing unpermitted chemical additives from unauthorized suppliers . chemicals such as colorants may also be added to mask the poor quality of cheap materials . due to the conditions under which street foods are sold , there is concern that food may be contaminated with heavy metals and pesticide residues . these contaminants may come from the utensils , raw materials , or transport methods used and may also occur due to the lack of appropriate storage facilities . a study carried out in ghana revealed that street food vendors source their pots and other utensils from both formal and informal manufacturers / retailers . some of the street food samples had higher levels of lead , cadmium , arsenic , mercury , and copper than average food samples , suggesting possible leaching from the utensils . further tests showed that lead from the pots obtained from informal manufacturers could leach into the food . these pots are manufactured using scrap metal that could come from diverse sources such as derelict cars , car batteries , and industrial machinery , which are obviously not suitable for use with foods . this was attributed to the fact that when police raid these vendors , they usually confiscate their wares , including the pots and utensils . for fear of losing their more expensive pots , the vendors resort to using informally fabricated pots , thereby exposing consumers to the possibility of food contamination by heavy metals . further work must be done in order to reduce the exposure of consumers to heavy metals and pesticide residues through street - vended food [ 29 , 36 ] . purchasing ready - to - eat foods and ingredients from street / market vendors poses a considerable risk to public health , especially due to the poor hygienic practices . in most cases , the vendors do not have adequate washing facilities , and some vendors started their duties without taking a proper bath . some of the vendors sleep at the vending sites in order to protect their wares . foods and ingredients are also subjected to repeated contamination from unwashed hands and the materials used for wrapping , such as leaves , old newspapers , and reusable polyethylene bags . however , many vendors are aware of the need to wear clean and appropriate clothes . some of the female vendors wear headgear and aprons . after a few awareness - raising campaigns for vendors , the absence of water points near their work places and poor drainage facilities make them unable to practice good hygiene . moreover , some food handlers washed their hands in the same bucket used for cleaning utensils , which may lead to the contamination of food with faecal matter . on the other hand , most food vendors operate their business without health certificates or licenses , which poses additional concern and required extensive training programme on food preparation and handling techniques . street food vendors use cheap bar soap than liquid soap , which may be more effective , to clean their utensils , but as they use cold water , resulting in inefficient cleaning . furthermore , washed plates are often stored in an unclean corner , plastic bowl , or cardboard box , leading to recontamination of the plates . this leads to an increased pest population and resulted in an increased risk of food contamination . in many instances , the vending sites are not included within the city or town plans , and therefore amenities such as refuse collection are not available . city authorities are often faced with the dilemma that if they provide services to illegal operations , this will imply recognition of these operations . at the same time , because the vending operations are illegal and vendors do not contribute anything towards the maintenance of infrastructure or provision of public services , therefore , they are not entitled to the service . this contributes to further deterioration of the hygienic condition of the area where the foods are vended . poor sanitary conditions in the area where foods are vended also contribute to poor food storage and transport conditions . street food vendors in some cities obtain their vegetables , maize meal , and other condiments from licensed shops , and therefore there is less concern regarding the safety of these raw materials . however , most of the vendors have no fixed stalls where they can store their raw materials on site . they usually store their goods at home overnight and transport them the following day , often improperly covered , to their operating sites . thus , the food becomes prone to contamination during transportation [ 18 , 19 ] . a recent review of the situation in asia found great diversity among the legal instruments developed to control the street food trade . some countries had no specific legislation or control systems at all [ 19 , 22 ] . in those countries where street food activities were regulated by law , the regulations or by - laws affecting the street food trade were part of a larger body of legislation dealing with food , health , or environmental sanitation . licensing or registration systems , inspection systems , and codes of practice are other forms of regulation that are in effect in some countries . a number of pieces of legislation relating to the preparation and sale of safe street foods have been established by the bangladesh government . the bangladesh pure food ordinance 1959 ( revised 2005 ) has several sections dealing with the safety of street food : adulteration of food ; prohibition of calcium carbide , formalin , and insecticide ; selling unwholesome food ; uncovered foods ; and unhygienic premises and violations of the health code . the other relevant legal measures related to safe street foods are the bangladesh standards and testing institution ( bsti ) ordinance 37 of 1985 ; the consumers rights preservation act 2009 ; and the penal code of 1860 , sections 272276 . a mobile court to monitor street food vendors a number of civil society organizations have emerged in recent years in bangladesh to promote safe street foods and overall food safety . for example , the consumers association of bangladesh ( cab ) , vocta ( consumer ) , which has conducted street food surveys and organized awareness , campaigns through rallies , seminars , workshops , and policy advocacy . the electronic and print media are also involved in providing public awareness of safe street foods . in contrast , key constraints to the effective management of street foods are the lack of awareness of personal hygiene and safe food among street food vendors and consumers ; insufficient awareness among the consumers about the consumer rights act ; lack of clarity in existing legislation and standards on street food ; no specific regulatory body for licensing , provision of i d card , medical fitness , or dress codes concerning street food vendors ; no demarcation of specific areas by local municipalities for street food vendors ; insufficient number of sanitary inspectors ; the inability of sanitary inspectors to take penal actions against street food vendors ; and absence of appropriate training and supervision of street food vendors . the quality and safety of street foods is determined by numerous factors such as the business organization , regulatory aspects , technical aspects related to the preparation , preservation and display of food sold in the streets , the consumer perspective , and educational programs . in order to improve the conditions of street food vendors and to make sure that the food sold does not jeopardize public health , the first and foremost necessity is to build awareness that food vendor should maintain certain quality standard . in many areas , street foods are sold and food safety issues are not taken into consideration neither on the producer nor on the consumer side . consumers tend to look mostly at the price and might be already accustomed to the taste of unhealthy meals . vendors , on the other hand , have a very small margin of profit and are incentivized to keep expenses low by utilizing low quality ingredients and disregarding costly hygienic practices . to break this vicious cycle , governments need to embrace street food vendors as a dynamic economic sector . with their adaptability to the frenetic life in the global cities , street food vendors have a huge potential to quickly fill niches , greatly improving urban access to food . while excessive regulation of the sector carries the risk of suffocating this adaptability and would just shift the problem to a new informal sector consisting of those dodging the regulation , certain minimum standards , especially related to food quality , need to be enforced . vendors should be given some basic training on how to safely prepare and store food and businesses should be certified accordingly . while some proposed the application of haccp standards , others argued against it stressing the need for much simpler guidelines such as the five keys to safer food . in addition , municipalities should provide vendors with appropriate infrastructure like access to clean water and sewage systems . street food vendors should be encouraged to partake in awareness raising programmes and given access to microcredit . in order to improve the vendors standing , strengthening their overall position vis - - vis authorities , promoting their organization into cooperatives has been identified as a path to follow . in addition to helping vendors run their business in a more efficient and safe manner , cooperatives would also ease the authorities work in enforcing hygienic and business standards . in general , interventions and programmes can only be successful if they do not focus on one aspect alone . tackling only food quality , for instance , can not ensure that street food vendors play the most positive role in realizing food security of the urban population . it is important not to forget that the street foods constitute a very heterogeneous sector and the interventions need to be carefully planned by keeping different aspects such as gender , secondary audience , and local customs into consideration . it is also necessary to differentiate between vendors selling freshly prepared food on the spot or hawking dishes prepared earlier at home , with the second practice being much more risky in terms of foodborne pathogen and spores . needless to say , general education levels also play an important role in ensuring safe street foods . the more both vendors and patrons will be educated and the more they will know about issues such as nutrition and food safety , the more they will be interested in having the business as clean and the products as healthy as possible .

the street foods play an important socioeconomic role in meeting food and nutritional requirements of city consumers at affordable prices to the lower and middle income people . the number of food poisoning notifications rose steadily worldwide since the inception of e. coli o157:h7 outbreak in the 1980s to date . this may be partly attributed to improved surveillance , increased global trade and travel , changes in modern food production , the impact of modern lifestyles , changes in food consumption , and the emergence of new pathogens . consumer 's knowledge and attitude may influence food safety behavior and practice . for the sake of public health , it is important to understand the epidemiology of foodborne illnesses that help in prevention and control efforts , appropriately allocating resources to control foodborne illness , monitoring and evaluation of food safety measures , development of new food safety standards , and assessment of the cost - effectiveness of interventions . this review paper described the sociodemographic characteristics , common hazards , and occupational hazards of street food vendors , microbial risk associated with street food , food safety interventions and control measures , regulatory aspects and legal requirements , financial constraints , and attitudes .

1. Introduction 2. Sociodemographic Characteristics of Street Food Vendors 3. Working Conditions and Occupational Hazards of Street Food Vendors 4. Growing Demand of Street Food 5. Consumers of Street Food 6. Safety of Street Foods 7. Food Legislation and Regulatory Aspects 8. Conclusion

the street foods play an important socioeconomic role in meeting food and nutritional requirements of city consumers at affordable prices to the lower and middle income groups and are appreciated for their unique flavors and convenience [ 13 ] . street foods also assure food security for low income urban population and livelihood for a significant proportion of the population in many developing countries . while street vended foods are appreciated for their unique flavors as well as their convenience , they are also important in contributing to the nutritional status of the population . in contrast to these potential benefits , it is also recognized that street food vendors are often poor , uneducated , and lack knowledge in safe food handling , environment , sanitation and hygiene , mode of food display , food service and hand washing , sources of raw materials , and use of potable water . consequently , street foods are perceived to be a major public health risk . foodborne illnesses of microbial origin are a major health problem associated with street foods [ 68 ] . in addition , resistance of foodborne microorganisms in multi - drug made the food safety situation more vulnerable in public health . the diseases are caused by either toxin from the microbe or by the human body 's reactions to the microbe . the traditional processing methods that are used in the preparation , inappropriate holding temperature , and poor personal hygiene of food handlers are some of the main causes of contamination of street foods [ 11 , 12 ] . in bangladesh , street foods are mostly prepared and processed manually and sold to the public at various lorry terminals , by the roadside or by itinerant vendors . a study of the socioeconomic conditions and determination of the hygienic and sanitary practices of street food vendors in dhaka city corporation was carried out by fao 2010 . the study result demonstrated that 25% street food vendors are illiterate and can not write their names and have no formal education . as street food business requires low investment , most of the vendors ( 88% ) were found to own the business . for the sake of public health , it is important to understand the epidemiology of foodborne illnesses because it will help in prevention and control efforts , appropriately allocating resources to control foodborne illness , monitoring , and evaluation of food safety measures , development of new food safety standards , and assessment of the cost - effectiveness of interventions . the purpose of this study is to see the microbial risk of food poisoning associated with street food in order to determine the magnitude of the problem , risk factors , monitoring and surveillance , and measures of control . the street food vendors of bangladesh are not enumerated in the formal sector of country 's economy . they are identified as the informal sector where their businesses are conducted as a form of irregular , unstable , and marginal economic activities . as such there is no systematic documentation of the numbers of street food vendors , their scale of businesses , or the viability of their pursuits . after rickshaw - pulling , street vending is probably the second most important employment opportunity for the urban poor in bangladesh , and particularly important for young and middle - aged men who have migrated to dhaka in the past five to ten years . dhaka is among the world 's cities with the highest number of hawkers : in asia , only mumbai ( ~250,000 ) , delhi ( ~200,000 ) , calcutta ( ~150,000 ) , and bangkok ( ~100,000 ) have similarly large numbers of street vendors . however , benjamin conducted a survey on street food vendors in dhaka , over a period of three years ( 2007 to 2010 ) . this survey and official labour statistics demonstrated that between 90,000 and 100,000 street vendors sell prepared food items , and around 418,000 people or 2.9 percent of dhaka 's total population depend on the income generated by street food vendors . this implies that almost eight million people or 55 percent of the population of dhaka take some street food everyday . the significance of street food system of dhaka is beyond doubt and selling street food is not a marginal economic activity , but a normal yet highly visible social practice , that is , economically efficient and deeply embedded in the urban economy and in urban life [ 2 , 4 , 20 ] . a glimpse of the socioeconomic background of the vendors is presented below to help understand who the street food vendors are.both males and females and married and unmarried operate as street food vendors . their age range is between 25 and 60 years with a majority being in the age group of 3040 years.many street food vendors and their families have their origin in rural backgrounds or have moved to urban centers at a later stage or else live in rural areas and travel daily to the city for their business operations.the level of education achieved by the street food vendors is comparatively low and in the case of a majority , education levels varied between grades 5 and 8.many street food vendors are constrained by the unstable socioeconomic backgrounds in their families.employment history of the street food vendors shows their previous involvement in several urban - based , irregular , and low - paid income generating activities , which required hard manual labor , prior to their involvement in the street food business . many street food vendors and their families have their origin in rural backgrounds or have moved to urban centers at a later stage or else live in rural areas and travel daily to the city for their business operations . the level of education achieved by the street food vendors is comparatively low and in the case of a majority , education levels varied between grades 5 and 8 . employment history of the street food vendors shows their previous involvement in several urban - based , irregular , and low - paid income generating activities , which required hard manual labor , prior to their involvement in the street food business . street food vendors are a self - employed category of small entrepreneurs who are not dependent on any institutional structures to find their livelihoods . as such , these economic activities of the street food vendors have not only provided a source of livelihood to the vendors and their dependent family members but also have reduced the plight of their becoming an economic and social burden on the state . street food , therefore , not only meets the food requirements particularly of those of the low income categories but also the busy customers who do not have much time either to prepare their own food or to go to other eating houses where probably the food is more expensive and servicing is time consuming . street vendors face unique kinds of livelihood risks because of the legal , physical , and sociocultural environment in which they work . given the growing numbers of the street food vendors and the customers who patronize them , the issues and problems the vendors encounter need special attention of the authorities concerned . a vendor with a fixed structure in a designated market , for example , may be more likely to hold a license or permit , and in turn would be less exposed to certain kinds of risks . the number of people living in the country 's capital dhaka almost doubled from 5.3 to 9.3 million . this development has led to an increase in the demand for relatively inexpensive and ready - to - eat foods as many urban residents spend most of the day outside of the house and have little time and money to spend on food . rapid urbanization also turned street - food vending into an important business ; in dhaka alone , around 200,000 people earn their living by selling street foods [ 4 , 23 ] . the low cost , accessibility , and convenience are the key factors for the growing popularity of street foods . women play a very vital role in the street food sector through their direct and/or indirect involvement in the business . additionally a significant number of street vendors are woman - headed households [ 1 , 19 ] . the diversity that exists among street food vendors is reflected in the type of food they prepare / sell , the scale of their business , the mode in which they are operating , the locations in which they prepare and sell food , the type of clients to whom they sell food , and so forth . there are so many varieties that it is impossible to provide a menu of all the different street foods consumed around the world . in bangladesh , street foods include chola boot ( chickpeas ) , bhelpuri ( puffed rice with potatoes ) , and samucha ( deep - fried dough stuffed with vegetables and/or meat ) as well as drinks like sugar - cane juice and lassi ( yoghurt and water ) . the customer surveys undertaken by fao 2006 and other investigators revealed that the main consumers of street foods in most countries were other members of the informal sector , such as fellow hawkers and hustlers and casual wage laborers . the studies also found that street foods were consumed across all income groups and the proportion of the daily household food budget spent on street foods was high , ranging from 25 percent in bogor to 47 percent in chonburi , thailand . the frequency and regularity of consumption were variable : in some countries , street foods were bought daily and formed an integral part of the diet ; in others , notably in bangladesh , they appeared supplementary and few customers bought them daily [ 6 , 26 ] . some categories of consumer ( students , itinerant unskilled laborers , and the homeless ) were found to buy almost all their food from vendors . the cost of street foods is usually competitive compared with that of foods purchased from larger food establishments , such as restaurants and fast food outlets . also , due to the sometimes high costs of fuel and ingredients in urban contexts , economies of scale can create a street food cheaper than the same food prepared at home . the hygienic aspects of street food vending are a major concern for food control officers . improved safety of street foods can be achieved through awareness raising programmes involving several partners such as local authorities , the food vendors , government departments , consumer organizations , standard setting bodies , and some nongovernmental organizations . for example , in a survey of street food vendors in lusaka and harare , the vendors indicated that they would be willing to pay for basic facilities such as running water and electricity but would want the local authorities to provide the water points , refuse receptacles , and washing facilities . a viable partnership involving local authorities , vendors and policy makers is therefore encouraged as this should lead to the improvement of business conditions and allow for the improvement of the livelihoods of vendors and their families . the battle against foodborne diseases is facing new challenges due to the globalization of the food market , climate change , and changing patterns of human consumption as fresh and convenient foods are currently preferred . as food is biological in nature , it is capable of supporting the growth of microorganisms and foodborne diseases result from the ingestion of contaminated foods and food products . more than 250 different types of viruses , bacteria , parasites , toxins , metals , and prions are associated with foodborne diseases in humans . although viruses are more responsible for more than 50% of all foodborne illnesses ; generally hospitalizations and deaths associated with foodborne infections are due to bacterial agents . the infections range from mild gastroenteritis to life - threatening neurologic , hepatic , and renal syndromes caused by either toxin from the disease - causing microbe or by the human body 's reaction to the microbe itself . of the many thousands different bacterial species , more than 90% of food - poisoning illnesses are caused by species of staphylococcus , salmonella , clostridium , campylobacter , listeria , vibrio , bacillus , and enteropathogenic escherichia coli . for instance , in the us and france , in the last decade of the 20th century , salmonella was the most frequent cause of bacterial foodborne illness ( 5,70010,200 cases ) , followed by campylobacter ( 2,6003,500 cases ) and listeria ( 304 cases ) . therefore , from october 2012 to the present , our laboratory conducted a series of experiments to assess the microbial quality of street foods of bangladesh . more than 100 street foods samples of 20 kinds including singara , jhal - muri , chatpati , chetoi pitha , chola / bengal gram , jilapi , jar drinking water , pickles , amra , tehari , vegetable rolls , sugarcane juice , raw cucumber slices , milk , other juices , beverages , and bread were analyzed for major foodborne pathogens including , salmonella spp . , escherichia coli o157 , o111 , o26 , and other e. coli , other coliforms , listeria spp . the presence of all the above mentioned pathogenic organisms found in the street foods was presented in table 2 . in addition , the isolated pathogenic organisms were found resistant to at least 7 antibiotics . these studies demonstrated that foods sold in the street of dhaka city constitute a potential microbial hazard to human health . reported that among different items of street vended foods , 54% of sliced fruits samples , 59% of jhalmuri samples , 29% of chotpotis samples , 53% of vajavuji samples , and all ( 100% ) sharbat samples were found unsatisfactory microbial quality . this finding also reflected poor microbiological quality of the school - based street vended foods indicating a health threat to the school children of dhaka city [ 1 , 8 ] . nonfood grade chemical additives , such as colorants and preservatives , and contaminants , such as pesticide residues , have also been found in street foods . a chemical analysis of street foods in bogor found unpermitted coloring agents such as textile dyes and also pesticide residues . proper use of salt , spices , nitrates , and sugar is an important means of preventing food spoilage , but the drive to keep prices low may lead to the purchase of cheap ingredients containing unpermitted chemical additives from unauthorized suppliers . due to the conditions under which street foods are sold , there is concern that food may be contaminated with heavy metals and pesticide residues . a study carried out in ghana revealed that street food vendors source their pots and other utensils from both formal and informal manufacturers / retailers . some of the street food samples had higher levels of lead , cadmium , arsenic , mercury , and copper than average food samples , suggesting possible leaching from the utensils . this was attributed to the fact that when police raid these vendors , they usually confiscate their wares , including the pots and utensils . for fear of losing their more expensive pots , the vendors resort to using informally fabricated pots , thereby exposing consumers to the possibility of food contamination by heavy metals . purchasing ready - to - eat foods and ingredients from street / market vendors poses a considerable risk to public health , especially due to the poor hygienic practices . in most cases , the vendors do not have adequate washing facilities , and some vendors started their duties without taking a proper bath . foods and ingredients are also subjected to repeated contamination from unwashed hands and the materials used for wrapping , such as leaves , old newspapers , and reusable polyethylene bags . after a few awareness - raising campaigns for vendors , the absence of water points near their work places and poor drainage facilities make them unable to practice good hygiene . moreover , some food handlers washed their hands in the same bucket used for cleaning utensils , which may lead to the contamination of food with faecal matter . street food vendors use cheap bar soap than liquid soap , which may be more effective , to clean their utensils , but as they use cold water , resulting in inefficient cleaning . in many instances , the vending sites are not included within the city or town plans , and therefore amenities such as refuse collection are not available . at the same time , because the vending operations are illegal and vendors do not contribute anything towards the maintenance of infrastructure or provision of public services , therefore , they are not entitled to the service . street food vendors in some cities obtain their vegetables , maize meal , and other condiments from licensed shops , and therefore there is less concern regarding the safety of these raw materials . a recent review of the situation in asia found great diversity among the legal instruments developed to control the street food trade . in those countries where street food activities were regulated by law , the regulations or by - laws affecting the street food trade were part of a larger body of legislation dealing with food , health , or environmental sanitation . a number of pieces of legislation relating to the preparation and sale of safe street foods have been established by the bangladesh government . the bangladesh pure food ordinance 1959 ( revised 2005 ) has several sections dealing with the safety of street food : adulteration of food ; prohibition of calcium carbide , formalin , and insecticide ; selling unwholesome food ; uncovered foods ; and unhygienic premises and violations of the health code . the other relevant legal measures related to safe street foods are the bangladesh standards and testing institution ( bsti ) ordinance 37 of 1985 ; the consumers rights preservation act 2009 ; and the penal code of 1860 , sections 272276 . a mobile court to monitor street food vendors a number of civil society organizations have emerged in recent years in bangladesh to promote safe street foods and overall food safety . for example , the consumers association of bangladesh ( cab ) , vocta ( consumer ) , which has conducted street food surveys and organized awareness , campaigns through rallies , seminars , workshops , and policy advocacy . in contrast , key constraints to the effective management of street foods are the lack of awareness of personal hygiene and safe food among street food vendors and consumers ; insufficient awareness among the consumers about the consumer rights act ; lack of clarity in existing legislation and standards on street food ; no specific regulatory body for licensing , provision of i d card , medical fitness , or dress codes concerning street food vendors ; no demarcation of specific areas by local municipalities for street food vendors ; insufficient number of sanitary inspectors ; the inability of sanitary inspectors to take penal actions against street food vendors ; and absence of appropriate training and supervision of street food vendors . the quality and safety of street foods is determined by numerous factors such as the business organization , regulatory aspects , technical aspects related to the preparation , preservation and display of food sold in the streets , the consumer perspective , and educational programs . in order to improve the conditions of street food vendors and to make sure that the food sold does not jeopardize public health , the first and foremost necessity is to build awareness that food vendor should maintain certain quality standard . in many areas , street foods are sold and food safety issues are not taken into consideration neither on the producer nor on the consumer side . to break this vicious cycle , governments need to embrace street food vendors as a dynamic economic sector . with their adaptability to the frenetic life in the global cities , street food vendors have a huge potential to quickly fill niches , greatly improving urban access to food . while excessive regulation of the sector carries the risk of suffocating this adaptability and would just shift the problem to a new informal sector consisting of those dodging the regulation , certain minimum standards , especially related to food quality , need to be enforced . street food vendors should be encouraged to partake in awareness raising programmes and given access to microcredit . in general , interventions and programmes can only be successful if they do not focus on one aspect alone . tackling only food quality , for instance , can not ensure that street food vendors play the most positive role in realizing food security of the urban population . it is important not to forget that the street foods constitute a very heterogeneous sector and the interventions need to be carefully planned by keeping different aspects such as gender , secondary audience , and local customs into consideration . it is also necessary to differentiate between vendors selling freshly prepared food on the spot or hawking dishes prepared earlier at home , with the second practice being much more risky in terms of foodborne pathogen and spores . needless to say , general education levels also play an important role in ensuring safe street foods . the more both vendors and patrons will be educated and the more they will know about issues such as nutrition and food safety , the more they will be interested in having the business as clean and the products as healthy as possible .

more than 2 billion people globally are estimated to suffer from micronutrient deficiencies ( von grebmer et al . iron , zinc , vitamin a , iodine , folate and other vitamin b deficiencies are among the most widespread global micronutrient deficiencies ( muthayya et al . factors that contribute to poor micronutrient intake and absorption include poor dietary diversity , poor nutrient density of staplebased complementary foods , antinutritional factors in plantbased foods and environmental enteropathy . disease conditions resulting from infections can further aggravate micronutrient deficiencies ( katona & katonaapte 2008 ) . strategies such as targeted supplementation with vitamin a , or universal salt iodization , oil fortification with vitamin a , or flour fortification with iron or folic acid , are often part of national strategies to address the problem of micronutrient malnutrition among vulnerable groups throughout the life cycle . overcoming deficiencies of micronutrients can have a potential benefit in terms of young child survival , growth and development and preventing intrauterine growth restriction and low birth weight ( black et al . reviews such as the copenhagen consensus have consistently ranked micronutrients as the most costeffective development intervention ( copenhagen consensus panel 2012 ) . economic analyses suggest that fortification and/or targeted supplementation with , for instance , iron , zinc , vitamin a , folic acid and iodine can be highly costeffective ( horton 2006 ; horton et al . fortification will not reach all individuals but can be a costeffective strategy provided that a centrally processed , affordable fortified food vehicle is available and either the deficiency is widespread or only a small group is affected but the adverse effects because of the deficiency are very costly ( horton 2006 ) . targeted micronutrient programs such as home fortification or periodic highdose supplements have the advantage to deliver micronutrients of concern to the vulnerable subpopulation(s ) without unnecessarily exposing other groups in the population . in order for supplementation to be costeffective , the defined target group should be readily reached with little compliance issues ( horton 2006 ) . when implementing micronutrient programs there is the risk of providing insufficient or excessive amounts , missing those at risk or redundant coverage ( coverage of individuals with adequate intake , or coverage by more programs than necessary ) , potentially resulting in inadequate or excessive intakes and poor costeffectiveness . designing an effective and safe fortification or supplementation program imposes the dual challenge of reaching the target population groups at most risk of micronutrient deficiency while avoiding overexposure of individuals at the high end of the intake distribution curves ( european food safety authority ( efsa ) 2010 ) . this may pose particular challenges regarding micronutrient interventions that exert benefits in the target population but unintentionally adverse effects in a subgroup of this target population with already high intake or status of the micronutrient ( e.g. targeted vitamin a supplementation ) ( european food safety authority ( efsa ) 2010 ) . increasing micronutrient intakes through universal fortification can deliver potential benefits to deficient population groups , but sometimes also risks in other population groups when consumed in excess ( e.g. folic acid fortification to prevent neural tube defects in the unborn child versus masking of vitamin b12 deficiency in elderly ) ( european food safety authority ( efsa ) 2010 ) . values have been set for the intake of each micronutrient below which the risk for adverse health effects is likely to increase . the risk of an inadequate intake increases as an individual 's intake falls further below the recommended intake ( food and nutrition board & institute of medicine ( iom ) 2000 ) . at two standard deviations below the recommended intake , i.e. the estimated average requirement ( ear ) , adverse health consequences because of a micronutrient deficiency condition increase in incidence and/or severity the further the usual intake falls below the ear ( renwick et al . 2004 ) . therefore , reducing the number of individuals with inadequate micronutrient intakes can be expected to have clear benefits on micronutrient deficiencyrelated morbidity especially when the morbidity is severe and/or chronic . however , the adverse health consequences of deficiency are far better understood than the evidence base related to the consequences of excess , if any . the excess level of intake is neither well understood and defined for many micronutrients , nor do biomarkers exist that can detect early adverse effects . threshold for intake or a biomarker , above which the risk of adverse effects may increase and the dose response relationship ( renwick et al . 2004 ) . the tolerable upper intake level ( ul ) is the highest daily intake still considered to be safe for almost all healthy individuals in a specified group . depending on the magnitude of the evidence , the ul is obtained by downward adjustment of the noobservedadverseeffect level ( if known ) or the lowestobservedadverseeffect level by applying an uncertainty factor . this has resulted for a number of micronutrients in an ul that is close to the recommended nutrient intake because of large uncertainty factors used to set this ul ( european commission 2006 ) . for some micronutrients , no evidence of risk of adverse effects is established , and hence no safe tolerable upper intake level ( ul ) is defined ( i.e. biotin , pantothenic acid , chromium , vitamin b1 , b2 , b12 and k ) . for other micronutrients , the risk of exceeding the ul is low ( i.e. vitamin b6 , c , d and e , niacin , molybdenum , phosphorus and selenium ) . for some micronutrients , a potential risk of exceeding the maximum safe ul exists ( i.e. vitamin a as preformed retinol , zinc , iron , iodine , copper and calcium ) . for the latter micronutrients , programs should be cautiously designed and monitored to ensure at risk population groups are reached without any appreciable risk of adverse effects in subgroups that consume high amounts of the micronutrient . important elements of the program cycle include the gathering of information to assess the nutrition situation , development of a national policy and strategy , program design , implementation and monitoring , review and evaluation , and adjustment if necessary . before implementing a micronutrient program it is critical to assess the nutrition situation by collecting data among the different population groups that can be referred to as the abcd of nutrition assessment ; anthropometric assessments , biomarker or biochemical assessments , clinical assessments ( morbidity and mortality ) and dietary assessment . these data allow understanding the magnitude of the health problem and the vulnerable population group(s ) at risk of micronutrient deficiency . information on food and micronutrient intake distribution in the different population groups will help predict how micronutrient interventions will work in a variety of contexts . another critical element in planning ( cost ) effective and safe programs is the ongoing monitoring and evaluation of the nutrition situation through collection of data . it provides an indication of the effectiveness and safety of the intervention and allows adjusting the type of intervention , amount of provided micronutrients or geographic distribution , if needed . some countries have implemented one or multiple programs , focusing mostly on vitamin a , iodine and iron . however , availability of data on biomarkers predictive of nutrient intake , status and adverse health effects ( combs et al . 2013 ; raiten & combs 2015 ) is still sparse , and few countries have conducted detailed nutrition surveys , as their collection can be time and resourceintensive . often multiple programs with micronutrients are implemented to reach a greater proportion of the atrisk population . in particular , implementation of multiple micronutrient interventions requires coordination and monitoring to ensure that these programs are complementary and not overlapping , which may result in unnecessarily exposure of vulnerable groups . collecting dietary intake data and biomarkers can be used to correct program designs to ensure that intake ranges in the different lifestage groups are effective and safe . dietary intake data can be used in software modelling to predict the shift in micronutrient adequacy in different population groups for a selected food vehicle and fortification level . until recently , dietary intake modelling tools were not able to predict the effect of intake from combined fortification and supplementation programs . as addressed in this symposium report , a new simulation approach allows to predict the effect of implementing fortification combined with supplementation on intakes below the ear and above the ul and related program cost versus the savings ( englestone et al . other software tools have been developed that simulate the effect of changing food or nutrient intakes on prevention of the related burden of disease ( i.e. the benefits ) . in risk benefit approaches , the same principle is used to assess both the preventable burden of disease and the risks related to excessive food or nutrient intake , provided that sufficient evidence is available to simulate the adverse health response . in order to use a risk benefit approach , a better understanding is needed of the relation between micronutrient intake , status and morbidity / mortality outcomes . for some micronutrients the link between micronutrient intake , status and morbidity / mortality is sufficiently established to predict health benefits when overcoming its deficiency ( horton 2006 ) . however , with the current dearth of data in many countries on micronutrient intake , status and related disease incidence , program modelling to predict the benefits , and even more so the risks , is still a challenge . it is nevertheless encouraging that in many countries the collection of nutrition data is increasing , which will further help program planners and policymakers to design costeffective and safe micronutrient interventions . minimum and maximum intake values for micronutrients are relevant for population groups , but do not necessarily reflect adequate or safe intake values for all individuals in that group . the requirement and safety limit for a micronutrient not only varies by age , gender and lifestage , but also by health condition , medication use and genetic profile , making it even more challenging to develop a safe and effective program tailored to all at risk individuals . for instance , chronic disease conditions or genetic polymorphisms may lead to higher micronutrient requirements . micronutrients do not necessarily provide benefits in all deficiency conditions ; complex interactions may exist between the delivered micronutrients , micronutrient status , infections and medication use . complicating factors include ( 1 ) interactions between micronutrients , for instance iron and zinc reducing each other 's absorption ( lnnerdal 2004 ) , ( 2 ) micronutrient drug interactions ; micronutrients reducing the absorption or efficacy of a drug or vice versa ( scaglione & panzavolta 2014 ; hathcock 1985 ) , or ( 3 ) antagonistic interactions between micronutrients and infections ( e.g. iron and malaria ) . the benefits and risks of some micronutrients therefore depend on the setting in which they are provided . for instance , the effect of certain micronutrients may not only depend on whether the target population is deficient or not , but also on whether malaria control strategies are implemented in malaria endemic regions . this is exemplified by the pemba study , in which children living on malariaendemic pemba island who received iron and folic acid supplements , were more likely to die or need treatment in hospital for an adverse event ( sazawal et al . however , in a substudy of the trial in which children were given malariareduction measures , children who were iron deficient and anaemic at baseline , showed a significant reduction in adverse events , including malaria episodes , suggesting that supplementation with iron and folic acid may especially confer benefits to those who are deficient . these issues , altogether , pose a challenge to program planners in designing micronutrient intervention programs that meet the requirements of the majority of the population at risk while still being safe . there is a need for tools that assist is designing micronutrient intervention programs that are effective in reaching the target groups at risk of developing micronutrient deficiencies , efficient , in terms of reaching the target group and not unnecessarily exposing nontarget groups , and safe , in terms of not overexposing a segment of the population that is already more than adequate , especially vulnerable target groups . there is a need to understand how specific micronutrients , mainly as folic acid and iron , may interact with medication and infection , and how they exert beneficial or antagonistic effects under these conditions . this raises the question of how to achieve the maximum possible public health benefits of a micronutrient intervention strategy with the least risks . monitoring of data ( dietary intake , program coverage , biomarkers and morbidity ) among vulnerable population groups is essential to optimize effectiveness , safety , and efficiency of micronutrient programs.modelling tools are needed that predict the effectiveness , safety , and efficiency of micronutrient programs by integrating above data.research is needed to identify biomarkers predictive of micronutrient exposure , status , and potential health consequences of inadequate and especially excessive micronutrient exposure . this may help setting the appropriate noobservedadverseeffect or lowestadverseeffect level , currently often based on scant data , particularly for young children.more insight is needed into the antagonistic effects resulting from complex interactions between micronutrients , drugs , and infections . monitoring of data ( dietary intake , program coverage , biomarkers and morbidity ) among vulnerable population groups is essential to optimize effectiveness , safety , and efficiency of micronutrient programs . modelling tools are needed that predict the effectiveness , safety , and efficiency of micronutrient programs by integrating above data . research is needed to identify biomarkers predictive of micronutrient exposure , status , and potential health consequences of inadequate and especially excessive micronutrient exposure . this may help setting the appropriate noobservedadverseeffect or lowestadverseeffect level , currently often based on scant data , particularly for young children . more insight is needed into the antagonistic effects resulting from complex interactions between micronutrients , drugs , and infections . until recently , dietary intake modelling tools were not able to predict the effect of intake from combined fortification and supplementation programs . as addressed in this symposium report , a new simulation approach allows to predict the effect of implementing fortification combined with supplementation on intakes below the ear and above the ul and related program cost versus the savings ( englestone et al . other software tools have been developed that simulate the effect of changing food or nutrient intakes on prevention of the related burden of disease ( i.e. the benefits ) . in risk benefit approaches , the same principle is used to assess both the preventable burden of disease and the risks related to excessive food or nutrient intake , provided that sufficient evidence is available to simulate the adverse health response . in order to use a risk benefit approach , a better understanding is needed of the relation between micronutrient intake , status and morbidity / mortality outcomes . for some micronutrients the link between micronutrient intake , status and morbidity / mortality is sufficiently established to predict health benefits when overcoming its deficiency ( horton 2006 ) . however , with the current dearth of data in many countries on micronutrient intake , status and related disease incidence , program modelling to predict the benefits , and even more so the risks , is still a challenge . it is nevertheless encouraging that in many countries the collection of nutrition data is increasing , which will further help program planners and policymakers to design costeffective and safe micronutrient interventions . minimum and maximum intake values for micronutrients are relevant for population groups , but do not necessarily reflect adequate or safe intake values for all individuals in that group . the requirement and safety limit for a micronutrient not only varies by age , gender and lifestage , but also by health condition , medication use and genetic profile , making it even more challenging to develop a safe and effective program tailored to all at risk individuals . for instance , chronic disease conditions or genetic polymorphisms may lead to higher micronutrient requirements . micronutrients do not necessarily provide benefits in all deficiency conditions ; complex interactions may exist between the delivered micronutrients , micronutrient status , infections and medication use . complicating factors include ( 1 ) interactions between micronutrients , for instance iron and zinc reducing each other 's absorption ( lnnerdal 2004 ) , ( 2 ) micronutrient drug interactions ; micronutrients reducing the absorption or efficacy of a drug or vice versa ( scaglione & panzavolta 2014 ; hathcock 1985 ) , or ( 3 ) antagonistic interactions between micronutrients and infections ( e.g. iron and malaria ) . the benefits and risks of some micronutrients therefore depend on the setting in which they are provided . for instance , the effect of certain micronutrients may not only depend on whether the target population is deficient or not , but also on whether malaria control strategies are implemented in malaria endemic regions . this is exemplified by the pemba study , in which children living on malariaendemic pemba island who received iron and folic acid supplements , were more likely to die or need treatment in hospital for an adverse event ( sazawal et al . however , in a substudy of the trial in which children were given malariareduction measures , children who were iron deficient and anaemic at baseline , showed a significant reduction in adverse events , including malaria episodes , suggesting that supplementation with iron and folic acid may especially confer benefits to those who are deficient . these issues , altogether , pose a challenge to program planners in designing micronutrient intervention programs that meet the requirements of the majority of the population at risk while still being safe . there is a need for tools that assist is designing micronutrient intervention programs that are effective in reaching the target groups at risk of developing micronutrient deficiencies , efficient , in terms of reaching the target group and not unnecessarily exposing nontarget groups , and safe , in terms of not overexposing a segment of the population that is already more than adequate , especially vulnerable target groups . there is a need to understand how specific micronutrients , mainly as folic acid and iron , may interact with medication and infection , and how they exert beneficial or antagonistic effects under these conditions . this raises the question of how to achieve the maximum possible public health benefits of a micronutrient intervention strategy with the least risks . monitoring of data ( dietary intake , program coverage , biomarkers and morbidity ) among vulnerable population groups is essential to optimize effectiveness , safety , and efficiency of micronutrient programs.modelling tools are needed that predict the effectiveness , safety , and efficiency of micronutrient programs by integrating above data.research is needed to identify biomarkers predictive of micronutrient exposure , status , and potential health consequences of inadequate and especially excessive micronutrient exposure . this may help setting the appropriate noobservedadverseeffect or lowestadverseeffect level , currently often based on scant data , particularly for young children.more insight is needed into the antagonistic effects resulting from complex interactions between micronutrients , drugs , and infections . monitoring of data ( dietary intake , program coverage , biomarkers and morbidity ) among vulnerable population groups is essential to optimize effectiveness , safety , and efficiency of micronutrient programs . modelling tools are needed that predict the effectiveness , safety , and efficiency of micronutrient programs by integrating above data . research is needed to identify biomarkers predictive of micronutrient exposure , status , and potential health consequences of inadequate and especially excessive micronutrient exposure . this may help setting the appropriate noobservedadverseeffect or lowestadverseeffect level , currently often based on scant data , particularly for young children . more insight is needed into the antagonistic effects resulting from complex interactions between micronutrients , drugs , and infections . during the global summit on food fortification on 911 september 2015 in arusha , one of the sessions included a special session coorganized by sight and life and unicef , entitled effective and safe micronutrient interventions : weighing the risks against the benefits. the aim of the session was to identify existing tools and needs to assist policy makers in designing effective micronutrient programs with the highest public health benefits and least risks . the presentations in the session addressed this challenge , based on a number of case studies related to folic acid , iodine and vitamin a. maaike bruins ( dsm biotechnology center , delft , the netherlands ) , with the title assessing the risks and benefits of micronutrients interventions. in order to understand the risks of inadequate and excessive intake of a micronutrient , information is required on the intake distribution of the micronutrient in the population . this enables to assess the proportion of a population lifestage group with intakes below their requirements and above their ul . software is available , such as pcside and imapp , for the estimation and modelling of food and micronutrient intake distributions , which can assist in selecting optimal food vehicles and fortification levels ( iowa state university 2015 ) . other tools make it possible to estimate the preventable public disease burden because of a micronutrient intervention program , expressed for instance as preventable disabilityadjusted life years ( dalys ) . the daly is a quantitative measure of overall disease burden and can serve as important guide to decisionmaking . the daly is expressed as the number of years lost because of early death or morbidity ( e.g. infections , anaemia , motor and cognitive impairments , or blindness ) . the daly metric is composed of mortality and incidence , duration and disability of the morbidity ( related to micronutrient deficiency ) . for instance , preventing dalys by overcoming maternal folate deficiency may have a large public health impact ; the preventable lifelong and severe disabilities resulting from of neural tube defects are substantial , even if a few neural tube defects in 1000 births could be prevented . the world health organization ( who ) has developed a source of estimates for costeffectiveness of micronutrient interventions is the choosing interventions that are cost effective ( whochoice ) database ( world health organization ( who ) 2016 ) . the estimates are based on known intervention costs and effectiveness ( in terms of micronutrient status ) , and links between micronutrient status and morbidity / mortality outcomes . the who has also developed tools that can assist program planners in simulating the public health impact of an increase in micronutrient intake in terms of dalys gained ( world health organization ( who ) 2015 ) . this requires ( 1 ) dietary intake data in different population groups and ( 2 ) evidence for a dose response relationship between micronutrient intake and morbidity because of deficiency . with the introduction of a micronutrient intervention , for instance , when implementing mandatory fortification , the benefits of preventing frequent , or severe , or lifelong disabilities should be balanced against the possible risks of excess intake in other population groups ( hoekstra et al . benefit assessment software provides the option to quantify the public health risks and benefits , and balance them , using for instance dalys as a common metric ( ellis & aspurger 2000 ) . even though simulating changes in dalys resulting from micronutrient intervention programs can provide useful information to policy makers to set investment priorities , moreover , for many micronutrients the dose response relationship between excessive intake and risks is not well known . even though simulating changes in dalys resulting from micronutrient intervention programs can provide useful information to policy makers to set investment priorities , next to these sophisticated modelling tools , more userfriendly tools are needed that integrate information on program coverage , dietary intake distribution , biomarkers and morbidity to design effective and safe micronutrient programs in terms of public health impact . increasing micronutrient intake may confer benefits on those who have inadequate intakes . for instance , in some studies iron supplementation has been associated with an increased risk of malaria and death in children living in malariaendemic regions , but not when regular malaria surveillance and treatment services are provided ( world health organization ( who ) 2015 ) . another example of a micronutrient that may not necessarily confer benefits under all circumstances is , for instance , folic acid . folic acid supplementation may be beneficial to the folatedeficient child , but may confer risks when highdose supplements are implemented in malariaendemic settings alongside antimalarial drugs ( kupka 2015 ) . the who calls for micronutrient powder ( mnp ) programs to be implemented alongside malaria control strategies in malaria endemic regions . current mnp formulations generally provide folic acid and other micronutrients at the recommended nutrient intake level . however , the benefits and risks of providing supplemental folic acid are largely unknown . the limited data available suggest that folate deficiency may not be a major public health problem among children living in subsaharan africa ; as a result , supplemental folic acid may not confer health benefits . furthermore , folic acid provided at supraphysiological , and possibly even physiological , levels may favour the growth of the parasite plasmodium falciparum ( responsible for 85% of malaria cases ) , inhibit clearance of the parasite by sulphadoxinepyrimethamine ( sp)treated malaria and increase subsequent recrudescence ( nzila et al . 2014 ) . limiting prophylactic sp use or promoting the use of insecticidetreated bed nets may render the use of folic acid in mnp programs safer , but programmatic barriers to these approaches may remain . the use of 5methyltetrahydrofolic acid concomitantly with sp may be a promising alternative , as it does not affect sp efficacy , although its stability in food and costinuse remain to be confirmed ( scaglione & panzavolta 2014 ) . the presentation concluded that more work is needed to characterize the prevalence of folate deficiency among young children worldwide , and to optimize the benefit risk ratio of mnp programs in subsaharan africa . michael zimmerman from the eth zrich ( switzerland ) discussed the risks and benefits of salt iodization programs . in general , the relatively small risks of iodine excess are far outweighed by the substantial risks of iodine deficiency ( zimmermann 2008 ) . iodine deficiency has multiple adverse effects on growth and development because of inadequate thyroid hormone production , which are termed the iodine deficiency disorders ( idd ) . idd remains one of the most common causes of preventable mental impairment worldwide . in nearly all iodinedeficient countries , the best strategy to control idd is salt iodization , one of the most costeffective ways of contributing to economic and social development ( zimmermann 2008 ) . in areas of severe iodine deficiency , iodine repletion in pregnant women iodine repletion in newborns reduces infant mortality , and may improve cognitive development and growth . even in areas of mildtomoderate iodine deficiency , repletion in schoolaged children improves cognition and fine motor skills ( zimmermann 2007 ) . on the other hand , the introduction of iodized salt to regions with chronic idd may transiently increase the incidence of thyroid disorders , such as iodineinduced hyperthyroidism and autoimmune hypothyroidism , and programs should therefore include monitoring for both iodine deficiency and excess . more data on the epidemiology of thyroid disorders caused by differences in iodine intake are needed , and should be the focus of future research . the adverse effects of vitamin a deficiency include childhood blindness , and the increased risk of mortality from measles and diarrhoea ( stevens et al . vitamin a supplementation and fortification programs can be a cheap and effective way to reduce morbidity and mortality because of vitamin a deficiency ( horton 2006 ) . these programs have significantly improved millions of lives among preschool children in many parts of the world ( stevens et al . longterm excessive vitamin a intakes , on the other hand , can lead to adverse effects , such as hepatotoxicity . generally , the substantial risks of vitamin a deficiency can be expected to far outweigh the relatively small risks of vitamin a excess ( allen & haskell 2002 ) . the lack of coordination of multiple vitamin a interventions and many overlapping schemes in some areas has increased concern about the risks of these programs , as some children could theoretically receive well above their recommended daily dose of the vitamin . the uncertainty around the level of excess for vitamin a has resulted in a small margin of safe intake for young children , between the recommended intake levels and maximum safe ul georg lietz from newcastle university ( newcastle upon tyne , uk ) discussed how to best assess the window of benefit for vitamin a intake . in his presentation , dr . lietz highlighted the need for reliable , robust and affordable biomarkers of inadequate and excess status , to enable policy and program makers to adjust the correct level and coverage of their interventions . the appropriate noobservedadverseeffect level or the lowestobservedadverseeffect level for children up to 5 years of age needs to be urgently determined and the uls revised as most uls for children are extrapolated from uls for adults . dr . lietz also discussed the benefits and problems associated with the most recent methods to have been developed to enable the monitoring of both fortification and supplementation programs , including the application of stable isotope dilution techniques , and the potential for labonchip technologies for future monitoring . he ended his presentation by highlighting the need to coordinate different methods of risk assessment , to make the monitoring process more efficient , reliable and costeffective . reina englestone from the university of california ( davis , ca , usa ) presented a dietary modelling approach to assess the risk of inadequate and excessive intakes under different program scenarios , and their applications to program planning . her presentation provided an overview of the methodology for the use of dietary data to examine the risk of inadequate and excessive micronutrient intakes using dietary reference values , and given information on current dietary patterns and the reach of program delivery platforms . examples from cameroon illustrated the use of dietary modelling to set fortification levels , by predicting the effects of each new or modified program on dietary intake ( englestone et al . this dietary modelling tool can be used to predict the effect of fortification of different food vehicles and overlapping micronutrient programs on the percentage of the population with intakes below ear or above the ul ( brown et al . 2015 ) . finally , the role of complementary modelling tools was discussed , including the ( 1 ) lives saved tool ( list ) , which predicts the mortality reduction from some micronutrient interventions delivered to mothers and children ( johns hopkins school of public health 2015 ) , ( 2 ) models assessing liver vitamin a concentration , which can assist in the interpretation of risk of dietary intakes above the upper intake level in young children , and ( 3 ) program coverage surveys which reveal absent or redundant program coverage and thus help identify subpopulations ( e.g. regions ) at risk of inadequate or excessive intake ( aaron 2014 ; spohrer 2015 ) . population dietary intake data can be used to model the effect of different food vehicles and micronutrient levels to optimize coverage and minimize the risks of inadequate and excessive intakes in relation to dietary cutoff points . monitoring intake data will enable policy makers to adjust to the correct food vehicle , micronutrient level , and coverage of intervention programs . however , the success of these programs has , inevitably , to be confirmed by applying biomarkers indicative of micronutrient status that are affordable , sensitive and specific , and can be applied in population settings ( combs et al . some methods which are currently applied are inadequate , because of their lack of precision and accuracy , to detect nutrient concentrations in atrisk population groups , or because of insensitivity to changes in status within a certain range ( e.g. serum retinol ) , or alterations during inflammation ( zinc , retinol , ferritin ) . thus , improved biomarkers are urgently needed which will enable policy makers to understand the benefits and risks in vulnerable population groups related to increasing micronutrient intakes . tools are available which assess or predict the effects of micronutrient programs , in terms of dietary intakes against dietary cutoff points . however , there is still need for standardized , userfriendly tools , which predict the public health impact made by micronutrient interventions , by integrating benefits and possible risk . more research is warranted in order to understand the concerns regarding the safety of iron and folic acid supplementation among young children in malaria endemic areas , and their interaction with antimalarials and infections . research is needed to assess the minimum and maximum intake range for vitamin a outside which intakes might cause concern . in general , the small risks of micronutrient excess are far outweighed by the substantial benefits of overcoming the deficiency . however , there is an urgent need to monitor overlapping micronutrient supplementation and fortification programs , and to evaluate the benefits and risks ( if any ) of increasing population micronutrient intakes for each setting ( population group , region , choice of food vehicle , micronutrient level in food , micronutrient status of the population group , concomitant food and medications , etc . ) . klaus kraemer is the director of sight and life foundation , a nutrition think tank primarily funded by dsm . the authors declare that they have all participated in drafting of the manuscript , and that they have approved the final version .

abstractinterventions to address micronutrient deficiencies have large potential to reduce the related disease and economic burden . however , the potential risks of excessive micronutrient intakes are often not well determined . during the global summit on food fortification , 911 september 2015 , in arusha , a symposium was organized on micronutrient risk benefit assessments . using case studies on folic acid , iodine and vitamin a , the presenters discussed how to maximize the benefits and minimize the risks of intervention programs to address micronutrient malnutrition . preimplementation assessment of dietary intake , and/or biomarkers of micronutrient exposure , status and morbidity / mortality is critical in identifying the population segments at risk of inadequate and excessive intake . dietary intake models allow to predict the effect of micronutrient interventions and their combinations , e.g. fortified food and supplements , on the proportion of the population with intakes below adequate and above safe thresholds . continuous monitoring of micronutrient intake and biomarkers is critical to identify whether the target population is actually reached , whether subgroups receive excessive amounts , and inform program adjustments . however , the relation between regular high intake and adverse health consequences is neither well understood for many micronutrients , nor do biomarkers exist that can detect them . more accurate and reliable biomarkers predictive of micronutrient exposure , status and function are needed to ensure effective and safe intake ranges for vulnerable population groups such as young children and pregnant women . modelling tools that integrate information on program coverage , dietary intake distribution and biomarkers will further enable program makers to design effective , efficient and safe programs .

Introduction The need for data modelling to predict efficacy and safety in micronutrient programs Interindividual variations and micronutrient interactions Objective Session summary Source of funding Conflicts of interest Contributor statement

iron , zinc , vitamin a , iodine , folate and other vitamin b deficiencies are among the most widespread global micronutrient deficiencies ( muthayya et al . strategies such as targeted supplementation with vitamin a , or universal salt iodization , oil fortification with vitamin a , or flour fortification with iron or folic acid , are often part of national strategies to address the problem of micronutrient malnutrition among vulnerable groups throughout the life cycle . economic analyses suggest that fortification and/or targeted supplementation with , for instance , iron , zinc , vitamin a , folic acid and iodine can be highly costeffective ( horton 2006 ; horton et al . when implementing micronutrient programs there is the risk of providing insufficient or excessive amounts , missing those at risk or redundant coverage ( coverage of individuals with adequate intake , or coverage by more programs than necessary ) , potentially resulting in inadequate or excessive intakes and poor costeffectiveness . designing an effective and safe fortification or supplementation program imposes the dual challenge of reaching the target population groups at most risk of micronutrient deficiency while avoiding overexposure of individuals at the high end of the intake distribution curves ( european food safety authority ( efsa ) 2010 ) . this may pose particular challenges regarding micronutrient interventions that exert benefits in the target population but unintentionally adverse effects in a subgroup of this target population with already high intake or status of the micronutrient ( e.g. increasing micronutrient intakes through universal fortification can deliver potential benefits to deficient population groups , but sometimes also risks in other population groups when consumed in excess ( e.g. however , the adverse health consequences of deficiency are far better understood than the evidence base related to the consequences of excess , if any . the excess level of intake is neither well understood and defined for many micronutrients , nor do biomarkers exist that can detect early adverse effects . for some micronutrients , no evidence of risk of adverse effects is established , and hence no safe tolerable upper intake level ( ul ) is defined ( i.e. for other micronutrients , the risk of exceeding the ul is low ( i.e. for some micronutrients , a potential risk of exceeding the maximum safe ul exists ( i.e. for the latter micronutrients , programs should be cautiously designed and monitored to ensure at risk population groups are reached without any appreciable risk of adverse effects in subgroups that consume high amounts of the micronutrient . before implementing a micronutrient program it is critical to assess the nutrition situation by collecting data among the different population groups that can be referred to as the abcd of nutrition assessment ; anthropometric assessments , biomarker or biochemical assessments , clinical assessments ( morbidity and mortality ) and dietary assessment . these data allow understanding the magnitude of the health problem and the vulnerable population group(s ) at risk of micronutrient deficiency . information on food and micronutrient intake distribution in the different population groups will help predict how micronutrient interventions will work in a variety of contexts . another critical element in planning ( cost ) effective and safe programs is the ongoing monitoring and evaluation of the nutrition situation through collection of data . some countries have implemented one or multiple programs , focusing mostly on vitamin a , iodine and iron . however , availability of data on biomarkers predictive of nutrient intake , status and adverse health effects ( combs et al . collecting dietary intake data and biomarkers can be used to correct program designs to ensure that intake ranges in the different lifestage groups are effective and safe . dietary intake data can be used in software modelling to predict the shift in micronutrient adequacy in different population groups for a selected food vehicle and fortification level . until recently , dietary intake modelling tools were not able to predict the effect of intake from combined fortification and supplementation programs . as addressed in this symposium report , a new simulation approach allows to predict the effect of implementing fortification combined with supplementation on intakes below the ear and above the ul and related program cost versus the savings ( englestone et al . other software tools have been developed that simulate the effect of changing food or nutrient intakes on prevention of the related burden of disease ( i.e. in risk benefit approaches , the same principle is used to assess both the preventable burden of disease and the risks related to excessive food or nutrient intake , provided that sufficient evidence is available to simulate the adverse health response . in order to use a risk benefit approach , a better understanding is needed of the relation between micronutrient intake , status and morbidity / mortality outcomes . for some micronutrients the link between micronutrient intake , status and morbidity / mortality is sufficiently established to predict health benefits when overcoming its deficiency ( horton 2006 ) . however , with the current dearth of data in many countries on micronutrient intake , status and related disease incidence , program modelling to predict the benefits , and even more so the risks , is still a challenge . it is nevertheless encouraging that in many countries the collection of nutrition data is increasing , which will further help program planners and policymakers to design costeffective and safe micronutrient interventions . the benefits and risks of some micronutrients therefore depend on the setting in which they are provided . for instance , the effect of certain micronutrients may not only depend on whether the target population is deficient or not , but also on whether malaria control strategies are implemented in malaria endemic regions . this is exemplified by the pemba study , in which children living on malariaendemic pemba island who received iron and folic acid supplements , were more likely to die or need treatment in hospital for an adverse event ( sazawal et al . however , in a substudy of the trial in which children were given malariareduction measures , children who were iron deficient and anaemic at baseline , showed a significant reduction in adverse events , including malaria episodes , suggesting that supplementation with iron and folic acid may especially confer benefits to those who are deficient . these issues , altogether , pose a challenge to program planners in designing micronutrient intervention programs that meet the requirements of the majority of the population at risk while still being safe . there is a need for tools that assist is designing micronutrient intervention programs that are effective in reaching the target groups at risk of developing micronutrient deficiencies , efficient , in terms of reaching the target group and not unnecessarily exposing nontarget groups , and safe , in terms of not overexposing a segment of the population that is already more than adequate , especially vulnerable target groups . there is a need to understand how specific micronutrients , mainly as folic acid and iron , may interact with medication and infection , and how they exert beneficial or antagonistic effects under these conditions . monitoring of data ( dietary intake , program coverage , biomarkers and morbidity ) among vulnerable population groups is essential to optimize effectiveness , safety , and efficiency of micronutrient programs.modelling tools are needed that predict the effectiveness , safety , and efficiency of micronutrient programs by integrating above data.research is needed to identify biomarkers predictive of micronutrient exposure , status , and potential health consequences of inadequate and especially excessive micronutrient exposure . this may help setting the appropriate noobservedadverseeffect or lowestadverseeffect level , currently often based on scant data , particularly for young children.more insight is needed into the antagonistic effects resulting from complex interactions between micronutrients , drugs , and infections . monitoring of data ( dietary intake , program coverage , biomarkers and morbidity ) among vulnerable population groups is essential to optimize effectiveness , safety , and efficiency of micronutrient programs . modelling tools are needed that predict the effectiveness , safety , and efficiency of micronutrient programs by integrating above data . research is needed to identify biomarkers predictive of micronutrient exposure , status , and potential health consequences of inadequate and especially excessive micronutrient exposure . until recently , dietary intake modelling tools were not able to predict the effect of intake from combined fortification and supplementation programs . as addressed in this symposium report , a new simulation approach allows to predict the effect of implementing fortification combined with supplementation on intakes below the ear and above the ul and related program cost versus the savings ( englestone et al . other software tools have been developed that simulate the effect of changing food or nutrient intakes on prevention of the related burden of disease ( i.e. in risk benefit approaches , the same principle is used to assess both the preventable burden of disease and the risks related to excessive food or nutrient intake , provided that sufficient evidence is available to simulate the adverse health response . in order to use a risk benefit approach , a better understanding is needed of the relation between micronutrient intake , status and morbidity / mortality outcomes . for some micronutrients the link between micronutrient intake , status and morbidity / mortality is sufficiently established to predict health benefits when overcoming its deficiency ( horton 2006 ) . however , with the current dearth of data in many countries on micronutrient intake , status and related disease incidence , program modelling to predict the benefits , and even more so the risks , is still a challenge . it is nevertheless encouraging that in many countries the collection of nutrition data is increasing , which will further help program planners and policymakers to design costeffective and safe micronutrient interventions . the benefits and risks of some micronutrients therefore depend on the setting in which they are provided . for instance , the effect of certain micronutrients may not only depend on whether the target population is deficient or not , but also on whether malaria control strategies are implemented in malaria endemic regions . however , in a substudy of the trial in which children were given malariareduction measures , children who were iron deficient and anaemic at baseline , showed a significant reduction in adverse events , including malaria episodes , suggesting that supplementation with iron and folic acid may especially confer benefits to those who are deficient . these issues , altogether , pose a challenge to program planners in designing micronutrient intervention programs that meet the requirements of the majority of the population at risk while still being safe . there is a need for tools that assist is designing micronutrient intervention programs that are effective in reaching the target groups at risk of developing micronutrient deficiencies , efficient , in terms of reaching the target group and not unnecessarily exposing nontarget groups , and safe , in terms of not overexposing a segment of the population that is already more than adequate , especially vulnerable target groups . there is a need to understand how specific micronutrients , mainly as folic acid and iron , may interact with medication and infection , and how they exert beneficial or antagonistic effects under these conditions . monitoring of data ( dietary intake , program coverage , biomarkers and morbidity ) among vulnerable population groups is essential to optimize effectiveness , safety , and efficiency of micronutrient programs.modelling tools are needed that predict the effectiveness , safety , and efficiency of micronutrient programs by integrating above data.research is needed to identify biomarkers predictive of micronutrient exposure , status , and potential health consequences of inadequate and especially excessive micronutrient exposure . this may help setting the appropriate noobservedadverseeffect or lowestadverseeffect level , currently often based on scant data , particularly for young children.more insight is needed into the antagonistic effects resulting from complex interactions between micronutrients , drugs , and infections . monitoring of data ( dietary intake , program coverage , biomarkers and morbidity ) among vulnerable population groups is essential to optimize effectiveness , safety , and efficiency of micronutrient programs . modelling tools are needed that predict the effectiveness , safety , and efficiency of micronutrient programs by integrating above data . research is needed to identify biomarkers predictive of micronutrient exposure , status , and potential health consequences of inadequate and especially excessive micronutrient exposure . during the global summit on food fortification on 911 september 2015 in arusha , one of the sessions included a special session coorganized by sight and life and unicef , entitled effective and safe micronutrient interventions : weighing the risks against the benefits. the aim of the session was to identify existing tools and needs to assist policy makers in designing effective micronutrient programs with the highest public health benefits and least risks . the presentations in the session addressed this challenge , based on a number of case studies related to folic acid , iodine and vitamin a. maaike bruins ( dsm biotechnology center , delft , the netherlands ) , with the title assessing the risks and benefits of micronutrients interventions. in order to understand the risks of inadequate and excessive intake of a micronutrient , information is required on the intake distribution of the micronutrient in the population . this enables to assess the proportion of a population lifestage group with intakes below their requirements and above their ul . software is available , such as pcside and imapp , for the estimation and modelling of food and micronutrient intake distributions , which can assist in selecting optimal food vehicles and fortification levels ( iowa state university 2015 ) . the estimates are based on known intervention costs and effectiveness ( in terms of micronutrient status ) , and links between micronutrient status and morbidity / mortality outcomes . the who has also developed tools that can assist program planners in simulating the public health impact of an increase in micronutrient intake in terms of dalys gained ( world health organization ( who ) 2015 ) . this requires ( 1 ) dietary intake data in different population groups and ( 2 ) evidence for a dose response relationship between micronutrient intake and morbidity because of deficiency . with the introduction of a micronutrient intervention , for instance , when implementing mandatory fortification , the benefits of preventing frequent , or severe , or lifelong disabilities should be balanced against the possible risks of excess intake in other population groups ( hoekstra et al . even though simulating changes in dalys resulting from micronutrient intervention programs can provide useful information to policy makers to set investment priorities , moreover , for many micronutrients the dose response relationship between excessive intake and risks is not well known . even though simulating changes in dalys resulting from micronutrient intervention programs can provide useful information to policy makers to set investment priorities , next to these sophisticated modelling tools , more userfriendly tools are needed that integrate information on program coverage , dietary intake distribution , biomarkers and morbidity to design effective and safe micronutrient programs in terms of public health impact . however , the benefits and risks of providing supplemental folic acid are largely unknown . furthermore , folic acid provided at supraphysiological , and possibly even physiological , levels may favour the growth of the parasite plasmodium falciparum ( responsible for 85% of malaria cases ) , inhibit clearance of the parasite by sulphadoxinepyrimethamine ( sp)treated malaria and increase subsequent recrudescence ( nzila et al . on the other hand , the introduction of iodized salt to regions with chronic idd may transiently increase the incidence of thyroid disorders , such as iodineinduced hyperthyroidism and autoimmune hypothyroidism , and programs should therefore include monitoring for both iodine deficiency and excess . more data on the epidemiology of thyroid disorders caused by differences in iodine intake are needed , and should be the focus of future research . the adverse effects of vitamin a deficiency include childhood blindness , and the increased risk of mortality from measles and diarrhoea ( stevens et al . longterm excessive vitamin a intakes , on the other hand , can lead to adverse effects , such as hepatotoxicity . generally , the substantial risks of vitamin a deficiency can be expected to far outweigh the relatively small risks of vitamin a excess ( allen & haskell 2002 ) . the lack of coordination of multiple vitamin a interventions and many overlapping schemes in some areas has increased concern about the risks of these programs , as some children could theoretically receive well above their recommended daily dose of the vitamin . the uncertainty around the level of excess for vitamin a has resulted in a small margin of safe intake for young children , between the recommended intake levels and maximum safe ul georg lietz from newcastle university ( newcastle upon tyne , uk ) discussed how to best assess the window of benefit for vitamin a intake . lietz highlighted the need for reliable , robust and affordable biomarkers of inadequate and excess status , to enable policy and program makers to adjust the correct level and coverage of their interventions . lietz also discussed the benefits and problems associated with the most recent methods to have been developed to enable the monitoring of both fortification and supplementation programs , including the application of stable isotope dilution techniques , and the potential for labonchip technologies for future monitoring . reina englestone from the university of california ( davis , ca , usa ) presented a dietary modelling approach to assess the risk of inadequate and excessive intakes under different program scenarios , and their applications to program planning . her presentation provided an overview of the methodology for the use of dietary data to examine the risk of inadequate and excessive micronutrient intakes using dietary reference values , and given information on current dietary patterns and the reach of program delivery platforms . this dietary modelling tool can be used to predict the effect of fortification of different food vehicles and overlapping micronutrient programs on the percentage of the population with intakes below ear or above the ul ( brown et al . finally , the role of complementary modelling tools was discussed , including the ( 1 ) lives saved tool ( list ) , which predicts the mortality reduction from some micronutrient interventions delivered to mothers and children ( johns hopkins school of public health 2015 ) , ( 2 ) models assessing liver vitamin a concentration , which can assist in the interpretation of risk of dietary intakes above the upper intake level in young children , and ( 3 ) program coverage surveys which reveal absent or redundant program coverage and thus help identify subpopulations ( e.g. regions ) at risk of inadequate or excessive intake ( aaron 2014 ; spohrer 2015 ) . population dietary intake data can be used to model the effect of different food vehicles and micronutrient levels to optimize coverage and minimize the risks of inadequate and excessive intakes in relation to dietary cutoff points . monitoring intake data will enable policy makers to adjust to the correct food vehicle , micronutrient level , and coverage of intervention programs . however , the success of these programs has , inevitably , to be confirmed by applying biomarkers indicative of micronutrient status that are affordable , sensitive and specific , and can be applied in population settings ( combs et al . thus , improved biomarkers are urgently needed which will enable policy makers to understand the benefits and risks in vulnerable population groups related to increasing micronutrient intakes . tools are available which assess or predict the effects of micronutrient programs , in terms of dietary intakes against dietary cutoff points . however , there is still need for standardized , userfriendly tools , which predict the public health impact made by micronutrient interventions , by integrating benefits and possible risk . more research is warranted in order to understand the concerns regarding the safety of iron and folic acid supplementation among young children in malaria endemic areas , and their interaction with antimalarials and infections . in general , the small risks of micronutrient excess are far outweighed by the substantial benefits of overcoming the deficiency . however , there is an urgent need to monitor overlapping micronutrient supplementation and fortification programs , and to evaluate the benefits and risks ( if any ) of increasing population micronutrient intakes for each setting ( population group , region , choice of food vehicle , micronutrient level in food , micronutrient status of the population group , concomitant food and medications , etc . )

expenditures for the medicaid program grew at an average annual rate of nearly 20 percent between 1989 and 1992 , from $ 58 billion to $ 113 billion ( coughlin , ku , and holahan , 1994 ) . moreover , medicaid spending growth significantly outpaced other categories of national health spending in the early 1990s . from 1990 to 1992 , medicaid grew at an average annual rate of 28 percent , while private health expenditures and medicare expenditures were growing at less than one - half that rate ( 7.2 percent and 10.7 percent , respectively ) ( coughlin , ku , and holahan , 1994 ) . these dramatic increases in medicaid expenditures constituted a significant fiscal burden to the federal government and the states , each of which bears part of the cost of the program . the states asserted that the medicaid program was straining their budgets and crowding out some states even predicted bankruptcy as a result of medicaid spending growth ( rovner , 1991 ) . the states largely blamed federal eligibility expansions for creating enrollment and , consequently , expenditure growth that the states could not control . the federal government asserted that the expenditure growth was largely attributable to states ' use of revenue - enhancing strategies , specifically , provider - specific tax and voluntary donation ( t&d ) programs ( executive office of the president , 1991 ) . the states typically return these receipts to donor institutions in the form of increased reimbursement rates or lump - sum payments ( i.e. , disproportionate share payments ) that are subsequently matched by federal funds . thus , without any real increase in state expenditures , federal matching revenues are generated . the federal government and the states were particularly concerned because such significant growth in medicaid expenditures was not anticipated . the office of management and budget ( 1991 ) predicted a 12-percent increase in medicaid expenditures from 1991 to 1992 . the statistics on medicaid expenditures raise a critical question : what caused spending to grow so dramatically ? the mandated expansions of medicaid eligibility to low - income infants , children , and pregnant women identified by the states could have been a significant factor . the expansions represented a significant departure from the traditional medicaid eligibility criteria that were linked to eligibility for aid to families with dependent children ( afdc ) and supplemental security income ( ssi ) . because of the large number of poor persons in the united states , particularly poor children , the low - income eligibility expansions unquestionably had the potential to increase medicaid enrollment and , consequently , medicaid expenditures . the revenue - enhancing strategies identified by the federal government could also have been a significant factor . state t&d revenues grew from $ 184 million in 1989 to $ 7.8 billion in 1992 . six states had programs in 1990 . in 1991 and 1992 , respectively , 31 and 39 states had programs ( coughlin , ku , and holahan , 1994 ) . first , two federal medicaid policies , the boren amendment and the nursing home provisions of the omnibus budget reconciliation act ( obra ) of 1987 , may have contributed directly to medicaid expenditure growth . reasonable payment rates for nursing facilities ( nfs ) and hospitals . since its enactment , it has been the basis of more than 30 lawsuits against states . in most of the resolved cases , providers have prevailed . in those instances , states were usually required to raise the level of medicaid payments and , in some instances , to make retroactive payments to facilities . moreover , states in which boren amendment cases have not been filed may be motivated to increase their reimbursement levels in anticipation of possible suits . the legislation effectively required intermediate care facilities ( icfs ) to meet the higher standards of skilled nursing facilities ( snfs ) . moreover , it required states to account for the cost of institutions ' compliance with the legislation in their payment rates . there were also federal policies implemented that would have affected expenditures through enrollment of groups other than low - income infants , children , and pregnant women . for example , the medicare catastrophic coverage act of 1988 required states to assume the medicare liabilities of low - income qualified medicare beneficiaries ( qmbs ) . in addition , the zebley decision retroactively expanded the ssi eligibility criteria and , consequently , medicaid coverage for disabled children . because the disabled are substantially more costly to the medicaid program than non - disabled children or pregnant women , the zebley decision had the potential to significantly increase program expenditures . finally , other factors , such as the economy and the prevalence of aids , may have contributed to expenditure growth . ( 1993 ) argue that some of the increase in medicaid enrollment and expenditures was due to higher unemployment during recessionary periods and , therefore , greater reliance upon public assistance , including medicaid . moreover , the treatment costs for a person with aids are high approximately $ 38,000 per year ( congressional research service , 1993 ) . thus , aids may also have contributed significantly to medicaid expenditure growth . while these factors are posited to have contributed to medicaid expenditure growth , their relative contribution , if any , has not been systematically analyzed in the literature . this article attempts to contribute to the literature by empirically analyzing the determinants of medicaid expenditure growth from 1984 to 1992 . the analysis uses cross - sectional time - series data from 49 states and the district of columbia to estimate a model of medicaid expenditures with fixed state effects . the analysis separately estimates models of expenditures for four categories of medicaid enrollees : adults ( under 65 years of age ) , children , the blind and disabled , and the elderly . the following section reviews the literature 's theoretical framework for analyzing medicaid expenditures and its empirical results . the next sections present the methodology and data for analysis and discuss the results of the empirical analysis . the analysis confirms that enrollment , federal and state medicaid policy , and the prevalence of aids are among the factors significantly related to medicaid expenditures . the median voter model is the theoretical basis for most prior empirical literature on medicaid expenditures . holahan and cohen ( 1986 ) include key elements of the median voter model in their empirical analysis , though they do not refer to it explicitly . the median voter model hypothesizes that the quantity of public goods ( such as medicaid services ) provided is determined by the preferences of the median voter or taxpayer . the median voter derives utility from providing public services as well as from the voter 's own private consumption of goods and services . the median voter maximizes utility ( u ) , or overall satisfaction , subject to a budget constraint , where x is the quantity of medicaid services provided and px is the tax price to the median voter of an additional unit of service provided to a medicaid enrollee . w represents a composite bundle of private goods and services demanded by the median voter , and pw is the price of the private bundle . z is a set of exogenous or external factors that affect the median voter 's preferences for providing medicaid services versus private consumption . in this model , the provision of public services is a function of the median voter 's income or tax capacity and the tax price of providing services . the model predicts that the amount or quantity of public services provided will decrease as the price to the median voter of providing services increases and that expenditures will increase as the median voter 's tax capacity or income increases . cromwell et al . ( 1984 ) , grannemann ( 1979 ) , and holahan and cohen 's ( 1986 ) analyses of state - level cross - sectional time - series data find key variables from the median voter model to be significantly related to medicaid expenditures . tax price and medicaid expenditures are consistently found to have a negative relationship . tax capacity , or income , and medicaid expenditures are consistently found to have a positive relationship . cromwell et al . ( 1984 ) and grannemann ( 1979 ) also find other state characteristics , notably demographic factors , to be significantly related to expenditures . unfortunately , the empirical analyses from the prior literature are based on data that precede the recent dramatic increases in medicaid expenditures as well as the policy changes that are hypothesized to have contributed to those increases . thus , they can not be used to explain recent expenditure patterns . the analysis presented in this article uses more recent data and incorporates t&d programs , boren amendment cases , and other important changes in the medicaid program . in addition , this analysis attempts to improve methodologically upon the prior literature in two ways . first , medicaid enrollment is treated as endogenous , i.e. , simultaneously determined with expenditure levels . second , this analysis attempts to improve on prior measures of the tax price of providing medicaid services by explicitly accounting for tax exportation . exported taxes are defined as funds from sources other than the federal government or a state 's taxpayers that could be used to finance a medicaid program . this analysis separately models medicaid expenditures for four groups of enrollees : adults , children , the blind and disabled , and the aged . these groups differ with respect to their basis of eligibility , service use and expenditure patterns , and policies or other exogenous factors affecting them . for example , the prevalence of aids might affect expenditures for adults or the blind and disabled . however , it is not expected to affect expenditures for the aged . for each group of enrollees , the model is used to analyze total expenditures and expenditures for categories of service relevant for each group . the general form of the empirical model for analyzing medicaid expenditures is : the coefficients to be estimated are , i , t , and j . i , is the intercept term for state i. t is the coefficient for the time - trend variable , t. j is the change in expenditures associated with an increase of 1 unit in independent variable j. the dependent variable is yit , medicaid expenditures in state i in year t. xijt is the value of independent variable j in state i in year t. it is an error term . the dependent variables for this analysis are state - level total expenditures and expenditures by type of service for adult , child , aged , and blind and disabled medicaid enrollees from 1984 - 92 . these data are from hcfa form-2082 , which contains aggregate data on medicaid recipients , enrollees , and expenditures . the analysis includes three broad categories of independent variables : federal medicaid policy , state policy , and enrollment and other factors . table 2 contains the variables ' definition , their hypothesized relationship to total expenditures , means , and standard deviations . the federal policy variables represent obra 1987 's nursing home provisions and the boren amendment , the two federal policy variables expected to directly affect expenditures . for example , the nurse staffing level required per snf bed is greater than the level required per icf bed . the additional costs would result in an upward shift in expenditures that is approximately proportional to the prior number of icf beds . therefore , the obra 1987 variable is the number of icf beds in a state prior to the legislation 's implementation . the boren amendment variable assumes a value of 1 in the year in which a case was decided in favor of provider institutions in a state and in all years thereafter . it is expected to be positively related to total medicaid expenditures and to inpatient and nf expenditures . the boren amendment is not expected to have influenced expenditures for other categories of service . there are two ways in which state policy could be incorporated into the analysis of medicaid expenditures . the alternative approach would assess the impacts of the underlying determinants of state policy on expenditures . the latter approach is most consistent with the focus of this analysis on the exogenous determinants of medicaid expenditures . therefore , the analysis focuses on exogenous determinants of state policy derived from the median voter model : tax price , tax capacity , and the governor 's political party ( as a measure of a state 's political ideology ) . these determinants are hypothesized to influence state medicaid policy which , in turn , is hypothesized to influence medicaid expenditures . the measure of tax capacity is median household income and is expected to be positively related to expenditures . the measure of tax price is an estimate of the median voter 's share of the unit cost of providing an additional unit of service to a medicaid enrollee . the tax price to the median voter of providing medicaid services is reduced by funds from other sources . the literature indicates that severance taxes ( those based on the value of extracted natural resources ) and corporate taxes are among the most important sources of tax exports . for this analysis , the specific components of the tax price of providing medicaid services are the federal matching rate ( match ) , the unit cost of providing the service ( cost ) , the tax exportation rate ( export ) , and the size of the taxpayer population ( taxpayers ) . algebraically , the tax price is : the measure of exports is the share of state tax revenues from exports . tax exports include corporate net income , severance , amusement , and other selective tax revenues . the measure of the size of the taxpayer population is the number of persons 18 years of age or over . we note that the governor 's political party is a crude measure of political ideology . unfortunately , more refined measures ( for example , those based on congressional voting records ) are not comparable over time . therefore , they were not considered for this analysis . the model also includes an indicator of whether a state has a t&d program . this state policy variable is explicitly included because of policymakers ' significant concerns about the programs ' contribution to medicaid expenditure growth . based on coughlin , ku , and holahan ( 1994 ) , the models of expenditures for adult and for blind and disabled enrollees also include a measure of the prevalence of aids . one concern is that medicaid enrollment is endogenous , i.e. , jointly determined with expenditures . in this case , we conducted a hausman ( 1978 ) test for endogeneity , which indicated that enrollment should be treated as endogenous . therefore , the analysis uses a two - stage least squares estimation procedure to generate unbiased coefficient estimates . the first stage estimates medicaid enrollment for each group . predicted enrollment from the first stage ( rather than actual enrollment ) is used as an independent variable in the second stage estimation of the expenditure model . enrollment models were estimated for adults , children , the blind and disabled , and the aged using state - level data from 1984 - 92 . the enrollment models ' independent variables included : federal policy variables representing the low - income eligibility expansions , the zebley decision , and the qmb program ; state policy determinants , specifically the median voter 's tax price , the governor 's political party , and the size of the relevant poverty population ; fixed state effects ; and a time - trend variable . the models of adult and child enrollment also included determinants of afdc participation , specifically the unemployment rate and wage rates . from the afdc participation literature , finally , the prevalence of aids was also included in the model for the blind and disabled . the blind and disabled are the groups through which the literature hypothesizes that aids would impact medicaid enrollment . the results of the enrollment model estimation indicated that federal policy variables , notably low - income eligibility expansions and the qmb program , were significantly related to enrollment . in addition , tax price , the size of the relevant poverty population , afdc participation variables , and aids were found to be significantly related to enrollment . however , because a hausman test did not indicate endogeneity , the programs were treated as exogenous . the lack of statistical evidence of endogeneity is consistent with the anecdotal evidence that the adoption of this financing innovation was primarily related to knowledge of its existence rather than factors that determine medicaid spending . the second concern is unobserved or unmeasured state characteristics that may affect medicaid expenditures . to the extent that these factors are correlated with independent variables included in the model , ols estimates of the model parameters would be biased . the fixed - effects approach includes an intercept term for each state in the expenditure models . the intercept term effectively controls for time - invariant state characteristics that may affect medicaid enrollment , but which are not otherwise included in the model . the ols estimates of the fixed - effects model parameters can be interpreted as representing the relationship between changes in the dependent variable and changes in the independent variable . it is important to note that this fixed - effects model primarily uses variation over time within states rather than cross - sectional variation to estimate model coefficients . discussions about the growth in medicaid expenditures in the late 1980s and early 1990s often focus on increases in total medicaid spending at the national level . table 3 shows that state - level medicaid spending grew dramatically but varied significantly across states . average annual growth rates from 1989 to 1992 ranged from a low of 10 percent in pennsylvania and rhode island to a high of 78 percent in new hampshire . these state - level statistics underscore the question raised by the national statistics : what determines medicaid expenditure growth ? in addition , t&d programs and the prevalence of aids are also found to be significantly related to expenditures . table 4 presents the estimated effects of the independent variables on total expenditures for each enrollment group . table 5 summarizes the results for the federal policy variables that were hypothesized to be related to expenditures for specific categories of service . the results indicate that total medicaid expenditures for each enrollment group and almost all categories of expenditures significantly increase as the group 's enrollment increases . the estimated coefficients are consistent with the facts that adults are more expensive than children and that blind and disabled enrollees and elderly enrollees are significantly more expensive than adults or children . given the potential importance of federally mandated eligibility expansions to enrollment , one relevant question is how much federal eligibility expansions contributed to expenditure growth . coughlin , ku , and holahan ( 1994 ) estimated that the eligibility expansions affecting low - income infants , children , and pregnant women accounted for 45 percent of the growth in total medicaid recipients from 1988 to 1992 . however , these groups accounted for only 9 percent of expenditure growth . while not insubstantial , this figure does not support the contention that the eligibility expansions were the driving force behind the dramatic growth in medicaid expenditures . holahan , liska , and obermaier ( 1994 ) report that , from 1992 to 1993 , enrollment growth accounted for 67 percent of expenditure growth . in contrast , from 1991 to 1992 , enrollment accounted for just over 30 percent of expenditure growth . the estimation results also indicate that other federal medicaid policies made a statistically significant contribution to medicaid expenditure growth from 1984 to 1992 . obra 1987 's provisions to improve nf quality are associated with increased nf expenditures and total expenditures for blind and disabled and aged enroilees . the coefficients suggest that the cost of maintaining an icf bed at a snf level is approximately $ 5,000 per year . boren amendment cases are associated with increases in inpatient , nf , and total expenditures , except in the model of acute - care expenditures for the aged . the lack of significance in this case is probably attributable to the fact that medicare covers these services for the aged . the results indicate that the boren amendment 's impact on total expenditures for the blind and disabled and the aged is greater than for adults and children . this reflects the fact that inpatient and nf expenditures ( i.e. , those directly affected by boren ) account for substantially more of total expenditures for the blind and disabled and the aged than for the other groups . the coefficient of the tax price variable is usually negative , indicating that total expenditures increase as the tax price decreases . the coefficient of the tax capacity variable , median household income , is usually positive , indicating that total expenditures increase as a state 's income ( or tax capacity ) increases . the influence of state policy determinants is more evident for category of service expenditures than for total expenditures ( table 6 ) . except in the case of children we interpret this result to indicate that tax capacity influences state medicaid policy which , in turn , influences medicaid expenditures . in addition , provider programs are significant in the models of inpatient expenditures for adult and blind and disabled enrollees and in the model of acute - care expenditures for aged enrollees . provider taxes and donations are revenue sources for the state and must be expended in order to qualify for federal matching . the results by type of service are consistent with the literature 's reports that funds from provider programs were expended as hospital payments ( coughlin , ku , and holahan , 1994 ) . it is noteworthy that the variable for t&d programs was statistically significant in any of the models . in fixed - effects models of the type estimated here , finding statistically significant relationships since the effects of t&d programs are based upon data for only 2 years , we might not have expected the estimated coefficients to be significant . it should be noted that the medicaid voluntary contribution and provider - specific tax amendments of 1991 effectively capped these programs beginning in 1993 . the estimation results suggest that a republican governorship is associated with significantly greater expenditures than a democratic governorship and is statistically significant only in the expenditure models for aged enrollees . this result raises questions about the commonly accepted supposition that democrats are big spenders and that republicans are not . similar results are reported in kronebusch ( 1993 ) . using a more precise measure of political ideology than the governor 's political party , kronebusch found that ideologically conservative states had higher levels of medicaid spending per recipient than liberal states . this result was significant in spending models for the aged , blind and disabled , and children . kronebusch offers two possible explanations for the positive relationship between medicaid spending per recipient and conservative ideology in a state . first , a conservative ideology may support higher reimbursement rates for providers than a liberal ideology . second , conservatives may be willing to spend relatively generously on groups that they have determined deserve or merit public support , such as the aged . finally , the prevalence of aids was found to be positively and significantly related to expenditures for adult enrollees . the result for adults reflects the prevalence of aids among the afdc - eligible population . the result for blind and disabled enrollees reflects the cost impact of aids patients who become eligible for medicaid as disabled after depleting their resources . the relatively larger coefficient for aids in the blind and disabled model may reflect that , on average , these individuals tend to be at a later and more costly phase of their illness when they qualify for medicaid than afdc - eligible enrollees who have aids . the latter group is composed of afdc recipients who contract aids but whose eligibility is based on the afdc designation which typically predates the onset of the disease . the dependent variables for this analysis are state - level total expenditures and expenditures by type of service for adult , child , aged , and blind and disabled medicaid enrollees from 1984 - 92 . these data are from hcfa form-2082 , which contains aggregate data on medicaid recipients , enrollees , and expenditures . the analysis includes three broad categories of independent variables : federal medicaid policy , state policy , and enrollment and other factors . table 2 contains the variables ' definition , their hypothesized relationship to total expenditures , means , and standard deviations . the federal policy variables represent obra 1987 's nursing home provisions and the boren amendment , the two federal policy variables expected to directly affect expenditures . for example , the nurse staffing level required per snf bed is greater than the level required per icf bed . the additional costs would result in an upward shift in expenditures that is approximately proportional to the prior number of icf beds . therefore , the obra 1987 variable is the number of icf beds in a state prior to the legislation 's implementation . the boren amendment variable assumes a value of 1 in the year in which a case was decided in favor of provider institutions in a state and in all years thereafter . it is expected to be positively related to total medicaid expenditures and to inpatient and nf expenditures . the boren amendment is not expected to have influenced expenditures for other categories of service . there are two ways in which state policy could be incorporated into the analysis of medicaid expenditures . the alternative approach would assess the impacts of the underlying determinants of state policy on expenditures . the latter approach is most consistent with the focus of this analysis on the exogenous determinants of medicaid expenditures . therefore , the analysis focuses on exogenous determinants of state policy derived from the median voter model : tax price , tax capacity , and the governor 's political party ( as a measure of a state 's political ideology ) . these determinants are hypothesized to influence state medicaid policy which , in turn , is hypothesized to influence medicaid expenditures . the measure of tax capacity is median household income and is expected to be positively related to expenditures . the measure of tax price is an estimate of the median voter 's share of the unit cost of providing an additional unit of service to a medicaid enrollee . the tax price to the median voter of providing medicaid services is reduced by funds from other sources . the literature indicates that severance taxes ( those based on the value of extracted natural resources ) and corporate taxes are among the most important sources of tax exports . for this analysis , the specific components of the tax price of providing medicaid services are the federal matching rate ( match ) , the unit cost of providing the service ( cost ) , the tax exportation rate ( export ) , and the size of the taxpayer population ( taxpayers ) . algebraically , the tax price is : the measure of exports is the share of state tax revenues from exports . tax exports include corporate net income , severance , amusement , and other selective tax revenues . the measure of the size of the taxpayer population is the number of persons 18 years of age or over . we note that the governor 's political party is a crude measure of political ideology . unfortunately , more refined measures ( for example , those based on congressional voting records ) are not comparable over time . the model also includes an indicator of whether a state has a t&d program . this state policy variable is explicitly included because of policymakers ' significant concerns about the programs ' contribution to medicaid expenditure growth . based on coughlin , ku , and holahan ( 1994 ) , the models of expenditures for adult and for blind and disabled enrollees also include a measure of the prevalence of aids . one concern is that medicaid enrollment is endogenous , i.e. , jointly determined with expenditures . in this case , we conducted a hausman ( 1978 ) test for endogeneity , which indicated that enrollment should be treated as endogenous . therefore , the analysis uses a two - stage least squares estimation procedure to generate unbiased coefficient estimates . the first stage estimates medicaid enrollment for each group . predicted enrollment from the first stage ( rather than actual enrollment ) is used as an independent variable in the second stage estimation of the expenditure model . enrollment models were estimated for adults , children , the blind and disabled , and the aged using state - level data from 1984 - 92 . the enrollment models ' independent variables included : federal policy variables representing the low - income eligibility expansions , the zebley decision , and the qmb program ; state policy determinants , specifically the median voter 's tax price , the governor 's political party , and the size of the relevant poverty population ; fixed state effects ; and a time - trend variable . the models of adult and child enrollment also included determinants of afdc participation , specifically the unemployment rate and wage rates . from the afdc participation literature , finally , the prevalence of aids was also included in the model for the blind and disabled . the blind and disabled are the groups through which the literature hypothesizes that aids would impact medicaid enrollment . the results of the enrollment model estimation indicated that federal policy variables , notably low - income eligibility expansions and the qmb program , were significantly related to enrollment . in addition , tax price , the size of the relevant poverty population , afdc participation variables , and aids were found to be significantly related to enrollment . however , because a hausman test did not indicate endogeneity , the programs were treated as exogenous . the lack of statistical evidence of endogeneity is consistent with the anecdotal evidence that the adoption of this financing innovation was primarily related to knowledge of its existence rather than factors that determine medicaid spending . the second concern is unobserved or unmeasured state characteristics that may affect medicaid expenditures . to the extent that these factors are correlated with independent variables included in the model , ols estimates of the model parameters the fixed - effects approach includes an intercept term for each state in the expenditure models . the intercept term effectively controls for time - invariant state characteristics that may affect medicaid enrollment , but which are not otherwise included in the model . the ols estimates of the fixed - effects model parameters can be interpreted as representing the relationship between changes in the dependent variable and changes in the independent variable . it is important to note that this fixed - effects model primarily uses variation over time within states rather than cross - sectional variation to estimate model coefficients . discussions about the growth in medicaid expenditures in the late 1980s and early 1990s often focus on increases in total medicaid spending at the national level . table 3 shows that state - level medicaid spending grew dramatically but varied significantly across states . average annual growth rates from 1989 to 1992 ranged from a low of 10 percent in pennsylvania and rhode island to a high of 78 percent in new hampshire . these state - level statistics underscore the question raised by the national statistics : what determines medicaid expenditure growth ? in addition , t&d programs and the prevalence of aids are also found to be significantly related to expenditures . table 4 presents the estimated effects of the independent variables on total expenditures for each enrollment group . table 5 summarizes the results for the federal policy variables that were hypothesized to be related to expenditures for specific categories of service . the results indicate that total medicaid expenditures for each enrollment group and almost all categories of expenditures significantly increase as the group 's enrollment increases . the estimated coefficients are consistent with the facts that adults are more expensive than children and that blind and disabled enrollees and elderly enrollees are significantly more expensive than adults or children . given the potential importance of federally mandated eligibility expansions to enrollment , one relevant question is how much federal eligibility expansions contributed to expenditure growth . coughlin , ku , and holahan ( 1994 ) estimated that the eligibility expansions affecting low - income infants , children , and pregnant women accounted for 45 percent of the growth in total medicaid recipients from 1988 to 1992 . however , these groups accounted for only 9 percent of expenditure growth . while not insubstantial , this figure does not support the contention that the eligibility expansions were the driving force behind the dramatic growth in medicaid expenditures . holahan , liska , and obermaier ( 1994 ) report that , from 1992 to 1993 , enrollment growth accounted for 67 percent of expenditure growth . in contrast , from 1991 to 1992 , enrollment accounted for just over 30 percent of expenditure growth . the estimation results also indicate that other federal medicaid policies made a statistically significant contribution to medicaid expenditure growth from 1984 to 1992 . obra 1987 's provisions to improve nf quality are associated with increased nf expenditures and total expenditures for blind and disabled and aged enroilees . the coefficients suggest that the cost of maintaining an icf bed at a snf level is approximately $ 5,000 per year . boren amendment cases are associated with increases in inpatient , nf , and total expenditures , except in the model of acute - care expenditures for the aged . the lack of significance in this case is probably attributable to the fact that medicare covers these services for the aged . the results indicate that the boren amendment 's impact on total expenditures for the blind and disabled and the aged is greater than for adults and children . this reflects the fact that inpatient and nf expenditures ( i.e. , those directly affected by boren ) account for substantially more of total expenditures for the blind and disabled and the aged than for the other groups . the coefficient of the tax price variable is usually negative , indicating that total expenditures increase as the tax price decreases . the coefficient of the tax capacity variable , median household income , is usually positive , indicating that total expenditures increase as a state 's income ( or tax capacity ) increases . the influence of state policy determinants is more evident for category of service expenditures than for total expenditures ( table 6 ) . except in the case of children we interpret this result to indicate that tax capacity influences state medicaid policy which , in turn , influences medicaid expenditures . in addition , provider programs are significant in the models of inpatient expenditures for adult and blind and disabled enrollees and in the model of acute - care expenditures for aged enrollees . provider taxes and donations are revenue sources for the state and must be expended in order to qualify for federal matching . the results by type of service are consistent with the literature 's reports that funds from provider programs were expended as hospital payments ( coughlin , ku , and holahan , 1994 ) . it is noteworthy that the variable for t&d programs was statistically significant in any of the models . in fixed - effects models of the type estimated here , finding statistically significant relationships largely depends on variation over time within a state . since the effects of t&d programs are based upon data for only 2 years , we might not have expected the estimated coefficients to be significant . it should be noted that the medicaid voluntary contribution and provider - specific tax amendments of 1991 effectively capped these programs beginning in 1993 . the estimation results suggest that a republican governorship is associated with significantly greater expenditures than a democratic governorship and is statistically significant only in the expenditure models for aged enrollees . this result raises questions about the commonly accepted supposition that democrats are big spenders and that republicans are not . similar results are reported in kronebusch ( 1993 ) . using a more precise measure of political ideology than the governor 's political party , kronebusch found that ideologically conservative states had higher levels of medicaid spending per recipient than liberal states . this result was significant in spending models for the aged , blind and disabled , and children . kronebusch offers two possible explanations for the positive relationship between medicaid spending per recipient and conservative ideology in a state . first , a conservative ideology may support higher reimbursement rates for providers than a liberal ideology . second , conservatives may be willing to spend relatively generously on groups that they have determined deserve or merit public support , such as the aged . finally , the prevalence of aids was found to be positively and significantly related to expenditures for adult enrollees . the result for adults reflects the prevalence of aids among the afdc - eligible population . the result for blind and disabled enrollees reflects the cost impact of aids patients who become eligible for medicaid as disabled after depleting their resources . the relatively larger coefficient for aids in the blind and disabled model may reflect that , on average , these individuals tend to be at a later and more costly phase of their illness when they qualify for medicaid than afdc - eligible enrollees who have aids . the latter group is composed of afdc recipients who contract aids but whose eligibility is based on the afdc designation which typically predates the onset of the disease . many of the factors that the literature hypothesizes to have contributed to medicaid expenditure growth in recent years were found to be significantly related to expenditure growth from 1984 to 1992 . first , federal medicaid policies directly affecting expenditures , i.e. , obra 1987 and boren amendment cases , were found to be significantly related to expenditure growth . third , this analysis suggests that state policy determinants , i.e. , tax capacity , affected expenditure growth . in addition , the results indicated a significant relationship between state t&d programs and expenditures for inpatient services . the analysis separately modeled medicaid expenditures for four groups of enrollees to allow for the differential impact of various factors . for example , provider programs were significantly related to inpatient expenditures but not to expenditures for other types of services . these results underscore the importance of considering the diverse groups that comprise the medicaid population . for example , the medical needs of aged enrollees ( many of whom are institutionalized ) are quite different from those of children ( who are relatively healthy ) . the service use and expenditure patterns also vary for these enrollment groups . thus , policy interventions and other exogenous factors may have different effects on expenditures , depending on the enrollment groups that they affect . the results suggest that the dramatic rates of increase in medicaid expenditures should not be expected to continue , because the impact of some factors should be subsiding . for example : the mandated eligibility expansions for low - income infants , children , and pregnant women are largely implemented ; states ' economic performance is improving ; and the potential for growth in t&d programs was curtailed by the medicaid voluntary contribution and provider - specific tax amendments of 1991 . the expected reduction in expenditure growth is supported by recent data indicating medicaid expenditures grew by 10 percent from 1992 to 1993 , nearly one - half the average annual growth rate from 1989 - 92 . moreover , the relative contribution of various factors to expenditure growth is shifting . enrollment growth accounts for substantially more of expenditure growth from 1992 to 1993 than from 1990 to 1992 , and real expenditures per enrollee account for less ( holahan , liska , and obermaier , 1994 ) . however , although medicaid expenditure growth is not expected to continue at average annual rates of more than 20 percent , it may be somewhat higher than the low growth rates of the early and middle 1980s . some policies are not fully phased - in and may continue to fuel expenditure growth . for example , in future years , obra 1990 expands states ' coverage of medicare part b premiums to beneficiaries with higher income levels than are currently eligible . this act also phases in eligibility for children with household incomes less than 100 percent of the fpl , until all children under 19 years of age are covered in the year 2002 . while this analysis begins to enlighten our understanding of the causes of medicaid expenditure growth , it also suggests areas for future research . for example , future research might consider the effects of specific state policy variables rather than state policy determinants . in addition , in fixed - effects models of the type estimated here , much of the variation in expenditures is essentially absorbed by the state intercepts .

expenditures for the medicaid program grew at the alarming and unexpected average annual rate of nearly 20 percent from 1989 ( $ 58 billion ) to 1992 ( $ 113 billion ) . these statistics raise a critical question : what caused spending to grow so dramatically ? using state - level data from 1984 - 92 , this analysis examines the determinants of medicaid expenditure growth . the results indicate that medicaid enrollment , federal medicaid policy , and state policy are significantly related to medicaid expenditure growth . the analysis also finds the prevalence of acquired immunodeficiency syndrome ( aids ) to be significantly related to medicaid expenditures .

Introduction Literature Review Model Specification and Estimation Dependent Variables Independent Variables Estimation Results Summary and Policy Implications

expenditures for the medicaid program grew at an average annual rate of nearly 20 percent between 1989 and 1992 , from $ 58 billion to $ 113 billion ( coughlin , ku , and holahan , 1994 ) . from 1990 to 1992 , medicaid grew at an average annual rate of 28 percent , while private health expenditures and medicare expenditures were growing at less than one - half that rate ( 7.2 percent and 10.7 percent , respectively ) ( coughlin , ku , and holahan , 1994 ) . the states asserted that the medicaid program was straining their budgets and crowding out some states even predicted bankruptcy as a result of medicaid spending growth ( rovner , 1991 ) . the federal government and the states were particularly concerned because such significant growth in medicaid expenditures was not anticipated . the office of management and budget ( 1991 ) predicted a 12-percent increase in medicaid expenditures from 1991 to 1992 . the statistics on medicaid expenditures raise a critical question : what caused spending to grow so dramatically ? the mandated expansions of medicaid eligibility to low - income infants , children , and pregnant women identified by the states could have been a significant factor . because of the large number of poor persons in the united states , particularly poor children , the low - income eligibility expansions unquestionably had the potential to increase medicaid enrollment and , consequently , medicaid expenditures . in 1991 and 1992 , respectively , 31 and 39 states had programs ( coughlin , ku , and holahan , 1994 ) . first , two federal medicaid policies , the boren amendment and the nursing home provisions of the omnibus budget reconciliation act ( obra ) of 1987 , may have contributed directly to medicaid expenditure growth . moreover , it required states to account for the cost of institutions ' compliance with the legislation in their payment rates . because the disabled are substantially more costly to the medicaid program than non - disabled children or pregnant women , the zebley decision had the potential to significantly increase program expenditures . finally , other factors , such as the economy and the prevalence of aids , may have contributed to expenditure growth . ( 1993 ) argue that some of the increase in medicaid enrollment and expenditures was due to higher unemployment during recessionary periods and , therefore , greater reliance upon public assistance , including medicaid . thus , aids may also have contributed significantly to medicaid expenditure growth . while these factors are posited to have contributed to medicaid expenditure growth , their relative contribution , if any , has not been systematically analyzed in the literature . this article attempts to contribute to the literature by empirically analyzing the determinants of medicaid expenditure growth from 1984 to 1992 . the analysis uses cross - sectional time - series data from 49 states and the district of columbia to estimate a model of medicaid expenditures with fixed state effects . the analysis separately estimates models of expenditures for four categories of medicaid enrollees : adults ( under 65 years of age ) , children , the blind and disabled , and the elderly . the analysis confirms that enrollment , federal and state medicaid policy , and the prevalence of aids are among the factors significantly related to medicaid expenditures . the median voter model is the theoretical basis for most prior empirical literature on medicaid expenditures . the median voter maximizes utility ( u ) , or overall satisfaction , subject to a budget constraint , where x is the quantity of medicaid services provided and px is the tax price to the median voter of an additional unit of service provided to a medicaid enrollee . ( 1984 ) , grannemann ( 1979 ) , and holahan and cohen 's ( 1986 ) analyses of state - level cross - sectional time - series data find key variables from the median voter model to be significantly related to medicaid expenditures . tax capacity , or income , and medicaid expenditures are consistently found to have a positive relationship . ( 1984 ) and grannemann ( 1979 ) also find other state characteristics , notably demographic factors , to be significantly related to expenditures . the analysis presented in this article uses more recent data and incorporates t&d programs , boren amendment cases , and other important changes in the medicaid program . in addition , this analysis attempts to improve methodologically upon the prior literature in two ways . first , medicaid enrollment is treated as endogenous , i.e. second , this analysis attempts to improve on prior measures of the tax price of providing medicaid services by explicitly accounting for tax exportation . this analysis separately models medicaid expenditures for four groups of enrollees : adults , children , the blind and disabled , and the aged . for example , the prevalence of aids might affect expenditures for adults or the blind and disabled . however , it is not expected to affect expenditures for the aged . the general form of the empirical model for analyzing medicaid expenditures is : the coefficients to be estimated are , i , t , and j . i , is the intercept term for state i. t is the coefficient for the time - trend variable , t. j is the change in expenditures associated with an increase of 1 unit in independent variable j. the dependent variable is yit , medicaid expenditures in state i in year t. xijt is the value of independent variable j in state i in year t. it is an error term . the dependent variables for this analysis are state - level total expenditures and expenditures by type of service for adult , child , aged , and blind and disabled medicaid enrollees from 1984 - 92 . these data are from hcfa form-2082 , which contains aggregate data on medicaid recipients , enrollees , and expenditures . the analysis includes three broad categories of independent variables : federal medicaid policy , state policy , and enrollment and other factors . it is expected to be positively related to total medicaid expenditures and to inpatient and nf expenditures . the boren amendment is not expected to have influenced expenditures for other categories of service . there are two ways in which state policy could be incorporated into the analysis of medicaid expenditures . the alternative approach would assess the impacts of the underlying determinants of state policy on expenditures . the latter approach is most consistent with the focus of this analysis on the exogenous determinants of medicaid expenditures . therefore , the analysis focuses on exogenous determinants of state policy derived from the median voter model : tax price , tax capacity , and the governor 's political party ( as a measure of a state 's political ideology ) . these determinants are hypothesized to influence state medicaid policy which , in turn , is hypothesized to influence medicaid expenditures . the measure of tax capacity is median household income and is expected to be positively related to expenditures . for this analysis , the specific components of the tax price of providing medicaid services are the federal matching rate ( match ) , the unit cost of providing the service ( cost ) , the tax exportation rate ( export ) , and the size of the taxpayer population ( taxpayers ) . tax exports include corporate net income , severance , amusement , and other selective tax revenues . therefore , they were not considered for this analysis . this state policy variable is explicitly included because of policymakers ' significant concerns about the programs ' contribution to medicaid expenditure growth . based on coughlin , ku , and holahan ( 1994 ) , the models of expenditures for adult and for blind and disabled enrollees also include a measure of the prevalence of aids . one concern is that medicaid enrollment is endogenous , i.e. therefore , the analysis uses a two - stage least squares estimation procedure to generate unbiased coefficient estimates . enrollment models were estimated for adults , children , the blind and disabled , and the aged using state - level data from 1984 - 92 . the enrollment models ' independent variables included : federal policy variables representing the low - income eligibility expansions , the zebley decision , and the qmb program ; state policy determinants , specifically the median voter 's tax price , the governor 's political party , and the size of the relevant poverty population ; fixed state effects ; and a time - trend variable . from the afdc participation literature , finally , the prevalence of aids was also included in the model for the blind and disabled . the results of the enrollment model estimation indicated that federal policy variables , notably low - income eligibility expansions and the qmb program , were significantly related to enrollment . in addition , tax price , the size of the relevant poverty population , afdc participation variables , and aids were found to be significantly related to enrollment . the second concern is unobserved or unmeasured state characteristics that may affect medicaid expenditures . the intercept term effectively controls for time - invariant state characteristics that may affect medicaid enrollment , but which are not otherwise included in the model . discussions about the growth in medicaid expenditures in the late 1980s and early 1990s often focus on increases in total medicaid spending at the national level . table 3 shows that state - level medicaid spending grew dramatically but varied significantly across states . average annual growth rates from 1989 to 1992 ranged from a low of 10 percent in pennsylvania and rhode island to a high of 78 percent in new hampshire . these state - level statistics underscore the question raised by the national statistics : what determines medicaid expenditure growth ? in addition , t&d programs and the prevalence of aids are also found to be significantly related to expenditures . table 5 summarizes the results for the federal policy variables that were hypothesized to be related to expenditures for specific categories of service . the results indicate that total medicaid expenditures for each enrollment group and almost all categories of expenditures significantly increase as the group 's enrollment increases . given the potential importance of federally mandated eligibility expansions to enrollment , one relevant question is how much federal eligibility expansions contributed to expenditure growth . coughlin , ku , and holahan ( 1994 ) estimated that the eligibility expansions affecting low - income infants , children , and pregnant women accounted for 45 percent of the growth in total medicaid recipients from 1988 to 1992 . however , these groups accounted for only 9 percent of expenditure growth . while not insubstantial , this figure does not support the contention that the eligibility expansions were the driving force behind the dramatic growth in medicaid expenditures . holahan , liska , and obermaier ( 1994 ) report that , from 1992 to 1993 , enrollment growth accounted for 67 percent of expenditure growth . in contrast , from 1991 to 1992 , enrollment accounted for just over 30 percent of expenditure growth . the estimation results also indicate that other federal medicaid policies made a statistically significant contribution to medicaid expenditure growth from 1984 to 1992 . boren amendment cases are associated with increases in inpatient , nf , and total expenditures , except in the model of acute - care expenditures for the aged . the results indicate that the boren amendment 's impact on total expenditures for the blind and disabled and the aged is greater than for adults and children . , those directly affected by boren ) account for substantially more of total expenditures for the blind and disabled and the aged than for the other groups . except in the case of children we interpret this result to indicate that tax capacity influences state medicaid policy which , in turn , influences medicaid expenditures . the results by type of service are consistent with the literature 's reports that funds from provider programs were expended as hospital payments ( coughlin , ku , and holahan , 1994 ) . this result was significant in spending models for the aged , blind and disabled , and children . kronebusch offers two possible explanations for the positive relationship between medicaid spending per recipient and conservative ideology in a state . finally , the prevalence of aids was found to be positively and significantly related to expenditures for adult enrollees . the result for adults reflects the prevalence of aids among the afdc - eligible population . the relatively larger coefficient for aids in the blind and disabled model may reflect that , on average , these individuals tend to be at a later and more costly phase of their illness when they qualify for medicaid than afdc - eligible enrollees who have aids . the dependent variables for this analysis are state - level total expenditures and expenditures by type of service for adult , child , aged , and blind and disabled medicaid enrollees from 1984 - 92 . these data are from hcfa form-2082 , which contains aggregate data on medicaid recipients , enrollees , and expenditures . the analysis includes three broad categories of independent variables : federal medicaid policy , state policy , and enrollment and other factors . table 2 contains the variables ' definition , their hypothesized relationship to total expenditures , means , and standard deviations . it is expected to be positively related to total medicaid expenditures and to inpatient and nf expenditures . there are two ways in which state policy could be incorporated into the analysis of medicaid expenditures . the alternative approach would assess the impacts of the underlying determinants of state policy on expenditures . the latter approach is most consistent with the focus of this analysis on the exogenous determinants of medicaid expenditures . therefore , the analysis focuses on exogenous determinants of state policy derived from the median voter model : tax price , tax capacity , and the governor 's political party ( as a measure of a state 's political ideology ) . these determinants are hypothesized to influence state medicaid policy which , in turn , is hypothesized to influence medicaid expenditures . the measure of tax capacity is median household income and is expected to be positively related to expenditures . for this analysis , the specific components of the tax price of providing medicaid services are the federal matching rate ( match ) , the unit cost of providing the service ( cost ) , the tax exportation rate ( export ) , and the size of the taxpayer population ( taxpayers ) . this state policy variable is explicitly included because of policymakers ' significant concerns about the programs ' contribution to medicaid expenditure growth . based on coughlin , ku , and holahan ( 1994 ) , the models of expenditures for adult and for blind and disabled enrollees also include a measure of the prevalence of aids . one concern is that medicaid enrollment is endogenous , i.e. enrollment models were estimated for adults , children , the blind and disabled , and the aged using state - level data from 1984 - 92 . the enrollment models ' independent variables included : federal policy variables representing the low - income eligibility expansions , the zebley decision , and the qmb program ; state policy determinants , specifically the median voter 's tax price , the governor 's political party , and the size of the relevant poverty population ; fixed state effects ; and a time - trend variable . the models of adult and child enrollment also included determinants of afdc participation , specifically the unemployment rate and wage rates . from the afdc participation literature , finally , the prevalence of aids was also included in the model for the blind and disabled . the results of the enrollment model estimation indicated that federal policy variables , notably low - income eligibility expansions and the qmb program , were significantly related to enrollment . in addition , tax price , the size of the relevant poverty population , afdc participation variables , and aids were found to be significantly related to enrollment . the lack of statistical evidence of endogeneity is consistent with the anecdotal evidence that the adoption of this financing innovation was primarily related to knowledge of its existence rather than factors that determine medicaid spending . the intercept term effectively controls for time - invariant state characteristics that may affect medicaid enrollment , but which are not otherwise included in the model . discussions about the growth in medicaid expenditures in the late 1980s and early 1990s often focus on increases in total medicaid spending at the national level . table 3 shows that state - level medicaid spending grew dramatically but varied significantly across states . average annual growth rates from 1989 to 1992 ranged from a low of 10 percent in pennsylvania and rhode island to a high of 78 percent in new hampshire . these state - level statistics underscore the question raised by the national statistics : what determines medicaid expenditure growth ? in addition , t&d programs and the prevalence of aids are also found to be significantly related to expenditures . table 5 summarizes the results for the federal policy variables that were hypothesized to be related to expenditures for specific categories of service . the results indicate that total medicaid expenditures for each enrollment group and almost all categories of expenditures significantly increase as the group 's enrollment increases . given the potential importance of federally mandated eligibility expansions to enrollment , one relevant question is how much federal eligibility expansions contributed to expenditure growth . coughlin , ku , and holahan ( 1994 ) estimated that the eligibility expansions affecting low - income infants , children , and pregnant women accounted for 45 percent of the growth in total medicaid recipients from 1988 to 1992 . while not insubstantial , this figure does not support the contention that the eligibility expansions were the driving force behind the dramatic growth in medicaid expenditures . holahan , liska , and obermaier ( 1994 ) report that , from 1992 to 1993 , enrollment growth accounted for 67 percent of expenditure growth . in contrast , from 1991 to 1992 , enrollment accounted for just over 30 percent of expenditure growth . the estimation results also indicate that other federal medicaid policies made a statistically significant contribution to medicaid expenditure growth from 1984 to 1992 . boren amendment cases are associated with increases in inpatient , nf , and total expenditures , except in the model of acute - care expenditures for the aged . the results indicate that the boren amendment 's impact on total expenditures for the blind and disabled and the aged is greater than for adults and children . , those directly affected by boren ) account for substantially more of total expenditures for the blind and disabled and the aged than for the other groups . except in the case of children we interpret this result to indicate that tax capacity influences state medicaid policy which , in turn , influences medicaid expenditures . the results by type of service are consistent with the literature 's reports that funds from provider programs were expended as hospital payments ( coughlin , ku , and holahan , 1994 ) . this result was significant in spending models for the aged , blind and disabled , and children . finally , the prevalence of aids was found to be positively and significantly related to expenditures for adult enrollees . the result for adults reflects the prevalence of aids among the afdc - eligible population . many of the factors that the literature hypothesizes to have contributed to medicaid expenditure growth in recent years were found to be significantly related to expenditure growth from 1984 to 1992 . first , federal medicaid policies directly affecting expenditures , i.e. , obra 1987 and boren amendment cases , were found to be significantly related to expenditure growth . third , this analysis suggests that state policy determinants , i.e. , tax capacity , affected expenditure growth . in addition , the results indicated a significant relationship between state t&d programs and expenditures for inpatient services . the analysis separately modeled medicaid expenditures for four groups of enrollees to allow for the differential impact of various factors . for example , provider programs were significantly related to inpatient expenditures but not to expenditures for other types of services . the results suggest that the dramatic rates of increase in medicaid expenditures should not be expected to continue , because the impact of some factors should be subsiding . for example : the mandated eligibility expansions for low - income infants , children , and pregnant women are largely implemented ; states ' economic performance is improving ; and the potential for growth in t&d programs was curtailed by the medicaid voluntary contribution and provider - specific tax amendments of 1991 . the expected reduction in expenditure growth is supported by recent data indicating medicaid expenditures grew by 10 percent from 1992 to 1993 , nearly one - half the average annual growth rate from 1989 - 92 . enrollment growth accounts for substantially more of expenditure growth from 1992 to 1993 than from 1990 to 1992 , and real expenditures per enrollee account for less ( holahan , liska , and obermaier , 1994 ) . however , although medicaid expenditure growth is not expected to continue at average annual rates of more than 20 percent , it may be somewhat higher than the low growth rates of the early and middle 1980s . some policies are not fully phased - in and may continue to fuel expenditure growth . while this analysis begins to enlighten our understanding of the causes of medicaid expenditure growth , it also suggests areas for future research .

the temperature - induced phase transition of poly(n - isopropylacrylamide ) , pnipam , in aqueous solutions , first reported by heskins and guillet in 1968 , has been an inspiration to an enormous amount of scientific research as well as to many suggestions of application during the recent decades . in dilute aqueous solution , pnipam undergoes a coil - to - globule transition at its lower critical solution temperature , lcst , of ca . the preparation of thermoresponsive colloidal particles was pioneered by pelton and chibante in the case of n - isopropylacrylamide , nipam , either alone or with styrene by pelton and by kawaguchi and coworkers [ 58 ] in the case of emulsion copolymerization of styrene with various n - substituted acrylamides . in aqueous systems particle production from nipam was shown to proceed via a precipitation polymerization mechanism as the reaction was conducted above the lcst and in the presence of crosslinking monomer , n , n - methylenebisacrylamide ( mba ) . since the pioneers , several groups have studied submicron - sized core - shell - like particles prepared of styrene and nipam . [ 10 , 11 ] focused on studying the effect of polymerization process on particle morphology . the particles were synthesized either by batch or by two - step surfactant - free emulsion copolymerization of styrene , nipam , and a cationic amino - containing comonomer with or without an added crosslinking monomer . characteristics such as particle size , electrokinetic properties , and colloidal stability of the particles were studied , as well as covalent and noncovalent binding of an antibody onto the particles . ballauff and coworkers [ 1418 ] have studied the phase transition of crosslinked pnipam as a shell on a polystyrene - based core . they prepared the core particles by conventional emulsion polymerization of styrene and nipam ( 95/5 wt% ) , and as a second step , they copolymerized nipam with the crosslinking monomer , n , n - methylenebisacrylamide , in the presence of the core particles [ 14 , 15 ] . phase behavior of the thermosensitive shell in aqueous particle dispersions was studied by combining the data from different scattering methods such as dynamic light scattering ( dls ) , small - angle neutron scattering ( sans ) [ 16 , 17 ] , and small - angle x - ray scattering ( saxs ) [ 14 , 17 ] . the effect of temperature on the rheological properties of the particle dispersions was also studied . typically , the core particles in aqueous dispersion were of approximately 100 nm in diameter and the crosslinked pnipam shell was of 1050 nm in thickness depending on the temperature . we have synthesized particles very similar to those reported by ballauff and coworkers , and studied the phase transition of the crosslinked pnipam shell on polystyrene core of ca . 50 nm , in addition to the dynamic light scattering by h nmr spectroscopy and by high sensitivity differential scanning microcalorimetry . to our knowledge , there are no reports of such phase transition studies employing the latter two methods for this type of pnipam microgel structures . however , few investigations of linear pnipam grafted or adsorbed on solid core particles have been published . zhu and napper pioneered the experimental studies on coil - to - globule transitions of linear pnipam chains attached to the surfaces of polystyrene latex particles . the kinetics of the transition was investigated in the case of chains grafted on polystyrene particles of ca . furthermore , a considerable change in the coil - to - globule transition of interfacial chains was observed as the molecular weight ( mw ) of the attached chains was varied from 310 to 210 g / mol . the broadening of the transition towards lower temperatures was more pronounced with the shortest polymer chains in question . the broad overall transition of the interfacial chains was explained to consist of two components . upon heating , first , the inner region of each chain adopts its globular form , whereas the segments of chains in coronal layer exist in the coil - like conformation until the lcst is reached . the phase transition of low molecular weight pnipam ( ca . 5,000 g / mol ) , covalently bound to the surface of gold clusters of few nanometer in size , have been investigated with microcalorimetry by shan et al . . the pnipam chains in these monolayer protected gold clusters exhibited two separate transition endotherms of dehydration around the lcst . the first transition with a sharp and narrow endothermic peak was observed at lower temperature while the second one with broader peak was observed at higher temperature . as in the case of zhu and napper , it was suggested that the inner segments of chains close to the particle surface are densely packed and less hydrated showing the first transition , while the segments in coronal layer are more hydrated showing the second transition . as methods sensitive to dynamic properties in the phase transition on molecular level , both microcalorimetry and nmr spectroscopy have been employed to investigate the high molecular weight ( 3.510 g / mol ) pnipam adsorbed on silica particles . in this case , the effect of the solid surface was an increased transition temperature and a broadening of the transition . the increase of the transition temperature was concluded to be due to increased motional constraints of the chains on the solid surface . the increase of the transition width was explained by the assumption of motional heterogeneity in the pnipam layer . at lower surface coverage , the system is dominated by immobile segments close to silica ; while at higher coverage , more mobile segments with a smaller broadening contributed to the transition . the introduced studies of phase transition show that interfacial pnipam exhibits very different and more complex dynamic properties compared to volume systems . this report describes the case of pnipam microgel on polystyrene core in comparison to colloidal microgel particles . styrene ( merck ) and acrylic acid ( aa ; merck ) were distilled under reduced pressure before use . n - isopropylacrylamide ( nipam ; acros ) was recrystallized from n - hexane and dried in vacuum . sodium dodecylsulfate ( sds ; merck ) , potassium peroxydisulfate ( kps ; merck ) , and n , n - methylenebisacrylamide ( mba ; aldrich ) were used as received . filter papers ( whatman , 2v ) used were of pore size 8 m . cellulose membrane tubings ( cellusep t4 ) with nominal molecular weight cut off of 12,00014,000 g / mol were used as membranes in particle purification by dialysis . a summary of the conditions of the syntheses and the abbreviations for the particles are shown in table 1 . polymerizations were carried out in a sealed round - bottom flask equipped with a magnetic stirrer and an oil bath to control the reaction temperature . the seed particles , ps1 , and ps2 were prepared by radical polymerization of styrene in aqueous emulsion . surfactant , sds , was dissolved in water in the reaction flask and , after which , styrene was added and the flask was sealed with a septum . the mixture was purged with nitrogen and stirred at room temperature for 20 min . the nitrogen inlet and outlet were removed and the flask was placed into a preheated oil bath at 60c . polymerization was initiated after 20 min by injecting kps dissolved in 1 ml of water to the reaction mixture . after which , reaction was allowed to proceed for 3 h with stirring ( 600700 rpm ) . the reaction was stopped by unsealing the flask and cooling the product to room temperature . the product was filtered through a filter paper and purified by dialyzing for 7 days against distilled water that was refreshed daily . the dialyzed aqueous particle dispersion was extracted several times with n - hexane and stored as such in the refrigerator . table 1summary of the synthesesparticleh2o / gseedsdskpsstyrenenipaammbaaaps1300.30.062.25ps2300.30.062.25ps1n20 0.010.20.013ps1na20 0.010.20.0130.004ps2n12.517.5 0.0150.30.02n1200.0020.010.30.02n2500.0050.030.750.025the components of the reaction mixtures are given as grams ps1 particle dispersion ( particle concentration 2.8 wt% ) ps2 particle dispersion ( particle concentration 3.2 wt% ) summary of the syntheses the components of the reaction mixtures are given as grams ps1 particle dispersion ( particle concentration 2.8 wt% ) ps2 particle dispersion ( particle concentration 3.2 wt% ) the two - stage particles were prepared by the following manner . in the synthesis of particle ps1n , nipam and mba were separately dissolved in aqueous seed particle dispersion , ps1 , and both solutions were transferred to a reaction flask . the flask was sealed with a septum and polymerization was initiated and carried out by following the procedure described for the synthesis of seed particles . in the synthesis of particle ps1na , acrylic acid was , in addition , injected to the reaction mixture before sealing the flask . for the synthesis of ps2n , the purified seed particle dispersion , ps2 , was diluted with 12.5 ml of water before use in polymerization . in the second stage polymerization , microgel particles , n1 and n2 , were prepared similarly to two - stage particles except of using dilute aqueous solution of sds as a reaction medium instead of aqueous seed particle dispersion . the flask was sealed with a septum and polymerization was initiated and carried out by following the procedure ( n2 , kps ) described for the synthesis of seed particles . the reaction time in the microgel particle syntheses was 4 h. the two - stage particles and the microgel particles were purified by dialysis for 7 days against distilled water that was refreshed daily . polymer contents of aqueous particle dispersions were obtained by drying weighed samples of aqueous dispersions to equilibrium weight in vacuum . ftir - spectra were measured from freeze - dried particles with perkinelmer spectrum one ft - ir - spectrometer , spectrum one ft - ir -software , and universal atr sampling accessory . h nmr spectra of the particles in an organic solvent were recorded with a 200 mhz varian gemini 2000 spectrometer . the samples were prepared from freeze - dried particles by dissolving in deuterated chloroform ( 30 mg / ml ) . h nmr spectra and relaxation measurements of the particles in d2o were measured using a varian unityinova spectrometer operating at 300 mhz for protons equipped with a temperature control unit . the nmr samples were prepared by redispersing 20 mg of freeze - dried particles in 1 ml of d2o and filtered through a filter paper . measurements were carried out with controlled heating and cooling steps , allowing the samples to equilibrate for 30 min at each incremented temperature . for the h t1 relaxation measurements of the polymer protons , a series of 30 spectra were collected using the standard inversion recovery sequence varying the delay time from 0.001 to 10 s. the h t2 relaxation measurements of the polymer protons were made using the carr gill spin echo sequence using an array of 20 values ranging from 0.002 to 1 s. the t1 and t2 relaxation times were obtained by fitting a monoexponential decay to the relaxation data . the size distributions of particles were obtained with dynamic light scattering ( dls ) by using the instrument of bookhaven instruments ( bi-200sm goniometer , bi-9000at digital correlator ) equipped with lexel 85 1w laser at wavelength 514.5 . scattering was measured at 90 angle and the obtained time correlation functions were analyzed by contin laplace - inversion program . the polymer concentration in the sample was 0.01 mg / ml obtained by diluting the particle dispersion with deionized water . hs dsc ( high sensitivity differential scanning calorimetry ) measurements were performed with a vp - dsc microcalorimeter ( microcal ) at an external pressure of ca . scans were recorded from 10 to 80c at heating rates 30 and 60c / h . before each scan the sample was kept at 10c for 15 min and each scan was repeated 3 times . the raw data from hs - dsc was manipulated by subtracting the baseline and with corrections to the sample concentration terms , where the sample concentration was given as molar concentration of nipam units in the particle sample . the samples were prepared from dialyzed particle dispersions by diluting with deionized water . also , some samples prepared from freeze - dried particles were measured in h2o and d2o . styrene ( merck ) and acrylic acid ( aa ; merck ) were distilled under reduced pressure before use . n - isopropylacrylamide ( nipam ; acros ) was recrystallized from n - hexane and dried in vacuum . sodium dodecylsulfate ( sds ; merck ) , potassium peroxydisulfate ( kps ; merck ) , and n , n - methylenebisacrylamide ( mba ; aldrich ) were used as received . filter papers ( whatman , 2v ) used were of pore size 8 m . cellulose membrane tubings ( cellusep t4 ) with nominal molecular weight cut off of 12,00014,000 g / mol were used as membranes in particle purification by dialysis . a summary of the conditions of the syntheses and the abbreviations for the particles are shown in table 1 . polymerizations were carried out in a sealed round - bottom flask equipped with a magnetic stirrer and an oil bath to control the reaction temperature . the seed particles , ps1 , and ps2 were prepared by radical polymerization of styrene in aqueous emulsion . surfactant , sds , was dissolved in water in the reaction flask and , after which , styrene was added and the flask was sealed with a septum . the mixture was purged with nitrogen and stirred at room temperature for 20 min . the nitrogen inlet and outlet were removed and the flask was placed into a preheated oil bath at 60c . polymerization was initiated after 20 min by injecting kps dissolved in 1 ml of water to the reaction mixture . after which , reaction was allowed to proceed for 3 h with stirring ( 600700 rpm ) . the reaction was stopped by unsealing the flask and cooling the product to room temperature . the product was filtered through a filter paper and purified by dialyzing for 7 days against distilled water that was refreshed daily . the dialyzed aqueous particle dispersion was extracted several times with n - hexane and stored as such in the refrigerator . table 1summary of the synthesesparticleh2o / gseedsdskpsstyrenenipaammbaaaps1300.30.062.25ps2300.30.062.25ps1n20 0.010.20.013ps1na20 0.010.20.0130.004ps2n12.517.5 0.0150.30.02n1200.0020.010.30.02n2500.0050.030.750.025the components of the reaction mixtures are given as grams ps1 particle dispersion ( particle concentration 2.8 wt% ) ps2 particle dispersion ( particle concentration 3.2 wt% ) summary of the syntheses the components of the reaction mixtures are given as grams ps1 particle dispersion ( particle concentration 2.8 wt% ) ps2 particle dispersion ( particle concentration 3.2 wt% ) the two - stage particles were prepared by the following manner . in the synthesis of particle ps1n , nipam and mba were separately dissolved in aqueous seed particle dispersion , ps1 , and both solutions were transferred to a reaction flask . the flask was sealed with a septum and polymerization was initiated and carried out by following the procedure described for the synthesis of seed particles . in the synthesis of particle ps1na , acrylic acid was , in addition , injected to the reaction mixture before sealing the flask . for the synthesis of ps2n , the purified seed particle dispersion , ps2 , was diluted with 12.5 ml of water before use in polymerization . in the second stage polymerization , microgel particles , n1 and n2 , were prepared similarly to two - stage particles except of using dilute aqueous solution of sds as a reaction medium instead of aqueous seed particle dispersion . the flask was sealed with a septum and polymerization was initiated and carried out by following the procedure ( n2 , kps ) described for the synthesis of seed particles . the reaction time in the microgel particle syntheses was 4 h. the two - stage particles and the microgel particles were purified by dialysis for 7 days against distilled water that was refreshed daily . polymer contents of aqueous particle dispersions were obtained by drying weighed samples of aqueous dispersions to equilibrium weight in vacuum . ftir - spectra were measured from freeze - dried particles with perkinelmer spectrum one ft - ir - spectrometer , spectrum one ft - ir -software , and universal atr sampling accessory . h nmr spectra of the particles in an organic solvent were recorded with a 200 mhz varian gemini 2000 spectrometer . the samples were prepared from freeze - dried particles by dissolving in deuterated chloroform ( 30 mg / ml ) . h nmr spectra and relaxation measurements of the particles in d2o were measured using a varian unityinova spectrometer operating at 300 mhz for protons equipped with a temperature control unit . the nmr samples were prepared by redispersing 20 mg of freeze - dried particles in 1 ml of d2o and filtered through a filter paper . measurements were carried out with controlled heating and cooling steps , allowing the samples to equilibrate for 30 min at each incremented temperature . for the h t1 relaxation measurements of the polymer protons , a series of 30 spectra were collected using the standard inversion recovery sequence varying the delay time from 0.001 to 10 s. the h t2 relaxation measurements of the polymer protons were made using the carr gill spin echo sequence using an array of 20 values ranging from 0.002 to 1 s. the t1 and t2 relaxation times were obtained by fitting a monoexponential decay to the relaxation data . the size distributions of particles were obtained with dynamic light scattering ( dls ) by using the instrument of bookhaven instruments ( bi-200sm goniometer , bi-9000at digital correlator ) equipped with lexel 85 1w laser at wavelength 514.5 . scattering was measured at 90 angle and the obtained time correlation functions were analyzed by contin laplace - inversion program . the polymer concentration in the sample was 0.01 mg / ml obtained by diluting the particle dispersion with deionized water . hs dsc ( high sensitivity differential scanning calorimetry ) measurements were performed with a vp - dsc microcalorimeter ( microcal ) at an external pressure of ca . scans were recorded from 10 to 80c at heating rates 30 and 60c / h . before each scan the sample was kept at 10c for 15 min and each scan was repeated 3 times . the raw data from hs - dsc was manipulated by subtracting the baseline and with corrections to the sample concentration terms , where the sample concentration was given as molar concentration of nipam units in the particle sample . the samples were prepared from dialyzed particle dispersions by diluting with deionized water . also , some samples prepared from freeze - dried particles were measured in h2o and d2o . the characteristic peaks of both the polystyrene seed and the crosslinked pnipam components were present in the ftir - spectra ( not shown ) of the two - stage particles . in the case of ps1na , only one clear characteristic peak corresponding to carboxylic acid functionality ( ca . 1,702 cm ) could be observed as a tiny shoulder on the strong peak corresponding to the amide functionality of pnipam ( ca . the quantitative h nmr - spectra ( not shown ) of the two - stage particles were measured in deuterated chloroform which is a good solvent for the both components and gives a clear solution . the analysis of integrated spectra gave the amounts of nipam units of 23 , 17 , and 44 mol% in the two - stage particles ps1n , ps1na , and ps2n , respectively . the small amount of crosslinker , mba , was ignored in the composition calculations because its weak proton signals could not be properly analyzed due to the complete overlap by the proton signals originating from the structurally similar nipam units . the mean hydrodynamic diameters and the size distributions in aqueous dispersions of the particles were obtained from the dls data . the largest 2-stage particle , ps2n , was synthesized by using a larger monomer to seed particle mass ratio compared to the syntheses of ps1n and ps1na . the 2-stage particles show monomodal size distributions that are broader than the size distributions obtained for their seed particles . the mean hydrodynamic diameters of the 2-stage particles vs temperature at range 1550c ( heating rate ca . the mean hydrodynamic radius vs temperature for pnipam microgel particle , n1 , is plotted in the same figure for comparison . the microgel particle deswells rapidly within a temperature range 2535c and its radius decreases nearly 50% upon heating at the studied temperature range . the 2-stage particles deswell gradually through the whole studied temperature range showing nearly linear deswelling . as an exception , a clear deviation from linearity is observed for ps2n at temperature range 3040c . behavior of ps2n differs from the other particles because its total volume , decreased upon heating , is very small compared to its original size at 20c . the size distributions in all cases remained monomodal and particle coagulation was not observed during the measurements at and above the phase transition temperature . dls data upon cooling was also measured ( not shown ) , and for all particles , the deswelling was observed to be reversible . the particles are most likely stabilized against coagulation at elevated temperatures by electrostatic repulsion between the anionic surface groups originating from the polymerization initiator fragments , residual surfactant , and in addition , from the dissociated carboxylic acid groups in the case of ps1na . the mean hydrodynamic radius of the microgel particle , n1 , is plotted in the figure for comparison the intensity - weighed size distributions of particles in aqueous dispersions at 20c the mean hydrodynamic diameters of the 2-stage particles vs temperature . the mean hydrodynamic radius of the microgel particle , n1 , is plotted in the figure for comparison the phase transition of the crosslinked pnipam was studied more closely on molecular level by quantitative h nmr spectroscopy in d2o . h nmr spectra of the microgel sample n2 in d2o above and below the critical temperature are shown in fig . 3 . figure 4 shows the curves of the normalized pnipam proton to solvent proton ( 4.8 ppm ) spectral intensity ratios vs temperature ( 2250c ) for the different protons of the main chain and the side chain n - isopropyl group in microgel particle , n1 ( 20 mg / ml ) . protons decrease considerably already at 2530c and the signals disappear completely at 50c . instead , the methyl protons ( ca . 1 ppm ) and the lone proton ( ca . 4 ppm ) of the n - isopropyl group show corresponding rapid response at temperatures closer to the cloud point of pnipam at 3235c and the side chain proton signals can be detected even above 50c as small ones . at temperatures above 35c , we are most likely observing proton signals only from the surface of the collapsed particle . fig . 3 h nmr spectra of the microgel , n2 , recorded at 22 and 40c in d2ofig . 4normalized ratios of h nmr signal intensity ( i ) of the pnipam protons to the solvent ( d2o ) protons vs temperature as measured for the protons of the n - isopropyl group ( methyl protons ca . 1 ppm and the lone proton ca . 4 ppm ) and the protons of the main chain ( methylene protons ca . 1.5 2 ppm ) in the microgel sample , n1 h nmr spectra of the microgel , n2 , recorded at 22 and 40c in d2o normalized ratios of h nmr signal intensity ( i ) of the pnipam protons to the solvent ( d2o ) protons vs temperature as measured for the protons of the n - isopropyl group ( methyl protons ca . 4 ppm ) and the protons of the main chain ( methylene protons ca . 2 ppm ) in the microgel sample , n1 due to the dramatic decrease of the pnipam proton signal intensities during the phase transition , the strongest signal of the proton spectra at ca . 1 ppm , corresponding to the methyl protons of the side chain , was chosen for comparing the transitions of crosslinked pnipam in different particles . figure 5 shows the curves of the normalized methyl proton to solvent proton ( 4.8 ppm ) spectral intensity ratios vs temperature ( 2250c ) for different particles . it appears that of the 2-stage particles , ps2n shows the closest resemblance to the microgels n1 and n2 , although for ps2n the rapid decrease of the methyl proton signal is observed at 3540c . it is concluded that the phase transition of crosslinked pnipam in this particle shell has shifted towards higher temperatures when compared to the microgels and also that the temperature range of the transition is broader . the 2-stage particle ps1n shows more gradual decrease of methyl proton signal through the whole studied temperature range . poly(nipam - co - acrylic acid ) microgel structure in the 2-stage particle ps1na appears to be quite swollen in d2o even at 50c due to the presence of the hydrophilic comonomer units . 5normalized ratios of the h nmr signal intensity ( i ) of the methyl protons ( ca . 1 ppm ) to the solvent ( d2o ) protons vs temperature in different particles normalized ratios of the h nmr signal intensity ( i ) of the methyl protons ( ca . 1 ppm ) to the solvent ( d2o ) protons vs temperature in different particles a noticeable increase of the signal intensities in fig . the increase of intensities could be interpreted as being due to increasing mobility with increasing temperature below the phase transition temperature . corresponding observations have been reported by zhu and napper for c nmr signals from linear pnipam chains both in solution and attached to polystyrene latex particles . increase of the signal intensities was observed to be more pronounced in the case of the interfacial chains . dynamic behavior of pnipam microgel in the particles was studied by measuring spin lattice ( t1 ) and spin spin ( t2 ) relaxation times for the side chain methyl and the main chain methyne protons at temperatures 2250c in d2o . the accuracy of the relaxation times is low at temperatures above 35c due to decreasing proton signals and therefore drawing conclusions from the data obtained at higher temperatures is avoided . figure 6 shows the temperature dependence of the t1 for protons of the side chain methyl groups in the studied particles . it appears that t1 of the side chain methyl protons is not , at least in this case , highly sensitive to the particle structure because the t1 values measured for different particles at the same temperature deviate from each other only with some tens of milliseconds . within this small deviation range , larger differences between the particles are observed when looking at the corresponding t1 data for the main chain methyne protons in fig . in general the 2-stage particles show lower values of t1 for main chain methyne protons than the reference microgels and the sample n1 shows again the highest t1 values . this may be an indication of more mobile structures of pnipaam in the 2-stage particles than in the microgel samples . some samples show increasing t1 values above 35c but any conclusions about clear overall trends of t1 with temperature during the phase transition can not be drawn from the data of the relaxation times taking into account the accuracy problems at high temperatures . figure 8 shows the temperature dependence of the t2 for protons of the main chain methyne groups in the studied particles . at temperatures below 35c these higher t2 values ( 0.030.04 s ) are close to the ones ( 0.030.05 s ) obtained earlier in our laboratory for a linear pnipam ( mw 150,000 g / mol , 2 mg / ml in d20 at 2735c ) in a similar experiment using the same instrumentation ( unpublished data , 2005 ) . higher t2 values refer again to more mobile components existing in 2-stage particle samples and possible explanation is the looser and/or more heterogeneous network structure of pnipam compared to the microgel samples . lower values of t2 for the microgel particles originate most likely from the motional restriction brought by the crosslinking . however , the relaxation decays were monoexponential in all cases , indicating that the signal is coming from the most mobile segments in the systems ( the most mobile segments between the crosslinks and dangling chains ) . this means that signal from the significantly less mobile parts of the polymers is not reflected in the relaxation data due to a very quick relaxation . 6spin lattice relaxation times ( t1 ) of the methyl protons ( side chain ) vs temperature for different particles in d2ofig . 7spin lattice relaxation times ( t1 ) of the methyne protons ( main chain ) vs temperature for different particles in d2ofig . 8spin spin relaxation times ( t2 ) of the methyne protons ( main chain ) vs temperature for different particles in d2o spin lattice relaxation times ( t1 ) of the methyl protons ( side chain ) vs temperature for different particles in d2o spin lattice relaxation times ( t1 ) of the methyne protons ( main chain ) vs temperature for different particles in d2o spin spin relaxation times ( t2 ) of the methyne protons ( main chain ) vs temperature for different particles in d2o the calorimetrically obtained transition temperature of pnipam - based polymers is often defined as the onset of the dsc transition endotherm , tonset ( the intersection of the baseline and the leading edge of the endotherm ) . the temperature of the maximum heat capacity , tmax , is also used to describe the transition temperature . for a sharp peak in a dsc thermogram however , for broad peaks , the determination of tonset is more difficult due to inaccuracy in setting the baseline and , in such cases , it appears to be more reliable to determine the tmax in comparative studies . microcalorimetric endotherms for aqueous dispersions of n1 , ps1n , and ps2n are shown in fig . broader endotherms are observed for crosslinked pnipam in the 2-stage particles compared to the reference microgel sample , n1 . especially in the case of ps2n , a clear increase of tmax can be observed . this finding is supported by the obtained nmr data of the methyl h - signal intensities upon heating in d2o ( fig . 5 ) showing that the phase transition of crosslinked pnipam in this particle structure has shifted towards higher temperatures when compared to the reference microgels , and that the temperature range of the transition is broader . the broadest endotherm ( not shown ) as indicated by the highest value of t1/2 ( the width of the endotherm at the half - height of the peak ) in table 2 was obtained for the particle containing small amount of acrylic acid . in this case , the transition is evidently broadened by the presence of both the core particle and the hydrophilic groups as was observed clearly from the h - signal intensities upon heating ( fig . 5 ) . h2o was used as a solvent in the microcalorimetric measurements . when comparing the nmr results to the calorimetric results , it is useful to keep in mind that h2o and d2o differ in their physical properties and that a hydrogen bond in d2o is ca . pnipam coils are suggested to be more extented in d2o below the critical temperature and there may be a higher level of ordering of d2o associated with a polymer chain . kujawa and winnik reported for linear pnipam ( of molecular weight ca . 300,000 g / mol ) a very similar microcalorimetric endotherm in general features in d2o compared to the corresponding endotherm in h2o . but in comparison to h2o , an increase of tmax by 0.6c and a doubling of the change of heat capacity occurring upon the phase transition were observed in d2o . as control samples in d2o , we measured thermograms ( data not shown ) of n1 ( 2 and 20 mg / ml ) . the differences observed between d2o and h2o in our microgel case were in agreement with the observations of kujawa and winnik for the linear pnipam . 9microcalorimetric endotherms ( heating rate 30c / h ) for aqueous dispersions of the microgel ( n1 : solid line ) and the 2-stage particles ( ps1n : dotted line , ps2n : dashed line ) . for more information , see table 2 table 2microcalorimetric datasamplen1 ( 2 g / l)n2 ( 2 g / l)ps1n ( 20 g / l)ps2n ( 2 g / l)ps1na ( 20 g / l ) t max ( c)34.033.034.837.836.6 t 12 ( c)3.72.313.56.124.1h cal ( kcal / mole)1.011.330.521.210.53heating rate 30c / h , h calculated per moles of nipaam units microcalorimetric endotherms ( heating rate 30c / h ) for aqueous dispersions of the microgel ( n1 : solid line ) and the 2-stage particles ( ps1n : dotted line , ps2n : dashed line ) . for more information , see table 2 microcalorimetric data heating rate 30c / h , h calculated per moles of nipaam units how are the characteristics of the phase transition observed by microcalorimetry connected to the structure of the pnipam microgel system ? woodward et al . investigated the volume phase transition of colloidal pnipam microgels prepared with varying crosslinker concentrations ( 0.2530% of mba in monomer mixture ) by surfactant - free emulsion polymerization . in this case , the system varies from a loose pnipam - network to a more compact and rigid particle structure with increasing crosslinking density . dsc analyzes of these aqueous microgel particles showed that the tmax and the width of the volume phase transition increase with increasing crosslinker concentration . for example , tmax of the calorimetric endotherm increased by about 2c when the amount of mba in the monomer mixture was increased from 0.5 to 5% . the calorimetric enthalpy change of the transition decreased with increasing crosslinker concentration . the increased temperature of transition could indicate increased stability of the microgel within the solvent . however , the polarity of the interior of the microgels was studied with fluorescence spectroscopy by using pyrene as a probe . the results indicated that the hydrophobicity sensed by the probe was increasing with increasing crosslinker concentration . the calorimetric results were explained as being due to relatively more rigid structure of the microgel at higher crosslinker concentrations , and , as a result , the volume phase transition broadens and it is pushed towards higher temperatures . our observations by microcalorimetry ( data in table 2 ) for the microgel particles , n1 ( 6 wt% of mba in monomer feed ) and n2 ( 3 wt% of mba in monomer feed ) , appear to be in accordance with their results . wu et al . studied the effect of mba on the swelling ability of pnipam microgels . it is assumed that there is a concentration gradient of the crosslinker in these microgels and the crosslinking density is decreasing from the particle core towards the surface . when studying the heterogeneous structures of thermosensitive microgels of poly(n - isopropylmethacrylamide ) produced with mba . assuming the existence of a core - shell - like structure , it was suggested that the relative size of the rigid core is increased upon increasing crosslinker concentration . this type of microgel particles are heterogeneous systems in which the crosslinking density determines whether the response to temperature is dominated by the phase transition of linear parts in the system or the volume phase transition of the gel structure . in our case , the feed of the crosslinking monomer was kept constant ( 6 wt% of the monomers in the reaction batch ) in the syntheses of the 2-stage particles and the microgel n1 . but how does the presence of the polystyrene seed particles affect to the structure of the forming pnipam network ? it appears that the pnipam network is firmly attached to the core particles because the samples showed good endurance to heating cooling cycles during the measurements . precipitation of pnipam to the surface of the seed particles evidently occurs in the used synthesis conditions , but the effect of chain transfer from pnipam chains to the seed polymer and resulting chain growth from the core surface is unknown . however , the precipitation polymerization of nipam in the presence of the crosslinking monomer produces a system locked to a network structure in a shrunken state . close packing of pnipam globules onto the surface of the seed particles during the synthesis must have an effect on the swelling abilities of the produced network . ballauff and coworkers [ 1418 ] analyzed the similar system of crosslinked pnipam shell on poly(styrene 95 wt%-co - nipam 5 wt% ) core particles by dls , sans , and saxs in aqueous media . it was shown that the pnipam shell in swollen state exhibited static and dynamic inhomogeneities , but clear evidences of radial distribution of crosslinks or inhomogeneities in the network were not found . investigations by dls revealed that a small number of chains extended beyond the network yielding a hairy surface . the volume phase transition of the shell was found to be continuous and the degree of shrinking was found to be much less than what is observed for macroscopic networks of a similar degree of crosslinking . this was suggested to be due to the fact that macroscopic networks shrink along three directions whereas the shell networks on a rigid core can only shrink in the dimension along the surface normal . in our case , the 2-stage particles showed continuous , nearly linear deswelling and , in the case of ps2n , it appears that the swelling and the collapse of pnipaam network is significantly hindered as shown in fig . 2 . evidently , severe motional restrictions affect to the volume phase transition of the network . on the other hand , the relaxation data refers to higher mobility in comparison to the microgel samples with high capacity to swell . a logical explanation could be , taking into account the results on linear polymers [ 2022 ] , a densely packed and fairly immobile pnipam layer close to the seed particle surface accompanied by highly mobile coronal pnipam layer . because the obtained relaxation times showed clearly only one main component , we were monitoring only the most mobile pnipam segments of the samples in the t1 and t2 measurements . in the case of ps1n , containing a thinner layer of crosslinked pnipam , the broad transition and the low enthalpy of dehydration refer to a more pronounced effect of the core particle through the whole pnipam layer . analogously , to linear pnipam attached to solid particles , the factors affecting to the phase transition of crosslinked pnipam on a solid particle appear to be the distance of the polymer segment from the core ( the thickness of the pnipam layer ) and the local concentration / packing of the polymer segments close to the core surface . increased motional restrictions are brought by the crosslinking in our case . certain analogy can also be observed when comparing the calorimetric results obtained in our case of crosslinked pnipam on a solid polystyrene particle to the results of woodward et al . which deals with the effect of increasing amount of crosslinker to the temperature and the width of the volume phase transition . although in their case , the rigid core was prepared by dense crosslinking of pnipam instead of using a solid polystyrene particle . according to our results from microcalorimetry and h nmr spectroscopy , the phase transition of crosslinked pnipam - shell on solid polystyrene particle shifts towards higher temperatures when compared to the results obtained for pnipam microgels and , also , that the temperature range of the transition is broader . the broadest transition was observed for the particle containing small amount of acrylic acid . in this case , the transition is evidently broadened by the presence of both the core particle and the hydrophilic groups . the results from dynamic light scattering showed a surprisingly small change of hydrodynamic size with temperature in the case of a thick pnipam layer referring to strong motional restrictions . however , the relaxation times of pnipam protons refer to components with higher mobility existing in 2-stage particle samples and possible explanation is a looser and/or more heterogeneous structure of pnipam network compared to the microgel samples . the relaxation decays were monoexponential in all cases , including the microgels , indicating that we were monitoring only the most mobile segments in all the systems . the broadening of the transition in the core - shell particles compared to microgels is interpreted as being due to more heterogeneous structure of the pnipam network . the increase of the transition temperature is concluded to be due to increased motional constraints of the polymer segments on the solid surface .

thermoresponsive colloidal particles were prepared by seeded precipitation polymerization of n - isopropylacrylamide ( nipam ) in the presence of a crosslinking monomer , n , n - methylenebisacrylamide ( mba ) , using polystyrene latex particles ( ca . 50 nm in diameter ) as seeds in aqueous dispersion . phase transitions of the prepared poly(n - isopropylacrylamide ) , pnipam , shells on polystyrene cores were studied in comparison to colloidal pnipam microgel particles , in h2o and/or in d2o by dynamic light scattering , microcalorimetry and by 1h nmr spectroscopy including the measurements of spin lattice ( t1 ) and spin spin ( t2 ) relaxation times for the protons of pnipam . as expected , the seed particles grew in hydrodynamic size during the crosslinking polymerization of nipam , and a larger nipam to seed mass ratio in the polymerization batch led to a larger increase of particle size indicating a product coated with a thicker pnipam shell . broader microcalorimetric endotherms of dehydration were observed for crosslinked pnipam on the solid cores compared to the pnipam microgels and also an increase of the transition temperature was observed . the calorimetric results were complemented by the nmr spectroscopy data of the 1h - signal intensities upon heating in d2o , showing that the phase transition of crosslinked pnipam on polystyrene core shifts towards higher temperatures when compared to the microgels , and also that the temperature range of the transition is broader .

Introduction Experimental Materials Particle syntheses Characterization methods Results and discussion Conclusion

the temperature - induced phase transition of poly(n - isopropylacrylamide ) , pnipam , in aqueous solutions , first reported by heskins and guillet in 1968 , has been an inspiration to an enormous amount of scientific research as well as to many suggestions of application during the recent decades . the preparation of thermoresponsive colloidal particles was pioneered by pelton and chibante in the case of n - isopropylacrylamide , nipam , either alone or with styrene by pelton and by kawaguchi and coworkers [ 58 ] in the case of emulsion copolymerization of styrene with various n - substituted acrylamides . in aqueous systems particle production from nipam was shown to proceed via a precipitation polymerization mechanism as the reaction was conducted above the lcst and in the presence of crosslinking monomer , n , n - methylenebisacrylamide ( mba ) . the particles were synthesized either by batch or by two - step surfactant - free emulsion copolymerization of styrene , nipam , and a cationic amino - containing comonomer with or without an added crosslinking monomer . characteristics such as particle size , electrokinetic properties , and colloidal stability of the particles were studied , as well as covalent and noncovalent binding of an antibody onto the particles . ballauff and coworkers [ 1418 ] have studied the phase transition of crosslinked pnipam as a shell on a polystyrene - based core . they prepared the core particles by conventional emulsion polymerization of styrene and nipam ( 95/5 wt% ) , and as a second step , they copolymerized nipam with the crosslinking monomer , n , n - methylenebisacrylamide , in the presence of the core particles [ 14 , 15 ] . phase behavior of the thermosensitive shell in aqueous particle dispersions was studied by combining the data from different scattering methods such as dynamic light scattering ( dls ) , small - angle neutron scattering ( sans ) [ 16 , 17 ] , and small - angle x - ray scattering ( saxs ) [ 14 , 17 ] . typically , the core particles in aqueous dispersion were of approximately 100 nm in diameter and the crosslinked pnipam shell was of 1050 nm in thickness depending on the temperature . we have synthesized particles very similar to those reported by ballauff and coworkers , and studied the phase transition of the crosslinked pnipam shell on polystyrene core of ca . 50 nm , in addition to the dynamic light scattering by h nmr spectroscopy and by high sensitivity differential scanning microcalorimetry . the kinetics of the transition was investigated in the case of chains grafted on polystyrene particles of ca . furthermore , a considerable change in the coil - to - globule transition of interfacial chains was observed as the molecular weight ( mw ) of the attached chains was varied from 310 to 210 g / mol . the phase transition of low molecular weight pnipam ( ca . as methods sensitive to dynamic properties in the phase transition on molecular level , both microcalorimetry and nmr spectroscopy have been employed to investigate the high molecular weight ( 3.510 g / mol ) pnipam adsorbed on silica particles . in this case , the effect of the solid surface was an increased transition temperature and a broadening of the transition . the increase of the transition temperature was concluded to be due to increased motional constraints of the chains on the solid surface . the increase of the transition width was explained by the assumption of motional heterogeneity in the pnipam layer . at lower surface coverage , the system is dominated by immobile segments close to silica ; while at higher coverage , more mobile segments with a smaller broadening contributed to the transition . this report describes the case of pnipam microgel on polystyrene core in comparison to colloidal microgel particles . n - isopropylacrylamide ( nipam ; acros ) was recrystallized from n - hexane and dried in vacuum . sodium dodecylsulfate ( sds ; merck ) , potassium peroxydisulfate ( kps ; merck ) , and n , n - methylenebisacrylamide ( mba ; aldrich ) were used as received . the seed particles , ps1 , and ps2 were prepared by radical polymerization of styrene in aqueous emulsion . table 1summary of the synthesesparticleh2o / gseedsdskpsstyrenenipaammbaaaps1300.30.062.25ps2300.30.062.25ps1n20 0.010.20.013ps1na20 0.010.20.0130.004ps2n12.517.5 0.0150.30.02n1200.0020.010.30.02n2500.0050.030.750.025the components of the reaction mixtures are given as grams ps1 particle dispersion ( particle concentration 2.8 wt% ) ps2 particle dispersion ( particle concentration 3.2 wt% ) summary of the syntheses the components of the reaction mixtures are given as grams ps1 particle dispersion ( particle concentration 2.8 wt% ) ps2 particle dispersion ( particle concentration 3.2 wt% ) the two - stage particles were prepared by the following manner . in the synthesis of particle ps1n , nipam and mba were separately dissolved in aqueous seed particle dispersion , ps1 , and both solutions were transferred to a reaction flask . in the synthesis of particle ps1na , acrylic acid was , in addition , injected to the reaction mixture before sealing the flask . for the h t1 relaxation measurements of the polymer protons , a series of 30 spectra were collected using the standard inversion recovery sequence varying the delay time from 0.001 to 10 s. the h t2 relaxation measurements of the polymer protons were made using the carr gill spin echo sequence using an array of 20 values ranging from 0.002 to 1 s. the t1 and t2 relaxation times were obtained by fitting a monoexponential decay to the relaxation data . sodium dodecylsulfate ( sds ; merck ) , potassium peroxydisulfate ( kps ; merck ) , and n , n - methylenebisacrylamide ( mba ; aldrich ) were used as received . the seed particles , ps1 , and ps2 were prepared by radical polymerization of styrene in aqueous emulsion . table 1summary of the synthesesparticleh2o / gseedsdskpsstyrenenipaammbaaaps1300.30.062.25ps2300.30.062.25ps1n20 0.010.20.013ps1na20 0.010.20.0130.004ps2n12.517.5 0.0150.30.02n1200.0020.010.30.02n2500.0050.030.750.025the components of the reaction mixtures are given as grams ps1 particle dispersion ( particle concentration 2.8 wt% ) ps2 particle dispersion ( particle concentration 3.2 wt% ) summary of the syntheses the components of the reaction mixtures are given as grams ps1 particle dispersion ( particle concentration 2.8 wt% ) ps2 particle dispersion ( particle concentration 3.2 wt% ) the two - stage particles were prepared by the following manner . in the synthesis of particle ps1n , nipam and mba were separately dissolved in aqueous seed particle dispersion , ps1 , and both solutions were transferred to a reaction flask . for the h t1 relaxation measurements of the polymer protons , a series of 30 spectra were collected using the standard inversion recovery sequence varying the delay time from 0.001 to 10 s. the h t2 relaxation measurements of the polymer protons were made using the carr gill spin echo sequence using an array of 20 values ranging from 0.002 to 1 s. the t1 and t2 relaxation times were obtained by fitting a monoexponential decay to the relaxation data . 1,702 cm ) could be observed as a tiny shoulder on the strong peak corresponding to the amide functionality of pnipam ( ca . the largest 2-stage particle , ps2n , was synthesized by using a larger monomer to seed particle mass ratio compared to the syntheses of ps1n and ps1na . the size distributions in all cases remained monomodal and particle coagulation was not observed during the measurements at and above the phase transition temperature . dls data upon cooling was also measured ( not shown ) , and for all particles , the deswelling was observed to be reversible . the mean hydrodynamic radius of the microgel particle , n1 , is plotted in the figure for comparison the phase transition of the crosslinked pnipam was studied more closely on molecular level by quantitative h nmr spectroscopy in d2o . 4normalized ratios of h nmr signal intensity ( i ) of the pnipam protons to the solvent ( d2o ) protons vs temperature as measured for the protons of the n - isopropyl group ( methyl protons ca . 1.5 2 ppm ) in the microgel sample , n1 h nmr spectra of the microgel , n2 , recorded at 22 and 40c in d2o normalized ratios of h nmr signal intensity ( i ) of the pnipam protons to the solvent ( d2o ) protons vs temperature as measured for the protons of the n - isopropyl group ( methyl protons ca . 2 ppm ) in the microgel sample , n1 due to the dramatic decrease of the pnipam proton signal intensities during the phase transition , the strongest signal of the proton spectra at ca . 1 ppm , corresponding to the methyl protons of the side chain , was chosen for comparing the transitions of crosslinked pnipam in different particles . it is concluded that the phase transition of crosslinked pnipam in this particle shell has shifted towards higher temperatures when compared to the microgels and also that the temperature range of the transition is broader . poly(nipam - co - acrylic acid ) microgel structure in the 2-stage particle ps1na appears to be quite swollen in d2o even at 50c due to the presence of the hydrophilic comonomer units . 1 ppm ) to the solvent ( d2o ) protons vs temperature in different particles a noticeable increase of the signal intensities in fig . increase of the signal intensities was observed to be more pronounced in the case of the interfacial chains . dynamic behavior of pnipam microgel in the particles was studied by measuring spin lattice ( t1 ) and spin spin ( t2 ) relaxation times for the side chain methyl and the main chain methyne protons at temperatures 2250c in d2o . some samples show increasing t1 values above 35c but any conclusions about clear overall trends of t1 with temperature during the phase transition can not be drawn from the data of the relaxation times taking into account the accuracy problems at high temperatures . figure 8 shows the temperature dependence of the t2 for protons of the main chain methyne groups in the studied particles . 6spin lattice relaxation times ( t1 ) of the methyl protons ( side chain ) vs temperature for different particles in d2ofig . 8spin spin relaxation times ( t2 ) of the methyne protons ( main chain ) vs temperature for different particles in d2o spin lattice relaxation times ( t1 ) of the methyl protons ( side chain ) vs temperature for different particles in d2o spin lattice relaxation times ( t1 ) of the methyne protons ( main chain ) vs temperature for different particles in d2o spin spin relaxation times ( t2 ) of the methyne protons ( main chain ) vs temperature for different particles in d2o the calorimetrically obtained transition temperature of pnipam - based polymers is often defined as the onset of the dsc transition endotherm , tonset ( the intersection of the baseline and the leading edge of the endotherm ) . broader endotherms are observed for crosslinked pnipam in the 2-stage particles compared to the reference microgel sample , n1 . this finding is supported by the obtained nmr data of the methyl h - signal intensities upon heating in d2o ( fig . 5 ) showing that the phase transition of crosslinked pnipam in this particle structure has shifted towards higher temperatures when compared to the reference microgels , and that the temperature range of the transition is broader . the broadest endotherm ( not shown ) as indicated by the highest value of t1/2 ( the width of the endotherm at the half - height of the peak ) in table 2 was obtained for the particle containing small amount of acrylic acid . in this case , the transition is evidently broadened by the presence of both the core particle and the hydrophilic groups as was observed clearly from the h - signal intensities upon heating ( fig . when comparing the nmr results to the calorimetric results , it is useful to keep in mind that h2o and d2o differ in their physical properties and that a hydrogen bond in d2o is ca . 300,000 g / mol ) a very similar microcalorimetric endotherm in general features in d2o compared to the corresponding endotherm in h2o . but in comparison to h2o , an increase of tmax by 0.6c and a doubling of the change of heat capacity occurring upon the phase transition were observed in d2o . for more information , see table 2 microcalorimetric data heating rate 30c / h , h calculated per moles of nipaam units how are the characteristics of the phase transition observed by microcalorimetry connected to the structure of the pnipam microgel system ? the calorimetric results were explained as being due to relatively more rigid structure of the microgel at higher crosslinker concentrations , and , as a result , the volume phase transition broadens and it is pushed towards higher temperatures . our observations by microcalorimetry ( data in table 2 ) for the microgel particles , n1 ( 6 wt% of mba in monomer feed ) and n2 ( 3 wt% of mba in monomer feed ) , appear to be in accordance with their results . this type of microgel particles are heterogeneous systems in which the crosslinking density determines whether the response to temperature is dominated by the phase transition of linear parts in the system or the volume phase transition of the gel structure . in our case , the feed of the crosslinking monomer was kept constant ( 6 wt% of the monomers in the reaction batch ) in the syntheses of the 2-stage particles and the microgel n1 . but how does the presence of the polystyrene seed particles affect to the structure of the forming pnipam network ? it appears that the pnipam network is firmly attached to the core particles because the samples showed good endurance to heating cooling cycles during the measurements . precipitation of pnipam to the surface of the seed particles evidently occurs in the used synthesis conditions , but the effect of chain transfer from pnipam chains to the seed polymer and resulting chain growth from the core surface is unknown . however , the precipitation polymerization of nipam in the presence of the crosslinking monomer produces a system locked to a network structure in a shrunken state . close packing of pnipam globules onto the surface of the seed particles during the synthesis must have an effect on the swelling abilities of the produced network . ballauff and coworkers [ 1418 ] analyzed the similar system of crosslinked pnipam shell on poly(styrene 95 wt%-co - nipam 5 wt% ) core particles by dls , sans , and saxs in aqueous media . it was shown that the pnipam shell in swollen state exhibited static and dynamic inhomogeneities , but clear evidences of radial distribution of crosslinks or inhomogeneities in the network were not found . the volume phase transition of the shell was found to be continuous and the degree of shrinking was found to be much less than what is observed for macroscopic networks of a similar degree of crosslinking . in our case , the 2-stage particles showed continuous , nearly linear deswelling and , in the case of ps2n , it appears that the swelling and the collapse of pnipaam network is significantly hindered as shown in fig . on the other hand , the relaxation data refers to higher mobility in comparison to the microgel samples with high capacity to swell . in the case of ps1n , containing a thinner layer of crosslinked pnipam , the broad transition and the low enthalpy of dehydration refer to a more pronounced effect of the core particle through the whole pnipam layer . analogously , to linear pnipam attached to solid particles , the factors affecting to the phase transition of crosslinked pnipam on a solid particle appear to be the distance of the polymer segment from the core ( the thickness of the pnipam layer ) and the local concentration / packing of the polymer segments close to the core surface . certain analogy can also be observed when comparing the calorimetric results obtained in our case of crosslinked pnipam on a solid polystyrene particle to the results of woodward et al . according to our results from microcalorimetry and h nmr spectroscopy , the phase transition of crosslinked pnipam - shell on solid polystyrene particle shifts towards higher temperatures when compared to the results obtained for pnipam microgels and , also , that the temperature range of the transition is broader . in this case , the transition is evidently broadened by the presence of both the core particle and the hydrophilic groups . the results from dynamic light scattering showed a surprisingly small change of hydrodynamic size with temperature in the case of a thick pnipam layer referring to strong motional restrictions . however , the relaxation times of pnipam protons refer to components with higher mobility existing in 2-stage particle samples and possible explanation is a looser and/or more heterogeneous structure of pnipam network compared to the microgel samples . the broadening of the transition in the core - shell particles compared to microgels is interpreted as being due to more heterogeneous structure of the pnipam network . the increase of the transition temperature is concluded to be due to increased motional constraints of the polymer segments on the solid surface .

different from total globozoospermia , which is characterized by 100% round - headed sperm , partial globozoospermia has a large , but not total , proportion of round - headed sperm and even has morphologically normal sperm in one sperm sample . in daily clinical practice , partial globozoospermia is more commonly found than total globozoospermia , and it might be correlated with a larger number of couples infertility . so far , total globozoospermia has been well - studied , especially its morphological characteristics , fertilizing capacity , oocyte activation capacity and even genetic background . for partial globozoospermia , current studies have mainly focused on structural characteristics and fertilizing capacity and the general view is that intracytoplasmic sperm injection ( icsi ) is a more suitable method than in - vitro fertilization ( ivf ) to obtain an acceptable fertilization rate . in total globozoospermia , icsi can not achieve an ideal fertilization rate because of the very low oocyte activation capacity of round - headed sperm . however , there are different proportion of acrosomeless sperm and even different proportion of morphologically normal sperm in partial globozoospermia . whether the different proportion of acrosomeless sperm or different proportion of morphologically normal sperm implies different oocyte activation capacity ? or whether there exists some correlation between icsi fertilization rate and the sperm morphology ? these questions remain open . in this study , we investigated the fertilization rate of ivf or icsi in 34 cases that were diagnosed with partial globozoospermia according to the criteria of more than 25% round - headed sperm in the ejaculate . simultaneously , 60 cases accepting ivf or icsi treatment in our reproductive center in november 2013 were considered as the control group after being matched for confounding conditions . fertilization rate , embryo quality , embryo implantation rate , and clinical pregnancy rate were calculated . this study was to describe the morphological characteristics of partial globozoospermia and to demonstrate if there is any correlation between icsi fertilization rate and the sperm morphology in patients with partial globozoospermia . thirty - four infertile couples in which the male partners had partial globozoospermia accepting ivf or icsi treatment in our reproductive center from january 2009 to november 2013 were recruited as our subjects . among the 34 female partners , 9 had obstruction of fallopian tube , 3 had polycystic ovarian syndrome , 2 had endometriosis , 1 had obesity ( body mass index > 30 kg / m ) , and other 19 had no obvious abnormalities . five couples achieved spontaneous pregnancy in the past , and the other 29 were diagnosed with primary infertility . ten out of the 34 couples ( five from secondary infertility and the other five from primary infertility ) had accepted at least two cycles of intrauterine insemination treatment before , but none had achieved successful pregnancy . sixty cases accepting ivf or icsi treatment in november 2013 were chosen as the control group based on the following matching criteria : female age between 31 and 33 years ; a standard long protocol ; the first ivf or icsi cycle without sperm or oocyte donation ; none of the male partners suffered from ( partial ) globozoospermia or azoospermia ; early or late rescue icsi for total ivf failure was also excluded . among the 60 cases , 45 cases accepted ivf alone , 10 cases accepted icsi alone , and 5 cases accepted split ivf / icsi . fertilization and embryo results were compared between the 34 couples and 60 control cases . at the same time , 125 sperm samples of nonglobozoospermia were collected from the semen analysis laboratory . here , 125 sperm samples included 25 samples of normozoospermia ( sperm routine parameters and morphology are both normal ) , 25 samples of oligozoospermia , 25 samples of asthenospermia , 25 samples of teratozoospermia and 25 samples of oligoasthenoteratozoospermia . sperm routine parameters and morphology were compared between 34 sperm samples of partial globozoospermia and 125 sperm samples of nonglobozoospermia . the study protocol was approved by the medical ethics committees of sir run run shaw hospital , and all the volunteers who supported for the scientific research signed informed consent forms . malefree ( bred life science , shenzhen , china ) was the container of semen sample , which consisted of one sharp - bottomed centrifuge tube and one funnel . sperm concentration and progressive motility were performed by the computer - assisted sperm analysis system , sca ( version 5.0 , microptic s.l , spain ) according to the world health organization ( who ) standard andrology criteria . the staining kit was purchased from one chinese company ( bred life science , shenzhen , china ) . first , a drop of semen was smeared on a slide and air - dried , then the slide was immersed in fixative solution for 15 s , rapid stain solution 1 for 10 s , rapid stain solution 2 for 5 s and running tap water 1015 times to remove excess stain . the evaluation of sperm morphology was performed according to criteria of who laboratory manual for the examination and processing of human semen ( 5 edition ) . acrosomeless sperm , abnormal - acrosomal sperm , and morphologically normal sperm were separately recorded . we calculated the percentage of round - headed sperm , abnormal - acrosomal sperm , or morphologically normal sperm according to the following formula : percentage of round - headed sperm = number of round - headed sperm/200 100% . percentage of abnormal - acrosomal sperm = number of abnormal - acrosomal sperm/200 100% . the standard long protocol involved down - regulation with subcutaneous injection of 0.05 mg gnrh agonist triptorelin ( ferring , wittland , germany ) starting on day 20 of the previous cycle , followed by recombinant gonadotrophin stimulation with either injection of puregon ( organon , oss , the netherlands ) or injection of gonal f ( merck serono , geneva , switzerland ) . when a group of follicles reached 16 mm in diameter , ultrasound - guided transvaginal ovum pick - up ( opu ) was performed 36 h after 5000 iu urinary hcg pregnyl ( organon , oss , the netherlands ) administration . finally , oocytes were cultured in bd falcon dishes ( becton dickinson , nj , usa ) , and the culture density was three oocytes per drop ( volume of each drop was 75 l ) . the culture medium for oocytes was g - ivf ( vitrolife , gteborg , sweden ) plus 10% ( v / v ) serum substitute supplement ( irvinescientific , california , usa ) and the incubation system was carbon dioxide incubator ( thermo fisher scientific , ohio , usa ) at 37c , with 6% co2 . on the day of opu , semen sample of the male partner was collected by masturbation into sterile plastic container and then centrifuged with gradient liquid isolate ( invinescientific , california , usa ) . finally , the sediment was diluted into suspension with a sperm concentration of 15 10/ml and then the suspension was cultured in an incubator at 37c with 6% co2 for ivf or icsi use . of the 34 cases , ivf alone was carried out for asking of 2 couples with secondary infertility , and split ivf / icsi was carried out for asking of 6 couples who met two preconditions more than 10 oocytes achieved and more than 1 10 progressive sperm in the stripped suspension . split ivf / icsi meant that for the same infertile couple ivf was carried in several oocytes while icsi was carried in the remaining oocytes . the other 26 cases accepted icsi treatment alone . for ivf , 30,000 sperm were added to one 75 l culture drop that meant the sperm concentration in the fertilizing drop was 4 10/ml . for icsi , about 1 l sperm suspension was added into polyvinylpyrrolidone ( invitrocare , maryland , usa ) and then icsi was performed as described previously by palermo et al . icsi instrument was narishige micromanipulation system ( nt-88ne - n2 , nikon , japan ) . indications of icsi in the control group include : progressive motility was under 10% ; normal morphology rate was below 1% ; sperm total number after washing process was below 1 10 . embryo transfer was performed on day 3 or day 5 after opu only if the conditions were suitable for transfer , otherwise transfer was canceled and the embryos were frozen . blood -hcg was measured on day 15 after opu , and an ultrasound was made 35 weeks after embryo transfer . indication of a successful clinical pregnancy was one or more intrauterine gestational sacs with fetal heartbeat . the normally distributed data were expressed as means standard deviation , and the skewed data were expressed as the median ( quartile range ) . test was used to compare the rate between two groups , and the one - way anova test was used to compare multiple samples . all p values were based on two - sided comparisons , and p < 0.05 was considered as statistically significant . thirty - four infertile couples in which the male partners had partial globozoospermia accepting ivf or icsi treatment in our reproductive center from january 2009 to november 2013 were recruited as our subjects . among the 34 female partners , 9 had obstruction of fallopian tube , 3 had polycystic ovarian syndrome , 2 had endometriosis , 1 had obesity ( body mass index > 30 kg / m ) , and other 19 had no obvious abnormalities . five couples achieved spontaneous pregnancy in the past , and the other 29 were diagnosed with primary infertility . ten out of the 34 couples ( five from secondary infertility and the other five from primary infertility ) had accepted at least two cycles of intrauterine insemination treatment before , but none had achieved successful pregnancy . sixty cases accepting ivf or icsi treatment in november 2013 were chosen as the control group based on the following matching criteria : female age between 31 and 33 years ; a standard long protocol ; the first ivf or icsi cycle without sperm or oocyte donation ; none of the male partners suffered from ( partial ) globozoospermia or azoospermia ; early or late rescue icsi for total ivf failure was also excluded . among the 60 cases , 45 cases accepted ivf alone , 10 cases accepted icsi alone , and 5 cases accepted split ivf / icsi . fertilization and embryo results were compared between the 34 couples and 60 control cases . at the same time , 125 sperm samples of nonglobozoospermia were collected from the semen analysis laboratory . here , 125 sperm samples included 25 samples of normozoospermia ( sperm routine parameters and morphology are both normal ) , 25 samples of oligozoospermia , 25 samples of asthenospermia , 25 samples of teratozoospermia and 25 samples of oligoasthenoteratozoospermia . sperm routine parameters and morphology were compared between 34 sperm samples of partial globozoospermia and 125 sperm samples of nonglobozoospermia . the study protocol was approved by the medical ethics committees of sir run run shaw hospital , and all the volunteers who supported for the scientific research signed informed consent forms . malefree ( bred life science , shenzhen , china ) was the container of semen sample , which consisted of one sharp - bottomed centrifuge tube and one funnel . sperm concentration and progressive motility were performed by the computer - assisted sperm analysis system , sca ( version 5.0 , microptic s.l , spain ) according to the world health organization ( who ) standard andrology criteria . the staining kit was purchased from one chinese company ( bred life science , shenzhen , china ) . first , a drop of semen was smeared on a slide and air - dried , then the slide was immersed in fixative solution for 15 s , rapid stain solution 1 for 10 s , rapid stain solution 2 for 5 s and running tap water 1015 times to remove excess stain . the evaluation of sperm morphology was performed according to criteria of who laboratory manual for the examination and processing of human semen ( 5 edition ) . acrosomeless sperm , abnormal - acrosomal sperm , and morphologically normal sperm were separately recorded . we calculated the percentage of round - headed sperm , abnormal - acrosomal sperm , or morphologically normal sperm according to the following formula : percentage of round - headed sperm = number of round - headed sperm/200 100% . percentage of abnormal - acrosomal sperm = number of abnormal - acrosomal sperm/200 100% . the standard long protocol involved down - regulation with subcutaneous injection of 0.05 mg gnrh agonist triptorelin ( ferring , wittland , germany ) starting on day 20 of the previous cycle , followed by recombinant gonadotrophin stimulation with either injection of puregon ( organon , oss , the netherlands ) or injection of gonal f ( merck serono , geneva , switzerland ) . when a group of follicles reached 16 mm in diameter , ultrasound - guided transvaginal ovum pick - up ( opu ) was performed 36 h after 5000 iu urinary hcg pregnyl ( organon , oss , the netherlands ) administration . finally , oocytes were cultured in bd falcon dishes ( becton dickinson , nj , usa ) , and the culture density was three oocytes per drop ( volume of each drop was 75 l ) . the culture medium for oocytes was g - ivf ( vitrolife , gteborg , sweden ) plus 10% ( v / v ) serum substitute supplement ( irvinescientific , california , usa ) and the incubation system was carbon dioxide incubator ( thermo fisher scientific , ohio , usa ) at 37c , with 6% co2 . on the day of opu , semen sample of the male partner was collected by masturbation into sterile plastic container and then centrifuged with gradient liquid isolate ( invinescientific , california , usa ) . finally , the sediment was diluted into suspension with a sperm concentration of 15 10/ml and then the suspension was cultured in an incubator at 37c with 6% co2 for ivf or icsi use . of the 34 cases , ivf alone was carried out for asking of 2 couples with secondary infertility , and split ivf / icsi was carried out for asking of 6 couples who met two preconditions more than 10 oocytes achieved and more than 1 10 progressive sperm in the stripped suspension . split ivf / icsi meant that for the same infertile couple ivf was carried in several oocytes while icsi was carried in the remaining oocytes . the other 26 cases accepted icsi treatment alone . for ivf , 30,000 sperm were added to one 75 l culture drop that meant the sperm concentration in the fertilizing drop was 4 10/ml . for icsi , about 1 l sperm suspension was added into polyvinylpyrrolidone ( invitrocare , maryland , usa ) and then icsi was performed as described previously by palermo et al . icsi instrument was narishige micromanipulation system ( nt-88ne - n2 , nikon , japan ) . indications of icsi in the control group include : progressive motility was under 10% ; normal morphology rate was below 1% ; sperm total number after washing process was below 1 10 . embryo transfer was performed on day 3 or day 5 after opu only if the conditions were suitable for transfer , otherwise transfer was canceled and the embryos were frozen . blood -hcg was measured on day 15 after opu , and an ultrasound was made 35 weeks after embryo transfer . indication of a successful clinical pregnancy was one or more intrauterine gestational sacs with fetal heartbeat . the normally distributed data were expressed as means standard deviation , and the skewed data were expressed as the median ( quartile range ) . test was used to compare the rate between two groups , and the one - way anova test was used to compare multiple samples . all p values were based on two - sided comparisons , and p < 0.05 was considered as statistically significant . under the oil immersion lens , we found in partial globozoospermia not only a large number of acrosomeless sperm , but also lots of sperm with small acrosomes . the head of acrosomeless sperm looked like a ball ( red arrow , figure 1 ) , and the head of small - acrosomal sperm was thin and pointy , which made it look like a sunflower seed ( yellow arrow , figure 1 ) . morphologically normal sperm was also seen in partial globozoospermia ( green arrow , figure 1 ) . morphological characteristics of normozoospermia , partial golobozoospermia , and total globozoospermia under the oil immersion lens . ( a and b ) showed that round - headed sperm , small - acrosomal sperm , and morphologically normal sperm could be seen simultaneously in normozoospermia and partial globozoospermia ( red arrow meant round - headed sperm , yellow arrow meant small - acrosomal sperm , and green meant morphologically normal sperm ) ; ( c ) showed partial globozoospermia with no morphologically normal sperm ; ( d ) showed that all the sperm were round headed . both the percentage of acrosomeless sperm and the percentage of small - acrosomal sperm in partial globozoospermia were significantly higher than nonglobozoospermia the percentage of morphologically normal sperm in partial globozoospermia was the lowest at about 0.3% , which was significantly lower than normozoospermia , oligozoospermia and asthenospermia ( p < 0.001 ) , lower than teratozoospermia ( p < 0.05 ) , and had no difference compared with oligoasthenoteratozoospermia ( p > 0.05 ) . proportion of acrosomeless sperm , small - acrosomal sperm , or morphologically normal sperm in different types of semen samples * proportion of acrosomeless sperm in partial globozoospermia was significantly higher than five types of non - globozoospermia ( p < 0.001 ) ; proportion of small - acrosomal sperm in partial globozoospermia was significantly higher than five types of nonglobozoospermia ( p < 0.001 ) ; proportion of morphologically normal sperm in partial globozoospermia was significantly lower than normozoospermia , oligozoospermia , and asthenospermia ( p < 0.001 ) , lower than teratozoospermia ( p < 0.01 ) , and had no difference compared with oligoasthenoteratozoospermia ( p > 0.05 ) . of the 34 cases of partial globozoospermia , 2 cases accepted ivf alone , 26 cases accepted icsi alone , and the other 6 cases accepted split ivf / icsi . for split ivf / icsi , 56 oocytes accepted ivf , and 60 oocytes accepted icsi . total ivf failure happened in two cases ( one from ivf alone , and the other from split ivf / icsi ) . fertilization rate of ivf ( ivf alone plus split ivf ) in partial globozoospermia was significantly lower than that of the control group ( p < 0.01 ) , but icsi ( icsi plus split icsi ) achieved a satisfying fertilization rate compared with the control group ( p > 0.05 ) [ table 2 ] . treatment characteristics of partial globozoospermia cases and the control group the results are expressed as the means sd . * fertilization rate of ivf in partial globozoospermia was significantly lower than that of the control group ( p < 0.01 ) . sd : standard deviation ; icsi : intracytoplasmic sperm injection ; ivf : in - vitro fertilization . pearson correlation test showed that there were no significant correlations between the fertilization rate of ivf or icsi and the proportion of round - headed sperm , small - acrosomal sperm , or morphologically normal sperm ( p > 0.05 ) [ figure 2 ] . correlations between the fertilization rate of in - vitro fertilization ( ivf ) or intracytoplasmic sperm injection ( icsi ) and the proportion of acrosomeless sperm , small - acrosomal sperm or morphologically normal sperm . ( a - c ) showed the correlations between the fertilization rate of ivf and the proportion of acrosomeless sperm , small - acrosomal sperm or morphologically normal sperm , and ( d - f ) showed the correlations between the fertilization rate of icsi and the proportion of acrosomeless sperm , small - acrosomal sperm or morphologically normal sperm . under the oil immersion lens , we found in partial globozoospermia not only a large number of acrosomeless sperm , but also lots of sperm with small acrosomes . the head of acrosomeless sperm looked like a ball ( red arrow , figure 1 ) , and the head of small - acrosomal sperm was thin and pointy , which made it look like a sunflower seed ( yellow arrow , figure 1 ) . morphologically normal sperm was also seen in partial globozoospermia ( green arrow , figure 1 ) . morphological characteristics of normozoospermia , partial golobozoospermia , and total globozoospermia under the oil immersion lens . ( a and b ) showed that round - headed sperm , small - acrosomal sperm , and morphologically normal sperm could be seen simultaneously in normozoospermia and partial globozoospermia ( red arrow meant round - headed sperm , yellow arrow meant small - acrosomal sperm , and green meant morphologically normal sperm ) ; ( c ) showed partial globozoospermia with no morphologically normal sperm ; ( d ) showed that all the sperm were round headed . both the percentage of acrosomeless sperm and the percentage of small - acrosomal sperm in partial globozoospermia were significantly higher than nonglobozoospermia the percentage of morphologically normal sperm in partial globozoospermia was the lowest at about 0.3% , which was significantly lower than normozoospermia , oligozoospermia and asthenospermia ( p < 0.001 ) , lower than teratozoospermia ( p < 0.05 ) , and had no difference compared with oligoasthenoteratozoospermia ( p > 0.05 ) . proportion of acrosomeless sperm , small - acrosomal sperm , or morphologically normal sperm in different types of semen samples * proportion of acrosomeless sperm in partial globozoospermia was significantly higher than five types of non - globozoospermia ( p < 0.001 ) ; proportion of small - acrosomal sperm in partial globozoospermia was significantly higher than five types of nonglobozoospermia ( p < 0.001 ) ; proportion of morphologically normal sperm in partial globozoospermia was significantly lower than normozoospermia , oligozoospermia , and asthenospermia ( p < 0.001 ) , lower than teratozoospermia ( p < 0.01 ) , and had no difference compared with oligoasthenoteratozoospermia ( p > 0.05 ) . of the 34 cases of partial globozoospermia , 2 cases accepted ivf alone , 26 cases accepted icsi alone , and the other 6 cases accepted split ivf / icsi . for split ivf / icsi , 56 oocytes accepted ivf , and 60 oocytes accepted icsi . total ivf failure happened in two cases ( one from ivf alone , and the other from split ivf / icsi ) . fertilization rate of ivf ( ivf alone plus split ivf ) in partial globozoospermia was significantly lower than that of the control group ( p < 0.01 ) , but icsi ( icsi plus split icsi ) achieved a satisfying fertilization rate compared with the control group ( p > 0.05 ) [ table 2 ] . treatment characteristics of partial globozoospermia cases and the control group the results are expressed as the means sd . * fertilization rate of ivf in partial globozoospermia was significantly lower than that of the control group ( p < 0.01 ) . sd : standard deviation ; icsi : intracytoplasmic sperm injection ; ivf : in - vitro fertilization . pearson correlation test showed that there were no significant correlations between the fertilization rate of ivf or icsi and the proportion of round - headed sperm , small - acrosomal sperm , or morphologically normal sperm ( p > 0.05 ) [ figure 2 ] . correlations between the fertilization rate of in - vitro fertilization ( ivf ) or intracytoplasmic sperm injection ( icsi ) and the proportion of acrosomeless sperm , small - acrosomal sperm or morphologically normal sperm . ( a - c ) showed the correlations between the fertilization rate of ivf and the proportion of acrosomeless sperm , small - acrosomal sperm or morphologically normal sperm , and ( d - f ) showed the correlations between the fertilization rate of icsi and the proportion of acrosomeless sperm , small - acrosomal sperm or morphologically normal sperm . it is well - known that large , but not total , proportion of round - headed sperm is the basic feature of partial globozoospermia . in this study , we investigated 34 cases of partial globozoospermia and further demonstrated two features of partial globozoospermia 's fertilizing capacity satisfying fertilization rate of icsi and unsatisfing fertilization rate of ivf . in addition , we also found a high proportion of small - acrosomal sperm in partial globozoospermia . to explain the absence of acrosome in globozoospermia , dam et al . concluded four possible mechanisms including acrosome being lost in the sertoli cell , acrosome being lost in the cytoplasm , acrosome degeneration and absence , or malfunction of the caudal manchette . although the exact mechanism of acrosomal absence was not clear , one common view is that globozoospermia likely originates during spermiogenesis . here , we found that acrosomeless sperm and small - acrosomal sperm were the overwhelming types of cells in partial globozoospermia . we postulated that the small - acrosomal sperm could be the same as those dysmorphic sperm described by dam et al . more or less oval head shapes with less condensed chromatin , partially present and even invaginated acrosomes . these morphological findings suggested that partial globozoospermia might have serious agenesis or even agensis of acrosomes . very high proportion of acrosomeless sperm , very high proportion of small - acrosomal sperm , and very low proportion of morphologically normal sperm were considered as the main reason for very low ivf capacity of partial globozoospermia . as we know , normal fertilizing capacity of sperm depends on normal acrosomal reaction and normal functions of acrosomal enzymes . round - headed sperm can not fertilize mature oocytes because of the absence of acrosome and acrosomal enzymes . a partially present and even invaginated acrosome makes the space between the sperm head membranes smaller and then the whole acrosomal volume smaller , which makes the amount of acrosomal enzymes in partial globozoospermia less than that in sperm with normal acrosome volume . meanwhile , as low as 0.3% of normal morphology rate might not be able to ensure ideal ivf capacity . from the above reasons , it might explain that the ivf capacity of partial globozoospermia was significantly lower than that of the control group in our study . fortunately , just because that there were still some oval and even morphologically normal sperm , partial globozoospermia did not lose fertilizing capacity completely unlike total globozoospermia . this might be why there is no spontaneous fertilization occurring in total globozoospermia , but some successful spontaneous pregnancies in partial globozoospermia . actually , although most couples with partial globozoospermia in our study suffered from primary infertility , five cases had conceived successfully before . in total globozoospermia , icsi is considered as the optimal method to achieve successful fertilization although the fertilization results are not good enough because of poor oocyte activation capability of round - headed sperm . unlike total globozoospermia , partial globozoospermia could achieve an ideal icsi fertilization rate , which suggested that sperm from partial globozoospermia chosen for injection have normal oocyte activation capability . therefore , we could conclude that the differences between partial globozoospermia and total globozoospermia include not only different proportion of round - headed sperm , but also different oocyte activation capability . this point of view is in consistent with dam 's point , which stated that partial globozoospermia is a distinctive sperm malformation that exists separately from total globozoospermia . in the present study , the pearson correlation test showed that there were no significant correlations between icsi fertilization rate and the proportion of round - headed sperm , small - acrosomal sperm , or even morphologically normal sperm ( p > 0.05 ) . this implied that icsi fertilization rate might not depend on the proportion of the three types of sperm cells . in an actual process of icsi for partial globozoospermia , oval sperm or even morphologically normal sperm are preferred for injection , and this selection process might be the key to achieve an ideal icsi fertilization rate . because , there is a very low percentage of morphologically normal sperm in partial globozoospermia , it takes more time and needs professional skills to select normal or nearly normal sperm . pearson correlation test also indicated that there were no significant correlations between the fertilization rate of ivf and the proportion of round - headed sperm , small - acrosomal sperm , or even morphologically normal sperm ( p > 0.05 ) . this result needs to be clarified by further studies with a larger sample size . in conclusion , we observed sperm morphology by diff - quik staining and found a very high proportion of small - acrosomal sperm in partial globozoospermia . a total of 34 cases of partial globozoospermia accepted ivf or icsi treatment and achieved low ivf rate and comparable icsi fertilization rate . we considered that icsi might be the preferable method to treat partial globozoospermia , and found that icsi fertilization rate might not depend on the proportion of round - headed sperm , small - acrosomal sperm , or morphologically normal sperm .

background : generally , intracytoplasmic sperm injection ( icsi ) may be the preferable method to treat partial globozoospermia , but whether there exist some correlations between icsi fertilization rate and the proportion of round - headed sperm or morphologically normal sperm remains open . this study was to explore the correlation between icsi fertilization rate and the sperm morphology in patients with partial globozoospermia.methods:thirty-four patients diagnosed with partial globozoospermia accepted the following assisted fertilization treatments 2 cases accepted in - vitro fertilization ( ivf ) alone , 26 cases accepted icsi alone , and 6 accepted split ivf / icsi . detailed morphological characteristics were described using diff - quik rapid staining . sixty cases accepting ivf or icsi treatment in our reproductive center were considered as the control group after being matched by relevant criteria . fertilization rate , embryo quality , embryo implantation rate and clinical pregnancy rate were calculated.results:besides very high proportion of round - headed sperm , partial globozoospermia also showed very high proportion of small - acrosomal sperm and very low proportion of morphologically normal sperm . fertilization rate of ivf ( ivf alone plus split ivf ) was very low in partial globozoospermia ( 25.4% 17.4% ) , but icsi ( icsi alone plus split icsi ) achieved satisfying fertilization rate compared with the control group ( 66.2% 22.5% vs. 68.8% 29.4% , p > 0.05 ) . in patients with partial globozoospermia , there were no correlations between icsi fertilization rate and the proportion of round - headed sperm , small - acrosomal sperm , or morphologically normal sperm.conclusions:there was high proportion of small - acrosomal sperm in partial globozoospermia . for patients with partial globozoospermia , icsi is more preferable than ivf . icsi fertilization rate does not depend on the proportion of round - headed sperm , small - acrosomal sperm , or morphologically normal sperm .

I M Subjects Analysis of sperm routine parameters Analysis of sperm morphology None Statistical analysis R Sperm morphological characteristics under the oil immersion lens Proportion of acrosomeless sperm, small-acrosomal sperm, or morphologically normal sperm in partial globozoospermia and nonglobozoospermia None Correlations between the fertilization rate of D

different from total globozoospermia , which is characterized by 100% round - headed sperm , partial globozoospermia has a large , but not total , proportion of round - headed sperm and even has morphologically normal sperm in one sperm sample . in daily clinical practice , partial globozoospermia is more commonly found than total globozoospermia , and it might be correlated with a larger number of couples infertility . for partial globozoospermia , current studies have mainly focused on structural characteristics and fertilizing capacity and the general view is that intracytoplasmic sperm injection ( icsi ) is a more suitable method than in - vitro fertilization ( ivf ) to obtain an acceptable fertilization rate . in total globozoospermia , icsi can not achieve an ideal fertilization rate because of the very low oocyte activation capacity of round - headed sperm . however , there are different proportion of acrosomeless sperm and even different proportion of morphologically normal sperm in partial globozoospermia . whether the different proportion of acrosomeless sperm or different proportion of morphologically normal sperm implies different oocyte activation capacity ? or whether there exists some correlation between icsi fertilization rate and the sperm morphology ? in this study , we investigated the fertilization rate of ivf or icsi in 34 cases that were diagnosed with partial globozoospermia according to the criteria of more than 25% round - headed sperm in the ejaculate . simultaneously , 60 cases accepting ivf or icsi treatment in our reproductive center in november 2013 were considered as the control group after being matched for confounding conditions . fertilization rate , embryo quality , embryo implantation rate , and clinical pregnancy rate were calculated . this study was to describe the morphological characteristics of partial globozoospermia and to demonstrate if there is any correlation between icsi fertilization rate and the sperm morphology in patients with partial globozoospermia . thirty - four infertile couples in which the male partners had partial globozoospermia accepting ivf or icsi treatment in our reproductive center from january 2009 to november 2013 were recruited as our subjects . five couples achieved spontaneous pregnancy in the past , and the other 29 were diagnosed with primary infertility . sixty cases accepting ivf or icsi treatment in november 2013 were chosen as the control group based on the following matching criteria : female age between 31 and 33 years ; a standard long protocol ; the first ivf or icsi cycle without sperm or oocyte donation ; none of the male partners suffered from ( partial ) globozoospermia or azoospermia ; early or late rescue icsi for total ivf failure was also excluded . among the 60 cases , 45 cases accepted ivf alone , 10 cases accepted icsi alone , and 5 cases accepted split ivf / icsi . acrosomeless sperm , abnormal - acrosomal sperm , and morphologically normal sperm were separately recorded . we calculated the percentage of round - headed sperm , abnormal - acrosomal sperm , or morphologically normal sperm according to the following formula : percentage of round - headed sperm = number of round - headed sperm/200 100% . finally , oocytes were cultured in bd falcon dishes ( becton dickinson , nj , usa ) , and the culture density was three oocytes per drop ( volume of each drop was 75 l ) . of the 34 cases , ivf alone was carried out for asking of 2 couples with secondary infertility , and split ivf / icsi was carried out for asking of 6 couples who met two preconditions more than 10 oocytes achieved and more than 1 10 progressive sperm in the stripped suspension . the other 26 cases accepted icsi treatment alone . all p values were based on two - sided comparisons , and p < 0.05 was considered as statistically significant . thirty - four infertile couples in which the male partners had partial globozoospermia accepting ivf or icsi treatment in our reproductive center from january 2009 to november 2013 were recruited as our subjects . sixty cases accepting ivf or icsi treatment in november 2013 were chosen as the control group based on the following matching criteria : female age between 31 and 33 years ; a standard long protocol ; the first ivf or icsi cycle without sperm or oocyte donation ; none of the male partners suffered from ( partial ) globozoospermia or azoospermia ; early or late rescue icsi for total ivf failure was also excluded . among the 60 cases , 45 cases accepted ivf alone , 10 cases accepted icsi alone , and 5 cases accepted split ivf / icsi . acrosomeless sperm , abnormal - acrosomal sperm , and morphologically normal sperm were separately recorded . we calculated the percentage of round - headed sperm , abnormal - acrosomal sperm , or morphologically normal sperm according to the following formula : percentage of round - headed sperm = number of round - headed sperm/200 100% . finally , oocytes were cultured in bd falcon dishes ( becton dickinson , nj , usa ) , and the culture density was three oocytes per drop ( volume of each drop was 75 l ) . of the 34 cases , ivf alone was carried out for asking of 2 couples with secondary infertility , and split ivf / icsi was carried out for asking of 6 couples who met two preconditions more than 10 oocytes achieved and more than 1 10 progressive sperm in the stripped suspension . the other 26 cases accepted icsi treatment alone . the normally distributed data were expressed as means standard deviation , and the skewed data were expressed as the median ( quartile range ) . all p values were based on two - sided comparisons , and p < 0.05 was considered as statistically significant . under the oil immersion lens , we found in partial globozoospermia not only a large number of acrosomeless sperm , but also lots of sperm with small acrosomes . the head of acrosomeless sperm looked like a ball ( red arrow , figure 1 ) , and the head of small - acrosomal sperm was thin and pointy , which made it look like a sunflower seed ( yellow arrow , figure 1 ) . morphologically normal sperm was also seen in partial globozoospermia ( green arrow , figure 1 ) . morphological characteristics of normozoospermia , partial golobozoospermia , and total globozoospermia under the oil immersion lens . ( a and b ) showed that round - headed sperm , small - acrosomal sperm , and morphologically normal sperm could be seen simultaneously in normozoospermia and partial globozoospermia ( red arrow meant round - headed sperm , yellow arrow meant small - acrosomal sperm , and green meant morphologically normal sperm ) ; ( c ) showed partial globozoospermia with no morphologically normal sperm ; ( d ) showed that all the sperm were round headed . both the percentage of acrosomeless sperm and the percentage of small - acrosomal sperm in partial globozoospermia were significantly higher than nonglobozoospermia the percentage of morphologically normal sperm in partial globozoospermia was the lowest at about 0.3% , which was significantly lower than normozoospermia , oligozoospermia and asthenospermia ( p < 0.001 ) , lower than teratozoospermia ( p < 0.05 ) , and had no difference compared with oligoasthenoteratozoospermia ( p > 0.05 ) . proportion of acrosomeless sperm , small - acrosomal sperm , or morphologically normal sperm in different types of semen samples * proportion of acrosomeless sperm in partial globozoospermia was significantly higher than five types of non - globozoospermia ( p < 0.001 ) ; proportion of small - acrosomal sperm in partial globozoospermia was significantly higher than five types of nonglobozoospermia ( p < 0.001 ) ; proportion of morphologically normal sperm in partial globozoospermia was significantly lower than normozoospermia , oligozoospermia , and asthenospermia ( p < 0.001 ) , lower than teratozoospermia ( p < 0.01 ) , and had no difference compared with oligoasthenoteratozoospermia ( p > 0.05 ) . of the 34 cases of partial globozoospermia , 2 cases accepted ivf alone , 26 cases accepted icsi alone , and the other 6 cases accepted split ivf / icsi . for split ivf / icsi , 56 oocytes accepted ivf , and 60 oocytes accepted icsi . total ivf failure happened in two cases ( one from ivf alone , and the other from split ivf / icsi ) . fertilization rate of ivf ( ivf alone plus split ivf ) in partial globozoospermia was significantly lower than that of the control group ( p < 0.01 ) , but icsi ( icsi plus split icsi ) achieved a satisfying fertilization rate compared with the control group ( p > 0.05 ) [ table 2 ] . treatment characteristics of partial globozoospermia cases and the control group the results are expressed as the means sd . * fertilization rate of ivf in partial globozoospermia was significantly lower than that of the control group ( p < 0.01 ) . sd : standard deviation ; icsi : intracytoplasmic sperm injection ; ivf : in - vitro fertilization . pearson correlation test showed that there were no significant correlations between the fertilization rate of ivf or icsi and the proportion of round - headed sperm , small - acrosomal sperm , or morphologically normal sperm ( p > 0.05 ) [ figure 2 ] . correlations between the fertilization rate of in - vitro fertilization ( ivf ) or intracytoplasmic sperm injection ( icsi ) and the proportion of acrosomeless sperm , small - acrosomal sperm or morphologically normal sperm . ( a - c ) showed the correlations between the fertilization rate of ivf and the proportion of acrosomeless sperm , small - acrosomal sperm or morphologically normal sperm , and ( d - f ) showed the correlations between the fertilization rate of icsi and the proportion of acrosomeless sperm , small - acrosomal sperm or morphologically normal sperm . under the oil immersion lens , we found in partial globozoospermia not only a large number of acrosomeless sperm , but also lots of sperm with small acrosomes . the head of acrosomeless sperm looked like a ball ( red arrow , figure 1 ) , and the head of small - acrosomal sperm was thin and pointy , which made it look like a sunflower seed ( yellow arrow , figure 1 ) . morphologically normal sperm was also seen in partial globozoospermia ( green arrow , figure 1 ) . morphological characteristics of normozoospermia , partial golobozoospermia , and total globozoospermia under the oil immersion lens . ( a and b ) showed that round - headed sperm , small - acrosomal sperm , and morphologically normal sperm could be seen simultaneously in normozoospermia and partial globozoospermia ( red arrow meant round - headed sperm , yellow arrow meant small - acrosomal sperm , and green meant morphologically normal sperm ) ; ( c ) showed partial globozoospermia with no morphologically normal sperm ; ( d ) showed that all the sperm were round headed . both the percentage of acrosomeless sperm and the percentage of small - acrosomal sperm in partial globozoospermia were significantly higher than nonglobozoospermia the percentage of morphologically normal sperm in partial globozoospermia was the lowest at about 0.3% , which was significantly lower than normozoospermia , oligozoospermia and asthenospermia ( p < 0.001 ) , lower than teratozoospermia ( p < 0.05 ) , and had no difference compared with oligoasthenoteratozoospermia ( p > 0.05 ) . proportion of acrosomeless sperm , small - acrosomal sperm , or morphologically normal sperm in different types of semen samples * proportion of acrosomeless sperm in partial globozoospermia was significantly higher than five types of non - globozoospermia ( p < 0.001 ) ; proportion of small - acrosomal sperm in partial globozoospermia was significantly higher than five types of nonglobozoospermia ( p < 0.001 ) ; proportion of morphologically normal sperm in partial globozoospermia was significantly lower than normozoospermia , oligozoospermia , and asthenospermia ( p < 0.001 ) , lower than teratozoospermia ( p < 0.01 ) , and had no difference compared with oligoasthenoteratozoospermia ( p > 0.05 ) . of the 34 cases of partial globozoospermia , 2 cases accepted ivf alone , 26 cases accepted icsi alone , and the other 6 cases accepted split ivf / icsi . for split ivf / icsi , 56 oocytes accepted ivf , and 60 oocytes accepted icsi . total ivf failure happened in two cases ( one from ivf alone , and the other from split ivf / icsi ) . fertilization rate of ivf ( ivf alone plus split ivf ) in partial globozoospermia was significantly lower than that of the control group ( p < 0.01 ) , but icsi ( icsi plus split icsi ) achieved a satisfying fertilization rate compared with the control group ( p > 0.05 ) [ table 2 ] . treatment characteristics of partial globozoospermia cases and the control group the results are expressed as the means sd . * fertilization rate of ivf in partial globozoospermia was significantly lower than that of the control group ( p < 0.01 ) . sd : standard deviation ; icsi : intracytoplasmic sperm injection ; ivf : in - vitro fertilization . pearson correlation test showed that there were no significant correlations between the fertilization rate of ivf or icsi and the proportion of round - headed sperm , small - acrosomal sperm , or morphologically normal sperm ( p > 0.05 ) [ figure 2 ] . correlations between the fertilization rate of in - vitro fertilization ( ivf ) or intracytoplasmic sperm injection ( icsi ) and the proportion of acrosomeless sperm , small - acrosomal sperm or morphologically normal sperm . ( a - c ) showed the correlations between the fertilization rate of ivf and the proportion of acrosomeless sperm , small - acrosomal sperm or morphologically normal sperm , and ( d - f ) showed the correlations between the fertilization rate of icsi and the proportion of acrosomeless sperm , small - acrosomal sperm or morphologically normal sperm . it is well - known that large , but not total , proportion of round - headed sperm is the basic feature of partial globozoospermia . in this study , we investigated 34 cases of partial globozoospermia and further demonstrated two features of partial globozoospermia 's fertilizing capacity satisfying fertilization rate of icsi and unsatisfing fertilization rate of ivf . in addition , we also found a high proportion of small - acrosomal sperm in partial globozoospermia . here , we found that acrosomeless sperm and small - acrosomal sperm were the overwhelming types of cells in partial globozoospermia . we postulated that the small - acrosomal sperm could be the same as those dysmorphic sperm described by dam et al . very high proportion of acrosomeless sperm , very high proportion of small - acrosomal sperm , and very low proportion of morphologically normal sperm were considered as the main reason for very low ivf capacity of partial globozoospermia . round - headed sperm can not fertilize mature oocytes because of the absence of acrosome and acrosomal enzymes . a partially present and even invaginated acrosome makes the space between the sperm head membranes smaller and then the whole acrosomal volume smaller , which makes the amount of acrosomal enzymes in partial globozoospermia less than that in sperm with normal acrosome volume . from the above reasons , it might explain that the ivf capacity of partial globozoospermia was significantly lower than that of the control group in our study . fortunately , just because that there were still some oval and even morphologically normal sperm , partial globozoospermia did not lose fertilizing capacity completely unlike total globozoospermia . this might be why there is no spontaneous fertilization occurring in total globozoospermia , but some successful spontaneous pregnancies in partial globozoospermia . in total globozoospermia , icsi is considered as the optimal method to achieve successful fertilization although the fertilization results are not good enough because of poor oocyte activation capability of round - headed sperm . unlike total globozoospermia , partial globozoospermia could achieve an ideal icsi fertilization rate , which suggested that sperm from partial globozoospermia chosen for injection have normal oocyte activation capability . therefore , we could conclude that the differences between partial globozoospermia and total globozoospermia include not only different proportion of round - headed sperm , but also different oocyte activation capability . in the present study , the pearson correlation test showed that there were no significant correlations between icsi fertilization rate and the proportion of round - headed sperm , small - acrosomal sperm , or even morphologically normal sperm ( p > 0.05 ) . this implied that icsi fertilization rate might not depend on the proportion of the three types of sperm cells . in an actual process of icsi for partial globozoospermia , oval sperm or even morphologically normal sperm are preferred for injection , and this selection process might be the key to achieve an ideal icsi fertilization rate . because , there is a very low percentage of morphologically normal sperm in partial globozoospermia , it takes more time and needs professional skills to select normal or nearly normal sperm . pearson correlation test also indicated that there were no significant correlations between the fertilization rate of ivf and the proportion of round - headed sperm , small - acrosomal sperm , or even morphologically normal sperm ( p > 0.05 ) . in conclusion , we observed sperm morphology by diff - quik staining and found a very high proportion of small - acrosomal sperm in partial globozoospermia . a total of 34 cases of partial globozoospermia accepted ivf or icsi treatment and achieved low ivf rate and comparable icsi fertilization rate . we considered that icsi might be the preferable method to treat partial globozoospermia , and found that icsi fertilization rate might not depend on the proportion of round - headed sperm , small - acrosomal sperm , or morphologically normal sperm .

children 's understanding that people may hold and act on false beliefs , such as looking for the keys in the wrong place , is a milestone in their development towards an adult - like theory - of - mind . to attribute a false belief to someone , children must understand that there can be representations of the world that differ from their own accurate understanding ( dennett , 1978 ) . thus , false belief tasks are benchmarks of children 's acquisition of a representational theory - of - mind , without which they are left unable to understand mistaken expectations , misguided actions , or misleading appearances . on a typical false belief task , children are asked to predict where a puppet , jill , thinks her chocolate is . the chocolate has been surreptitiously moved from where jill originally put it , creating a false belief . a typical question could be , where does jill think the chocolate is ? the correct answer is that jill thinks the chocolate is in the original location . yet children below 4 years typically answer that jill thinks that the chocolate is in the new and actual location ( wimmer and perner , 1983 ) . children 's failure is striking because children actually remember the crucial pieces of information , i.e. , they know where jill put the chocolate and that she did not see it being moved . even more striking is that children as young as 15 months reveal a tacit understanding of false belief on new nonverbal tasks ( clements and perner , 1994 ; garnham and ruffman , 2001 ; onishi and baillargeon , 2005 ) . nevertheless , overwhelming confirmation of the original results in hundreds of false belief studies across many cultures and languages has generally led to the conclusion that children do not develop the cognitive foundation required to understand mental representation until their fifth year ( perner , 1991 ; gopnik and wellman , 1994 ; wellman et al . , 2001 ) . i propose an alternative account based on the conversational logic involved in talking about certain mental states . in certain contexts , two speakers may not have direct knowledge about the identity or the location of an object . instead , this could be appearance , e.g. , what is this ? -it looks like an apple , it could be where a third , absent , person thinks the object is , e.g. , where is the chocolate ? it follows that the question , where does jill think the chocolate is ? could be interpreted as a request for indirect knowledge about the actual location of the chocolate in certain contexts , rather than always being a unique request for jill 's belief . if children make this interpretation on false belief tasks , everyday conversational logic directs them to respond with the true location rather than with jill 's false belief . a firefighter arrives on the scene of a smoking building and asks an office worker , where does your manager think the fire is ? this is a plausible way to obtain indirect knowledge about the location of a fire . crucially , answering such a request is an unremarkable event when neither the first person ( the firefighter ) nor the second person ( the worker ) knows the location of the fire . but when the second person knows better than the absent , third person ( the manager ) , everyday conversational logic complicates the answer . suppose the worker knows that the fire is in fact in the basement , even though his manager believes that it is in the kitchen . though a literally correct answer , it would be uncooperative and even misleading of the worker to answer , my manager thinks the fire is in the kitchen . suppose the firefighter instead arrived on the scene and asked , where will your manager look for the fire ? this is another plausible way to request the likely true location of the fire , if the firefighter also here assumes that the office worker does not know from personal experience . but if he knows that the fire is in the basement , he would have sent the firefighter to the wrong place . because people 's actions reflect their thoughts , an action question can function pragmatically as a belief question . while the context in the firefighter example is clearly different from that of a false belief task , the point is to illustrate that situations exist where the question , where does x think y is , is not meant to include , or is interpreted as including , somebody 's false belief . , young children may not be sufficiently sensitive to the context to invoke precisely the meaning that is the premise of the false belief task . thus , the current study experimentally manipulates the conversational context so that it is less equivocal to young children . people must often solicit absent people 's knowledge to learn about the world on the assumption that none of the present speakers knows the truth from personal experience , e.g. , what would piaget say ? they are also likely to be familiar to children , e.g. , please go ask your mom where the keys are , or , does your mom know where the keys are ? , a child would not reply that his mother said that the keys are in the kitchen if the child knows that they are in the basement . or to the latter question solely give the literal answer , such answers would be considered uncooperative , jocular , or a sign of undeveloped pragmatic understanding . a request for indirect knowledge may simultaneously constitute an indirect request . a classic example of an indirect request is asking someone if she can pass the salt . the point is normally not to inquire about the respondent 's physical ability , but to actually request the salt ( e.g. , searle , 1975 ) . in a similar vein , the child in the preceding two examples is not directly requested to report his mother 's belief about the location of the keys , but most listeners would nevertheless interpret the questions in this way . however , the focus of this paper is not on indirect requests in searle 's sense but on the conversational logic of requests for indirect knowledge , such as asking about appearances as a path to the identity of an object or requesting someone 's belief about the location of an object as a path to its actual location . the analysis rests on two interrelated insights into language use from the field of psycholinguistics . first , interpreting a belief question as a request for indirect knowledge requires the assumption that the questioner lacks knowledge . any conversational context partly consists of a set of assumptions about the other speakers state of knowledge , termed the common ground ( stalnaker , 1978 ) . at any point in a conversation lack of knowledge about a location may be indicated explicitly , e.g. , verbally with questions such as , where are the keys , or nonverbally by searching . it may also be assumed implicitly by the listener ( clark et al . , 1983 ) . i will consider it the ordinary context because it depends on the least number of conditionals . it would be redundant to interpret the question , where does jill think the keys are ? as a request for indirect knowledge because the location is already known . second , certain answers will be deemed uncooperative if they do not fulfill the intended meaning with the request . this is captured by grice 's account of everyday conversational logic ( grice , 1975 ) . people make inferences , termed conversational implicatures , about a speaker 's meaning based on the assumption that speakers adhere to four maxims that prescribe how to communicate most efficiently and rationally . the maxims belong to a general cooperative principle , under which people recognize a common purpose of a conversation and match answers to requests . reporting beliefs as indirect knowledge is mainly regulated by the maxim of quantity : make a contribution no more or no less informative than required . in the exchange , son , where are the keys ? -well , mom thinks they are in the cabinet , the son 's seemingly irrelevant answer is nevertheless intelligible because people may infer that he cooperates with the request , but even at his most informative can only provide indirect knowledge about the location . suppose he knows that the keys are on the table but that his mother falsely thinks they are in the cabinet . he would be literally correct in saying , mom thinks the keys are in the cabinet . but this information would be superfluous and therefore uncooperative . people 's false beliefs are uninformative when the common purpose of the conversation is to discover the true location . suppose young children presume the ordinary context of lack of knowledge about reality on the part of the experimenter , in the sense of , ( i 'm looking for the chocolate and i assume you do n't know from personal experience . ) where does jill think the chocolate is ? the question then implies that the experimenter seeks indirect information about the likely location third - hand , through jill 's belief . but the point of the false belief task is that children know better than jill . so if they wish to cooperate with the presumed request for the likely location and avoid violating the maxim of quantity , children must skip the uninformative step of jill 's false belief and respond with reality . conversational logic compels children to say the truth if they know it , and leads them to fail the tasks . to pass false belief tasks , , it would violate the maxim of quantity to interpret the test question as a request for indirect knowledge about the likely location because both experimenter and child already know it . it can be argued that children should already be aware of the conditional context . after all , the child and the experimenter jointly witnessed that the object was transferred to a new location . however , children may interpret the test question locally and invoke the ordinary context for several reasons . first , the pragmatic implication of asking an honest question is lack of knowledge on the part of the experimenter , which for young children may override any shared knowledge and indicate the ordinary context . according to a related view ( e.g. , siegal and peterson , 1994 ) , young children have yet to fully understand the conversational requirements of academic questioning where the questioner already knows the answer . the current discourse - based account explicates the conversational logic of certain mental - state expressions which is a novel extension of this more general view . second , the shell game - like atmosphere of the false belief task , in particular the location task , may suggest to young children that the context remains one of following the ball . a major challenge for researchers , then , is to design false belief tasks that block the possibility of presuming the local , ordinary context by clearly linking the test question to the preceding discourse . some related accounts that integrate the concept of common ground and the gricean maxims to explain certain mental - state verbs can be found in the linguistic and philosophical literature . they are based on an original proposition by kiparsky and kiparsky ( 1970 ) . these authors proposed categorizing cognitive verbs which take that complements into two broad categories . factives are verbs where the complement can be presupposed to be true , e.g. , know and forget , as in non - factives are verbs where there is no presupposition as to whether the complement is true or not , e.g. , think , believe , and say , as in , kempson ( 1975 ) pointed out that for factive verbs the implication of truth of the complement does not necessarily hold . her analysis of factivity based on the gricean maxims and the concept of common ground showed that the implication of truth of the complement is context - dependent rather than a semantic property of the sentence in question . conversely , in the case of non - factive verbs , karttunen ( 1973 ) points out that non - factives such as say can in fact function as factives under certain conditions . while these proposals support the context - dependent aspects of the discourse - based account in a general manner , it is important to note that to date , the fields of linguistics and philosophy are grappling with the issue of factivity . numerous authors have identified flaws in the original semantic and logical explanations of the kiparskys proposal and have criticized the accuracy and scope of the distinction itself ( for an overview , see hazlett , 2010 ) . some authors instead favor a gricean account like that of kempson ( e.g. , karttunen , 1998 ; stalnaker , 1998 ; hazlett , 2010 ) . however , the versions of the factivity theory that include a gricean analysis are presently too narrow to account for our phenomenon of interest . to properly cover children 's understanding of the test questions on a variety of theory - of - mind tasks , such an account must not only include mental - state verbs that take that complements , but all types of expressions used on false belief tasks and appearance - reality tasks such as thinks , says , looks for , and looks like . from a developmental point of view , abbeduto and rosenberg ( 1985 ) they showed that before the age of 4 years , children treat non - factives as factives . the results confirm that the interpretation of thinks and believes may be difficult for 3-year - olds under circumstances other than the false belief task , but the study is nevertheless of limited relevance to the current account . the authors acknowledged the pragmatic weaknesses of the kiparskys original analysis but opted for ignoring them , thus not allowing a comparison with the broader view of the conversational context that is presented here . the discourse - based account receives empirical support from its application to another major theory - of - mind task : the appearance - reality task . on this task children are introduced to a deceptive object , such as a sponge that looks like a rock . at test , children below 4 years of age typically fail , strikingly saying that the object not only is a sponge but also looks like a sponge . this suggests that they can not distinguish appearances from reality ( flavell et al . , 1986 ) . similarly to the false belief task , the key test question , what does this look like ? may be interpreted as a request for indirect knowledge about object identity in an ordinary context , e.g. , ( austin , 1962 ) . as the answer to the last question will provide indirect knowledge about the identity , it is subject to the conversational logic outlined above . for deceptive objects , answering with appearance , children must understand that the test question is embedded in the conditional context that both speakers already share knowledge of the identity of the object , which frees the child to answer with appearance . on three appearance - reality tasks that emphasized the conditional context , even 3-year - old children gave nearly all - correct appearance responses while they failed the standard versions of the tasks ( hansen and markman , 2005 ) . young children should be similarly successful on false belief tasks that emphasize the conditional context . this would be strong evidence that children 's problems with theory - of - mind tasks are due to difficulties with talking about mental states rather than with understanding the mind . this hypothesis was tested on three major false belief tasks : ( 1 ) a location false belief task ; ( 2 ) a contents false belief task ; and ( 3 ) a representational change false belief task . on all tasks , the shared knowledge was emphasized in connection with the test question by simply restating it , e.g. , you and i know that the chocolate is in the basket , where does jill think it is ? compared to standard versions of each task , young children should do better in the emphasized context condition and thus demonstrate their knowledge of false belief . participants were 73 preschoolers from sydney , australia ( mean : 3 years and 11 months , range : 3 years and 5 months to 4 years and 7 months ) . there were 34 boys and 39 girls , all english - speaking and primarily of caucasian , middle socio - economic background . all participated in two conditions , except 11 children who only participated in one condition to complete the design . the research was carried out in accordance with the ethical standards of the american psychological association for the treatment of research participants . there were three types of false belief tasks : location ( n = 54 ) , contents ( n = 36 ) , and representational change ( n = 45 ) . each type of task had three conditions with equal numbers of participants in each : standard , emphasized context , and frame control . each condition contained two similar scenarios acted out by the experimenter with various dolls and containers . because of the relative similarity of tasks , conditions , and scenarios , children did not participate in all three types of tasks or all three types of conditions . each child participated in two different types of tasks with an unrelated distractor task in between , and only in one of the conditions from each task , with the constraint the conditions were not the same . the combination of tasks and conditions was specified on six lists that were cycled through . this yielded a sufficiently counter - balanced pattern of the types of tasks , types of conditions , scenarios , and their respective orders . for example , a child might participate in a block of two similar scenarios of the standard condition of the location task , then receive a distractor task , and then participate in a block of two similar scenarios of the emphasized context condition of the contents task . elmo played with a ball in front of three cups . while holding the ball , he then turned all three cups upside - down and hid the ball in one of them , and then left the stage . the experimenter then explained that elmo wanted to play with the ball again . children in the standard condition were asked , where does elmo think the ball is ? children in the emphasized context condition were asked , you and i know that the ball is in the < color of > cup , where does elmo think it is ? it is conceivable that the question frame itself invited children to use a low - level opposites strategy in the emphasized context condition such as , you say one thing , so i say the other . this tendency might be enhanced by the fact that the two mental - state words think and know may be somewhat difficult to distinguish for children of 3 to 5 years of age ( for a review , see papafragou et al . , 2007 ) . therefore , the location task included three locations , so even if children keyed off the location mentioned in the question , they could not use this information to select between the remaining two answers . children saw elmo turn over the three cups while holding the ball , hide the ball in one cup , go outside and play , and return to find the ball . as elmo returned , children were asked : elmo thinks the ball is in the yellow cup , where do you and i know it is ? as there was only a true belief , the correct answer was the same location as in the question , i.e. , the yellow cup . because the phenomenon under investigation is contrastive , a control condition using exactly the same contrastive frame as the emphasized context condition but calling for a non - contrastive answer would be pragmatically infelicitous . asking with a contrast where none can be found , for example in the case of a true belief setup , would not lead to a clear prediction for children 's answers . recall that on the discourse - based explanation , stating what somebody knows has implications for how to interpret the following think . however , the reverse should not be true so the solution was to simply reverse the order of these mental - state words in the test question of the frame control condition . if children were using an opposites strategy , blindly keying off the option mentioned in the test question in combination with uncertainty about the exact meaning of the mental - state words , they should tend to choose the incorrect answer options . task 2 was a contents false belief task ( hogrefe et al . , 1986 ) . the experimenter closed the box and introduced the character dorothy , explaining that dorothy had never looked inside the box before . children in the standard condition were asked , what does dorothy think is inside the box ? children typically fail answering crayons rather than band - aids , children in the emphasized context condition were asked , you and i know that there are crayons inside , what does dorothy think is inside the box ? the frame control condition did not include false belief : dorothy inspected the box with the lid open , so she could see the true contents . dorothy thinks there are crayons in the box , what do you and i know is inside ? the correct answer was the same contents as mentioned in the test question , i.e. , crayons . if children were guided by an opposites strategy , they should choose the other possible answer , i.e. , band - aids . task 3 was a representational change false belief task ( gopnik and astington , 1988 ) . the task is based on the contents false belief task but rather than reporting someone else 's false belief , this type of task requires children to report their own previous false belief . the introduction is the same as in the previous task , but after the experimenter closes the band - aid box , children in the standard condition are asked , seemingly unaware of their own previous false belief . on a discourse - based account , a question about someone 's past belief can be interpreted as a request for indirect knowledge . consider the example of a judge asking a police officer , we have to find the money . where did you think it was ? if the police officer knows better than she previously did , it would be uncooperative of her to just report her previous belief and withhold the truth . to ensure that children did not misread the context as one of searching for indirect knowledge in the way of a past , but presumably still valid , belief , children in the emphasized context condition were therefore asked , you and i know there are crayons inside , what did you think was in the box before you opened it ? in the frame control condition , children initially saw the band - aid box with the lid open and were asked , what do you think is inside the box ? all answered , crayons . before you thought there were crayons inside the box , what do you and i know is inside ? since the child had seen the actual contents , there was no false belief and the correct answer was the same contents as mentioned in the test question , i.e. , crayons . but if children were guided by an opposites strategy , they should not say crayons . participants were 73 preschoolers from sydney , australia ( mean : 3 years and 11 months , range : 3 years and 5 months to 4 years and 7 months ) . there were 34 boys and 39 girls , all english - speaking and primarily of caucasian , middle socio - economic background . all participated in two conditions , except 11 children who only participated in one condition to complete the design . the research was carried out in accordance with the ethical standards of the american psychological association for the treatment of research participants . there were three types of false belief tasks : location ( n = 54 ) , contents ( n = 36 ) , and representational change ( n = 45 ) . each type of task had three conditions with equal numbers of participants in each : standard , emphasized context , and frame control . each condition contained two similar scenarios acted out by the experimenter with various dolls and containers . because of the relative similarity of tasks , conditions , and scenarios , children did not participate in all three types of tasks or all three types of conditions . each child participated in two different types of tasks with an unrelated distractor task in between , and only in one of the conditions from each task , with the constraint the conditions were not the same . the combination of tasks and conditions was specified on six lists that were cycled through . this yielded a sufficiently counter - balanced pattern of the types of tasks , types of conditions , scenarios , and their respective orders . for example , a child might participate in a block of two similar scenarios of the standard condition of the location task , then receive a distractor task , and then participate in a block of two similar scenarios of the emphasized context condition of the contents task . elmo played with a ball in front of three cups . while holding the ball , he then turned all three cups upside - down and hid the ball in one of them , and then left the stage . the experimenter then explained that elmo wanted to play with the ball again . children in the standard condition were asked , where does elmo think the ball is ? children in the emphasized context condition were asked , you and i know that the ball is in the < color of > cup , where does elmo think it is ? it is conceivable that the question frame itself invited children to use a low - level opposites strategy in the emphasized context condition such as , you say one thing , so i say the other . this tendency might be enhanced by the fact that the two mental - state words think and know may be somewhat difficult to distinguish for children of 3 to 5 years of age ( for a review , see papafragou et al . , 2007 ) . therefore , the location task included three locations , so even if children keyed off the location mentioned in the question , they could not use this information to select between the remaining two answers . children saw elmo turn over the three cups while holding the ball , hide the ball in one cup , go outside and play , and return to find the ball . elmo thinks the ball is in the yellow cup , where do you and i know it is ? as there was only a true belief , the correct answer was the same location as in the question , i.e. , the yellow cup . because the phenomenon under investigation is contrastive , a control condition using exactly the same contrastive frame as the emphasized context condition but calling for a non - contrastive answer would be pragmatically infelicitous . asking with a contrast where none can be found , for example in the case of a true belief setup recall that on the discourse - based explanation , stating what somebody knows has implications for how to interpret the following think . however , the reverse should not be true so the solution was to simply reverse the order of these mental - state words in the test question of the frame control condition . if children were using an opposites strategy , blindly keying off the option mentioned in the test question in combination with uncertainty about the exact meaning of the mental - state words , they should tend to choose the incorrect answer options . task 2 was a contents false belief task ( hogrefe et al . , 1986 ) . what do you think is inside this box ? most children answered band - aids . the experimenter closed the box and introduced the character dorothy , explaining that dorothy had never looked inside the box before . children in the standard condition were asked , what does dorothy think is inside the box ? children typically fail answering crayons rather than band - aids , children in the emphasized context condition were asked , you and i know that there are crayons inside , what does dorothy think is inside the box ? the frame control condition did not include false belief : dorothy inspected the box with the lid open , so she could see the true contents . dorothy thinks there are crayons in the box , what do you and i know is inside ? the correct answer was the same contents as mentioned in the test question , i.e. , crayons . if children were guided by an opposites strategy , they should choose the other possible answer , i.e. , band - aids . task 3 was a representational change false belief task ( gopnik and astington , 1988 ) . the task is based on the contents false belief task but rather than reporting someone else 's false belief , this type of task requires children to report their own previous false belief . the introduction is the same as in the previous task , but after the experimenter closes the band - aid box , children in the standard condition are asked , seemingly unaware of their own previous false belief . on a discourse - based account , a question about someone 's past belief can be interpreted as a request for indirect knowledge . consider the example of a judge asking a police officer , we have to find the money . where did you think it was ? if the police officer knows better than she previously did , it would be uncooperative of her to just report her previous belief and withhold the truth . to ensure that children did not misread the context as one of searching for indirect knowledge in the way of a past , but presumably still valid , belief , children in the emphasized context condition were therefore asked , you and i know there are crayons inside , what did you think was in the box before you opened it ? in the frame control condition , children initially saw the band - aid box with the lid open and were asked , what do you think is inside the box ? all answered , crayons . before you thought there were crayons inside the box , what do you and i know is inside ? since the child had seen the actual contents , there was no false belief and the correct answer was the same contents as mentioned in the test question , i.e. , crayons . but if children were guided by an opposites strategy , they should not say crayons . children 's responses to the test questions in task 1 to 3 were scored online by the experimenter as correct or incorrect . the analyses were done on the number of correct answers . because there were two scenarios within each condition , the maximum number of correct answers in each condition is 2 . preliminary analyses showed no effects of task , order , gender , or age ( split at mean age ) . as predicted , children 's performance improved significantly on all three tasks when the conversational context of the test question was made clear . mean number of correct answers out of 2 to test questions in task 1 to 3 . on the location false belief task , children in the emphasized context condition gave 1.17 correct answers ( 59% ) compared to 0.44 correct answers ( 22% ) in the standard condition , t(34 ) = 2.43 , p = 0.02 . even under the complex verbal circumstances in the emphasized context condition , children performed better than chance , set at 33% , t(17 ) = 2.18 , p = 0.04 . on the contents and representational change false belief tasks , children in the emphasized context condition gave 1.25 ( 63% ) and 1.40 ( 70% ) correct answers , respectively , compared to 0.08 ( 4% ) and 0.40 ( 20% ) , respectively , in the standard condition . both differences were significant at t(22 ) = 4.01 , p = 0.001 and t(28 ) = 3.49 , p = 0.002 , respectively . children 's performance in the emphasized context condition was better than chance , set at 50% , on the representational change task , t - one - tailed(14 ) = 1.87 , p = 0.04 , but not on the contents false belief task . this may be due to the fact that children 's performance in the standard conditions of these two tasks was already low . it was below chance , t(14 ) = 3.15 , p = 0.007 , and t(11 ) = 11.00 , p = 0.001 , respectively , which was not the case for the location false belief task . additionally , correct performance in the standard condition of all three tasks was generally on the low side of the average proportion of correct answers in wellman et al . children 's performance nevertheless fell within the range of the results that were included in the meta - analysis . overall , it is impressive that younger children performed better than the standard condition on all three tasks and above chance on two of the three tasks ( location and representational change ) , given the minimal modifications to the inherently pragmatically infelicitous standard tasks . despite there being only two answer options on the contents and representational change tasks , children 's performance is likely not attributable to a low - level opposites strategy . this is partly because children nearly aced the control condition at an average of 1.84 correct answers out of 2 across the three tasks , despite a question frame that might plausibly elicit an opposites answer . in planned contrasts , this differed significantly from the above - mentioned chance levels for each task , t(42 ) = 24.92 , p = 0.001 . thus , children are likely not guided by a conceivable tendency to automatically choose the opposite answer of the one mentioned in the test question . however , the frame control condition was not intended to stand alone but to be considered within the overall pattern of results . the quite similar location false belief task had not just two but three answer options , between which children still choose correctly . furthermore , on all three tasks children gave an average of 36% incorrect answers in the enhanced context condition which they would be unlikely to do on an automatic opposites interpretation . standard false belief tasks may mislead young children because questions about other people 's thoughts on reality ( including their intended actions which reflect their thoughts ) are a perfectly fine way to obtain indirect knowledge about reality in certain contexts such as the ordinary context , e.g. , i 'm looking for the chocolate . where does your mom think it is ? such questions require answering with the truth , not beliefs known to be false . if children assume the ordinary context on the false belief task , it places them in a pragmatic bind because they know the truth about the location better than the absent , third person . it would be awkward not to say the truth , so young children do , and fail the standard task . young children may assume the ordinary context , perhaps even by default , for a number of reasons . first and foremost , the test question expresses ignorance on the part of the experimenter if interpreted as an honest question , which young children have been argued to do on a number of standard developmental tasks ( e.g. , siegal and peterson , 1994 ) . the perception of an honest question indicates the ordinary context rather than the conditional context . second , the ordinary context of looking for knowledge about reality is supported by the follow - the - ball atmosphere of the test scenario , especially in the case of the location false belief task . finally , the ordinary context depends on fewer inferences . the analysis of the discourse of the false belief task suggested that interpreting the test question as it is meant requires complex pragmatic inferences . crucially , children must consider how the state of mind of the experimenter is factored into the discourse . in order to understand the test question correctly , children must integrate into their interpretation that the experimenter already knows the location , here called the conditional context . he can therefore not intend to gain indirect knowledge about it from the child , because it would violate normal rules of conversation to ask about something he already knows . to help young children achieve better integration of the discourse of the false belief task , the experimenter in the present study clearly stated that he was already aware of the location as part of the test question . with this slight modification to the discourse , 3-year - olds were able to pass three false belief tasks , while failing the standard versions . because the intervention was minimal no new information was added and the structure of the task was unchanged these results are quite strong evidence that contrary to current theories ( e.g. , perner , 1991 ; gopnik and wellman , 1994 ) , at least older 3-year - olds have the cognitive foundation needed to understand beliefs , which is being masked by the standard false belief tasks because they rely on context - dependent mental - state verbs . in contrast , clearly non - reality - based mental - state verbs such as pretend , dream , and fantasize do not semantically lend themselves to interpretations as requests for indirect knowledge ( see morgan , 1969 ; kempson , 1975 ) . indeed , young children pass some false belief tasks couched in terms of pretense ( flavell et al . , 1987 ; the discourse - based account then solves the puzzle of why even 1-year - olds seem to understand pretend play , which implicates some understanding of mental representation , and pass new nonverbal tests , while young children still fail standard false belief tasks . a remaining challenge is to account for the developmental change that enables children above 4 years to pass the standard false belief tasks . the results hint that the core problem for younger children may lie in understanding the pragmatics of how and when to connect current and preceding discourse , in particular factoring the common ground of shared knowledge into their interpretation of the test question . older children may have achieved a better understanding that the experimenter 's seemingly honest question does not negate his previous indication that he already knows the truth . in contrast , younger children may not realize that this shared information is sustained throughout a conversation and even trumps contradictory pragmatic indicators . two sources of evidence point to a related development between 3 and 5 years of age in children 's ability to sustain preceding pragmatic information when interpreting ambiguous statements . in one study , avrutin and coopmans ( 2000 ) investigated which inferences 3- to 5-year - old children made to bridge an ambiguous statement with preceding utterances . for example , children saw a picture of a boy with red pants and a girl with green pants . children 's task was to judge whether the puppet 's sentence was true or false . five - year - olds would bridge the definite reference back to the boy and conclude that the sentence was false , whereas 3-year - olds identified the sentence as true or false at chance . in subsequent studies , the authors found that it took only a slight re - arrangement of the sentence to increase the performance of 3-year - olds . making the topic of the initial phrase more salient by moving it to the beginning of the sentence , e.g. , a boy is eating . the pants are green , helped most children correctly identify the sentence as false in relation to the picture . another set of studies investigated whether children used the implicit pragmatic suggestions of the discourse setting to infer the topic of a conversation ( shatz , 1978 ; bacharach and luszcz , 1979 ; allen , 1991 ) . here , 3- to 5-year - olds saw pictures that invited comments on an aspect of the picture . for example , bacharach and luszcz ( 1979 ) , would preface a picture with , horses can run . children 's answers showed that 5-year - olds , but not 3-year - olds , matched the action setting with action answers and the information setting with information answers . while the studies of bridging inferences and referential ambiguity are not direct analogues to the current account of the false belief task , the results give some reason to think that younger children may in fact struggle with making the required links to preceding discourse which older children more readily accomplish . more basic cognitive achievements such as general information processing abilities may contribute to this pragmatic development . increased processing capacity and an improved ability to inhibit knowledge about current information in favor of more distant information have previously been found to correlate with the standard false belief task ( frye et al . , 1995 ; german and leslie , 2000 ; carlson and moses , 2001 ; andrews et al . , 2003 ) . while pragmatic inferences require understanding people 's communicative intentions and thus can be described in theory - of - mind terms , the present results do not support the radical change in children 's theory - of - mind at the late age of 4 years proposed by current theories . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

whether young children understand that others may hold false beliefs is a hotly debated topic in psychology and neuroscience . much evidence suggests that children do not pass this milestone in their understanding of other people until the age of 5 years . other evidence suggests that they understand already in their second year . this study proposes a novel account of the logic of conversations about certain mental states . by modifying the discourse accordingly , children passed three false belief tasks at 3 years of age while they failed standard false belief tasks . the results support the view that even young children construe other people in adult - like psychological terms .

Introduction Materials and Methods Participants Design and materials Procedure Results Discussion Conflict of Interest Statement

children 's understanding that people may hold and act on false beliefs , such as looking for the keys in the wrong place , is a milestone in their development towards an adult - like theory - of - mind . to attribute a false belief to someone , children must understand that there can be representations of the world that differ from their own accurate understanding ( dennett , 1978 ) . thus , false belief tasks are benchmarks of children 's acquisition of a representational theory - of - mind , without which they are left unable to understand mistaken expectations , misguided actions , or misleading appearances . even more striking is that children as young as 15 months reveal a tacit understanding of false belief on new nonverbal tasks ( clements and perner , 1994 ; garnham and ruffman , 2001 ; onishi and baillargeon , 2005 ) . nevertheless , overwhelming confirmation of the original results in hundreds of false belief studies across many cultures and languages has generally led to the conclusion that children do not develop the cognitive foundation required to understand mental representation until their fifth year ( perner , 1991 ; gopnik and wellman , 1994 ; wellman et al . i propose an alternative account based on the conversational logic involved in talking about certain mental states . if children make this interpretation on false belief tasks , everyday conversational logic directs them to respond with the true location rather than with jill 's false belief . , young children may not be sufficiently sensitive to the context to invoke precisely the meaning that is the premise of the false belief task . people 's false beliefs are uninformative when the common purpose of the conversation is to discover the true location . but the point of the false belief task is that children know better than jill . so if they wish to cooperate with the presumed request for the likely location and avoid violating the maxim of quantity , children must skip the uninformative step of jill 's false belief and respond with reality . it can be argued that children should already be aware of the conditional context . first , the pragmatic implication of asking an honest question is lack of knowledge on the part of the experimenter , which for young children may override any shared knowledge and indicate the ordinary context . the current discourse - based account explicates the conversational logic of certain mental - state expressions which is a novel extension of this more general view . second , the shell game - like atmosphere of the false belief task , in particular the location task , may suggest to young children that the context remains one of following the ball . while these proposals support the context - dependent aspects of the discourse - based account in a general manner , it is important to note that to date , the fields of linguistics and philosophy are grappling with the issue of factivity . to properly cover children 's understanding of the test questions on a variety of theory - of - mind tasks , such an account must not only include mental - state verbs that take that complements , but all types of expressions used on false belief tasks and appearance - reality tasks such as thinks , says , looks for , and looks like . from a developmental point of view , abbeduto and rosenberg ( 1985 ) they showed that before the age of 4 years , children treat non - factives as factives . the results confirm that the interpretation of thinks and believes may be difficult for 3-year - olds under circumstances other than the false belief task , but the study is nevertheless of limited relevance to the current account . at test , children below 4 years of age typically fail , strikingly saying that the object not only is a sponge but also looks like a sponge . for deceptive objects , answering with appearance , children must understand that the test question is embedded in the conditional context that both speakers already share knowledge of the identity of the object , which frees the child to answer with appearance . on three appearance - reality tasks that emphasized the conditional context , even 3-year - old children gave nearly all - correct appearance responses while they failed the standard versions of the tasks ( hansen and markman , 2005 ) . young children should be similarly successful on false belief tasks that emphasize the conditional context . this hypothesis was tested on three major false belief tasks : ( 1 ) a location false belief task ; ( 2 ) a contents false belief task ; and ( 3 ) a representational change false belief task . compared to standard versions of each task , young children should do better in the emphasized context condition and thus demonstrate their knowledge of false belief . there were three types of false belief tasks : location ( n = 54 ) , contents ( n = 36 ) , and representational change ( n = 45 ) . because of the relative similarity of tasks , conditions , and scenarios , children did not participate in all three types of tasks or all three types of conditions . this tendency might be enhanced by the fact that the two mental - state words think and know may be somewhat difficult to distinguish for children of 3 to 5 years of age ( for a review , see papafragou et al . there were three types of false belief tasks : location ( n = 54 ) , contents ( n = 36 ) , and representational change ( n = 45 ) . because of the relative similarity of tasks , conditions , and scenarios , children did not participate in all three types of tasks or all three types of conditions . this tendency might be enhanced by the fact that the two mental - state words think and know may be somewhat difficult to distinguish for children of 3 to 5 years of age ( for a review , see papafragou et al . the introduction is the same as in the previous task , but after the experimenter closes the band - aid box , children in the standard condition are asked , seemingly unaware of their own previous false belief . to ensure that children did not misread the context as one of searching for indirect knowledge in the way of a past , but presumably still valid , belief , children in the emphasized context condition were therefore asked , you and i know there are crayons inside , what did you think was in the box before you opened it ? on the location false belief task , children in the emphasized context condition gave 1.17 correct answers ( 59% ) compared to 0.44 correct answers ( 22% ) in the standard condition , t(34 ) = 2.43 , p = 0.02 . on the contents and representational change false belief tasks , children in the emphasized context condition gave 1.25 ( 63% ) and 1.40 ( 70% ) correct answers , respectively , compared to 0.08 ( 4% ) and 0.40 ( 20% ) , respectively , in the standard condition . children 's performance nevertheless fell within the range of the results that were included in the meta - analysis . standard false belief tasks may mislead young children because questions about other people 's thoughts on reality ( including their intended actions which reflect their thoughts ) are a perfectly fine way to obtain indirect knowledge about reality in certain contexts such as the ordinary context , e.g. it would be awkward not to say the truth , so young children do , and fail the standard task . first and foremost , the test question expresses ignorance on the part of the experimenter if interpreted as an honest question , which young children have been argued to do on a number of standard developmental tasks ( e.g. the analysis of the discourse of the false belief task suggested that interpreting the test question as it is meant requires complex pragmatic inferences . crucially , children must consider how the state of mind of the experimenter is factored into the discourse . to help young children achieve better integration of the discourse of the false belief task , the experimenter in the present study clearly stated that he was already aware of the location as part of the test question . with this slight modification to the discourse , 3-year - olds were able to pass three false belief tasks , while failing the standard versions . , perner , 1991 ; gopnik and wellman , 1994 ) , at least older 3-year - olds have the cognitive foundation needed to understand beliefs , which is being masked by the standard false belief tasks because they rely on context - dependent mental - state verbs . indeed , young children pass some false belief tasks couched in terms of pretense ( flavell et al . , 1987 ; the discourse - based account then solves the puzzle of why even 1-year - olds seem to understand pretend play , which implicates some understanding of mental representation , and pass new nonverbal tests , while young children still fail standard false belief tasks . a remaining challenge is to account for the developmental change that enables children above 4 years to pass the standard false belief tasks . two sources of evidence point to a related development between 3 and 5 years of age in children 's ability to sustain preceding pragmatic information when interpreting ambiguous statements . while the studies of bridging inferences and referential ambiguity are not direct analogues to the current account of the false belief task , the results give some reason to think that younger children may in fact struggle with making the required links to preceding discourse which older children more readily accomplish . while pragmatic inferences require understanding people 's communicative intentions and thus can be described in theory - of - mind terms , the present results do not support the radical change in children 's theory - of - mind at the late age of 4 years proposed by current theories .

models describing the development rates of blowfly larvae are the primary tools used by forensic entomologists to estimate a minimum time of death or minimum post - mortem interval ( pmimin ) in cases of suspicious death . there are numerous biotic factors that may affect the development rates of blowfly larvae , and an understanding of these effects is necessary in order to make accurate corrections to a resulting pmimin estimate [ 2 , 8 , 12 ] . one such factor is the effect of diet on the development of immature blowflies and this has been the subject of several recent studies [ 57 , 9 , 10 ] . the study by kaneshrajah and turner was the first significant contribution to this area and reported differences in the development of calliphora vicina larvae of up to 2 days , after the first 5 days of development at 20 c , when larvae reared on pig 's liver were compared to those reared on pig 's brain , heart , kidney or lung tissue . several notable studies in 2006 looked at a range of diets including the effects of : different organs of one species of animal ; the same organ of different animals ; different treatments of one organ type , i.e. frozen / thawed vs fresh ; and larval densities on a range of different food sources . all these studies have added significant insight into the effects of blowfly larval diet on development , but more work is needed . it is well documented that blowflies colonise a body soon after death and it is for this reason that they are a good tool to use to estimate pmimin [ 1 , 4 , 11 ] . however , these insects may not always feed on fresh tissue , e.g. in cases where access to a body is delayed , or second and subsequent waves of insect colonisers are used to establish a timeline of events in a case . in these scenarios , the insects will be feeding on decomposed tissue which may have a reduced nutritional value . if this is the case , then it is likely that the development will be retarded , and failure to account for this may have a significant effect on resultant estimates . this paper investigates the differences in development rates of c. vicina larvae reared on fresh minced pig 's liver and whole pig 's liver that was either fresh , thawed from frozen or decomposed . adult c. vicina larvae were trapped in the wildlife garden of the natural history museum and then maintained on sugar and water ad libitum at 21 c ( 2 c ) . they were fed 24 ml of blood each day for 10 days , after which , they were provided with a 250-g pork chop as an oviposition medium . within 1 h , numerous eggs were laid , which hatched within 24 h. fresh pig 's liver ( 20 g ) was placed into each of ten plastic cups ( 250 ml ) . these cups were then placed into a lms ( series a1 80 l ) incubator set at 23 c ( mean = 23.2 c ; n = 673 ; sd = 0.2 ) . five larvae , one from each of five cups , were randomly sampled every 6 h for the first 84 h , after which , five larvae were sampled once a day until pupariation . larvae were killed by immersing them in boiling water for at least 30 s , after which , the length and instar were recorded at each sampling event using a dinolight pro2 digital microscope and a wild m5 light microscope , respectively . approximately 5 cm depth of dampened soil was added into each experimental cup , as a pupariation medium , once larvae started showing signs of entering the wandering phase , e.g. emptying of the crop and/or regularly crawling up the sides of the experimental cups . the food and larvae of each cup were placed in a petri dish , in the experimental cup , on top of the soil . half of the experimental cups were used to continue sampling larval development , in the manner described above , while the other half of the experimental cups were used to assess pupariation and eclosion . this was achieved by counting the number of larvae , pupae and eclosed adults in one experimental cup at each sampling event . this sampling protocol limits the summary statistic for developmental events data to median data rather than average data . the average time taken to reach maximum larval size , the median times taken to reach first ecdysis and second ecdysis , the onset of pupariation and the onset of eclosion were calculated . these data and those detailing the development rates of larvae ( calculated from length data ) were analysed in a krusal wallis ( one - way ) anova using the statistical software package statistica 9 . the procedure described above was replicated three times , and the whole experiment was repeated using frozen / thawed pig 's liver , fresh minced pig 's liver and pig 's liver decomposed for 7 days ( at 21 2 c ) . gases generated by the decomposition processes caused the packaging to balloon to its maximum possible size by day 7 . there was no significant difference between the three replicates within treatments for all four diet treatments , i.e. fresh whole liver ( f = 1.32 ; degrees of freedom ( df ) = 30 ; p > 0.13 ) , fresh minced liver ( f = 1.02 ; df = 30 ; p > 0.45 ) , frozen / thawed liver ( f = 1.77 ; df = 30 ; p > 0.12 ) and decomposed liver ( f = 1.08 ; df = 34 ; p > 0.37 ) . therefore , all replicates within treatments were combined ( n = 15 for each sample time per diet treatment ) and used to test significant differences between treatments . there was a significant difference in the duration of development for larvae reared on different diets ( f = 38.1 ; df = 45 ; p > 0.00 ) . a tukey post - hoc test indicated that larval development was significantly slower on decomposed liver than on the other three diets . this significant difference amounted to as much as 30 h of development or 55.6 % of total development time for mid - sized larvae ( approx . 7 mm long ) , but was a consistent difference of 1 day for feeding larvae that were 2 days or older . no significant differences in length data were observed between the other three diets , similar to the findings of day and wallman ( fig . 1larval development of c. vicina on fresh whole ( empty circle ) , frozen / thawed ( filled square ) , fresh minced ( filled diamond ) and decomposed ( for 7 days ) ( empty triangle ) pig 's liver up to 126 h. error bars represent + 1 standard deviation . the filled and empty arrows on the graph represent the timing of the first and second ecdysis respectively for larvae reared on the ( a ) fresh whole liver , ( b ) frozen / thawed liver , ( c ) fresh minced liver and ( d ) decomposed liver larval development of c. vicina on fresh whole ( empty circle ) , frozen / thawed ( filled square ) , fresh minced ( filled diamond ) and decomposed ( for 7 days ) ( empty triangle ) pig 's liver up to 126 h. error bars represent + 1 standard deviation . the filled and empty arrows on the graph represent the timing of the first and second ecdysis respectively for larvae reared on the ( a ) fresh whole liver , ( b ) frozen / thawed liver , ( c ) fresh minced liver and ( d ) decomposed liver the development rates of larvae ( derived from length data ) up to second ecdysis were very similar for larvae reared on whole fresh liver ( slope = 0.19 ) , fresh minced liver ( slope = 0.18 ) and frozen / thawed liver ( slope = 0.17 ) ( fig . 1 ) ( although those reared on fresh liver were statistically significantly faster ( f3,8 = 66.61 ; p < 0.02 ) than those reared on frozen / thawed liver , we consider this difference biologically insignificant because they all reached second ecdysis within 5 h of one another ( table 1 ) ) . however , larvae , up to second ecdysis , reared on decomposed liver ( slope = 0.11 ) developed statistically and biologically significantly slower ( f3,8 = 66.61 ; p < 0.00 ) than those reared on the other diets ( fig . 1 ) . on the other hand , the development rates of feeding third instar larvae , larger than 8 mm , reared on all diets were not significantly different ( f3,8 = 2.02 ; p > 0.18 ) from one another : whole fresh liver ( slope = 0.28 ) , fresh minced liver ( slope = 0.29 ) , frozen / thawed liver ( slope = 0.28 ) and on decomposed liver ( slope = 0.28 ) ( fig . 1).table 1accumulative median times taken for development of c. vicina to five developmental eventsdevelopmental eventdevelopment time ( h ) on different dietsfresh wholefrozen / thawedfresh minceddecomposedfirst ecdysis13101010second ecdysis33283140maximum length708484112pupariation130143140201eclosion397430422455accumulative median times are the time for 50 % of the experimental population to reach a developmental event ; durations to all developmental events exclude egg incubation ( n = 15 ; pooled data from three replicates of five samples each ) . significant difference within rows ( p < 0.05 ) was calculated using one - way anova and denoted by superscript letters accordingly accumulative median times taken for development of c. vicina to five developmental events accumulative median times are the time for 50 % of the experimental population to reach a developmental event ; durations to all developmental events exclude egg incubation ( n = 15 ; pooled data from three replicates of five samples each ) . significant difference within rows ( p < 0.05 ) was calculated using one - way anova and denoted by superscript letters accordingly these data suggest that first , second and early third instar larvae are the larval feeding stages that are adversely affected when reared on decomposed liver , while larvae larger than 8 mm have the ability to develop equally well on decomposed liver as on all other diets tested . a reason for this might be because larger ( i.e. oldest ) larvae are the most likely to be naturally exposed to decomposed diets and are perhaps best adapted to tolerate them . larvae reared on the four different diets reached first ecdysis within 3 h of one another . larvae reared on fresh whole , frozen / thawed and minced liver reached second ecdysis within 5 h of one another , while those reared on decomposed liver took significantly longer ( another 7 h ) ( f3,8 = 7.88 ; p < 0.00 ) . development times to reach the maximum larval length were the same for larvae reared on frozen / thawed and minced liver and differed by only 3 h ( 2.1 % of total development time ) and 8 h ( 1.9 % ) for pupariation and eclosion . on the other hand , larvae developing on whole fresh liver were significantly ( 14.3 and 7.6 % ) quicker than larvae reared on other food sources in reaching the maximum larval length ( f3,8 = 310.33 ; p < 0.00 ) and eclosion ( f3,8 = 27.22 ; p < 0.00 ) , respectively . larvae reared on decomposed liver took significantly longer to reach these developmental landmarks , including pupariation ( f3,8 = 161.56 ; p < 0.00 ) , when compared to those reared on the other diets ( table 1 ) . in particular , larvae reared on decomposed liver took 71 h ( 35.0 % ) and 58 h ( 14.6 % ) longer , respectively , to reach pupariation and eclosion when compared to those reared on whole fresh liver ( table 1 ) . the duration of second instar ( f3,8 = 7.41 ; p < 0.01 ) , feeding third instar ( f3,8 = 53.53 ; p < 0.00 ) and the wandering phase ( f3,8 = 50.58 ; p < 0.00 ) were all significantly longer for larvae reared on decomposed liver than the other three diets tested . however , the duration of pupariation ( f3,8 = 24.76 ; p < 0.00 ) for larvae reared on decomposed liver was significantly shorter than those larvae reared on fresh minced liver ( p < 0.00 ) and frozen / thawed liver ( p < 0.00 ) , but not significantly different from those reared on whole fresh liver ( p > 0.05 ) ( table 2).table 2median duration of each developmental stage for c. vicina ( n = 15 ; pooled data from three replicates of five samples each)developmental stagedevelopment time ( h ) on different dietsfresh wholefrozen / thawedfresh minceddecomposedfirst instar13101010second instar20182130feeding third instar37565372wandering phase60595689pupariation267287282254significant difference within rows ( p < 0.05 ) calculated by one - way anova and denoted by superscript letters accordingly median duration of each developmental stage for c. vicina ( n = 15 ; pooled data from three replicates of five samples each ) significant difference within rows ( p < 0.05 ) calculated by one - way anova and denoted by superscript letters accordingly the reason for a lack of effect of decomposed liver on the duration of pupariation may be due to the fact that metamorphosis is not regulated by diet , as the larvae had already finished feeding , but rather by other developmental process . why the post - feeding larvae were affected after a diet of decomposed liver is presently unknown there was a significant difference in the duration of development for larvae reared on different diets ( f = 38.1 ; df = 45 ; p > 0.00 ) . a tukey post - hoc test indicated that larval development was significantly slower on decomposed liver than on the other three diets . this significant difference amounted to as much as 30 h of development or 55.6 % of total development time for mid - sized larvae ( approx . 7 mm long ) , but was a consistent difference of 1 day for feeding larvae that were 2 days or older . no significant differences in length data were observed between the other three diets , similar to the findings of day and wallman ( fig . 1larval development of c. vicina on fresh whole ( empty circle ) , frozen / thawed ( filled square ) , fresh minced ( filled diamond ) and decomposed ( for 7 days ) ( empty triangle ) pig 's liver up to 126 h. error bars represent + 1 standard deviation . the filled and empty arrows on the graph represent the timing of the first and second ecdysis respectively for larvae reared on the ( a ) fresh whole liver , ( b ) frozen / thawed liver , ( c ) fresh minced liver and ( d ) decomposed liver larval development of c. vicina on fresh whole ( empty circle ) , frozen / thawed ( filled square ) , fresh minced ( filled diamond ) and decomposed ( for 7 days ) ( empty triangle ) pig 's liver up to 126 h. error bars represent + 1 standard deviation . the filled and empty arrows on the graph represent the timing of the first and second ecdysis respectively for larvae reared on the ( a ) fresh whole liver , ( b ) frozen / thawed liver , ( c ) fresh minced liver and ( d ) decomposed liver the development rates of larvae ( derived from length data ) up to second ecdysis were very similar for larvae reared on whole fresh liver ( slope = 0.19 ) , fresh minced liver ( slope = 0.18 ) and frozen / thawed liver ( slope = 0.17 ) ( fig . 1 ) ( although those reared on fresh liver were statistically significantly faster ( f3,8 = 66.61 ; p < 0.02 ) than those reared on frozen / thawed liver , we consider this difference biologically insignificant because they all reached second ecdysis within 5 h of one another ( table 1 ) ) . however , larvae , up to second ecdysis , reared on decomposed liver ( slope = 0.11 ) developed statistically and biologically significantly slower ( f3,8 = 66.61 ; p < 0.00 ) than those reared on the other diets ( fig . 1 ) . on the other hand , the development rates of feeding third instar larvae , larger than 8 mm , reared on all diets were not significantly different ( f3,8 = 2.02 ; p > 0.18 ) from one another : whole fresh liver ( slope = 0.28 ) , fresh minced liver ( slope = 0.29 ) , frozen / thawed liver ( slope = 0.28 ) and on decomposed liver ( slope = 0.28 ) ( fig . 1).table 1accumulative median times taken for development of c. vicina to five developmental eventsdevelopmental eventdevelopment time ( h ) on different dietsfresh wholefrozen / thawedfresh minceddecomposedfirst ecdysis13101010second ecdysis33283140maximum length708484112pupariation130143140201eclosion397430422455accumulative median times are the time for 50 % of the experimental population to reach a developmental event ; durations to all developmental events exclude egg incubation ( n = 15 ; pooled data from three replicates of five samples each ) . significant difference within rows ( p < 0.05 ) was calculated using one - way anova and denoted by superscript letters accordingly accumulative median times taken for development of c. vicina to five developmental events accumulative median times are the time for 50 % of the experimental population to reach a developmental event ; durations to all developmental events exclude egg incubation ( n = 15 ; pooled data from three replicates of five samples each ) . significant difference within rows ( p < 0.05 ) was calculated using one - way anova and denoted by superscript letters accordingly these data suggest that first , second and early third instar larvae are the larval feeding stages that are adversely affected when reared on decomposed liver , while larvae larger than 8 mm have the ability to develop equally well on decomposed liver as on all other diets tested . a reason for this might be because larger ( i.e. oldest ) larvae are the most likely to be naturally exposed to decomposed diets and are perhaps best adapted to tolerate them . larvae reared on the four different diets reached first ecdysis within 3 h of one another . larvae reared on fresh whole , frozen / thawed and minced liver reached second ecdysis within 5 h of one another , while those reared on decomposed liver took significantly longer ( another 7 h ) ( f3,8 = 7.88 ; p < 0.00 ) . development times to reach the maximum larval length were the same for larvae reared on frozen / thawed and minced liver and differed by only 3 h ( 2.1 % of total development time ) and 8 h ( 1.9 % ) for pupariation and eclosion . on the other hand , larvae developing on whole fresh liver were significantly ( 14.3 and 7.6 % ) quicker than larvae reared on other food sources in reaching the maximum larval length ( f3,8 = 310.33 ; p < 0.00 ) and eclosion ( f3,8 = 27.22 ; p < 0.00 ) , respectively . larvae reared on decomposed liver took significantly longer to reach these developmental landmarks , including pupariation ( f3,8 = 161.56 ; p < 0.00 ) , when compared to those reared on the other diets ( table 1 ) . in particular , larvae reared on decomposed liver took 71 h ( 35.0 % ) and 58 h ( 14.6 % ) longer , respectively , to reach pupariation and eclosion when compared to those reared on whole fresh liver ( table 1 ) . the duration of second instar ( f3,8 = 7.41 ; p < 0.01 ) , feeding third instar ( f3,8 = 53.53 ; p < 0.00 ) and the wandering phase ( f3,8 = 50.58 ; p < 0.00 ) were all significantly longer for larvae reared on decomposed liver than the other three diets tested . however , the duration of pupariation ( f3,8 = 24.76 ; p < 0.00 ) for larvae reared on decomposed liver was significantly shorter than those larvae reared on fresh minced liver ( p < 0.00 ) and frozen / thawed liver ( p < 0.00 ) , but not significantly different from those reared on whole fresh liver ( p > 0.05 ) ( table 2).table 2median duration of each developmental stage for c. vicina ( n = 15 ; pooled data from three replicates of five samples each)developmental stagedevelopment time ( h ) on different dietsfresh wholefrozen / thawedfresh minceddecomposedfirst instar13101010second instar20182130feeding third instar37565372wandering phase60595689pupariation267287282254significant difference within rows ( p < 0.05 ) calculated by one - way anova and denoted by superscript letters accordingly median duration of each developmental stage for c. vicina ( n = 15 ; pooled data from three replicates of five samples each ) significant difference within rows ( p < 0.05 ) calculated by one - way anova and denoted by superscript letters accordingly the reason for a lack of effect of decomposed liver on the duration of pupariation may be due to the fact that metamorphosis is not regulated by diet , as the larvae had already finished feeding , but rather by other developmental process . why the post - feeding larvae were affected after a diet of decomposed liver is presently unknown decomposed liver had a significantly adverse effect on the development rate of c. vicina , when compared to the development on fresh whole liver , frozen / thawed liver and minced liver . it is important to note that , in most cases , these differences in rates of development will be unlikely to affect estimates of pmimin , because larvae or other stages ( i.e. the oldest specimens ) used to estimate pmimin will be developing on fresh bodies due to the promptness with which adult parent blowflies can locate a dead body . however , estimates in cases where there was a significant delay in colonisation of the body or those used to establish a timeline of events , e.g. the date of relocation of a badly decomposed body , could result in an over estimate of up to 30 h ( 55.6 % ) if using larvae and up to 71 h ( 35.0 % ) and 58 h ( 14.6 % ) if using times to the onset of pupariation and eclosion , respectively . it is assumed that the decomposition of pig 's liver in this study was primarily due to anaerobic bacteria residing in the liver due to the sealed packaging in which the liver was stored . it is possible that decomposition by aerobic bacteria might have a different effect on the development rates of blowflies , but this was outside the scope of this study . this is something that could be included in future studies , as well as assessing the effects of different stages of decomposition on the development of blowflies and other forensically important insects and determining what factors in decomposed liver are responsible for the observed effects . larvae reared on fresh liver required the least amount of time to reach maximum larval length , pupariation and eclosion than those larvae reared on all other diets . therefore , altering the food substance that blowfly larvae feed on , by either bacterial ( represented by the decomposed treatment ) or mechanical means ( represented by the minced and frozen / thawed treatment ) appears to have a negative effect of development .

the development rate of immature calliphora vicina reared on decomposed liver was significantly slower , by as much as 30 h ( 55.4 % of total development time ) for mid - sized larvae , and 71 h ( 35.0 % ) and 58 h ( 14.6 % ) if using times to the onset of pupariation and eclosion , respectively , than those of immatures that developed on fresh whole pig 's liver . development rates of larvae reared on decomposed liver were also slower than those of larvae reared on minced pig 's liver and frozen / thawed pig 's liver . these results suggest that any estimate of minimum post - mortem interval may result in an over estimate if the blowflies used were developing on an already decomposed body .

Introduction Materials and methods Results and discussion Differences between length data for larvae reared on different diets Differences between the duration to developmental events for larvae reared on different diets Conclusions

models describing the development rates of blowfly larvae are the primary tools used by forensic entomologists to estimate a minimum time of death or minimum post - mortem interval ( pmimin ) in cases of suspicious death . there are numerous biotic factors that may affect the development rates of blowfly larvae , and an understanding of these effects is necessary in order to make accurate corrections to a resulting pmimin estimate [ 2 , 8 , 12 ] . one such factor is the effect of diet on the development of immature blowflies and this has been the subject of several recent studies [ 57 , 9 , 10 ] . the study by kaneshrajah and turner was the first significant contribution to this area and reported differences in the development of calliphora vicina larvae of up to 2 days , after the first 5 days of development at 20 c , when larvae reared on pig 's liver were compared to those reared on pig 's brain , heart , kidney or lung tissue . frozen / thawed vs fresh ; and larval densities on a range of different food sources . in these scenarios , the insects will be feeding on decomposed tissue which may have a reduced nutritional value . if this is the case , then it is likely that the development will be retarded , and failure to account for this may have a significant effect on resultant estimates . this paper investigates the differences in development rates of c. vicina larvae reared on fresh minced pig 's liver and whole pig 's liver that was either fresh , thawed from frozen or decomposed . within 1 h , numerous eggs were laid , which hatched within 24 h. fresh pig 's liver ( 20 g ) was placed into each of ten plastic cups ( 250 ml ) . half of the experimental cups were used to continue sampling larval development , in the manner described above , while the other half of the experimental cups were used to assess pupariation and eclosion . this was achieved by counting the number of larvae , pupae and eclosed adults in one experimental cup at each sampling event . the average time taken to reach maximum larval size , the median times taken to reach first ecdysis and second ecdysis , the onset of pupariation and the onset of eclosion were calculated . these data and those detailing the development rates of larvae ( calculated from length data ) were analysed in a krusal wallis ( one - way ) anova using the statistical software package statistica 9 . the procedure described above was replicated three times , and the whole experiment was repeated using frozen / thawed pig 's liver , fresh minced pig 's liver and pig 's liver decomposed for 7 days ( at 21 2 c ) . fresh whole liver ( f = 1.32 ; degrees of freedom ( df ) = 30 ; p > 0.13 ) , fresh minced liver ( f = 1.02 ; df = 30 ; p > 0.45 ) , frozen / thawed liver ( f = 1.77 ; df = 30 ; p > 0.12 ) and decomposed liver ( f = 1.08 ; df = 34 ; p > 0.37 ) . there was a significant difference in the duration of development for larvae reared on different diets ( f = 38.1 ; df = 45 ; p > 0.00 ) . a tukey post - hoc test indicated that larval development was significantly slower on decomposed liver than on the other three diets . this significant difference amounted to as much as 30 h of development or 55.6 % of total development time for mid - sized larvae ( approx . 1larval development of c. vicina on fresh whole ( empty circle ) , frozen / thawed ( filled square ) , fresh minced ( filled diamond ) and decomposed ( for 7 days ) ( empty triangle ) pig 's liver up to 126 h. error bars represent + 1 standard deviation . the filled and empty arrows on the graph represent the timing of the first and second ecdysis respectively for larvae reared on the ( a ) fresh whole liver , ( b ) frozen / thawed liver , ( c ) fresh minced liver and ( d ) decomposed liver larval development of c. vicina on fresh whole ( empty circle ) , frozen / thawed ( filled square ) , fresh minced ( filled diamond ) and decomposed ( for 7 days ) ( empty triangle ) pig 's liver up to 126 h. error bars represent + 1 standard deviation . the filled and empty arrows on the graph represent the timing of the first and second ecdysis respectively for larvae reared on the ( a ) fresh whole liver , ( b ) frozen / thawed liver , ( c ) fresh minced liver and ( d ) decomposed liver the development rates of larvae ( derived from length data ) up to second ecdysis were very similar for larvae reared on whole fresh liver ( slope = 0.19 ) , fresh minced liver ( slope = 0.18 ) and frozen / thawed liver ( slope = 0.17 ) ( fig . 1 ) ( although those reared on fresh liver were statistically significantly faster ( f3,8 = 66.61 ; p < 0.02 ) than those reared on frozen / thawed liver , we consider this difference biologically insignificant because they all reached second ecdysis within 5 h of one another ( table 1 ) ) . however , larvae , up to second ecdysis , reared on decomposed liver ( slope = 0.11 ) developed statistically and biologically significantly slower ( f3,8 = 66.61 ; p < 0.00 ) than those reared on the other diets ( fig . on the other hand , the development rates of feeding third instar larvae , larger than 8 mm , reared on all diets were not significantly different ( f3,8 = 2.02 ; p > 0.18 ) from one another : whole fresh liver ( slope = 0.28 ) , fresh minced liver ( slope = 0.29 ) , frozen / thawed liver ( slope = 0.28 ) and on decomposed liver ( slope = 0.28 ) ( fig . significant difference within rows ( p < 0.05 ) was calculated using one - way anova and denoted by superscript letters accordingly these data suggest that first , second and early third instar larvae are the larval feeding stages that are adversely affected when reared on decomposed liver , while larvae larger than 8 mm have the ability to develop equally well on decomposed liver as on all other diets tested . larvae reared on the four different diets reached first ecdysis within 3 h of one another . larvae reared on fresh whole , frozen / thawed and minced liver reached second ecdysis within 5 h of one another , while those reared on decomposed liver took significantly longer ( another 7 h ) ( f3,8 = 7.88 ; p < 0.00 ) . development times to reach the maximum larval length were the same for larvae reared on frozen / thawed and minced liver and differed by only 3 h ( 2.1 % of total development time ) and 8 h ( 1.9 % ) for pupariation and eclosion . on the other hand , larvae developing on whole fresh liver were significantly ( 14.3 and 7.6 % ) quicker than larvae reared on other food sources in reaching the maximum larval length ( f3,8 = 310.33 ; p < 0.00 ) and eclosion ( f3,8 = 27.22 ; p < 0.00 ) , respectively . larvae reared on decomposed liver took significantly longer to reach these developmental landmarks , including pupariation ( f3,8 = 161.56 ; p < 0.00 ) , when compared to those reared on the other diets ( table 1 ) . in particular , larvae reared on decomposed liver took 71 h ( 35.0 % ) and 58 h ( 14.6 % ) longer , respectively , to reach pupariation and eclosion when compared to those reared on whole fresh liver ( table 1 ) . the duration of second instar ( f3,8 = 7.41 ; p < 0.01 ) , feeding third instar ( f3,8 = 53.53 ; p < 0.00 ) and the wandering phase ( f3,8 = 50.58 ; p < 0.00 ) were all significantly longer for larvae reared on decomposed liver than the other three diets tested . however , the duration of pupariation ( f3,8 = 24.76 ; p < 0.00 ) for larvae reared on decomposed liver was significantly shorter than those larvae reared on fresh minced liver ( p < 0.00 ) and frozen / thawed liver ( p < 0.00 ) , but not significantly different from those reared on whole fresh liver ( p > 0.05 ) ( table 2).table 2median duration of each developmental stage for c. vicina ( n = 15 ; pooled data from three replicates of five samples each)developmental stagedevelopment time ( h ) on different dietsfresh wholefrozen / thawedfresh minceddecomposedfirst instar13101010second instar20182130feeding third instar37565372wandering phase60595689pupariation267287282254significant difference within rows ( p < 0.05 ) calculated by one - way anova and denoted by superscript letters accordingly median duration of each developmental stage for c. vicina ( n = 15 ; pooled data from three replicates of five samples each ) significant difference within rows ( p < 0.05 ) calculated by one - way anova and denoted by superscript letters accordingly the reason for a lack of effect of decomposed liver on the duration of pupariation may be due to the fact that metamorphosis is not regulated by diet , as the larvae had already finished feeding , but rather by other developmental process . why the post - feeding larvae were affected after a diet of decomposed liver is presently unknown there was a significant difference in the duration of development for larvae reared on different diets ( f = 38.1 ; df = 45 ; p > 0.00 ) . a tukey post - hoc test indicated that larval development was significantly slower on decomposed liver than on the other three diets . this significant difference amounted to as much as 30 h of development or 55.6 % of total development time for mid - sized larvae ( approx . 1larval development of c. vicina on fresh whole ( empty circle ) , frozen / thawed ( filled square ) , fresh minced ( filled diamond ) and decomposed ( for 7 days ) ( empty triangle ) pig 's liver up to 126 h. error bars represent + 1 standard deviation . the filled and empty arrows on the graph represent the timing of the first and second ecdysis respectively for larvae reared on the ( a ) fresh whole liver , ( b ) frozen / thawed liver , ( c ) fresh minced liver and ( d ) decomposed liver larval development of c. vicina on fresh whole ( empty circle ) , frozen / thawed ( filled square ) , fresh minced ( filled diamond ) and decomposed ( for 7 days ) ( empty triangle ) pig 's liver up to 126 h. error bars represent + 1 standard deviation . the filled and empty arrows on the graph represent the timing of the first and second ecdysis respectively for larvae reared on the ( a ) fresh whole liver , ( b ) frozen / thawed liver , ( c ) fresh minced liver and ( d ) decomposed liver the development rates of larvae ( derived from length data ) up to second ecdysis were very similar for larvae reared on whole fresh liver ( slope = 0.19 ) , fresh minced liver ( slope = 0.18 ) and frozen / thawed liver ( slope = 0.17 ) ( fig . 1 ) ( although those reared on fresh liver were statistically significantly faster ( f3,8 = 66.61 ; p < 0.02 ) than those reared on frozen / thawed liver , we consider this difference biologically insignificant because they all reached second ecdysis within 5 h of one another ( table 1 ) ) . however , larvae , up to second ecdysis , reared on decomposed liver ( slope = 0.11 ) developed statistically and biologically significantly slower ( f3,8 = 66.61 ; p < 0.00 ) than those reared on the other diets ( fig . on the other hand , the development rates of feeding third instar larvae , larger than 8 mm , reared on all diets were not significantly different ( f3,8 = 2.02 ; p > 0.18 ) from one another : whole fresh liver ( slope = 0.28 ) , fresh minced liver ( slope = 0.29 ) , frozen / thawed liver ( slope = 0.28 ) and on decomposed liver ( slope = 0.28 ) ( fig . significant difference within rows ( p < 0.05 ) was calculated using one - way anova and denoted by superscript letters accordingly these data suggest that first , second and early third instar larvae are the larval feeding stages that are adversely affected when reared on decomposed liver , while larvae larger than 8 mm have the ability to develop equally well on decomposed liver as on all other diets tested . larvae reared on the four different diets reached first ecdysis within 3 h of one another . larvae reared on fresh whole , frozen / thawed and minced liver reached second ecdysis within 5 h of one another , while those reared on decomposed liver took significantly longer ( another 7 h ) ( f3,8 = 7.88 ; p < 0.00 ) . development times to reach the maximum larval length were the same for larvae reared on frozen / thawed and minced liver and differed by only 3 h ( 2.1 % of total development time ) and 8 h ( 1.9 % ) for pupariation and eclosion . on the other hand , larvae developing on whole fresh liver were significantly ( 14.3 and 7.6 % ) quicker than larvae reared on other food sources in reaching the maximum larval length ( f3,8 = 310.33 ; p < 0.00 ) and eclosion ( f3,8 = 27.22 ; p < 0.00 ) , respectively . larvae reared on decomposed liver took significantly longer to reach these developmental landmarks , including pupariation ( f3,8 = 161.56 ; p < 0.00 ) , when compared to those reared on the other diets ( table 1 ) . in particular , larvae reared on decomposed liver took 71 h ( 35.0 % ) and 58 h ( 14.6 % ) longer , respectively , to reach pupariation and eclosion when compared to those reared on whole fresh liver ( table 1 ) . the duration of second instar ( f3,8 = 7.41 ; p < 0.01 ) , feeding third instar ( f3,8 = 53.53 ; p < 0.00 ) and the wandering phase ( f3,8 = 50.58 ; p < 0.00 ) were all significantly longer for larvae reared on decomposed liver than the other three diets tested . however , the duration of pupariation ( f3,8 = 24.76 ; p < 0.00 ) for larvae reared on decomposed liver was significantly shorter than those larvae reared on fresh minced liver ( p < 0.00 ) and frozen / thawed liver ( p < 0.00 ) , but not significantly different from those reared on whole fresh liver ( p > 0.05 ) ( table 2).table 2median duration of each developmental stage for c. vicina ( n = 15 ; pooled data from three replicates of five samples each)developmental stagedevelopment time ( h ) on different dietsfresh wholefrozen / thawedfresh minceddecomposedfirst instar13101010second instar20182130feeding third instar37565372wandering phase60595689pupariation267287282254significant difference within rows ( p < 0.05 ) calculated by one - way anova and denoted by superscript letters accordingly median duration of each developmental stage for c. vicina ( n = 15 ; pooled data from three replicates of five samples each ) significant difference within rows ( p < 0.05 ) calculated by one - way anova and denoted by superscript letters accordingly the reason for a lack of effect of decomposed liver on the duration of pupariation may be due to the fact that metamorphosis is not regulated by diet , as the larvae had already finished feeding , but rather by other developmental process . why the post - feeding larvae were affected after a diet of decomposed liver is presently unknown decomposed liver had a significantly adverse effect on the development rate of c. vicina , when compared to the development on fresh whole liver , frozen / thawed liver and minced liver . the oldest specimens ) used to estimate pmimin will be developing on fresh bodies due to the promptness with which adult parent blowflies can locate a dead body . the date of relocation of a badly decomposed body , could result in an over estimate of up to 30 h ( 55.6 % ) if using larvae and up to 71 h ( 35.0 % ) and 58 h ( 14.6 % ) if using times to the onset of pupariation and eclosion , respectively . it is assumed that the decomposition of pig 's liver in this study was primarily due to anaerobic bacteria residing in the liver due to the sealed packaging in which the liver was stored . it is possible that decomposition by aerobic bacteria might have a different effect on the development rates of blowflies , but this was outside the scope of this study . this is something that could be included in future studies , as well as assessing the effects of different stages of decomposition on the development of blowflies and other forensically important insects and determining what factors in decomposed liver are responsible for the observed effects . larvae reared on fresh liver required the least amount of time to reach maximum larval length , pupariation and eclosion than those larvae reared on all other diets . therefore , altering the food substance that blowfly larvae feed on , by either bacterial ( represented by the decomposed treatment ) or mechanical means ( represented by the minced and frozen / thawed treatment ) appears to have a negative effect of development .

acetaldehyde ( ethanal ) is carcinogenic in animal experiments [ 1 , 2 ] and was classified by the international agency for research on cancer ( iarc ) in 1999 as possibly carcinogenic to humans only recently , acetaldehyde in association with alcohol consumption has been upgraded by iarc into group 1 ( i.e. , the highest level of evidence ) as carcinogenic to humans . the carcinogenicity is considered to be caused by a genotoxic mechanism as several acetaldehyde - dna adducts were found in vitro and in vivo [ 59 ] . for this reason , it is currently not possible to suggest a clear threshold or maximum tolerable limit for foods , but the margin of exposure model has to be used for risk assessment [ 1013 ] . this requires to have robust occurrence data for exposure assessment . in foods , acetaldehyde may occur either naturally or because of intentional addition as flavour compound . other sources of human exposure to acetaldehyde may be cosmetic products or environmental exposure from burning fossil fuels , but the major sources are tobacco smoke and exposure from ethanol oxidation following alcoholic beverage consumption [ 11 , 13 , 15 ] . in vivo , acetaldehyde may also be formed endogenously , but especially in the gastrointestinal tract by bacteria that metabolize ethanol or carbohydrates . in foods , the highest concentrations of acetaldehyde were determined in vinegar ( 1.06 g / kg ) , but also in milk products and diverse fruits and vegetables [ 16 , 17 ] . the netherlands organization for applied scientific research ( tno ) database volatile compounds in foods ( vcf ) lists numerous studies on the occurrence of acetaldehyde but most of these were from the 1980s or earlier . an absence of current data about the occurrence of acetaldehyde in food can be noted , so that the aim of this study was to develop a methodology to most efficiently analyze acetaldehyde in a wide variety of food matrices , and also provide an overview on the occurrence in the most susceptible food groups . the most simplistic procedure for acetaldehyde analysis is the direct injection of a sample solution into a gas chromatograph with flame ionization detection ( fid ) . such a procedure can be used for analysis of alcoholic beverages without any further sample preparation and is also included in the eu reference methods for the analysis of spirits . our laboratory had participated in the interlaboratory trial , in which the eu procedure was evaluated for the first time , and has used it since then with success ( as documented by regular participation in international interlaboratory trials ) , and our previous studies on acetaldehyde occurrence in alcoholic beverages were all based on this procedure [ 2126 ] . as we are now interested to analyze other foods ( besides alcoholic beverages ) , the reference procedure for spirits can not be directly applied . the major problem is to obtain sample extracts without losses of the very volatile analyte that can be injected into the gc system . in the literature , several methods were suggested for acetaldehyde analysis in foods , which include photometric , fluorimetric , chromatographic , and enzymatic methods . however , some of the methods are either expensive or lack sensitivity ( enzymatic method ) , include time - consuming sample preparation steps or lack in specificity ( spectrophotometric methods ) . the extraction of acetaldehyde with steam distillation is generally judged as being problematic , as considerable losses may occur ( up to 30% in unpublished studies by the authors ) . furthermore , this procedure is leading to a dilution of the analyte , which may facilitate the need for an additional extraction step [ 28 , 29 ] . ott et al . also stressed the fact that too strong warming during sample preparation must be prohibited , as this leads not only to volatilization but also increases the reactivity of acetaldehyde . a strong heating ( e.g. , in the headspace oven ) may also lead to artefactual formation of acetaldehyde from ethanol , which may explain reports of very high concentrations in early studies . prior to chromatographic measurement of acetaldehyde , derivatization using 2,4-dinitrophenylhydrazine was suggested , and the formed hydrazone can be measured using gas chromatography ( gc ) or high - performance liquid chromatography ( hplc ) . another derivatization reagent ( cysteamine - hcl ) was suggested by miyake and shibamoto , which , however , needs careful adjustment of ph and a further extraction step . a simpler procedure is the analysis of underivatized acetaldehyde using headspace gas chromatography ( hs - gc ) . the headspace injection can be used instead of the liquid injection while all other parameters of the eu reference method can be used . we have previously used such a hs - gc - fid procedure for the analysis of acetaldehyde in saliva samples [ 13 , 34 ] , and this work reports the modifications needed for analyzing all kinds of foodstuffs , including a validation of the procedure . similar to our previous procedure , we use simple static hs injection , as it was previously shown that the dynamic variant ( purge and trap ) is not possible as acetaldehyde is only insufficiently adsorbed into the usual materials ( e.g. , tenax ) . we have set focus on providing a sample preparation without losses as well as an improved acid digestion that simulates physiological conditions of the human stomach and therefore allows to estimate the exposure after oral consumption of foods . sodium chloride was from riedel - de - haen , and pepsin from porcine gastric mucosa ( 8002500 u / mg protein ) was from sigma - aldrich . hydrochloric acid ( 37% ) and ethanol ( > 99.9% ) were obtained from merck . the simulated gastric fluid ( sgf ) was prepared according to usp 32 . to compensate for the dilution , which occurs during sample preparation , the sgf was concentrated by a factor of 4 . for this , 4.0 g of sodium chloride were dissolved in 400 ml of distilled water and 4 ml of hydrochloric acid ( 37% ) were added , and finally 6.4 g of pepsin were added . after complete dissolution of the pepsin , the now slightly yellow coloured solution was filled up with distilled water to 500 ml . to prepare an acetaldehyde stock solution with a concentration of 3.0 g / l , a 100 ml measuring flask is filled with about 80 ml of distilled water , which is temperated at exactly 20c , and 300 mg of acetaldehyde are weighed into the flask . the flask is filled with distilled water to 100 ml and stored at 58c . the solution is stable for not more than 10 days . from this stock solution , the calibration curve was prepared by filling up 20 , 270 , 520 , and 720 l of the stock solution with distilled water in a 10 ml measuring flask ( 5.4240 mg 800 l of stock solution were filled up with distilled water in a 50 ml measuring flask ( 48 mg all standard and spiking solutions were stored in a water bath at 20c till use . for basic calibration , 200 l of the standard solution , 1.25 ml of sgf and 3.55 ml of distilled water were given in a 20 ml headspace vial , which is immediately tightly sealed using a silicon / ptfe septum . the basic calibration was measured at least once weekly to check the performance of the gc system . the sample types were selected according to risk oriented principles based on previously published acetaldehyde contents and the typical food intake in germany . we excluded alcoholic beverages , as this group was previously analyzed in detail . from the 140 products analyzed in total , we focused especially on dairy products ( n = 43 ) , fruits ( n = 37 ) , vegetables ( n = 18 ) , and alcohol - free beverages ( n = 33 ) . the samples were stored at 58c and analyzed in fresh condition , or , for packaged foods , before the expiration of the dependent on consistency , the samples were weighed with help of a 20 ml disposable plastic syringe or using an eppendorf pipette , with a tip that was cut off with scissors to facilitate the pipetting of semisolid samples . for quantification with standard addition , five aliquots ( in the range of 1.22.0 g ) of the sample were weighed with an accuracy of 10 mg into 20 ml headspace vials . after addition of 1.25 ml of sgf and the required acetaldehyde spiking , distilled water up to a total volume of 5 ml was added . solid foods were homogenized in a standard household mixer ( magic maxx , ds - produkte gmbh , gallin ) . for fruits and vegetables only the edible parts the homogenized samples were weighed similar to the liquid foods described above . only completely dry or highly viscous samples were weighed using a spatula . the total time for sample preparation of the solid foods was 1525 min per sample . the prepared headspace vials were stored at 58c and generally analyzed on the same day , but never later than on the next day after preparation . the hs - gc - fid system used for analysis was an agilent model 6890n gas chromatograph in combination with a ctc combi pal autosampler . to simulate the physiological conditions inside the stomach , the samples were incubated for 60 min at 37c under constant stirring in the oven of the autosampler . after that , 500 l of headspace were injected into the gc system at 500 l / sec with a temperature of the transfer syringe of 80c . substances were separated on a capillary column ( db - wax , 58 m 0.32 mm i.d . , temperature program : 30c hold for 8 min , 14c / min up to 200c , hold for 10 min . the temperatures for the injection port and fid were set at 140c and 210c , respectively . splitless injection mode and helium with a flow rate of 2.0 ml / min as carrier gas was used . data acquisition and peak integration were performed using chromeleon 6.8 chromatography data system ( dionex corporation , sunnyvale , usa ) . the data analysis of the standard additions to calculate the concentrations was conducted using valoo 2.3 ( analytik software , leer , germany ) . the standard additions were only evaluated in the case of a correlation coefficient with a minimum of 0.9995 and a coefficient of variation ( cv ) of not more than 3% . per calibration one outlier was tolerated and eliminated . if the criteria were not fulfilled after elimination of this one outlier , the results were discarded and the sample preparation and measurement were repeated from the beginning . the limits of detection and quantification were determined according to german norm 32645 using the calibration curve method . the limits are extrapolated based on the tolerance interval of a calibration curve , which is measured with concentrations surrounding the limits . this method gives more realistic limits than extrapolation from blank measurements ( signal / noise ratios ) . l with equidistant calibrators ( n = 10 ) . the precision ( expressed as coefficient as variation ) can be directly calculated for each sample from the calibration curve resulting from standard addition ( 5 aliquots measured per sample ) . furthermore , we have measured one yoghurt sample several times with different amounts of sample weight ( 1.2 , 1.4 , and 1.6 g ) . the storage stability was evaluated by preparing three standard addition series of the same yoghurt sample and measuring them after 2 , 9 , and 16 days after preparation ( the prepared headspace vials were stored at 58c in the meantime ) . to test for artefactual formation of acetaldehyde from ethanol during sample preparation or analysis , two standard addition series of an apple sample were prepared with and without addition of ethanol ( 250 g ethanol per sample vial ) . possible losses during sample preparation were tested as follows : ( 1 ) 50 ml of an acetaldehyde stock solution in a 100 ml measuring flask was left to stand open ( i.e. , without stopper on the flask ) for 65 min in the 20c water bath ( normally , the flask are directly sealed after the pipetting of the standard , of course ) . ( 2 ) 100 ml of standard solution were filled into the mixer used for homogenization and mixed for 20 s similar to the samples . during initial method development , it was noted that buttermilk did not contain any detectable acetaldehyde in the headspace if aqueous samples are analyzed . after addition of sgf , considerable amounts of acetaldehyde were found , however . to research the influence of sgf on the matrix , light microscopy was conducted ( axiostar plus , camera : axiocam icc1 , carl zeiss gmbh , oberkochen ) . preliminary experiments had shown that the differences in matrix composition have massive influences on the recovery of acetaldehyde in the headspace . for this reason , external calibration with aqueous standards is not possible . due to the diversity of matrices we wanted to analyze , it would also not have been possible to conduct calibration in matrix , with the additional problem of finding acetaldehyde - free matrices for spiking . it was also not possible to find a suitable internal standard with similar behaviour to acetaldehyde , and the use of mass spectrometry with the possibility to use isotopically labelled acetaldehyde was not possible for instrumental restrictions and cost reasons . for all these reasons , we decided to use standard addition according to the german norm 32633:1998 . for this we used 5 aliquots of each sample , from which one was measured without spiking and four were spiked with increasing amounts of acetaldehyde in equidistant concentrations ( generally we spiked 0 , 4 , 8 , 12 , and 16 g per g of sample weight ) . all sample aliquots were filled up to the same volume ( 5 ml ) with water . the selection of 5 aliquots results from a compromise between precision and work efforts , because a smaller number of aliquots would considerably increase the measurement error . while the standard addition sounds to be an incredible amount of work , we nevertheless judged it to be superior to all other methods . first , it must be noted that all steps besides the weighing of the samples are conducted automatically on the autosampler . an extraction using solid - phase extraction or liquid - liquid extraction would have been a similarly large effort for the preparation , with additional problems of loss of the analyte . a further search for an internal standard with same volatility , solubility , reactivity , and headspace behaviour as acetaldehyde was also judged to be pointless , especially as free retention time windows in the complex food matrices are very restricted . the basic calibration using external standards showed an acceptable linearity and precision in the working range ( r = 0.9997 , cv 1.47% ) . the homogeneity of variances shows that the method is equally precise for the whole working range ( figure 1 ) . mg / l , which is in the same order of magnitude as the limits determined in previous research [ 28 , 40 , 41 ] the matrix effect leads to a drastic influence on the slopes of the calibration lines , which necessitates the use of standard addition for quantification . it must be noted that the matrix effect depends on the sample weight , and can be reduced by decreasing the weight . however , this may pose the problem that the response falls below the quantification limit . on the other hand , the sample amount should not be selected too high , because the increase may even result in reduced response , as demonstrated for an apple sample shown in figure 3 . in yoghurt and banana samples , which were analyzed in the same fashion as the apple sample , this effect was not observed , however . with the exception of a single sample of roast coffee powder ( r = 0.9987 , cv 3.28% ) , all samples fulfilled our requirements for precision ( r > 0.9990 , cv < 3% ) . apart from 8 samples ( peas , roast coffee , apple soft drink , orange soft drink , paprika , and 3 bananas ) , we even had r of > 0.9995 and the cvs were typically below 2% . the yoghurt sample that was independently measured for 3 times , had a cv of 2.59% ( average 6.18 mg / kg , standard deviation 0.16 mg / kg ) . only one sample ( a radish ) was not measurable at all , because there was a large interference near the retention time of acetaldehyde , which overlapped its peak and hindered correct integration , probably derived from a glucosinolate or glucosinolate degradation product . ms detection would be required in such cases . during the storage stability experiment , no significant difference in the results was seen between the yoghurt samples stored for 2 , 9 , or 16 days . the overall mean was 16.91 mg / kg ( standard deviation 0.49 mg / kg , cv 2.90% ) . no artefactual formation of acetaldehyde was detected in the apple sample series with spiked ethanol ( 2.39 0.47 mg / kg without ethanol ; 2.35 0.32 mg / kg with ethanol ) . regarding the losses during sample preparation , the highest influence had the storage of the stock solution without stopper ( 4% loss of acetaldehyde during 65 min ) , while during homogenization only a minor loss of 2% occurred . the samples were stored in a fridge and the temperature in the samples during mixing was increased by a maximum of 7c ( in the case of a cheddar cheese ) . the loss during sample preparation of solid matrices in the mixer is therefore deemed as acceptable , but unavoidable . our validation results show that the method has an acceptable performance for the use of analyzing food matrices . due to the volatility of acetaldehyde the regression curves for different spiking levels of acetaldehyde in buttermilk are shown in figure 4 . without the use of sgf , apparently , the matrix needs to be saturated till the excess of acetaldehyde can diffuse into the headspace . interestingly , this phenomenon was only noted for buttermilk , but not for other matrices , for example , yoghurt . macroscopically , both solutions ( with and without sgf ) had the same colour after colouring with schiff reagent . microscopically , the buttermilk without sgf shows large particulate agglomerations , in which the colour is concentrated . if the sample is treated with sgf , the agglomerations disappear and the colour becomes evenly distributed . according to the literature , milk fat globule membranes , which are broken down during buttermilk making , can interact with casein - micelles and form globular aggregates [ 42 , 43 ] . in neutral aqueous solution , these aggregates are stabile and obviously bind acetaldehyde very effectively . in other milk products ( i.e. , yoghurt ) , where the milk fat globule membranes are occurring in intact form , acetaldehyde - binding aggregates are apparently not yet formed . as the sgf preparation adequately frees the acetaldehyde , we have refrained from performing further experiments , while from a scientific standpoint it would be interesting to further study the binding behaviour of acetaldehyde in buttermilk , for example using transmission electron microscopy . the maximum content of foods for direct consumption was found in a yoghurt ( 17.42 mg / kg ) . in food ingredients , 26.3 mg / kg were found in a baking flavour , while an industrial orange flavour contained 1416 mg / kg of acetaldehyde , which was the maximum of all analysed samples . in milk products , goat milk products had lower acetaldehyde contents than cow milk products . this can be explained by its higher glycin concentration , which acts as inhibitor of threonine - aldolase , which may produce acetaldehyde from threonine . in fruits , the highest acetaldehyde contents were found in bananas and in citrus fruits . some apple varieties ( granny smith , elstar ) showed higher contents than the other varieties , but there could also be an influence of other factors not controlled , for example , environment during storage , climate , country of origin , and so forth . it would be interesting , however , to further investigate if certain varieties of apples are especially susceptible for acetaldehyde content . while all fruits were generally analyzed in fresh state , we made an experiment with bananas and followed the acetaldehyde content during ripening ( figure 7 ) . the bananas for this experiment came from the same hand and were stored in a fridge up to 22 days . similar to the blackening of the colour , the acetaldehyde content rose up to an increase of 80% compared to the initial content . causative could be on the one hand losses of the volatile compound during pressing or concentration of the juice , as well as a dilution effect in products with less than 100% fruit content . the content in direct juices was for the same reason higher than in juices from concentrate . strikingly high acetaldehyde contents were detected in lemonades or soft drinks that only contain low amounts of fruit juice ( apple drink 7.5 mg / kg , orange soft drink 15 mg / kg ) . in view of the results found naturally in the fruits , these contents can only be explained if acetaldehyde has been added as flavour compound , which is consistent with the labelling of the products ( flavour was given in the ingredients list ) . compared to the literature , our survey results were generally consistent compared to the previous data . the exception are the results of lund et al . for orange juice , which contained 50130 mg / l while grapefruit juice contained 40230 the plausibility of these values is questionable as other authors never reported acetaldehyde contents this high in these fruit juices again [ 45 , 47 , 48 ] . another inconsistency in the literature is the reporting of a maximum acetaldehyde content of 400 mg / kg in peas in the vcf database . this is clearly an input data error , as the original reference reported 400 mg / kg not for acetaldehyde but for ethanol . if this value is deleted , the range for acetaldehyde in peas would be 0.562.4 mg / kg . very high values were also reported for vinegar ( 1.91060 mg / kg ) . this can be traced back to the publication of jones and greenshield , who reported a range of 201060 mg / kg for malt vinegar . in our opinion , conventional table vinegar contains significantly less acetaldehyde . finally , the vcf database reported higher values for yoghurt , than what was found in our study ( 0.776 mg / kg ) . however , it must be pointed out that all references reporting contents above 20 mg / kg were from 1982 and earlier , so that these probably resulted from analytical deficiencies ( artefactual formation ) or represent technological changes . the joint fao / who expert committee on food additives ( jecfa ) has estimated the acetaldehyde amount , which is ingested due to its use as food flavour additive , in the range of 9.711 mg per person per day . the food safety commission of japan has estimated a similar range between 9.618 mg ( europe ) and 19.211 mg ( usa ) , which was assumed to be 20% of the acetaldehyde that is contained in foods while the other 80% can be traced as natural occurrence . from these data a total acetaldehyde exposure of 4896 mg / day ( 0.641.28 mg / kg bodyweight ( bw)/day ) can be extrapolated ( see also ) . the us flavor and extract manufacturers association ( fema ) has estimated the possible average daily intake as 35 mg ( 0.47 mg / kg bw / day ) while morris et al . estimated a range of 4080 mg ( 0.531.07 mg / kg bw / day ) , with worst case levels up to 200 mg ( 2.67 mg / kg bw / day ) . from the acetaldehyde content found in our survey for each food group and the estimated intake of each group for a selected population , the acetaldehyde exposure can be estimated . regarding the exposure to acetaldehyde on a population basis , the food intake assessed during the german national nutrition survey ii can be taken as basis . the exposure can be estimated by multiplying the daily consumption amount of each food group with the acetaldehyde content of this food group found in our survey . we estimate that the major factors for acetaldehyde exposure are alcohol - free beverages , especially for men who have a higher consumption of this group than women . women compensate this , however , by their higher consumption of milk products , fruits , and vegetables ( figure 8) . the average exposure from food ( without alcoholic beverages ) would be around 40 g / kg bw / day for the german population . this exposure estimated in our study is considerably lower than the previous assumptions ( e.g. , from jecfa or fema ) , which were derived from very old occurrence data or industrial production amounts for the use as food flavour additive . this shows the need for further research on acetaldehyde in foods , as the exposure situation appears to be far from well characterized . nevertheless , the margin of exposure ( moe ) calculated according to our previous studies [ 10 , 11 ] for the exposure estimated in this study would be 1175 , which is in a similar region to the moes of other food carcinogens such as acrylamide , furan , aflatoxins , or nitrosamines [ 5760 ] . of course , this risk assessment must be treated as preliminary as we only have analyzed certain food groups ; however , these were selected according to the risk of containing acetaldehyde . moes above 10000 are normally judged as of low relevance for health by the european food safety authority ( efsa ) , but our calculation for acetaldehyde is below this threshold . furthermore , this assessment disregards genetic polymorphisms in subgroups of the population that could lead to an accumulation of acetaldehyde by reduced metabolic activity [ 62 , 63 ] . we think that this preliminary risk assessment justifies further studies into acetaldehyde exposure from food , and risk managers should also consider the possibility to reduce the exposure by disallowing the practice of acetaldehyde addition as a flavour compound .

acetaldehyde ( ethanal ) is a genotoxic carcinogen , which may occur naturally or as an added flavour in foods . we have developed an efficient method to analyze the compound in a wide variety of food matrices . the analysis is conducted using headspace ( hs ) gas chromatography ( gc ) with flame ionization detector . using a robot autosampler , the samples are digested in full automation with simulated gastric fluid ( 1 h at 37c ) under shaking , which frees acetaldehyde loosely bound to matrix compounds . afterwards , an aliquot of the hs is injected into the gc system . standard addition was applied for quantification to compensate for matrix effects . the precision of the method was sufficient ( < 3% coefficient of variation ) . the limit of detection was 0.01 mg / l and the limit of quantification was 0.04 mg / l . 140 authentic samples were analyzed . the acetaldehyde content in apples was 0.97 0.80 mg / kg , orange juice contained 3.86 2.88 mg / kg . the highest concentration was determined in a yoghurt ( 17 mg / kg ) . a first - exposure estimation resulted in a daily acetaldehyde intake of less than 0.1 mg / kg bodyweight from food , which is considerably lower than the exposures from alcohol consumption or tobacco smoking .

1. Introduction 2. Experimental 3. Results and Discussion 4. Conclusions

acetaldehyde ( ethanal ) is carcinogenic in animal experiments [ 1 , 2 ] and was classified by the international agency for research on cancer ( iarc ) in 1999 as possibly carcinogenic to humans only recently , acetaldehyde in association with alcohol consumption has been upgraded by iarc into group 1 ( i.e. , the highest level of evidence ) as carcinogenic to humans . the carcinogenicity is considered to be caused by a genotoxic mechanism as several acetaldehyde - dna adducts were found in vitro and in vivo [ 59 ] . in foods , acetaldehyde may occur either naturally or because of intentional addition as flavour compound . in foods , the highest concentrations of acetaldehyde were determined in vinegar ( 1.06 g / kg ) , but also in milk products and diverse fruits and vegetables [ 16 , 17 ] . the netherlands organization for applied scientific research ( tno ) database volatile compounds in foods ( vcf ) lists numerous studies on the occurrence of acetaldehyde but most of these were from the 1980s or earlier . an absence of current data about the occurrence of acetaldehyde in food can be noted , so that the aim of this study was to develop a methodology to most efficiently analyze acetaldehyde in a wide variety of food matrices , and also provide an overview on the occurrence in the most susceptible food groups . the most simplistic procedure for acetaldehyde analysis is the direct injection of a sample solution into a gas chromatograph with flame ionization detection ( fid ) . such a procedure can be used for analysis of alcoholic beverages without any further sample preparation and is also included in the eu reference methods for the analysis of spirits . as we are now interested to analyze other foods ( besides alcoholic beverages ) , the reference procedure for spirits can not be directly applied . the major problem is to obtain sample extracts without losses of the very volatile analyte that can be injected into the gc system . in the literature , several methods were suggested for acetaldehyde analysis in foods , which include photometric , fluorimetric , chromatographic , and enzymatic methods . however , some of the methods are either expensive or lack sensitivity ( enzymatic method ) , include time - consuming sample preparation steps or lack in specificity ( spectrophotometric methods ) . furthermore , this procedure is leading to a dilution of the analyte , which may facilitate the need for an additional extraction step [ 28 , 29 ] . , in the headspace oven ) may also lead to artefactual formation of acetaldehyde from ethanol , which may explain reports of very high concentrations in early studies . prior to chromatographic measurement of acetaldehyde , derivatization using 2,4-dinitrophenylhydrazine was suggested , and the formed hydrazone can be measured using gas chromatography ( gc ) or high - performance liquid chromatography ( hplc ) . another derivatization reagent ( cysteamine - hcl ) was suggested by miyake and shibamoto , which , however , needs careful adjustment of ph and a further extraction step . a simpler procedure is the analysis of underivatized acetaldehyde using headspace gas chromatography ( hs - gc ) . the headspace injection can be used instead of the liquid injection while all other parameters of the eu reference method can be used . we have previously used such a hs - gc - fid procedure for the analysis of acetaldehyde in saliva samples [ 13 , 34 ] , and this work reports the modifications needed for analyzing all kinds of foodstuffs , including a validation of the procedure . similar to our previous procedure , we use simple static hs injection , as it was previously shown that the dynamic variant ( purge and trap ) is not possible as acetaldehyde is only insufficiently adsorbed into the usual materials ( e.g. we have set focus on providing a sample preparation without losses as well as an improved acid digestion that simulates physiological conditions of the human stomach and therefore allows to estimate the exposure after oral consumption of foods . the simulated gastric fluid ( sgf ) was prepared according to usp 32 . to compensate for the dilution , which occurs during sample preparation , the sgf was concentrated by a factor of 4 . after complete dissolution of the pepsin , the now slightly yellow coloured solution was filled up with distilled water to 500 ml . to prepare an acetaldehyde stock solution with a concentration of 3.0 g / l , a 100 ml measuring flask is filled with about 80 ml of distilled water , which is temperated at exactly 20c , and 300 mg of acetaldehyde are weighed into the flask . from this stock solution , the calibration curve was prepared by filling up 20 , 270 , 520 , and 720 l of the stock solution with distilled water in a 10 ml measuring flask ( 5.4240 mg 800 l of stock solution were filled up with distilled water in a 50 ml measuring flask ( 48 mg all standard and spiking solutions were stored in a water bath at 20c till use . for basic calibration , 200 l of the standard solution , 1.25 ml of sgf and 3.55 ml of distilled water were given in a 20 ml headspace vial , which is immediately tightly sealed using a silicon / ptfe septum . the basic calibration was measured at least once weekly to check the performance of the gc system . the sample types were selected according to risk oriented principles based on previously published acetaldehyde contents and the typical food intake in germany . the samples were stored at 58c and analyzed in fresh condition , or , for packaged foods , before the expiration of the dependent on consistency , the samples were weighed with help of a 20 ml disposable plastic syringe or using an eppendorf pipette , with a tip that was cut off with scissors to facilitate the pipetting of semisolid samples . for quantification with standard addition , five aliquots ( in the range of 1.22.0 g ) of the sample were weighed with an accuracy of 10 mg into 20 ml headspace vials . after addition of 1.25 ml of sgf and the required acetaldehyde spiking , distilled water up to a total volume of 5 ml was added . solid foods were homogenized in a standard household mixer ( magic maxx , ds - produkte gmbh , gallin ) . for fruits and vegetables only the edible parts the homogenized samples were weighed similar to the liquid foods described above . only completely dry or highly viscous samples were weighed using a spatula . the total time for sample preparation of the solid foods was 1525 min per sample . to simulate the physiological conditions inside the stomach , the samples were incubated for 60 min at 37c under constant stirring in the oven of the autosampler . after that , 500 l of headspace were injected into the gc system at 500 l / sec with a temperature of the transfer syringe of 80c . the data analysis of the standard additions to calculate the concentrations was conducted using valoo 2.3 ( analytik software , leer , germany ) . the standard additions were only evaluated in the case of a correlation coefficient with a minimum of 0.9995 and a coefficient of variation ( cv ) of not more than 3% . if the criteria were not fulfilled after elimination of this one outlier , the results were discarded and the sample preparation and measurement were repeated from the beginning . the limits of detection and quantification were determined according to german norm 32645 using the calibration curve method . the limits are extrapolated based on the tolerance interval of a calibration curve , which is measured with concentrations surrounding the limits . the precision ( expressed as coefficient as variation ) can be directly calculated for each sample from the calibration curve resulting from standard addition ( 5 aliquots measured per sample ) . furthermore , we have measured one yoghurt sample several times with different amounts of sample weight ( 1.2 , 1.4 , and 1.6 g ) . the storage stability was evaluated by preparing three standard addition series of the same yoghurt sample and measuring them after 2 , 9 , and 16 days after preparation ( the prepared headspace vials were stored at 58c in the meantime ) . to test for artefactual formation of acetaldehyde from ethanol during sample preparation or analysis , two standard addition series of an apple sample were prepared with and without addition of ethanol ( 250 g ethanol per sample vial ) . possible losses during sample preparation were tested as follows : ( 1 ) 50 ml of an acetaldehyde stock solution in a 100 ml measuring flask was left to stand open ( i.e. , without stopper on the flask ) for 65 min in the 20c water bath ( normally , the flask are directly sealed after the pipetting of the standard , of course ) . ( 2 ) 100 ml of standard solution were filled into the mixer used for homogenization and mixed for 20 s similar to the samples . during initial method development , it was noted that buttermilk did not contain any detectable acetaldehyde in the headspace if aqueous samples are analyzed . due to the diversity of matrices we wanted to analyze , it would also not have been possible to conduct calibration in matrix , with the additional problem of finding acetaldehyde - free matrices for spiking . it was also not possible to find a suitable internal standard with similar behaviour to acetaldehyde , and the use of mass spectrometry with the possibility to use isotopically labelled acetaldehyde was not possible for instrumental restrictions and cost reasons . for all these reasons , we decided to use standard addition according to the german norm 32633:1998 . all sample aliquots were filled up to the same volume ( 5 ml ) with water . while the standard addition sounds to be an incredible amount of work , we nevertheless judged it to be superior to all other methods . first , it must be noted that all steps besides the weighing of the samples are conducted automatically on the autosampler . a further search for an internal standard with same volatility , solubility , reactivity , and headspace behaviour as acetaldehyde was also judged to be pointless , especially as free retention time windows in the complex food matrices are very restricted . the homogeneity of variances shows that the method is equally precise for the whole working range ( figure 1 ) . mg / l , which is in the same order of magnitude as the limits determined in previous research [ 28 , 40 , 41 ] the matrix effect leads to a drastic influence on the slopes of the calibration lines , which necessitates the use of standard addition for quantification . in yoghurt and banana samples , which were analyzed in the same fashion as the apple sample , this effect was not observed , however . with the exception of a single sample of roast coffee powder ( r = 0.9987 , cv 3.28% ) , all samples fulfilled our requirements for precision ( r > 0.9990 , cv < 3% ) . apart from 8 samples ( peas , roast coffee , apple soft drink , orange soft drink , paprika , and 3 bananas ) , we even had r of > 0.9995 and the cvs were typically below 2% . the yoghurt sample that was independently measured for 3 times , had a cv of 2.59% ( average 6.18 mg / kg , standard deviation 0.16 mg / kg ) . only one sample ( a radish ) was not measurable at all , because there was a large interference near the retention time of acetaldehyde , which overlapped its peak and hindered correct integration , probably derived from a glucosinolate or glucosinolate degradation product . the overall mean was 16.91 mg / kg ( standard deviation 0.49 mg / kg , cv 2.90% ) . no artefactual formation of acetaldehyde was detected in the apple sample series with spiked ethanol ( 2.39 0.47 mg / kg without ethanol ; 2.35 0.32 mg / kg with ethanol ) . regarding the losses during sample preparation , the highest influence had the storage of the stock solution without stopper ( 4% loss of acetaldehyde during 65 min ) , while during homogenization only a minor loss of 2% occurred . the samples were stored in a fridge and the temperature in the samples during mixing was increased by a maximum of 7c ( in the case of a cheddar cheese ) . our validation results show that the method has an acceptable performance for the use of analyzing food matrices . without the use of sgf , apparently , the matrix needs to be saturated till the excess of acetaldehyde can diffuse into the headspace . microscopically , the buttermilk without sgf shows large particulate agglomerations , in which the colour is concentrated . if the sample is treated with sgf , the agglomerations disappear and the colour becomes evenly distributed . according to the literature , milk fat globule membranes , which are broken down during buttermilk making , can interact with casein - micelles and form globular aggregates [ 42 , 43 ] . as the sgf preparation adequately frees the acetaldehyde , we have refrained from performing further experiments , while from a scientific standpoint it would be interesting to further study the binding behaviour of acetaldehyde in buttermilk , for example using transmission electron microscopy . the maximum content of foods for direct consumption was found in a yoghurt ( 17.42 mg / kg ) . in food ingredients , 26.3 mg / kg were found in a baking flavour , while an industrial orange flavour contained 1416 mg / kg of acetaldehyde , which was the maximum of all analysed samples . this can be explained by its higher glycin concentration , which acts as inhibitor of threonine - aldolase , which may produce acetaldehyde from threonine . in fruits , the highest acetaldehyde contents were found in bananas and in citrus fruits . some apple varieties ( granny smith , elstar ) showed higher contents than the other varieties , but there could also be an influence of other factors not controlled , for example , environment during storage , climate , country of origin , and so forth . it would be interesting , however , to further investigate if certain varieties of apples are especially susceptible for acetaldehyde content . while all fruits were generally analyzed in fresh state , we made an experiment with bananas and followed the acetaldehyde content during ripening ( figure 7 ) . the bananas for this experiment came from the same hand and were stored in a fridge up to 22 days . similar to the blackening of the colour , the acetaldehyde content rose up to an increase of 80% compared to the initial content . causative could be on the one hand losses of the volatile compound during pressing or concentration of the juice , as well as a dilution effect in products with less than 100% fruit content . the content in direct juices was for the same reason higher than in juices from concentrate . strikingly high acetaldehyde contents were detected in lemonades or soft drinks that only contain low amounts of fruit juice ( apple drink 7.5 mg / kg , orange soft drink 15 mg / kg ) . in view of the results found naturally in the fruits , these contents can only be explained if acetaldehyde has been added as flavour compound , which is consistent with the labelling of the products ( flavour was given in the ingredients list ) . for orange juice , which contained 50130 mg / l while grapefruit juice contained 40230 the plausibility of these values is questionable as other authors never reported acetaldehyde contents this high in these fruit juices again [ 45 , 47 , 48 ] . another inconsistency in the literature is the reporting of a maximum acetaldehyde content of 400 mg / kg in peas in the vcf database . this is clearly an input data error , as the original reference reported 400 mg / kg not for acetaldehyde but for ethanol . if this value is deleted , the range for acetaldehyde in peas would be 0.562.4 mg / kg . very high values were also reported for vinegar ( 1.91060 mg / kg ) . this can be traced back to the publication of jones and greenshield , who reported a range of 201060 mg / kg for malt vinegar . finally , the vcf database reported higher values for yoghurt , than what was found in our study ( 0.776 mg / kg ) . however , it must be pointed out that all references reporting contents above 20 mg / kg were from 1982 and earlier , so that these probably resulted from analytical deficiencies ( artefactual formation ) or represent technological changes . the joint fao / who expert committee on food additives ( jecfa ) has estimated the acetaldehyde amount , which is ingested due to its use as food flavour additive , in the range of 9.711 mg per person per day . the food safety commission of japan has estimated a similar range between 9.618 mg ( europe ) and 19.211 mg ( usa ) , which was assumed to be 20% of the acetaldehyde that is contained in foods while the other 80% can be traced as natural occurrence . from these data a total acetaldehyde exposure of 4896 mg / day ( 0.641.28 mg / kg bodyweight ( bw)/day ) can be extrapolated ( see also ) . the us flavor and extract manufacturers association ( fema ) has estimated the possible average daily intake as 35 mg ( 0.47 mg / kg bw / day ) while morris et al . estimated a range of 4080 mg ( 0.531.07 mg / kg bw / day ) , with worst case levels up to 200 mg ( 2.67 mg / kg bw / day ) . from the acetaldehyde content found in our survey for each food group and the estimated intake of each group for a selected population , the acetaldehyde exposure can be estimated . regarding the exposure to acetaldehyde on a population basis , the food intake assessed during the german national nutrition survey ii can be taken as basis . the exposure can be estimated by multiplying the daily consumption amount of each food group with the acetaldehyde content of this food group found in our survey . the average exposure from food ( without alcoholic beverages ) would be around 40 g / kg bw / day for the german population . this exposure estimated in our study is considerably lower than the previous assumptions ( e.g. this shows the need for further research on acetaldehyde in foods , as the exposure situation appears to be far from well characterized . nevertheless , the margin of exposure ( moe ) calculated according to our previous studies [ 10 , 11 ] for the exposure estimated in this study would be 1175 , which is in a similar region to the moes of other food carcinogens such as acrylamide , furan , aflatoxins , or nitrosamines [ 5760 ] . furthermore , this assessment disregards genetic polymorphisms in subgroups of the population that could lead to an accumulation of acetaldehyde by reduced metabolic activity [ 62 , 63 ] . we think that this preliminary risk assessment justifies further studies into acetaldehyde exposure from food , and risk managers should also consider the possibility to reduce the exposure by disallowing the practice of acetaldehyde addition as a flavour compound .

yttria - stabilized zro2 ( ysz ) is already commonly used as a solid oxide fuel cell ( sofc ) electrolyte and anode component . however , despite its technological importance , ysz itself is a very complex material , adopting different crystal structures ( monoclinic , tetragonal , and cubic ) with varying y content below 20 mol % . of all the associated mixed oxide compounds , ysz containing 8 mol % yttria is the most technologically important one . therefore , on the one hand , the understanding of molecular processes on its surface is highly desirable , and on the other hand , insights into the surface reactivity of ysz are also expected from in - depth studies of the two chemical constituents of the ysz structure , that is , y2o3 and zro2 . the aim for a better understanding of the surface chemistry of the latter two oxides is also fueled by their technological relevance , comprising the use as ceramic materials or as catalysts for a number of chemical reactions . however , despite their importance , the use of ysz and zro2 strongly depends on the particular polymorph used . the most common modification of zro2 is the monoclinic structure that is the only thermodynamically stable phase until 1400 k , where a phase change to a tetragonal modification takes place . above a temperature of 2600 these polymorphic forms exhibit strikingly different chemical properties , giving rise to also different catalytic behavior . the tetragonal and the cubic phases are catalytically more active than their monoclinic counterpart . doping zro2 with y2o3 stabilizes either the cubic or tetragonal phase ( strongly depending on production technique ) over a large temperature range and creates oxygen vacancies . thus , a lot of progress has been made especially regarding characterization of the surface chemistry of a variety of different zro2 samples . materialwise , this essentially refers to supported metal / oxide systems and sulfated zro2 samples . most data are available for pure zro2 , with the exception of comparative adsorption studies of tetragonal and monoclinic zro2 . strikingly different , information on the surface chemistry of y2o3 and ysz mixed oxide phases is virtually nonexistent , and adsorption studies essentially represent no - man s land . information on y2o3 is restricted to ft - ir studies of supported ru and pd / y2o3 systems and er - doped y2o3 , being used as transparent ceramics . studies on ysz by ft - ir spectroscopy are almost exclusively focused on investigating its crystal structure . one notable exception in that respect is the studies on zro2 , doped with 3 mol % yttria , which usually serves as a compound to simulate tetragonal zro2 . the aim of our studies therefore is to provide a first comparison of the surface chemical properties of technically used ysz with 8 mol % yttria content and its separated chemical constituents y2o3 and zro2 . as representative probe molecules , co and co2 were chosen , due to their importance as reactants in a variety of chemical reactions , including the water gas shift reaction or other c1 reactions relevant to anodic fuel conversion in solid oxide fuel cells . as the perfect method for monitoring the adsorbed species under technically relevant conditions , a dedicated in situ ft - ir spectroscopy setup was used , allowing static and flowing treatments up to the bar range and elevated temperatures up to 773 k. exceeding the scientific input from the simple comparison between the three oxidic materials , we also anticipate in - depth information on the reactivity of more complex reactions and materials . special attention was also paid to a comparison of the determined surface reactivity to literature data , as especially oxide pretreatments are expected to strongly alter the adsorption behavior . regarding surface studies on oxides , this essentially refers to the hydroxylation state of the surface . commercial powders of y2o3 , zro2 , and ysz were used as starting materials . cubic ( bcc ) y2o3 ( yttrium(iii)oxide , nanopowder , < 50 nm particle size ) and tetragonal ysz ( zirconium(iv)oxide - yttria stabilized , nanopowder , containing 8 mol % y2o3 as stabilizer , from now on called ysz-8 ) were supplied by sigma aldrich and monoclinic zro2 ( with a small amount of tetragonal phase after thermal annealing at 1173 k , zirconium(iv)oxide , 99.978% ) by alfa aesar . all samples were pretreated by calcination at 1173 k in air and subsequently checked by xrd for structural changes upon annealing . the surface area after pretreatment was determined by nitrogen adsorption at 77 k according to the bet method as 21.7 m / g ( y2o3 ) , 31.6 m / g ( ysz-8 ) , and 10.4 m / g ( zro2 ) . for bet measurements , a quantachrome nova 2000 surface area and pore size analyzer was used . ft - ir spectra were recorded in transmission mode on a perkin - elmer ft - ir system 2000 spectrometer . the powder samples were pressed into thin pellets using a pressure of 2 t ( sample mass about 100 mg each ) . this cell setup allows treatments under static and flowing conditions up to pressures of 1 bar and temperatures up to 873 k. the temperature is controlled by a thermocouple placed next to the pellet . pretreatment of the powders was performed outside the cell ( annealing up to 1173 k ) , and the pellet was mounted hot into the ir cell . calcium fluoride is used as window material allowing access to wavelength ranges above 1000 cm . to ensure identical starting conditions , samples were oxidized with 20% oxygen seeded in helium at 873 k for one hour . to ensure a minimal degree of hydroxylation , during the flow mode measurements the gases are cleaned and dried using two liquid nitrogen or liquid nitrogen / ethanol cooling traps . flowing measurements were performed under ambient conditions . in static mode , the gases are preadsorbed on a 5 zeolite trap binding water sufficiently strongly , before the dry gases are desorbed into the evacuated and degassed cell . all reported spectra are corrected with the spectrum of the dry preoxidized oxide pellet prior to adsorption . figure 1 shows an overview of effects associated with the static adsorption of dry co2 on y2o3 at room temperature using co2 pressures up to 10 mbar . infrared spectra of static adsorption of co2 on pure y2o3 at room temperature at pressures up to 10 mbar . as it can be clearly seen , the adsorption leads to formation of a number of distinct ( bi)carbonate and hydroxyl species . the assignment of the carbonate species ( detailed representation in figure 3 ) was based on recent investigations on carbonate formation on different ga2o3 polymorphs , where both experimental and theoretical information is most abundant and thus the assignment in the present case is most straightforward . the structure of the most likely analogous ( bi)carbonate species is highlighted in figure s1 ( supporting information ) . a closer look at figure 3 reveals that at a pressure of 3 10 mbar almost no formation of carbonates is observed , whereas raising the pressure up to 1 mbar leads to the formation of a dominant species with signals at 1674 , 1397 , and 1223 cm . during further pressure increase to 10 mbar , another species with signals at 1652 , 1419 , and 1223 cm dominates the spectra . on the basis of ref ( 15 ) , these peaks can be attributed to the as , s , and oh vibration modes of monodentate and bidentate bicarbonates . the intensification of the latter peaks correlates with the decrease of signals at 3702 and 3676 cm and an increase of the associated modes at 3658 and 3625 cm ( figures 2 and 3 ) . details of the room - temperature - collected infrared spectra of adsorbed co2 on y2o3 shown in figure 1 in the wavenumber range between 3750 and 3540 cm at pressures up to 10 mbar in the region of the hydroxyl stretching modes . assignment of the infrared signals between 1900 and 1160 cm of the observed ( bi)carbonates on y2o3 at room temperature at pressures up to 10 mbar . figure 2 shows that , according to refs ( 16 and 17 ) , the negative peaks are related to (oh ) modes of isolated surface hydroxyl groups of the oxide ( more than one peak is visible due to the presence of differently coordinated hydroxyl groups ) , which become consumed by bicarbonate formation , and the accordingly positive signals can be associated with the (oh ) modes of the newly formed hydroxyl groups of the bicarbonates . the intensity of the hydroxyl group at 3658 cm correlates with the rise of the signals of the monodentate bicarbonate , whereas the signal at 3625 cm matches the bidentate bicarbonate species . the analogous appearance of two different bicarbonate species was also observed on ceria samples . the broad feature below 3500 cm can be assigned to interacting hydroxyl groups of the formed bicarbonates . hence , the main process associated with chemisorption of a co2 molecule on y2o3 is the insertion into a basic surface hydroxyl group leading to the formation of bicarbonates . note that the experiments probably do not represent true thermodynamically equilibrated processes since the waiting time to reach an adsorption equilibrium had to be balanced against possible contamination of the surface especially at prolonged times . following refs ( 15 and 18 ) the assignment to the different ( bi)carbonate species is shown in figure 3 . concerning the pattern of the peaks it is apparent that with increasing coordination of the ( bi)carbonates a superposition of signals of the different adsorbed features occurs . as pointed out in ref ( 19 ) such an effect is due to the presence of different adsorption sites arising from different coordination of the metal atoms on the surface , especially on a powder sample . also an interaction between different adsorbed species can not be excluded . to test the thermal stability of the adsorbates , figure 4 illustrates the thermal evolution of the ( bi)carbonate species on y2o3 in 10 mbar co2 up to 873 k under static conditions . infrared spectra collected on y2o3 after exposure of 10 mbar static co2 upon heating to 873 k. infrared spectra have been taken in 100 k steps . the bicarbonate species persist up to a temperature of 373 k , whereas the intensity of the peaks attributed to bidentate species decreases first . at 473 k the bicarbonates have vanished , and the remaining main species is the bridged carbonate , which subsequently is converted into polydentate carbonate species upon further temperature increase . thus , at 873 k only signals of polydentate carbonate species are present . hence , during heating the more weakly bound adsorbates are converted into more strongly bound carbonates . summarizing , the process of this conversion , i.e. , the binding strength of the different kinds of co2 adsorbates with increasing temperature on y2o3 , follows the sequence monodentate bicarbonate bidentate bicarbonate bridged carbonate polydentate carbonate . adsorbates on y2o3 , resulting from chemisorption of co2 , can therefore not be removed entirely by heating up to 873 k. additional experiments ( not shown ) show that a carbonate - poisoned y2o3 surface can only be successfully recovered by heating in dry oxygen up to 873 k , likely because of efficient surface reoxidation . a final comment on the spectroscopic requirements for studying carbonate formation on y2o3 following co2 adsorption should be added . as y2o3 is very prone to hydroxylation , even by traces of h2o , carbonate formation is in turn highly favorable , but subsequent attribution to distinct carbonate species is not possible due to low resolution of the resulting spectra . this effect was observed in particular under flowing co2 conditions , which under the chosen experimental conditions are less dry . consequently , only static co2 adsorption under as dry as possible conditions , using a zeolite trap close to the ir cell for removing all traces of water from co2 , permits collecting highly resolved carbonate spectra . experiments on yttria - stabilized zro2 ( ysz-8 ) under flowing conditions ( co2 flow = 0.4 ml s ) show a behavior similar to pure y2o3 at room temperature . in the case of ysz-8 , flowing conditions were chosen to achieve more intense and thus more reliably interpretable signals because on ysz-8 the carbonate adsorbates are generally more weakly bound as compared to on y2o3 . as apparent from figure 5 , showing also the thermal evolution of the carbonate species , the formation of y2o3-analogous bi- and bridged carbonate species can also be observed . infrared spectra collected during heating ysz-8 in dry flowing co2 ( co2 flow = 0.4 ml / s ) up to 873 k together with the assignment of the observed ( bi- ) carbonates at room temperature . the broad features located at 1305 cm can be ascribed to the s mode of the bridged carbonate , and the other main adsorbate signals at 1646 , 1429 , and 1225 cm represent the corresponding bidentate carbonate . we note that the adsorption behavior of ysz-8 very much resembles that of pure y2o3 ( cf . figure 3 ) and is crucially different from spectra obtained on ysz phases with a lower amount of y , i.e. , 3 mol % . tetragonal zro2 in the literature and also adsorption - wise closely matches pure tetragonal zro2 . in contrast to what was observed on pure y2o3 , ( bi)carbonates on a ysz-8 surface can be easily removed by heating , and almost no signals arising from polydentate carbonate species can be detected . above a temperature of 573 k , these adsorbates decompose completely ( figure 5 ) , although in this experiment flowing conditions with a pressure of 1 bar were chosen . on pure monoclinic zro2 , only very weak adsorption signals of co2 were observed after annealing in co2 pressures up to 12 mbar ( figure 6 ) . infrared spectra taken after static adsorption of co2 on zro2 at room temperature at pressures up to 12 mbar . static conditions with pressures below 12 mbar were enough to saturate the surface with chemisorbed species . therefore , higher pressures or even flowing conditions do not intensify the very weak ( bi)carbonate signals . nevertheless , the vibrations at 1628 , 1431 , and 1223 cm can be associated with a bicarbonate species on the surface . these signals very much coincide with literature - reported spectra taken on pure monoclinic zro2 . however , in our case only very weak signals are observed , which is essentially due to the strong dehydroxylation treatment , conducted prior to the ft - ir measurements . spectra shown in ref ( 11 ) ( figure 2 of that reference ) clearly show peaks of surface hydroxyl groups , which are almost absent in our case . the starting pressure of 2 mbar at room temperature was enough to saturate the surface , and thus any further increase of the co2 pressure did not influence the spectra . this corroborates the assumption that hydroxyl groups are of crucial importance for the chemisorption of co2 . the following heating of zro2 in co2 did not lead to reliably interpretable spectra due to the low signal - to - noise ratio . all oxides were pretreated by heating in air up to 1173 k , which leads to effective dehydroxylation of the surface . only in the case of pure zro2 , no rehydroxylation was observed , even in the presence of water vapor at temperatures up to 873 k ( see figure s2 , supporting information , also showing a comparison of the three oxides upon h2o adsorption ) . as a result , zro2 remains almost unreactive for co2 adsorption . this emphasizes the fact that surface hydroxyl species are important for effective chemisorption of co2 . the comparison of the three oxides y2o3 , ysz-8 , and zro2 during exposure to co2 at room temperature and 873 k is highlighted in figure 7 ( concerning the assignment to ( bi)carbonate species , see table 1 ) . comparison of the collected infrared spectra upon co2 adsorption at room temperature ( lower panel ) and 873 k ( upper panel ) on the surfaces of the oxides y2o3 , ysz-8 , and zro2 . at room temperature , y2o3 and ysz-8 show a very similar behavior ( with the exception of a different distribution of the ( bi)carbonate species ) , suggesting that the hydroxylated yttrium centers in ysz-8 are the reactive sites of the mixed oxide . upon heating ysz-8 to at least 673 k , the adsorbed carbonate species are completely removed , indicating weaker bonding than on pure y2o3 , which is also reflected by the eventual absence of strongly bound polydentate carbonate species . as a conclusion from measurements on pure y2o3 , the monodentate bicarbonate must be more strongly bound than the bidentate carbonate , and the conversion to the associated bridged species bound to two yttrium atoms is the essential step toward even more strongly bound species , which are hard to remove . most likely , the monodentate bicarbonate is not only bound to an yttrium atom via one of its oxygen atoms but also stabilized through a hydrogen bond with a neighboring hydroxyl group . if water binds dissociatively onto a defect caused by a y in the zro2 lattice , a ho bound to the yttrium ( because of its basicity ) is formed . this is the adsorption site for a co2 molecule to subsequently form a ( bi-)carbonate species . in the case of a bridged carbonate , this species is more likely bound to a yttrium and a zirconium atom than to two adjacent y centers , and this seems to make the difference in the binding strength of this particular species . comparing the carbonate spectra provided by pokrovski et al . and baeza et al . obtained on different hydroxylated zro2 polymorphs , we note remarkable similarities in the overall features of the carbonate regions but a slightly different population of the different ( bi)carbonate species . on pure y2o3 , the bicarbonate species are dominating , whereas on ysz-8 , the relative fraction of bidentate and bridged carbonate species is higher . in close correlation , the spectra of pokrovski et al . show a higher fraction of bridged species . most important , the spectra obtained on tetragonal zro2 are strikingly different from those observed on ysz-8 . the main species on tetragonal zro2 , as reported by baeza et al . and however , despite the different distribution of bicarbonate , bidentate , and bridged carbonates , no polydentate carbonates are found on ysz-8 , further confirming that the adsorption of ysz is basically dominated by the hydroxylated y centers . besides molecular adsorption as intact co , for the adsorption of a co molecule and following reaction to co2 on an oxide surface , two mechanisms are in principle possible : ( i ) the co molecule reacts with an oxygen atom of the lattice to form co2 and leaves an oxygen vacancy behind or ( ii ) it inserts into a surface hydroxyl group , forms a formate , and this intermediate subsequently decomposes into co2 ( or reacts backward to co and oh ) . figure s3 ( supporting information ) shows the possible formate species on an oxide surface . figure 8 shows y2o3 heated in 290 mbar co under static measurement conditions ( main panel ) . infrared spectra taken upon heating y2o3 in 290 mbar co under static measurement conditions up to 773 k ( upper panel ) and with details of the wavenumber region above 2700 cm ( right lower panel ) and below 1700 cm ( left lower panel ) . at room temperature , no adsorption or reaction takes place , but during heating , co2 formation starts at 373 k. as an intermediate of this reaction , signals of formates can be detected , with an intensity maximum reached at 573 k. the corresponding vibrations for the as and the s mode of the formate are at 1595 and 1381 cm , and the related (ch ) vibration is located at 2856 cm . also the decrease of surface hydroxyl groups ( left lower panel ) can be detected starting from a temperature of 373 k at 3704 and 3672 cm , which illustrates the analogy to the above - mentioned observations regarding co2 adsorption / carbonate formation . because of the formation of co2 , vibration modes for carbonates are observed as well especially in analogy to the measurements in co2 at 473 k the bridged carbonate is the main species . at 773 k the polydentate carbonate is again predominant ( right lower panel ) . accompanied by the already mentioned formate signals , combination modes , namely , 1(combi ) ( as(co2 + (ch ) ) at 2972 cm and 2(combi ) ( s(co2 ) + (ch ) ) at 2727 cm , are observed ( lower left panel ) . on the basis of refs ( 18 and 21 ) the observed formate species starting from room temperature , weak signals are observed at 1630 and 1294 cm , which may be arising from monodentate formate species . comparing our results to those of the literature , it is generally assumed that the monodentate species is the more reactive and less stable one . therefore , the bidentate or bridged formate species could be mainly a spectator below 773 k. figure 9 shows ysz-8 heated in flowing co ( co flow = 0.2 ml s ) . infrared spectra collected upon heating ysz-8 in flowing co ( co flow = 0.2 ml s ) up to 873 k. infrared spectra have been taken in 100 k steps . as for co2 , flowing conditions were chosen for ysz-8 because of better interpretable spectra due to increased adsorption of surface species . at room temperature , however , the processes on ysz-8 are again quite similar to those observed on y2o3 , with the exception that the spectra are more easy to interpret because of no disturbing ( bi)carbonates . co2 formation starts at 573 k , and in - parallel typical formate peaks at 2875 cm for the (ch ) mode and 1581 , 1384 , and 1357 cm representing the as(oco ) , the (ch ) , and the as(oco ) mode of the formate arise . these formate vibration modes are visible up to a temperature of 675 k , and above 773 k the same spectra fingerprint as for co2 adsorption is observed . in other words , a decrease of signals at 3690 , 3643 , and 3613 cm , attributed to surface hydroxyl groups , occurs . interestingly , on ysz-8 no signals for monodentate formate species are visible . to ensure full correlation with the co2 measurements , pure dehydroxylated zro2 did not show significant signals attributable to surface adsorbates in static co ( figure 10 ) . infrared spectra collected upon heating zro2 following static adsorption of 248 mbar co up to 873 k. infrared spectra have been taken in 100 k steps . this essentially verifies that no defects ( which are usually titrated by molecular co adsorption ) and no substantial amount of hydroxyl groups are present because of the oxidizing high - temperature pretreatment of the sample . however , during heating in 248 mbar co at 573 k , very weak peaks at 2361 and 2875 cm being attributable to (ch ) and/or combination modes of formates are observed ( see left inset ) . the associated broad signals in the region below 1600 cm are too weak for an appropriate interpretation . we note that all oxides were pretreated by heating up to 1173 k in air to reduce impurities , and in the case of zro2 no rehydroxylation takes place . because of the lack of surface hydroxyl groups only a minute amount of formates can in turn be formed . the comparison of the adsorption behavior during exposure to co at maximum formate signal , namely , at 573 k , is highlighted in figure 11 . comparison of the infrared spectra of the oxides y2o3 , ysz-8 , and zro2 at maximum formate signal ( 573 k ) . on none of the three studied oxides were signals of adsorbed ( physisorbed ) co detected , as was previously shown for more defective zro2 samples . we might anticipate that all defects are therefore quenched upon the preannealing in oxygen to 1173 k. note that our results strongly suggest that in line with the initial question about the mechanism of co2 formation following co adsorption a mechanism including the participation of surface oh - groups is found to be most likely prevailing ( mechanism ii ) . substantial reduction of the oxides is therefore less likely , and this assumption is also corroborated by additional electric impedance measurements , which only show the reversible formation of thermally excited charge carriers but no substantial formation of surface defects ( figure s4 , supporting information ) . like in the case of co2 adsorption , the comparison of the oxides shows that the yttrium amount in ysz-8 is important for chemisorption reactivity because in contrast to pure zro2 the surface of the mixed oxide can be easily reactivated after dehydroxylation . this again corroborates the assumption that the basic surface hydroxyl species on y - centers are also important for the conversion of co toward formates . on ysz-8 , the carbonates resulting from adsorption of the formed co2are not bound as strongly as on y2o3 and thus do not interfere with the formates . using ft - ir spectroscopy investigations , surface reaction - induced adsorbates of co2 and co on the technically relevant oxides y2o3 , ysz-8 , and zro2 could be identified . y2o3 and ysz-8 show much higher reactivity toward both co2 and co adsorption than zro2 . the reversible formation and basic presence of surface hydroxyl groups is a crucial factor of an active surface , and the yttrium centers in ysz-8 seem to essentially exhibit this quality . thus , they are the c1-reactive sites of the mixed oxide ysz-8 . on y2o3 and ysz-8 , several kinds of carbonates are detected , whereby in general on y2o3 the adsorbates are more difficult to remove because of obviously easier conversion to more strongly bound , higher - coordinated species . the mechanism of chemisorption of co2 most likely proceeds via an insertion of the co2 molecule into a surface hydroxyl group and the following formation of a bicarbonate . further conversion into more strongly bound carbonate species is observed at higher temperatures . in the case of co oxidation the formation of formate intermediates is observed on all oxides , but the absence of a significant amount of hydroxyl groups on zro2 leads to very weak signals on this oxide . the present ft - ir spectroscopic study moreover suggests that sofc - related c1 reactions ( water gas shift reaction , intermediates of ch4 reforming ) on ysz - based cermet anodes may be mechanistically / kinetically influenced by the basic character and by the pronounced reversibility and degree of hydroxylation of the y - centers in ysz-8 .

in situ ft - ir spectroscopy was exploited to study the adsorption of co2 and co on commercially available yttria - stabilized zro2 ( 8 mol % y , ysz-8 ) , y2o3 , and zro2 . all three oxides were pretreated at high temperatures ( 1173 k ) in air , which leads to effective dehydroxylation of pure zro2 . both y2o3 and ysz-8 show a much higher reactivity toward co and co2 adsorption than zro2 because of more facile rehydroxylation of y - containing phases . several different carbonate species have been observed following co2 adsorption on y2o3 and ysz-8 , which are much more strongly bound on the former , due to formation of higher - coordinated polydentate carbonate species upon annealing . as the crucial factor governing the formation of carbonates , the presence of reactive ( basic ) surface hydroxyl groups on y - centers was identified . therefore , chemisorption of co2 most likely includes insertion of the co2 molecule into a reactive surface hydroxyl group and the subsequent formation of a bicarbonate species . formate formation following co adsorption has been observed on all three oxides but is less pronounced on zro2 due to effective dehydroxylation of the surface during high - temperature treatment . the latter generally causes suppression of the surface reactivity of zro2 samples regarding reactions involving co or co2 as reaction intermediates .

Introduction Experimental Section Results and Discussion Conclusion

yttria - stabilized zro2 ( ysz ) is already commonly used as a solid oxide fuel cell ( sofc ) electrolyte and anode component . therefore , on the one hand , the understanding of molecular processes on its surface is highly desirable , and on the other hand , insights into the surface reactivity of ysz are also expected from in - depth studies of the two chemical constituents of the ysz structure , that is , y2o3 and zro2 . the aim for a better understanding of the surface chemistry of the latter two oxides is also fueled by their technological relevance , comprising the use as ceramic materials or as catalysts for a number of chemical reactions . however , despite their importance , the use of ysz and zro2 strongly depends on the particular polymorph used . thus , a lot of progress has been made especially regarding characterization of the surface chemistry of a variety of different zro2 samples . strikingly different , information on the surface chemistry of y2o3 and ysz mixed oxide phases is virtually nonexistent , and adsorption studies essentially represent no - man s land . information on y2o3 is restricted to ft - ir studies of supported ru and pd / y2o3 systems and er - doped y2o3 , being used as transparent ceramics . studies on ysz by ft - ir spectroscopy are almost exclusively focused on investigating its crystal structure . one notable exception in that respect is the studies on zro2 , doped with 3 mol % yttria , which usually serves as a compound to simulate tetragonal zro2 . the aim of our studies therefore is to provide a first comparison of the surface chemical properties of technically used ysz with 8 mol % yttria content and its separated chemical constituents y2o3 and zro2 . as representative probe molecules , co and co2 were chosen , due to their importance as reactants in a variety of chemical reactions , including the water gas shift reaction or other c1 reactions relevant to anodic fuel conversion in solid oxide fuel cells . as the perfect method for monitoring the adsorbed species under technically relevant conditions , a dedicated in situ ft - ir spectroscopy setup was used , allowing static and flowing treatments up to the bar range and elevated temperatures up to 773 k. exceeding the scientific input from the simple comparison between the three oxidic materials , we also anticipate in - depth information on the reactivity of more complex reactions and materials . special attention was also paid to a comparison of the determined surface reactivity to literature data , as especially oxide pretreatments are expected to strongly alter the adsorption behavior . cubic ( bcc ) y2o3 ( yttrium(iii)oxide , nanopowder , < 50 nm particle size ) and tetragonal ysz ( zirconium(iv)oxide - yttria stabilized , nanopowder , containing 8 mol % y2o3 as stabilizer , from now on called ysz-8 ) were supplied by sigma aldrich and monoclinic zro2 ( with a small amount of tetragonal phase after thermal annealing at 1173 k , zirconium(iv)oxide , 99.978% ) by alfa aesar . all samples were pretreated by calcination at 1173 k in air and subsequently checked by xrd for structural changes upon annealing . the surface area after pretreatment was determined by nitrogen adsorption at 77 k according to the bet method as 21.7 m / g ( y2o3 ) , 31.6 m / g ( ysz-8 ) , and 10.4 m / g ( zro2 ) . pretreatment of the powders was performed outside the cell ( annealing up to 1173 k ) , and the pellet was mounted hot into the ir cell . as it can be clearly seen , the adsorption leads to formation of a number of distinct ( bi)carbonate and hydroxyl species . a closer look at figure 3 reveals that at a pressure of 3 10 mbar almost no formation of carbonates is observed , whereas raising the pressure up to 1 mbar leads to the formation of a dominant species with signals at 1674 , 1397 , and 1223 cm . on the basis of ref ( 15 ) , these peaks can be attributed to the as , s , and oh vibration modes of monodentate and bidentate bicarbonates . details of the room - temperature - collected infrared spectra of adsorbed co2 on y2o3 shown in figure 1 in the wavenumber range between 3750 and 3540 cm at pressures up to 10 mbar in the region of the hydroxyl stretching modes . assignment of the infrared signals between 1900 and 1160 cm of the observed ( bi)carbonates on y2o3 at room temperature at pressures up to 10 mbar . figure 2 shows that , according to refs ( 16 and 17 ) , the negative peaks are related to (oh ) modes of isolated surface hydroxyl groups of the oxide ( more than one peak is visible due to the presence of differently coordinated hydroxyl groups ) , which become consumed by bicarbonate formation , and the accordingly positive signals can be associated with the (oh ) modes of the newly formed hydroxyl groups of the bicarbonates . the intensity of the hydroxyl group at 3658 cm correlates with the rise of the signals of the monodentate bicarbonate , whereas the signal at 3625 cm matches the bidentate bicarbonate species . hence , the main process associated with chemisorption of a co2 molecule on y2o3 is the insertion into a basic surface hydroxyl group leading to the formation of bicarbonates . as pointed out in ref ( 19 ) such an effect is due to the presence of different adsorption sites arising from different coordination of the metal atoms on the surface , especially on a powder sample . at 473 k the bicarbonates have vanished , and the remaining main species is the bridged carbonate , which subsequently is converted into polydentate carbonate species upon further temperature increase . , the binding strength of the different kinds of co2 adsorbates with increasing temperature on y2o3 , follows the sequence monodentate bicarbonate bidentate bicarbonate bridged carbonate polydentate carbonate . adsorbates on y2o3 , resulting from chemisorption of co2 , can therefore not be removed entirely by heating up to 873 k. additional experiments ( not shown ) show that a carbonate - poisoned y2o3 surface can only be successfully recovered by heating in dry oxygen up to 873 k , likely because of efficient surface reoxidation . a final comment on the spectroscopic requirements for studying carbonate formation on y2o3 following co2 adsorption should be added . as y2o3 is very prone to hydroxylation , even by traces of h2o , carbonate formation is in turn highly favorable , but subsequent attribution to distinct carbonate species is not possible due to low resolution of the resulting spectra . experiments on yttria - stabilized zro2 ( ysz-8 ) under flowing conditions ( co2 flow = 0.4 ml s ) show a behavior similar to pure y2o3 at room temperature . as apparent from figure 5 , showing also the thermal evolution of the carbonate species , the formation of y2o3-analogous bi- and bridged carbonate species can also be observed . the broad features located at 1305 cm can be ascribed to the s mode of the bridged carbonate , and the other main adsorbate signals at 1646 , 1429 , and 1225 cm represent the corresponding bidentate carbonate . in contrast to what was observed on pure y2o3 , ( bi)carbonates on a ysz-8 surface can be easily removed by heating , and almost no signals arising from polydentate carbonate species can be detected . infrared spectra taken after static adsorption of co2 on zro2 at room temperature at pressures up to 12 mbar . nevertheless , the vibrations at 1628 , 1431 , and 1223 cm can be associated with a bicarbonate species on the surface . however , in our case only very weak signals are observed , which is essentially due to the strong dehydroxylation treatment , conducted prior to the ft - ir measurements . spectra shown in ref ( 11 ) ( figure 2 of that reference ) clearly show peaks of surface hydroxyl groups , which are almost absent in our case . the starting pressure of 2 mbar at room temperature was enough to saturate the surface , and thus any further increase of the co2 pressure did not influence the spectra . this corroborates the assumption that hydroxyl groups are of crucial importance for the chemisorption of co2 . all oxides were pretreated by heating in air up to 1173 k , which leads to effective dehydroxylation of the surface . only in the case of pure zro2 , no rehydroxylation was observed , even in the presence of water vapor at temperatures up to 873 k ( see figure s2 , supporting information , also showing a comparison of the three oxides upon h2o adsorption ) . this emphasizes the fact that surface hydroxyl species are important for effective chemisorption of co2 . the comparison of the three oxides y2o3 , ysz-8 , and zro2 during exposure to co2 at room temperature and 873 k is highlighted in figure 7 ( concerning the assignment to ( bi)carbonate species , see table 1 ) . comparison of the collected infrared spectra upon co2 adsorption at room temperature ( lower panel ) and 873 k ( upper panel ) on the surfaces of the oxides y2o3 , ysz-8 , and zro2 . at room temperature , y2o3 and ysz-8 show a very similar behavior ( with the exception of a different distribution of the ( bi)carbonate species ) , suggesting that the hydroxylated yttrium centers in ysz-8 are the reactive sites of the mixed oxide . upon heating ysz-8 to at least 673 k , the adsorbed carbonate species are completely removed , indicating weaker bonding than on pure y2o3 , which is also reflected by the eventual absence of strongly bound polydentate carbonate species . as a conclusion from measurements on pure y2o3 , the monodentate bicarbonate must be more strongly bound than the bidentate carbonate , and the conversion to the associated bridged species bound to two yttrium atoms is the essential step toward even more strongly bound species , which are hard to remove . most likely , the monodentate bicarbonate is not only bound to an yttrium atom via one of its oxygen atoms but also stabilized through a hydrogen bond with a neighboring hydroxyl group . on pure y2o3 , the bicarbonate species are dominating , whereas on ysz-8 , the relative fraction of bidentate and bridged carbonate species is higher . and however , despite the different distribution of bicarbonate , bidentate , and bridged carbonates , no polydentate carbonates are found on ysz-8 , further confirming that the adsorption of ysz is basically dominated by the hydroxylated y centers . besides molecular adsorption as intact co , for the adsorption of a co molecule and following reaction to co2 on an oxide surface , two mechanisms are in principle possible : ( i ) the co molecule reacts with an oxygen atom of the lattice to form co2 and leaves an oxygen vacancy behind or ( ii ) it inserts into a surface hydroxyl group , forms a formate , and this intermediate subsequently decomposes into co2 ( or reacts backward to co and oh ) . at room temperature , no adsorption or reaction takes place , but during heating , co2 formation starts at 373 k. as an intermediate of this reaction , signals of formates can be detected , with an intensity maximum reached at 573 k. the corresponding vibrations for the as and the s mode of the formate are at 1595 and 1381 cm , and the related (ch ) vibration is located at 2856 cm . also the decrease of surface hydroxyl groups ( left lower panel ) can be detected starting from a temperature of 373 k at 3704 and 3672 cm , which illustrates the analogy to the above - mentioned observations regarding co2 adsorption / carbonate formation . because of the formation of co2 , vibration modes for carbonates are observed as well especially in analogy to the measurements in co2 at 473 k the bridged carbonate is the main species . as for co2 , flowing conditions were chosen for ysz-8 because of better interpretable spectra due to increased adsorption of surface species . at room temperature , however , the processes on ysz-8 are again quite similar to those observed on y2o3 , with the exception that the spectra are more easy to interpret because of no disturbing ( bi)carbonates . co2 formation starts at 573 k , and in - parallel typical formate peaks at 2875 cm for the (ch ) mode and 1581 , 1384 , and 1357 cm representing the as(oco ) , the (ch ) , and the as(oco ) mode of the formate arise . in other words , a decrease of signals at 3690 , 3643 , and 3613 cm , attributed to surface hydroxyl groups , occurs . this essentially verifies that no defects ( which are usually titrated by molecular co adsorption ) and no substantial amount of hydroxyl groups are present because of the oxidizing high - temperature pretreatment of the sample . we note that all oxides were pretreated by heating up to 1173 k in air to reduce impurities , and in the case of zro2 no rehydroxylation takes place . because of the lack of surface hydroxyl groups only a minute amount of formates can in turn be formed . comparison of the infrared spectra of the oxides y2o3 , ysz-8 , and zro2 at maximum formate signal ( 573 k ) . on none of the three studied oxides were signals of adsorbed ( physisorbed ) co detected , as was previously shown for more defective zro2 samples . we might anticipate that all defects are therefore quenched upon the preannealing in oxygen to 1173 k. note that our results strongly suggest that in line with the initial question about the mechanism of co2 formation following co adsorption a mechanism including the participation of surface oh - groups is found to be most likely prevailing ( mechanism ii ) . substantial reduction of the oxides is therefore less likely , and this assumption is also corroborated by additional electric impedance measurements , which only show the reversible formation of thermally excited charge carriers but no substantial formation of surface defects ( figure s4 , supporting information ) . like in the case of co2 adsorption , the comparison of the oxides shows that the yttrium amount in ysz-8 is important for chemisorption reactivity because in contrast to pure zro2 the surface of the mixed oxide can be easily reactivated after dehydroxylation . this again corroborates the assumption that the basic surface hydroxyl species on y - centers are also important for the conversion of co toward formates . on ysz-8 , the carbonates resulting from adsorption of the formed co2are not bound as strongly as on y2o3 and thus do not interfere with the formates . using ft - ir spectroscopy investigations , surface reaction - induced adsorbates of co2 and co on the technically relevant oxides y2o3 , ysz-8 , and zro2 could be identified . y2o3 and ysz-8 show much higher reactivity toward both co2 and co adsorption than zro2 . the reversible formation and basic presence of surface hydroxyl groups is a crucial factor of an active surface , and the yttrium centers in ysz-8 seem to essentially exhibit this quality . on y2o3 and ysz-8 , several kinds of carbonates are detected , whereby in general on y2o3 the adsorbates are more difficult to remove because of obviously easier conversion to more strongly bound , higher - coordinated species . the mechanism of chemisorption of co2 most likely proceeds via an insertion of the co2 molecule into a surface hydroxyl group and the following formation of a bicarbonate . further conversion into more strongly bound carbonate species is observed at higher temperatures . in the case of co oxidation the formation of formate intermediates is observed on all oxides , but the absence of a significant amount of hydroxyl groups on zro2 leads to very weak signals on this oxide . the present ft - ir spectroscopic study moreover suggests that sofc - related c1 reactions ( water gas shift reaction , intermediates of ch4 reforming ) on ysz - based cermet anodes may be mechanistically / kinetically influenced by the basic character and by the pronounced reversibility and degree of hydroxylation of the y - centers in ysz-8 .

pns precede clinical manifestation of ovarian tumors and enable their diagnosis at an early stage.paraneoplastic cerebellar degeneration ( pcd ) can coexist with ovarian carcinoma.anti-nmdar antibodies detected in patient affected with encephalitis are highly suggestive of ovarian teratoma.ovarian tumors and nervous tissue share common antigens in pns ( e.g. , cdr2 , nmda receptor)the concept of cross - presentation , however , may not be sufficient to explain an emergence of pns.cell-mediated immune response plays a role in the pathogenesis of pns.antibody-mediated immune response is a major mechanism of anti - nmdar encephalitis and other nsas.in patients with pns associated potentially with ovarian tumors , transvaginal ultrasound examination and pelvic ct scan are indicated if negative , pet imaging is required.if imaging studies remain normal , explorative laparoscopy / laparotomy should be considered.an early resection of ovarian tumors is a significant part of pns management and improves the outcome . pns precede clinical manifestation of ovarian tumors and enable their diagnosis at an early stage . anti - nmdar antibodies detected in patient affected with encephalitis are highly suggestive of ovarian teratoma . ovarian tumors and nervous tissue share common antigens in pns ( e.g. , cdr2 , nmda receptor)the concept of cross - presentation , however , may not be sufficient to explain an emergence of pns . antibody - mediated immune response is a major mechanism of anti - nmdar encephalitis and other nsas . in patients with pns associated potentially with ovarian tumors , transvaginal ultrasound examination and pelvic ct scan are indicated if negative , pet imaging is required . an early resection of ovarian tumors is a significant part of pns management and improves the outcome . paraneoplastic neurological syndromes ( pns ) are defined as the pathologic involvement of the nervous system in the course of malignancy . this entity does not include tumor infiltration , compression or metastasis of the nervous system ( graus et al . pns often precede clinical manifestation of a tumor and enable diagnosis at an early stage . interestingly , neoplasms that appear in patients with pns are limited in size and metastasize less commonly than those without pns ( hetzel et al . ovarian tumors account for about 10 % of malignancies associated with pns ( giometto et al . paraneoplastic cerebellar degeneration ( pcd ) , the most common paraneoplastic neurological syndrome , may coexist with ovarian carcinoma ( giometto et al . 2010 ) . recently , a group of neurologic disorders , neuronal surface antibodies syndromes ( nsas ) , has been defined ( zuliani et al . they usually affect the central nervous system in the form of encephalitis that manifests mainly as behavioral disorder and seizures . however , they are rarely related to malignancy . one of nsas , however , anti - nmdar encephalitis , appears typically in young women with a benign tumor , ovarian teratoma . the diagnosis of pns is based on criteria defined in graus et al . this takes into account neurological symptoms , presence of a tumor and antibodies associated with this condition , referred to as onconeural antibodies ( graus et al . the diagnosis of pns is a complex process that always requires exclusion of primary etiology of neurologic symptoms , including among others vascular disorders , infection , nervous tissue tumors or hereditary syndromes . the classification of pns distinguishes classical syndromes and well - characterized onconeural antibodies that are the most specific for pns ( tables 1 , 2 ) . the term definite pns in contrast to possible pns indicates higher probability of the paraneoplastic nature of a disorder ( table 3 ) . this differentiation is important since definite pns requires a more intense search for underlying tumor . hence , once the diagnosis of anti - nmdar encephalitis is established , it is classified as definite nsas if it is responsive to immunomodulatory agents such as steroid and intravenous immunoglobulin and as probable nsas if not responsive ( zuliani et al . 2012).table 1classical paraneoplastic syndromesclassical paraneoplastic neurological syndromesencephalomyelitislimbic encephalitis subacute cerebellar degeneration opsoclonus myoclonus subacute sensory neuronopathy chronic gastrointestinal pseudo - obstructionlambert eaton myasthenic syndromedermatomyositispns the most commonly associated with ovarian tumors are in bold ( graus et al . 2004)table 2onconeural and neuronal surface antibodiesonconeural and neuronal surface antibodieswell characterizedpartly characterizedneuronal surface antibodiesanti - hu anti - yo anti - ri anti - cv2anti - ma anti - amphiphysin anti - tranti - zic4anti - mglur1anna3pca2anti - vgkc complex antigens ( lgi1 or caspr2 ) anti - nmdar anti - amparanti - gababranti - glyranti - vgcc - abanti - mglur1anti - mglur5antibodies detected commonly in pns associated with ovarian tumors are in bold ( graus et al . 2012)table 3definite and possible pns according to diagnostic criteria as published by graus et al . ( 2004)definite pnspossible pns definite and possible diagnosis of pns 1 . a classical syndrome and cancer that develop within 5 years of the diagnosis of the neurological disorder1 . a classical syndrome , no onconeural antibodies , no cancer is diagnosed , but at high risk for having an underlying tumor2 . a non - classical syndrome that resolves or significantly improves after cancer treatment without concomitant immunotherapy provided that the syndrome is not susceptible to spontaneous remission2 . a neurological syndrome ( classical or not ) with partially characterized onconeural antibodies and no cancer3 . a non - classical syndrome with onconeural antibodies ( well characterized or not ) and cancer that develops within 5 years of the diagnosis of the neurological disorder3 . a non - classical syndrome , no onconeural antibodies and cancer present within 2 years of diagnosis4 . a neurological syndrome ( classical or not ) with well - characterized onconeural antibodies ( anti - hu , yo , cv2 , ri , ma2 or amphiphysin ) and no cancer neurological syndrome with well - characterized antibodies ( in bold ) is a clinical setting that requires intense search for underlying malignancy and often enables its detection at an early stage . reproduced from recommended diagnostic criteria for paraneoplastic neurological syndromes ( graus et al . classical paraneoplastic syndromes pns the most commonly associated with ovarian tumors are in bold ( graus et al . 2004 ) onconeural and neuronal surface antibodies antibodies detected commonly in pns associated with ovarian tumors are in bold ( graus et al . ( 2004 ) neurological syndrome with well - characterized antibodies ( in bold ) is a clinical setting that requires intense search for underlying malignancy and often enables its detection at an early stage . reproduced from recommended diagnostic criteria for paraneoplastic neurological syndromes ( graus et al . paraneoplastic syndromes are rare in cancer patients . in the group of 1,465 unselected patients with various tumors , the history and physical examination revealed neurologic abnormalities in 96 individuals ( croft and wilkinson 1965 ) . in 55 women with ovarian cancer , neurologic disorder these data should , however , be interpreted with caution as since the time this study was performed the diagnostic criteria of pns have evolved markedly . it is estimated that paraneoplastic neurological syndromes appear in about 1 % of all malignancies ( rees 2004 ) . the diagnosis , however , becomes more probable in particular contexts approximately half of patients with non - familial cerebellar syndrome of subacute onset suffer from some malignancy , primarily ovarian or lung cancer . the criteria for nsas diagnosis were clearly defined in 2012 ( zuliani et al . 2012 ) . antibodies in anti - nmdar encephalitis typically associated with ovarian teratoma were described for the first time in 2005 ( vitaliani et al . 2005 ) . in over 3 years , more than 400 cases of nsas have been reported ( dalmau et al . 2011 ) . it is worth mentioning that three cases of anti - nmdar encephalitis occurred during pregnancy complicated with ovarian teratoma ( kumar et al . 2010 ) . though pns are still considered rare , their estimates of incidence increase with the improvement in diagnostic tools it is worth stressing that a combination of classical pns and the detection of well - characterized antibodies are known to be involved in pns , even without established diagnosis of malignancy is also referred to as definite paraneoplastic disorder ( table 3 ) . in such cases the neurological symptoms antedated the diagnosis of a neoplasm in 34 individuals ( peterson et al . 1992 ) . in 18 out of 19 women with gynecologic cancer , primarily ovarian carcinoma , neurologic pathology preceded the detection of malignancy ( hetzel et al . the time between the onset of paraneoplastic syndrome and diagnosis of a tumor varies from a few weeks to months or even years ( hetzel et al . rarely , paraneoplastic neurological syndrome may emerge after completed treatment of ovarian cancer ( forgy et al . unfortunately , the paraneoplastic reaction is sometimes so intense that it itself worsens significantly the patient s clinical outcome and leads to disability ( cao et al . by contrast , anti - nmdar encephalitis that affects women with ovarian teratoma responds well to tumor resection and subsequent immunotherapy which may lead to almost complete recovery . the onset of signs and symptoms of paraneoplastic syndrome is usually subacute and develops within a few weeks . the patient suffers from incoordination of movements ( ataxia ) , balance and gait disturbances , speech disorder ( dysarthria ) and altered ocular movements ( nystagmus , often in a downbeat form ) ( peterson et al . paraneoplastic neurological syndromes associated with ovarian tumors may also appear as a peripheral polyneuropathy with diffuse paresthesia and anesthesia ( cao et al . young women or children with ovarian teratoma can be affected with encephalitis that manifests as psychosis , memory loss and behavior disorder . it subsequently develops into seizures , dyskinesias and autonomic instability ( florance et al . 2009 ; tanyi et al . 2012 ; zuliani et al . 2012 ) . neurologic pathology is often highly debilitating and renders many patients either wheelchair bound or bedridden ( frings et al . 2005 ) . they are detected by means of an indirect immunofluorescence and confirmatory western or line blot ( monstad et al . the most common type of antibody found in pcd is anti - cdr2 ( cerebellar degeneration protein-2 antibody ) ( also known as anti - yo ) ( anderson et al . anti - yo antibody seropositive status suggests ovarian cancer , breast cancer or other gynecologic malignancies ( peterson et al . 1992 ) . infrequently , it can be related to small cell lung cancer ( greenlee and lipton 1986 ) . recently , new antibodies , termed anti - cdr2-like ( cdr2l ) , have been described ( eichler et al . they are reactive against an antigen similar to the cdr2 protein , which instead of being cytoplasmic is within the cell membrane . paraneoplastic cerebellar syndrome may also be associated with anti - hu antibodies which raises suspicion of small cell lung cancer . the detection of anti - ri , in turn , suggests an underlying breast cancer . clinician should also take into consideration the diagnosis of lymphoma , especially if anti - tr or anti - mglur1 antibodies are identified ( shamsili et al . 2003 ) . interestingly , pcd related to anti - yo antibodies is associated with a worse survival due to cancer as compared to other antibodies ( giometto et al . 2009 ; zuliani et al . 2012 ) . a substantial number of patients , however , manifest paraneoplastic syndromes unrelated to detection of any of the above antibodies ( giometto et al . in addition , onconeuronal antibodies appear in some patients with ovarian tumors who do not suffer from any neurological symptoms . in a group of 181 women with ovarian cancer without paraneoplastic syndrome , there were four who had anti - yo and seven who had anti - ri onconeuronal antibodies ( drlicek et al . the detection of anti - cdr2 antibodies in ovarian cancer can be improved by in vitro transcription translation ( itt ) of radiolabelled cdr2 protein and immunoprecipitation assay ( monstad et al . 2006 ; eichler et al . anti - nmdar antibodies were detected in six out 21 women with teratoma ( michalak et al . 2010 ) . in another study , however , anti - nmdar antibodies were not found among 20 neurologically asymptomatic patients with ovarian teratoma ( mangler et al . in general , ovarian tumors have high potential to mount an immune response , since anti - ovarian autoantibodies are commonly found in patients affected with these neoplasms ( szubert et al . the detection of antibodies is a major reason to raise the suspicion of paraneoplastic syndrome in a patient who manifests neurologic symptoms . the type of antibody indicates the most probable tumor location , including ovarian cancer and ovarian teratoma . an analysis of cerebrospinal fluid ( csf ) may display pleocytosis ( with a high fraction of lymphocytes ) , elevated protein or oligoclonal bands ( frings et al . 2005 ; rubello et al . inflammatory changes and anti - nmdar antibodies in csf are frequently present in anti - nmdar encephalitis ( vitaliani et al . in some patients with pcd , however , no abnormalities have been detected in the csf ( peterson et al . though the analysis of csf often reveals disturbances in pns , it is more informative in anti - nmdar encephalitis than in pcd . magnetic resonance imaging ( mri ) in pcd can reveal cerebellar atrophy ( frings et al . 2005 ) . this is consistent with pathological postmortem examination , which demonstrates significant loss of purkinje cells and sometimes coexistent infiltrates within cerebellum . in some pcd patients , , in turn , imaging most commonly reveals cerebellar hypometabolism in pcd ( basu and alavi 2008 ) . rarely , increased fdg uptake , consistent with hypermetabolism , is found in the cerebellar region ( choi et al . they help to exclude other conditions related to neoplasm that may give rise to neurologic symptoms , such as metastases , infiltration or vascular complications . mri in anti - nmdr encephalitis in 50 % of patients shows hyperintensity in various brain regions , including hippocampus , corpus callosum , temporal and frontal lobes , while in other patients the brain image appears normal ( dalmau et al . though imaging studies are not key to establish the diagnosis of pns so far , they are indispensable in excluding other conditions responsible for neurologic symptoms . abnormally slow and disorganized activity is usually not associated with anomalous movements and is unresponsive to antiepileptic therapy ( dalmau et al . the onset of signs and symptoms of paraneoplastic syndrome is usually subacute and develops within a few weeks . the patient suffers from incoordination of movements ( ataxia ) , balance and gait disturbances , speech disorder ( dysarthria ) and altered ocular movements ( nystagmus , often in a downbeat form ) ( peterson et al . 1992 ; dalmau and rosenfeld 2008 ) . paraneoplastic neurological syndromes associated with ovarian tumors may also appear as a peripheral polyneuropathy with diffuse paresthesia and anesthesia ( cao et al . young women or children with ovarian teratoma can be affected with encephalitis that manifests as psychosis , memory loss and behavior disorder . it subsequently develops into seizures , dyskinesias and autonomic instability ( florance et al . 2009 ; tanyi et al . 2012 ; zuliani et al . 2012 ) . neurologic pathology is often highly debilitating and renders many patients either wheelchair bound or bedridden ( frings et al . they are detected by means of an indirect immunofluorescence and confirmatory western or line blot ( monstad et al . the most common type of antibody found in pcd is anti - cdr2 ( cerebellar degeneration protein-2 antibody ) ( also known as anti - yo ) ( anderson et al . anti - yo antibody seropositive status suggests ovarian cancer , breast cancer or other gynecologic malignancies ( peterson et al . 1992 ) . infrequently , it can be related to small cell lung cancer ( greenlee and lipton 1986 ) . recently , new antibodies , termed anti - cdr2-like ( cdr2l ) , have been described ( eichler et al . they are reactive against an antigen similar to the cdr2 protein , which instead of being cytoplasmic is within the cell membrane . paraneoplastic cerebellar syndrome may also be associated with anti - hu antibodies which raises suspicion of small cell lung cancer . the detection of anti - ri , in turn , suggests an underlying breast cancer . clinician should also take into consideration the diagnosis of lymphoma , especially if anti - tr or anti - mglur1 antibodies are identified ( shamsili et al . interestingly , pcd related to anti - yo antibodies is associated with a worse survival due to cancer as compared to other antibodies ( giometto et al . 2009 ; zuliani et al . 2012 ) . a substantial number of patients , however , manifest paraneoplastic syndromes unrelated to detection of any of the above antibodies ( giometto et al . 2010 ) . in addition , onconeuronal antibodies appear in some patients with ovarian tumors who do not suffer from any neurological symptoms . in a group of 181 women with ovarian cancer without paraneoplastic syndrome , there were four who had anti - yo and seven who had anti - ri onconeuronal antibodies ( drlicek et al . the detection of anti - cdr2 antibodies in ovarian cancer can be improved by in vitro transcription translation ( itt ) of radiolabelled cdr2 protein and immunoprecipitation assay ( monstad et al . anti - nmdar antibodies were detected in six out 21 women with teratoma ( michalak et al . 2010 ) . in another study , however , anti - nmdar antibodies were not found among 20 neurologically asymptomatic patients with ovarian teratoma ( mangler et al . 2013 ) . in general , ovarian tumors have high potential to mount an immune response , since anti - ovarian autoantibodies are commonly found in patients affected with these neoplasms ( szubert et al . the detection of antibodies is a major reason to raise the suspicion of paraneoplastic syndrome in a patient who manifests neurologic symptoms . the type of antibody indicates the most probable tumor location , including ovarian cancer and ovarian teratoma . an analysis of cerebrospinal fluid ( csf ) may display pleocytosis ( with a high fraction of lymphocytes ) , elevated protein or oligoclonal bands ( frings et al . 2005 ; rubello et al . 2005 ; tanyi et al . 2012 ) . inflammatory changes and anti - nmdar antibodies in csf are frequently present in anti - nmdar encephalitis ( vitaliani et al . , however , no abnormalities have been detected in the csf ( peterson et al . though the analysis of csf often reveals disturbances in pns , it is more informative in anti - nmdar encephalitis than in pcd . magnetic resonance imaging ( mri ) in pcd can reveal cerebellar atrophy ( frings et al . 2005 ) . this is consistent with pathological postmortem examination , which demonstrates significant loss of purkinje cells and sometimes coexistent infiltrates within cerebellum . in some pcd patients , however , both mri and ct scan can appear normal ( negishi et al . 2013 ) . the fluorodeoxyglucose positron - emission tomography ( fdg - pet ) , in turn , imaging most commonly reveals cerebellar hypometabolism in pcd ( basu and alavi 2008 ) . rarely , increased fdg uptake , consistent with hypermetabolism , is found in the cerebellar region ( choi et al . they help to exclude other conditions related to neoplasm that may give rise to neurologic symptoms , such as metastases , infiltration or vascular complications . mri in anti - nmdr encephalitis in 50 % of patients shows hyperintensity in various brain regions , including hippocampus , corpus callosum , temporal and frontal lobes , while in other patients the brain image appears normal ( dalmau et al . though imaging studies are not key to establish the diagnosis of pns so far , they are indispensable in excluding other conditions responsible for neurologic symptoms . abnormally slow and disorganized activity is usually not associated with anomalous movements and is unresponsive to antiepileptic therapy ( dalmau et al . pns can provide an opportunity to investigate naturally occurring antitumor immunity ( albert and darnell 2004 ) . in hu syndrome associated with small cell lung cancer , the beneficial effect on survival of early detection due to pns symptoms is well proven and several cases of tumor regression have been reported ( darnell and deangelis 1993 ; graus et al . it is a question of debate , however , whether such a phenomenon applies to all tumors , including ovarian cancer . specific cytotoxic t lymphocytes active against cdr2 antigen expressed by ovarian cancer and purkinje cells were identified in peripheral blood in patients with pcd ( albert et al . 1998 ) . in some patients with ovarian cancer coexisting with pcd , tumors are limited in size and only discovered by microscopic examination ( peterson et al . 1992 ) and have fewer secondary foci in comparison with patients without pcd ( hetzel et al . these tumors are characterized as lesion with intense lymphocyte infiltration characteristic of an immune response ( peterson et al . 1992 ; cao et al . 1999 ) . on the other hand , in another study , metastatic disease was discovered in 15 out of 18 patients with ovarian cancer and pcd , with a median survival of 22 months comparable to patients with this form of cancer without pcd ( rojas et al . . however , it can be hard to estimate an effect of antitumor response on overall survival as nearly half of the patients with pns die from neurologic pathology ( rojas et al . currently , in pcd associated with ovarian cancer , there is no study that explicitly corroborates an effective antitumor response in terms of prognosis . further , the presence of anti - yo antibodies in neurologically normal patients with ovarian cancer had no influence on survival ( drlicek et al . hence , the antitumor reaction is probably effective at the very beginning of tumor development , so it less advanced at the time of diagnosis . for unknown reason , this response fails to eliminate cancer cells and the tumor progresses in a natural aggressive way . ovarian tumors associated with anti - nmdar encephalitis include mainly teratomas ( dalmau et al . 2007 , 2011 ) . among 91 women with anti - nmdar encephalitis , 49 had ovarian teratoma , including 17 immature tumors and eight bilateral ovarian lesions ( dalmau et al . cases of sex cord stromal tumors coexistent with anti - nmdar encephalitis were also reported ( tanyi et al . 2012 ) . pathological studies revealed the presence of nervous tissue with the expression of nr2 subunit of nmda receptor within all teratomas ( dalmau et al . significant inflammatory infiltrates were also demonstrated in tumors associated with anti - nmdar encephalitis ( tzn et al . the strong link between anti - nmdar encephalitis and ovarian teratomas incidence can be well explained by the cross - presentation of the same antigen . autoimmune processes undoubtedly take part in pathogenesis of pns ( darnell and posner 2003 ) . the major hypothesis about the origins of pns states that tumors express antigens that are normally present almost exclusively in nervous tissue ( fig . the presentation of neuronal antigens by a neoplasm then mounts an intense immune response against the tumor which cross - reacts with the nervous tissue . to study this hypothesis , sera of patients affected with pcd were incubated with ovarian cancer tissue from individuals without neurologic pathology ( darnell et al . 2000 ) . in 13 out of 20 tissues , an antigen cdr2 ( also called yo protein ) was detected in both cerebellar neurons and ovarian tumors . this cdr2 antigen was also expressed in 2 out of 9 breast cancer specimens as well . in one study , it appears that many ovarian cancers express this protein , irrespective of the presence of anti - yo antibodies or manifestation of pcd in patients ( darnell et al . the same relationship was observed in paraneoplastic neurological syndromes that accompany other malignancies , for example , with all small cell lung cancers possessing the hu antigen and only some patients developing paraneoplastic hu syndrome ( manley et al . thus , the presence of onconeuronal antigens and antibodies against them does not completely correlate with the origin of paraneoplastic reaction.fig . the same cdr2 antigen is a intracellular protein in purkinje cell in cerebellum . as a result , this mechanism represents the prevailing view on the pathogenesis of paraneoplastic cerebellar degeneration ( pcd ) related to ovarian cancer . the hallmarks of immune reaction , however , are not detected in all patients ovarian cancer cell expresses cdr 2 antigen that triggers immune response against malignancy . the same cdr2 antigen is a intracellular protein in purkinje cell in cerebellum . as a result , this mechanism represents the prevailing view on the pathogenesis of paraneoplastic cerebellar degeneration ( pcd ) related to ovarian cancer . the hallmarks of immune reaction , however , are not detected in all patients a detection of onconeuronal antibodies is important for the syndrome diagnosis , even though it is not compulsory according to diagnostic criteria ( graus et al . the importance of this humoral response in the pathomechanism of the disease , however , is unclear . the passive transfer of antibodies to onconeuronal antigens did not lead to neurologic pathology in mouse model ( tanaka et al . further , immunization of mice with human cdr2 antigen induced only antibody production in the absence of nervous tissue damage ( tanaka et al . it appears that lymphocytes taken from patients blood samples were also active against the same antigens as antibodies used in diagnosis ( albert et al . specific cytotoxic t lymphocytes have been found both in acute and chronic phase of pcd . in pcd associated with the anti - yo antibody , a high frequency of hla a24 has been demonstrated ( tanaka and tanaka 1996 ) . this observation suggests that patients in this group are more susceptible to autoimmune disorder , which is consistent with the involvement of cytotoxic t lymphocytes . it seems , on the other hand , that cell - mediated reaction is not always involved in neuronal damage in the course of pns , as pathology studies do not always reveal infiltrates that accompany significant neuronal loss ( peterson et al . 1992 ) and a number of patients with pcd lack antibodies to onconeuronal antigens ( anderson et al . cerebellar degeneration with purkinje cell loss has been described without any detectable onconeuronal antibodies ( michalak 2008 ) . the recent identification of anti - cdr2l antibodies may shed new light on the mechanism involved in pcd emergence ( eichler et al . 2013 ) , and other , as yet unidentified , onconeural antigens may still be discovered . in contrast to the cdr2 protein that is cytoplasmic , cdr2l antigen is present at the cell membrane which may make it more accessible for antibody - mediated cytotoxicity . moreover , when both present , anti - cdr2 and anti - cdr2l antibodies had much higher avidity than when detected alone . it was consistent with the fact that only patients with both antibody types developed definite pns in contrast to individuals with either anti - cdr2 or anti - cdr2l alone . cdr2l antigen is expressed both in purkinje cells in cerebellum and in ovarian tumor tissue . the role of anti - cdr2l in the mechanism of pcd certainly requires further exploration . taken together , the immune reaction , especially cell - mediated response , is an important player in the origins of pcd . contradictory results of research in animal models , however , suggest that other factors potentially deleterious to nervous tissue may also be involved . cdr2 is involved in the inhibition of c - myc oncoprotein that is widely expressed in many tumors , including ovarian cancer ( okano et al . cdr2 expression presumably represses cell proliferation through this mechanism , functioning as a tumor suppressor . elevated expression of cdr2 in a tumor tissue may lead to immune responses that are also active against nervous tissue . over - expression of cdr2 could explain why pcd appears in neoplasms that have a less aggressive course , since suppressive mechanisms mediated by cdr2 are constitutively more effective . since in pns , tumors have often intense infiltrates and the significance of cell - mediated immune response has been revealed , it is probable that immune responses eliminate cancer cells . by contrast , the pathogenic role of antibodies seems well proven in anti - nmdar encephalitis ( fig . 2 ) . in vitro studies revealed that rat neurons incubated with patients sera containing anti - nmdar antibodies had reduced numbers of nmda receptors in postsynaptic dendrites . moreover , in anti - nmdar encephalitis , clinical improvement is associated with a decrease in nmdr antibody titers ( dalmau et al . pathological studies have demonstrated numerous antibody deposits and scarce cell infiltrates in affected regions of brain ( tzn et al . 2009 ) . notably , the nmdr antigens for these antibodies were identified mainly in hippocampus ( vitaliani et al . in contrast to intracellular onconeuronal antigens , the target protein was detected within the plasma membrane which makes it more accessible to antibodies . a favorable clinical response to plasma exchange also provides an argument in favor of antibody - mediated mechanism of disease . nmdar antigen has been found in all ovarian teratomas excised in the course of anti - nmdar encephalitis ( tzn et al . these observations suggest that the cross - presentation of nmdar antigen in neurons and ovarian tumor leads to the anti - nmdar antibodies production , which induces encephalitis.fig . 2nmda receptor ( nmdar ) is expressed on the surface of ovarian teratoma cells . as a result of immune reaction , anti - the same receptor is present at the dendrites of neurons in many region of central nervous system . anti - nmdar antibodies enter nervous tissue through the vasculature and react against the target protein . consequently , neural signaling mediated by nmda receptor is considerably disturbed leading to both psychiatric and neurologic symptoms nmda receptor ( nmdar ) is expressed on the surface of ovarian teratoma cells . as a result of immune reaction , the same receptor is present at the dendrites of neurons in many region of central nervous system . anti - nmdar antibodies enter nervous tissue through the vasculature and react against the target protein . consequently , neural signaling mediated by nmda receptor is considerably disturbed leading to both psychiatric and neurologic symptoms diagnosis of pcd should always be associated with a search for a primary origin of malignancy . this nervous system pathology is most commonly seen in ovarian , breast and small cell lung cancers or lymphoma ( peterson et al . 1992 ) . in some patients , an ovarian tumor can be visualized by transvaginal ultrasound or by pelvic ct scan ( negishi et al . the neoplastic disease , however , is frequently at a very early stage and consequently a conventional diagnostic procedure may appear inconclusive . a number of studies show that the use of fdg - pet imaging is more sensitive and justified in this clinical context ( frings et al . 2005 ) . an ovarian tumor that was not visible in transvaginal ultrasound appeared evident on the integrated pet surgery confirmed the diagnosis of a papillar serous adenocarcinoma of an ovary at stage iib . an annual follow - up of another patient with pcd uncovered hyperactivity of an axillary region 5 years after the onset of neurologic symptoms ( mathew et al . combined fdg - pet imaging is the only one that can reveal the location of the malignancy ( rubello et al . it is rather the antigenic specificity than particular neurologic syndromes that is more indicative for tumor location ( titulaer et al . the most specific for diagnosis , however , is the combination of both . in patients with anti - yo if anti - nmdar antibody is detected , it suggests coexistence of teratoma , including its immature type . if it is negative , pelvic computed tomography or magnetic resonance imaging should be performed . in patients with antibodies that suggest ovarian cancer , an integrated fdg - pet / it is important to perform it as soon as possible as combined pet / ct scan used in this context may significantly reduce the delay time to the surgery ( frings et al . 2005 ) . if initial investigations do not detect any tumor , it is recommended to repeat them every 6 months for 4 years . thus , it is not surprising that the neurological outcome in pcd may improve after tumor excision . subsequent systemic immune therapy in the form of intravenous immunoglobulin , steroids and tacrolimus has also proven beneficial ( negishi et al . 2013 ) . in young women affected with nmdr encephalitis associated with teratoma , surgical removal of the tumor in combination with plasma exchange , steroid and intravenous immunoglobulin may yield almost complete recovery ( tanyi et al . 2012 ) . in some patients , a second - line immunotherapy ( rituximab or cyclophosphamide ) it has been proven that early surgery combined with immunotherapy markedly improves outcome ( dalmau et al . the question arises whether the diagnosis of paraneoplastic reaction commonly associated with ovarian tumor may itself constitute an indication for surgical management . exhaustive laboratory and imaging investigations often remain inconclusive as to the presence and location of the tumor . some reports suggest , however , that explorative surgery may reveal an occult neoplasm . in a group of 19 patients with both ovarian cancer and paraneoplastic cerebellar syndrome , , there were no tumor symptoms and the laboratory as well as imaging investigation was normal prior to the surgery . the decision on operative management was taken exclusively based on the detection of onconeuronal antibodies . surprisingly , all these patients had high - grade ovarian adenocarcinoma . another remarkable observation made by the authors was either the lack of or limited volume of peritoneal invasion that are typical for advance ovarian cancer . in another study , laparotomy performed in some patients with pcd uncovered solely microscopic foci of ovarian cancer ( peterson et al . these findings are consistent with a report of postmortem examination of a patient who died due to very severe paraneoplastic reaction affecting the nervous system ( brain et al . 1951 ) . it revealed two very small ovarian tumors the size of which did not exceed 2 cm in diameter . the history of a patient with pcd and abnormal ca-125 levels at postmenopausal age has also been reported ( mason et al . though imaging studies did not suggest ovarian pathology , both laparoscopic salpingo - oophorectomy and endometrial biopsy were performed . the pathological examination did not confirm malignancy ; however , the ca-125 level decreased to normal limits after ovarian resection . another report presented the history of a patient with pcd whose retroperitoneal lymph node was enlarged to the size of 2.2 cm and ca125 was highly elevated . though during laparotomy adnexa were normal macroscopically , pathological examination revealed the presence of a microscopic adenocarcinoma of the fallopian tube ( frings et al . 2005 ) . in a 53-year - old patient with anti - nmdar encephalitis whose neurologic and psychiatric condition was deteriorating , the decision of abdominal laparoscopy was made in spite of normal abdominal and pelvic ct scan . though both adnexa were macroscopically normal , the pathologic examination revealed a sex cord tumor ( tanyi et al . 2012 ) . in a group of adult women affected with anti - nmdar encephalitis , it should be noted that this fraction was significantly lower in adolescents and children ( 31 % ) . ovarian resection in patients with pns without clear diagnosis of cancer can be considered effective in terms of both malignancy and neurologic outcome . this can have considerable value in neoplasm therapy , for often the surgery reveals very early stage disease . it is also suggested by some authors that an early management of malignancy may prevent a more severe neurologic deterioration that may remain irreversible and highly debilitating otherwise ( vedeler et al . it has been confirmed that antitumor treatment improves overall survival in these patients ( shamsili et al . , an emergent surgery contributes substantially to the final outcome and may enable even almost complete recovery ( dalmau et al . 2007 ; florance et al . 2009 ; tanyi et al . 2012 ) . even if serial ct or ultrasound is not suggestive of teratoma affected with anti - nmdar encephalitis , blind oophorectomy was performed revealing an underlying teratoma and the resection was associated with significant neurological improvement ( johnson et al . in contrast , an excision of ovaries in combination with immunotherapy led to clinical improvement in another patient , but surprisingly the pathological examination was normal ( parratt et al . , it seems reasonable to perform an explorative laparoscopy or laparotomy in patients with paraneoplastic cerebellar syndrome or anti - nmdar encephalitis whose imaging studies appear normal . after precise exclusion of other neurological aetiologies and considering the patient s age , the salpingo - oophorectomy of macroscopically normal adnexa may appear beneficial . the clinical manifestation of ovarian tumors can be preceded by neurological deficit , particularly in cases with ovarian cancer or teratoma . the detection of onconeural antibodies ( e.g. , anti - yo ) strongly indicates the presence of ovarian tumor in patients with neurological deficit . if the tumor is not identified , an obligation for systematic screening exists . surgery and immunomodulatory treatment are considered as the most important management among such a group of patients .

introductionparaneoplastic neurological syndromes ( pns ) are neurologic deficits triggered by an underlying remote tumor . pns can antedate clinical manifestation of ovarian malignancy and enable its diagnosis at an early stage . interestingly , neoplasms associated with pns are less advanced and metastasize less commonly than those without pns . this suggests that pns may be associated with a naturally occurring antitumor response.methodswe review the literature on the diagnosis , pathogenesis and management of pns associated with ovarian tumors : paraneoplastic cerebellar degeneration ( pcd ) and anti - n - methyl - d - aspartate receptor ( anti - nmdar ) encephalitis . an approach to the diagnostic workup of underlying tumors is discussed.resultspcd can precede the manifestation of ovarian carcinoma . anti - nmdar encephalitis in young women appears often as a result of ovarian teratoma . since ovarian tumors and nervous tissue share common antigens ( e.g. , cdr2 , nmdar ) , autoimmune etiology is a probable mechanism of these neurologic disorders . the concept of cross - presentation , however , seems insufficient to explain entirely the emergence of pns . early resection of ovarian tumors is a significant part of pns management and improves the outcome.conclusionsthe diagnosis of pns potentially associated with ovarian tumor indicates a need for a thorough diagnostic procedure in search of the neoplasm . in some patients , explorative laparoscopy / laparotomy can be considered .

Key points Definition Incidence Diagnosis Clinical presentation Antibodies CSF analysis Imaging studies Electroencephalogram (EEG) Ovarian tumors Pathogenesis Management Summary Conflict of interest

pns precede clinical manifestation of ovarian tumors and enable their diagnosis at an early stage.paraneoplastic cerebellar degeneration ( pcd ) can coexist with ovarian carcinoma.anti-nmdar antibodies detected in patient affected with encephalitis are highly suggestive of ovarian teratoma.ovarian tumors and nervous tissue share common antigens in pns ( e.g. , cdr2 , nmda receptor)the concept of cross - presentation , however , may not be sufficient to explain an emergence of pns.cell-mediated immune response plays a role in the pathogenesis of pns.antibody-mediated immune response is a major mechanism of anti - nmdar encephalitis and other nsas.in patients with pns associated potentially with ovarian tumors , transvaginal ultrasound examination and pelvic ct scan are indicated if negative , pet imaging is required.if imaging studies remain normal , explorative laparoscopy / laparotomy should be considered.an early resection of ovarian tumors is a significant part of pns management and improves the outcome . pns precede clinical manifestation of ovarian tumors and enable their diagnosis at an early stage . anti - nmdar antibodies detected in patient affected with encephalitis are highly suggestive of ovarian teratoma . ovarian tumors and nervous tissue share common antigens in pns ( e.g. , cdr2 , nmda receptor)the concept of cross - presentation , however , may not be sufficient to explain an emergence of pns . antibody - mediated immune response is a major mechanism of anti - nmdar encephalitis and other nsas . in patients with pns associated potentially with ovarian tumors , transvaginal ultrasound examination and pelvic ct scan are indicated if negative , pet imaging is required . an early resection of ovarian tumors is a significant part of pns management and improves the outcome . paraneoplastic neurological syndromes ( pns ) are defined as the pathologic involvement of the nervous system in the course of malignancy . pns often precede clinical manifestation of a tumor and enable diagnosis at an early stage . interestingly , neoplasms that appear in patients with pns are limited in size and metastasize less commonly than those without pns ( hetzel et al . ovarian tumors account for about 10 % of malignancies associated with pns ( giometto et al . paraneoplastic cerebellar degeneration ( pcd ) , the most common paraneoplastic neurological syndrome , may coexist with ovarian carcinoma ( giometto et al . recently , a group of neurologic disorders , neuronal surface antibodies syndromes ( nsas ) , has been defined ( zuliani et al . one of nsas , however , anti - nmdar encephalitis , appears typically in young women with a benign tumor , ovarian teratoma . the diagnosis of pns is based on criteria defined in graus et al . the diagnosis of pns is a complex process that always requires exclusion of primary etiology of neurologic symptoms , including among others vascular disorders , infection , nervous tissue tumors or hereditary syndromes . hence , once the diagnosis of anti - nmdar encephalitis is established , it is classified as definite nsas if it is responsive to immunomodulatory agents such as steroid and intravenous immunoglobulin and as probable nsas if not responsive ( zuliani et al . 2012).table 1classical paraneoplastic syndromesclassical paraneoplastic neurological syndromesencephalomyelitislimbic encephalitis subacute cerebellar degeneration opsoclonus myoclonus subacute sensory neuronopathy chronic gastrointestinal pseudo - obstructionlambert eaton myasthenic syndromedermatomyositispns the most commonly associated with ovarian tumors are in bold ( graus et al . 2004)table 2onconeural and neuronal surface antibodiesonconeural and neuronal surface antibodieswell characterizedpartly characterizedneuronal surface antibodiesanti - hu anti - yo anti - ri anti - cv2anti - ma anti - amphiphysin anti - tranti - zic4anti - mglur1anna3pca2anti - vgkc complex antigens ( lgi1 or caspr2 ) anti - nmdar anti - amparanti - gababranti - glyranti - vgcc - abanti - mglur1anti - mglur5antibodies detected commonly in pns associated with ovarian tumors are in bold ( graus et al . a classical syndrome and cancer that develop within 5 years of the diagnosis of the neurological disorder1 . a non - classical syndrome with onconeural antibodies ( well characterized or not ) and cancer that develops within 5 years of the diagnosis of the neurological disorder3 . a neurological syndrome ( classical or not ) with well - characterized onconeural antibodies ( anti - hu , yo , cv2 , ri , ma2 or amphiphysin ) and no cancer neurological syndrome with well - characterized antibodies ( in bold ) is a clinical setting that requires intense search for underlying malignancy and often enables its detection at an early stage . reproduced from recommended diagnostic criteria for paraneoplastic neurological syndromes ( graus et al . classical paraneoplastic syndromes pns the most commonly associated with ovarian tumors are in bold ( graus et al . 2004 ) onconeural and neuronal surface antibodies antibodies detected commonly in pns associated with ovarian tumors are in bold ( graus et al . ( 2004 ) neurological syndrome with well - characterized antibodies ( in bold ) is a clinical setting that requires intense search for underlying malignancy and often enables its detection at an early stage . in 55 women with ovarian cancer , neurologic disorder these data should , however , be interpreted with caution as since the time this study was performed the diagnostic criteria of pns have evolved markedly . the diagnosis , however , becomes more probable in particular contexts approximately half of patients with non - familial cerebellar syndrome of subacute onset suffer from some malignancy , primarily ovarian or lung cancer . antibodies in anti - nmdar encephalitis typically associated with ovarian teratoma were described for the first time in 2005 ( vitaliani et al . it is worth mentioning that three cases of anti - nmdar encephalitis occurred during pregnancy complicated with ovarian teratoma ( kumar et al . though pns are still considered rare , their estimates of incidence increase with the improvement in diagnostic tools it is worth stressing that a combination of classical pns and the detection of well - characterized antibodies are known to be involved in pns , even without established diagnosis of malignancy is also referred to as definite paraneoplastic disorder ( table 3 ) . in such cases the neurological symptoms antedated the diagnosis of a neoplasm in 34 individuals ( peterson et al . by contrast , anti - nmdar encephalitis that affects women with ovarian teratoma responds well to tumor resection and subsequent immunotherapy which may lead to almost complete recovery . paraneoplastic neurological syndromes associated with ovarian tumors may also appear as a peripheral polyneuropathy with diffuse paresthesia and anesthesia ( cao et al . young women or children with ovarian teratoma can be affected with encephalitis that manifests as psychosis , memory loss and behavior disorder . paraneoplastic cerebellar syndrome may also be associated with anti - hu antibodies which raises suspicion of small cell lung cancer . clinician should also take into consideration the diagnosis of lymphoma , especially if anti - tr or anti - mglur1 antibodies are identified ( shamsili et al . interestingly , pcd related to anti - yo antibodies is associated with a worse survival due to cancer as compared to other antibodies ( giometto et al . a substantial number of patients , however , manifest paraneoplastic syndromes unrelated to detection of any of the above antibodies ( giometto et al . in addition , onconeuronal antibodies appear in some patients with ovarian tumors who do not suffer from any neurological symptoms . the detection of anti - cdr2 antibodies in ovarian cancer can be improved by in vitro transcription translation ( itt ) of radiolabelled cdr2 protein and immunoprecipitation assay ( monstad et al . in another study , however , anti - nmdar antibodies were not found among 20 neurologically asymptomatic patients with ovarian teratoma ( mangler et al . inflammatory changes and anti - nmdar antibodies in csf are frequently present in anti - nmdar encephalitis ( vitaliani et al . in some patients with pcd , however , no abnormalities have been detected in the csf ( peterson et al . though the analysis of csf often reveals disturbances in pns , it is more informative in anti - nmdar encephalitis than in pcd . though imaging studies are not key to establish the diagnosis of pns so far , they are indispensable in excluding other conditions responsible for neurologic symptoms . paraneoplastic neurological syndromes associated with ovarian tumors may also appear as a peripheral polyneuropathy with diffuse paresthesia and anesthesia ( cao et al . young women or children with ovarian teratoma can be affected with encephalitis that manifests as psychosis , memory loss and behavior disorder . paraneoplastic cerebellar syndrome may also be associated with anti - hu antibodies which raises suspicion of small cell lung cancer . the detection of anti - ri , in turn , suggests an underlying breast cancer . clinician should also take into consideration the diagnosis of lymphoma , especially if anti - tr or anti - mglur1 antibodies are identified ( shamsili et al . interestingly , pcd related to anti - yo antibodies is associated with a worse survival due to cancer as compared to other antibodies ( giometto et al . a substantial number of patients , however , manifest paraneoplastic syndromes unrelated to detection of any of the above antibodies ( giometto et al . in addition , onconeuronal antibodies appear in some patients with ovarian tumors who do not suffer from any neurological symptoms . the detection of anti - cdr2 antibodies in ovarian cancer can be improved by in vitro transcription translation ( itt ) of radiolabelled cdr2 protein and immunoprecipitation assay ( monstad et al . in another study , however , anti - nmdar antibodies were not found among 20 neurologically asymptomatic patients with ovarian teratoma ( mangler et al . an analysis of cerebrospinal fluid ( csf ) may display pleocytosis ( with a high fraction of lymphocytes ) , elevated protein or oligoclonal bands ( frings et al . inflammatory changes and anti - nmdar antibodies in csf are frequently present in anti - nmdar encephalitis ( vitaliani et al . though the analysis of csf often reveals disturbances in pns , it is more informative in anti - nmdar encephalitis than in pcd . in some pcd patients , however , both mri and ct scan can appear normal ( negishi et al . mri in anti - nmdr encephalitis in 50 % of patients shows hyperintensity in various brain regions , including hippocampus , corpus callosum , temporal and frontal lobes , while in other patients the brain image appears normal ( dalmau et al . though imaging studies are not key to establish the diagnosis of pns so far , they are indispensable in excluding other conditions responsible for neurologic symptoms . pns can provide an opportunity to investigate naturally occurring antitumor immunity ( albert and darnell 2004 ) . it is a question of debate , however , whether such a phenomenon applies to all tumors , including ovarian cancer . in some patients with ovarian cancer coexisting with pcd , tumors are limited in size and only discovered by microscopic examination ( peterson et al . on the other hand , in another study , metastatic disease was discovered in 15 out of 18 patients with ovarian cancer and pcd , with a median survival of 22 months comparable to patients with this form of cancer without pcd ( rojas et al . however , it can be hard to estimate an effect of antitumor response on overall survival as nearly half of the patients with pns die from neurologic pathology ( rojas et al . currently , in pcd associated with ovarian cancer , there is no study that explicitly corroborates an effective antitumor response in terms of prognosis . ovarian tumors associated with anti - nmdar encephalitis include mainly teratomas ( dalmau et al . among 91 women with anti - nmdar encephalitis , 49 had ovarian teratoma , including 17 immature tumors and eight bilateral ovarian lesions ( dalmau et al . cases of sex cord stromal tumors coexistent with anti - nmdar encephalitis were also reported ( tanyi et al . significant inflammatory infiltrates were also demonstrated in tumors associated with anti - nmdar encephalitis ( tzn et al . the strong link between anti - nmdar encephalitis and ovarian teratomas incidence can be well explained by the cross - presentation of the same antigen . in one study , it appears that many ovarian cancers express this protein , irrespective of the presence of anti - yo antibodies or manifestation of pcd in patients ( darnell et al . the same relationship was observed in paraneoplastic neurological syndromes that accompany other malignancies , for example , with all small cell lung cancers possessing the hu antigen and only some patients developing paraneoplastic hu syndrome ( manley et al . as a result , this mechanism represents the prevailing view on the pathogenesis of paraneoplastic cerebellar degeneration ( pcd ) related to ovarian cancer . the hallmarks of immune reaction , however , are not detected in all patients ovarian cancer cell expresses cdr 2 antigen that triggers immune response against malignancy . as a result , this mechanism represents the prevailing view on the pathogenesis of paraneoplastic cerebellar degeneration ( pcd ) related to ovarian cancer . the hallmarks of immune reaction , however , are not detected in all patients a detection of onconeuronal antibodies is important for the syndrome diagnosis , even though it is not compulsory according to diagnostic criteria ( graus et al . the importance of this humoral response in the pathomechanism of the disease , however , is unclear . in pcd associated with the anti - yo antibody , a high frequency of hla a24 has been demonstrated ( tanaka and tanaka 1996 ) . it seems , on the other hand , that cell - mediated reaction is not always involved in neuronal damage in the course of pns , as pathology studies do not always reveal infiltrates that accompany significant neuronal loss ( peterson et al . the recent identification of anti - cdr2l antibodies may shed new light on the mechanism involved in pcd emergence ( eichler et al . contradictory results of research in animal models , however , suggest that other factors potentially deleterious to nervous tissue may also be involved . by contrast , the pathogenic role of antibodies seems well proven in anti - nmdar encephalitis ( fig . moreover , in anti - nmdar encephalitis , clinical improvement is associated with a decrease in nmdr antibody titers ( dalmau et al . nmdar antigen has been found in all ovarian teratomas excised in the course of anti - nmdar encephalitis ( tzn et al . these observations suggest that the cross - presentation of nmdar antigen in neurons and ovarian tumor leads to the anti - nmdar antibodies production , which induces encephalitis.fig . 2nmda receptor ( nmdar ) is expressed on the surface of ovarian teratoma cells . as a result of immune reaction , anti - the same receptor is present at the dendrites of neurons in many region of central nervous system . anti - nmdar antibodies enter nervous tissue through the vasculature and react against the target protein . consequently , neural signaling mediated by nmda receptor is considerably disturbed leading to both psychiatric and neurologic symptoms nmda receptor ( nmdar ) is expressed on the surface of ovarian teratoma cells . as a result of immune reaction , the same receptor is present at the dendrites of neurons in many region of central nervous system . anti - nmdar antibodies enter nervous tissue through the vasculature and react against the target protein . consequently , neural signaling mediated by nmda receptor is considerably disturbed leading to both psychiatric and neurologic symptoms diagnosis of pcd should always be associated with a search for a primary origin of malignancy . in some patients , an ovarian tumor can be visualized by transvaginal ultrasound or by pelvic ct scan ( negishi et al . the neoplastic disease , however , is frequently at a very early stage and consequently a conventional diagnostic procedure may appear inconclusive . an ovarian tumor that was not visible in transvaginal ultrasound appeared evident on the integrated pet surgery confirmed the diagnosis of a papillar serous adenocarcinoma of an ovary at stage iib . the most specific for diagnosis , however , is the combination of both . in young women affected with nmdr encephalitis associated with teratoma , surgical removal of the tumor in combination with plasma exchange , steroid and intravenous immunoglobulin may yield almost complete recovery ( tanyi et al . in some patients , a second - line immunotherapy ( rituximab or cyclophosphamide ) it has been proven that early surgery combined with immunotherapy markedly improves outcome ( dalmau et al . the question arises whether the diagnosis of paraneoplastic reaction commonly associated with ovarian tumor may itself constitute an indication for surgical management . exhaustive laboratory and imaging investigations often remain inconclusive as to the presence and location of the tumor . in another study , laparotomy performed in some patients with pcd uncovered solely microscopic foci of ovarian cancer ( peterson et al . in a 53-year - old patient with anti - nmdar encephalitis whose neurologic and psychiatric condition was deteriorating , the decision of abdominal laparoscopy was made in spite of normal abdominal and pelvic ct scan . in a group of adult women affected with anti - nmdar encephalitis , it should be noted that this fraction was significantly lower in adolescents and children ( 31 % ) . ovarian resection in patients with pns without clear diagnosis of cancer can be considered effective in terms of both malignancy and neurologic outcome . it is also suggested by some authors that an early management of malignancy may prevent a more severe neurologic deterioration that may remain irreversible and highly debilitating otherwise ( vedeler et al . even if serial ct or ultrasound is not suggestive of teratoma affected with anti - nmdar encephalitis , blind oophorectomy was performed revealing an underlying teratoma and the resection was associated with significant neurological improvement ( johnson et al . , it seems reasonable to perform an explorative laparoscopy or laparotomy in patients with paraneoplastic cerebellar syndrome or anti - nmdar encephalitis whose imaging studies appear normal . the clinical manifestation of ovarian tumors can be preceded by neurological deficit , particularly in cases with ovarian cancer or teratoma . , anti - yo ) strongly indicates the presence of ovarian tumor in patients with neurological deficit .

migraine is a chronic debilitating neurological condition affecting 1220% of the population worldwide.1 preventive treatments are available , decreasing the number and severity of headache attacks , and improving health outcomes and quality of life ; half of the patients show 50% reduction in attack frequency.2 although migraine prophylaxis guidelines from north american , south american and european societies are available , only 35% of patients receive adequate preventive therapy.3 public surveys report that migraineurs are among the most dissatisfied patients.4 5 about half of the patients with migraine stop seeking care for their headaches , partly because of their side effect profile.6 most guidelines recommend topiramate , divalproex / sodium valproate , propranolol and metoprolol as having the highest level of evidence.7 newer effective and tolerable options for migraine prophylaxis are necessary to reduce this gap and improve patients ' quality of life . melatonin use in migraine prevention is supported by several biological effects.8 melatonin levels are decreased in patients with migraine ; it has been studied for migraine prophylaxis with conflicting results.911 we aimed to study the effect of melatonin in a double - blind , placebo controlled trial with an active comparator . patients with migraine were eligible for the study if they met inclusion criteria : age of 1865 years ; migraine with or without aura criteria according to the international classification of headache disorders , third edition , -version12 for at least 1 year , age of onset before 50 years , at least three migraine headache attacks or four migraine headache days ( defined as any occurrence of migraine headache pain of at least 30 min in duration with acute treatment ) per month , presents with migraine or non - migraine headache attacks < 15 days per month during each of the 3 months prior to the screening visit and the reference period . women were eligible if they were unable to bear children or if they were not pregnant and using adequate contraception . patients were excluded if they had a history of psychiatric disorder ( in the past or present ) ; ergotamine , triptan , opioid , or combination medication intake for > 10 days per month , or simple analgesic intake for > 15 days per month for > 3 months ; in use of preventive medications such as -blockers , tricyclic antidepressants , calcium channel blockers , antiepileptic drugs , bupropion , serotonergic norepinephrine reuptake inhibitors ; and were unable to discontinue the treatment ; had previously taken melatonin , amitriptyline or agomelatine ; or had uncontrolled hypertension ( ie , sitting systolic blood pressure > 160 mm hg or sitting diastolic blood pressure > 90 mm hg ) at the screening visit or at randomisation . patients were recruited from the general population , primary care , advertising and social media . if inclusion and exclusion criteria were met , they were invited for a screening visit . patients / service users / carers / laypeople were not involved in the study design . outcome measures were defined by scientific criteria , designed to better meet patients ' preferences , priorities and experience . results will be disseminated to patients / carers / laypeople by publication in the clinicaltrials.org database and to press / social media . patients with migraine were eligible for the study if they met inclusion criteria : age of 1865 years ; migraine with or without aura criteria according to the international classification of headache disorders , third edition , -version12 for at least 1 year , age of onset before 50 years , at least three migraine headache attacks or four migraine headache days ( defined as any occurrence of migraine headache pain of at least 30 min in duration with acute treatment ) per month , presents with migraine or non - migraine headache attacks < 15 days per month during each of the 3 months prior to the screening visit and the reference period . women were eligible if they were unable to bear children or if they were not pregnant and using adequate contraception . patients were excluded if they had a history of psychiatric disorder ( in the past or present ) ; ergotamine , triptan , opioid , or combination medication intake for > 10 days per month , or simple analgesic intake for > 15 days per month for > 3 months ; in use of preventive medications such as -blockers , tricyclic antidepressants , calcium channel blockers , antiepileptic drugs , bupropion , serotonergic norepinephrine reuptake inhibitors ; and were unable to discontinue the treatment ; had previously taken melatonin , amitriptyline or agomelatine ; or had uncontrolled hypertension ( ie , sitting systolic blood pressure > 160 mm hg or sitting diastolic blood pressure > 90 mm hg ) at the screening visit or at randomisation . patients were recruited from the general population , primary care , advertising and social media . if inclusion and exclusion criteria were met , they were invited for a screening visit . patients / service users / carers / laypeople were not involved in the study design . outcome measures were defined by scientific criteria , designed to better meet patients ' preferences , priorities and experience . results will be disseminated to patients / carers / laypeople by publication in the clinicaltrials.org database and to press / social media . randomisation was performed centrally with the use of randomisation lists with randomly permuted block lengths stratified according to centre . study medications were also blinded and delivered to the investigators by the pharmacy which prepared the three study medications equally in design , shape , and colour . the trial conformed to the declaration of helsinki , good clinical practice guidelines and the international organization for standardization standards and was approved by the ethics committee for each participating centre . the trial was funded by fapesp , fundao de amparo a pesquisa de so paulo , a brazilian governmental funding agency without any role in manuscript preparation . the study consisted of a 4-week period to established baseline measures followed by a 12-week treatment period . after eligibility had been determined , patients were randomised to treatment groups ( figure 1 ) . patients meeting eligibility criteria were randomised 1:1:1 to one of the three groups : placebo , melatonin 3 mg or amitriptyline 25 mg , all taken at bedtime . patients recorded information about migraine or non - migraine headache occurrence , mean migraine headache attack intensity , duration , analgesics taken , ( type of medication , number of days of medication use , number of doses ) and associated symptoms in a paper - based diary . trained neurologists in headache abstracted the information , which was double - checked by another investigator and uploaded into a spreadsheet . any migraine headache during at least 30 min was defined as a migraine headache day . the variables were collected from the diaries and defined at the end of the baseline period , and at the first ( weeks 14 ) , second ( weeks 58 ) and third ( weeks 912 ) months after treatment . the primary efficacy outcome measure was frequency in number of migraine headache days per month comparing baseline with the past 4 weeks of treatment . secondary end points included reduction in migraine intensity , attack duration , number of analgesics used and percentages of patients with greater than 50% reductions in migraine headache days . an adverse event was defined as any medical occurrence reported by a patient or noted by a clinician during the study , regardless of its suspected cause . tolerability measures included the incidences of adverse events , including those that led to the premature withdrawal of the study and serious adverse events ( defined as death , disability or incapacity ; were life - threatening ; or required hospitalisation ) . adverse events data were summarised for the safety population , defined as randomised patients who were administered at least one dose of the study medication . the randomisation ratio of 1:1:1 for placebo , and the two treatment groups , 59 participants in each groups were determined to provide 80% power to detect a significant difference in the primary endpoint . this estimate was based on a two - sided analysis of variance ( anova ) , an level of 0.05 and an sd of 4.0 . efficacy data were analysed for the intention - to - treat population , defined as randomised patients who received at least one dose of the study medication and provided at least one postbaseline efficacy assessment . the first method extended the calendar earlier into the treatment period until 28 days of non - missing data contributed to the count of migraine headache days . the second method proportionately adjusted the number of migraine headache days ( multiplied by 28 and divided by the number of non - missing days ) . the third method treated all missing days as non - migraine headache days ( used for the primary end point ) . an analysis of covariance ( ancova ) model was used to test the null hypothesis of no difference between placebo and the average of the values for the three groups . results were summarised using the adjusted mean and se for each treatment group , a 95% ci for the change from baseline for each treatment group , a model estimate of the difference between each active treatment group and placebo , a 95% ci for the difference , and an associated p value and adjusted p value for the difference . analysis of the primary end point was carried out using a combination of a sequential method and a hochberg procedure to maintain the experiment - wise level of 0.05 . the study consisted of a 4-week period to established baseline measures followed by a 12-week treatment period . after eligibility had been determined , patients were randomised to treatment groups ( figure 1 ) . patients meeting eligibility criteria were randomised 1:1:1 to one of the three groups : placebo , melatonin 3 mg or amitriptyline 25 mg , all taken at bedtime . patients recorded information about migraine or non - migraine headache occurrence , mean migraine headache attack intensity , duration , analgesics taken , ( type of medication , number of days of medication use , number of doses ) and associated symptoms in a paper - based diary . trained neurologists in headache abstracted the information , which was double - checked by another investigator and uploaded into a spreadsheet . any migraine headache during at least 30 min was defined as a migraine headache day . the variables were collected from the diaries and defined at the end of the baseline period , and at the first ( weeks 14 ) , second ( weeks 58 ) and third ( weeks 912 ) months after treatment . the primary efficacy outcome measure was frequency in number of migraine headache days per month comparing baseline with the past 4 weeks of treatment . secondary end points included reduction in migraine intensity , attack duration , number of analgesics used and percentages of patients with greater than 50% reductions in migraine headache days . an adverse event was defined as any medical occurrence reported by a patient or noted by a clinician during the study , regardless of its suspected cause . tolerability measures included the incidences of adverse events , including those that led to the premature withdrawal of the study and serious adverse events ( defined as death , disability or incapacity ; were life - threatening ; or required hospitalisation ) . adverse events data were summarised for the safety population , defined as randomised patients who were administered at least one dose of the study medication . the primary efficacy outcome measure was frequency in number of migraine headache days per month comparing baseline with the past 4 weeks of treatment . secondary end points included reduction in migraine intensity , attack duration , number of analgesics used and percentages of patients with greater than 50% reductions in migraine headache days . an adverse event was defined as any medical occurrence reported by a patient or noted by a clinician during the study , regardless of its suspected cause . tolerability measures included the incidences of adverse events , including those that led to the premature withdrawal of the study and serious adverse events ( defined as death , disability or incapacity ; were life - threatening ; or required hospitalisation ) . adverse events data were summarised for the safety population , defined as randomised patients who were administered at least one dose of the study medication . the randomisation ratio of 1:1:1 for placebo , and the two treatment groups , 59 participants in each groups were determined to provide 80% power to detect a significant difference in the primary endpoint . this estimate was based on a two - sided analysis of variance ( anova ) , an level of 0.05 and an sd of 4.0 . efficacy data were analysed for the intention - to - treat population , defined as randomised patients who received at least one dose of the study medication and provided at least one postbaseline efficacy assessment . the first method extended the calendar earlier into the treatment period until 28 days of non - missing data contributed to the count of migraine headache days . the second method proportionately adjusted the number of migraine headache days ( multiplied by 28 and divided by the number of non - missing days ) . the third method treated all missing days as non - migraine headache days ( used for the primary end point ) . an analysis of covariance ( ancova ) model was used to test the null hypothesis of no difference between placebo and the average of the values for the three groups . results were summarised using the adjusted mean and se for each treatment group , a 95% ci for the change from baseline for each treatment group , a model estimate of the difference between each active treatment group and placebo , a 95% ci for the difference , and an associated p value and adjusted p value for the difference . analysis of the primary end point was carried out using a combination of a sequential method and a hochberg procedure to maintain the experiment - wise level of 0.05 . the number of patients randomised to treatment was 196 after 438 were recruited and assessed for eligibility . eighteen randomised patients were excluded from the study , the analysis was performed in the remaining completed cases , 178 patients ( 59 placebo , 60 melatonin and 59 amitriptyline . adverse events occurrence was the most common reason for premature withdrawal for all randomised patients ( figure 2 ) . characteristics of study patients at baseline and of completers at the end of the trial * at baseline ( 4 weeks before start of study medication ) . the primary efficacy end point was frequency in number of migraine headache days per month . melatonin 3 mg and amitriptyline 25 mg efficiencies were superior to placebo ( p<0.05 ) when comparing baseline with the last month of observation ( table 2 and figure 3 ) . observed means and differences ( sd ) , effect sizes and ci for primary and secondary outcomes in baseline versus months 1 , 2 and 3 primary end point number of migraine headache days at baseline and at months 1 , 2 and 3 . were more effective to placebo reducing the number of analgesics taken , migraine headache attacks duration and intensity . melatonin and amitriptyline were equally effective for the primary end point , but for the secondary end point number of responders ( patients with a higher than 50% improvement in headache frequency ) melatonin was superior to amitriptyline ( p<0.05 ) and placebo ( p<0.01 ) ( table 2 and figure 4 ) . proportion of responders ( patients with a higher than 50% improvement in headache frequency , number of migraine headache days ) comparing baseline vs last month of treatment . over the 3-month course of treatment , 77 adverse events were reported by 60 participants , 46 reports in the amitriptyline group , 16 in the melatonin group and 17 in the placebo group . the majority of adverse events were either mild or moderate in intensity and occurred more commonly in the amitriptyline group compared with melatonin and placebo ( p<0.03 ) , whereas the melatonin and placebo groups had similar numbers ( p value = not significant ) . the most common adverse events were daytime sleepiness , dry mouth , epigastralgia , weight gain and constipation ( table 3 ) . number of patients reporting side effects in melatonin , amitriptyline and placebo groups we also evaluated weight variation in all groups . surprisingly , patients taking melatonin had on average a weight reduction ( mean 0.1400.156 kg ) , whereas those taking placebo and amitriptyline had a weight gain ( mean 0.4320.247 kg , and 0.9710.359 kg ) , respectively . mean weight variation comparing baseline with past 4 weeks of treatment in all groups . the number of patients randomised to treatment was 196 after 438 were recruited and assessed for eligibility . eighteen randomised patients were excluded from the study , the analysis was performed in the remaining completed cases , 178 patients ( 59 placebo , 60 melatonin and 59 amitriptyline . adverse events occurrence was the most common reason for premature withdrawal for all randomised patients ( figure 2 ) . characteristics of study patients at baseline and of completers at the end of the trial * at baseline ( 4 weeks before start of study medication ) . the primary efficacy end point was frequency in number of migraine headache days per month . melatonin 3 mg and amitriptyline 25 mg efficiencies were superior to placebo ( p<0.05 ) when comparing baseline with the last month of observation ( table 2 and figure 3 ) . observed means and differences ( sd ) , effect sizes and ci for primary and secondary outcomes in baseline versus months 1 , 2 and 3 primary end point number of migraine headache days at baseline and at months 1 , 2 and 3 . were more effective to placebo reducing the number of analgesics taken , migraine headache attacks duration and intensity . melatonin and amitriptyline were equally effective for the primary end point , but for the secondary end point number of responders ( patients with a higher than 50% improvement in headache frequency ) melatonin was superior to amitriptyline ( p<0.05 ) and placebo ( p<0.01 ) ( table 2 and figure 4 ) . proportion of responders ( patients with a higher than 50% improvement in headache frequency , number of migraine headache days ) comparing baseline vs last month of treatment . over the 3-month course of treatment , 77 adverse events were reported by 60 participants , 46 reports in the amitriptyline group , 16 in the melatonin group and 17 in the placebo group . the majority of adverse events were either mild or moderate in intensity and occurred more commonly in the amitriptyline group compared with melatonin and placebo ( p<0.03 ) , whereas the melatonin and placebo groups had similar numbers ( p value = not significant ) . the most common adverse events were daytime sleepiness , dry mouth , epigastralgia , weight gain and constipation ( table 3 ) . number of patients reporting side effects in melatonin , amitriptyline and placebo groups we also evaluated weight variation in all groups . surprisingly , patients taking melatonin had on average a weight reduction ( mean 0.1400.156 kg ) , whereas those taking placebo and amitriptyline had a weight gain ( mean 0.4320.247 kg , and 0.9710.359 kg ) , respectively . mean weight variation comparing baseline with past 4 weeks of treatment in all groups . the results from this study support the efficacy and tolerability of melatonin 3 mg as a preventive therapy for migraine . melatonin was more effective than placebo in the primary end point and all other end points studied . although melatonin was as effective as amitriptyline 25 mg in the primary end point , in the secondary end point , proportion of patients who improved > 50% in headache frequency , melatonin was superior to both placebo and amitriptyline . the placebo rate in our study was 20.4% , similar to the rates reported in the meta - analysis of pharmacological migraine prevention studies 22% ( 0.17% to 0.28% ) . a pilot open - label study was published in 2004,9 showing efficacy and good tolerability of the same melatonin dose as was studied in this trial . a placebo controlled study comparing a different melatonin compound and dose ( extended - release melatonin 2.0 mg , circadin ( neurim pharmaceuticals , tel - aviv , israel ) ) with placebo showed negative results.10 several methodological issues including the short trial length ( only 8 weeks ) , high placebo response rate and low melatonin dosage and patient population limit this trial interpretation . nevertheless , in this trial , the melatonin response rate was 44% , similar to several other positive migraine prophylaxis trials . the mean decrease in headache frequency was also similar to the most recent and well - designed preventive trials.11 we designed the study according to the international headache society guidelines for clinical trials,13 including an active comparator , in order to warrant a better blinding of treatment groups . amitriptyline 25 mg is a lower than usually prescribed dose in the usa and studied in trials , but the usual dose in many countries , including brazil . one study showed that topiramate and amitriptyline were equally effective with a mean daily dose of 74.8 and 76.5 mg , respectively ; the amitriptyline group reported an unacceptable side effect profile , at least in our culture , for our patient population profile : dry mouth ( 35.5% ) , fatigue ( 24.3% ) , somnolence ( 17.8% ) , weight increase ( 13.6% ) and dizziness ( 10.7% ) . higher doses of amitriptyline could give better results but certainly a lot higher dropout rates in our population.14 although our focus was to study melatonin efficacy , and the study was not designed for non - inferiority , this trial also gives support for the use of amitriptyline in lower doses for migraine prophylaxis . weight loss was found in the melatonin group as opposed to a slight weight gain in placebo and a significant one in amitriptyline group . there is substantial experimental evidence in the literature indicating the role of melatonin in the control of food intake , energy balance and body weight . an inhibitory action of melatonin in preadipocytes differentiation into adipocytes , reducing the number of cells in the visceral adipose tissue has been demonstrated.15 melatonin treatment reduced body weight gain , visceral adiposity , blood triglycerides and insulin resistance in a model of high - calorie diet - induced metabolic syndrome.16 likewise , a single daily administration of melatonin decreased visceral fat in middle - aged mice,17 and after melatonin treatment aged rats showed weight reduction , preceded by an increase in insulin signalling in the central nervous system ( cns ) and peripheral tissues.18 in addition , melatonin might have a direct anorexigenic action regulating hypothalamic pro - opiomelanocortin ( pomc ) gene expression.19 in ageing animals , melatonin and moderate exercise training induced a reduction in food intake associated with a reduction in body weight and amount of visceral adipose tissue depot.20 melatonin should be considered as regulating the other side of energy balance increasing the energy expenditure by its ability to convert white adipose tissue into brown adipose tissue increasing its metabolic rate.21 the reduction in body weight of patients in the melatonin treated group is , to the best of our knowledge , the first demonstration of the putative effect of melatonin in body weight reduction in humans . melatonin mechanism of action in migraine prevention could be due to one of its several effects , including : membrane stabilisation , anti - inflammatory properties , inhibition of dopamine release , modulation of serotonin , gamma amino butyric acid ( gama ) and glutamate neurotransmission , scavenging toxic - free radicals and cerebrovascular regulation.22 melatonin also potentiates opioid analgesia ; therefore , it should be used with caution in patients taking and/or overusing opioids.23 patients with diabetes and hypertensive patients should be monitored since melatonin may decrease blood pressure24 and glucose levels.25 owing to its favourable side effect profile and efficacy , melatonin could be an option for patients sensitive to other drugs or with a preference for natural products . with the same efficacy level compared with other treatments and a low cost different melatonin doses ( lower and higher ) should be studied , as well as its effect in other migraine types and comorbidities . the use of other chronobiotic agents , including melatonin analogues , could be tested in migraine prevention . melatonin is as effective as amitriptyline 25 mg in the primary end point , but better than amitriptyline in the secondary end point ( 50% responder rate ) . melatonin is as effective as amitriptyline 25 mg in the primary end point , but better than amitriptyline in the secondary end point ( 50% responder rate ) .

introductionmelatonin has been studied in headache disorders . amitriptyline is efficacious for migraine prevention , but its unfavourable side effect profile limits its use.methodsa randomised , double - blind , placebo - controlled study was carried out . men and women , aged 1865 years , with migraine with or without aura , experiencing 28 attacks per month , were enrolled . after a 4-week baseline phase , 196 participants were randomised to placebo , amitriptyline 25 mg or melatonin 3 mg , and 178 took a study medication and were followed for 3 months ( 12 weeks ) . the primary outcome was the number of migraine headache days per month at baseline versus last month . secondary end points were responder rate , migraine intensity , duration and analgesic use . tolerability was also compared between groups.resultsmean headache frequency reduction was 2.7 migraine headache days in the melatonin group , 2.2 for amitriptyline and 1.1 for placebo . melatonin significantly reduced headache frequency compared with placebo ( p=0.009 ) , but not to amitriptyline ( p=0.19 ) . melatonin was superior to amitriptyline in the percentage of patients with a greater than 50% reduction in migraine frequency . melatonin was better tolerated than amitriptyline . weight loss was found in the melatonin group , a slight weight gain in placebo and significantly for amitriptyline users.conclusionsmelatonin 3 mg is better than placebo for migraine prevention , more tolerable than amitriptyline and as effective as amitriptyline 25 mg .

Introduction Methods Patients Patients involvement Study design Procedures Outcome measures Efficacy Tolerability and safety Statistical analysis Results Patients Tolerability Discussion Conclusion

migraine is a chronic debilitating neurological condition affecting 1220% of the population worldwide.1 preventive treatments are available , decreasing the number and severity of headache attacks , and improving health outcomes and quality of life ; half of the patients show 50% reduction in attack frequency.2 although migraine prophylaxis guidelines from north american , south american and european societies are available , only 35% of patients receive adequate preventive therapy.3 public surveys report that migraineurs are among the most dissatisfied patients.4 5 about half of the patients with migraine stop seeking care for their headaches , partly because of their side effect profile.6 most guidelines recommend topiramate , divalproex / sodium valproate , propranolol and metoprolol as having the highest level of evidence.7 newer effective and tolerable options for migraine prophylaxis are necessary to reduce this gap and improve patients ' quality of life . melatonin use in migraine prevention is supported by several biological effects.8 melatonin levels are decreased in patients with migraine ; it has been studied for migraine prophylaxis with conflicting results.911 we aimed to study the effect of melatonin in a double - blind , placebo controlled trial with an active comparator . patients with migraine were eligible for the study if they met inclusion criteria : age of 1865 years ; migraine with or without aura criteria according to the international classification of headache disorders , third edition , -version12 for at least 1 year , age of onset before 50 years , at least three migraine headache attacks or four migraine headache days ( defined as any occurrence of migraine headache pain of at least 30 min in duration with acute treatment ) per month , presents with migraine or non - migraine headache attacks < 15 days per month during each of the 3 months prior to the screening visit and the reference period . patients were excluded if they had a history of psychiatric disorder ( in the past or present ) ; ergotamine , triptan , opioid , or combination medication intake for > 10 days per month , or simple analgesic intake for > 15 days per month for > 3 months ; in use of preventive medications such as -blockers , tricyclic antidepressants , calcium channel blockers , antiepileptic drugs , bupropion , serotonergic norepinephrine reuptake inhibitors ; and were unable to discontinue the treatment ; had previously taken melatonin , amitriptyline or agomelatine ; or had uncontrolled hypertension ( ie , sitting systolic blood pressure > 160 mm hg or sitting diastolic blood pressure > 90 mm hg ) at the screening visit or at randomisation . results will be disseminated to patients / carers / laypeople by publication in the clinicaltrials.org database and to press / social media . patients with migraine were eligible for the study if they met inclusion criteria : age of 1865 years ; migraine with or without aura criteria according to the international classification of headache disorders , third edition , -version12 for at least 1 year , age of onset before 50 years , at least three migraine headache attacks or four migraine headache days ( defined as any occurrence of migraine headache pain of at least 30 min in duration with acute treatment ) per month , presents with migraine or non - migraine headache attacks < 15 days per month during each of the 3 months prior to the screening visit and the reference period . patients were excluded if they had a history of psychiatric disorder ( in the past or present ) ; ergotamine , triptan , opioid , or combination medication intake for > 10 days per month , or simple analgesic intake for > 15 days per month for > 3 months ; in use of preventive medications such as -blockers , tricyclic antidepressants , calcium channel blockers , antiepileptic drugs , bupropion , serotonergic norepinephrine reuptake inhibitors ; and were unable to discontinue the treatment ; had previously taken melatonin , amitriptyline or agomelatine ; or had uncontrolled hypertension ( ie , sitting systolic blood pressure > 160 mm hg or sitting diastolic blood pressure > 90 mm hg ) at the screening visit or at randomisation . patients / service users / carers / laypeople were not involved in the study design . the trial was funded by fapesp , fundao de amparo a pesquisa de so paulo , a brazilian governmental funding agency without any role in manuscript preparation . after eligibility had been determined , patients were randomised to treatment groups ( figure 1 ) . patients meeting eligibility criteria were randomised 1:1:1 to one of the three groups : placebo , melatonin 3 mg or amitriptyline 25 mg , all taken at bedtime . patients recorded information about migraine or non - migraine headache occurrence , mean migraine headache attack intensity , duration , analgesics taken , ( type of medication , number of days of medication use , number of doses ) and associated symptoms in a paper - based diary . trained neurologists in headache abstracted the information , which was double - checked by another investigator and uploaded into a spreadsheet . the variables were collected from the diaries and defined at the end of the baseline period , and at the first ( weeks 14 ) , second ( weeks 58 ) and third ( weeks 912 ) months after treatment . the primary efficacy outcome measure was frequency in number of migraine headache days per month comparing baseline with the past 4 weeks of treatment . secondary end points included reduction in migraine intensity , attack duration , number of analgesics used and percentages of patients with greater than 50% reductions in migraine headache days . the randomisation ratio of 1:1:1 for placebo , and the two treatment groups , 59 participants in each groups were determined to provide 80% power to detect a significant difference in the primary endpoint . efficacy data were analysed for the intention - to - treat population , defined as randomised patients who received at least one dose of the study medication and provided at least one postbaseline efficacy assessment . the first method extended the calendar earlier into the treatment period until 28 days of non - missing data contributed to the count of migraine headache days . the second method proportionately adjusted the number of migraine headache days ( multiplied by 28 and divided by the number of non - missing days ) . the third method treated all missing days as non - migraine headache days ( used for the primary end point ) . results were summarised using the adjusted mean and se for each treatment group , a 95% ci for the change from baseline for each treatment group , a model estimate of the difference between each active treatment group and placebo , a 95% ci for the difference , and an associated p value and adjusted p value for the difference . analysis of the primary end point was carried out using a combination of a sequential method and a hochberg procedure to maintain the experiment - wise level of 0.05 . after eligibility had been determined , patients were randomised to treatment groups ( figure 1 ) . patients meeting eligibility criteria were randomised 1:1:1 to one of the three groups : placebo , melatonin 3 mg or amitriptyline 25 mg , all taken at bedtime . patients recorded information about migraine or non - migraine headache occurrence , mean migraine headache attack intensity , duration , analgesics taken , ( type of medication , number of days of medication use , number of doses ) and associated symptoms in a paper - based diary . trained neurologists in headache abstracted the information , which was double - checked by another investigator and uploaded into a spreadsheet . the variables were collected from the diaries and defined at the end of the baseline period , and at the first ( weeks 14 ) , second ( weeks 58 ) and third ( weeks 912 ) months after treatment . the primary efficacy outcome measure was frequency in number of migraine headache days per month comparing baseline with the past 4 weeks of treatment . secondary end points included reduction in migraine intensity , attack duration , number of analgesics used and percentages of patients with greater than 50% reductions in migraine headache days . the primary efficacy outcome measure was frequency in number of migraine headache days per month comparing baseline with the past 4 weeks of treatment . secondary end points included reduction in migraine intensity , attack duration , number of analgesics used and percentages of patients with greater than 50% reductions in migraine headache days . the randomisation ratio of 1:1:1 for placebo , and the two treatment groups , 59 participants in each groups were determined to provide 80% power to detect a significant difference in the primary endpoint . efficacy data were analysed for the intention - to - treat population , defined as randomised patients who received at least one dose of the study medication and provided at least one postbaseline efficacy assessment . the first method extended the calendar earlier into the treatment period until 28 days of non - missing data contributed to the count of migraine headache days . the second method proportionately adjusted the number of migraine headache days ( multiplied by 28 and divided by the number of non - missing days ) . the third method treated all missing days as non - migraine headache days ( used for the primary end point ) . results were summarised using the adjusted mean and se for each treatment group , a 95% ci for the change from baseline for each treatment group , a model estimate of the difference between each active treatment group and placebo , a 95% ci for the difference , and an associated p value and adjusted p value for the difference . analysis of the primary end point was carried out using a combination of a sequential method and a hochberg procedure to maintain the experiment - wise level of 0.05 . the number of patients randomised to treatment was 196 after 438 were recruited and assessed for eligibility . eighteen randomised patients were excluded from the study , the analysis was performed in the remaining completed cases , 178 patients ( 59 placebo , 60 melatonin and 59 amitriptyline . adverse events occurrence was the most common reason for premature withdrawal for all randomised patients ( figure 2 ) . characteristics of study patients at baseline and of completers at the end of the trial * at baseline ( 4 weeks before start of study medication ) . the primary efficacy end point was frequency in number of migraine headache days per month . melatonin 3 mg and amitriptyline 25 mg efficiencies were superior to placebo ( p<0.05 ) when comparing baseline with the last month of observation ( table 2 and figure 3 ) . observed means and differences ( sd ) , effect sizes and ci for primary and secondary outcomes in baseline versus months 1 , 2 and 3 primary end point number of migraine headache days at baseline and at months 1 , 2 and 3 . were more effective to placebo reducing the number of analgesics taken , migraine headache attacks duration and intensity . melatonin and amitriptyline were equally effective for the primary end point , but for the secondary end point number of responders ( patients with a higher than 50% improvement in headache frequency ) melatonin was superior to amitriptyline ( p<0.05 ) and placebo ( p<0.01 ) ( table 2 and figure 4 ) . proportion of responders ( patients with a higher than 50% improvement in headache frequency , number of migraine headache days ) comparing baseline vs last month of treatment . over the 3-month course of treatment , 77 adverse events were reported by 60 participants , 46 reports in the amitriptyline group , 16 in the melatonin group and 17 in the placebo group . the majority of adverse events were either mild or moderate in intensity and occurred more commonly in the amitriptyline group compared with melatonin and placebo ( p<0.03 ) , whereas the melatonin and placebo groups had similar numbers ( p value = not significant ) . the most common adverse events were daytime sleepiness , dry mouth , epigastralgia , weight gain and constipation ( table 3 ) . number of patients reporting side effects in melatonin , amitriptyline and placebo groups we also evaluated weight variation in all groups . surprisingly , patients taking melatonin had on average a weight reduction ( mean 0.1400.156 kg ) , whereas those taking placebo and amitriptyline had a weight gain ( mean 0.4320.247 kg , and 0.9710.359 kg ) , respectively . the number of patients randomised to treatment was 196 after 438 were recruited and assessed for eligibility . eighteen randomised patients were excluded from the study , the analysis was performed in the remaining completed cases , 178 patients ( 59 placebo , 60 melatonin and 59 amitriptyline . characteristics of study patients at baseline and of completers at the end of the trial * at baseline ( 4 weeks before start of study medication ) . the primary efficacy end point was frequency in number of migraine headache days per month . melatonin 3 mg and amitriptyline 25 mg efficiencies were superior to placebo ( p<0.05 ) when comparing baseline with the last month of observation ( table 2 and figure 3 ) . observed means and differences ( sd ) , effect sizes and ci for primary and secondary outcomes in baseline versus months 1 , 2 and 3 primary end point number of migraine headache days at baseline and at months 1 , 2 and 3 . were more effective to placebo reducing the number of analgesics taken , migraine headache attacks duration and intensity . melatonin and amitriptyline were equally effective for the primary end point , but for the secondary end point number of responders ( patients with a higher than 50% improvement in headache frequency ) melatonin was superior to amitriptyline ( p<0.05 ) and placebo ( p<0.01 ) ( table 2 and figure 4 ) . proportion of responders ( patients with a higher than 50% improvement in headache frequency , number of migraine headache days ) comparing baseline vs last month of treatment . over the 3-month course of treatment , 77 adverse events were reported by 60 participants , 46 reports in the amitriptyline group , 16 in the melatonin group and 17 in the placebo group . the majority of adverse events were either mild or moderate in intensity and occurred more commonly in the amitriptyline group compared with melatonin and placebo ( p<0.03 ) , whereas the melatonin and placebo groups had similar numbers ( p value = not significant ) . number of patients reporting side effects in melatonin , amitriptyline and placebo groups we also evaluated weight variation in all groups . surprisingly , patients taking melatonin had on average a weight reduction ( mean 0.1400.156 kg ) , whereas those taking placebo and amitriptyline had a weight gain ( mean 0.4320.247 kg , and 0.9710.359 kg ) , respectively . the results from this study support the efficacy and tolerability of melatonin 3 mg as a preventive therapy for migraine . melatonin was more effective than placebo in the primary end point and all other end points studied . although melatonin was as effective as amitriptyline 25 mg in the primary end point , in the secondary end point , proportion of patients who improved > 50% in headache frequency , melatonin was superior to both placebo and amitriptyline . the placebo rate in our study was 20.4% , similar to the rates reported in the meta - analysis of pharmacological migraine prevention studies 22% ( 0.17% to 0.28% ) . a pilot open - label study was published in 2004,9 showing efficacy and good tolerability of the same melatonin dose as was studied in this trial . a placebo controlled study comparing a different melatonin compound and dose ( extended - release melatonin 2.0 mg , circadin ( neurim pharmaceuticals , tel - aviv , israel ) ) with placebo showed negative results.10 several methodological issues including the short trial length ( only 8 weeks ) , high placebo response rate and low melatonin dosage and patient population limit this trial interpretation . the mean decrease in headache frequency was also similar to the most recent and well - designed preventive trials.11 we designed the study according to the international headache society guidelines for clinical trials,13 including an active comparator , in order to warrant a better blinding of treatment groups . amitriptyline 25 mg is a lower than usually prescribed dose in the usa and studied in trials , but the usual dose in many countries , including brazil . one study showed that topiramate and amitriptyline were equally effective with a mean daily dose of 74.8 and 76.5 mg , respectively ; the amitriptyline group reported an unacceptable side effect profile , at least in our culture , for our patient population profile : dry mouth ( 35.5% ) , fatigue ( 24.3% ) , somnolence ( 17.8% ) , weight increase ( 13.6% ) and dizziness ( 10.7% ) . higher doses of amitriptyline could give better results but certainly a lot higher dropout rates in our population.14 although our focus was to study melatonin efficacy , and the study was not designed for non - inferiority , this trial also gives support for the use of amitriptyline in lower doses for migraine prophylaxis . weight loss was found in the melatonin group as opposed to a slight weight gain in placebo and a significant one in amitriptyline group . an inhibitory action of melatonin in preadipocytes differentiation into adipocytes , reducing the number of cells in the visceral adipose tissue has been demonstrated.15 melatonin treatment reduced body weight gain , visceral adiposity , blood triglycerides and insulin resistance in a model of high - calorie diet - induced metabolic syndrome.16 likewise , a single daily administration of melatonin decreased visceral fat in middle - aged mice,17 and after melatonin treatment aged rats showed weight reduction , preceded by an increase in insulin signalling in the central nervous system ( cns ) and peripheral tissues.18 in addition , melatonin might have a direct anorexigenic action regulating hypothalamic pro - opiomelanocortin ( pomc ) gene expression.19 in ageing animals , melatonin and moderate exercise training induced a reduction in food intake associated with a reduction in body weight and amount of visceral adipose tissue depot.20 melatonin should be considered as regulating the other side of energy balance increasing the energy expenditure by its ability to convert white adipose tissue into brown adipose tissue increasing its metabolic rate.21 the reduction in body weight of patients in the melatonin treated group is , to the best of our knowledge , the first demonstration of the putative effect of melatonin in body weight reduction in humans . melatonin mechanism of action in migraine prevention could be due to one of its several effects , including : membrane stabilisation , anti - inflammatory properties , inhibition of dopamine release , modulation of serotonin , gamma amino butyric acid ( gama ) and glutamate neurotransmission , scavenging toxic - free radicals and cerebrovascular regulation.22 melatonin also potentiates opioid analgesia ; therefore , it should be used with caution in patients taking and/or overusing opioids.23 patients with diabetes and hypertensive patients should be monitored since melatonin may decrease blood pressure24 and glucose levels.25 owing to its favourable side effect profile and efficacy , melatonin could be an option for patients sensitive to other drugs or with a preference for natural products . the use of other chronobiotic agents , including melatonin analogues , could be tested in migraine prevention . melatonin is as effective as amitriptyline 25 mg in the primary end point , but better than amitriptyline in the secondary end point ( 50% responder rate ) . melatonin is as effective as amitriptyline 25 mg in the primary end point , but better than amitriptyline in the secondary end point ( 50% responder rate ) .

increasing findings have evidenced that cerebral ischemia plays a critical role in the pathogenesis of vascular cognitive impairment , and the reduction of cerebral blood flow correlates with the severity of cognitive impairment.1,2 various mechanisms of neuronal injury suffered from cerebral ischemia have been proposed , including formation of free radicals , oxidative stress,3,4 mitochondrial dysfunction,5,6 inflammatory processes,7 genetic factors , environmental impact factors,8,9 apoptosis,10 and so on . these factors may interact with and amplify each other in a vicious cycle of toxicity , leading to neuronal dysfunction and cognitive impairment . the transcription factor cyclic amp response element binding protein ( creb ) and neurotrophin brain - derived neurotrophic factor ( bdnf ) have emerged as molecules that may play an important role in modulating mood , behavior , and memory.1113 creb and bdnf are known to be dysregulated in animal models and in patients suffering from cerebral ischemia , and are deemed to be therapeutic targets of cerebral ischemia.14,15 minocycline , a tetracycline derivative , protects against cerebral ischemia via inhibiting inflammation , oxidative stress , and apoptosis.16,17 previously , we have found that minocycline retarded astrocytic reactivation , and restrained oxidative stress and neuroinflammation in the hippocampus of cerebral ischemia rats.18,19 in the present study , we observed the expression of creb , phosphorylated creb ( pcreb ) , and bdnf in the hippocampus of cerebral ischemia rats with cognitive impairment by permanent bilateral occlusion of both common carotid arteries , and explored the neuroprotective mechanism of minocycline for the treatment of cerebral ischemia injury . we found that creb , pcreb , and bdnf were downregulated after permanent bilateral occlusion of both common carotid arteries in a model group , and minocycline attenuated cognitive impairment and upregulated creb , pcreb , and bdnf in the hippocampus of rats with permanent bilateral occlusion of both common carotid arteries . therefore , a hypothesis was made that minocycline upregulated creb , pcreb , and bdnf and improved cognitive impairment from cerebral vascular factors . wistar rats ( 10 weeks old , female , quality 200250 g , from the field zoology research institute of the third military medical university of the people s republic of china ) were randomly divided into sham - operated group ( s ) ( with a mean survival time of 16 weeks ) , ischemia model group ( m ) ( with permanent bilateral occlusion of both common carotid arteries ) , and minocycline treatment group ( mt ) ( beginning treatment after 4 days from permanent bilateral occlusion of both common carotid arteries , minocycline was administered by douche via the stomach for 4 weeks ) . m and mt groups were separately subdivided into 4- , 8- , and 16-week groups . each group had six animals . the animal model of cerebral ischemia was established with permanent bilateral occlusion of both common carotid arteries for chronic bilateral common carotid artery occlusion ( bccao).20,21 rats were anesthetized with 10% chloral hydrate ( 350 mg / kg , intraperitoneally ) and breathed normally throughout the surgical procedure . both common carotid arteries were exposed via a midline cervical incision and doubly - ligated with silk suture . sham - operated animals were treated in the same manner , except that the common arteries were not ligated . the investigation was performed according to the guide for the care and use of laboratory animals published by the us national institutes of health.22 the animal experiments were performed according to internationally followed ethical standards and approved by the research ethics committee of chongqing medical university , chongqing , people s republic of china . minocycline ( 100 mg / capsule ; huishi pharmaceutical limited company , shanghai , the people s republic of china ) was diluted to 0.5 mg / ml density by normal saline . s and m groups were given the same volume of normal saline through douche via the stomach . mt group was given 50 mg / kg / d minocycline through douche via the stomach . the minocycline dosage used for animals was as described elsewhere.16,17 the morris water maze task ( chinese academy of medical sciences , people s republic of china ) includes a place navigation test and spatial probe test , and is widely used in behavioral neuroscience to study spatial learning and memory.23 the rats were placed in a large circular pool with an invisible platform that allows them to escape the water . the time it took a rat to find the platform and escape was measured for up to four trials a day for 5 days . the time it took to find the platform after training was complete , the spatial probe trial was conducted in which the escape platform was removed from the pool and the animal allowed to swim for 120 seconds . the spatial probe test was for the measurement of preservation - of - experience ( memory ) capacity , ie , looking for the platform position . the time it took to find the unmoved platform ( learning latency ) and the times a rat crossed the corresponding position of the removed platform in 120 seconds ( memory latency ) were recorded . prior to immunohistochemistry assay , frozen sections were prepared with a cryostat ( facs caliber ; becton dickinson , franklin lakes , nj , usa ) at 20c , dried at room temperature , and fixed with acetone . the peripheral blood mononuclear cells were routinely isolated and the slides were prepared with a cytospin . the avidin - biotin - peroxidase complex immunohistochemical assay was carried out according to the protocols we described before anti - creb ( sigma - aldrich co. , st louis , mo , usa ) , anti - pcreb ( sigma - aldrich co. ) , and anti - bdnf ( santa cruz , la , ca , usa ) were prepared . the second antibody , a goat anti - mouse igg labeled with biotin , was purchased from vector co. ( burlingame , ca , usa ) . two hundred cells were counted and the intensity of staining for each of those cells was adjusted . five grades were employed to express the degrees of staining , which represent five reaction coefficients , respectively . the five products of every coefficient and the corresponding cell number were added up , which resulted in the value of a positive score.16,18 all slides were measured in duplicate . those samples with a positive score over 10 or a frequency over 5% were considered as positive . rat tissues were dissected and homogenized in tissue protein extraction reagent ( t - per ) buffer in the presence of protease inhibitors . after homogenization , the lysates were centrifuged at 100,000 g , and the supernatants were saved for western blotting . equal amounts of lysates were subject to sodium dodecyl sulfate - polyacrylamide gel electrophoresis and western blotting analysis using antibodies specific for the following : creb ( 1:1,000 ; biosource international , inc . , usa ) , pcreb ( 1:1,000 ; biosource international , inc . ) , bdnf ( 1:500 ; sigma - aldrich co. , st louis , mo , usa ) , and -tublin ( 1:200 ; biosource international inc . , the optical densities of the specific bands were scanned and measured by image analysis software ( tongji qianping company , wuhan , hubei province , people s republic of china ) . , chicago , il , usa ) and student s t - test for intergroup analysis . keuls test was performed when variance was equal , and games howell test was performed when variance was not equal . wistar rats ( 10 weeks old , female , quality 200250 g , from the field zoology research institute of the third military medical university of the people s republic of china ) were randomly divided into sham - operated group ( s ) ( with a mean survival time of 16 weeks ) , ischemia model group ( m ) ( with permanent bilateral occlusion of both common carotid arteries ) , and minocycline treatment group ( mt ) ( beginning treatment after 4 days from permanent bilateral occlusion of both common carotid arteries , minocycline was administered by douche via the stomach for 4 weeks ) . m and mt groups were separately subdivided into 4- , 8- , and 16-week groups . each group had six animals . the animal model of cerebral ischemia was established with permanent bilateral occlusion of both common carotid arteries for chronic bilateral common carotid artery occlusion ( bccao).20,21 rats were anesthetized with 10% chloral hydrate ( 350 mg / kg , intraperitoneally ) and breathed normally throughout the surgical procedure . both common carotid arteries were exposed via a midline cervical incision and doubly - ligated with silk suture . sham - operated animals were treated in the same manner , except that the common arteries were not ligated . the investigation was performed according to the guide for the care and use of laboratory animals published by the us national institutes of health.22 the animal experiments were performed according to internationally followed ethical standards and approved by the research ethics committee of chongqing medical university , chongqing , people s republic of china . minocycline ( 100 mg / capsule ; huishi pharmaceutical limited company , shanghai , the people s republic of china ) was diluted to 0.5 mg / ml density by normal saline . s and m groups were given the same volume of normal saline through douche via the stomach . mt group was given 50 mg / kg / d minocycline through douche via the stomach . the morris water maze task ( chinese academy of medical sciences , people s republic of china ) includes a place navigation test and spatial probe test , and is widely used in behavioral neuroscience to study spatial learning and memory.23 the rats were placed in a large circular pool with an invisible platform that allows them to escape the water . the time it took a rat to find the platform and escape was measured for up to four trials a day for 5 days . the time it took to find the platform is referred to as escape latency ( the earliest learning measure ) . after training was complete , the spatial probe trial was conducted in which the escape platform was removed from the pool and the animal allowed to swim for 120 seconds . the spatial probe test was for the measurement of preservation - of - experience ( memory ) capacity , ie , looking for the platform position . the time it took to find the unmoved platform ( learning latency ) and the times a rat crossed the corresponding position of the removed platform in 120 seconds ( memory latency ) were recorded . tissue samples were collected after surgery and immediately frozen with liquid nitrogen . prior to immunohistochemistry assay , frozen sections were prepared with a cryostat ( facs caliber ; becton dickinson , franklin lakes , nj , usa ) at 20c , dried at room temperature , and fixed with acetone . the peripheral blood mononuclear cells were routinely isolated and the slides were prepared with a cytospin . the avidin - biotin - peroxidase complex immunohistochemical assay was carried out according to the protocols we described before anti - creb ( sigma - aldrich co. , st louis , mo , usa ) , anti - pcreb ( sigma - aldrich co. ) , and anti - bdnf ( santa cruz , la , ca , usa ) were prepared . the second antibody , a goat anti - mouse igg labeled with biotin , was purchased from vector co. ( burlingame , ca , usa ) . two hundred cells were counted and the intensity of staining for each of those cells was adjusted . five grades were employed to express the degrees of staining , which represent five reaction coefficients , respectively . the five products of every coefficient and the corresponding cell number were added up , which resulted in the value of a positive score.16,18 all slides were measured in duplicate . those samples with a positive score over 10 or a frequency over 5% were considered as positive . rat tissues were dissected and homogenized in tissue protein extraction reagent ( t - per ) buffer in the presence of protease inhibitors . after homogenization , the lysates were centrifuged at 100,000 g , and the supernatants were saved for western blotting . equal amounts of lysates were subject to sodium dodecyl sulfate - polyacrylamide gel electrophoresis and western blotting analysis using antibodies specific for the following : creb ( 1:1,000 ; biosource international , inc . , usa ) , pcreb ( 1:1,000 ; biosource international , inc . ) , bdnf ( 1:500 ; sigma - aldrich co. , st louis , mo , usa ) , and -tublin ( 1:200 ; biosource international inc . , the optical densities of the specific bands were scanned and measured by image analysis software ( tongji qianping company , wuhan , hubei province , people s republic of china ) . all statistical analyses used the spss software for windows 13.0 ( spss , inc . , chicago , il , usa ) and student s t - test for intergroup analysis . keuls test was performed when variance was equal , and games howell test was performed when variance was not equal . cerebral ischemia was induced in 10-week - old wistar rats by bccao as described previously.18,19 after the performance of bccao , rats were subjected to the morris water maze . escape latency decreased after 1 day of training . on days 3 , 4 , and 5 , s animals immediately swam toward the platforms in the water maze , whereas m rats swam longer distances before finding the platform ( figure 1a ) . in general , escape latency decreased with bccao duration ( p=0.0004 ) , the m rats , after minocycline treatment , swam shorter distances than control rats before finding the platform ( p=0.0007 ) ( figure 1a ) . in the probe trials , the number of times the platform position was crossed for the s group was more than for the bccao rats groups in the corresponding platform location ( p=0.0000 ) ( figure 1b ) . the number of times the platform position was crossed for mt animals was significantly increased compared to bccao rat groups in the corresponding platform position ( p=0.0016 ) ( figure 1b ) . the results of immunohistochemistry and western blotting showed that expression of creb and pcreb in the mt group was significantly higher than that of the m group at the corresponding time . expression of creb and pcreb by immunohistochemistry in the m groups was more decreased than in the control group ( p=0.0009 ; p=0.0023 ) , whereas expression of creb and pcreb in the mt groups was more increased than in the control groups ( p=0.0001 ; p=0.0005 ) ( figure 2 ) . expression of creb and pcreb by western blotting in the m groups was lowered more than in the control group ( p=0.0010 ; p=0.0031 ) , whereas expression of creb and pcreb in the mt groups was more enhanced than in the control groups ( p=0.0004 ; p=0.0003 ) ( figure 2 ) . the results of immunohistochemistry showed that expression of bdnf in the mt animals was higher than that of the m ones ( p=0.0005 ) , whereas expression of bdnf in the m groups was decreased compared to the control group ( p=0.0001 ) . western blotting analysis found that bdnf in the mt groups was higher than that of the m group ( p=0.0006 ) , while expression of bdnf in the m groups was more reduced than in the control group ( p=0.0000 ) ( figure 3 ) . the number of times the platform position was crossed during the probe trial for the s group was higher than for m rats ( p=0.0021 ) . therefore , we determined correlation analysis between the number of times the platform position was crossed and creb , pcreb , and bdnf expression . linear correlation analysis shows that the optical density ( od ) values of immunoblotting protein for creb , pcreb , and bdnf were negatively correlated with the number of platform position crossings ( r=0.314 , p=0.0062 ; r=0.352 , p=0.0004 ; r=0.381 , p=0.0031 ) respectively ( figure 4 ) . we inferred that downregulation of creb , pcreb , and bdnf contributed to cognitive impairment from chronic cerebral ischemia . cerebral ischemia was induced in 10-week - old wistar rats by bccao as described previously.18,19 after the performance of bccao , rats were subjected to the morris water maze . escape latency decreased after 1 day of training . on days 3 , 4 , and 5 , s animals immediately swam toward the platforms in the water maze , whereas m rats swam longer distances before finding the platform ( figure 1a ) . in general , escape latency decreased with bccao duration ( p=0.0004 ) , the m rats , after minocycline treatment , swam shorter distances than control rats before finding the platform ( p=0.0007 ) ( figure 1a ) . in the probe trials , the number of times the platform position was crossed for the s group was more than for the bccao rats groups in the corresponding platform location ( p=0.0000 ) ( figure 1b ) . the number of times the platform position was crossed for mt animals was significantly increased compared to bccao rat groups in the corresponding platform position ( p=0.0016 ) ( figure 1b ) . the results of immunohistochemistry and western blotting showed that expression of creb and pcreb in the mt group was significantly higher than that of the m group at the corresponding time . expression of creb and pcreb by immunohistochemistry in the m groups was more decreased than in the control group ( p=0.0009 ; p=0.0023 ) , whereas expression of creb and pcreb in the mt groups was more increased than in the control groups ( p=0.0001 ; p=0.0005 ) ( figure 2 ) . expression of creb and pcreb by western blotting in the m groups was lowered more than in the control group ( p=0.0010 ; p=0.0031 ) , whereas expression of creb and pcreb in the mt groups was more enhanced than in the control groups ( p=0.0004 ; p=0.0003 ) ( figure 2 ) . the results of immunohistochemistry showed that expression of bdnf in the mt animals was higher than that of the m ones ( p=0.0005 ) , whereas expression of bdnf in the m groups was decreased compared to the control group ( p=0.0001 ) . western blotting analysis found that bdnf in the mt groups was higher than that of the m group ( p=0.0006 ) , while expression of bdnf in the m groups was more reduced than in the control group ( p=0.0000 ) ( figure 3 ) . the number of times the platform position was crossed during the probe trial for the s group was higher than for m rats ( p=0.0021 ) . therefore , we determined correlation analysis between the number of times the platform position was crossed and creb , pcreb , and bdnf expression . linear correlation analysis shows that the optical density ( od ) values of immunoblotting protein for creb , pcreb , and bdnf were negatively correlated with the number of platform position crossings ( r=0.314 , p=0.0062 ; r=0.352 , p=0.0004 ; r=0.381 , p=0.0031 ) respectively ( figure 4 ) . we inferred that downregulation of creb , pcreb , and bdnf contributed to cognitive impairment from chronic cerebral ischemia . minocycline , a semisynthetic tetracycline antibiotic that effectively crosses the blood brain barrier , has been reported to have significant neuroprotective effects in cognitive impairment,24,25 schizophrenia,26 cerebral ischemia,27 amyotrophic lateral sclerosis,28 alzheimer s disease,25,29 huntington s disease,30,31 and parkinson s diseases.32 minocycline can inhibit ischemic - induced inflammation,33,34 astrocyte reactivation,35 microglia activation,32 oxidative stress,36,37 apoptosis,37,38 and so on . one common manifestation after brain ischemic damage is cognitive impairment . in this present study , we established the cerebral ischemia model by a permanent bilateral occlusion of both common carotid arteries . the results from morris water maze test showed that cognitive impairment occurred with the ischemic brain damage model , and cognitive impairment of control animals had been attenuated after minocycline administration . furthermore , minocycline increased the levels of creb , pcreb , and bdnf in the hippocampus of rats by a permanent bilateral occlusion of both common carotid arteries . the results from the morris water maze test showed that cognitive impairment occurred with chronic cerebral ischemia injury , and minocycline reduced cognitive impairment caused by permanent bilateral occlusion of both common carotid arteries . to further examine the mechanism by which cognitive impairment occurred with chronic cerebral ischemia and by which minocycline improved behavioral deficits , the expression of creb , a bio - marker of memory,39 was examined in the hippocampus tissue of rats . creb , belonging to the family of leucine zipper transcription factors , is critical to induce its effects at phosphorylation of a serine residue ( s133 ) in its kinase - inducible domain . phosphorylation of creb can be accomplished by a number of upstream signaling cascades.40,41 studies indicate that these pathways are perturbed in patients suffering from cognitive impairment and they are also known to be influenced by anti - cognitive impairment treatment.42,43 creb has a role to play in the pathogenesis of cognitive impairment and in anti - cognitive impairment action.39,44,45 in the present study , both creb and pcreb were downregulated after cerebral ischemia injury , and downregulation of creb and pcreb contributed to cognitive impairment from cerebral ischemia injury by correlation analysis . in addition , the camp creb signaling cascade is critical to the generation of new neurons in the rodent hippocampus , and also facilitates their subsequent morphological maturation . thus , creb s neuroprotective and survival - enhancing properties can act in a manner analogous to that of anti - cognitive impairment . therefore , minocycline can mediate overexpression of creb in the hippocampus and has an anti - cognitive impairment - like effect in the process of cerebral ischemia injury . to further clarify the mechanism by which minocycline improved behavioral deficits , the expression of bdnf ( a neurotrophin that play a critical role in the development of the brain and continues to have a seminal role in shaping plasticity in the mature nervous system ) was investigated . bdnf is the most widely expressed member of the nerve growth factor family of growth regulators , collectively termed the neurotrophins.4648 the neurotrophins play a critical role in the development of the brain and continue to have a seminal role in shaping plasticity in the mature nervous system.49 bdnf has also been shown to elicit rapid action potentials , thus influencing neuronal excitability , and it has a demonstrable role in activity - dependent synaptic plasticity events like long - term potentiation , learning tasks , and memory.50,51 bdnf is involved in structural remodeling , neuronal plasticity , and synaptic restructuring,52,53 and is promising as a candidate molecule underlying the structural changes associated with cerebral ischemia damage , and as a potential target for cerebral ischemia damage.54 in the present study , bdnf was downregulated in the hippocampus tissue after chronic cerebral ischemia injury , whereas bdnf was upregulated after minocycline administration . thus , it is speculated that minocycline has an anti - cognitive impairment - like effect in behavioral models of vascular cognitive impairment through enhancing the expression of bdnf in the hippocampus . the hippocampus is a key limbic region whose structure and function is compromised in cognition disorders . in the hippocampus , bdnf cascade results in anti - cognition responses.13,55,56 hippocampal overexpression of bdnf and creb is capable of mimicking both the structural consequences of sustained anti - cognition treatment as well as exerting anti - cognition - like behavioral effects.56 activation of the camp creb cascade results in increased neurogenesis of dentate granule cell progenitors , and increased dendritic length and branching . it is possible that creb , a transcriptional activator of bdnf , recruits this neurotrophin to mediate its effects on structural plasticity.57,58 bdnf , in addition to being a target of creb , can itself recruit this particular transcription factor by activating the map kinase cascade , thus setting up a potential positive feedback loop . taken together , elevated creb bdnf , through its protective influences on vulnerable hippocampal neurons and ability to directly promote structural reorganization , could result in repair of the region known to be damaged in cognitive impairment . moreover , the well - established role of bdnf and creb in hippocampal - dependent learning and memory may play a critical role in ameliorating the cognitive symptoms.13,59 in the present study , minocycline efficiently improved behavioral deficits and increased creb , pcreb , and bdnf that had been down - regulated by cerebral ischemia . it is possible that minocycline recruits creb bdnf cascade to mediate its effects on structural plasticity and set up a potential positive creb bdnf activity by minocycline , through its protective influences on vulnerable hippocampal neurons and ability to directly promote structural reorganization , could result in repair of the region known to be damaged in cognitive impairment . in conclusion , this study is the first to evaluate the influence of minocycline on the transcription factors ( creb , pcreb , and bdnf ) as potential key players in the treatment of vascular cognitive impairment in the process of cerebral ischemia injury . from a clinical point of view , the ability of minocycline to modulate cognitive impairment may be of great importance in the selection of neuroprotective agents , especially in chronic cerebral ischemia procedures .

background and purposethe camp response element binding protein ( creb ) plays an important role in the mechanism of cognitive impairment and is also pivotal in the switch from short - term to long - term memory . brain - derived neurotrophic factor ( bdnf ) seems a promising avenue in the treatment of cerebral ischemia injury since this neurotrophin could stimulate structural plasticity and repair cognitive impairment . several findings have displayed that the dysregulation of the creb bdnf cascade has been involved in cognitive impairment . the aim of this study was to investigate the effect of cerebral ischemia on learning and memory as well as on the levels of creb , phosphorylated creb ( pcreb ) , and bdnf , and to determine the effect of minocycline on creb , pcreb , bdnf , and behavioral functional recovery after cerebral ischemia.methodsthe animal model was established by permanent bilateral occlusion of both common carotid arteries . behavior was evaluated 5 days before decapitation with morris water maze and open - field task . four days after permanent bilateral occlusion of both common carotid arteries , minocycline was administered by douche via the stomach for 4 weeks . creb and pcreb were examined by western blotting , reverse transcription polymerase chain reaction , and immunohistochemistry . bdnf was measured by immunohistochemistry and western blotting.resultsthe model rats after minocycline treatment swam shorter distances than control rats before finding the platform ( p=0.0007 ) . the number of times the platform position was crossed for sham - operation rats was more than that of the model groups in the corresponding platform location ( p=0.0021 ) . the number of times the platform position was crossed for minocycline treatment animals was significantly increased compared to the model groups in the corresponding platform position ( p=0.0016 ) . creb , pcreb , and bdnf were downregulated after permanent bilateral occlusion of both common carotid arteries in the model group . minocycline increased the expression of creb , pcreb , and bdnf , and improved cognitive suffered from impairment of permanent bilateral occlusion of both common carotid arteries.conclusionminocycline improved cognitive impairment from cerebral ischemia via enhancing creb , pcreb , and bdnf activity in the hippocampus .

Introduction Materials and methods Animal and drug Morris water maze task Immunohistochemical assay Western blotting Statistical analysis Results Minocycline improved behavioral deficits Minocycline upregulated CREB and pCREB Minocycline enhanced BDNF activity Correlation between cognition and CREB, pCREB, and BDNF Discussion Conclusion

increasing findings have evidenced that cerebral ischemia plays a critical role in the pathogenesis of vascular cognitive impairment , and the reduction of cerebral blood flow correlates with the severity of cognitive impairment.1,2 various mechanisms of neuronal injury suffered from cerebral ischemia have been proposed , including formation of free radicals , oxidative stress,3,4 mitochondrial dysfunction,5,6 inflammatory processes,7 genetic factors , environmental impact factors,8,9 apoptosis,10 and so on . the transcription factor cyclic amp response element binding protein ( creb ) and neurotrophin brain - derived neurotrophic factor ( bdnf ) have emerged as molecules that may play an important role in modulating mood , behavior , and memory.1113 creb and bdnf are known to be dysregulated in animal models and in patients suffering from cerebral ischemia , and are deemed to be therapeutic targets of cerebral ischemia.14,15 minocycline , a tetracycline derivative , protects against cerebral ischemia via inhibiting inflammation , oxidative stress , and apoptosis.16,17 previously , we have found that minocycline retarded astrocytic reactivation , and restrained oxidative stress and neuroinflammation in the hippocampus of cerebral ischemia rats.18,19 in the present study , we observed the expression of creb , phosphorylated creb ( pcreb ) , and bdnf in the hippocampus of cerebral ischemia rats with cognitive impairment by permanent bilateral occlusion of both common carotid arteries , and explored the neuroprotective mechanism of minocycline for the treatment of cerebral ischemia injury . we found that creb , pcreb , and bdnf were downregulated after permanent bilateral occlusion of both common carotid arteries in a model group , and minocycline attenuated cognitive impairment and upregulated creb , pcreb , and bdnf in the hippocampus of rats with permanent bilateral occlusion of both common carotid arteries . therefore , a hypothesis was made that minocycline upregulated creb , pcreb , and bdnf and improved cognitive impairment from cerebral vascular factors . wistar rats ( 10 weeks old , female , quality 200250 g , from the field zoology research institute of the third military medical university of the people s republic of china ) were randomly divided into sham - operated group ( s ) ( with a mean survival time of 16 weeks ) , ischemia model group ( m ) ( with permanent bilateral occlusion of both common carotid arteries ) , and minocycline treatment group ( mt ) ( beginning treatment after 4 days from permanent bilateral occlusion of both common carotid arteries , minocycline was administered by douche via the stomach for 4 weeks ) . the animal model of cerebral ischemia was established with permanent bilateral occlusion of both common carotid arteries for chronic bilateral common carotid artery occlusion ( bccao).20,21 rats were anesthetized with 10% chloral hydrate ( 350 mg / kg , intraperitoneally ) and breathed normally throughout the surgical procedure . both common carotid arteries were exposed via a midline cervical incision and doubly - ligated with silk suture . sham - operated animals were treated in the same manner , except that the common arteries were not ligated . s and m groups were given the same volume of normal saline through douche via the stomach . the minocycline dosage used for animals was as described elsewhere.16,17 the morris water maze task ( chinese academy of medical sciences , people s republic of china ) includes a place navigation test and spatial probe test , and is widely used in behavioral neuroscience to study spatial learning and memory.23 the rats were placed in a large circular pool with an invisible platform that allows them to escape the water . the time it took a rat to find the platform and escape was measured for up to four trials a day for 5 days . the time it took to find the unmoved platform ( learning latency ) and the times a rat crossed the corresponding position of the removed platform in 120 seconds ( memory latency ) were recorded . the avidin - biotin - peroxidase complex immunohistochemical assay was carried out according to the protocols we described before anti - creb ( sigma - aldrich co. , st louis , mo , usa ) , anti - pcreb ( sigma - aldrich co. ) , and anti - bdnf ( santa cruz , la , ca , usa ) were prepared . , bdnf ( 1:500 ; sigma - aldrich co. , st louis , mo , usa ) , and -tublin ( 1:200 ; biosource international inc . wistar rats ( 10 weeks old , female , quality 200250 g , from the field zoology research institute of the third military medical university of the people s republic of china ) were randomly divided into sham - operated group ( s ) ( with a mean survival time of 16 weeks ) , ischemia model group ( m ) ( with permanent bilateral occlusion of both common carotid arteries ) , and minocycline treatment group ( mt ) ( beginning treatment after 4 days from permanent bilateral occlusion of both common carotid arteries , minocycline was administered by douche via the stomach for 4 weeks ) . the animal model of cerebral ischemia was established with permanent bilateral occlusion of both common carotid arteries for chronic bilateral common carotid artery occlusion ( bccao).20,21 rats were anesthetized with 10% chloral hydrate ( 350 mg / kg , intraperitoneally ) and breathed normally throughout the surgical procedure . s and m groups were given the same volume of normal saline through douche via the stomach . mt group was given 50 mg / kg / d minocycline through douche via the stomach . the morris water maze task ( chinese academy of medical sciences , people s republic of china ) includes a place navigation test and spatial probe test , and is widely used in behavioral neuroscience to study spatial learning and memory.23 the rats were placed in a large circular pool with an invisible platform that allows them to escape the water . the avidin - biotin - peroxidase complex immunohistochemical assay was carried out according to the protocols we described before anti - creb ( sigma - aldrich co. , st louis , mo , usa ) , anti - pcreb ( sigma - aldrich co. ) , and anti - bdnf ( santa cruz , la , ca , usa ) were prepared . equal amounts of lysates were subject to sodium dodecyl sulfate - polyacrylamide gel electrophoresis and western blotting analysis using antibodies specific for the following : creb ( 1:1,000 ; biosource international , inc . , bdnf ( 1:500 ; sigma - aldrich co. , st louis , mo , usa ) , and -tublin ( 1:200 ; biosource international inc . cerebral ischemia was induced in 10-week - old wistar rats by bccao as described previously.18,19 after the performance of bccao , rats were subjected to the morris water maze . on days 3 , 4 , and 5 , s animals immediately swam toward the platforms in the water maze , whereas m rats swam longer distances before finding the platform ( figure 1a ) . in general , escape latency decreased with bccao duration ( p=0.0004 ) , the m rats , after minocycline treatment , swam shorter distances than control rats before finding the platform ( p=0.0007 ) ( figure 1a ) . in the probe trials , the number of times the platform position was crossed for the s group was more than for the bccao rats groups in the corresponding platform location ( p=0.0000 ) ( figure 1b ) . the number of times the platform position was crossed for mt animals was significantly increased compared to bccao rat groups in the corresponding platform position ( p=0.0016 ) ( figure 1b ) . the results of immunohistochemistry and western blotting showed that expression of creb and pcreb in the mt group was significantly higher than that of the m group at the corresponding time . expression of creb and pcreb by immunohistochemistry in the m groups was more decreased than in the control group ( p=0.0009 ; p=0.0023 ) , whereas expression of creb and pcreb in the mt groups was more increased than in the control groups ( p=0.0001 ; p=0.0005 ) ( figure 2 ) . expression of creb and pcreb by western blotting in the m groups was lowered more than in the control group ( p=0.0010 ; p=0.0031 ) , whereas expression of creb and pcreb in the mt groups was more enhanced than in the control groups ( p=0.0004 ; p=0.0003 ) ( figure 2 ) . the results of immunohistochemistry showed that expression of bdnf in the mt animals was higher than that of the m ones ( p=0.0005 ) , whereas expression of bdnf in the m groups was decreased compared to the control group ( p=0.0001 ) . western blotting analysis found that bdnf in the mt groups was higher than that of the m group ( p=0.0006 ) , while expression of bdnf in the m groups was more reduced than in the control group ( p=0.0000 ) ( figure 3 ) . the number of times the platform position was crossed during the probe trial for the s group was higher than for m rats ( p=0.0021 ) . therefore , we determined correlation analysis between the number of times the platform position was crossed and creb , pcreb , and bdnf expression . linear correlation analysis shows that the optical density ( od ) values of immunoblotting protein for creb , pcreb , and bdnf were negatively correlated with the number of platform position crossings ( r=0.314 , p=0.0062 ; r=0.352 , p=0.0004 ; r=0.381 , p=0.0031 ) respectively ( figure 4 ) . we inferred that downregulation of creb , pcreb , and bdnf contributed to cognitive impairment from chronic cerebral ischemia . cerebral ischemia was induced in 10-week - old wistar rats by bccao as described previously.18,19 after the performance of bccao , rats were subjected to the morris water maze . on days 3 , 4 , and 5 , s animals immediately swam toward the platforms in the water maze , whereas m rats swam longer distances before finding the platform ( figure 1a ) . in general , escape latency decreased with bccao duration ( p=0.0004 ) , the m rats , after minocycline treatment , swam shorter distances than control rats before finding the platform ( p=0.0007 ) ( figure 1a ) . in the probe trials , the number of times the platform position was crossed for the s group was more than for the bccao rats groups in the corresponding platform location ( p=0.0000 ) ( figure 1b ) . the number of times the platform position was crossed for mt animals was significantly increased compared to bccao rat groups in the corresponding platform position ( p=0.0016 ) ( figure 1b ) . the results of immunohistochemistry and western blotting showed that expression of creb and pcreb in the mt group was significantly higher than that of the m group at the corresponding time . expression of creb and pcreb by immunohistochemistry in the m groups was more decreased than in the control group ( p=0.0009 ; p=0.0023 ) , whereas expression of creb and pcreb in the mt groups was more increased than in the control groups ( p=0.0001 ; p=0.0005 ) ( figure 2 ) . expression of creb and pcreb by western blotting in the m groups was lowered more than in the control group ( p=0.0010 ; p=0.0031 ) , whereas expression of creb and pcreb in the mt groups was more enhanced than in the control groups ( p=0.0004 ; p=0.0003 ) ( figure 2 ) . the results of immunohistochemistry showed that expression of bdnf in the mt animals was higher than that of the m ones ( p=0.0005 ) , whereas expression of bdnf in the m groups was decreased compared to the control group ( p=0.0001 ) . western blotting analysis found that bdnf in the mt groups was higher than that of the m group ( p=0.0006 ) , while expression of bdnf in the m groups was more reduced than in the control group ( p=0.0000 ) ( figure 3 ) . the number of times the platform position was crossed during the probe trial for the s group was higher than for m rats ( p=0.0021 ) . therefore , we determined correlation analysis between the number of times the platform position was crossed and creb , pcreb , and bdnf expression . linear correlation analysis shows that the optical density ( od ) values of immunoblotting protein for creb , pcreb , and bdnf were negatively correlated with the number of platform position crossings ( r=0.314 , p=0.0062 ; r=0.352 , p=0.0004 ; r=0.381 , p=0.0031 ) respectively ( figure 4 ) . we inferred that downregulation of creb , pcreb , and bdnf contributed to cognitive impairment from chronic cerebral ischemia . in this present study , we established the cerebral ischemia model by a permanent bilateral occlusion of both common carotid arteries . the results from morris water maze test showed that cognitive impairment occurred with the ischemic brain damage model , and cognitive impairment of control animals had been attenuated after minocycline administration . furthermore , minocycline increased the levels of creb , pcreb , and bdnf in the hippocampus of rats by a permanent bilateral occlusion of both common carotid arteries . the results from the morris water maze test showed that cognitive impairment occurred with chronic cerebral ischemia injury , and minocycline reduced cognitive impairment caused by permanent bilateral occlusion of both common carotid arteries . to further examine the mechanism by which cognitive impairment occurred with chronic cerebral ischemia and by which minocycline improved behavioral deficits , the expression of creb , a bio - marker of memory,39 was examined in the hippocampus tissue of rats . phosphorylation of creb can be accomplished by a number of upstream signaling cascades.40,41 studies indicate that these pathways are perturbed in patients suffering from cognitive impairment and they are also known to be influenced by anti - cognitive impairment treatment.42,43 creb has a role to play in the pathogenesis of cognitive impairment and in anti - cognitive impairment action.39,44,45 in the present study , both creb and pcreb were downregulated after cerebral ischemia injury , and downregulation of creb and pcreb contributed to cognitive impairment from cerebral ischemia injury by correlation analysis . in addition , the camp creb signaling cascade is critical to the generation of new neurons in the rodent hippocampus , and also facilitates their subsequent morphological maturation . therefore , minocycline can mediate overexpression of creb in the hippocampus and has an anti - cognitive impairment - like effect in the process of cerebral ischemia injury . to further clarify the mechanism by which minocycline improved behavioral deficits , the expression of bdnf ( a neurotrophin that play a critical role in the development of the brain and continues to have a seminal role in shaping plasticity in the mature nervous system ) was investigated . bdnf is the most widely expressed member of the nerve growth factor family of growth regulators , collectively termed the neurotrophins.4648 the neurotrophins play a critical role in the development of the brain and continue to have a seminal role in shaping plasticity in the mature nervous system.49 bdnf has also been shown to elicit rapid action potentials , thus influencing neuronal excitability , and it has a demonstrable role in activity - dependent synaptic plasticity events like long - term potentiation , learning tasks , and memory.50,51 bdnf is involved in structural remodeling , neuronal plasticity , and synaptic restructuring,52,53 and is promising as a candidate molecule underlying the structural changes associated with cerebral ischemia damage , and as a potential target for cerebral ischemia damage.54 in the present study , bdnf was downregulated in the hippocampus tissue after chronic cerebral ischemia injury , whereas bdnf was upregulated after minocycline administration . thus , it is speculated that minocycline has an anti - cognitive impairment - like effect in behavioral models of vascular cognitive impairment through enhancing the expression of bdnf in the hippocampus . in the hippocampus , bdnf cascade results in anti - cognition responses.13,55,56 hippocampal overexpression of bdnf and creb is capable of mimicking both the structural consequences of sustained anti - cognition treatment as well as exerting anti - cognition - like behavioral effects.56 activation of the camp creb cascade results in increased neurogenesis of dentate granule cell progenitors , and increased dendritic length and branching . it is possible that creb , a transcriptional activator of bdnf , recruits this neurotrophin to mediate its effects on structural plasticity.57,58 bdnf , in addition to being a target of creb , can itself recruit this particular transcription factor by activating the map kinase cascade , thus setting up a potential positive feedback loop . taken together , elevated creb bdnf , through its protective influences on vulnerable hippocampal neurons and ability to directly promote structural reorganization , could result in repair of the region known to be damaged in cognitive impairment . moreover , the well - established role of bdnf and creb in hippocampal - dependent learning and memory may play a critical role in ameliorating the cognitive symptoms.13,59 in the present study , minocycline efficiently improved behavioral deficits and increased creb , pcreb , and bdnf that had been down - regulated by cerebral ischemia . it is possible that minocycline recruits creb bdnf cascade to mediate its effects on structural plasticity and set up a potential positive creb bdnf activity by minocycline , through its protective influences on vulnerable hippocampal neurons and ability to directly promote structural reorganization , could result in repair of the region known to be damaged in cognitive impairment . in conclusion , this study is the first to evaluate the influence of minocycline on the transcription factors ( creb , pcreb , and bdnf ) as potential key players in the treatment of vascular cognitive impairment in the process of cerebral ischemia injury . from a clinical point of view , the ability of minocycline to modulate cognitive impairment may be of great importance in the selection of neuroprotective agents , especially in chronic cerebral ischemia procedures .

body image is the mental image formed from the size and shape of one s own body . it is a multidimensional construct that encompasses feelings , thoughts and behavior concerning physical attributes [ 2 , 3 ] . it can also be considered as the relationship between the body of an individual and body - related cognitive processes ( beliefs , values and attitudes ) [ 4 , 5 ] . the growing urbanization and technological development of the modern world has imposed important changes in people s lifestyle . sedentary behavior , which decreases physical activity levels , in conjunction with poor eating habits and increased intake of industrialized and hypercaloric food have contributed to the rise in problems related to excess body weight . a number of studies suggest that a significant increase in body mass index ( bmi ) , waist circumference ( wc ) and body fat percentage ( bfp ) are strong indicators of body image dissatisfaction ( bid ) [ 8 - 10 ] . although there are anthropometric values suggested by the world health organization to maintain physical fitness related to health , the ideal physical type is determined by culture . in this respect , the current esthetic beauty standards accepted by society and constantly promoted by the media , are thin women and muscular men [ 14 - 16 ] , which generates excessive concern with body image and influences the growing dissatisfaction of people with their appearance [ 18 , 19 ] . thus , people compulsively strive for the perfect body and the esthetic ideal of beauty in order to follow these standards and be socially accepted [ 20 - 22 ] . this imposition aims to meet the demands of the beauty industry [ 23 , 24 ] , triggering increased consumption in medications , clothes , gym services , and weight loss clinics [ 21 , 22 ] , among other stakeholders in this expanding market . moreover , these factors may contribute directly to the incidence of eating disorders [ 25 , 26 ] . the body stereotype proposed is not always attainable due to different physical structures of the population , causing an increase in bid , which can be understood as a negative assessment of one s body . this is normally diagnosed using questionnaires and silhouette scales , which emphasize concerns with weight , shape and bfp . however , when the perception domain is investigated , silhouette figures are more frequently used . dissatisfaction with body image , resulting from not meeting the esthetic - cultural standards of contemporary society is highly prevalent in both genders and in different age groups . it is more common in young people , who are more vulnerable to behavioral changes , as well as in health professionals who value physical appearance [ 32 , 33 ] . depending on its severity , this dissatisfaction may hinder some aspects of life related to eating behavior , self - esteem as well as psychosocial , physical and cognitive performance [ 19 , 34 - 37 ] . given that psychological well - being in current society is significantly associated with body measurements , a negative self - concept of body image can decrease quality of life [ 45 - 47 ] and is a risk factor for psychiatric symptoms such as depression or mood changes [ 37 - 40 ] , anxiety [ 41 , 42 ] and stress [ 43 , 44 ] . there is wide evidence showing that impaired body image perception may contribute to an increase in suicide attempts [ 48 , 49 ] . thus , is bid related to anthropometric profile ? moreover , does bid influence mental health measures , irrespective of anthropometric profile ? the present study aimed to identify the prevalence of bid in university students , as well as to compare the mean values obtained in anthropometric tests and instruments related to mental health between individuals satisfied and dissatisfied with body image . this is a cross - sectional study with a convenience sample composed of 140 physical education students ( 75 men and 65 women ) from the veiga de almeiga university in rio de janeiro state ( uva - rj ) . undergraduates were excluded from the sample who were absent from the class on data collection days ; did not complete all the anthropometric assessment tests ; refused to participate ; did not correctly fill out the questionnaires . individuals were instructed regarding the study procedures and gave their informed consent in accordance to the 196/96 resolution of the national health council and the declaration of helsinki . the project was approved by the human research ethics committee from the universidade veiga de almeida ( uva rj ) ( no . they were explained the goals and ethical procedures and gave their informed consent . trained evaluators performed data collection at two different moments . on the first day , the individuals responded to the instruments ( stai , poms , sf-36 and the silhouette scale ) and on the second day , anthropometric variables were collected ( body composition - bia , weight , height and waist circumference ) . all study individuals received the same verbal instructions and doubts were clarified before the questionnaires were filled out . the instruments also contained written instructions on how to complete them . during the application of the instruments , there was no communication between individuals and there was no time limit imposed . the following anthropometric and body composition variables were collected : a ) height ( cm ) using a stadiometer with a 0.1 cm accuracy ( sanny es 2020 , brazil ) ; b ) weight using a digital scale with a 0.01 kg accuracy ( filizola pl 180 , brazil ) ; c ) waist circumference ( wc ) was measured using a steel anthropometric tape ( cescorf , brazil ) , with a 2 m extension and 0.01 m intervals , at the narrowest point between the external surface of the last rib and the anterior superior iliac spine ; d ) bfp using a bipolar bioelectrical impedance analysis ( bia ) device ( omron hbf-306 , usa ) . to complete the bia analysis , participants completed the following procedures : ( 1 ) did not use diuretic drugs in the previous 7 days ; ( 2 ) fasting for at least 4 h ; ( 3 ) did not consume alcoholic beverages in the previous 48 h ; ( 4 ) did not engage in intense physical activity in the last 24 h ; ( 5 ) urinated at least 30 min before the measurement ; ( 6 ) removed body adornments such as earrings , bracelets , necklaces and piercings ; and ( 7 ) remained at absolute rest for at least 8 - 10 min in the supine position before the measurement . all anthropometric measurements were made in accordance to the recommendations of the international society for the advancement of kinanthropometry . body mass index ( bmi ) was computed using height and weight measures ( weight / height ) . bmi was classified as normal ( < 25 kg / m ) and overweight ( 25 the cutoff points used for wc were based on the degree of risk for cardiovascular diseases , namely moderate risk for women ( wc > 80 cm ) and men ( wc > 94 cm ) , and high risk for women ( wc > 88 cm ) and men ( wc > 102 cm ) . with respect to bfp , the cut off points for obesity or excess fat were 25% for men and this consisted of male and female figures representing different body shapes numbered from 1 to 9 . the individuals marked the silhouette that was most similar to their current body image and which one they would like to have . if the difference was zero , the individuals were classified as satisfied , and if different from zero then they were classified as dissatisfied . the 42 items on the short form are divided into six subscales : tension ( t ) , depression ( d ) , hostility ( h ) , vigor ( v ) , fatigue ( f ) and mental confusion ( c ) . each subscale contains four items rated using a likert - type scale ( not at all - 0 ; a little - 1 ; moderately - 2 ; quite a bit - 3 ; extremely - 4 ) , with sub - scores ranging from 0 to 16 . subscales t , d , h , f and c are considered negative factors of mood , and v a positive factor . total mood disturbance ( tmd ) is calculated by the sum of negative factors , subtracting the positive factor score . anxiety was measured by the self - rated stai scales ( state and trait anxiety inventory ) . the state scale ( stai s ) requires individuals to express how they feel at the moment in relation to the 20 items contained on a 4-point likert scale ( not at all - 1 ; somewhat - 2 ; moderately so - 3 ; very much so - 4 ) . the trait scale ( stai t ) also has 20 items , but individuals are instructed to respond how they generally feel on a different 4-point likert scale ( almost never - 1 ; sometimes - 2 ; often - 3 ; almost always - 4 ) . the total score of each scale can vary from 20 to 80 points , with scores below 30 indicating a low degree of anxiety , 31 - 49 a medium level and 50 or more a high degree [ 55 , 56 ] . the sf-36 questionnaire ( medical outcomes study 36 item short - form health survey ) was used to assess quality of life . this instrument contains 36 items , 35 of which encompass eight domains , grouped into two large components : physical ( domains : functional capacity , physical aspects , pain and general health status ) and mental ( domains : mental health , vitality , social aspects and emotional aspects ) . each of these domains received scores between 0 and 100 , where zero corresponds to the lowest score and 100 to the highest [ 57 , 58 ] . the sample was characterized using descriptive statistics , namely central tendency ( mean ) and dispersion ( standard deviation ) measures . the shapiro - wilk and levene tests were used to verify the sample s normality and homogeneity of variances , respectively . the student s t - test was applied to compare means from the anthropometric measures and mental health scores . a p - value < 0.05 was considered significant . for purposes of the present study , the d score was defined as the difference in scores between groups ( i.e. , body satisfied and body dissatisfied ) expressed in standard deviation units ( mgroup1-mgroup2/sdpooled across groups ) . the most commonly used cohen s d formula has a slight bias as it overestimates the sample estimate effect size in small samples . thereby , we completed the hedges correction which removes this bias using a simple correction , yielding an unbiased estimate of the effect size . effect sizes were classified as minimal to small ( d < 0.2 ) , small to moderate ( d = 0.2 0.5 ) , moderate to large ( d = 0.5 0.8 ) and large ( d > 0.8 ) . this is a cross - sectional study with a convenience sample composed of 140 physical education students ( 75 men and 65 women ) from the veiga de almeiga university in rio de janeiro state ( uva - rj ) . undergraduates were excluded from the sample who were absent from the class on data collection days ; did not complete all the anthropometric assessment tests ; refused to participate ; did not correctly fill out the questionnaires . individuals were instructed regarding the study procedures and gave their informed consent in accordance to the 196/96 resolution of the national health council and the declaration of helsinki . the project was approved by the human research ethics committee from the universidade veiga de almeida ( uva rj ) ( no . they were explained the goals and ethical procedures and gave their informed consent . trained evaluators performed data collection at two different moments . on the first day , the individuals responded to the instruments ( stai , poms , sf-36 and the silhouette scale ) and on the second day , anthropometric variables were collected ( body composition - bia , weight , height and waist circumference ) . all study individuals received the same verbal instructions and doubts were clarified before the questionnaires were filled out . the instruments also contained written instructions on how to complete them . during the application of the instruments , there was no communication between individuals and there was no time limit imposed . the following anthropometric and body composition variables were collected : a ) height ( cm ) using a stadiometer with a 0.1 cm accuracy ( sanny es 2020 , brazil ) ; b ) weight using a digital scale with a 0.01 kg accuracy ( filizola pl 180 , brazil ) ; c ) waist circumference ( wc ) was measured using a steel anthropometric tape ( cescorf , brazil ) , with a 2 m extension and 0.01 m intervals , at the narrowest point between the external surface of the last rib and the anterior superior iliac spine ; d ) bfp using a bipolar bioelectrical impedance analysis ( bia ) device ( omron hbf-306 , usa ) . to complete the bia analysis , participants completed the following procedures : ( 1 ) did not use diuretic drugs in the previous 7 days ; ( 2 ) fasting for at least 4 h ; ( 3 ) did not consume alcoholic beverages in the previous 48 h ; ( 4 ) did not engage in intense physical activity in the last 24 h ; ( 5 ) urinated at least 30 min before the measurement ; ( 6 ) removed body adornments such as earrings , bracelets , necklaces and piercings ; and ( 7 ) remained at absolute rest for at least 8 - 10 min in the supine position before the measurement . all anthropometric measurements were made in accordance to the recommendations of the international society for the advancement of kinanthropometry . body mass index ( bmi ) was computed using height and weight measures ( weight / height ) . bmi was classified as normal ( < 25 kg / m ) and overweight ( 25 the cutoff points used for wc were based on the degree of risk for cardiovascular diseases , namely moderate risk for women ( wc > 80 cm ) and men ( wc > 94 cm ) , and high risk for women ( wc > 88 cm ) and men ( wc > 102 cm ) . with respect to bfp , the cut off points for obesity or excess fat were 25% for men and the silhouette scale was used to estimate body image satisfaction . this consisted of male and female figures representing different body shapes numbered from 1 to 9 . the individuals marked the silhouette that was most similar to their current body image and which one they would like to have . if the difference was zero , the individuals were classified as satisfied , and if different from zero then they were classified as dissatisfied . the 42 items on the short form are divided into six subscales : tension ( t ) , depression ( d ) , hostility ( h ) , vigor ( v ) , fatigue ( f ) and mental confusion ( c ) . each subscale contains four items rated using a likert - type scale ( not at all - 0 ; a little - 1 ; moderately - 2 ; quite a bit - 3 ; extremely - 4 ) , with sub - scores ranging from 0 to 16 . subscales t , d , h , f and c are considered negative factors of mood , and v a positive factor . total mood disturbance ( tmd ) is calculated by the sum of negative factors , subtracting the positive factor score . anxiety was measured by the self - rated stai scales ( state and trait anxiety inventory ) . the state scale ( stai s ) requires individuals to express how they feel at the moment in relation to the 20 items contained on a 4-point likert scale ( not at all - 1 ; somewhat - 2 ; moderately so - 3 ; very much so - 4 ) . the trait scale ( stai t ) also has 20 items , but individuals are instructed to respond how they generally feel on a different 4-point likert scale ( almost never - 1 ; sometimes - 2 ; often - 3 ; almost always - 4 ) . the total score of each scale can vary from 20 to 80 points , with scores below 30 indicating a low degree of anxiety , 31 - 49 a medium level and 50 or more a high degree [ 55 , 56 ] . the sf-36 questionnaire ( medical outcomes study 36 item short - form health survey ) was used to assess quality of life . this instrument contains 36 items , 35 of which encompass eight domains , grouped into two large components : physical ( domains : functional capacity , physical aspects , pain and general health status ) and mental ( domains : mental health , vitality , social aspects and emotional aspects ) . each of these domains received scores between 0 and 100 , where zero corresponds to the lowest score and 100 to the highest [ 57 , 58 ] . the sample was characterized using descriptive statistics , namely central tendency ( mean ) and dispersion ( standard deviation ) measures . the shapiro - wilk and levene tests were used to verify the sample s normality and homogeneity of variances , respectively . the student s t - test was applied to compare means from the anthropometric measures and mental health scores . a p - value < 0.05 was considered significant . for purposes of the present study , the d score was defined as the difference in scores between groups ( i.e. , body satisfied and body dissatisfied ) expressed in standard deviation units ( mgroup1-mgroup2/sdpooled across groups ) . the most commonly used cohen s d formula has a slight bias as it overestimates the sample estimate effect size in small samples . thereby , we completed the hedges correction which removes this bias using a simple correction , yielding an unbiased estimate of the effect size . effect sizes were classified as minimal to small ( d < 0.2 ) , small to moderate ( d = 0.2 0.5 ) , moderate to large ( d = 0.5 0.8 ) and large ( d > 0.8 ) . a total of 140 physical education undergraduate students , aged 23.6 3.7 years , were participated in the study . the mean height , body weight , body fat percentage ( bfp ) , bmi and wc were 70.7 14.3 kg , 169.4 1.1 cm , 19.5 7.1% , 24.5 3.5 the prevalence of bid in the students was 67.1% , with 80.4% of women and 64.2% of men aiming to reduce their body measures . there was no difference in bmi ( p = 0.067 ) between satisfied ( 23.7 2.9 kg / m ) and dissatisfied individuals ( 24.9 3.7 kg / m ) . a significant difference ( t = -2.061 ; p = 0.041 ) in bf was observed between the satisfied and dissatisfied individuals ( 20.3 7.7% vs. 17.9 5.4% ) . mean wc was significantly higher ( t = -1.993 ; p = 0.048 ) in dissatisfied students than in their satisfied counterparts ( 77.9 10.0 cm vs.74.4 8.7 cm ) . although there were no significant differences in bmi between groups , the reported effect size was small to moderate ( d = 0.330 ) , just as for wc ( d = 0.357 ) and bfp ( d = 0.328 ) . the poms compared satisfied and dissatisfied individuals , respectively , finding significant differences between anger ( 8.4 4.0 vs. 10.2 3.9 ; t = -2.490 ; p = 0.014 ) , depression ( 2.6 3.8 vs. 5.0 5.6 ; t = -2.582 ; p = 0.011 ) , hostility ( 3.1 3.6 vs. 5.0 3.9 ; t = -2.815 ; p = 0.006 ) , fatigue ( 5.4 4.0 vs. 7.4 4.6 ; t = -2.526 ; p = 0.013 ) and mental confusion ( 7.1 2.5 vs. 8.2 2.7 ; t = -2.331 ; p = 0.021 ) . in relation to tmd , the dissatisfied obtained a significantly higher score ( 120.5 18.0 vs. 110.1 14.5 ; t = -3.422 ; p = 0.001 ) . all the domains from poms had small to moderate or moderate to large effect sizes , with the tmd score having the largest ( d = 0.612 ) . the mental aspect score of quality of life ( qol ) was significantly higher ( t = 3.444 ; p = 0.001 ) in satisfied students ( 75.1 12.1 ) when compared to the dissatisfied ( 66.8 15.9 ) , with a small to moderate difference between - groups ( d = 0.491 ) . the aim of the present study was to assess bid of university students , and to compare anthropometric measures and mental health scores between satisfied and unsatisfied students . it is important to underline that this value is significantly high , since approximately two out of three students expressed the desire to have a body shape different from the one currently perceived . similar results were found in studies with physical education students or participants from other study domains . miranda et al . found that 76.6% of university students recruited from several study domains were dissatisfied with their body image ( 20.82 3.03 years ) . in addition , gonalves et al . identified bid in 75.8% and 78.2% of nutrition and physical education undergraduates , respectively . a research project conducted by claumann et al . found that 79.2% of physical education freshmen expressed bid . in another study carried out with 236 undergraduates of both genders , similarly , an investigation performed with 294 physical education students from the state university of ponta grossa ( uepg ) found that 61.2% of individuals were dissatisfied with perceived body image . due to their high prevalence of bid , physical education undergraduates are highly susceptible to developing mental disorders , including eating disorders . the physical and esthetic demands inherent to the course and profession they chose to pursue may be a possible explanation for the high percentage of dissatisfaction observed . thereby , these future professionals face pressure to follow socially accepted esthetic standards , generating exaggerated concerns with body appearance that can influence body image satisfaction levels [ 41 , 64 ] . a number of authors agree that cultural pressures ultimately define idealized values , leading men to demonstrate a consensual desire to reduce body fat , increase muscle mass , develop broad shoulders , strong arms , a thin waist and hips , as well as a flat stomach . by contrast , women crave a thin and tall stereotype , with slim thighs , buttocks , and waist , in addition to a flat stomach [ 12 , 65 ] . cross - sectional studies have been conducted in order to establish the relationship between bid and anthropometric measures . the findings of the present study showed no difference in bmi ( p = 0.067 ) between undergraduates satisfied and dissatisfied with their body image , differing from those that reported an increase in bid with the rise in bmi [ 10 , 22 , 33 , 34 , 63 , 66 - 74 ] . some studies also found no significant association between these variables [ 12 , 69 , 75 ] . the results presented here suggest significant differences in the anthropometric variables bfp ( p = 0.041 ) and wc ( p = 0.048 ) between the two groups analyzed . observed an association between bid and bfp in a study conducted on 180 females aged between 10 an 17 years . studied individuals that engaged in activities at the pontifical university of parana ( puc - pr ) sports center and using multiple linear regression they observed that higher bfp values represented greater body image dissatisfaction . there is also an evidence associating bid with sum of skinfolds , an alternative anthropometric measure to assess body adiposity [ 10 , 66 ] . in a sample of 220 undergraduates , no association was found between wc and bid , a measure that can be used to estimate central adiposity . these findings suggest that bfp and wc , just like bmi , may be reliable anthropometric indicators of bid in university students . it is also important to notice that several researchers highlight the limitations related to the use of bmi , which does not differentiate between adipose tissue and lean mass [ 10 , 33 ] . thereby , it seems that bid is more interrelated to the amount of subcutaneous adipose tissue than total body weight . the current esthetic standard of beauty accepted by society and constantly promoted by the media may exert some influence on body image satisfaction and the mental health of individuals . these results corroborate those obtained for women in a study by birkeland et al . , where 138 female undergraduates demonstrated bid and a negative mood in the presence of a model . negative feelings about their bodies contribute to the increased prevalence of depression symptoms and decreased self - esteem in adolescent women . excessive media exposure of ideal bodies ( thin models ) in our society may trigger a more negative mood and lower food intake in women , contributing to the onset of eating disorders such as anorexia and bulimia . this low self - esteem in female adolescents may be attributed to overweight and obesity , which generates bid , negatively affecting mental health . el ansari et al . analyzed body image differences in 1,384 university undergraduates in two european countries and found that students who perceived themselves as being overweight reported lower quality of life . better body image was also related to an increase in self - esteem , optimism and social support among women , which are mental health related components of quality of life . quality of life seems to have a positive influence on how individuals perceive their body image , but the opposite is equally likely . there is a need to acknowledge and increase our understanding of bid as a public health problem . a total of 5,255 australian women , aged between 18 and 42 years , filled out specific questionnaires on body image and mental health . the results demonstrate that most individuals ( 86.9% ) reported some level of dissatisfaction with their weight , and more than one - third ( 39.4% ) relate moderate dissatisfaction . higher levels of bid were associated with poor quality of life , primarily with the mental health and psychosocial dimensions . individuals with third - degree obesity exhibit concerns with body image and high frequency of binge eating episodes , as well as severe depression symptoms and anxiety . depression symptoms were detected in 100% of these individuals , and severe symptomatology in 84% . the frequency of anxiety as a trait was 70% , as a state 54% , and 76% of all patients reported discomfort regarding their body image . first , it has a cross - sectional design , which does not allow establishing a causal relationship between the assessed variables . furthermore , body silhouette scales are two - dimensional and do not allow to fully represent the individual , their body fat distribution and other anthropometric measures important to body image . future studies should make efforts to include new variables to analyze the interaction between the biological , physiological , emotional and social aspects that encompass body image understanding . furthermore , individuals with greater fat accumulation in the visceral region and a higher percentage of body fat were more likely to exhibit bid . finally , bid was related with poorer quality of life and mood state in these university students .

backgroundthe prevalence of body image dissatisfaction ( bid ) is currently high . given that psychological well - being is associated with the body measurements imposed by esthetic standards , bid is an important risk factor for mental disorders.objective identify the prevalence of bid , and compare anthropometric and mental health parameters between individuals satisfied and dissatisfied with their body image.methoda total of 140 university students completed the silhouette scale to screen for bid . anthropometric measures , body mass index ( bmi ) , waist circumference ( wc ) and body fat percentage ( bfp ) were used . to investigate mental health , the state - trait anxiety inventories ( stai - s and stai - t ) , profile of mood states ( poms ) scale and quality of life ( qol-36 ) questionnaire were used to investigate mental health . the student s t - test was applied to compare anthropometric and mental health parameters . results67.1% of university students exhibited bid . there was a significant difference ( p = 0.041 ) in bf and wc ( p = 0.048 ) between dissatisfied and satisfied individuals . with respect to mood states , significant differences were observed for anger ( p = 0.014 ) , depression ( p = 0.011 ) , hostility ( p = 0.006 ) , fatigue ( p = 0.013 ) , mental confusion ( p = 0.021 ) and total mood disturbance ( tmd ) ( p = 0.001 ) . the mental aspect of qol was significantly higher ( p = 0.001 ) in satisfied university students compared to their dissatisfied counterparts.conclusion bid was high and it seems to be influenced by anthropometric measures related to the amount and distribution of body fat . this dissatisfaction may have a negative effect on the quality of life and mood state of young adults .

INTRODUCTION METHODS Sample Data Collection Procedures Anthropometric Variables Body Image Mood State State-Trait Anxiety Quality of Life Statistical Analysis RESULTS DISCUSSION LIMITATIONS CONCLUSION CONFLICT OF INTEREST

body image is the mental image formed from the size and shape of one s own body . it can also be considered as the relationship between the body of an individual and body - related cognitive processes ( beliefs , values and attitudes ) [ 4 , 5 ] . sedentary behavior , which decreases physical activity levels , in conjunction with poor eating habits and increased intake of industrialized and hypercaloric food have contributed to the rise in problems related to excess body weight . a number of studies suggest that a significant increase in body mass index ( bmi ) , waist circumference ( wc ) and body fat percentage ( bfp ) are strong indicators of body image dissatisfaction ( bid ) [ 8 - 10 ] . although there are anthropometric values suggested by the world health organization to maintain physical fitness related to health , the ideal physical type is determined by culture . in this respect , the current esthetic beauty standards accepted by society and constantly promoted by the media , are thin women and muscular men [ 14 - 16 ] , which generates excessive concern with body image and influences the growing dissatisfaction of people with their appearance [ 18 , 19 ] . the body stereotype proposed is not always attainable due to different physical structures of the population , causing an increase in bid , which can be understood as a negative assessment of one s body . depending on its severity , this dissatisfaction may hinder some aspects of life related to eating behavior , self - esteem as well as psychosocial , physical and cognitive performance [ 19 , 34 - 37 ] . given that psychological well - being in current society is significantly associated with body measurements , a negative self - concept of body image can decrease quality of life [ 45 - 47 ] and is a risk factor for psychiatric symptoms such as depression or mood changes [ 37 - 40 ] , anxiety [ 41 , 42 ] and stress [ 43 , 44 ] . moreover , does bid influence mental health measures , irrespective of anthropometric profile ? the present study aimed to identify the prevalence of bid in university students , as well as to compare the mean values obtained in anthropometric tests and instruments related to mental health between individuals satisfied and dissatisfied with body image . individuals were instructed regarding the study procedures and gave their informed consent in accordance to the 196/96 resolution of the national health council and the declaration of helsinki . on the first day , the individuals responded to the instruments ( stai , poms , sf-36 and the silhouette scale ) and on the second day , anthropometric variables were collected ( body composition - bia , weight , height and waist circumference ) . during the application of the instruments , there was no communication between individuals and there was no time limit imposed . the following anthropometric and body composition variables were collected : a ) height ( cm ) using a stadiometer with a 0.1 cm accuracy ( sanny es 2020 , brazil ) ; b ) weight using a digital scale with a 0.01 kg accuracy ( filizola pl 180 , brazil ) ; c ) waist circumference ( wc ) was measured using a steel anthropometric tape ( cescorf , brazil ) , with a 2 m extension and 0.01 m intervals , at the narrowest point between the external surface of the last rib and the anterior superior iliac spine ; d ) bfp using a bipolar bioelectrical impedance analysis ( bia ) device ( omron hbf-306 , usa ) . to complete the bia analysis , participants completed the following procedures : ( 1 ) did not use diuretic drugs in the previous 7 days ; ( 2 ) fasting for at least 4 h ; ( 3 ) did not consume alcoholic beverages in the previous 48 h ; ( 4 ) did not engage in intense physical activity in the last 24 h ; ( 5 ) urinated at least 30 min before the measurement ; ( 6 ) removed body adornments such as earrings , bracelets , necklaces and piercings ; and ( 7 ) remained at absolute rest for at least 8 - 10 min in the supine position before the measurement . body mass index ( bmi ) was computed using height and weight measures ( weight / height ) . bmi was classified as normal ( < 25 kg / m ) and overweight ( 25 the cutoff points used for wc were based on the degree of risk for cardiovascular diseases , namely moderate risk for women ( wc > 80 cm ) and men ( wc > 94 cm ) , and high risk for women ( wc > 88 cm ) and men ( wc > 102 cm ) . with respect to bfp , the cut off points for obesity or excess fat were 25% for men and this consisted of male and female figures representing different body shapes numbered from 1 to 9 . the individuals marked the silhouette that was most similar to their current body image and which one they would like to have . if the difference was zero , the individuals were classified as satisfied , and if different from zero then they were classified as dissatisfied . the 42 items on the short form are divided into six subscales : tension ( t ) , depression ( d ) , hostility ( h ) , vigor ( v ) , fatigue ( f ) and mental confusion ( c ) . each subscale contains four items rated using a likert - type scale ( not at all - 0 ; a little - 1 ; moderately - 2 ; quite a bit - 3 ; extremely - 4 ) , with sub - scores ranging from 0 to 16 . subscales t , d , h , f and c are considered negative factors of mood , and v a positive factor . total mood disturbance ( tmd ) is calculated by the sum of negative factors , subtracting the positive factor score . the state scale ( stai s ) requires individuals to express how they feel at the moment in relation to the 20 items contained on a 4-point likert scale ( not at all - 1 ; somewhat - 2 ; moderately so - 3 ; very much so - 4 ) . the trait scale ( stai t ) also has 20 items , but individuals are instructed to respond how they generally feel on a different 4-point likert scale ( almost never - 1 ; sometimes - 2 ; often - 3 ; almost always - 4 ) . the sf-36 questionnaire ( medical outcomes study 36 item short - form health survey ) was used to assess quality of life . this instrument contains 36 items , 35 of which encompass eight domains , grouped into two large components : physical ( domains : functional capacity , physical aspects , pain and general health status ) and mental ( domains : mental health , vitality , social aspects and emotional aspects ) . the sample was characterized using descriptive statistics , namely central tendency ( mean ) and dispersion ( standard deviation ) measures . the shapiro - wilk and levene tests were used to verify the sample s normality and homogeneity of variances , respectively . the student s t - test was applied to compare means from the anthropometric measures and mental health scores . , body satisfied and body dissatisfied ) expressed in standard deviation units ( mgroup1-mgroup2/sdpooled across groups ) . thereby , we completed the hedges correction which removes this bias using a simple correction , yielding an unbiased estimate of the effect size . effect sizes were classified as minimal to small ( d < 0.2 ) , small to moderate ( d = 0.2 0.5 ) , moderate to large ( d = 0.5 0.8 ) and large ( d > 0.8 ) . on the first day , the individuals responded to the instruments ( stai , poms , sf-36 and the silhouette scale ) and on the second day , anthropometric variables were collected ( body composition - bia , weight , height and waist circumference ) . during the application of the instruments , there was no communication between individuals and there was no time limit imposed . the following anthropometric and body composition variables were collected : a ) height ( cm ) using a stadiometer with a 0.1 cm accuracy ( sanny es 2020 , brazil ) ; b ) weight using a digital scale with a 0.01 kg accuracy ( filizola pl 180 , brazil ) ; c ) waist circumference ( wc ) was measured using a steel anthropometric tape ( cescorf , brazil ) , with a 2 m extension and 0.01 m intervals , at the narrowest point between the external surface of the last rib and the anterior superior iliac spine ; d ) bfp using a bipolar bioelectrical impedance analysis ( bia ) device ( omron hbf-306 , usa ) . to complete the bia analysis , participants completed the following procedures : ( 1 ) did not use diuretic drugs in the previous 7 days ; ( 2 ) fasting for at least 4 h ; ( 3 ) did not consume alcoholic beverages in the previous 48 h ; ( 4 ) did not engage in intense physical activity in the last 24 h ; ( 5 ) urinated at least 30 min before the measurement ; ( 6 ) removed body adornments such as earrings , bracelets , necklaces and piercings ; and ( 7 ) remained at absolute rest for at least 8 - 10 min in the supine position before the measurement . body mass index ( bmi ) was computed using height and weight measures ( weight / height ) . bmi was classified as normal ( < 25 kg / m ) and overweight ( 25 the cutoff points used for wc were based on the degree of risk for cardiovascular diseases , namely moderate risk for women ( wc > 80 cm ) and men ( wc > 94 cm ) , and high risk for women ( wc > 88 cm ) and men ( wc > 102 cm ) . with respect to bfp , the cut off points for obesity or excess fat were 25% for men and the silhouette scale was used to estimate body image satisfaction . the individuals marked the silhouette that was most similar to their current body image and which one they would like to have . if the difference was zero , the individuals were classified as satisfied , and if different from zero then they were classified as dissatisfied . the 42 items on the short form are divided into six subscales : tension ( t ) , depression ( d ) , hostility ( h ) , vigor ( v ) , fatigue ( f ) and mental confusion ( c ) . subscales t , d , h , f and c are considered negative factors of mood , and v a positive factor . total mood disturbance ( tmd ) is calculated by the sum of negative factors , subtracting the positive factor score . anxiety was measured by the self - rated stai scales ( state and trait anxiety inventory ) . the state scale ( stai s ) requires individuals to express how they feel at the moment in relation to the 20 items contained on a 4-point likert scale ( not at all - 1 ; somewhat - 2 ; moderately so - 3 ; very much so - 4 ) . the trait scale ( stai t ) also has 20 items , but individuals are instructed to respond how they generally feel on a different 4-point likert scale ( almost never - 1 ; sometimes - 2 ; often - 3 ; almost always - 4 ) . the sf-36 questionnaire ( medical outcomes study 36 item short - form health survey ) was used to assess quality of life . this instrument contains 36 items , 35 of which encompass eight domains , grouped into two large components : physical ( domains : functional capacity , physical aspects , pain and general health status ) and mental ( domains : mental health , vitality , social aspects and emotional aspects ) . each of these domains received scores between 0 and 100 , where zero corresponds to the lowest score and 100 to the highest [ 57 , 58 ] . the sample was characterized using descriptive statistics , namely central tendency ( mean ) and dispersion ( standard deviation ) measures . the shapiro - wilk and levene tests were used to verify the sample s normality and homogeneity of variances , respectively . the student s t - test was applied to compare means from the anthropometric measures and mental health scores . , body satisfied and body dissatisfied ) expressed in standard deviation units ( mgroup1-mgroup2/sdpooled across groups ) . effect sizes were classified as minimal to small ( d < 0.2 ) , small to moderate ( d = 0.2 0.5 ) , moderate to large ( d = 0.5 0.8 ) and large ( d > 0.8 ) . a total of 140 physical education undergraduate students , aged 23.6 3.7 years , were participated in the study . the mean height , body weight , body fat percentage ( bfp ) , bmi and wc were 70.7 14.3 kg , 169.4 1.1 cm , 19.5 7.1% , 24.5 3.5 the prevalence of bid in the students was 67.1% , with 80.4% of women and 64.2% of men aiming to reduce their body measures . there was no difference in bmi ( p = 0.067 ) between satisfied ( 23.7 2.9 kg / m ) and dissatisfied individuals ( 24.9 3.7 kg / m ) . a significant difference ( t = -2.061 ; p = 0.041 ) in bf was observed between the satisfied and dissatisfied individuals ( 20.3 7.7% vs. 17.9 5.4% ) . mean wc was significantly higher ( t = -1.993 ; p = 0.048 ) in dissatisfied students than in their satisfied counterparts ( 77.9 10.0 cm vs.74.4 8.7 cm ) . although there were no significant differences in bmi between groups , the reported effect size was small to moderate ( d = 0.330 ) , just as for wc ( d = 0.357 ) and bfp ( d = 0.328 ) . the poms compared satisfied and dissatisfied individuals , respectively , finding significant differences between anger ( 8.4 4.0 vs. 10.2 3.9 ; t = -2.490 ; p = 0.014 ) , depression ( 2.6 3.8 vs. 5.0 5.6 ; t = -2.582 ; p = 0.011 ) , hostility ( 3.1 3.6 vs. 5.0 3.9 ; t = -2.815 ; p = 0.006 ) , fatigue ( 5.4 4.0 vs. 7.4 4.6 ; t = -2.526 ; p = 0.013 ) and mental confusion ( 7.1 2.5 vs. 8.2 2.7 ; t = -2.331 ; p = 0.021 ) . in relation to tmd , the dissatisfied obtained a significantly higher score ( 120.5 18.0 vs. 110.1 14.5 ; t = -3.422 ; p = 0.001 ) . all the domains from poms had small to moderate or moderate to large effect sizes , with the tmd score having the largest ( d = 0.612 ) . the mental aspect score of quality of life ( qol ) was significantly higher ( t = 3.444 ; p = 0.001 ) in satisfied students ( 75.1 12.1 ) when compared to the dissatisfied ( 66.8 15.9 ) , with a small to moderate difference between - groups ( d = 0.491 ) . the aim of the present study was to assess bid of university students , and to compare anthropometric measures and mental health scores between satisfied and unsatisfied students . found that 76.6% of university students recruited from several study domains were dissatisfied with their body image ( 20.82 3.03 years ) . in another study carried out with 236 undergraduates of both genders , similarly , an investigation performed with 294 physical education students from the state university of ponta grossa ( uepg ) found that 61.2% of individuals were dissatisfied with perceived body image . due to their high prevalence of bid , physical education undergraduates are highly susceptible to developing mental disorders , including eating disorders . the physical and esthetic demands inherent to the course and profession they chose to pursue may be a possible explanation for the high percentage of dissatisfaction observed . thereby , these future professionals face pressure to follow socially accepted esthetic standards , generating exaggerated concerns with body appearance that can influence body image satisfaction levels [ 41 , 64 ] . the findings of the present study showed no difference in bmi ( p = 0.067 ) between undergraduates satisfied and dissatisfied with their body image , differing from those that reported an increase in bid with the rise in bmi [ 10 , 22 , 33 , 34 , 63 , 66 - 74 ] . the results presented here suggest significant differences in the anthropometric variables bfp ( p = 0.041 ) and wc ( p = 0.048 ) between the two groups analyzed . studied individuals that engaged in activities at the pontifical university of parana ( puc - pr ) sports center and using multiple linear regression they observed that higher bfp values represented greater body image dissatisfaction . in a sample of 220 undergraduates , no association was found between wc and bid , a measure that can be used to estimate central adiposity . these findings suggest that bfp and wc , just like bmi , may be reliable anthropometric indicators of bid in university students . it is also important to notice that several researchers highlight the limitations related to the use of bmi , which does not differentiate between adipose tissue and lean mass [ 10 , 33 ] . thereby , it seems that bid is more interrelated to the amount of subcutaneous adipose tissue than total body weight . the current esthetic standard of beauty accepted by society and constantly promoted by the media may exert some influence on body image satisfaction and the mental health of individuals . negative feelings about their bodies contribute to the increased prevalence of depression symptoms and decreased self - esteem in adolescent women . excessive media exposure of ideal bodies ( thin models ) in our society may trigger a more negative mood and lower food intake in women , contributing to the onset of eating disorders such as anorexia and bulimia . this low self - esteem in female adolescents may be attributed to overweight and obesity , which generates bid , negatively affecting mental health . analyzed body image differences in 1,384 university undergraduates in two european countries and found that students who perceived themselves as being overweight reported lower quality of life . better body image was also related to an increase in self - esteem , optimism and social support among women , which are mental health related components of quality of life . quality of life seems to have a positive influence on how individuals perceive their body image , but the opposite is equally likely . there is a need to acknowledge and increase our understanding of bid as a public health problem . a total of 5,255 australian women , aged between 18 and 42 years , filled out specific questionnaires on body image and mental health . the results demonstrate that most individuals ( 86.9% ) reported some level of dissatisfaction with their weight , and more than one - third ( 39.4% ) relate moderate dissatisfaction . higher levels of bid were associated with poor quality of life , primarily with the mental health and psychosocial dimensions . individuals with third - degree obesity exhibit concerns with body image and high frequency of binge eating episodes , as well as severe depression symptoms and anxiety . the frequency of anxiety as a trait was 70% , as a state 54% , and 76% of all patients reported discomfort regarding their body image . furthermore , body silhouette scales are two - dimensional and do not allow to fully represent the individual , their body fat distribution and other anthropometric measures important to body image . furthermore , individuals with greater fat accumulation in the visceral region and a higher percentage of body fat were more likely to exhibit bid . finally , bid was related with poorer quality of life and mood state in these university students .

human immunodeficiency virus ( hiv ) infiltrates the blood - brain barrier and infects macrophages / microglia and astrocytes in the brain . during disease progression , patients show various levels of neurocognitive impairment , which is collectively termed hiv - associated neurocognitive disorders ( hand ) . thus , neuronal dysfunction has been linked to the indirect effects of factors released by hiv - infected or activated glial cells . nitric oxide ( no ) , generated by nitric oxide synthase , is an important cellular messenger in signal transduction . in the hiv - infected brain , the expression of inducible nitric oxide synthase is increased , leading to elevated amounts of no . additionally , an imbalance between oxidants and reductants is observed . no is known to react with superoxide to form peroxynitrite , which nitrates protein tyrosine and tryptophan residues through multiple mechanisms . nitration of tyrosine can change the activity of enzymes and interfere with inter- and intramolecular interactions . nitration of tryptophan was discovered more recently , and its functional consequences are not well - defined . tyrosine nitration has been implicated in various neurodegenerative diseases such as alzheimer s disease , amyotrophic lateral sclerosis , and parkinson s disease . moreover the majority of patients with hand show hiv encephalitis at autopsy , and elevated inducible nitric oxide synthase levels are correlated with the severity of hand . the immunohistochemical analysis of brain tissues from demented and nondemented hiv - infected patients indicated an increased level of nitrotyrosine in the demented group . nitrated l - prostaglandin d synthase with decreased activity upon modification was found in the cerebrospinal fluid of hiv - infected patients and was proposed as a hand biomarker . however , other nitrated proteins in the brain have not been identified , and their role in hand progression has not been investigated . the two main methods used in nitrated protein identification are two - dimensional gel electrophoresis ( 2de ) separation and immunoprecipitation followed by mass spectrometric analysis . 2de does not always have the resolving power to separate proteins with similar sizes and chemical properties . thus , an immunoreactive gel spot detected by western blot analysis with the antinitrotyrosine antibody may not always correspond to a single protein . additionally , hydrophobic proteins can not be easily extracted from gel spots , biasing the method toward hydrophilic proteins . two antinitrotyrosine antibodies were shown to bind to tyrosine and tryptophan structures similar to 3-nitrotyrosine . detecting low - abundance nitrated peptides on the identified proteins is essential for the identification of nitrated proteins . the nanoflow liquid chromatography has a higher sensitivity and enables the detection of low - abundance peptides in samples when the amounts are limited . a nanoflow liquid chromatography system coupled to a high - resolution tandem mass spectrometer is ideal for the detection of low - abundance nitrated peptides and identifying the modification sites . determining the modification site ensures the correct identification of nitrated proteins and provides insight into the possible molecular and functional changes upon nitration . in this study , we identified nitrated proteins and the sites of nitration in postmortem human brain tissues from individuals without hiv infection ( control ) , with hiv infection but without encephalitis ( hiv ) , or with hiv infection with encephalitis ( hiv - e ) . immunoprecipitation was utilized for the enrichment of nitrated proteins in each brain lysate . a nano - hplc system coupled to high - resolution orbitrap velos mass spectrometer was used for analysis . we identified the nitrated proteins and the sites of modifications , and we used molecular modeling to predict the biological role of these post - translational modifications during hiv infection of the brain . frozen postmortem human brain tissues ( parietal cortex ) from individuals with hiv infection but without encephalitis ( n = 2 ) and hiv infection with encephalitis ( n = 2 ) were obtained from the university of california , los angeles ( case ids : 1093 , 6081 , 5007 , and 5008 ) . frozen control human brains ( n = 4 ) from a matching site of the brain were obtained from the university of maryland , baltimore ( case ids : 5125 , 5343 , 5346 , and 5189 ) ( supporting information table 1 ) . the tissues were washed with ice - cold buffer ( 20 mm tris / hcl , ph 6.8 , 100 mm nacl , 1 mm edta , and 1 mm dithiothreitol containing roche complete protease inhibitor cocktail ) and then homogenized using a pestle in the same buffer ( 1:5 w / v ) with the addition of 1% ( v / v ) triton - x 100 and 0.1% ( w / v ) sds . the lysate was centrifuged at 18 000 g for 20 min to remove insoluble cellular components . the bca assay ( pierce ) was used according to the manufacturer s directions to determine protein concentrations . the samples were cleaned using detergent removal spin columns ( pierce ) , 0.5 ml , according to the manufacturer s directions . the columns were initially equilibrated with dulbecco s phosphate buffered saline ( pbs ) . the samples were incubated with monoclonal anti-3-nitrotyrosine antibody ( clone 1a6 ) cross - linked to protein g the following day , the flow through was collected using microcentrifuge spin filters ( pierce ) with a 30 m filter . the immunoprecipitate was washed three times with 1 pbs , and the proteins were eluted using 1 , 2 , and 5% formic acid ( fa ) ( v / v ) . the immunoprecipitation procedure was repeated twice , and all of the eluents from a single sample were combined , their ph was neutralized , and they were dried ( scheme 1 ) . we termed the proteins pulled down with the anti-3-nitroyrosine antibody protein g agarose as immunoprecipitate . the immunoprecipitated proteins were called nitrated only if their nitrated peptides were detected in the ms / ms analysis . brain samples from the parietal cortex of hiv - negative controls , hiv - infected individuals without encephalitis , and hiv - infected individuals with encephalitis were immunoprecipitated with anti-3-nitrotyrosine antibody conjugated to protein g agarose . the peptides were separated on a reverse - phase nano - lc system and analyzed by high - resolution tandem mass spectrometry . the immunoprecipitated samples were reconstituted in 100 mm ammonium bicarbonate , reduced with 5 mm dithiothreitol , and alkylated with 15 mm iodoacetamide . the proteins were digested with trypsin ( sigma ) at a 1:20 trypsin - to - protein ratio ( w / w ) for 18 h at 37 c . the tryptic peptides were enriched and separated on an eksigent nanoflow lc system coupled to a ltq - orbitrap velos mass spectrometer ( thermo scientific ) . a 2 cm long trap column ( ymc gel ods - a s-10 m ) and a 75 m 10 cm analytical column containing magic aq c18 material , 5 m , 100 ( michrom bioresources ) were utilized . the peptides were separated on a 70 min linear gradient and directly introduced to the ltq - orbitrap velos at a flow rate of 300 nl / min and a spray voltage of 2.0 kv . data - dependent tandem ms analysis was employed in the orbitrap , with a 30 000 resolution for ms and 7500 resolution for ms / ms . full scans were acquired from m / z 3002000 with up to the 15 most intense ions isolated using a 1.9 da window . the peptide ions were fragmented using a collision energy of 35% in the hcd cell with a dynamic exclusion of 30 s. the first mass value was fixed at m / z 140 , and the minimum signal for triggering an ms / ms scan was set to 2000 . an ambient air - lock mass was set at m / z 371.10123 for real - time calibration . ms and ms / ms data were searched using proteome discoverer ( v. 1.3.0.339 ) with the mascot ( v. 1.27 ) algorithm . database searching of ms / ms spectra was performed using the national center for biotechnology information nonredundant database ( 2012 ) . homo sapiens was selected for the taxonomy , and 230 236 protein sequences were searched . the scan event filter had the following criteria : ms2 for ms order , hcd for activation type , and full for scan type . trypsin was selected as the enzyme with the allowance of a maximum of two missed cleavages . the anti-3-nitrotyrosine antibody , clone 1a6 ( millipore ) , has been reported to bind to nitrohydroxytryptophan also because of the similarity in the chemical structures of these modified amino acids . the variable modifications included oxidation ( m , + 15.99492 ) , deamidation ( n , q , + 0.98402 ) , acetylation ( protein n terminal , + 42.01057 ) , nitration ( y , + 44.98508 ) , and nitrohydroxylation ( w , + 60.97999 ) ( figure 1 ) . the precursor mass tolerance was set to 20 ppm , and the fragment tolerance was set to 0.05 da . peptide validator was applied to the mascot results to search against a decoy database and to obtain false discovery rates ( strict , 0.01 ; relaxed , 0.05 ) . the results were filtered for minimum medium peptide confidence for peptide identification and differentiable protein groups for protein group identification . chemical structures of 3-nitrotyrosine and nitrohydroxy tryptophan . the exact location of nitro and hydroxyl groups on the tryptophan can not be determined by mass spectrometric approaches . the anti-3-nitrotyrosine antibody has previously been shown to be capable of binding to both of these modified amino acids . a comparative model of immunoglobulin light and heavy chains was obtained using the hiv - neutralizing antibodies ( pdb identifier : 2cmr and 4jdt ) as templates with the molecular modeling program moe ( chemical computing group inc . , version 2012.10 ) . in this model , the loop between the 4 and 5 strands that differs in size and has a disordered structure in the templates was not modeled . because the modified residues were either on the complementarity - determining region ( cdr ) or were two amino acids away from the cdr , our model gives insights into the molecular changes that take place upon nitration and nitrohydroxylation as well as their potential effect on antibody structure and antigen recognition . in the current study , a total of 253 differentiable proteins ( supporting information table 2 ) were identified in the immunoprecipitates of all samples with at least one unique peptide . among these proteins , 24 were present only in the control , 11 , only in hiv , and 128 , only in the hiv - e sample sets ( figure 2 ) . the number of proteins identified in both control and hiv - e samples was four , in both the control and hiv sample sets was 23 , and in both the hiv - e and hiv sample sets was 15 . because the vast majority of the immunoprecipated proteins were in the hiv - e samples , it suggests that these protein modifications did not occur as a result of sample preparation but were specific for the underlying biological processes in hiv - e . the proteins identified in the control samples likely indicate either nonspecific binding to the anti-3-nitrotyrosine antibody or proteins that were not related with hiv infection . thus , efforts were focused on the proteins that were identified only in the hiv sample sets ( both the hiv and hiv - e sample sets ) and only in the hiv - e sample set . number of proteins identified in the sample sets and the distribution of proteins identified exclusively in the hiv - e brain . ( a ) venn diagram representation of differentiable proteins in the parietal cortex brain tissue immunoprecipitates . the majority of the proteins are found exclusively in the hiv - e sample set . ( b ) distribution of proteins found in the immunoprecipitates from hiv - e samples . ninety proteins are immunoglobulin variable regions , of which five proteins are immunoglobulins against hiv envelope proteins . the proteins that were identified only in the immunoprecipitates from the hiv sample set included small nuclear ribonucleoprotein sm d1 , proteolipid protein 1 , nadph dehydrogenase 1 alpha subcomplex 6 , apo - human serum transferrin , and pitp - alpha complexed to phosphatidylinositol . thirteen out of the 15 proteins that were identified in both hiv and hiv - e sample sets were also immunoglobulins . the other two proteins were cytochrome c oxidase subunit 5a and gamma - synuclein . the ncbi nr database has a large number of immunoglobulin sequences predicted from the genbank . a total of 128 proteins were identified only in the immunoprecipitate from the hiv - e sample set . interestingly , 90 of these proteins were immunoglobulin variable regions , and five of them were immunoglobulins against hiv-1 envelope proteins ( figure 2 ) . we also identified glyoxalase double mutant , carbonic anhydrase , cold agglutinin , and cd47 . the other 36 proteins were either immunoglobulins without region specification or unnamed protein products . next , we analyzed the nitrated peptides to confirm that the identified proteins were nitrated . using mascot , 18 peptides with nitrated tyrosines and 10 peptides with nitrohydroxylated tryptophan modifications were identified ( table 1 ) . the lowest mascot ion score was 38 , and the highest e - value was 5.09 10 . an investigation of murine and human immunoglobulin heavy chain cdr3 repertoire has shown that they exhibit differences in amino acid sequence and structure . the data was searched using rodentia taxonomy as well to ensure that the modified peptides were not coming from the monoclonal anti-3-nitrotyrosine antibody used during the immunoprecipitation . the modified peptide qac , deyno2nasvsvpdssgper could be part of an unnamed protein product in mus musculus as well as heterogeneous nuclear ribonucleoprotein k in h. sapiens . the rest of the modified peptides reported were found in the h. sapiens database but not in rodentia . hiv , brain samples from individuals with hiv infection without encephalitis ; hiv - e , brain samples from individuals with hiv infection and encephalitis ; psm , peptide spectrum match ; hv , heavy chain variable region ; and lv , light chain variable region on immunoglobulins . on the peptide sequences , nitrotyrosines and nitrohydroxytryptophans are shown in red , deamidated residues are indicated in green , carbamidomethylated cysteines are colored green , and the oxidized methionine is shown in purple . the complete peptide identification information can be found in supporting information table 4 . the 32llvvypwno2ohtvr41 peptide from beta globin was detected in seven of the samples except for one of the hiv brain samples ( table 1 ) . the 85lqqgyno2ndeatgfsqggflr102 peptide from palmitoyl - protein thioesterase 1 chain a was present only in one of the four control samples , whereas the 72qac , deyno2nasvsvpdssgper86 peptide from human heterogeneous nuclear ribonucleoprotein k was found in two of the control samples and one hiv brain sample ( figure 3 ) . the majority of the nitrotyrosine and nitrohydroxytryptophan containing peptides were identified in the tryptic digests of the immunoprecipitated proteins from the hiv - infected with encephalitis sample set . the nitrated peptides that were identified only in the immunoprecipitates from hiv and/or hiv - e sample sets may reflect the effect of hiv infection on the human proteins . the 196vyno2adevthqglsspvtk212 peptide from antitubulin igg1 kappa vl chain was found in both hiv and hiv - e samples and not in the controls . the 66lliyno2aapslqdesgipsr81 peptide from immunoglobulin kappa light chain variable region was present in one hiv brain sample . twenty three of the nitrated and nitrohydroxylated peptides were found only on peptides from hiv - e brain immunoprecipitates , and 21 of them were on immunoglobulin variable regions ( table 1 ) . among these peptides , 108nfdhwo2ohgrgtlvtvssastk126 may belong to anti - hiv-1 gp41 immunoglobulin heavy chain ( gi : 212675107 ) and 23agqsissndeyno2lawyqqkpgqapr44 may belong to anti - hiv-1 gp120 immunoglobulin kappa chain variable region ( gi : 299742 ) . the high amino acid level variation observed in immunoglobulin variable regions and the sequence homology in the immunoglobulin protein family made protein identification challenging by shotgun proteomic methods . the ms / ms spectra of the modified peptides can be found in supporting information figures 114 . the ms / ms spectra for m / z 1600.75139 , 1660.78826 , 2069.02292 , 2102.96147 , 2155.08268 , 2307.07646 , 2423.1778 , and 2552.19048 were matched to more than one peptide sequence ( supporting information table 4 ) ; therefore , the identity of these peptides was ambiguous . these eight spectra were found only in the hiv - e sample set , and the peptide sequences belonged to immunoglobulin variable regions ( five belonged to the heavy chain and three belonged to the light chain ) . despite the ambiguity in the exact sequence , the identified human protein sequences had high sequence homology , and the modified residues were mostly conserved ( figure 4 ) . amino acid sequence alignment of immunoglobulin variable regions identified only in the hiv - e immunoprecipitates . the national center for biotechnology constraint - based multiple alignment tool ( cobalt ) was used . highly conserved residues are in red , and mostly conserved residues are in blue . the nitrated residues , shown with an arrow , are conserved among different immunoglobulin variable regions . ( b ) immunoglobulin heavy chain variable region sequences . among the nitrated and nitrohydroxylated peptides that were observed in the hiv - e sample set , 17 belonged to immunoglobulin heavy chain variable regions and four belonged to immunoglobulin light chains ( table 1 ) . although a higher number of nitrated tyrosine- and nitrohydroxylated tryptophan - containing peptides were found in the hiv - e sample set , the same modified peptide sequences were not present in both hiv - e samples . we observed that despite the variation in the peptide sequences from immunoglobulin variable regions the modified tyrosine and tryptophan residues were conserved among the nitrated peptides . thus , we aligned the amino acid sequences of the 42 immunoglobulin heavy chain variable region proteins and 48 immunoglobulin light chain variable region proteins that were identified in the immunoprecipitates ( figure 4 ) . again , these residues were mostly conserved in the human immunoglobulin variable regions , which suggests that these modifications may be of functional significance . additionally , two tyrosines are found on the cdr of the immunoglobulin heavy chain variable region , and four of the modified residues are two amino acids away from the cdr , further suggesting biological significance of the nitration at these sites . we modeled immunoglobulin light and heavy chains using the crystal structures of neutralizing antibody with gp41 innercore and gp120 ( pdb code : 2cmr and 4jdt ) to determine the potential molecular changes that take place upon the nitration of the tyrosine and tryptophan residues ( figure 5a ) . although we do not know the antigens of the modified antibodies and can not determine the number of modifications on a single antibody using shotgun proteomics methods , this model demonstrates the potential interactions that the modified residues may be involved in . for convenience , we used the residue numbers from the neutralizing antibody from the 2cmr crystal structure . the nitration of heavy chain cdr residues y32 and y59 may affect antigen recognition . in this case , nitration of y32 could lead to h - bonding with the h199 of the gp41 innercore ( figure 5b ) . similarly , nitrated y59 may have a favorable intermolecular interaction with r214 of the gp41 innercore ( figure 5c ) . conserved w36 is located one residue away from the cdr in the strand of the immunoglobulin heavy chain , and the nitrohydroxylated form can hydrogen bond with the q6 and c22 ( supporting information figure 15a ) . however , modified w105 is found at the interface between the heavy and the light chains of the immunoglobulin . this region is hydrophobic and can not accommodate the nitro group ( supporting information figure 15b ) . thus , the nitrohydroxylation of this residue may interfere with the intramolecular interactions of the two chains . nitrated y91 is also located at the interface between the heavy and the light chains , but its nitration would have minor effects on antibody shape ( data not shown ) . interestingly , the nitrated immunoglobulin light chain y36 fits well into the interface between the two chains . the nitro group can be well - accommodated by the h - bonds with heavy chain w105 and a backbone amine ( figure 5d ) . finally , light chain y49 does not interact directly with the antigen but is found below the flexible loop that binds to the antigen ( figure 5e ) . thus , by interacting with the loop residues , it may change the shape of cdr3 . the model of immunoglobulin with gp120 did not show any significant change in antigen recognition upon nitration of tyrosine and tryptophan , although the changes in intramolecular interactions were similar ( data not shown ) . as explained above , the modification of each residue may have unique effects on antigen binding and chain interactions . model of the complex between immunoglobulin light and heavy chains and the hiv envelope protein gp41 innercore obtained using the crystal structure of the neutralizing antibody as templates . ( a ) the cartoon of the heavy and light chains of the immunoglobulin are shown in yellow and green , respectively . the solvent - accessible surface of gp41 is drawn semitransparent and is colored according to the electrostatic potential ( red , negative ; blue , positive ) around the cartoon of the receptor ( colored in red ) . nitrated y32 , y36 , y49 and nitrohydroxylated w36 and w105 of the immunoglobulin as well as h199 and r214 of the receptor are drawn in a ball - and - stick representation . the positions of the nitro- and hydroxyl - group substitutions were chosen to minimize steric conflicts with the immunoglobulin . ( c ) heavy chain y59 is also solvent - exposed , and its nitration may have a positive effect on gp41 binding through the favorable interaction between the nitro group and r214 . ( d ) light chain y36 resides at the interface between the heavy and the light chains . interestingly , the nitro group can be accommodated well at this position through h - bonds with heavy chain w105 and a backbone amine . ( e ) light chain y49 resides below the disordered loop ( dashed line ) between the heavy chain 4 and 5 strands , and nitration of this residue may affect this loop , which contains cdr3 and is critical for antigen binding . in this study , we identified nitrated proteins in the brain of hiv - infected individuals with neurocognitive disorders . the majority of the nitrated peptides belong to immunoglobulin heavy and light chain variable regions . despite the sequence variation , the nitrated tyrosine and nitrohydroxylated tryptophan residues were conserved in the human immunoglobulins detected in these samples and the previously reported hiv-1-neutralizing antibodies that bind to the hiv envelope glycoproteins gp41 and gp120 . the potential roles of tyrosine and tryptophan and their modified forms in antibody antigen binding are addressed here . nitrated proteins have been previously detected in various inflammatory conditions and in multiple tissues . thus , it is not surprising to detect the highest number of nitrated peptides in hiv - infected individuals with encephalitis . during the infection , reactive oxygen and nitrogen species are released from activated macrophages and microglia within the brain . peroxynitrite is the key species involved in the nitration of tyrosine and tryptophan residues . its conjugated acid can diffuse through membranes , and its anionic form can be transported by anion channels . how nitrohydroxylation takes place in the setting of hiv infection interestingly , we did not find any of the peptides from the variable region of the immunoglobulins listed in table 1 with only hydroxylation or nitration ( data not shown ) . thus , in our samples , these modifications were always present together on tryptophan residues . a likely possibility is that both of these modifications occur either simultaneously or in quick succession . peroxynitrite has been shown to both nitrate and hydroxylate tryptophan and may be part of the nitrohydroxylation process . in vitro nitration of tryptophan , however , yields a mixture of products depending on the type of nitrating agent , its concentration , and the reaction conditions . we can not exclude the possibility of artificial hydroxylation of nitrated tryptophan in the postmortem brain ; however , its presence in only the hiv - e sample set indicates relevance to the disease condition and is in agreement with previous reports on oxidative and nitrosative stress in hiv infection and hand progression . unlike enzymatic post - translational modifications such as phosphorylation , n - glycosylation , and ubiquitination , which have consensus sequences on their substrates , post - translational modifications based on radical reactions have not been found to have such a consensus sequence . interestingly , nitration selectivity has been linked to the proximity to the site where the nitrating agent is produced , the abundance of the protein and its primary sequence , the abundance of tyrosine , and the residue exposure . among the nitrated peptides listed in table 1 , 22 out of 28 are from immunoglobulin variable chains . although , this modification does not seem to be specific to only immunoglobulin variable chains in the brains from individuals with hiv - e , they seem to be favored . our observation is consistent with the previous reports of nitration because ( 1 ) oxidative and nitrosative species are present at hiv - infected cells , ( 2 ) immunoglobulins are expressed at elevated levels and are diversified during infection , ( 3 ) tyrosine and tryptophan residues are found at a high frequency in the cdr of immunoglobulins on the variable regions , and ( 4 ) tyrosines and tryptophan residues are predominantly located on the solvent - exposed antigen - binding surface of immunoglobulins . additionally , lack of methionine and cysteine residues in the close proximity to tyrosine has been determined to be important for nitration reactions . only three nitrated peptides out of the 22 ( table 1 ) have cysteine and methionine residues in the sequence . thus , although nitration is not specific to immunoglobulin variable regions in the hiv - infected brain , it seems to be favored . the structure , function , and properties of immunoglobulin binding fragments are widely studied because of their potential applications in vaccine design and therapeutics based on synthetic antibodies or peptides . the sequence similarities between antibodies isolated from patients and synthetic antibody libraries have been determined previously . both tyrosine and tryptophan were predicted to be ideal residues within antibody binding regions because of the following physical and chemical properties : hydrogen - bond formation , hydrophobic interactions , electrostatic interactions between positively charged groups and the ring structure , amphipathicity , large size for maximized intermolecular interaction , and enhanced mobility when adjacent to small residues such as glycine , alanine , and serine . the importance of tyrosine in synthetic antibody affinity and specificity has been reviewed . also , tyrosine accounts for 25% of the immunoglobulin side chains that interact with antigens in isolated antibodies . in our study , the majority of the proteins identified in the immunoprecipitate from hiv - e samples were immunoglobulin variable chains . immunoglobulins are produced by b - cells and may enter the brain either by the cerebrospinal fluid or through the blood - brain barrier , which may be disrupted during hiv infection . once in the brain , they can bind to viral antigens . in accordance with previous studies , the alignment of immunoglobulin light variable region and heavy variable region sequences of the identified proteins revealed that the modified tyrosine and tryptophan residues are mostly conserved ( figure 4 ) . the structural basis of broad neutralizing antibodies for hiv envelope proteins gp41 and gp120 has been investigated previously . often , the molecular effect of a modification depends on where the residue is located on the protein and the interacting residues in the milieu . in our structural model using two neutralizing - antibody crystal structures , we characterized potential antigen - binding effects of the immunoglobulin heavy chain y32 and y59 of the cdr . similarly , immunoglobulin light chain y49 may influence antigen interactions by affecting the flexible loop that contains the immunoglobulin cdr3 . the rest of the modified residues are located on the framework region ( fr ) of the variable region , internal to the heavy chain barrel ( w36 ) , or at the interface between the light and heavy chains ( l , y36 ; h , w105 ) . their nitration could modify the fold of each chain and the interaction between heavy and light chains . whereas the cdr residues are involved directly in antigen binding , the fr residues can influence the overall structure of the variable region , affecting the recognition . during immune activation a previous study found tyrosine sulfation on the amino terminus of ccr5 , which was shown to be critical for recognition of gp120 . furthermore , antibodies that mimic the coreceptor through tyrosine sulfation have been detected . this modification was suggested as a secondary level of antibody variation to enhance antigen recognition . the in vitro nitration of a monoclonal human immunoglobulin and its mass spectrometric analysis revealed tyrosine nitration on both light and heavy chains . an antibody - based breast cancer drug has been reported to contain tyrosine nitration . although the oxidation of a critical tryptophan residue on an humanized antibody was shown to lead to a loss of its binding activity to its antigen , the functional consequences of nitration and nitrohydroxylation on immunoglobulins are largely unknown . to our knowledge , our study is the first report of nitration on human immunoglobulin variable regions with site - specific information . because of structural similarity , nitrohydroxy tryptophan may serve similar functional roles as nitrated tyrosine . our data indicates a role of nitration in the immune system , and it may be a marker of encephalitis , which is commonly observed as a pathological correlate in patients with hand . our findings may have important implications in elucidating the immune response during hiv infection and in the development of novel therapeutics . there is widespread interest in antibodies for vaccine design and antibody - based therapeutics development as antibody drug conjugates . antibodies either are isolated from patients and characterized or synthetic antibody / peptide libraries are screened for affinity and specificity for antigens of interest . tyrosine has been identified as a critical residue in both isolated and synthetic antibodies . tyrosine sulfation was discovered on the cdr3 region of ccr5 mimetic gp9 and gp16 broadly neutralizing antibodies against hiv envelope proteins . furthermore , sulfated tyrosine containing ccr5 and cd4 mimetic peptide fused to a dimeric antibody constant domain showed enhanced potency . investigation of the role of nitration and nitrohydroxylation of the tyrosine and tryptophan residues on immunoglobulins is necessary to determine how they alter the function of immunoglobulins when compared to the previously reported role of tyrosine sulfation for hiv envelope protein binding . antibodies are one of the major sensors of the body , especially during an infection . after the introduction of antiretroviral therapy , hand nosology has changed with the addition of more prevalent , milder forms of hand . recognition of these forms of hand requires extensive neuropsychological testing , which may not be easily accomplished in a clinical practice setting . there is a need for developing biomarkers that correlate with the neurocognitive state of the hiv - infected patients , follow medication effects , and help to provide appropriate therapy . the exclusive presence of nitration on immunoglobulins in the hiv - e sample set makes them potential biomarkers if these findings can be confirmed in either cerebrospinal fluid or serum . there has been increased interest in post - translational modifications in biomarker studies because protein expression alone does not always reflect the change in the system during disease states . targeted and sensitive assays for detecting post - translational modifications on abundant proteins such as hemoglobin and albumin in biological fluids have been designed . cerebrospinal fluid , which is rich in immunoglobulins , can be analyzed without depletion of abundant proteins for the validation of nitrated tyrosine- or nitrohydroxylated tryptophan - containing peptides as hand biomarkers . we used autopsy tissue ; hence , some of the changes may have occurred terminally or postmortem . the study used a small sample size , and the samples were not perfectly matched for age and gender . despite these limitations , the differences between hiv - e and controls were quite striking , suggesting that the differences were most likely driven by the underlying biological processes associated with hiv - e , as described above . in this study , we identified nitrated proteins in control , hiv , and hiv - e brain lysate using an affinity - enrichment protocol followed by lc ms / ms analysis . the predominant presence of these modified peptides on immunoglobulin variable regions and on conserved tyrosine and tryptophan residues suggests potential functional roles . furthermore , the high frequencies of tyrosine on the antigen - binding fragments of antibodies and the function of tryptophan in inter- and intramolecular interactions make the modification of these residues significant . our results provide unique insight into hiv neuropathogenesis and have opened the door for future structure function studies .

hiv can infiltrate the brain and lead to hiv - associated neurocognitive disorders ( hand ) . the pathophysiology of hand is poorly understood , and there are no diagnostic biomarkers for it . previously , an increase in inducible nitric oxide synthase levels and protein tyrosine nitration in the brain were found to correlate with the severity of hand.1,2 in this study , we analyzed human brains from individuals who had hiv infection without encephalitis and with encephalitis / hand and compared them to the brains of healthy individuals . we identified the nitrated proteins and determined the sites of modification using affinity enrichment followed by high - resolution and high - mass - accuracy nanolc ms / ms . we found that nitrated proteins were predominantly present in the hiv - infected individuals with encephalitis , and , interestingly , the modifications were predominantly located on immunoglobulin variable regions . our molecular model indicated potential interactions with hiv envelope proteins and changes on the heavy and light chain interface upon the nitration and nitrohydroxylation of these residues . therefore , our findings suggest a role for these modifications in the immune response , which may have implications in disease pathogenesis .

Introduction Experimental Methods Results Discussion Conclusions

during disease progression , patients show various levels of neurocognitive impairment , which is collectively termed hiv - associated neurocognitive disorders ( hand ) . thus , neuronal dysfunction has been linked to the indirect effects of factors released by hiv - infected or activated glial cells . in the hiv - infected brain , the expression of inducible nitric oxide synthase is increased , leading to elevated amounts of no . moreover the majority of patients with hand show hiv encephalitis at autopsy , and elevated inducible nitric oxide synthase levels are correlated with the severity of hand . the immunohistochemical analysis of brain tissues from demented and nondemented hiv - infected patients indicated an increased level of nitrotyrosine in the demented group . nitrated l - prostaglandin d synthase with decreased activity upon modification was found in the cerebrospinal fluid of hiv - infected patients and was proposed as a hand biomarker . however , other nitrated proteins in the brain have not been identified , and their role in hand progression has not been investigated . detecting low - abundance nitrated peptides on the identified proteins is essential for the identification of nitrated proteins . determining the modification site ensures the correct identification of nitrated proteins and provides insight into the possible molecular and functional changes upon nitration . in this study , we identified nitrated proteins and the sites of nitration in postmortem human brain tissues from individuals without hiv infection ( control ) , with hiv infection but without encephalitis ( hiv ) , or with hiv infection with encephalitis ( hiv - e ) . we identified the nitrated proteins and the sites of modifications , and we used molecular modeling to predict the biological role of these post - translational modifications during hiv infection of the brain . frozen postmortem human brain tissues ( parietal cortex ) from individuals with hiv infection but without encephalitis ( n = 2 ) and hiv infection with encephalitis ( n = 2 ) were obtained from the university of california , los angeles ( case ids : 1093 , 6081 , 5007 , and 5008 ) . frozen control human brains ( n = 4 ) from a matching site of the brain were obtained from the university of maryland , baltimore ( case ids : 5125 , 5343 , 5346 , and 5189 ) ( supporting information table 1 ) . the tissues were washed with ice - cold buffer ( 20 mm tris / hcl , ph 6.8 , 100 mm nacl , 1 mm edta , and 1 mm dithiothreitol containing roche complete protease inhibitor cocktail ) and then homogenized using a pestle in the same buffer ( 1:5 w / v ) with the addition of 1% ( v / v ) triton - x 100 and 0.1% ( w / v ) sds . the immunoprecipitated proteins were called nitrated only if their nitrated peptides were detected in the ms / ms analysis . brain samples from the parietal cortex of hiv - negative controls , hiv - infected individuals without encephalitis , and hiv - infected individuals with encephalitis were immunoprecipitated with anti-3-nitrotyrosine antibody conjugated to protein g agarose . the peptides were separated on a reverse - phase nano - lc system and analyzed by high - resolution tandem mass spectrometry . data - dependent tandem ms analysis was employed in the orbitrap , with a 30 000 resolution for ms and 7500 resolution for ms / ms . the peptide ions were fragmented using a collision energy of 35% in the hcd cell with a dynamic exclusion of 30 s. the first mass value was fixed at m / z 140 , and the minimum signal for triggering an ms / ms scan was set to 2000 . ms and ms / ms data were searched using proteome discoverer ( v. 1.3.0.339 ) with the mascot ( v. 1.27 ) algorithm . a comparative model of immunoglobulin light and heavy chains was obtained using the hiv - neutralizing antibodies ( pdb identifier : 2cmr and 4jdt ) as templates with the molecular modeling program moe ( chemical computing group inc . in this model , the loop between the 4 and 5 strands that differs in size and has a disordered structure in the templates was not modeled . because the modified residues were either on the complementarity - determining region ( cdr ) or were two amino acids away from the cdr , our model gives insights into the molecular changes that take place upon nitration and nitrohydroxylation as well as their potential effect on antibody structure and antigen recognition . among these proteins , 24 were present only in the control , 11 , only in hiv , and 128 , only in the hiv - e sample sets ( figure 2 ) . the number of proteins identified in both control and hiv - e samples was four , in both the control and hiv sample sets was 23 , and in both the hiv - e and hiv sample sets was 15 . because the vast majority of the immunoprecipated proteins were in the hiv - e samples , it suggests that these protein modifications did not occur as a result of sample preparation but were specific for the underlying biological processes in hiv - e . the proteins identified in the control samples likely indicate either nonspecific binding to the anti-3-nitrotyrosine antibody or proteins that were not related with hiv infection . thus , efforts were focused on the proteins that were identified only in the hiv sample sets ( both the hiv and hiv - e sample sets ) and only in the hiv - e sample set . number of proteins identified in the sample sets and the distribution of proteins identified exclusively in the hiv - e brain . the majority of the proteins are found exclusively in the hiv - e sample set . ninety proteins are immunoglobulin variable regions , of which five proteins are immunoglobulins against hiv envelope proteins . the proteins that were identified only in the immunoprecipitates from the hiv sample set included small nuclear ribonucleoprotein sm d1 , proteolipid protein 1 , nadph dehydrogenase 1 alpha subcomplex 6 , apo - human serum transferrin , and pitp - alpha complexed to phosphatidylinositol . a total of 128 proteins were identified only in the immunoprecipitate from the hiv - e sample set . interestingly , 90 of these proteins were immunoglobulin variable regions , and five of them were immunoglobulins against hiv-1 envelope proteins ( figure 2 ) . next , we analyzed the nitrated peptides to confirm that the identified proteins were nitrated . hiv , brain samples from individuals with hiv infection without encephalitis ; hiv - e , brain samples from individuals with hiv infection and encephalitis ; psm , peptide spectrum match ; hv , heavy chain variable region ; and lv , light chain variable region on immunoglobulins . the majority of the nitrotyrosine and nitrohydroxytryptophan containing peptides were identified in the tryptic digests of the immunoprecipitated proteins from the hiv - infected with encephalitis sample set . the nitrated peptides that were identified only in the immunoprecipitates from hiv and/or hiv - e sample sets may reflect the effect of hiv infection on the human proteins . twenty three of the nitrated and nitrohydroxylated peptides were found only on peptides from hiv - e brain immunoprecipitates , and 21 of them were on immunoglobulin variable regions ( table 1 ) . the high amino acid level variation observed in immunoglobulin variable regions and the sequence homology in the immunoglobulin protein family made protein identification challenging by shotgun proteomic methods . the ms / ms spectra for m / z 1600.75139 , 1660.78826 , 2069.02292 , 2102.96147 , 2155.08268 , 2307.07646 , 2423.1778 , and 2552.19048 were matched to more than one peptide sequence ( supporting information table 4 ) ; therefore , the identity of these peptides was ambiguous . these eight spectra were found only in the hiv - e sample set , and the peptide sequences belonged to immunoglobulin variable regions ( five belonged to the heavy chain and three belonged to the light chain ) . despite the ambiguity in the exact sequence , the identified human protein sequences had high sequence homology , and the modified residues were mostly conserved ( figure 4 ) . amino acid sequence alignment of immunoglobulin variable regions identified only in the hiv - e immunoprecipitates . the nitrated residues , shown with an arrow , are conserved among different immunoglobulin variable regions . among the nitrated and nitrohydroxylated peptides that were observed in the hiv - e sample set , 17 belonged to immunoglobulin heavy chain variable regions and four belonged to immunoglobulin light chains ( table 1 ) . although a higher number of nitrated tyrosine- and nitrohydroxylated tryptophan - containing peptides were found in the hiv - e sample set , the same modified peptide sequences were not present in both hiv - e samples . we observed that despite the variation in the peptide sequences from immunoglobulin variable regions the modified tyrosine and tryptophan residues were conserved among the nitrated peptides . thus , we aligned the amino acid sequences of the 42 immunoglobulin heavy chain variable region proteins and 48 immunoglobulin light chain variable region proteins that were identified in the immunoprecipitates ( figure 4 ) . again , these residues were mostly conserved in the human immunoglobulin variable regions , which suggests that these modifications may be of functional significance . additionally , two tyrosines are found on the cdr of the immunoglobulin heavy chain variable region , and four of the modified residues are two amino acids away from the cdr , further suggesting biological significance of the nitration at these sites . in this case , nitration of y32 could lead to h - bonding with the h199 of the gp41 innercore ( figure 5b ) . conserved w36 is located one residue away from the cdr in the strand of the immunoglobulin heavy chain , and the nitrohydroxylated form can hydrogen bond with the q6 and c22 ( supporting information figure 15a ) . thus , the nitrohydroxylation of this residue may interfere with the intramolecular interactions of the two chains . interestingly , the nitrated immunoglobulin light chain y36 fits well into the interface between the two chains . finally , light chain y49 does not interact directly with the antigen but is found below the flexible loop that binds to the antigen ( figure 5e ) . model of the complex between immunoglobulin light and heavy chains and the hiv envelope protein gp41 innercore obtained using the crystal structure of the neutralizing antibody as templates . ( a ) the cartoon of the heavy and light chains of the immunoglobulin are shown in yellow and green , respectively . ( d ) light chain y36 resides at the interface between the heavy and the light chains . ( e ) light chain y49 resides below the disordered loop ( dashed line ) between the heavy chain 4 and 5 strands , and nitration of this residue may affect this loop , which contains cdr3 and is critical for antigen binding . in this study , we identified nitrated proteins in the brain of hiv - infected individuals with neurocognitive disorders . the majority of the nitrated peptides belong to immunoglobulin heavy and light chain variable regions . despite the sequence variation , the nitrated tyrosine and nitrohydroxylated tryptophan residues were conserved in the human immunoglobulins detected in these samples and the previously reported hiv-1-neutralizing antibodies that bind to the hiv envelope glycoproteins gp41 and gp120 . thus , it is not surprising to detect the highest number of nitrated peptides in hiv - infected individuals with encephalitis . how nitrohydroxylation takes place in the setting of hiv infection interestingly , we did not find any of the peptides from the variable region of the immunoglobulins listed in table 1 with only hydroxylation or nitration ( data not shown ) . we can not exclude the possibility of artificial hydroxylation of nitrated tryptophan in the postmortem brain ; however , its presence in only the hiv - e sample set indicates relevance to the disease condition and is in agreement with previous reports on oxidative and nitrosative stress in hiv infection and hand progression . unlike enzymatic post - translational modifications such as phosphorylation , n - glycosylation , and ubiquitination , which have consensus sequences on their substrates , post - translational modifications based on radical reactions have not been found to have such a consensus sequence . interestingly , nitration selectivity has been linked to the proximity to the site where the nitrating agent is produced , the abundance of the protein and its primary sequence , the abundance of tyrosine , and the residue exposure . although , this modification does not seem to be specific to only immunoglobulin variable chains in the brains from individuals with hiv - e , they seem to be favored . our observation is consistent with the previous reports of nitration because ( 1 ) oxidative and nitrosative species are present at hiv - infected cells , ( 2 ) immunoglobulins are expressed at elevated levels and are diversified during infection , ( 3 ) tyrosine and tryptophan residues are found at a high frequency in the cdr of immunoglobulins on the variable regions , and ( 4 ) tyrosines and tryptophan residues are predominantly located on the solvent - exposed antigen - binding surface of immunoglobulins . thus , although nitration is not specific to immunoglobulin variable regions in the hiv - infected brain , it seems to be favored . in our study , the majority of the proteins identified in the immunoprecipitate from hiv - e samples were immunoglobulin variable chains . immunoglobulins are produced by b - cells and may enter the brain either by the cerebrospinal fluid or through the blood - brain barrier , which may be disrupted during hiv infection . once in the brain , they can bind to viral antigens . often , the molecular effect of a modification depends on where the residue is located on the protein and the interacting residues in the milieu . the rest of the modified residues are located on the framework region ( fr ) of the variable region , internal to the heavy chain barrel ( w36 ) , or at the interface between the light and heavy chains ( l , y36 ; h , w105 ) . although the oxidation of a critical tryptophan residue on an humanized antibody was shown to lead to a loss of its binding activity to its antigen , the functional consequences of nitration and nitrohydroxylation on immunoglobulins are largely unknown . to our knowledge , our study is the first report of nitration on human immunoglobulin variable regions with site - specific information . our data indicates a role of nitration in the immune system , and it may be a marker of encephalitis , which is commonly observed as a pathological correlate in patients with hand . our findings may have important implications in elucidating the immune response during hiv infection and in the development of novel therapeutics . tyrosine sulfation was discovered on the cdr3 region of ccr5 mimetic gp9 and gp16 broadly neutralizing antibodies against hiv envelope proteins . investigation of the role of nitration and nitrohydroxylation of the tyrosine and tryptophan residues on immunoglobulins is necessary to determine how they alter the function of immunoglobulins when compared to the previously reported role of tyrosine sulfation for hiv envelope protein binding . recognition of these forms of hand requires extensive neuropsychological testing , which may not be easily accomplished in a clinical practice setting . there is a need for developing biomarkers that correlate with the neurocognitive state of the hiv - infected patients , follow medication effects , and help to provide appropriate therapy . the exclusive presence of nitration on immunoglobulins in the hiv - e sample set makes them potential biomarkers if these findings can be confirmed in either cerebrospinal fluid or serum . despite these limitations , the differences between hiv - e and controls were quite striking , suggesting that the differences were most likely driven by the underlying biological processes associated with hiv - e , as described above . in this study , we identified nitrated proteins in control , hiv , and hiv - e brain lysate using an affinity - enrichment protocol followed by lc ms / ms analysis . the predominant presence of these modified peptides on immunoglobulin variable regions and on conserved tyrosine and tryptophan residues suggests potential functional roles . furthermore , the high frequencies of tyrosine on the antigen - binding fragments of antibodies and the function of tryptophan in inter- and intramolecular interactions make the modification of these residues significant .

beginning with the alma ata declaration in 1978 , the need for comprehensive and integrated approaches to health care has been well acknowledged . however , for a long time , such approaches had been enmeshed in polarised debates about vertical versus horizontal health programmes and/or comprehensive versus selective primary health care ( mills , 2005 ; rifkin & walt , 1986 ) . the recent global calls to achieve universal health care and the millennium development goals ( mdgs ) in low- and middle - income countries have revived , at least rhetorically , the emphasis on strengthening health systems through comprehensive and integrated approaches to health care . in line with these global developments and after a protracted focus on disease - specific programmes , india has recently embarked on a path to revamp its public health delivery system . it seeks to revitalise the primary health care approach and put in place several architectural corrections in the basic health care system to guarantee equitable access to quality health care for the rural poor , women and children ( nrhm , 2005 ) . the nrhm sets out a horizontal and comprehensive approach to health care to undertake these architectural corrections . some of the core strategies of this horizontal approach include : integration of vertical programmes and structures ; integrating health with its broader determinants ; mainstreaming traditional systems of medicine and revitalising local health traditions ; decentralised health planning ; effective community participation and ownership of health ; and improved and effective public health management ( government of odisha , 2007 ; nrhm , 2005 ) . notions of integration in nrhm thus flow from the comprehensive primary health care approach , that reinforce principles of community participation , provision of comprehensive care ( curative , preventive and promotive ) inter - sectoral collaboration , decentralisation and equity ( nrhm mission document , 2005 ) . such an approach has been justified in light of india 's race towards achieving the goals of mdgs 4 and 5 ( reducing child mortality and improving maternal health , respectively ) . 1 ) defines integrated service delivery as management and delivery of health services so that clients receive a continuum of preventive and curative services , according to their needs , over time and across different levels of the health system. despite the wider policy acknowledgement of the need for such an integrated approach to health care , empirical evidence on how such integration operates on the ground is rather sparse and disparate ( atun , jongh , secci , ohiri , & adeyi , 2010 ; wallace , dietz , & cairns , 2009 ; who , 2008a ) . existing literature on global programmatic experiences with integration of health services tend to approach delivery of services as a technical and mechanistic process ( banerjee , elamon , & aggarwal , 2009 ; partapuri , steinglass , & sequiera , 2012 ) . integrated service delivery in this sense is an approach of combining services of multiple interrelated diseases to increase overall efficiency of the health system and patient convenience ( lenka & george , 2013 , p. 1 ) . for example , integration would include combining delivery of family planning messages during routine immunisation sessions or distribution of mosquito nets during post - partum visits . often referred to as service add on ( magtymova , 2007 ) , benefits of integration are assessed through data on the uptake of the specific health services reflected in an increase in contraceptive prevalence , improved immunisation coverage or uptake of mosquito nets ( banerjee et al . , 2009 ; partapuri et al . , 2012 ; usaid , fhi360 & progress , 2011 ) . feasibility of integration is weighed in terms of health system factors like availability of human resources , compatibility of services or supply chain management and infrastructure ( lenka & george , 2013 ) . the focus on supply - side health system factors , though important , assumes that community demand relies unproblematically on providers delivering services . commentaries on health sector and systems tend to focus on structural aspects and not on the actors who comprise the systems ( sheikh & george , 2010 , p. 2 ) . ethnographic evidence suggests that the demand / uptake of health services is linked to a host of factors , such as the community 's perceived vulnerability to a specific illness for which the health service is offered , previous experiences with other state health services , modes of health communication , interaction with health workers and broader political identities and perceptions of the state by the community ( leach & fairhead , 2005 ; mishra , flikke , nordfeldt , & nyirenda , 2013a ; nichter , 1995 ) . exclusive focus on individual health services and sites of delivery ignores larger social processes at work at the intersection of supply and demand , as well as providers and local communities , thus reflecting little on how integration is achieved or even the sustainability of integration measures . using nrhm as a case study , this article offers a grounded perspective on integration of health services through capturing the everyday experiences of community health workers located at the interface between the formal health system and the local communities . existing ethnographic studies show how the social experiences of health workers are important to capture in order to understand how policies are translated on the ground , communities are mobilised , care is delivered and raw data on the health status of populations and functioning of public health programmes are produced at the local level ( coutinho , bisht , & raje , 2000 ; george , 2010 ; mishra , hasija , & roalkvam , 2013b ; walker & gilson , 2004 ) . this article adds to the literature and discusses community health workers experiences with integrating immunisation with broader maternal and child health services through the village outreach sessions called health and nutrition days . this article draws on 8 months of fieldwork conducted in 20112012 in one of the southern districts in the state of odisha , india . the district is largely inhabited by indigenous communities ( referred to as adivasis and/or tribal by the communities themselves and others ) . the fieldwork was conducted as part of a larger ethnographic research study that sought to chart the growing focus on health system strengthening within global and national vaccination policies and explore how debates about health systems and strengthening are played out at global , national and local levels . this article is concerned with the local level , specifically , how health workers understand and translate their roles within the nrhm 's emphasis on strengthening integrated and comprehensive primary health care . additionally , the article draws on the author 's prior fieldwork experiences focusing on local immunisation practices conducted in the same region between 2009 and 2010 ( mishra et al . , 2013b ) . the fieldwork was conducted by the author along with two junior researchers trained in anthropology . data were collected through participant observation in two villages selected in the district , including during 8-monthly health and nutrition days ( outreach sessions ) in each village ; monthly supervisory meetings at the primary health centre which caters to the villages ; training sessions of health workers ; and panchayat ( local government ) level meetings . additionally , we observed rituals / healing sessions in which health workers participated . we conducted open - ended in - depth interviews with 12 health workers , and accompanied these health workers in all their routine activities beyond the outreach sessions for a period of 6 months , including when they accompanied a pregnant woman to the health centre , took a malnourished child for treatment , distributed iron syrups to the villagers or accompanied a villager to the traditional healer . we also conducted interviews with 18 sub - district level health officers and with 43 villagers ( both men and women ) . data were analysed through a grounded theory approach ( strauss , 1987 ) by developing detailed coding and constantly validating responses through juxtaposing different sources of data ( interviews with observations , data from interviews with health workers with responses from the communities ) . the coding was further refined to develop sub - themes and themes , and relationships between themes were further examined . this was analysed iteratively , by constantly going back and forth between these field data and secondary literature on the subject . apart from ethical approval at the host university ( university of oslo , norway ) , permission to undertake the study was obtained from the district health authorities in the state . at the community level , outreach sessions like village health and nutrition days ( also known as immunisation days ) are an important forum for provision of integrated and comprehensive care . the nrhm mandates that health and nutrition days , hereafter referred to as outreach sessions , are organised once a month in each village . these sessions aim to serve as an important mechanism under nrhm for the convergence of all health - related activities through inter - sectoral collaboration , thus bringing together department of health and family welfare , department of women and child development and community representatives including the panchayati raj ( local government ) members in the village ( nrhm mission document , 2005 ) . as per the nrhm guidelines , these sessions offer a package of services pertaining to maternal and child health , communicable and non - communicable diseases ; address social determinants of health including sanitation , nutrition and gender ; and facilitate the collection of data on specific needs of vulnerable populations , vital events including disease outbreaks and audits of maternal and child deaths ( nrhm , 2007 ) . this package of services is offered through direct delivery ( antenatal care [ anc ] , treatment of minor ailments , distribution of iron syrup ) ; identification and referral ( for symptoms of tuberculosis , malaria and others ) ; information and counselling on several issues including early signs of communicable diseases , existing gender - based laws , importance of a clean environment , locally available nutritious diet and prevention of tobacco , etc . the nrhm envisages that these sessions provide an effective platform for delivering first - contact primary health care , thus maximising the points of interaction between the community and the formal health system . three sets of community level female health workers are principally involved in mobilising and delivering health services during these outreach sessions , though the nrhm guidelines envisage the involvement of panchayat members , primary schoolteachers , and traditional birth attendants in supporting and facilitating these sessions ( nrhm , 2007 ) . the three health workers are composed of an auxiliary nurse midwife ( anm ) , an accredited social health activist ( asha ) and an anganwadi ( literally translated as courtyard shelter ) worker ( aww ) . each has different professional training and skills , performance incentives , professional trajectories and accountability mechanisms . the ashas ( who are addressed through this acronym by everyone , including the villagers ) are village - level health workers who have been recruited since the start of the nrhm in 2005 . they are recruited on a voluntary basis from the village by the local government village representatives and are supposedly accountable to the community . ashas receive about 3 weeks of training over five rounds , focusing on their role as a link between the health system and the village . they are responsible for mobilising the community to access public health services , including immunisation , hospital birth and anc ; identifying and referring people affected by tuberculosis , leprosy and malaria ; and promoting family planning programmes . apart from this , they have a broader role of being health activists in the community by creating awareness about health and its determinants , mobilising the community towards local - level planning and increasing utilisation and accountability of existing health services ( national institute of health and family welfare [ nihfw ] , government of india , 2005 ) . although they receive no fixed salary , they are paid performance - based cash incentives , the amount of which differs according to the nature of the service ( e.g. , 700 rs . or us$11 for mobilising and accompanying a pregnant woman for institutional delivery , 50 rs . , less than us$1 , for organising an immunisation day ) . unlike the ashas who are recent recruits , the anms ( also addressed through this acronym ) , have been a part of the health bureaucracy since the 1950s . an anm serves four or five villages ( a population of 5000 ) , constituting a sub - centre , the lowest of the three - tier primary health care organisation in india . the anms roles have changed over time , from being focused initially on midwifery to include broader maternal and child health services , family planning services , nutrition and health education , immunisation , treatment of minor ailments and epidemic tours during outbreaks . re - designated as multi - purpose workers , the anms are permanent government employees , and receive fixed salaries ( approximately 10,000 rs . or us$180 per month ) . they receive 1.5 to 2 years of training in midwifery , as well as periodic on - the - job training in different components of national health programmes and interventions . unlike the ashas instead , they travel to these villages on different days , particularly for outreach sessions . the anms are accountable to the local health bureaucracy ( report to a medical supervisor ) in the primary health centre headed by the medical officer . the awws , on the other hand , are recruited from the village ( one aww per one village , constituting of a population of about 1000 ) through the integrated child development service ( icds ) programme that the government of india introduced in the mid-1970s . this is organised under the department of women and child development , rather than the health bureaucracy , which is administered under the department of health and family welfare . they are responsible for new born care , as well as ensuring that all children below the age of six are immunised . they provide pre - school education to children between 3 and 5 years old and are also responsible for providing supplementary nutrition to both children below the age of six and pregnant and nursing women . awws receive monthly salaries , though well below that of anms ( 2000 rs . or us$36 ) . marital status ( married ) and residence ( residing in the village where she works ) are important considerations for recruitment of both ashas and awws . as discussed below , these differences in their professional training , status and role are important considerations in ways they conduct outreach sessions and engage with the community . while trying to operationalise the nrhm 's strategy of integrating delivery of services through outreach sessions , health workers in our study emphasised how effective teamwork was critical to their practice of delivering care , although they acknowledged the challenges posed by differential training , salary , status and role . health workers efforts to provide integrated health services and care were not restricted to either individual outreach sessions or health services . more critically , it was about building relationships with the community , an important trust building mechanism ( rowe & calnan , 2006 , p. 5 ) . thus , mobilisation , communication and delivery of services were embedded in the dynamics of relationship building at the interface between the health system ( mediated by individual health works ) and the community . an asha worker clarified what it means to be a good worker and to build a healthy relationship with the community : an asha should have the ability to cooperate with others . if a child suffers from diarrhoea , an asha should accompany the patient to the primary health centre rather than just showing the way to the centre.in a similar vein , another asha added : villagers trust me . asha sister is there so there will not be any problem during delivery. once a woman developed labour at night . i arranged an auto - rickshaw and accompanied her to the referral hospital , as she wanted to be taken there ( instead of the primary health centre ) . one of the important qualities of an asha is that one needs to talk to the people of the village regularly.an anm added , we need to be there when the community members want our help , you know , be one of them. such qualities of being there , being sisterly , responding to the villager 's needs and preferences are important elements of the meaning of trust that the health workers espouse . health workers experiences suggest that building relations of trust with the community are not an ad hoc event but rather a continuous process of eliciting and sustaining such trust . our field observations suggest that such trust can not be taken for granted but needs to be nurtured . a host of factors are at play in the trust - building process , beginning with the social status ( caste , ethnic , familial relations ) of individual health workers , modes of communication , ability to cater to community health and non - health needs and the community 's own prior experiences with other health interventions , including their perceptions of the state ( see roalkvam , this volume ) . if a child suffers from diarrhoea , an asha should accompany the patient to the primary health centre rather than just showing the way to the centre . asha sister is there so there will not be any problem during delivery. once a woman developed labour at night . i arranged an auto - rickshaw and accompanied her to the referral hospital , as she wanted to be taken there ( instead of the primary health centre ) . one of the important qualities of an asha is that one needs to talk to the people of the village regularly . for the asha and aww workers who mostly live in the villages they serve , creating demand for any health service implies eliciting trust in their roles , both as health workers and , by extension , state representatives , and as members of the community . for the tribal people in the villages studied , the state ( referred to as the government ) is represented by the health workers . vaccination or institutional delivery are not mere biomedical interventions , but rather reach the community as interventions provided by the state , such that for the local community , engaging with vaccines or anc entails an engagement with the state . more than any other programme , due to its scope , approach and delivery mechanisms , the nrhm has made the state distinctly visible in local communities through the everyday material practices of immunisation days , recruitment of health workers , disbursement of cash incentives and distribution of iron syrup and other medicines . for tribal people , this is reflected in the villagers statements : the government knows we are prone to diseases like diarrhoea , malaria and hence provides medicines through the asha sisters and the mobile clinics ; the government is helping our women to deliver in the hospital and provide cash incentives. communicating about state health services , as the workers shared ( particularly the ashas and awws ) , was enmeshed in everyday interactions taking place during routine work in the river , agricultural fields and community rituals . health workers engaged in the process of building trust with the communities in several ways . for instance , they repackaged the merits of health services in terms of the community 's existing health priorities , or ensured that these priorities were taken into account through relevant means . beneficial for overall health of children ( instead of how each vaccine helps in preventing a specific disease ) and even for curing malaria , fits , diarrhoea. such repackaging is justified on the grounds of addressing the community 's traditional concerns for protecting child health and the burden of common diseases . further , they tried to ensure that the gynaecological complaints of all village women rather than only the pregnant women who are targeted as part of the nrhm 's focus on reducing maternal mortality are addressed . in most of the cases we observed , the asha worker took to the doctor in the primary health centre ( 8 km away from one of the villages ) both pregnant women who were eligible for anc check - ups , as well as other women who had other complaints . the asha would not only accompany them , but also facilitated the interaction with the doctor , took care of the prescription and arranged the medicines from the chemist . by expanding their remit to cover all women and address their health needs , they seemed to successfully contribute to the process of building trust in health workers and formal state health services , as evidenced by a young married woman lamenting the death of her in - laws 7 years prior due to tuberculosis : had it been now , my in - laws would not have died , asha sister would have taken them to hospital and given medicines. similarly , another elderly woman attributed reduced mortality in her village to the asha 's intervention : deaths , suicide cases have reduced in the village as asha gives medicines , ( and ) takes us to hospital when necessary. apart from addressing the immediate health concerns of villagers , health workers also ensured that all state health services were moulded to accommodate local aetiologies of illness and remedies . among the tribal communities of odisha , health and illness are believed to be rooted in a number of factors including poverty , supernatural forces and lack of access to medicines . for example , the high number of stillbirths and the vulnerability of children to common illnesses are predominantly explained through the role of the dumas ( ancestors ) who need to be appeased periodically through rituals both at individual households and at community levels . a child ( till he / she turns a year old ) is considered to belong to the world of ancestors and becomes a social person only through these rituals . thus , protection of child health involves a number of traditional rituals beginning with the protection of the foetus in the womb to safe delivery . while promoting anc , immunisation and institutional delivery for pregnant women , asha and aww workers actively participated in these traditional rituals . for the community , care has a larger connotation that involves integrating local aetiologies of illness , notions of health and healing modalities with that of the state - provided biomedical health services . thus the traditional rituals of godh bhariba ( a ritual aimed at protecting the foetus in the womb ) , the ritual tying of sacred thread and bila sukheiba ( the ritual offerings to a tree representing the goddess responsible for ensuring healthy children ) need not conflict with immunisation , distribution of nutritious meals and treatment for malnourishment in the community health centre . the health workers promoted and subscribed to such notions by focusing as much on accompanying women to deliver in the public hospital ( for which they are paid cash incentives ) as on accompanying them to the female shaman to seek treatment for infertility and fits ( two clinically under - diagnosed complaints in the region and for which there is no linked cash incentives ) . however , these efforts were confined to the community space and never shared with senior health officials , lest this might be seen as promoting quackery . one of the ways nrhm seeks to promote integration is by revitalising local health traditions , though modalities of revitalisation are so far absent in the policy documents . hence , for the officials in the health bureaucracy , all forms of traditional healing are branded as quackery . the potential role of health workers in initiating critical dialogue between different systems of medicine and providers ( in the light of the community 's preferences , notions of efficacy , common complaints for which traditional rituals are sought ) is thus ignored ( scott & shankar , 2010 ) . another key element that health workers drew attention to in their experiences with integrating health services was the compatibility of curative and preventive services . malaria , diarrhoea , skin - related diseases and generic symptoms like fever , headache and body ache are common complaints in the villages . studies show how the credibility of primary health workers is linked to their ability to provide curative services and adequate supply of drugs ( nichter , 1995 ; paralato & favin , 1982 ) . villagers talk about a good or inefficient anm in terms of her contribution to diagnosing and , where necessary , giving adequate information about referral service . during outreach sessions , villagers often came to the anm with complaints of night blindness , fever or diarrhoea . the ashas and awws , on the other hand , ensured an adequate supply of drugs pertaining to more common complaints . the government of odisha facilitates the distribution of medicines through the primary health centres for combating the five priority diseases ( tuberculosis , malaria , pneumonia , diarrhoea and leprosy ) . the nrhm seeks to resolve the institutionalised dichotomy between curative and preventive services that has plagued the indian public health system for a long time . the outreach sessions are important forums for resolution of this dichotomy by addressing curative , preventive and promotive services . however , the community 's demand for preventive health services is largely mediated through their access to and experiences with curative services , particularly for the common diseases that become their immediate priority health needs ( mishra et al . hence , adequate supply of medicines catering to these health needs is a major concern for the health workers , often voiced during monthly supervisory meetings . an asha worker explained : the other day , the aww shared that four children were suffering from diarrhoea and i should bring medicines . how could we tell the mothers of these children to not bother about the diarrhoea or go to the phc on their own but come to the outreach session the next day to get their children weighed?adequate availability of medicines with the asha / aww is also a source of potential conflict among health workers and possible complaints against the anm specifically , who is supposed to facilitate supply of drugs ( at least for the ashas ) . social relations of trust between the health workers and the community however go beyond the delivery of specific health services . health workers provide information when demanded on government insurance schemes , for instance in the wake of a house fire , submission of relevant papers to the administrative office for availing of educational scholarships meant for eligible low - income families , etc . the other day , the aww shared that four children were suffering from diarrhoea and i should bring medicines . how could we tell the mothers of these children to not bother about the diarrhoea or go to the phc on their own but come to the outreach session the next day to get their children weighed ? health workers thus emphasised values of cooperation , continuous and open communication , personal and professional motivation and empathy as critical elements of trust , values like those gilson ( 2003 , p. 1461 ) argues are important to produce health through a trust - based health system . it is around the acknowledgement of such values , communities talk about an active / good / cooperative and inactive / non - cooperative / indifferent / rude health worker . our data indicate that relationships of trust and reciprocity work better where the asha worker is from the same community in a relatively ethnically homogenous village and is recruited through the involvement of village members . on the other hand , in villages where the asha does not reside in the village , or handles more than two or three villages and has been nominated by the anm without village involvement , such trust was missing and the asha was regarded merely as a subordinate of the anm . in a multi - caste or multi - tribe village , the caste and class status of the health worker and familial relations with other members of the village mediate in relationship - building and hence service delivery . during our visit to one such village , villagers even refused to lead us to the asha 's residence and later complained about how indifferent and inactive an asha she was . we learned that this asha 's recruitment had been controversial , as she was married to a rich contractor and a converted christian , pitting her as someone who is privileged and unfit to relate to the concerns of the villagers . in multi - caste villages , the process of trust - building with the community is thus fragile and requires much more concerted and careful efforts on the part of both the community and the health workers . in addition to trust , health workers also highlighted the role of effective teamwork in providing integrated and comprehensive care . the team dynamics we observed among the health workers are important not only for the success of individual episodes of outreach sessions but also its contribution to legitimating and sustaining their role as health workers . the vertical power hierarchy between the anm on the one hand and the asha and aww on the other is explicit though the ashas are supposed to be accountable to the local government / community . the anms power emanates from their training , salary and possession of technical skills to diagnose and treat minor ailments and put injections. the asha 's ability to fulfil her job responsibilities directly depends on the anm 's support . for example , it is the anm who distributes drugs to the asha ; provides help and advice for pregnant women , even at odd hours ; clears the papers to enable the ashas to avail of the cash incentive for taking women to institutional delivery ; provides curative care to those whom the asha and aww bring to the immunisation site . though the aww submits records to a supervisor in the icds , these records are validated and examined by the anm in the village . one aww explained , we cross check our records ( anm and aww ) and ensure that we are presenting the same set of data to our respective supervisors . these will be discussed together in the sub - district and the district level , if there is discrepancy , we will be in trouble. further , the anm validates and signs the document certifying the grade of a malnourished child for referral for which the aww gets cash incentives . both the technical and signing authority put the anms vertically above the ashas and awws . such power is acknowledged positively on the one hand , and yet subtly resisted . such resistance , however , for instance , ashas and awws complained to us that an anm often does n't take rounds of the villages and enquire about people 's health ; makes mistakes in names while recording names of eligible children for vaccination ; comes to the villages only for two fixed days outreach sessions ; keeps the medicines to herself and does not give it to us ; ( then ) villagers get upset when they realise that we do not have the stock ; and does n't cooperate to get the cash incentives. such resentment can be attributed to a number of factors such as unequal professional growth trajectories , fixed salaries versus incentives and professional vulnerability in the absence of signing and other symbols of authority . despite the resentment , all three of them realised the value of teamwork . as one anm clarified , taking care of pregnant women is necessarily group work . neither the asha nor the anm alone can do much ; it needs the cooperation and equal commitment from each one of us. an asha worker added we fall back on the anm for help in clarifying the usage of any medicine , responding to villagers queries regarding post - vaccination effects and submitting our records. values of cooperation were expressed in clarifying how each one needs the other . the asha 's role is critical to mobilise women for anc registration , institutional delivery and accessing the services in the outreach sessions . as we observed during an outreach session , one of the anms told an asha : . they will listen to you more than me. strategies to build teamwork include keeping each other 's contact phone numbers , meeting or talking to plan outreach sessions , trying to develop a common understanding of state health programmes , helping each other in filling out health and nutrition records and devising ways to cooperate to obtain cash incentives ( using the system creatively to draw mutual benefits ) . thus , during an outreach session , we observed that the health workers shared their common understanding of state health messages to discuss their adaptability to local contexts . for example , they communicated to the pregnant women how they could eat a nutritious diet that is locally available with them , gave examples of several such food items ( and hence healthy diet need not be a platter of fruits which they can not afford ) , and advised them that they could continue to work in the fields , provided they take care of their diet , monitor any warning signs and report to the health worker . backstage preparation is undertaken to ensure that the onstage state event the outreach session is successfully conducted . this is because the outreach sessions provide the raw data for constituting the success of public health programmes and more importantly , the output indicators such as number of children immunised are part of the evaluation of the health workers performance , as explained below . these events have unequivocal sanctity marked through joint signatures of all three health workers ( the proof that the events had taken place ) , records of names of pregnant women and children , weight measures and numbers of iron syrup bottles distributed , which give these sessions an objective , measurable character . our ethnographic data suggest that favourable team dynamics such as visible cooperation , a proactive role played by each of the three workers and constructive supervision by the anms often translated not merely into successful outreach sessions , but also community trust in state health services mediated by trust in health workers . villagers recounted positive experiences of timely care ( how the asha and the anm coordinated to ensure that the woman in labour reached the hospital on time and was referred swiftly when complications arose ) . similarly , negative experiences were also shared when such team dynamics failed . practical strategies to establish team spirit and teamwork serve the larger purpose of being a good health worker both for the community and that of the health system , though these expectations often conflict with each other , as discussed below . scott and shankar ( 2010 ) , in an article called tying their hands ? institutional obstacles to the success of the community health worker programme in rural north india show how the asha workers potential role as health activists are restricted by institutional obstacles like the rigid hierarchical health bureaucracy , and lack of flexible and creative remuneration structures . our study findings share these concerns . while the health workers subscribe to larger notions of integration inherent in a primary health care approach , such efforts conflict with the narrow monitoring indicators used for health system performance , health system 's privileging of statistical over experiential knowledge and its reliance on top - down channels of communication . attended by the ashas and anms , such meetings are held once a month in the primary health centre , which caters to a population of 20,000 to 30,000 . these meetings are an important forum for dissemination and monitoring of state health programmes by the senior officials in the primary health bureaucracy ( the medical officers and supervisors ) . during these meetings , health workers submit the records of activities for the previous month gathered at the outreach sessions . though nrhm has the broad objective of strengthening primary health care and the rural public health system , in practice , there is a tendency among sub - district and district health officials to restrict nrhm efforts to control the infant mortality rate and the maternal mortality ratio only . this could be due to the pressure from higher up the health bureaucracy at the state and national levels to contribute to achieving the mdgs on maternal and child health and more directly to the fact that the health system performance is evaluated through indicators on maternal and child health . thus , in all the monthly meetings , the asha workers and anms are asked to provide numerical evidence on number of pregnant women registered , offered anc care and accompanied for institutional delivery ( used as a proxy for maternal mortality ) , the number of malaria slides collected , the number of children immunised and the number of malnourished children referred further for treatment . this focus on indicator reporting means that such forums rarely provide any opportunity for the workers to share broader feedback from their practice . this is despite the fact that their experiences from the field would help to explain the mechanisms through which outputs are achieved ( or not ) and how . health workers , for example , had insights on immunisation programme drop out in peak agricultural season , reasons for seeking treatment for epileptic fits from a traditional healer , and pregnant women being taken to a district hospital rather than the recommended primary health centres . these insights , however , never get communicated in the monthly meetings . for the health workers , extras which need to be kept quiet or shared informally with peers , rather than evidence on whether and how nrhm is achieving its aims . the supervisory meetings become a site for top - down communication exclusively , where senior officials either demand reports and records or make specific announcements on recent orders passed down to them from further up the health bureaucracy . discussions in these meetings take the form of one - way communication : did immunisation take place last month ? did you collect malaria slides? such top - down communication dilutes the significance of the forum of the supervisory meetings , which are an important point of encounter between top - down and bottom - up planning . the block level health system ( the primary health centre ) thus fails to fulfil its key roles in responding to local needs and contexts in service provision as much as adapting national policy and guidelines to local circumstances . this is indeed a caricature of the nrhm 's emphasis on decentralised health planning from the village level to the block , district , state and national levels . the monthly project meetings of the icds where the aww submits the records ( attended by the icds officers and supervisors along with the medical officers ) followed a similar pattern . the focus on numerical compliance , lack of scope for open communication and supervision result in demoralisation among health workers ( coutinho et al . gaming refers to strategies to maximise performance in relation to the rewarded behaviour ( magrath & nichter , 2012 , p. 1780 ) . in the context of village - level health workers , rewarded behaviour includes tasks that are incentivised and for which the health workers performance is evaluated . gaming strategies among the health workers in our study included falsification of evidence on identification of malnourished children for referral and the number of institutional deliveries . institutional even when a woman delivered at home or on the way to the hospital , or recording a child as grade iii malnourished even in the absence of systematic data . while conforming to the pressure of reporting practices , health workers sought to devise creative ways of dealing with individual situations deciding to facilitate cash incentives for a woman who delivered at home on the ground that the family was poor . one of the consequences of such gaming strategies is that the health system actually has poor quality data about what is happening in the field . mainstream public health writings on delivery of integrated services tend to focus on the health services per se and modalities of their integration , assuming that effective supply chain management , infrastructure and human resources would achieve integration . these writings approach health service delivery itself as a technical and mechanistic process . based on ethnographic evidence , this article shows how building social relations of trust and teamwork are critical to health workers efforts in delivering integrated services . the role of trust in health care has traditionally been examined in relation to doctor patient relationships . however , recent anthropological literature has sought to bring social relations of trust to the centre stage in the study of health systems and policies ( gilson , 2003 , 2005 ; magrath & nichter , 2012 ; rowe & calnan , 2006 ; theide , 2005 ) . drawing on empirical evidence from a number of contexts , these studies have demonstrated that trust matters to health systems . these community health workers espouse an integrated approach to care by fostering relations of mutual trust , teamwork , cooperation , addressing community health and other needs , promoting a continuum of care from curative to preventive care and valuing the role of regular and effective communication with villagers and also amongst health workers themselves . these values are indeed the cornerstone of a primary health care ideology that promotes democracy , equity and participation ( nichter , 1986 ) . however the voices and experiences of health workers and other implementers are hardly taken into account and rarely thought to constitute evidence for public health policies and programmes . anthropologists suggest an urgent need for consideration of process variables that could track how outcome variables are achieved ( magrath & nichter , 2012 ) . such process evaluation needs to include inputs from different actors involved at different levels , including community health workers . we agree with walker and gilson ( 2004 ) , who based on their study of nurses in south africa , argue that discounting the perspectives and experiences of frontline health workers emanates from the methodological limitation of approaching policy implementation as a linear , top - down process . the vision of comprehensive primary health care that the nrhm 's health workers try to promote , and which is enshrined in policy documents , is in constant tension with other important elements of the indian public health system : the narrow indicators used for health system performance ; the highly hierarchical bureaucratic structure that rests on top - down communication and information ; the institutionalised privileging of statistical evidence over field - based experiences . these features curb the potential role of community health workers as agents of social change , cultural mediators and health promotors through effective community participation . a narrow focus on achieving mdgs 4 and 5 through cash incentives , regularising outreach sessions , stricter monitoring of indicators relating to these goals might result in better immunisation coverage and higher rates of institutional delivery , but these need not guarantee a robust , sustainable health system or indeed result in real improvement in health outcomes . beneficiaries of health care interventions , and their health , not just the prevention and treatment of biomedically defined illness , at the centre stage . a recent who summary of african countries experiences with revitalisation of primary health care documents many similar challenges to fulfilling the primary health care agenda to those identified here , including reforming monitoring and evaluation systems , changing the vertical focus of health service delivery and enhancing greater community participation ( who , 2008b ) . though the nrhm evokes the spirit of primary health care values , these are enforced through a health organisational bureaucracy that is deeply hierarchical . the nrhm is thus in danger of being looked upon as just another programme undermining the overhauling of public health ideologies in india . bhatia and rifkin ( 2010 ) rightly note that it is not only enough to revitalise primary health care , but also emphasise the need to reframe it in light of values of equity , community empowerment and determinants of health . this research has been funded by the norwegian research council 's global health and vaccination research programme ( globvac ) as part of a larger research project [ grant number 196382 ] on health systems strengthening within vaccination programmes : an ethnographic study .

a comprehensive and integrated approach to strengthen primary health care has been the major thrust of the national rural health mission ( nrhm ) that was launched in 2005 to revamp india 's rural public health system . though the logic of horizontal and integrated health care to strengthen health systems has long been acknowledged at policy level , empirical evidence on how such integration operates is rare . based on recent ( 20112012 ) ethnographic fieldwork in odisha , india , this article discusses community health workers ' experiences in integrated service delivery through village - level outreach sessions within the nrhm . it shows that for health workers , the notion of integration goes well beyond a technical lens of mixing different health services . crucially , they perceive teamwork and building trust with the community ( beyond trust in health services ) to be critical components of their practice . however , the comprehensive nrhm primary health care ideology which the health workers espouse is in constant tension with the exigencies of narrow indicators of health system performance . our ethnography shows how monitoring mechanisms , the institutionalised privileging of statistical evidence over field - based knowledge and the highly hierarchical health bureaucratic structure that rests on top - down communications mitigate efforts towards sustainable health system integration .

Introduction Methods Integration sites and actors: outreach sessions Eliciting and sustaining trust with the community Teamwork Tying their hands? Conclusion Funding

beginning with the alma ata declaration in 1978 , the need for comprehensive and integrated approaches to health care has been well acknowledged . the recent global calls to achieve universal health care and the millennium development goals ( mdgs ) in low- and middle - income countries have revived , at least rhetorically , the emphasis on strengthening health systems through comprehensive and integrated approaches to health care . in line with these global developments and after a protracted focus on disease - specific programmes , india has recently embarked on a path to revamp its public health delivery system . the nrhm sets out a horizontal and comprehensive approach to health care to undertake these architectural corrections . some of the core strategies of this horizontal approach include : integration of vertical programmes and structures ; integrating health with its broader determinants ; mainstreaming traditional systems of medicine and revitalising local health traditions ; decentralised health planning ; effective community participation and ownership of health ; and improved and effective public health management ( government of odisha , 2007 ; nrhm , 2005 ) . notions of integration in nrhm thus flow from the comprehensive primary health care approach , that reinforce principles of community participation , provision of comprehensive care ( curative , preventive and promotive ) inter - sectoral collaboration , decentralisation and equity ( nrhm mission document , 2005 ) . 1 ) defines integrated service delivery as management and delivery of health services so that clients receive a continuum of preventive and curative services , according to their needs , over time and across different levels of the health system. despite the wider policy acknowledgement of the need for such an integrated approach to health care , empirical evidence on how such integration operates on the ground is rather sparse and disparate ( atun , jongh , secci , ohiri , & adeyi , 2010 ; wallace , dietz , & cairns , 2009 ; who , 2008a ) . existing literature on global programmatic experiences with integration of health services tend to approach delivery of services as a technical and mechanistic process ( banerjee , elamon , & aggarwal , 2009 ; partapuri , steinglass , & sequiera , 2012 ) . integrated service delivery in this sense is an approach of combining services of multiple interrelated diseases to increase overall efficiency of the health system and patient convenience ( lenka & george , 2013 , p. 1 ) . often referred to as service add on ( magtymova , 2007 ) , benefits of integration are assessed through data on the uptake of the specific health services reflected in an increase in contraceptive prevalence , improved immunisation coverage or uptake of mosquito nets ( banerjee et al . feasibility of integration is weighed in terms of health system factors like availability of human resources , compatibility of services or supply chain management and infrastructure ( lenka & george , 2013 ) . ethnographic evidence suggests that the demand / uptake of health services is linked to a host of factors , such as the community 's perceived vulnerability to a specific illness for which the health service is offered , previous experiences with other state health services , modes of health communication , interaction with health workers and broader political identities and perceptions of the state by the community ( leach & fairhead , 2005 ; mishra , flikke , nordfeldt , & nyirenda , 2013a ; nichter , 1995 ) . exclusive focus on individual health services and sites of delivery ignores larger social processes at work at the intersection of supply and demand , as well as providers and local communities , thus reflecting little on how integration is achieved or even the sustainability of integration measures . using nrhm as a case study , this article offers a grounded perspective on integration of health services through capturing the everyday experiences of community health workers located at the interface between the formal health system and the local communities . existing ethnographic studies show how the social experiences of health workers are important to capture in order to understand how policies are translated on the ground , communities are mobilised , care is delivered and raw data on the health status of populations and functioning of public health programmes are produced at the local level ( coutinho , bisht , & raje , 2000 ; george , 2010 ; mishra , hasija , & roalkvam , 2013b ; walker & gilson , 2004 ) . this article adds to the literature and discusses community health workers experiences with integrating immunisation with broader maternal and child health services through the village outreach sessions called health and nutrition days . this article draws on 8 months of fieldwork conducted in 20112012 in one of the southern districts in the state of odisha , india . this article is concerned with the local level , specifically , how health workers understand and translate their roles within the nrhm 's emphasis on strengthening integrated and comprehensive primary health care . data were collected through participant observation in two villages selected in the district , including during 8-monthly health and nutrition days ( outreach sessions ) in each village ; monthly supervisory meetings at the primary health centre which caters to the villages ; training sessions of health workers ; and panchayat ( local government ) level meetings . we conducted open - ended in - depth interviews with 12 health workers , and accompanied these health workers in all their routine activities beyond the outreach sessions for a period of 6 months , including when they accompanied a pregnant woman to the health centre , took a malnourished child for treatment , distributed iron syrups to the villagers or accompanied a villager to the traditional healer . at the community level , outreach sessions like village health and nutrition days ( also known as immunisation days ) are an important forum for provision of integrated and comprehensive care . as per the nrhm guidelines , these sessions offer a package of services pertaining to maternal and child health , communicable and non - communicable diseases ; address social determinants of health including sanitation , nutrition and gender ; and facilitate the collection of data on specific needs of vulnerable populations , vital events including disease outbreaks and audits of maternal and child deaths ( nrhm , 2007 ) . the nrhm envisages that these sessions provide an effective platform for delivering first - contact primary health care , thus maximising the points of interaction between the community and the formal health system . three sets of community level female health workers are principally involved in mobilising and delivering health services during these outreach sessions , though the nrhm guidelines envisage the involvement of panchayat members , primary schoolteachers , and traditional birth attendants in supporting and facilitating these sessions ( nrhm , 2007 ) . the ashas ( who are addressed through this acronym by everyone , including the villagers ) are village - level health workers who have been recruited since the start of the nrhm in 2005 . they are responsible for mobilising the community to access public health services , including immunisation , hospital birth and anc ; identifying and referring people affected by tuberculosis , leprosy and malaria ; and promoting family planning programmes . apart from this , they have a broader role of being health activists in the community by creating awareness about health and its determinants , mobilising the community towards local - level planning and increasing utilisation and accountability of existing health services ( national institute of health and family welfare [ nihfw ] , government of india , 2005 ) . although they receive no fixed salary , they are paid performance - based cash incentives , the amount of which differs according to the nature of the service ( e.g. unlike the ashas who are recent recruits , the anms ( also addressed through this acronym ) , have been a part of the health bureaucracy since the 1950s . an anm serves four or five villages ( a population of 5000 ) , constituting a sub - centre , the lowest of the three - tier primary health care organisation in india . as discussed below , these differences in their professional training , status and role are important considerations in ways they conduct outreach sessions and engage with the community . while trying to operationalise the nrhm 's strategy of integrating delivery of services through outreach sessions , health workers in our study emphasised how effective teamwork was critical to their practice of delivering care , although they acknowledged the challenges posed by differential training , salary , status and role . health workers efforts to provide integrated health services and care were not restricted to either individual outreach sessions or health services . thus , mobilisation , communication and delivery of services were embedded in the dynamics of relationship building at the interface between the health system ( mediated by individual health works ) and the community . an asha worker clarified what it means to be a good worker and to build a healthy relationship with the community : an asha should have the ability to cooperate with others . such qualities of being there , being sisterly , responding to the villager 's needs and preferences are important elements of the meaning of trust that the health workers espouse . health workers experiences suggest that building relations of trust with the community are not an ad hoc event but rather a continuous process of eliciting and sustaining such trust . a host of factors are at play in the trust - building process , beginning with the social status ( caste , ethnic , familial relations ) of individual health workers , modes of communication , ability to cater to community health and non - health needs and the community 's own prior experiences with other health interventions , including their perceptions of the state ( see roalkvam , this volume ) . for the asha and aww workers who mostly live in the villages they serve , creating demand for any health service implies eliciting trust in their roles , both as health workers and , by extension , state representatives , and as members of the community . for the tribal people in the villages studied , the state ( referred to as the government ) is represented by the health workers . vaccination or institutional delivery are not mere biomedical interventions , but rather reach the community as interventions provided by the state , such that for the local community , engaging with vaccines or anc entails an engagement with the state . more than any other programme , due to its scope , approach and delivery mechanisms , the nrhm has made the state distinctly visible in local communities through the everyday material practices of immunisation days , recruitment of health workers , disbursement of cash incentives and distribution of iron syrup and other medicines . health workers engaged in the process of building trust with the communities in several ways . for instance , they repackaged the merits of health services in terms of the community 's existing health priorities , or ensured that these priorities were taken into account through relevant means . further , they tried to ensure that the gynaecological complaints of all village women rather than only the pregnant women who are targeted as part of the nrhm 's focus on reducing maternal mortality are addressed . by expanding their remit to cover all women and address their health needs , they seemed to successfully contribute to the process of building trust in health workers and formal state health services , as evidenced by a young married woman lamenting the death of her in - laws 7 years prior due to tuberculosis : had it been now , my in - laws would not have died , asha sister would have taken them to hospital and given medicines. for example , the high number of stillbirths and the vulnerability of children to common illnesses are predominantly explained through the role of the dumas ( ancestors ) who need to be appeased periodically through rituals both at individual households and at community levels . for the community , care has a larger connotation that involves integrating local aetiologies of illness , notions of health and healing modalities with that of the state - provided biomedical health services . thus the traditional rituals of godh bhariba ( a ritual aimed at protecting the foetus in the womb ) , the ritual tying of sacred thread and bila sukheiba ( the ritual offerings to a tree representing the goddess responsible for ensuring healthy children ) need not conflict with immunisation , distribution of nutritious meals and treatment for malnourishment in the community health centre . the potential role of health workers in initiating critical dialogue between different systems of medicine and providers ( in the light of the community 's preferences , notions of efficacy , common complaints for which traditional rituals are sought ) is thus ignored ( scott & shankar , 2010 ) . the nrhm seeks to resolve the institutionalised dichotomy between curative and preventive services that has plagued the indian public health system for a long time . however , the community 's demand for preventive health services is largely mediated through their access to and experiences with curative services , particularly for the common diseases that become their immediate priority health needs ( mishra et al . hence , adequate supply of medicines catering to these health needs is a major concern for the health workers , often voiced during monthly supervisory meetings . social relations of trust between the health workers and the community however go beyond the delivery of specific health services . on the other hand , in villages where the asha does not reside in the village , or handles more than two or three villages and has been nominated by the anm without village involvement , such trust was missing and the asha was regarded merely as a subordinate of the anm . in a multi - caste or multi - tribe village , the caste and class status of the health worker and familial relations with other members of the village mediate in relationship - building and hence service delivery . in multi - caste villages , the process of trust - building with the community is thus fragile and requires much more concerted and careful efforts on the part of both the community and the health workers . the team dynamics we observed among the health workers are important not only for the success of individual episodes of outreach sessions but also its contribution to legitimating and sustaining their role as health workers . the vertical power hierarchy between the anm on the one hand and the asha and aww on the other is explicit though the ashas are supposed to be accountable to the local government / community . for example , they communicated to the pregnant women how they could eat a nutritious diet that is locally available with them , gave examples of several such food items ( and hence healthy diet need not be a platter of fruits which they can not afford ) , and advised them that they could continue to work in the fields , provided they take care of their diet , monitor any warning signs and report to the health worker . this is because the outreach sessions provide the raw data for constituting the success of public health programmes and more importantly , the output indicators such as number of children immunised are part of the evaluation of the health workers performance , as explained below . our ethnographic data suggest that favourable team dynamics such as visible cooperation , a proactive role played by each of the three workers and constructive supervision by the anms often translated not merely into successful outreach sessions , but also community trust in state health services mediated by trust in health workers . practical strategies to establish team spirit and teamwork serve the larger purpose of being a good health worker both for the community and that of the health system , though these expectations often conflict with each other , as discussed below . institutional obstacles to the success of the community health worker programme in rural north india show how the asha workers potential role as health activists are restricted by institutional obstacles like the rigid hierarchical health bureaucracy , and lack of flexible and creative remuneration structures . while the health workers subscribe to larger notions of integration inherent in a primary health care approach , such efforts conflict with the narrow monitoring indicators used for health system performance , health system 's privileging of statistical over experiential knowledge and its reliance on top - down channels of communication . though nrhm has the broad objective of strengthening primary health care and the rural public health system , in practice , there is a tendency among sub - district and district health officials to restrict nrhm efforts to control the infant mortality rate and the maternal mortality ratio only . this could be due to the pressure from higher up the health bureaucracy at the state and national levels to contribute to achieving the mdgs on maternal and child health and more directly to the fact that the health system performance is evaluated through indicators on maternal and child health . health workers , for example , had insights on immunisation programme drop out in peak agricultural season , reasons for seeking treatment for epileptic fits from a traditional healer , and pregnant women being taken to a district hospital rather than the recommended primary health centres . for the health workers , extras which need to be kept quiet or shared informally with peers , rather than evidence on whether and how nrhm is achieving its aims . the supervisory meetings become a site for top - down communication exclusively , where senior officials either demand reports and records or make specific announcements on recent orders passed down to them from further up the health bureaucracy . such top - down communication dilutes the significance of the forum of the supervisory meetings , which are an important point of encounter between top - down and bottom - up planning . in the context of village - level health workers , rewarded behaviour includes tasks that are incentivised and for which the health workers performance is evaluated . gaming strategies among the health workers in our study included falsification of evidence on identification of malnourished children for referral and the number of institutional deliveries . one of the consequences of such gaming strategies is that the health system actually has poor quality data about what is happening in the field . mainstream public health writings on delivery of integrated services tend to focus on the health services per se and modalities of their integration , assuming that effective supply chain management , infrastructure and human resources would achieve integration . based on ethnographic evidence , this article shows how building social relations of trust and teamwork are critical to health workers efforts in delivering integrated services . the role of trust in health care has traditionally been examined in relation to doctor patient relationships . however , recent anthropological literature has sought to bring social relations of trust to the centre stage in the study of health systems and policies ( gilson , 2003 , 2005 ; magrath & nichter , 2012 ; rowe & calnan , 2006 ; theide , 2005 ) . these community health workers espouse an integrated approach to care by fostering relations of mutual trust , teamwork , cooperation , addressing community health and other needs , promoting a continuum of care from curative to preventive care and valuing the role of regular and effective communication with villagers and also amongst health workers themselves . these values are indeed the cornerstone of a primary health care ideology that promotes democracy , equity and participation ( nichter , 1986 ) . however the voices and experiences of health workers and other implementers are hardly taken into account and rarely thought to constitute evidence for public health policies and programmes . we agree with walker and gilson ( 2004 ) , who based on their study of nurses in south africa , argue that discounting the perspectives and experiences of frontline health workers emanates from the methodological limitation of approaching policy implementation as a linear , top - down process . the vision of comprehensive primary health care that the nrhm 's health workers try to promote , and which is enshrined in policy documents , is in constant tension with other important elements of the indian public health system : the narrow indicators used for health system performance ; the highly hierarchical bureaucratic structure that rests on top - down communication and information ; the institutionalised privileging of statistical evidence over field - based experiences . a narrow focus on achieving mdgs 4 and 5 through cash incentives , regularising outreach sessions , stricter monitoring of indicators relating to these goals might result in better immunisation coverage and higher rates of institutional delivery , but these need not guarantee a robust , sustainable health system or indeed result in real improvement in health outcomes . a recent who summary of african countries experiences with revitalisation of primary health care documents many similar challenges to fulfilling the primary health care agenda to those identified here , including reforming monitoring and evaluation systems , changing the vertical focus of health service delivery and enhancing greater community participation ( who , 2008b ) . though the nrhm evokes the spirit of primary health care values , these are enforced through a health organisational bureaucracy that is deeply hierarchical . bhatia and rifkin ( 2010 ) rightly note that it is not only enough to revitalise primary health care , but also emphasise the need to reframe it in light of values of equity , community empowerment and determinants of health .

in the late 1970s an increasing awareness of the importance of disease prevention in the u.s . culminated in healthy people : the surgeon general 's report on health promotion and disease prevention . this led to the healthy people program , a major nationwide effort to monitor and reduce death and disability from chronic disease , building on efforts in canada and europe . one of the consequences of this increased focus on chronic disease prevention has been an increased emphasis on having screening incorporated into routine primary care . the us preventive services task force is charged with assessing the costs and benefits of screening and developing guidelines for clinical preventive services [ 46 ] . gradually , clinical preventive services guidelines , such as routine screening for breast and cervical cancer for women in certain age groups , have been adopted nationwide . in spite of this , a recent study showed that from 40 to 60 per cent of americans ( depending on age , gender or other factors ) do not receive recommended preventive care . the impact of chronic disease is not limited to developed countries , but rather is increasingly a source of death and disability world - wide . thus , many of the efforts to develop screening guidelines in the u.s . and however , guidelines may need to be modified in low resource settings where both the screening and the referrals from screening are unaffordable . following the break - up of yugoslavia , and the balkan war , much of the health care infrastructure was damaged or destroyed and there was a severe shortage of health personnel due to outward migration . as part of the international assistance ( provided as part of the rebuilding effort ) , michael mcginnis who had been a developer of the healthy people program in the u.s.travelled to the republika srpska ( rs ) , one of the two entities that formed bosnia and herzegovina after the war.1 dr . milorad balaban ) to implement a program of health goals and monitoring for the rs , and thus a program that resembles healthy people was initiated . on july 26 , 2002 , the national assembly of the rs adopted the program of health policy and strategies for health in the rs by the year 2010 , with a goal of continuous improvement of the health status of the population and improvement of conditions that influence health . the four main objectives of the health policy are : decreasing inequalities in health status and improving accessibility to health careimproving health status and increasing accessibility to health services for vulnerable groupsreorienting health services towards disease preventionincreasing the efficiency and quality of health care . decreasing inequalities in health status and improving accessibility to health care improving health status and increasing accessibility to health services for vulnerable groups reorienting health services towards disease prevention increasing the efficiency and quality of health care . in addition , because chronic disease accounts for much of the disease burden in the rs ( see table 1 ) , an emphasis was put on increasing access to screening services for such conditions . in april 2003 , the rs government adopted the strategy for prevention and control of non - communicable diseases . this prevention strategy includes : a health - promotion program aimed at reducing risks;screening to detect risks for selected conditions or the presence of the conditions;early diagnosis and treatment . a health - promotion program aimed at reducing risks ; screening to detect risks for selected conditions or the presence of the conditions ; early diagnosis and treatment . more information on the program is contained in an rs government document and the article by atun et al . the conditions being emphasized are those with the highest mortality rates , including heart disease , cancer , and diabetes , with an overall goal of reducing mortality due to these diseases by 5% by 2010 . the specific 2010 objectives by disease category include : decreasing cardiovascular mortality by 10% in people under 65;decreasing cancer mortality by 5% in people under 65;decreasing diabetes - related complications ( blindness , amputations , renal failure ) by 20%;decreasing the number of adult smokers by 50% and the number of adolescent smokers by 80%;prohibiting smoking in the workplace , public places and in public transportation;decreasing the number of adults who regularly drink alcohol by 50% and the number of adolescent drinkers by 80% . decreasing cardiovascular mortality by 10% in people under 65 ; decreasing cancer mortality by 5% in people under 65 ; decreasing diabetes - related complications ( blindness , amputations , renal failure ) by 20% ; decreasing the number of adult smokers by 50% and the number of adolescent smokers by 80% ; prohibiting smoking in the workplace , public places and in public transportation ; decreasing the number of adults who regularly drink alcohol by 50% and the number of adolescent drinkers by 80% . to provide a context for the screening program , it is important to understand the financing and delivery of health services in the rs . the health care financing system in the rs is based on the concept of universal public health insurance ( the bismarck model ) . funds are provided by the rs health insurance fund , which collects money for premiums from employers ( a system that dates back to the days of the former yugoslavia ) . while health insurance is theoretically universal in bosnia and herzegovina , the health insurance funds in both entities ( including the rs ) are currently under - financed . this is true for many private sector employers , particularly small businesses , and for self - employed and unemployed individuals . the result is that many people remain uninsured and there is under - financing of the health insurance fund . health care delivery is the responsibility of the central rs government ministry of health and social welfare , which operates hospitals , and local governments which provide primary care in 63 dom zdravljas many health professionals left during the war , immigrating to europe or the united states , and have not returned . in spite of these challenges facing the health system , the rs government adopted plans to implement a screening program in order to achieve the objectives outlined above . the screening program was to begin on january 1 , 2004 and be implemented in all of the dzs . according to the government plan , each citizen of the rs was to be registered with a personal primary care provider at a dz who would provide age - appropriate screening at a regular physical examination , as shown in table 2 . an estimate of the size of the population to be served by each dz was developed , and some additional funds were provided to the dzs to accomplish their screening goals . as an integral part of the screening program , the ministry of health and social welfare required the family doctors at the dzs to keep a registry of people who were screened and found to be at high risk for one of the listed chronic diseases . they were then required to follow - up with those individuals on a regular basis . in 2005 , two years after the adoption of the prevention objectives , usaid provided funding through the urban institute to the economic institute of banja luka to perform an evaluation of the first year of the program . the purpose was to assess implementation and provide recommendations to the health ministry concerning possible program improvements , as well as to assess the adequacy of financing to the dzs for program implementation . to accomplish those goals the evaluation team , with a consultant from the urban institute , analyzed existing data from the rs government and collected new information from key stakeholders and citizens .2 the evaluation team at the economic institute of banja luka interviewed twenty - five key stakeholders using a semi - structured interview protocol . interviewers asked about the stakeholders ' perceptions of implementation of the prevention program ( both what has happened and how it is working ) ; their opinions about the feasibility of achieving the prevention program 's goals ; and issues concerning the adequacy of financing of the initiative . the individuals who were interviewed include the assistant minister for health of the rs ; the director of the public health institute ( a quasi - governmental organization that monitors quality of care ) ; four representatives of the health insurance fund that provides funding to the dzs ; and representatives from each of the dzs ( for example the directors and one or more physicians ) . to assess citizen awareness of and participation in the prevention program , partner marketing consulting conducted an in - person interview with a sample of citizens in their homes . the questionnaire contained 23 questions concerning several topics including : ( 1 ) the citizens ' familiarity with the screening program , and with disease prevention more generally ; ( 2 ) their willingness to receive preventive health check - ups and whether they have done so recently ; ( 3 ) whether they are enrolled with a doctor at a dz ; ( 4 ) whether they have health insurance ; ( 5 ) the types of media they are exposed to ; and ( 6 ) other socio - demographic characteristics . experienced interviewers , who had been trained in this and other similar demographic in - person surveys , collected the data . twenty per cent of interviews were back - checked by a field supervisor by phone or in person , for quality control purposes . the sample using methods adapted for polling in the rs was a convenience sample with a substantial random component to the selection of respondents . geographic sampling. each interviewer begins at a precise pre - specified location and walks a randomly determined direction and distance to a particular house ( or apartment house , where the interviewer randomly selects an apartment ) . they select one adult respondent per household , asking for the next adult who has a birthday . ( only individuals 18 years of age and older were included in the sample . ) usually there are two call - backs before a selected respondent is replaced ( from a randomly selected household near the same sampling point ) . each interviewer is asked to achieve a gender balance among those interviewed , so that after 80% of their interviews are conducted they check the gender distribution . they then continue interviewing only males or females in order to have approximately half of their interviews of each gender . given the resources for the study , 1004 households were interviewed from across the rs , with stratification according to the size of each of eight districts of the rs . the initial geographic locations for the interviews were chosen to represent urban , suburban , and rural areas . the size of the sample in each district was selected to be proportionate to the population size according to recent estimates from the world bank and election commission voter registration lists . approximately the same number of males and females were interviewed , and the age distribution of the sample approximated the estimated age distribution of adults in the rs . the questionnaire covered the following topics : demographic characteristics ( gender , age , education , income and employment)health insurance statusregistration with the dz ( whether and where)receipt of diagnosis of chronic diseaseawareness of prevention and of chronic diseaseawareness of the prevention programuse of any health services and use of prevention program servicesuse of media ( radio , tv , newspapers , internet ) demographic characteristics ( gender , age , education , income and employment ) health insurance status registration with the dz ( whether and where ) receipt of diagnosis of chronic disease awareness of prevention and of chronic disease awareness of the prevention program use of any health services and use of prevention program services use of media ( radio , tv , newspapers , internet ) there are several limitations to the methods of the study . the stakeholders who participated in the in - depth interviews do not represent the full range of stakeholders or a random sample of that group . also , because there is no full census of the population in the rs , it was necessary to use geographic sampling which under represents certain types of people , such as people who are rarely at home or do not have a fixed address . stakeholders believe that a prevention program is an obligation of the government , and that it is an important investment in population health . however , there are features of the current organization of the rs health system that make the implementation of the current prevention program very difficult . in particular , all interviewed stakeholders agree that the scope of the program ( for example , the number and frequency of screening services ) is overly - broad . first , in order for the program to succeed as currently designed , it would be necessary to register every citizen with a family medicine team at the local dz . however , most of the dzs are incapable of implementing the program due to a lack of enough medical personnel . as of april 2005 , only 30 of the 54 dzs in the rs had a family doctor on their staff , and only 16 had 25% or more of the population in the service area registered at the dz . most patients still come to the dz because of health problems , not for preventive care . thus it is not possible to screen people who are in the early stages of disease or to identify those at high risk . medical staff in most of the dzs is already overworked providing curative services to patients . in a typical dz , in order to accomplish the number of screenings necessary to cover the entire population served , it would be necessary to increase the number of patient encounters each day by about 50% according to one key informant . the extra time is not just the time to administer screening tests , but also to provide counseling to patients . for example one family medicine team might care for 30 sick cases in a day , and in addition would be asked to provide preventive screening for an additional 15 people . they commented that , practically , it is very difficult for a team to provide so many services each day during normal working hours . there are inadequate personnel to perform outreach to inform citizens of the program , or to conduct public information campaigns . the preventive care program also requires extra administrative time to track patients and document screening tests , as required by program guidelines . only one center ( the dom zdravlja laktai ) operates almost completely within the preventive care family medicine model . they estimate that 98% of the population of their service area is registered with a family doctor . they used three communication methods to advertise the prevention program : media ( radio and tv ) , direct mail , and telephone calls . all are effective , but the telephone calls while expensive are the most successful way to encourage registration and use of preventive care . in addition , financing for the program is inadequate , and stakeholders commented that there was not a sufficient prior analysis of the cost of the program and the feasibility of implementing it as designed before putting it into place . only insured people 's health needs are financed through the health insurance fund , but dzs are obligated to provide health services ( with some co - payments ) to anyone in their service area . according to key informants at the health insurance fund , preventive services for uninsured people ( approximately one - fourth of the population ) should be financed by some other mechanism . preventive care is not designated as a separate item in financial planning , so curative services are given priority in resource reallocation over preventive services . consequently , the health insurance fund can not provide adequate funds to each of the dzs to care for all individuals in their service area . these funds are needed to make the infrastructure improvements and to add personnel before the screening program can be thoroughly implemented . for example , representatives from only two of the eight dzs interviewed reported that they own the apparatus necessary for mammography screening . . however , many doctors practicing in the hospitals are unaware of the screening program . that the screening program reveals diseases requiring treatment , there must be sufficient resources for treatment at the hospitals , as well as closer coordination between the levels of care than is currently in place . beyond the cost of screening services , stakeholders had additional questions about the feasibility and acceptability of some of the services to their patients . finally , there has been little monitoring of program implementation either at the dz level or by the government . this is because there is little automation of data in the dzs , and collecting data for program monitoring and evaluation creates an additional burden for the already - stretched dz staff . when requested monitoring data were provided by the dzs , most reported very low rates of screening , likely due to underreporting in some instances . one reason for underreporting is that , even when dzs report on screenings provided during preventive visits , they usually can not identify those provided during curative visits , leading to additional underreporting and lack of comparability between dzs . the survey of rs citizens ' knowledge , attitudes , and practices of prevention enabled us to examine the degree to which citizens are aware of the importance of chronic disease prevention and control , whether they are informed about the prevention program in the dzs , and whether they have participated . an important factor in whether citizens use preventive care is their health insurance status . fully one - fifth of those surveyed report that they are not covered by the health insurance fund ( see figure 1 ) . such individuals will be requested to pay out - of - pocket for preventive care services , a strong deterrent to using them . as noted above , in order for the current prevention program to succeed , all citizens must be registered at a dz and with a particular family doctor . however , most of the people surveyed report that they are not registered with a family doctor . only 32% of them , a little less than one third , said that they have completed the registration process at a dz ( see figure 2 ) . survey results confirm much of the information gathered in the in - depth stakeholder interviews , in particular that there is a low level of awareness about the importance of chronic disease prevention and about the rs prevention program in particular . while awareness of the importance of prevention is lower than desirable , it is higher among insured citizens ( 78.1% ) ( see figure 3 ) . in addition , while a small minority of both insured and uninsured citizens is aware of the rs chronic disease prevention program , awareness is higher among the insured ( 31.2 per cent ) ( see figure 4 ) . as shown in figure 5 , even those who are registered with a family doctor at the dz are still usually unaware of the disease prevention program , showing that doctors are either not themselves aware of the program or are not educating their patients about it . confirming that cost is likely an issue in obtaining preventive care , insured citizens are more willing to obtain preventive examinations than uninsured citizens ( see figure 6 ) . rates of actual preventive care are low overall , but are higher among those who are registered with a family doctor ( 29.1% ) ( see figure 7 ) , and among insured citizens ( 24.1% ) ( see figure 8) . media play an important role in informing the public about the prevention program , since 44.2% of people who are aware of the program learn about it through the media . stakeholders believe that a prevention program is an obligation of the government , and that it is an important investment in population health . however , there are features of the current organization of the rs health system that make the implementation of the current prevention program very difficult . in particular , all interviewed stakeholders agree that the scope of the program ( for example , the number and frequency of screening services ) is overly - broad . first , in order for the program to succeed as currently designed , it would be necessary to register every citizen with a family medicine team at the local dz . however , most of the dzs are incapable of implementing the program due to a lack of enough medical personnel . as of april 2005 , only 30 of the 54 dzs in the rs had a family doctor on their staff , and only 16 had 25% or more of the population in the service area registered at the dz . most patients still come to the dz because of health problems , not for preventive care . thus it is not possible to screen people who are in the early stages of disease or to identify those at high risk . medical staff in most of the dzs is already overworked providing curative services to patients . in a typical dz , in order to accomplish the number of screenings necessary to cover the entire population served , it would be necessary to increase the number of patient encounters each day by about 50% according to one key informant . the extra time is not just the time to administer screening tests , but also to provide counseling to patients . for example one family medicine team might care for 30 sick cases in a day , and in addition would be asked to provide preventive screening for an additional 15 people . they commented that , practically , it is very difficult for a team to provide so many services each day during normal working hours . there are inadequate personnel to perform outreach to inform citizens of the program , or to conduct public information campaigns . the preventive care program also requires extra administrative time to track patients and document screening tests , as required by program guidelines . only one center ( the dom zdravlja laktai ) operates almost completely within the preventive care family medicine model . they estimate that 98% of the population of their service area is registered with a family doctor . they used three communication methods to advertise the prevention program : media ( radio and tv ) , direct mail , and telephone calls . all are effective , but the telephone calls while expensive are the most successful way to encourage registration and use of preventive care . in addition , financing for the program is inadequate , and stakeholders commented that there was not a sufficient prior analysis of the cost of the program and the feasibility of implementing it as designed before putting it into place . only insured people 's health needs are financed through the health insurance fund , but dzs are obligated to provide health services ( with some co - payments ) to anyone in their service area . according to key informants at the health insurance fund , preventive services for uninsured people ( approximately one - fourth of the population ) should be financed by some other mechanism . preventive care is not designated as a separate item in financial planning , so curative services are given priority in resource reallocation over preventive services . consequently , the health insurance fund can not provide adequate funds to each of the dzs to care for all individuals in their service area . these funds are needed to make the infrastructure improvements and to add personnel before the screening program can be thoroughly implemented . for example , representatives from only two of the eight dzs interviewed reported that they own the apparatus necessary for mammography screening . . however , many doctors practicing in the hospitals are unaware of the screening program . that the screening program reveals diseases requiring treatment , there must be sufficient resources for treatment at the hospitals , as well as closer coordination between the levels of care than is currently in place . beyond the cost of screening services , stakeholders had additional questions about the feasibility and acceptability of some of the services to their patients . finally , there has been little monitoring of program implementation either at the dz level or by the government . this is because there is little automation of data in the dzs , and collecting data for program monitoring and evaluation creates an additional burden for the already - stretched dz staff . when requested monitoring data were provided by the dzs , most reported very low rates of screening , likely due to underreporting in some instances . one reason for underreporting is that , even when dzs report on screenings provided during preventive visits , they usually can not identify those provided during curative visits , leading to additional underreporting and lack of comparability between dzs . the survey of rs citizens ' knowledge , attitudes , and practices of prevention enabled us to examine the degree to which citizens are aware of the importance of chronic disease prevention and control , whether they are informed about the prevention program in the dzs , and whether they have participated . an important factor in whether citizens use preventive care is their health insurance status . fully one - fifth of those surveyed report that they are not covered by the health insurance fund ( see figure 1 ) . such individuals will be requested to pay out - of - pocket for preventive care services , a strong deterrent to using them . as noted above , in order for the current prevention program to succeed , all citizens must be registered at a dz and with a particular family doctor . however , most of the people surveyed report that they are not registered with a family doctor . only 32% of them , a little less than one third , said that they have completed the registration process at a dz ( see figure 2 ) . survey results confirm much of the information gathered in the in - depth stakeholder interviews , in particular that there is a low level of awareness about the importance of chronic disease prevention and about the rs prevention program in particular . while awareness of the importance of prevention is lower than desirable , it is higher among insured citizens ( 78.1% ) ( see figure 3 ) . in addition , while a small minority of both insured and uninsured citizens is aware of the rs chronic disease prevention program , awareness is higher among the insured ( 31.2 per cent ) ( see figure 4 ) . as shown in figure 5 , even those who are registered with a family doctor at the dz are still usually unaware of the disease prevention program , showing that doctors are either not themselves aware of the program or are not educating their patients about it . confirming that cost is likely an issue in obtaining preventive care , insured citizens are more willing to obtain preventive examinations than uninsured citizens ( see figure 6 ) . rates of actual preventive care are low overall , but are higher among those who are registered with a family doctor ( 29.1% ) ( see figure 7 ) , and among insured citizens ( 24.1% ) ( see figure 8) . media play an important role in informing the public about the prevention program , since 44.2% of people who are aware of the program learn about it through the media . after one year of implementation , the rs strategy for prevention and control of non - communicable diseases still struggles with problems of awareness ( both among the medical personnel who must implement the program and the citizens who must use it ) and financing . in particular , the key factors slowing implementation are the following : there are insufficient financial resources to fund the facilities , equipment and personnel needed to fully implement the preventive screening program . for example , most of the dzs can not do mammograms on site.most rs citizens are unfamiliar with the program , and 68% of adults are not registered with a family doctor at the dz . over half of those not registered are not aware they are supposed to do so.most dzs do not have sufficient medical personnel . in addition , many doctors either do not know about the program , or are not informing their patients about the program.over 20% of the rs population has no health insurance . since the fund 's policy is to finance health care for insured citizens , this creates a strong financial disincentive to obtain preventive care.there is little cooperation between the dzs and the hospitals in providing services for the preventive care program . many doctors in the hospitals are unaware of the program , but often they are the only doctors with access to screening equipment and they are the ones to provide follow - up treatment.doctors in dzs are not motivated to implement preventive checkups due to time and cost constraints , since they receive no reimbursement for the additional administrative burden.evaluation of the program is very difficult , because it must be based on manual chart reviews due to a lack of automated data.finally , the survey shows that people are very receptive to information in various forms , especially from media ( radio and television ) . this provides a good way to reach many people , as the program moves forward . other strategies may be needed , since experience in one dz shows that a direct telephone call is the most effective way to reach people . there are insufficient financial resources to fund the facilities , equipment and personnel needed to fully implement the preventive screening program . most rs citizens are unfamiliar with the program , and 68% of adults are not registered with a family doctor at the dz . over half of those not registered in addition , many doctors either do not know about the program , or are not informing their patients about the program since the fund 's policy is to finance health care for insured citizens , there is currently no source of funding for the uninsured in the dzs . there is little cooperation between the dzs and the hospitals in providing services for the preventive care program . many doctors in the hospitals are unaware of the program , but often they are the only doctors with access to screening equipment and they are the ones to provide follow - up treatment . doctors in dzs are not motivated to implement preventive checkups due to time and cost constraints , since they receive no reimbursement for the additional administrative burden . evaluation of the program is very difficult , because it must be based on manual chart reviews due to a lack of automated data . finally , the survey shows that people are very receptive to information in various forms , especially from media ( radio and television ) . this provides a good way to reach many people , as the program moves forward . other strategies may be needed , since experience in one dz shows that a direct telephone call is the most effective way to reach people . as a result of this study , we have provided a series of recommendations to the government of the rs about how to improve the prevention program and its financing . first , we recommend that a broad coalition of stakeholders work together to improve the implementation of the preventive care program , including doctors at the primary and secondary level ; the dzs ; the ministry of health and social welfare ; the health insurance fund ; the ministry of finance ; the ministry of transport and communications ; the public health institute ; local authorities , ngos ; and private companies . each of these stakeholders has a great interest in improving the health of the citizens of the rs and preventing death and disability from chronic disease . however , up to the time of the study there was a lack of information about the prevention program among many of the key participants . we also recommend increasing targeted promotion of the program , both among citizens and among the medical personnel who must implement the program . the promotion strategy should use the experience gained in dom zdravlja laktai about how to reach out to citizens in order to educate them about the importance of prevention for their health and the importance of registering with a family doctor . finally , the most pressing issue facing the prevention program is a lack of financial resources . if the program is to continue , more funding must be identified , especially for care for uninsured patients at the dzs . some ideas for additional funds include a possible reallocation of health insurance fund towards the prevention program . however , because of financial shortages system - wide and the fact that the health insurance fund is designed to cover only insured citizens , new resources of revenue are needed . possible sources include a tax on the registration of motor vehicles , or excise taxes on tobacco products , alcohol and alcoholic beverages . the latter form of taxes could also potentially further the goals of the prevention program by deterring the use of tobacco and alcohol , especially among younger drinkers and smokers . these recommendations have contributed to improving the dialogue about how to prevent a continued increase in chronic diseases in the rs . lessons from this area of the world should also have implications for developing countries and for low resource areas of developed countries , for example economically deprived inner city areas of the u.s . katherine m. wilson , phd , mph , ches , division of cancer prevention and control , national center for chronic disease prevention and health promotion , centers for disease control and prevention , atlanta , usa robert lange , phd , head of the department laboratory medicine , immanuel diakonie group and secretary of the german society of disease management ( dgdm ) , bernau bei berlin , germany

introductionin 2002 the republika srpska of bosnia and herzegovina adopted goals for reducing the burden of chronic disease through a new screening program in its publicly funded health centers ( dom zdravljas ) . this study evaluated the first year of program implementation.methodsthe evaluation used in - depth interviews with 25 key stakeholders and in - person interviews with 1004 citizens.resultswe found that many health care providers and citizens were unaware of the program . in addition , there was inadequate financing for the program , because the health insurance fund does not collect revenue for uninsured citizens , more than 20 per cent of the population.conclusionwe recommend improved co - ordination among public and private organizations involved in implementation ; increased promotion of the program with health care providers and citizens ; and increased financial resources for providing screening for uninsured citizens .

Introduction Methods Findings Key informant interviews Citizens survey Conclusions and recommendations Reviewers

this led to the healthy people program , a major nationwide effort to monitor and reduce death and disability from chronic disease , building on efforts in canada and europe . one of the consequences of this increased focus on chronic disease prevention has been an increased emphasis on having screening incorporated into routine primary care . gradually , clinical preventive services guidelines , such as routine screening for breast and cervical cancer for women in certain age groups , have been adopted nationwide . in spite of this , a recent study showed that from 40 to 60 per cent of americans ( depending on age , gender or other factors ) do not receive recommended preventive care . the impact of chronic disease is not limited to developed countries , but rather is increasingly a source of death and disability world - wide . following the break - up of yugoslavia , and the balkan war , much of the health care infrastructure was damaged or destroyed and there was a severe shortage of health personnel due to outward migration . as part of the international assistance ( provided as part of the rebuilding effort ) , michael mcginnis who had been a developer of the healthy people program in the u.s.travelled to the republika srpska ( rs ) , one of the two entities that formed bosnia and herzegovina after the war.1 dr . on july 26 , 2002 , the national assembly of the rs adopted the program of health policy and strategies for health in the rs by the year 2010 , with a goal of continuous improvement of the health status of the population and improvement of conditions that influence health . the four main objectives of the health policy are : decreasing inequalities in health status and improving accessibility to health careimproving health status and increasing accessibility to health services for vulnerable groupsreorienting health services towards disease preventionincreasing the efficiency and quality of health care . in addition , because chronic disease accounts for much of the disease burden in the rs ( see table 1 ) , an emphasis was put on increasing access to screening services for such conditions . more information on the program is contained in an rs government document and the article by atun et al . the specific 2010 objectives by disease category include : decreasing cardiovascular mortality by 10% in people under 65;decreasing cancer mortality by 5% in people under 65;decreasing diabetes - related complications ( blindness , amputations , renal failure ) by 20%;decreasing the number of adult smokers by 50% and the number of adolescent smokers by 80%;prohibiting smoking in the workplace , public places and in public transportation;decreasing the number of adults who regularly drink alcohol by 50% and the number of adolescent drinkers by 80% . to provide a context for the screening program , it is important to understand the financing and delivery of health services in the rs . the health care financing system in the rs is based on the concept of universal public health insurance ( the bismarck model ) . funds are provided by the rs health insurance fund , which collects money for premiums from employers ( a system that dates back to the days of the former yugoslavia ) . while health insurance is theoretically universal in bosnia and herzegovina , the health insurance funds in both entities ( including the rs ) are currently under - financed . the result is that many people remain uninsured and there is under - financing of the health insurance fund . health care delivery is the responsibility of the central rs government ministry of health and social welfare , which operates hospitals , and local governments which provide primary care in 63 dom zdravljas many health professionals left during the war , immigrating to europe or the united states , and have not returned . in spite of these challenges facing the health system , the rs government adopted plans to implement a screening program in order to achieve the objectives outlined above . the screening program was to begin on january 1 , 2004 and be implemented in all of the dzs . according to the government plan , each citizen of the rs was to be registered with a personal primary care provider at a dz who would provide age - appropriate screening at a regular physical examination , as shown in table 2 . an estimate of the size of the population to be served by each dz was developed , and some additional funds were provided to the dzs to accomplish their screening goals . as an integral part of the screening program , the ministry of health and social welfare required the family doctors at the dzs to keep a registry of people who were screened and found to be at high risk for one of the listed chronic diseases . in 2005 , two years after the adoption of the prevention objectives , usaid provided funding through the urban institute to the economic institute of banja luka to perform an evaluation of the first year of the program . the purpose was to assess implementation and provide recommendations to the health ministry concerning possible program improvements , as well as to assess the adequacy of financing to the dzs for program implementation . to accomplish those goals the evaluation team , with a consultant from the urban institute , analyzed existing data from the rs government and collected new information from key stakeholders and citizens .2 the evaluation team at the economic institute of banja luka interviewed twenty - five key stakeholders using a semi - structured interview protocol . interviewers asked about the stakeholders ' perceptions of implementation of the prevention program ( both what has happened and how it is working ) ; their opinions about the feasibility of achieving the prevention program 's goals ; and issues concerning the adequacy of financing of the initiative . the individuals who were interviewed include the assistant minister for health of the rs ; the director of the public health institute ( a quasi - governmental organization that monitors quality of care ) ; four representatives of the health insurance fund that provides funding to the dzs ; and representatives from each of the dzs ( for example the directors and one or more physicians ) . to assess citizen awareness of and participation in the prevention program , partner marketing consulting conducted an in - person interview with a sample of citizens in their homes . the questionnaire contained 23 questions concerning several topics including : ( 1 ) the citizens ' familiarity with the screening program , and with disease prevention more generally ; ( 2 ) their willingness to receive preventive health check - ups and whether they have done so recently ; ( 3 ) whether they are enrolled with a doctor at a dz ; ( 4 ) whether they have health insurance ; ( 5 ) the types of media they are exposed to ; and ( 6 ) other socio - demographic characteristics . experienced interviewers , who had been trained in this and other similar demographic in - person surveys , collected the data . twenty per cent of interviews were back - checked by a field supervisor by phone or in person , for quality control purposes . given the resources for the study , 1004 households were interviewed from across the rs , with stratification according to the size of each of eight districts of the rs . the initial geographic locations for the interviews were chosen to represent urban , suburban , and rural areas . the size of the sample in each district was selected to be proportionate to the population size according to recent estimates from the world bank and election commission voter registration lists . the questionnaire covered the following topics : demographic characteristics ( gender , age , education , income and employment)health insurance statusregistration with the dz ( whether and where)receipt of diagnosis of chronic diseaseawareness of prevention and of chronic diseaseawareness of the prevention programuse of any health services and use of prevention program servicesuse of media ( radio , tv , newspapers , internet ) demographic characteristics ( gender , age , education , income and employment ) health insurance status registration with the dz ( whether and where ) receipt of diagnosis of chronic disease awareness of prevention and of chronic disease awareness of the prevention program use of any health services and use of prevention program services use of media ( radio , tv , newspapers , internet ) there are several limitations to the methods of the study . the stakeholders who participated in the in - depth interviews do not represent the full range of stakeholders or a random sample of that group . also , because there is no full census of the population in the rs , it was necessary to use geographic sampling which under represents certain types of people , such as people who are rarely at home or do not have a fixed address . however , there are features of the current organization of the rs health system that make the implementation of the current prevention program very difficult . in particular , all interviewed stakeholders agree that the scope of the program ( for example , the number and frequency of screening services ) is overly - broad . first , in order for the program to succeed as currently designed , it would be necessary to register every citizen with a family medicine team at the local dz . however , most of the dzs are incapable of implementing the program due to a lack of enough medical personnel . as of april 2005 , only 30 of the 54 dzs in the rs had a family doctor on their staff , and only 16 had 25% or more of the population in the service area registered at the dz . for example one family medicine team might care for 30 sick cases in a day , and in addition would be asked to provide preventive screening for an additional 15 people . there are inadequate personnel to perform outreach to inform citizens of the program , or to conduct public information campaigns . they estimate that 98% of the population of their service area is registered with a family doctor . in addition , financing for the program is inadequate , and stakeholders commented that there was not a sufficient prior analysis of the cost of the program and the feasibility of implementing it as designed before putting it into place . only insured people 's health needs are financed through the health insurance fund , but dzs are obligated to provide health services ( with some co - payments ) to anyone in their service area . according to key informants at the health insurance fund , preventive services for uninsured people ( approximately one - fourth of the population ) should be financed by some other mechanism . consequently , the health insurance fund can not provide adequate funds to each of the dzs to care for all individuals in their service area . for example , representatives from only two of the eight dzs interviewed reported that they own the apparatus necessary for mammography screening . however , many doctors practicing in the hospitals are unaware of the screening program . that the screening program reveals diseases requiring treatment , there must be sufficient resources for treatment at the hospitals , as well as closer coordination between the levels of care than is currently in place . finally , there has been little monitoring of program implementation either at the dz level or by the government . the survey of rs citizens ' knowledge , attitudes , and practices of prevention enabled us to examine the degree to which citizens are aware of the importance of chronic disease prevention and control , whether they are informed about the prevention program in the dzs , and whether they have participated . fully one - fifth of those surveyed report that they are not covered by the health insurance fund ( see figure 1 ) . survey results confirm much of the information gathered in the in - depth stakeholder interviews , in particular that there is a low level of awareness about the importance of chronic disease prevention and about the rs prevention program in particular . in addition , while a small minority of both insured and uninsured citizens is aware of the rs chronic disease prevention program , awareness is higher among the insured ( 31.2 per cent ) ( see figure 4 ) . as shown in figure 5 , even those who are registered with a family doctor at the dz are still usually unaware of the disease prevention program , showing that doctors are either not themselves aware of the program or are not educating their patients about it . media play an important role in informing the public about the prevention program , since 44.2% of people who are aware of the program learn about it through the media . however , there are features of the current organization of the rs health system that make the implementation of the current prevention program very difficult . in particular , all interviewed stakeholders agree that the scope of the program ( for example , the number and frequency of screening services ) is overly - broad . first , in order for the program to succeed as currently designed , it would be necessary to register every citizen with a family medicine team at the local dz . however , most of the dzs are incapable of implementing the program due to a lack of enough medical personnel . as of april 2005 , only 30 of the 54 dzs in the rs had a family doctor on their staff , and only 16 had 25% or more of the population in the service area registered at the dz . for example one family medicine team might care for 30 sick cases in a day , and in addition would be asked to provide preventive screening for an additional 15 people . there are inadequate personnel to perform outreach to inform citizens of the program , or to conduct public information campaigns . they estimate that 98% of the population of their service area is registered with a family doctor . in addition , financing for the program is inadequate , and stakeholders commented that there was not a sufficient prior analysis of the cost of the program and the feasibility of implementing it as designed before putting it into place . only insured people 's health needs are financed through the health insurance fund , but dzs are obligated to provide health services ( with some co - payments ) to anyone in their service area . according to key informants at the health insurance fund , preventive services for uninsured people ( approximately one - fourth of the population ) should be financed by some other mechanism . consequently , the health insurance fund can not provide adequate funds to each of the dzs to care for all individuals in their service area . for example , representatives from only two of the eight dzs interviewed reported that they own the apparatus necessary for mammography screening . however , many doctors practicing in the hospitals are unaware of the screening program . that the screening program reveals diseases requiring treatment , there must be sufficient resources for treatment at the hospitals , as well as closer coordination between the levels of care than is currently in place . beyond the cost of screening services , stakeholders had additional questions about the feasibility and acceptability of some of the services to their patients . finally , there has been little monitoring of program implementation either at the dz level or by the government . this is because there is little automation of data in the dzs , and collecting data for program monitoring and evaluation creates an additional burden for the already - stretched dz staff . the survey of rs citizens ' knowledge , attitudes , and practices of prevention enabled us to examine the degree to which citizens are aware of the importance of chronic disease prevention and control , whether they are informed about the prevention program in the dzs , and whether they have participated . fully one - fifth of those surveyed report that they are not covered by the health insurance fund ( see figure 1 ) . as noted above , in order for the current prevention program to succeed , all citizens must be registered at a dz and with a particular family doctor . survey results confirm much of the information gathered in the in - depth stakeholder interviews , in particular that there is a low level of awareness about the importance of chronic disease prevention and about the rs prevention program in particular . in addition , while a small minority of both insured and uninsured citizens is aware of the rs chronic disease prevention program , awareness is higher among the insured ( 31.2 per cent ) ( see figure 4 ) . as shown in figure 5 , even those who are registered with a family doctor at the dz are still usually unaware of the disease prevention program , showing that doctors are either not themselves aware of the program or are not educating their patients about it . confirming that cost is likely an issue in obtaining preventive care , insured citizens are more willing to obtain preventive examinations than uninsured citizens ( see figure 6 ) . media play an important role in informing the public about the prevention program , since 44.2% of people who are aware of the program learn about it through the media . after one year of implementation , the rs strategy for prevention and control of non - communicable diseases still struggles with problems of awareness ( both among the medical personnel who must implement the program and the citizens who must use it ) and financing . in particular , the key factors slowing implementation are the following : there are insufficient financial resources to fund the facilities , equipment and personnel needed to fully implement the preventive screening program . for example , most of the dzs can not do mammograms on site.most rs citizens are unfamiliar with the program , and 68% of adults are not registered with a family doctor at the dz . in addition , many doctors either do not know about the program , or are not informing their patients about the program.over 20% of the rs population has no health insurance . since the fund 's policy is to finance health care for insured citizens , this creates a strong financial disincentive to obtain preventive care.there is little cooperation between the dzs and the hospitals in providing services for the preventive care program . many doctors in the hospitals are unaware of the program , but often they are the only doctors with access to screening equipment and they are the ones to provide follow - up treatment.doctors in dzs are not motivated to implement preventive checkups due to time and cost constraints , since they receive no reimbursement for the additional administrative burden.evaluation of the program is very difficult , because it must be based on manual chart reviews due to a lack of automated data.finally , the survey shows that people are very receptive to information in various forms , especially from media ( radio and television ) . there are insufficient financial resources to fund the facilities , equipment and personnel needed to fully implement the preventive screening program . most rs citizens are unfamiliar with the program , and 68% of adults are not registered with a family doctor at the dz . over half of those not registered in addition , many doctors either do not know about the program , or are not informing their patients about the program since the fund 's policy is to finance health care for insured citizens , there is currently no source of funding for the uninsured in the dzs . many doctors in the hospitals are unaware of the program , but often they are the only doctors with access to screening equipment and they are the ones to provide follow - up treatment . evaluation of the program is very difficult , because it must be based on manual chart reviews due to a lack of automated data . as a result of this study , we have provided a series of recommendations to the government of the rs about how to improve the prevention program and its financing . first , we recommend that a broad coalition of stakeholders work together to improve the implementation of the preventive care program , including doctors at the primary and secondary level ; the dzs ; the ministry of health and social welfare ; the health insurance fund ; the ministry of finance ; the ministry of transport and communications ; the public health institute ; local authorities , ngos ; and private companies . each of these stakeholders has a great interest in improving the health of the citizens of the rs and preventing death and disability from chronic disease . however , up to the time of the study there was a lack of information about the prevention program among many of the key participants . we also recommend increasing targeted promotion of the program , both among citizens and among the medical personnel who must implement the program . if the program is to continue , more funding must be identified , especially for care for uninsured patients at the dzs . some ideas for additional funds include a possible reallocation of health insurance fund towards the prevention program . however , because of financial shortages system - wide and the fact that the health insurance fund is designed to cover only insured citizens , new resources of revenue are needed . katherine m. wilson , phd , mph , ches , division of cancer prevention and control , national center for chronic disease prevention and health promotion , centers for disease control and prevention , atlanta , usa robert lange , phd , head of the department laboratory medicine , immanuel diakonie group and secretary of the german society of disease management ( dgdm ) , bernau bei berlin , germany

acute respiratory distress syndrome ( ards ) is a syndrome characterized by progressive respiratory distress and refractory hypoxemia caused by severe intrapulmonary and extrapulmonary disease . it is one of the most common causes of icu inpatient admission . despite continuous advancements in intensive care technology , the case fatality rate in critical ards is still as high as 80100% . applying the low tidal volume pulmonary protection ventilation strategy does help improve patient prognosis , however , it is hard to achieve effective gas exchange in those patients with an oxygenation index < 100 via this strategy ; mechanical ventilation with high - pressure often leads to ventilator - induced lung injury ( vili ) . extracorporeal membrane oxygenation ( ecmo ) is a mobile extracorporeal life support device that provides temporary , complete cardiopulmonary function support . the respiratory support mode ( veno - venous ecmo ) gains valuable time to improve pulmonary functions and transition into isolated mechanical ventilation by enhancing oxygenation without increasing average alveolar pressure . positive results reported in a recent multi - central , randomized controlled trial and subsequent success during an influenza a / h1n1 panepidemic [ 79 ] has consistently demonstrated that an ecmo - based management protocol significantly improves survival compared to conventional mechanical ventilation . however , there has been relatively little research on treating critical ards with ecmo in developing countries , and likewise very few studies on predicting the impact of biochemical parameter evolution on survival to hospital discharge . in this study , we evaluated the effectiveness and safety of rescuing critical ards patients with ecmo when conventional treatment was ineffective , and identify the factors related to patient prognosis in a chinese ecmo referral center . this study was a retrospective review of a single institution s experience with severe ards adults ( > 18 years old ) who were treated with ecmo at our center from january 2009 to december 2015 . according to the extracorporeal life support organization ( elso ) , ecmo can be initiated when risk of death exceeds 80% , where pao2/fio2 is less than 100 mm hg on fio2 more than 90% , and murray score is 34 , or in the case of hypercapnia as indicated by paco2 over 80 mm hg or inability to achieve safe inflation pressures ( plateau pressure < 30 cm h2o ) . patients were excluded if they 1 ) had been on mechanical ventilation for more than 10 days , 2 ) showed contraindication to anticoagulation , 3 ) had irreversible multiple organ failure , 4 ) had severe chronic pulmonary parenchymal disease , or 5 ) were in the terminal stage of cancer . we considered the veno - venous mode to be the best choice for hemodynamically stable patients . systemic heparinization was performed to keep the activated clotting time ( act ) between 160 and 200 seconds , and blood flow was maintained between 3 l / minute to 4 l / minute . strategy following ecmo , which included pressure control ventilation , low tidal volume ( 4 ml / kg ) , low pressure ( peep 1015 cm h2o , peak airway pressure < 30 cm h2o ) , low frequency ( 812 times / minute ) and low oxygen concentration ( fio2 < 50% ) , and remained unchanged throughout the entire process . ecmo flow was gradually decreased until being removed when the flow reached 1 l / minute . the blood flow and anticoagulation intensity were kept unchanged , and respirator parameters were upregulated simultaneously as hemodynamic indexes and gas exchange conditions were closely monitored . the ecmo could be removed once arterial conditions , po2 > 60 mm hg and pco2 3045 mm hg , were constant for more than two hours . inter - hospital referral and transportation was used to indicate critical patients who were referred , accepted , and transferred to our center . the patients meeting these indications and able to tolerate the conventional transfer method were considered transferrable to our institute with the assistance of mechanical ventilation . patients with oxygenation or hemodynamic instability who were unable to tolerate conventional transfer were brought to the institute with ecmo support . the equipment for transport included a medtronic bio - console 550/560 centrifugal pump , pipe package and cannula , surgical instruments , oxygen cylinders , respirator ( savina respirator , drager co. germany ) , act detector , blood - gas analyzer , monitor , defibrillator , and 5% albumin . statistical analyses were performed using spss 16.0 ( chicago , illinois , usa ) and prism 6 ( graphpad prism 6.00 , san diego , california , usa ) . the comparison of the quantitative data between the two groups were determined via t - test or t test depending on its distribution . the differences in constituent ratio among various groups were compared via fisher s exact test , and univariate correlation analysis was conducted according to the spearman method . multiple longitudinal comparisons of parameters between survivors and nonsurvivors were made by two - way repeated - measures analysis of variance to test the influence of time on the variables . when a difference was detected , lsd method was used to adjust for multiple comparisons . this study was a retrospective review of a single institution s experience with severe ards adults ( > 18 years old ) who were treated with ecmo at our center from january 2009 to december 2015 . according to the extracorporeal life support organization ( elso ) , ecmo can be initiated when risk of death exceeds 80% , where pao2/fio2 is less than 100 mm hg on fio2 more than 90% , and murray score is 34 , or in the case of hypercapnia as indicated by paco2 over 80 mm hg or inability to achieve safe inflation pressures ( plateau pressure < 30 cm h2o ) . patients were excluded if they 1 ) had been on mechanical ventilation for more than 10 days , 2 ) showed contraindication to anticoagulation , 3 ) had irreversible multiple organ failure , 4 ) had severe chronic pulmonary parenchymal disease , or 5 ) were in the terminal stage of cancer . we considered the veno - venous mode to be the best choice for hemodynamically stable patients . systemic heparinization was performed to keep the activated clotting time ( act ) between 160 and 200 seconds , and blood flow was maintained between 3 l / minute to 4 l / minute . strategy following ecmo , which included pressure control ventilation , low tidal volume ( 4 ml / kg ) , low pressure ( peep 1015 cm h2o , peak airway pressure < 30 cm h2o ) , low frequency ( 812 times / minute ) and low oxygen concentration ( fio2 < 50% ) , and remained unchanged throughout the entire process . ecmo flow was gradually decreased until being removed when the flow reached 1 l / minute . the blood flow and anticoagulation intensity were kept unchanged , and respirator parameters were upregulated simultaneously as hemodynamic indexes and gas exchange conditions were closely monitored . the ecmo could be removed once arterial conditions , po2 > 60 mm hg and pco2 3045 mm hg , were constant for more than two hours . inter - hospital referral and transportation was used to indicate critical patients who were referred , accepted , and transferred to our center . the patients meeting these indications and able to tolerate the conventional transfer method were considered transferrable to our institute with the assistance of mechanical ventilation . patients with oxygenation or hemodynamic instability who were unable to tolerate conventional transfer were brought to the institute with ecmo support . the equipment for transport included a medtronic bio - console 550/560 centrifugal pump , pipe package and cannula , surgical instruments , oxygen cylinders , respirator ( savina respirator , drager co. germany ) , act detector , blood - gas analyzer , monitor , defibrillator , and 5% albumin . statistical analyses were performed using spss 16.0 ( chicago , illinois , usa ) and prism 6 ( graphpad prism 6.00 , san diego , california , usa ) . the comparison of the quantitative data between the two groups were determined via t - test or t test depending on its distribution . the differences in constituent ratio among various groups were compared via fisher s exact test , and univariate correlation analysis was conducted according to the spearman method . multiple longitudinal comparisons of parameters between survivors and nonsurvivors were made by two - way repeated - measures analysis of variance to test the influence of time on the variables . when a difference was detected , lsd method was used to adjust for multiple comparisons . twenty - three consecutive patients ( 82.6% were male ) were ultimately enrolled in this study . the number of cases and the survival rate of discharge annually since 2009 was shown in figure 1 . sixteen ( 69.6% ) patients were transferred by ambulance from peripheral hospitals , and notably , seven ( 30.4% ) patients were transported back to our institution on ecmo , including three cases where the patients were transferred from hebei province to tianjin . there were five patients ( 21.7% ) with influenza a ( h1n1 ) including two patients who had been transferred to our hospital immediately after cesarean procedures in another hospital . bilateral pneumothorax or mediastinal emphysema occurred in these two patients and four other patients who were given cardiopulmonary resuscitation before ecmo establishment ( table 1 ) . the median time for mechanical ventilation was 24.0 ( 4.056.8 ) hours before ecmo establishment , and the average time of ecmo assistance was 114.465.4 hours ( range 26305 hours ) . veno - venous mode was applied in eighteen cases ( 78.3% ) , veno - arterial mode in four ( 17.4% ) , and conversion from v v to v - a mode in one case ( 4.3% ) . the time for mechanical ventilation was 164.0 hours ( 105.6259.3 hours ) after ecmo establishment . complications included : membrane oxygenator replacement ( 4 cases ) , major hemorrhage , including surgical sites or intracranial hemorrhage ( 9 cases ) , arterial / venous system embolism ( 4 cases ) , and catheter- related septicopyemia ( 2 cases ) . the length of icu stay and the total length of hospital stay were ( 15.319.0 ) days and ( 20.619.3 ) days , respectively . the difference in apache ii scores , proportion of acute kidney injury ( defined by the standard from the acute kidney injury network ) , membrane oxygenator replacement between the survivor group and nonsurvivor group were statistically significant ( p<0.05 ) ( table 2 ) . the univariate correlation analysis showed that a high apache ii score ( r=0.439 , p=0.041 ) , occurrence of acute kidney injury ( r=0.574 , p=0.005 ) , membrane oxygenator replacement ( r=0.516 , p=0.014 ) were significantly negatively correlated with prognosis . the biochemical index evolution analysis showed that there were significant changes in blood platelet count , albumin , total bilirubin , blood urea nitrogen , oxygenation index ( po2/fio2 ) , and fibrinogen evolution in the overall group within the first 72 hours after ecmo establishment . the blood platelet counts and fibrinogen levels significantly decreased over time after assistance while the albumin , oxygenation index , total bilirubin , and blood urea nitrogen all significantly increased ( table 3 ) . further intergroup comparative analyses demonstrated that fibrinogen level at 24 hours and platelet count at 72 hours in the nonsurvivor group were much lower than those of the survivor group ; however , the level of blood urea nitrogen at 24 hours and 48 hours were significantly higher in the nonsurvivor group than the survivor group . sixteen patients referred for ecmo in outside hospitals were successfully transported to our institution by ambulance . nine were transferred on a standard ventilator and the other seven were transferred to our institution on ecmo , which was initiated by our team at the original hospital . we discovered that the patients were older ( though not statistically significant ) and with poorer murray scores and oxygenation indexes before transfer in the ecmo assistance group compared with those in the conventional transfer group , however , patient oxygenation indexes improved significantly and ventilator conditions were substantially downregulated after transfer in the case of an equivalent average transfer distance . hemodynamic indexes , such as heart rate and systolic pressure , also improved , although this observation may not constitute a statistical difference due to the small number of cases . twenty - three consecutive patients ( 82.6% were male ) were ultimately enrolled in this study . the number of cases and the survival rate of discharge annually since 2009 was shown in figure 1 . sixteen ( 69.6% ) patients were transferred by ambulance from peripheral hospitals , and notably , seven ( 30.4% ) patients were transported back to our institution on ecmo , including three cases where the patients were transferred from hebei province to tianjin . there were five patients ( 21.7% ) with influenza a ( h1n1 ) including two patients who had been transferred to our hospital immediately after cesarean procedures in another hospital . bilateral pneumothorax or mediastinal emphysema occurred in these two patients and four other patients who were given cardiopulmonary resuscitation before ecmo establishment ( table 1 ) . the median time for mechanical ventilation was 24.0 ( 4.056.8 ) hours before ecmo establishment , and the average time of ecmo assistance was 114.465.4 hours ( range 26305 hours ) . veno - venous mode was applied in eighteen cases ( 78.3% ) , veno - arterial mode in four ( 17.4% ) , and conversion from v v to v - a mode in one case ( 4.3% ) . the time for mechanical ventilation was 164.0 hours ( 105.6259.3 hours ) after ecmo establishment . complications included : membrane oxygenator replacement ( 4 cases ) , major hemorrhage , including surgical sites or intracranial hemorrhage ( 9 cases ) , arterial / venous system embolism ( 4 cases ) , and catheter- related septicopyemia ( 2 cases ) . the length of icu stay and the total length of hospital stay were ( 15.319.0 ) days and ( 20.619.3 ) days , respectively . the difference in apache ii scores , proportion of acute kidney injury ( defined by the standard from the acute kidney injury network ) , membrane oxygenator replacement between the survivor group and nonsurvivor group were statistically significant ( p<0.05 ) ( table 2 ) . the univariate correlation analysis showed that a high apache ii score ( r=0.439 , p=0.041 ) , occurrence of acute kidney injury ( r=0.574 , p=0.005 ) , membrane oxygenator replacement ( r=0.516 , p=0.014 ) were significantly negatively correlated with prognosis . the biochemical index evolution analysis showed that there were significant changes in blood platelet count , albumin , total bilirubin , blood urea nitrogen , oxygenation index ( po2/fio2 ) , and fibrinogen evolution in the overall group within the first 72 hours after ecmo establishment . the blood platelet counts and fibrinogen levels significantly decreased over time after assistance while the albumin , oxygenation index , total bilirubin , and blood urea nitrogen all significantly increased ( table 3 ) . further intergroup comparative analyses demonstrated that fibrinogen level at 24 hours and platelet count at 72 hours in the nonsurvivor group were much lower than those of the survivor group ; however , the level of blood urea nitrogen at 24 hours and 48 hours were significantly higher in the nonsurvivor group than the survivor group . sixteen patients referred for ecmo in outside hospitals were successfully transported to our institution by ambulance . nine were transferred on a standard ventilator and the other seven were transferred to our institution on ecmo , which was initiated by our team at the original hospital . we discovered that the patients were older ( though not statistically significant ) and with poorer murray scores and oxygenation indexes before transfer in the ecmo assistance group compared with those in the conventional transfer group , however , patient oxygenation indexes improved significantly and ventilator conditions were substantially downregulated after transfer in the case of an equivalent average transfer distance . hemodynamic indexes , such as heart rate and systolic pressure , also improved , although this observation may not constitute a statistical difference due to the small number of cases . following major improvements in ecmo technology and supportive evidence obtained in a series of recent clinical trials [ 69 ] , ecmo is , as of now , utilized frequently to treat severe ards patients . early application of ecmo may help to avoid substantial lung and sequential organ dysfunction via inhibited systemic release of inflammatory mediators induced by high concentrations of oxygen and high - volume ventilation [ 1214 ] . through a 15-year retrospective analysis , hemmila et al . found that the actual survival rate of severe ards patients given ecmo reached 52% , though it was expected to be less than 20% . the data we gathered in this study showed that a significant improvement in hemodynamic function and normalization of blood gases with simultaneous reduction of inotropic requirements and ventilator parameter settings occur within the first 24 hours of ecmo support . as an ecmo referral center , we have implemented ecmo support for severe ards patients since 2009 . our results demonstrate quite favorable results an over 50% survival rate after a mean support interval of approximately five days . notably , 16 cases of inter - hospital transportation were completed sucessfully in this series , including seven transports with mobile ecmo and the first domestic inter - provincial transport over a distance of 125 km by ground . although general guidelines for ecmo initiation do exist , it remains difficult to make a decision on cannulation in real - world clinical practice . early objective individual risk assessment is needed to aid the proper selection of candidates , compare success rates across different centers , and augment the predictive performance of prognosis . in the specific population investigated here , factors associated with poor outcomes according to the literature include old age [ 6,1522 ] , lengthy duration of mechanical ventilation prior to ecmo , high levels of organ failure [ 15,16,19,2022,24 ] , and immunosuppression . in this study , we found that patients less than 45 years of age had a markedly better prognosis . in fact , in younger patients , favorable outcomes were achieved even independent of other organ failure . based on this and information from a previous study with similar findings , we presumed that ecmo should not be contraindicated on the basis of organ dysfunction in young patients . lengthy duration of mechanical ventilation prior to ecmo , a well - known predictor of poor prognosis , inextricably indicates severe vili . two valuable predictive models both demonstrated mechanical ventilation more than seven days before ecmo support was the cutoff point for decline in prognosis , but that ecmo exerted the most beneficial effect when initiated early to within less than 48 hours . we also found that the nonsurvivor group had a longer interval of pre - ecmo mechanical ventilation , although the difference was not statistically significant . during the h1n1 pandemic ( 20092010 ) we received some young adults who had been given prolonged ventilation prior to arriving at our center , as they were young , previously fit ( some even having given birth just prior to treatment ) . the ecmo outcome was indeed disappointing for those who had been ventilated for more than seven days . the elso database also showed that the duration of mechanical ventilation was no longer associated with mortality in the most recently treated patients , which may be attributed to the fact that the number of patients treated after seven days of mechanical ventilation decreased with time . excessive organ failure ( marked by high apache or sofa score ) , as determined prior to administering ecmo or at day 1 after ecmo initiation , was consistently shown to be a strong predictor of poor prognosis . we discovered that the incidence of acute kidney injury during assistance was one of the mortality risk factors , however , interpreting the prognostic value of changes in hepatic functions proved highly complex . as a component of medical therapy , an abundance of albumin was transfused into the two groups which had different prognoses to maintain adequate colloid osmotic pressure ; accordingly , the albumin levels of both groups were shown to increase significantly within 72 hours after assistance began and there was no significant difference between the groups . the levels of blood urea nitrogen 24 hours and 48 hours after ecmo establishment in the nonsurvivor group were significantly higher than those of the survivor group , which indicated differing intensity of catabolism between the groups despite their shared trend of significant increase over time . we inferred that the balance between anabolism and catabolism at early stages may be an important factor in prognosis . we further suggest that the causes of the abnormal rise in total bilirubin level in the survivor group may be : 1 ) the total bilirubin levels at different points in time in the survivor group , including two patients with the etiology of sepsis , were significantly higher than those in the pneumonia group ; and 2 ) the three patients with multiple organ failure in the nonsurvivor group died within 72 hours after ecmo establishment ( which was excluded in the statistics ) . we also found that the occurrence of coagulopathy within 72 hours may be a valuable marker for predicting intrahospital prognosis . although both groups presented significant decreasing trends in platelet count and fibrinogen within the first 72 hours of ecmo support , said decrease occurred at a quicker rate in the nonsurvivor group compared to the survival group ; to be specific , the intergroup difference at the same time point reached statistical significance at 72 hours for platelet count and at 24 hours for fibrinogen . the fibrinogen level of the nonsurvivor group at any time point was much lower than that of the survivor group . our data confirmed the previous finding that high fibrinogen concentration following ecmo initiation has a protective effect and positive predictive value of treatment success , which may be attributable to its indication of effective immune response and relative scarcity of bleeding disorders . in addition to the predictors mentioned , our data also demonstrated that oxygenator replacement was a strong poor prognosis predictor . patients whose oxygenator had been replaced often showed higher likelihood to be obliged to withdraw ecmo prematurely due to other complications . similarly , a recent retrospective study on pediatric pneumonia managed with ecmo showed that any need to change ecmo circuit was a strong predictor of death . we speculate , to this effect , that the occurrence of mechanical ecmo complications may be a valuable predictor of poor prognosis . another challenge posed in clinical practice is that it is unclear how to best and most safely administer ecmo support over long - distance transport . severely ill patients should ideally be transported to an ecmo center before they can no longer be transported by conventional means , but this is not always possible because sometimes a patient s course can derteriorate extremely quickly . for this reason , inter - hospital transportation techniques for administering ecmo en route to an ecmo center are a valuable , and urgent , consideration . though inter - hospital transport on ecmo was first reported in 1986 , it was not widely launched until the 2000s [ 17,20,2932 ] . recently , researchers from a regional referral center found that a proportion of 69% of severe ards patients required ecmo during transport , and further , that ecmo treatment resulted in a 60% survival rate . in a series of 124 patients treated at a danish center , 85% of patients who received ecmo via mobile unit before being transferred to the referral hospital had a survival rate of 71% . similarly , in a german cohort study , adults with severe cardiopulmonary failure benefited from life - saving ecmo administration during long - distance inter - hospital ground transport . to the best of our knowledge , there is scant literature on ecmo support as it applies to severe respiratory failure patients from china , and there has been practically no research on mobile ecmo unit efficacy over long - distance inter - hospital transport domestically . transporting severe ards adults to centers where they receive specialized tertiary care has become routine our institution , for example , now has a referral radius of 125 km . transport on extracorporeal support is obviously more complicated and resource - dependent than conventional transport , as it requires larger vehicles and larger transport teams , and is more equipment - intensive . although worse oxygenation indices and murray scores before transport existed in patients transferred on ecmo in our study , the oxygenation index was significantly improved over the conventional transport group , and the ventilator parameters were much lower post - transport , likely attributable to the fact that the use of ecmo allowed a rapid correction of blood gases and safer transport . furthermore , the rapid solution of the hypoxia allowed us to adopt a protective ventilation strategy during transport . although the potential for complications during transport invariably exists , we have been pleased with the overall results in our initial experience . the group who were retrieved via a mobile ecmo team in our series had a comparable survival rate to conventional transport ( 57.1% vs. 44.4% , p=1.000 ) or the non - transport group treated with ecmo in our center ( 57.1% vs. 66.7% , p=1.000 ) , which is in agreement with other observational studies similar to this one . the utilization of ecmo treatment in severe ards in china is still in its initial stages . retrospective analysis from six domestic ecmo centers from 2002 to 2010 showed that ecmo was used in only 65 cases ( about 19% of the total ) for respiratory support , and the overall survival rate was not satisfactory ( approximately 26% ) . ventilator - associated lung injury secondary to long - term , high - condition mechanical ventilation is the achilles heel of the recovery of lung function . regardless , for properly selected severely ill patients , we continue to encourage our colleagues to recommend ecmo support in the future , and to consider transporting patients under ecmo support . . apache ii score on admission , occurrence of acute kidney injury , membrane oxygenator replacement during ecmo support , and that the evolution of levels of urea nitrogen , platelet , and fibrinogen within the three days of initial ecmo assistance may help to determine the prognosis . by establishing a well - trained mobile ecmo team , a long - distance ,

backgroundno definitive conclusions have been drawn from the available data about the utilization of extracorporeal membrane oxygenation ( ecmo ) to treat severe acute respiratory distress syndrome ( ards ) . the aim of this study was to review our center s experience with ecmo and determine predictors of outcome from our chinese center.material/methodswe retrospectively analyzed a total of 23 consecutive candidates who fulfilled the study entry criteria between january 2009 and december 2015 . detailed clinical data , ecmo flow , and respiratory parameters before and after the introduction of ecmo were compared among in - hospital survivors and nonsurvivors ; factors associated with mortality were investigated.resultshemodynamics and oxygenation parameters were significantly improved after ecmo initiation . thirteen patients survived to hospital discharge . univariate correlation analysis demonstrated that apache ii score ( r=0.463 , p=0.03 ) , acute kidney injury ( r=0.574 , p=0.005 ) , membrane oxygenator replacement ( r=0.516 , p=0.014 ) and total length of hospital stay ( r=0.526 , p=0.012 ) were significantly correlated with survival to hospital discharge , and that the evolution of the levels of urea nitrogen , platelet , and fibrinogen may help to determine patient prognosis . sixteen patients referred for ecmo from an outside hospital were successfully transported to our institution by ambulance , including seven transported under ecmo support . the survival rate of the ecmo - transport group was comparable to the conventional transport or the non - transport group ( both p=1.000).conclusionsecmo is an effective alternative option for severe ards . apache ii score on admission , onset of acute kidney injury , and membrane oxygenator replacement , and the evolution of levels of urea nitrogen , platelet , and fibrinogen during hospitalization may help to determine the in - hospital patient prognosis . by establishing a well - trained mobile ecmo team , a long - distance , inter - hospital transport can be administered safely .

Background Material and Methods Patient selection Inclusion and exclusion criteria Establishment and management of ECMO ECMO removal Inter-hospital referral and transportation Statistical analysis Results Demographic characteristics of study population Clinical data during ECMO bypass Clinical data among patients with different prognoses Patients transferred by different means Discussion Conclusions

acute respiratory distress syndrome ( ards ) is a syndrome characterized by progressive respiratory distress and refractory hypoxemia caused by severe intrapulmonary and extrapulmonary disease . applying the low tidal volume pulmonary protection ventilation strategy does help improve patient prognosis , however , it is hard to achieve effective gas exchange in those patients with an oxygenation index < 100 via this strategy ; mechanical ventilation with high - pressure often leads to ventilator - induced lung injury ( vili ) . extracorporeal membrane oxygenation ( ecmo ) is a mobile extracorporeal life support device that provides temporary , complete cardiopulmonary function support . positive results reported in a recent multi - central , randomized controlled trial and subsequent success during an influenza a / h1n1 panepidemic [ 79 ] has consistently demonstrated that an ecmo - based management protocol significantly improves survival compared to conventional mechanical ventilation . however , there has been relatively little research on treating critical ards with ecmo in developing countries , and likewise very few studies on predicting the impact of biochemical parameter evolution on survival to hospital discharge . in this study , we evaluated the effectiveness and safety of rescuing critical ards patients with ecmo when conventional treatment was ineffective , and identify the factors related to patient prognosis in a chinese ecmo referral center . this study was a retrospective review of a single institution s experience with severe ards adults ( > 18 years old ) who were treated with ecmo at our center from january 2009 to december 2015 . according to the extracorporeal life support organization ( elso ) , ecmo can be initiated when risk of death exceeds 80% , where pao2/fio2 is less than 100 mm hg on fio2 more than 90% , and murray score is 34 , or in the case of hypercapnia as indicated by paco2 over 80 mm hg or inability to achieve safe inflation pressures ( plateau pressure < 30 cm h2o ) . strategy following ecmo , which included pressure control ventilation , low tidal volume ( 4 ml / kg ) , low pressure ( peep 1015 cm h2o , peak airway pressure < 30 cm h2o ) , low frequency ( 812 times / minute ) and low oxygen concentration ( fio2 < 50% ) , and remained unchanged throughout the entire process . the blood flow and anticoagulation intensity were kept unchanged , and respirator parameters were upregulated simultaneously as hemodynamic indexes and gas exchange conditions were closely monitored . inter - hospital referral and transportation was used to indicate critical patients who were referred , accepted , and transferred to our center . the patients meeting these indications and able to tolerate the conventional transfer method were considered transferrable to our institute with the assistance of mechanical ventilation . patients with oxygenation or hemodynamic instability who were unable to tolerate conventional transfer were brought to the institute with ecmo support . the equipment for transport included a medtronic bio - console 550/560 centrifugal pump , pipe package and cannula , surgical instruments , oxygen cylinders , respirator ( savina respirator , drager co. germany ) , act detector , blood - gas analyzer , monitor , defibrillator , and 5% albumin . the differences in constituent ratio among various groups were compared via fisher s exact test , and univariate correlation analysis was conducted according to the spearman method . multiple longitudinal comparisons of parameters between survivors and nonsurvivors were made by two - way repeated - measures analysis of variance to test the influence of time on the variables . this study was a retrospective review of a single institution s experience with severe ards adults ( > 18 years old ) who were treated with ecmo at our center from january 2009 to december 2015 . according to the extracorporeal life support organization ( elso ) , ecmo can be initiated when risk of death exceeds 80% , where pao2/fio2 is less than 100 mm hg on fio2 more than 90% , and murray score is 34 , or in the case of hypercapnia as indicated by paco2 over 80 mm hg or inability to achieve safe inflation pressures ( plateau pressure < 30 cm h2o ) . strategy following ecmo , which included pressure control ventilation , low tidal volume ( 4 ml / kg ) , low pressure ( peep 1015 cm h2o , peak airway pressure < 30 cm h2o ) , low frequency ( 812 times / minute ) and low oxygen concentration ( fio2 < 50% ) , and remained unchanged throughout the entire process . the blood flow and anticoagulation intensity were kept unchanged , and respirator parameters were upregulated simultaneously as hemodynamic indexes and gas exchange conditions were closely monitored . inter - hospital referral and transportation was used to indicate critical patients who were referred , accepted , and transferred to our center . the patients meeting these indications and able to tolerate the conventional transfer method were considered transferrable to our institute with the assistance of mechanical ventilation . patients with oxygenation or hemodynamic instability who were unable to tolerate conventional transfer were brought to the institute with ecmo support . the equipment for transport included a medtronic bio - console 550/560 centrifugal pump , pipe package and cannula , surgical instruments , oxygen cylinders , respirator ( savina respirator , drager co. germany ) , act detector , blood - gas analyzer , monitor , defibrillator , and 5% albumin . the differences in constituent ratio among various groups were compared via fisher s exact test , and univariate correlation analysis was conducted according to the spearman method . multiple longitudinal comparisons of parameters between survivors and nonsurvivors were made by two - way repeated - measures analysis of variance to test the influence of time on the variables . twenty - three consecutive patients ( 82.6% were male ) were ultimately enrolled in this study . the number of cases and the survival rate of discharge annually since 2009 was shown in figure 1 . sixteen ( 69.6% ) patients were transferred by ambulance from peripheral hospitals , and notably , seven ( 30.4% ) patients were transported back to our institution on ecmo , including three cases where the patients were transferred from hebei province to tianjin . the median time for mechanical ventilation was 24.0 ( 4.056.8 ) hours before ecmo establishment , and the average time of ecmo assistance was 114.465.4 hours ( range 26305 hours ) . veno - venous mode was applied in eighteen cases ( 78.3% ) , veno - arterial mode in four ( 17.4% ) , and conversion from v v to v - a mode in one case ( 4.3% ) . complications included : membrane oxygenator replacement ( 4 cases ) , major hemorrhage , including surgical sites or intracranial hemorrhage ( 9 cases ) , arterial / venous system embolism ( 4 cases ) , and catheter- related septicopyemia ( 2 cases ) . the length of icu stay and the total length of hospital stay were ( 15.319.0 ) days and ( 20.619.3 ) days , respectively . the difference in apache ii scores , proportion of acute kidney injury ( defined by the standard from the acute kidney injury network ) , membrane oxygenator replacement between the survivor group and nonsurvivor group were statistically significant ( p<0.05 ) ( table 2 ) . the univariate correlation analysis showed that a high apache ii score ( r=0.439 , p=0.041 ) , occurrence of acute kidney injury ( r=0.574 , p=0.005 ) , membrane oxygenator replacement ( r=0.516 , p=0.014 ) were significantly negatively correlated with prognosis . the biochemical index evolution analysis showed that there were significant changes in blood platelet count , albumin , total bilirubin , blood urea nitrogen , oxygenation index ( po2/fio2 ) , and fibrinogen evolution in the overall group within the first 72 hours after ecmo establishment . the blood platelet counts and fibrinogen levels significantly decreased over time after assistance while the albumin , oxygenation index , total bilirubin , and blood urea nitrogen all significantly increased ( table 3 ) . further intergroup comparative analyses demonstrated that fibrinogen level at 24 hours and platelet count at 72 hours in the nonsurvivor group were much lower than those of the survivor group ; however , the level of blood urea nitrogen at 24 hours and 48 hours were significantly higher in the nonsurvivor group than the survivor group . sixteen patients referred for ecmo in outside hospitals were successfully transported to our institution by ambulance . nine were transferred on a standard ventilator and the other seven were transferred to our institution on ecmo , which was initiated by our team at the original hospital . we discovered that the patients were older ( though not statistically significant ) and with poorer murray scores and oxygenation indexes before transfer in the ecmo assistance group compared with those in the conventional transfer group , however , patient oxygenation indexes improved significantly and ventilator conditions were substantially downregulated after transfer in the case of an equivalent average transfer distance . twenty - three consecutive patients ( 82.6% were male ) were ultimately enrolled in this study . the number of cases and the survival rate of discharge annually since 2009 was shown in figure 1 . sixteen ( 69.6% ) patients were transferred by ambulance from peripheral hospitals , and notably , seven ( 30.4% ) patients were transported back to our institution on ecmo , including three cases where the patients were transferred from hebei province to tianjin . the median time for mechanical ventilation was 24.0 ( 4.056.8 ) hours before ecmo establishment , and the average time of ecmo assistance was 114.465.4 hours ( range 26305 hours ) . veno - venous mode was applied in eighteen cases ( 78.3% ) , veno - arterial mode in four ( 17.4% ) , and conversion from v v to v - a mode in one case ( 4.3% ) . complications included : membrane oxygenator replacement ( 4 cases ) , major hemorrhage , including surgical sites or intracranial hemorrhage ( 9 cases ) , arterial / venous system embolism ( 4 cases ) , and catheter- related septicopyemia ( 2 cases ) . the length of icu stay and the total length of hospital stay were ( 15.319.0 ) days and ( 20.619.3 ) days , respectively . the difference in apache ii scores , proportion of acute kidney injury ( defined by the standard from the acute kidney injury network ) , membrane oxygenator replacement between the survivor group and nonsurvivor group were statistically significant ( p<0.05 ) ( table 2 ) . the univariate correlation analysis showed that a high apache ii score ( r=0.439 , p=0.041 ) , occurrence of acute kidney injury ( r=0.574 , p=0.005 ) , membrane oxygenator replacement ( r=0.516 , p=0.014 ) were significantly negatively correlated with prognosis . the biochemical index evolution analysis showed that there were significant changes in blood platelet count , albumin , total bilirubin , blood urea nitrogen , oxygenation index ( po2/fio2 ) , and fibrinogen evolution in the overall group within the first 72 hours after ecmo establishment . the blood platelet counts and fibrinogen levels significantly decreased over time after assistance while the albumin , oxygenation index , total bilirubin , and blood urea nitrogen all significantly increased ( table 3 ) . further intergroup comparative analyses demonstrated that fibrinogen level at 24 hours and platelet count at 72 hours in the nonsurvivor group were much lower than those of the survivor group ; however , the level of blood urea nitrogen at 24 hours and 48 hours were significantly higher in the nonsurvivor group than the survivor group . sixteen patients referred for ecmo in outside hospitals were successfully transported to our institution by ambulance . nine were transferred on a standard ventilator and the other seven were transferred to our institution on ecmo , which was initiated by our team at the original hospital . we discovered that the patients were older ( though not statistically significant ) and with poorer murray scores and oxygenation indexes before transfer in the ecmo assistance group compared with those in the conventional transfer group , however , patient oxygenation indexes improved significantly and ventilator conditions were substantially downregulated after transfer in the case of an equivalent average transfer distance . following major improvements in ecmo technology and supportive evidence obtained in a series of recent clinical trials [ 69 ] , ecmo is , as of now , utilized frequently to treat severe ards patients . early application of ecmo may help to avoid substantial lung and sequential organ dysfunction via inhibited systemic release of inflammatory mediators induced by high concentrations of oxygen and high - volume ventilation [ 1214 ] . found that the actual survival rate of severe ards patients given ecmo reached 52% , though it was expected to be less than 20% . the data we gathered in this study showed that a significant improvement in hemodynamic function and normalization of blood gases with simultaneous reduction of inotropic requirements and ventilator parameter settings occur within the first 24 hours of ecmo support . as an ecmo referral center , we have implemented ecmo support for severe ards patients since 2009 . our results demonstrate quite favorable results an over 50% survival rate after a mean support interval of approximately five days . notably , 16 cases of inter - hospital transportation were completed sucessfully in this series , including seven transports with mobile ecmo and the first domestic inter - provincial transport over a distance of 125 km by ground . although general guidelines for ecmo initiation do exist , it remains difficult to make a decision on cannulation in real - world clinical practice . in the specific population investigated here , factors associated with poor outcomes according to the literature include old age [ 6,1522 ] , lengthy duration of mechanical ventilation prior to ecmo , high levels of organ failure [ 15,16,19,2022,24 ] , and immunosuppression . lengthy duration of mechanical ventilation prior to ecmo , a well - known predictor of poor prognosis , inextricably indicates severe vili . the elso database also showed that the duration of mechanical ventilation was no longer associated with mortality in the most recently treated patients , which may be attributed to the fact that the number of patients treated after seven days of mechanical ventilation decreased with time . excessive organ failure ( marked by high apache or sofa score ) , as determined prior to administering ecmo or at day 1 after ecmo initiation , was consistently shown to be a strong predictor of poor prognosis . we discovered that the incidence of acute kidney injury during assistance was one of the mortality risk factors , however , interpreting the prognostic value of changes in hepatic functions proved highly complex . the levels of blood urea nitrogen 24 hours and 48 hours after ecmo establishment in the nonsurvivor group were significantly higher than those of the survivor group , which indicated differing intensity of catabolism between the groups despite their shared trend of significant increase over time . we further suggest that the causes of the abnormal rise in total bilirubin level in the survivor group may be : 1 ) the total bilirubin levels at different points in time in the survivor group , including two patients with the etiology of sepsis , were significantly higher than those in the pneumonia group ; and 2 ) the three patients with multiple organ failure in the nonsurvivor group died within 72 hours after ecmo establishment ( which was excluded in the statistics ) . although both groups presented significant decreasing trends in platelet count and fibrinogen within the first 72 hours of ecmo support , said decrease occurred at a quicker rate in the nonsurvivor group compared to the survival group ; to be specific , the intergroup difference at the same time point reached statistical significance at 72 hours for platelet count and at 24 hours for fibrinogen . in addition to the predictors mentioned , our data also demonstrated that oxygenator replacement was a strong poor prognosis predictor . similarly , a recent retrospective study on pediatric pneumonia managed with ecmo showed that any need to change ecmo circuit was a strong predictor of death . another challenge posed in clinical practice is that it is unclear how to best and most safely administer ecmo support over long - distance transport . for this reason , inter - hospital transportation techniques for administering ecmo en route to an ecmo center are a valuable , and urgent , consideration . though inter - hospital transport on ecmo was first reported in 1986 , it was not widely launched until the 2000s [ 17,20,2932 ] . recently , researchers from a regional referral center found that a proportion of 69% of severe ards patients required ecmo during transport , and further , that ecmo treatment resulted in a 60% survival rate . in a series of 124 patients treated at a danish center , 85% of patients who received ecmo via mobile unit before being transferred to the referral hospital had a survival rate of 71% . similarly , in a german cohort study , adults with severe cardiopulmonary failure benefited from life - saving ecmo administration during long - distance inter - hospital ground transport . to the best of our knowledge , there is scant literature on ecmo support as it applies to severe respiratory failure patients from china , and there has been practically no research on mobile ecmo unit efficacy over long - distance inter - hospital transport domestically . transporting severe ards adults to centers where they receive specialized tertiary care has become routine our institution , for example , now has a referral radius of 125 km . transport on extracorporeal support is obviously more complicated and resource - dependent than conventional transport , as it requires larger vehicles and larger transport teams , and is more equipment - intensive . although worse oxygenation indices and murray scores before transport existed in patients transferred on ecmo in our study , the oxygenation index was significantly improved over the conventional transport group , and the ventilator parameters were much lower post - transport , likely attributable to the fact that the use of ecmo allowed a rapid correction of blood gases and safer transport . the group who were retrieved via a mobile ecmo team in our series had a comparable survival rate to conventional transport ( 57.1% vs. 44.4% , p=1.000 ) or the non - transport group treated with ecmo in our center ( 57.1% vs. 66.7% , p=1.000 ) , which is in agreement with other observational studies similar to this one . the utilization of ecmo treatment in severe ards in china is still in its initial stages . retrospective analysis from six domestic ecmo centers from 2002 to 2010 showed that ecmo was used in only 65 cases ( about 19% of the total ) for respiratory support , and the overall survival rate was not satisfactory ( approximately 26% ) . ventilator - associated lung injury secondary to long - term , high - condition mechanical ventilation is the achilles heel of the recovery of lung function . regardless , for properly selected severely ill patients , we continue to encourage our colleagues to recommend ecmo support in the future , and to consider transporting patients under ecmo support . apache ii score on admission , occurrence of acute kidney injury , membrane oxygenator replacement during ecmo support , and that the evolution of levels of urea nitrogen , platelet , and fibrinogen within the three days of initial ecmo assistance may help to determine the prognosis . by establishing a well - trained mobile ecmo team , a long - distance ,

cardiac resynchronization therapy ( crt ) is an important treatment for drug - refractory heart failure and left ventricular ( lv ) dyssynchrony . pacing leads have often been placed at the coronary vein and right ventricle to reduce lv dyssynchrony and to improve hemodynamics in patients with heart failure . in some patients , clinicians encounter difficulties when placing leads ; in approximately one - quarter of patients , there is an insufficient response to biventricular ( biv ) pacing , primarily because of difficulty in accurately placing the lv lead due to patients anatomical features , pacing thresholds , twitching , or pacing lead anchoring . some researchers have described other pacing sites that yield better hemodynamics and less dyssynchrony than biv pacing . for example , derval et al . attempted lateral lv wall pacing in patients with left bundle branch block pattern who were referred for crt device implantation . van gelder et al . reported that transseptal lead placement was useful in cases where there was difficulty in placing a coronary sinus ( cs ) lead . yoshida et al . reported that triventricular pacing , which uses two right ventricular leads and one lv lead , results in greater improvement in hemodynamics in patients with severe heart failure , when compared with bi - v pacing , sashida et al . reported improved lv function with his bundle pacing ( hbp ) in a patient with dilated cardiomyopathy due to atrial fibrillation without intraventricular conduction delay , however , whether these or other pacing sites are superior to conventional biv pacing remains unclear . some recent studies reported that biv pacing with lv endocardial stimulation sites yield better hemodynamics and lv synchrony , compared with conventional biv pacing . these procedures would have the benefit of lead placement in an extended area , regardless of coronary vein location , with better threshold and avoidance of twitching . on the other hand , it has been reported that the purkinje network can survive in patients with ischemic or idiopathic cardiomyopathy , and in patients with heart failure and left bundle branch block ( lbbb ) . in addition , idiopathic lv tachycardia that involves the purkinje network as the circuit is characterized as showing a narrow qrs . therefore , it is conceivable that direct pacing of the purkinje fiber or purkinje network may show a narrow qrs , comparable to conventional biv pacing in favor of shorter qrs duration , and lead to impulse conduction in the lv endocardium in patients with advanced heart failure . furthermore , purkinje fibers are widely distributed in the lv and are easy to detect . we believe that pp pacing is superior to other lv endocardial pacing in reproducibility and ability to detect the purkinje fiber as the pacing site , even in injured myocardium , but there has been no report of direct pacing of the purkinje network . we hypothesized that direct pacing of the peripheral network in patients with lbbb and heart failure might lead to more physiological pacing than conventional pacing . this study was done to verify that purkinje potential ( pp ) pacing is a promising strategy for resynchronization and for improving hemodynamics in such patients . the goal of the present study was to compare the effects of pp and biv pacing on hemodynamics in patients with drug - refractory heart failure . the study population comprised 11 patients ( eight men and three women ; mean age , 6214 years ) with new york heart association functional ( nyha ) class ii or iii heart failure despite optimal medical therapy . the echocardiographic lv ejection fraction , as determined on two - dimensional examination , was < 35% , and the qrs duration was > 120 ms . these patients were deemed likely to require resynchronization therapy in the near future according to guideline recommendations . all patients were on stable medical therapy for chronic heart failure , including diuretics ( n=10 ) , spironolactone ( n=7 ) , -blockers ( n=11 ) , angiotensin - converting enzyme ( ace ) inhibitors ( n=10 ) , and amiodarone ( n=1 ) . the medication regimen was not changed in any patient for at least 3 months prior to the study . cardiac catheterization was performed to assess the acute hemodynamic effects of biv pacing as a feasibility study for crt implantation . this study was approved by the local research ethics committee of hyogo college of medicine hospital , and patients provided written , informed consent to participate . left- and right - side cardiac catheterization was performed in all patients to assess lv function . a temporary electrode catheter was introduced into the high right atrium ( hra ) , and ventricular pacing catheters were placed at the right ventricular apex ( rva ) , coronary sinus , and lv epicardial wall . another electrode catheter was positioned in the lv endocardium to detect the pp via the aorta . a pp was defined as a sharp , brief , high frequency potential swing in the periphery ( fig . the pp pacing site was defined as the site at which the pp was detected ( fig . the lv lead was positioned mainly in the lateral branch of the coronary vein . a 5-fr high - fidelity micromanometer - tipped pigtail angiographic catheter ( millar instruments inc . , houston , tx , usa ) was placed in the lv cavity via the femoral artery approach in order to determine lv pressure , as previously reported , . lv pressure signals were digitized and analyzed on a computer system . pulmonary capillary wedge pressure ( pcwp ) and cardiac index ( ci ) were measured with a swan - ganz catheter placed in the proximal pulmonary artery via the femoral vein . six lv functional parameters were measured in the control state and during biv and pp pacing in each patient : maximum ( max ) dp / dt ( + dp / dt ) , minimum ( min ) dp / dt ( dp / dt ) , lv peak systolic pressure ( lvp ) , lv end - diastolic pressure ( lvedp ) , pcwp , and ci . typical surface electrocardiograms in the control state and during biv and pp pacing are shown in fig . the order of biv and pp pacing was switched with each case to avoid confounding factors . if the sinus rhythm rate was under 75 bpm , the control state was measured under atrial pacing ( ap : sinus rate+10 to 20 bpm ) , and the biv and pp pacing states were measured under the same pacing rate ( apvp : av sequential pacing ) . if the sinus rhythm rate was above 75 bpm , the control state was measured under sinus rhythm , and the biv and pp pacing states were measured under atrial sensing ventricular pacing ( asvp ) . measurements were collected in the non - paced state and at each pacing condition for 5 min , after 3 min of hemodynamic stabilization . av delay was generally 150 ms in patients with a normal pq duration , and ventricular pacing was performed with a shorter av delay ( < 150 ms ) in patients with a short pr duration . there was no adjustment for interventricular ( vv ) delay because of a limitation of time in this study . wilks test , were analyzed by unpaired t - tests and are expressed as means ( standard deviation ) . fixed factor analysis of variance ( anova ) for repeated measures was used for post hoc comparisons . all statistical analyses were performed with ezr ( easy r ) ( saitama medical center , jichi medical university , japan ) , a graphical user interface for r ( the r foundation for statistical computing , vienna , austria ) based on r commander ( version 1.6 - 3 ) . the study population comprised 11 patients ( eight men and three women ; mean age , 6214 years ) with new york heart association functional ( nyha ) class ii or iii heart failure despite optimal medical therapy . the echocardiographic lv ejection fraction , as determined on two - dimensional examination , was < 35% , and the qrs duration was > 120 ms . these patients were deemed likely to require resynchronization therapy in the near future according to guideline recommendations . all patients were on stable medical therapy for chronic heart failure , including diuretics ( n=10 ) , spironolactone ( n=7 ) , -blockers ( n=11 ) , angiotensin - converting enzyme ( ace ) inhibitors ( n=10 ) , and amiodarone ( n=1 ) . the medication regimen was not changed in any patient for at least 3 months prior to the study . cardiac catheterization was performed to assess the acute hemodynamic effects of biv pacing as a feasibility study for crt implantation . this study was approved by the local research ethics committee of hyogo college of medicine hospital , and patients provided written , informed consent to participate . left- and right - side cardiac catheterization was performed in all patients to assess lv function . a temporary electrode catheter was introduced into the high right atrium ( hra ) , and ventricular pacing catheters were placed at the right ventricular apex ( rva ) , coronary sinus , and lv epicardial wall . another electrode catheter was positioned in the lv endocardium to detect the pp via the aorta . a pp was defined as a sharp , brief , high frequency potential swing in the periphery ( fig . the pp pacing site was defined as the site at which the pp was detected ( fig . the lv lead was positioned mainly in the lateral branch of the coronary vein . a 5-fr high - fidelity micromanometer - tipped pigtail angiographic catheter ( millar instruments inc . , houston , tx , usa ) was placed in the lv cavity via the femoral artery approach in order to determine lv pressure , as previously reported , . lv pressure signals were digitized and analyzed on a computer system . pulmonary capillary wedge pressure ( pcwp ) and cardiac index ( ci ) were measured with a swan - ganz catheter placed in the proximal pulmonary artery via the femoral vein . six lv functional parameters were measured in the control state and during biv and pp pacing in each patient : maximum ( max ) dp / dt ( + dp / dt ) , minimum ( min ) dp / dt ( dp / dt ) , lv peak systolic pressure ( lvp ) , lv end - diastolic pressure ( lvedp ) , pcwp , and ci . typical surface electrocardiograms in the control state and during biv and pp pacing are shown in fig . the order of biv and pp pacing was switched with each case to avoid confounding factors . if the sinus rhythm rate was under 75 bpm , the control state was measured under atrial pacing ( ap : sinus rate+10 to 20 bpm ) , and the biv and pp pacing states were measured under the same pacing rate ( apvp : av sequential pacing ) . if the sinus rhythm rate was above 75 bpm , the control state was measured under sinus rhythm , and the biv and pp pacing states were measured under atrial sensing ventricular pacing ( asvp ) . measurements were collected in the non - paced state and at each pacing condition for 5 min , after 3 min of hemodynamic stabilization . av delay was generally 150 ms in patients with a normal pq duration , and ventricular pacing was performed with a shorter av delay ( < 150 ms ) in patients with a short pr duration . there was no adjustment for interventricular ( vv ) delay because of a limitation of time in this study . wilks test , were analyzed by unpaired t - tests and are expressed as means ( standard deviation ) . fixed factor analysis of variance ( anova ) for repeated measures was used for post hoc comparisons . all statistical analyses were performed with ezr ( easy r ) ( saitama medical center , jichi medical university , japan ) , a graphical user interface for r ( the r foundation for statistical computing , vienna , austria ) based on r commander ( version 1.6 - 3 ) . the etiology of heart failure was considered idiopathic cardiomyopathy in nine patients and ischemic cardiomyopathy in two . lv ejection fraction was 35% or lower in all patients ( mean , 286% ) . lv end - diastolic diameter ( lvdd ) was 669 mm , and qrs duration was 14236 ms . nine ( 81% ) of 11 patients had lbbb , and two had intraventricular conduction delay . maximum ( max ) dp / dt increased during biv pacing and pp pacing when compared with control ( 717171 mmhg / s vs. 917191 mmhg / s , p<0.05 ; and 717171 mmhg / s vs. 921199 mmhg / s , p<0.05 ) , but the difference between biv and pp pacing was not significant . there was no difference in hr , qrs duration , min dp / dt , lvedp , lvesp , pcwp , or ci when comparing these pacing sites . after this study , 10 of 11 patients received crt , and 6 had a satisfactory response . the etiology of heart failure was considered idiopathic cardiomyopathy in nine patients and ischemic cardiomyopathy in two . lv ejection fraction was 35% or lower in all patients ( mean , 286% ) . lv end - diastolic diameter ( lvdd ) was 669 mm , and qrs duration was 14236 ms . nine ( 81% ) of 11 patients had lbbb , and two had intraventricular conduction delay . maximum ( max ) dp / dt increased during biv pacing and pp pacing when compared with control ( 717171 mmhg / s vs. 917191 mmhg / s , p<0.05 ; and 717171 mmhg / s vs. 921199 mmhg / s , p<0.05 ) , but the difference between biv and pp pacing was not significant . there was no difference in hr , qrs duration , min dp / dt , lvedp , lvesp , pcwp , or ci when comparing these pacing sites . after this study , 10 of 11 patients received crt , and 6 had a satisfactory response . crt is an important therapeutic strategy for patients with drug - refractory heart failure and lv dyssynchrony . however , limitations to this modality include difficulty in placement of the lv lead and lack of response to biv pacing . some recent studies reported that biv pacing with lv endocardial stimulation sites yields better hemodynamics and lv synchrony , compared with conventional biv pacing . they also assessed the optimal lv pacing site to produce the most satisfactory hemodynamic parameters . these procedures have the benefit of lead placement in an extended area , with better threshold and avoidance of twitching , regardless of coronary vein location . we hypothesized that pp pacing is a good alternative to conventional biv pacing for patients with lbbb and heart failure . the purkiinje network can remain viable in patients with ischemic or idiopathic cardiomyopathy , and it is detected with relative ease , most typically at the left posterior fascicle of the left ventricle , and is even stable in the injured myocardium . because pp pacing is single site pacing , we can search for the optimal lv pacing site , without taking the positional relationship between lv and rv pacing sites into consideration . therefore , we assessed the efficacy of pacing at the lv left posterior fascicle in patients with advanced heart failure , and showed that stimulation at that site produced hemodynamics comparable to that of biv pacing . the present study showed that single lv endocardial pp pacing achieved acute hemodynamic effects , and even might be useful in the injured myocardium . this study compared measures of lv function during biv and pp pacing in patients with heart failure due to lv systolic dysfunction . max dp / dt increased during biv and pp pacing , when compared with control , but the difference was not significant . the improvements in lv function were similar , when comparing pp and biv pacing to control . the mean value of qrs duration did not differ significantly when comparing the three groups . in 4 of 11 cases , the qrs duration during pp pacing was longer than during sinus rhythm and bi - v pacing . however , in these cases max dp / dt also improved , regardless of wide qrs morphology . the cases with long qrs duration during pp pacing often showed right bundle branch block . the potentials during pp pacing , detected by the electrode at the coronary sinus as the terminal branch in the left ventricle , converged earlier than the end of the qrs morphology on the intracardiac electrogram . therefore , the long qrs duration may not have been caused by the failure of selective pp pacing ( i.e. , direct capture of the lv endocardium ) , but rather by delayed right bundle branch potentials . in the modern era of resynchronization therapy , alonso et al . however , although hbp produces a short qrs duration , it does not always result in improved lv function . thus , the relationship between lv function and qrs duration would benefit from further study , especially in patients with heart failure . purkinje fibers are often still viable in injured myocardium and patients with severe heart failure , and pp pacing in such patients might be effective for maintaining electrical conduction and improving lv function , regardless of qrs duration . furthermore , purkinje fibers are widely distributed in the lv and are easy to detect . consequently , pp pacing might be a good therapeutic strategy in patients with heart failure . recently , some researchers implanted lv leads transseptally . van gelder et al . reported transseptal lv endocardial pacing using standard techniques and equipment , without any dislodgement or thromboembolic events during follow - up . gamble et al . reported that the intraventricular transseptal approach for implantation of the lv lead would lower the risk of thromboembolism or valvular regurgitation . other approaches have been reported , such as transseptal , transaortic , or transapical , and further development of reliable and reproducible procedures and instrumentation might lead to safer and more effective lv endocardial pacing . with these procedures , left posterior fascicle permanent pacing has the potential for actual clinical use . the sample size was small , and only hemodynamic changes in the acute phase were assessed . we had estimated the parameters of pp pacing and performed cardiac catheterization to assess the acute hemodynamic effects of biv pacing before crt implantation . therefore we compared parameters among pp pacing , biv pacing , and controls over a short period . further studies that include more patients with longer - term follow - up are needed . furthermore , despite recently reported developments in lv endocardial pacing , these remain complex procedures , and are also associated with a risk of thromboembolism or valvular regurgitation , compared to conventional biv pacing . although some researchers have tried to lower the risks in the placement of lv endocardial pacing leads , the procedure remains challenging , and must be compared with conventional crt in the long term . therefore , this treatment should be restricted to those who are not candidates for or did not benefit from biv pacing . the hemodynamic outcome of pp pacing in the lv endocardium was comparable to that of biv pacing in patients with advanced heart failure . crt is an important therapeutic strategy for patients with drug - refractory heart failure and lv dyssynchrony . however , limitations to this modality include difficulty in placement of the lv lead and lack of response to biv pacing . some recent studies reported that biv pacing with lv endocardial stimulation sites yields better hemodynamics and lv synchrony , compared with conventional biv pacing . they also assessed the optimal lv pacing site to produce the most satisfactory hemodynamic parameters . these procedures have the benefit of lead placement in an extended area , with better threshold and avoidance of twitching , regardless of coronary vein location . we hypothesized that pp pacing is a good alternative to conventional biv pacing for patients with lbbb and heart failure . the purkiinje network can remain viable in patients with ischemic or idiopathic cardiomyopathy , and it is detected with relative ease , most typically at the left posterior fascicle of the left ventricle , and is even stable in the injured myocardium . because pp pacing is single site pacing , we can search for the optimal lv pacing site , without taking the positional relationship between lv and rv pacing sites into consideration . therefore , we assessed the efficacy of pacing at the lv left posterior fascicle in patients with advanced heart failure , and showed that stimulation at that site produced hemodynamics comparable to that of biv pacing . the present study showed that single lv endocardial pp pacing achieved acute hemodynamic effects , and even might be useful in the injured myocardium . this study compared measures of lv function during biv and pp pacing in patients with heart failure due to lv systolic dysfunction . max dp / dt increased during biv and pp pacing , when compared with control , but the difference was not significant . the improvements in lv function were similar , when comparing pp and biv pacing to control . the mean value of qrs duration did not differ significantly when comparing the three groups . in 4 of 11 cases , the qrs duration during pp pacing was longer than during sinus rhythm and bi - v pacing . however , in these cases max dp / dt also improved , regardless of wide qrs morphology . the cases with long qrs duration during pp pacing often showed right bundle branch block . the potentials during pp pacing , detected by the electrode at the coronary sinus as the terminal branch in the left ventricle , converged earlier than the end of the qrs morphology on the intracardiac electrogram . therefore , the long qrs duration may not have been caused by the failure of selective pp pacing ( i.e. , direct capture of the lv endocardium ) , but rather by delayed right bundle branch potentials . in the modern era of resynchronization therapy , alonso et al . reported that better pacing sites in the rv are associated with shorter qrs duration . however , although hbp produces a short qrs duration , it does not always result in improved lv function . thus , the relationship between lv function and qrs duration would benefit from further study , especially in patients with heart failure . purkinje fibers are often still viable in injured myocardium and patients with severe heart failure , and pp pacing in such patients might be effective for maintaining electrical conduction and improving lv function , regardless of qrs duration . furthermore , purkinje fibers are widely distributed in the lv and are easy to detect . consequently , pp pacing might be a good therapeutic strategy in patients with heart failure . van gelder et al . reported transseptal lv endocardial pacing using standard techniques and equipment , without any dislodgement or thromboembolic events during follow - up . gamble et al . reported that the intraventricular transseptal approach for implantation of the lv lead would lower the risk of thromboembolism or valvular regurgitation . other approaches have been reported , such as transseptal , transaortic , or transapical , and further development of reliable and reproducible procedures and instrumentation might lead to safer and more effective lv endocardial pacing . with these procedures , left posterior fascicle permanent pacing has the potential for actual clinical use . the sample size was small , and only hemodynamic changes in the acute phase were assessed . we had estimated the parameters of pp pacing and performed cardiac catheterization to assess the acute hemodynamic effects of biv pacing before crt implantation . therefore we compared parameters among pp pacing , biv pacing , and controls over a short period . further studies that include more patients with longer - term follow - up are needed . furthermore , despite recently reported developments in lv endocardial pacing , these remain complex procedures , and are also associated with a risk of thromboembolism or valvular regurgitation , compared to conventional biv pacing . although some researchers have tried to lower the risks in the placement of lv endocardial pacing leads , the procedure remains challenging , and must be compared with conventional crt in the long term . therefore , this treatment should be restricted to those who are not candidates for or did not benefit from biv pacing . the hemodynamic outcome of pp pacing in the lv endocardium was comparable to that of biv pacing in patients with advanced heart failure .

backgroundvarious difficulties can occur in patients who undergo cardiac resynchronization therapy for drug - refractory heart failure with respect to placement of the left ventricular ( lv ) lead , because of anatomical features , pacing thresholds , twitching , or pacing lead anchoring , possibly requiring other pacing sites . the goal of this study was to determine whether purkinje potential ( pp ) pacing could provide better hemodynamics in patients with left bundle branch block and heart failure than biventricular ( biv ) pacing.methodseleven patients with new york heart association functional class ii or iii heart failure despite optimal medical therapy were selected for this study . all patients underwent left- and right - sided cardiac catheterization for measurement of lv functional parameters in the control state during biv and pp pacing.resultsmaximum dp / dt increased during biv and pp pacing when compared with control measurements . this study compared parameters measured during biv pacing with pp pacing and non - paced beats as the control state in each patient ( 717171 mmhg / s vs. 917191 mmhg / s , p<0.05 ; and 921199 mmhg / s , p<0.005 ) ; however , the difference between pp pacing and biv pacing was not significant . there was no difference in heart rate , electrocardiographic wave complex duration , minimum dp / dt , left ventricular end - diastolic pressure , left ventricular end - systolic pressure , pulmonary capillary wedge pressure , or cardiac index when comparing biv pacing and pp pacing to control measurements.conclusionsthe hemodynamic outcome of pp pacing was comparable to that of biv pacing in patients with advanced heart failure .

Introduction Material and methods Study patients Cardiac catheterization Study protocol Statistical analysis Results Baseline characteristics Effect of PP pacing compared with control and BiV pacing Discussion LV endocardial pacing as an alternative, compared with LV epicardial pacing through the coronary sinus Effects of PP pacing Technical considerations in LV endocardial pacing Limitations of the study Conclusions Conflict of interest

cardiac resynchronization therapy ( crt ) is an important treatment for drug - refractory heart failure and left ventricular ( lv ) dyssynchrony . pacing leads have often been placed at the coronary vein and right ventricle to reduce lv dyssynchrony and to improve hemodynamics in patients with heart failure . in some patients , clinicians encounter difficulties when placing leads ; in approximately one - quarter of patients , there is an insufficient response to biventricular ( biv ) pacing , primarily because of difficulty in accurately placing the lv lead due to patients anatomical features , pacing thresholds , twitching , or pacing lead anchoring . some researchers have described other pacing sites that yield better hemodynamics and less dyssynchrony than biv pacing . attempted lateral lv wall pacing in patients with left bundle branch block pattern who were referred for crt device implantation . reported that transseptal lead placement was useful in cases where there was difficulty in placing a coronary sinus ( cs ) lead . reported that triventricular pacing , which uses two right ventricular leads and one lv lead , results in greater improvement in hemodynamics in patients with severe heart failure , when compared with bi - v pacing , sashida et al . reported improved lv function with his bundle pacing ( hbp ) in a patient with dilated cardiomyopathy due to atrial fibrillation without intraventricular conduction delay , however , whether these or other pacing sites are superior to conventional biv pacing remains unclear . some recent studies reported that biv pacing with lv endocardial stimulation sites yield better hemodynamics and lv synchrony , compared with conventional biv pacing . on the other hand , it has been reported that the purkinje network can survive in patients with ischemic or idiopathic cardiomyopathy , and in patients with heart failure and left bundle branch block ( lbbb ) . therefore , it is conceivable that direct pacing of the purkinje fiber or purkinje network may show a narrow qrs , comparable to conventional biv pacing in favor of shorter qrs duration , and lead to impulse conduction in the lv endocardium in patients with advanced heart failure . we believe that pp pacing is superior to other lv endocardial pacing in reproducibility and ability to detect the purkinje fiber as the pacing site , even in injured myocardium , but there has been no report of direct pacing of the purkinje network . we hypothesized that direct pacing of the peripheral network in patients with lbbb and heart failure might lead to more physiological pacing than conventional pacing . this study was done to verify that purkinje potential ( pp ) pacing is a promising strategy for resynchronization and for improving hemodynamics in such patients . the goal of the present study was to compare the effects of pp and biv pacing on hemodynamics in patients with drug - refractory heart failure . the study population comprised 11 patients ( eight men and three women ; mean age , 6214 years ) with new york heart association functional ( nyha ) class ii or iii heart failure despite optimal medical therapy . these patients were deemed likely to require resynchronization therapy in the near future according to guideline recommendations . all patients were on stable medical therapy for chronic heart failure , including diuretics ( n=10 ) , spironolactone ( n=7 ) , -blockers ( n=11 ) , angiotensin - converting enzyme ( ace ) inhibitors ( n=10 ) , and amiodarone ( n=1 ) . cardiac catheterization was performed to assess the acute hemodynamic effects of biv pacing as a feasibility study for crt implantation . this study was approved by the local research ethics committee of hyogo college of medicine hospital , and patients provided written , informed consent to participate . left- and right - side cardiac catheterization was performed in all patients to assess lv function . another electrode catheter was positioned in the lv endocardium to detect the pp via the aorta . the pp pacing site was defined as the site at which the pp was detected ( fig . the lv lead was positioned mainly in the lateral branch of the coronary vein . , houston , tx , usa ) was placed in the lv cavity via the femoral artery approach in order to determine lv pressure , as previously reported , . pulmonary capillary wedge pressure ( pcwp ) and cardiac index ( ci ) were measured with a swan - ganz catheter placed in the proximal pulmonary artery via the femoral vein . six lv functional parameters were measured in the control state and during biv and pp pacing in each patient : maximum ( max ) dp / dt ( + dp / dt ) , minimum ( min ) dp / dt ( dp / dt ) , lv peak systolic pressure ( lvp ) , lv end - diastolic pressure ( lvedp ) , pcwp , and ci . typical surface electrocardiograms in the control state and during biv and pp pacing are shown in fig . the order of biv and pp pacing was switched with each case to avoid confounding factors . if the sinus rhythm rate was under 75 bpm , the control state was measured under atrial pacing ( ap : sinus rate+10 to 20 bpm ) , and the biv and pp pacing states were measured under the same pacing rate ( apvp : av sequential pacing ) . if the sinus rhythm rate was above 75 bpm , the control state was measured under sinus rhythm , and the biv and pp pacing states were measured under atrial sensing ventricular pacing ( asvp ) . measurements were collected in the non - paced state and at each pacing condition for 5 min , after 3 min of hemodynamic stabilization . av delay was generally 150 ms in patients with a normal pq duration , and ventricular pacing was performed with a shorter av delay ( < 150 ms ) in patients with a short pr duration . there was no adjustment for interventricular ( vv ) delay because of a limitation of time in this study . the study population comprised 11 patients ( eight men and three women ; mean age , 6214 years ) with new york heart association functional ( nyha ) class ii or iii heart failure despite optimal medical therapy . these patients were deemed likely to require resynchronization therapy in the near future according to guideline recommendations . all patients were on stable medical therapy for chronic heart failure , including diuretics ( n=10 ) , spironolactone ( n=7 ) , -blockers ( n=11 ) , angiotensin - converting enzyme ( ace ) inhibitors ( n=10 ) , and amiodarone ( n=1 ) . the medication regimen was not changed in any patient for at least 3 months prior to the study . cardiac catheterization was performed to assess the acute hemodynamic effects of biv pacing as a feasibility study for crt implantation . this study was approved by the local research ethics committee of hyogo college of medicine hospital , and patients provided written , informed consent to participate . left- and right - side cardiac catheterization was performed in all patients to assess lv function . another electrode catheter was positioned in the lv endocardium to detect the pp via the aorta . the pp pacing site was defined as the site at which the pp was detected ( fig . the lv lead was positioned mainly in the lateral branch of the coronary vein . , houston , tx , usa ) was placed in the lv cavity via the femoral artery approach in order to determine lv pressure , as previously reported , . pulmonary capillary wedge pressure ( pcwp ) and cardiac index ( ci ) were measured with a swan - ganz catheter placed in the proximal pulmonary artery via the femoral vein . six lv functional parameters were measured in the control state and during biv and pp pacing in each patient : maximum ( max ) dp / dt ( + dp / dt ) , minimum ( min ) dp / dt ( dp / dt ) , lv peak systolic pressure ( lvp ) , lv end - diastolic pressure ( lvedp ) , pcwp , and ci . typical surface electrocardiograms in the control state and during biv and pp pacing are shown in fig . the order of biv and pp pacing was switched with each case to avoid confounding factors . if the sinus rhythm rate was under 75 bpm , the control state was measured under atrial pacing ( ap : sinus rate+10 to 20 bpm ) , and the biv and pp pacing states were measured under the same pacing rate ( apvp : av sequential pacing ) . if the sinus rhythm rate was above 75 bpm , the control state was measured under sinus rhythm , and the biv and pp pacing states were measured under atrial sensing ventricular pacing ( asvp ) . measurements were collected in the non - paced state and at each pacing condition for 5 min , after 3 min of hemodynamic stabilization . av delay was generally 150 ms in patients with a normal pq duration , and ventricular pacing was performed with a shorter av delay ( < 150 ms ) in patients with a short pr duration . there was no adjustment for interventricular ( vv ) delay because of a limitation of time in this study . lv end - diastolic diameter ( lvdd ) was 669 mm , and qrs duration was 14236 ms . maximum ( max ) dp / dt increased during biv pacing and pp pacing when compared with control ( 717171 mmhg / s vs. 917191 mmhg / s , p<0.05 ; and 717171 mmhg / s vs. 921199 mmhg / s , p<0.05 ) , but the difference between biv and pp pacing was not significant . there was no difference in hr , qrs duration , min dp / dt , lvedp , lvesp , pcwp , or ci when comparing these pacing sites . lv ejection fraction was 35% or lower in all patients ( mean , 286% ) . lv end - diastolic diameter ( lvdd ) was 669 mm , and qrs duration was 14236 ms . maximum ( max ) dp / dt increased during biv pacing and pp pacing when compared with control ( 717171 mmhg / s vs. 917191 mmhg / s , p<0.05 ; and 717171 mmhg / s vs. 921199 mmhg / s , p<0.05 ) , but the difference between biv and pp pacing was not significant . there was no difference in hr , qrs duration , min dp / dt , lvedp , lvesp , pcwp , or ci when comparing these pacing sites . after this study , 10 of 11 patients received crt , and 6 had a satisfactory response . crt is an important therapeutic strategy for patients with drug - refractory heart failure and lv dyssynchrony . however , limitations to this modality include difficulty in placement of the lv lead and lack of response to biv pacing . some recent studies reported that biv pacing with lv endocardial stimulation sites yields better hemodynamics and lv synchrony , compared with conventional biv pacing . these procedures have the benefit of lead placement in an extended area , with better threshold and avoidance of twitching , regardless of coronary vein location . we hypothesized that pp pacing is a good alternative to conventional biv pacing for patients with lbbb and heart failure . the purkiinje network can remain viable in patients with ischemic or idiopathic cardiomyopathy , and it is detected with relative ease , most typically at the left posterior fascicle of the left ventricle , and is even stable in the injured myocardium . because pp pacing is single site pacing , we can search for the optimal lv pacing site , without taking the positional relationship between lv and rv pacing sites into consideration . therefore , we assessed the efficacy of pacing at the lv left posterior fascicle in patients with advanced heart failure , and showed that stimulation at that site produced hemodynamics comparable to that of biv pacing . the present study showed that single lv endocardial pp pacing achieved acute hemodynamic effects , and even might be useful in the injured myocardium . this study compared measures of lv function during biv and pp pacing in patients with heart failure due to lv systolic dysfunction . max dp / dt increased during biv and pp pacing , when compared with control , but the difference was not significant . the improvements in lv function were similar , when comparing pp and biv pacing to control . in 4 of 11 cases , the qrs duration during pp pacing was longer than during sinus rhythm and bi - v pacing . however , in these cases max dp / dt also improved , regardless of wide qrs morphology . the cases with long qrs duration during pp pacing often showed right bundle branch block . the potentials during pp pacing , detected by the electrode at the coronary sinus as the terminal branch in the left ventricle , converged earlier than the end of the qrs morphology on the intracardiac electrogram . therefore , the long qrs duration may not have been caused by the failure of selective pp pacing ( i.e. , direct capture of the lv endocardium ) , but rather by delayed right bundle branch potentials . in the modern era of resynchronization therapy , alonso et al . however , although hbp produces a short qrs duration , it does not always result in improved lv function . thus , the relationship between lv function and qrs duration would benefit from further study , especially in patients with heart failure . purkinje fibers are often still viable in injured myocardium and patients with severe heart failure , and pp pacing in such patients might be effective for maintaining electrical conduction and improving lv function , regardless of qrs duration . consequently , pp pacing might be a good therapeutic strategy in patients with heart failure . the sample size was small , and only hemodynamic changes in the acute phase were assessed . we had estimated the parameters of pp pacing and performed cardiac catheterization to assess the acute hemodynamic effects of biv pacing before crt implantation . therefore we compared parameters among pp pacing , biv pacing , and controls over a short period . furthermore , despite recently reported developments in lv endocardial pacing , these remain complex procedures , and are also associated with a risk of thromboembolism or valvular regurgitation , compared to conventional biv pacing . although some researchers have tried to lower the risks in the placement of lv endocardial pacing leads , the procedure remains challenging , and must be compared with conventional crt in the long term . the hemodynamic outcome of pp pacing in the lv endocardium was comparable to that of biv pacing in patients with advanced heart failure . crt is an important therapeutic strategy for patients with drug - refractory heart failure and lv dyssynchrony . however , limitations to this modality include difficulty in placement of the lv lead and lack of response to biv pacing . some recent studies reported that biv pacing with lv endocardial stimulation sites yields better hemodynamics and lv synchrony , compared with conventional biv pacing . we hypothesized that pp pacing is a good alternative to conventional biv pacing for patients with lbbb and heart failure . the purkiinje network can remain viable in patients with ischemic or idiopathic cardiomyopathy , and it is detected with relative ease , most typically at the left posterior fascicle of the left ventricle , and is even stable in the injured myocardium . because pp pacing is single site pacing , we can search for the optimal lv pacing site , without taking the positional relationship between lv and rv pacing sites into consideration . therefore , we assessed the efficacy of pacing at the lv left posterior fascicle in patients with advanced heart failure , and showed that stimulation at that site produced hemodynamics comparable to that of biv pacing . the present study showed that single lv endocardial pp pacing achieved acute hemodynamic effects , and even might be useful in the injured myocardium . this study compared measures of lv function during biv and pp pacing in patients with heart failure due to lv systolic dysfunction . max dp / dt increased during biv and pp pacing , when compared with control , but the difference was not significant . the improvements in lv function were similar , when comparing pp and biv pacing to control . the mean value of qrs duration did not differ significantly when comparing the three groups . in 4 of 11 cases , the qrs duration during pp pacing was longer than during sinus rhythm and bi - v pacing . however , in these cases max dp / dt also improved , regardless of wide qrs morphology . the cases with long qrs duration during pp pacing often showed right bundle branch block . the potentials during pp pacing , detected by the electrode at the coronary sinus as the terminal branch in the left ventricle , converged earlier than the end of the qrs morphology on the intracardiac electrogram . therefore , the long qrs duration may not have been caused by the failure of selective pp pacing ( i.e. , direct capture of the lv endocardium ) , but rather by delayed right bundle branch potentials . in the modern era of resynchronization therapy , alonso et al . reported that better pacing sites in the rv are associated with shorter qrs duration . however , although hbp produces a short qrs duration , it does not always result in improved lv function . thus , the relationship between lv function and qrs duration would benefit from further study , especially in patients with heart failure . purkinje fibers are often still viable in injured myocardium and patients with severe heart failure , and pp pacing in such patients might be effective for maintaining electrical conduction and improving lv function , regardless of qrs duration . furthermore , purkinje fibers are widely distributed in the lv and are easy to detect . consequently , pp pacing might be a good therapeutic strategy in patients with heart failure . reported that the intraventricular transseptal approach for implantation of the lv lead would lower the risk of thromboembolism or valvular regurgitation . with these procedures , left posterior fascicle permanent pacing has the potential for actual clinical use . we had estimated the parameters of pp pacing and performed cardiac catheterization to assess the acute hemodynamic effects of biv pacing before crt implantation . therefore we compared parameters among pp pacing , biv pacing , and controls over a short period . furthermore , despite recently reported developments in lv endocardial pacing , these remain complex procedures , and are also associated with a risk of thromboembolism or valvular regurgitation , compared to conventional biv pacing . although some researchers have tried to lower the risks in the placement of lv endocardial pacing leads , the procedure remains challenging , and must be compared with conventional crt in the long term . the hemodynamic outcome of pp pacing in the lv endocardium was comparable to that of biv pacing in patients with advanced heart failure .