FIZIOLOGIA
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ARDELEAN AURELBĂDĂRĂU ANCA
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RÂCZ OLIVERRIGA DANSABĂU MARIUS
SAULEA I. AURELSIMIONESCU MAIASWYNGHEDAUW BERNARDTANGUAY M. ROBERTTATU ROMULUS FABIANVLAD AURELIANVOICU VICTORZĂGREAN LEON

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Fiziologia - Physiology • 2016.26.4(92)CONTENTS
1. Prevalence of the Burnout Syndrome Among Medical Staff .................................................................................................................4
Hogea L, Hogea B, Bredicean CA, Chiriac D
2. Momordica Charantia Restores Compromised Haematological Parameters in Streptozotocin-Induced
Diabetic Rats ............................................................................................................................................................................................8
Omobola Aderibigbe Komolafe, David Adesanya Ofusori, Samson Ayodeji Odukoya, Olarinde Stephen Adewole
3. Quality of Life in Haemophilia Patients with Knee Haemarthrosis .....................................................................................................13
Hogea B, Nussbaum L, Aungurence M, Hogea L
4. Body Mass Dependence of Reactive Oxygen Species Compartmental Distribution in the Rat Diabetic Kidneys ........................16
Vaduva A, Sturza A, Tiugan DA, Muntean D, Dema A
5. Bioprinting: Current Techniques and Applications ..............................................................................................................................21
Hurmuz M, Bojin F, Miu C, Ionac M, Tatu F
6. Histochemical and Heamatological Study of the Effects of Ethanolic Leaf Extract C.
Zambesicus on the Stomach in Diabetic Rats ...................................................................................................................................26
DA Ofusori, OA Komolafe, OS Adewole
7. Usefulness of Real-Time Shear Wave Elastography in the Diagnosis of Malignant Thyroid Nodules
in Patients with Autoimmune Thyroiditis ............................................................................................................................................32
Oprea MC, Lazar F
8. Risk Factors Associated with Chronic Venous Insufficiency in Central India ...................................................................................37
Samita R Sorte, Sachin B Rathod
9. Emotional regulation of children diagnosed with Autistic Spectrum Disorder .................................................................................43
Cristina Gianina Anghel, Liana Kobylinska, Mihaela Stancu, Iuliana Dobrescu

CUPRINS
1. Prevalența sindromului burnout in randul personalului medical ..........................................................................................................4
Hogea L, Hogea B, Bredicean CA, Chiriac D
2. Momordica Charantia restaureaza parametrii hematologici compromise la sobolanii cu diabet indus
de streptozotocin ....................................................................................................................................................................................8
Omobola Aderibigbe Komolafe, David Adesanya Ofusori, Samson Ayodeji Odukoya, Olarinde Stephen Adewole
3. Calitatea vietii la pacientii hemofilici cu hemartroza la genunchi .......................................................................................................13
Hogea B, Nussbaum L, Aungurence M, Hogea L
4. Masa corporala influenteaza distributia speciilor reactive de oxigen in diferite compartimente ale rinichiuluide sobolan diabetic ...............................................................................................................................................................................16
Vaduva A, Sturza A, Tiugan DA, Muntean D, Dema A
5. Bioimprimarea: Tehnologii actuale si aplicatii ......................................................................................................................................21
Hurmuz M, Bojin F, Miu C, Ionac M, Tatu F
6. Studii histochimice si hematologice asupra efectelor induse la nivelul stomacului sobolanilor diabetici
de catre extractul etanolic din frunze de C. Zambesicus ............................................................................................................... 26
DA Ofusori, OA Komolafe, OS Adewole
7. Utilitatea elastografiei shearwave in timp real in diagnosticul nodulilor tiroidieni maligni la pacientii cu tiroidita autoimuna ...32
Oprea MC, Lazar F
8. Factorii de risc asociati insuficientei venoase cornice in India Centrala ...........................................................................................37
Samita R Sorte, Sachin B Rathod
9. Emotional regulation of children diagnosed with Autistic Spectrum Disorder .................................................................................43
Cristina Gianina Anghel, Liana Kobylinska, Mihaela Stancu, Iuliana Dobrescu

Fiziologia - Physiology • 2016.26.4(92)3PREVALENCE OF THE BURNOUT SYNDROME
AMONG MEDICAL STAFF
LAVINIA HOGEA1, BOGDAN HOGEA2*, CRISTINA ANA BREDICEAN1, DANA CHIRIAC3
1Neuroscience Department, „Victor Babes“ University of Medicine and Pharmacy, Timisoara, Romania
2Ortopedics and Trauma Department, „Victor Babes“ University of Medicine and Pharmacy, Timisoara, Romania
3 Obstetrics-Gynecology Department, „Victor Babes“ University of Medicine and Pharmacy, Timisoara, Romania

ABSTRACTThe burnout syndrome got the attention of both researchers and practitioners because of its insidious effects on an individual,organizational and health department level. It is considered a public health problem because of its increased frequency but
mainly due to the negative consequences that it has regarding the health department. It hampers the health system, indirectly
by affecting the health of the people that have burnout syndrome and indirectly by the consequences that it has over the qualityof the medical caring.
The purpose of this thesis is to provide an analysis of the burnout syndrome inside a study group from the medical staff ofRomania. Following this purpose, 100 medics have been selected from different departments from hospitals in Timisoara withages between 30 and 55 years to whom a questionnaire for the measurement of the burnout syndrome has been applied. The
results showed the fact that selected medics confront themselves with this syndrome, a syndrome that affects their personal
performance, leading in time to exhaustion.
Keywords: burnout syndrome, stress, depersonalization syndrome

INTRODUCTION
Some specialists that dealt with the study of theburnout syndrome among the medical staff, got to theconclusion that the etiology of this system consists of theimperious need of the people in it to think that their lives
have a purpose in this world and that all of the things they
do are useful, valuable, important and even heroic [1].
Because of this, when the defeat, failing sensation
appears, the burnout also appears [2]. But this thing is not
generally true for everybody, it happens mostly to thepersons who chose this line of work because they thoughtthat they have a vocation for this sort of thing, and for thepeople that enter this work field realistically, but also very
motivated, the risk for the syndrome to appear is much
lower.
These observation and research have helped
strengthen the ideas presented by Maslach in 1988 related
to the differences between burnout and stress [3].
The medics from Romania are the most prone todevelop burnout, because they are always at risk to get
exhausted both emotional and physical. The accumulationof tiredness from the sleepless nights which the medic
spends trying to minimalize the suffering and the sickness ofa huge number of patients, the less and less free time that

he needs to relax and recover his energy combined with thenecessity to react promptly to the different solicitations leadto exhaustion, stress and finally to burnout.
Therefore, it is very important to identify the signs
associated with the burnout syndrome so they can bedifferentiated from the signs of stress, and assuming thatthese signs exist, measurements regarding its development
must be taken thus avoiding the critical faze with graveconsequences on an individual and institutional level.

MATERIAL AND METHODS
100 medics employed in the emergency clinics from
hospitals in Timisoara, aged between 30 and 55 years havetaken part in this study. 40 of them are females and 60 ofthem are males.
A questionnaire build based on the theory of Maslach
and Jackson, that measures the level of professional
exhaustion from this line of work, was used for the collectingof data. The questionnaire includes 25 terms that are
structured on 3 dimensions: emotional exhaustion (9items),
depersonalization (6items) and the reduction of personal
achievements (10items) [4, 5]. The Likert scale was use as

Received 15th of September 2016. Accepted 20th of October 2016. Address for correspondence: Bogdan Gheorghe Hogea, Department of Orthopedics andTrauma, “Victor Babes” University of Medicine and Pharmacy, Eftimie Murgu Square No. 2A, Timisoara, Romania; phone: +40747-025010; e-mail:hogeabg@yahoo.com

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Fiziologia - Physiology • 2016.26.4(92)a response method because it confers the advantage of agreater variety of answers.

and in time these have led to vulnerability towards stressors[Figure 2].

RESULTS
The studies made in the ranks of medical staff fromdifferent European countries have shown that approximately
a third of them experience the burnout phenomena [6-10].
The negative effects of these illness from which the
medical personnel suffers are being felt by the individual,
the institution as well as the patients. According to somestudies, the medics, the residents, and the medical
assistants who suffer from this syndrome are more prone
to substance usage [11], depression [12], insomnia [13],or show some higher and higher rates of suicidal ideation[1].
Some studies made in 2001 and 2009 have confirmed thefact that the burnout syndrome affects the performance of
hospitals by favoring the intentions to change jobs [2], theabsence from work [3], the increase of demotivation or evenby favoring the big amount of intentions to retire before term
[14].
The medium presence (41%) of the burnout syndrome
among medics owes itself to the fact that the medics must
permanently deal with emotional stimuli from with theirrelation with their patients as well as with physical stimuli
from the exhausting work that they must do in order to fill infor the lack of medical personnel. All of these are affecting
the professional performance and lead to exhaustion andstress [Figure 1].

Fig. 1. The total score of burnout syndrome

The most important aspect of the burnout syndrome isthe one regarding the emotional exhaustion and the results
show that 44.4% from the total number have a high level ofstress.
Emotional exhaustion represents the wasting of
emotional energy caused by the excessive psychologicalrequirements of the different tasks at the job.
The relations with the patients may be very demanding
because they require empathy and emotional involvement
Fiziologia - Physiology • 2016.26.4(92)Fig. 2. Distribution of subjects on the three domains of burnout
syndrome

Depersonalization is another dimension represented in
the figure number 2. Depersonalization refers to the attemptof people to detach themselves from others by associating
and treating them like objects. The aspects ofdepersonalization are reflected either by language, by theusage of labels to describe patients or their illnesses, either
by negativity or a cynical attitude.
Voluntarily or not, each of us witnessed once in our lives,
some of these treatments from medics and we judged themwithout thinking that they may suffer from thedepersonalization syndrome. 29.8% of the subjects from thisquestionnaire present symptoms of a depersonalization
syndrome [Figure 2].
The last component of the burnout syndrome is the
reduction of personal achievements and it is characterized
by the tendency of negatively evaluation of one’s
capacities, achievements or professional success (30.2%)[Figure 2].
These phenomena start faster at people who chose thisline of work because they considered that they have avocation towards it, and at the moment of the failure
apparition, one thinks of himself as professional
incompetent and incapable of reaching one’s initial goal.
Emotional exhaustion makes its presence felt at people
with a job experience of more than 5-10 years andrespectively over 20 years.
The lack of experience and the lack of means ofprotection may lead in time towards emotional exhaustion,
thus may be explained the higher score of people with lessthan 10 years of experience. Whereas people with morethan 20 years of experience are experiencing thissyndrome because they have accumulated a large numberof losses that they didn’t knew or couldn’t express or
manage [Figure 3].

5

CONCLUSIONS

Fig. 3. Correlation between emotional exhaustion and professional
experience

Regarding the case of the depersonalization syndrome,it can be observed in the figure bellow a directly proportional
increase between itself and the acquired professional
experience. If in the first years of experience only two out often people experienced the depersonalization phenomena,
their number also rose with 12 out of 35 people to manifest
this phenomenon [Figure 4].

The analysis of the results based on the questionnairecreated based on the theory of Maslach and Jackson,
confirmed the worrying reasons regarding the presence of
this syndrome among medics and it also helped toaccomplish the research objective, the identification of a
possible prevalence of the burnout syndrome among the
medics of Timisoara. The results of this study have shownthat the reduction of personal achievements anddepersonalization are of a medium level, whilst theemotional exhaustion presents a high score.
A brief or prolonged work experience presents a factor
that leads to an increased level of stress and implicitly to anincrease of the possibility to develop the burnout syndrome.
Schaufeli and Enzman identified in their researches
some affective signs on the medical personnel level likeirritability, over-sensibility, low empathy towards patients orincreased anger [15]. Simultaneously they also underlined
typical cognitive signs as cynical perceptions ordehumanization towards patients, negativity/pessimism
towards patients or their labeling in a derogatory way.
The behavior signs at an interpersonal level are violent
outbursts, aggressiveness towards patients, interpersonal
conflicts, the loss of interest or being indifferent towards
the patients and the mechanical responses for the patients[15].

REFERENCES

Fig. 4. Correlation between professional experience anddepersonalization

Persons with more than 20 years of experience are
more affected and they feel more the effects of the reductionof their personal achievements, declaring that they feel
unsatisfied on a professional plan [Figure 5].

Fig. 5. Correlation between the reduction of personal
achievements and professional experience6

1. Vela-Bueno, A., Moreno-Jiménez, B., Rodríguez-Muño, A.,
Olavarrieta-Bernardino, S., Fernández-Mendoza, J., De la
Cruz-Troca, J. J., & Vgontzas, A. N. (2008). Insomnia andsleep quality among primary care physicians with low and highburnout levels. Journal of Psychosomatic Research; 64:435442.
2. Leiter, M. P. & Maslach, C. Nurse turnover: The mediating roleof burnout. Journal of Nursing Management, 17, 331-339.
3. Davey, M. M., Cummings, G., Newburn-Cook, C. V., & Lo, E.
A. Predictors of nurse absenteeism in hospitals: A systematic
review. Journal of Nursing Management, 2009, 17, 312-330.
4. Maslach, C., & Jackson, S. E. The measurement ofexperienced burnout. Journal of Occupational Behaviour,
1981, 2, 99113.
5. Maslach, C. & Jackson, S. E. The Maslach Burnout Inventory(Research edition). Palo Alto, CA: Consulting Psychologists
Press, 1981.
6. Adam S., Gyoffy Z. & Susanszky, Physician Burnout in
Hungary: A potentiale Role for Work Family Conflict. J Health
Psychol. 2008,13:847-856.
7. Escribá-Agüir V., Martin-Baena D. & S. Pères-Hoyos.
Psychosocial work environment and burnout among
emergency medical and nursing staff. Int Arch Occup Environ
Health. 2006; 80:127-133.
8. Anagnostopoulos F. & Niakas D. Job Burnout, Health-Related
Quality of Life, and Sickness Absence in Greek HealthProfessionals. Eur Psychol. 2010; 15(2):132-141.

Fiziologia - Physiology • 2016.26.4(92)9. Chiron B., Michinov E., Olivier-Chiron E., Laffon M. & Rusch E.
Job Satisfaction, Life Satisfaction and Burnout in French
Anaesthetists. J Health Psychol. 2010; 15: 948.
10. Ringrose R., Houterman S., Koops W. & Oei G. Burnout inmedical residents: A questionnaire and interview study.
Psychol Health Med. 2009, Aug. 14; 4: 476-486.
11. Oreskovich, M. R, Kaups, K. L., Balch, C. M., Hanks, J. B.,
Satele, D., Sloan, & Shanafelt, T. D. Prevalence of alcohol use
disorders among American surgeons. Archives of Surgery.
2012; 147: 168-74.
12. Hakanen, J. J. & Schaufeli, W. B. Do burnout and work
engagement predict depressive symptoms and life satisfaction?
A three-wave seven-year prospective study. Journal of
Affective Disorders, 2012; 141: 415-424.
13. Shanafelt, T. D., Balch, C. M., Dyrbye, L., Bechamps, G.,

Russell, T., Satele, D., & Oreskovich, M. R. Special report:
suicidal ideation among American surgeons. Archives ofSurgery 2011; 146: 54-62.
14. Linzer, M., Visser, M. R. M., Oort, F. J., Smets, E. M. A.,
McMurray, J. E., & de Haes, H. C. J. M. Predicting andpreventing physician burnout: Results from the United States
and the Netherlands. The American Journal of Medicine, 2001;
111: 170-175.
15. Schaufeli, W. B. & Enzman, D. The Burnout companion to
study & practice. London: Taylor & Francis, 1998.
16. Maslach, C. Jackson, S. E. & Leiter, M. P. MBI: The Maslach
Burnout Inventory manual (3rd ed.). Palo Alto, CA: Consulting
Psychologists, 4, 1996.
17. World Health Organization. The European Health Report.
Health and health systems, 2009.

PREVALENȚA SINDROMULUI BURNOUT ÎN RÂNDULPERSONALULUI MEDICALREZUMAT
Sindromul burnout a captat interesul atat al cercatatorilor cat si al practicienilor din cauza efectelor insidiuase pe care le are la
nivel individual, organizational si a sistemului de sanatate. Este considerat o problema de sanatate publica din cauza frecventei
in crestere dar in principal din cauza consecintelor negative pe care le are asupra sistemului de sanatate. Ingreuneaza sistemule sanatate in mod indirect prin afectarea sanatatii persoanelor cu sindrom burnout si indirect prin consecintele pe care le areasupra calitatii ingrijirii medicale.
Scopul acestei lucrări este de a oferi o analiza a sindromului burnout in randul unui esantion din personalul medical din Romania.
In acest scop au fost selectati 100 de medici din diferite sectii din spitalele din Timisoara cu varste cuprinse intre 30-55 ani
carora li s-a aplicat un chestionar de masurare a sindromului burnout. Rezultatele au relevat faptul ca medici selectati se
confrunta cu acest sindrom, sindrom ce le afecteaza performanta profesionala, ducand in timp la extenuare.
Cuvinte cheie: sindromul burnout, stres, depersonalizare

Fiziologia - Physiology • 2016.26.4(92)7MOMORDICA CHARANTIA RESTORESCOMPROMISED HAEMATOLOGICAL PARAMETERS
IN STREPTOZOTOCIN-INDUCED DIABETIC RATSOMOBOLA ADERIBIGBE KOMOLAFE*, DAVID ADESANYA OFUSORI,
SAMSON AYODEJI ODUKOYA, OLARINDE STEPHEN ADEWOLE
Department of Anatomy and Cell Biology, Faculty of Basic Medical Sciences, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria

ABSTRACT
Background: The present study investigated the effects of M. charantia on haematological changes in streptozotocin-induced
diabetic Wistar rats.
Method: Forty healthy adult Wistar rats of both sexes were randomly assigned into five groups A, B, C, D and E of eight rats
each. Group A were the control (normal rats); B were the experimentally-induced diabetic rats; C were diabetic rats treated withmethanolic extracts of M. charantia for two weeks (withdrawal group); D were diabetic rats treated with methanolic extracts of
M. charantia for four weeks. E was diabetic rats treated with glimepiride for four weeks. Blood samples obtained by cardiac
puncture were used for haematological studies.
Results: Results showed an improvement in most RBC indices in groups D and E as compared with untreated diabetic group.
The groups treated with Momordica charantia and glimepiride enhanced WBC profiles as compared with the untreated diabetic
group. Platelet count showed a significant (p<0.05) increase in groups D and E as compared with the untreated diabetic group.
PDW and MPV were not significantly reduced in groups D and E as compared with the untreated diabetic group.
Conclusion: M. charantia restored some haematological indices in diabetic animals, and in a manner similar to glimepiride.
Keywords: Momordica charantia; Haematological; Streptozotocin; Diabetic RatsINTRODUCTION
Diabetes mellitus (DM) is a serious metabolic disorderwith micro- and macro-vascular complications that result insignificant morbidity and mortality. Increasing proportion of theaging population, consumption of calorie rich diet, obesity andsedentary lifestyle have led to a tremendous increase in thenumber of diabetics worldwide [1]. Thus researches arealways ongoing to increase and improve the understanding ofpathogenesis of the disorder, its associated complications
and develop better, safer and cheaper treatment. Momordica
charantia (cucurbitaceae) is one of the popular herbs thatgrow in different regions of Nigeria. It is believed to havebeneficial effects in prevention and treatment of many
diseases in folkloric medicine, especially in the treatment of
DM in individuals with non-insulin dependent diabetes. It has
hypoglycemic properties as it significantly suppressed the risein blood glucose concentrations in albino rats [2]. Bitter melon
grows in tropical areas, including parts of the Amazon, east
Africa, Asia, and the Caribbean, and is cultivated throughout
South America as a food and medicine. In the Amazon, local
people and indigenous tribes grow bitter melon in theirgardens for food and medicine. They add the fruit and/or
leaves to beans and soup for a bitter or sour flavor: Parboiling
it with a dash of salt may remove some of the bitter taste.
Medicinally, the plant has a long history of use by the indigenous
people of the Amazon. A leaf tea is used for diabetes, to expel
intestinal gas, to promote menstruation, and as an antiviral formeasles, hepatitis, and feverish conditions. It is used topicallyfor sores, wounds, and infections, internally and externally forworms and parasites [3].
Various parts of M. charantia such as the seed, fruit andeven the whole plant has been reported to have beneficial
effects in prevention and treatment of many diseases infolkloric medicine, especially in the treatment of DM inindividuals with non-insulin dependent diabetes [4,5]. It has
hypoglycaemic properties as it significantly suppressed therise in blood glucose concentrations in albino rats [4,2]. The
first clinical study into the influence of the fresh juice ofbittergourd on the management of DM was by Akhtar 1981[6].
These findings suggested the intervention would effectively
treat all symptoms of diabetes including polyuria, polydipsia,
and polyphagia. Sarkar et al. [7] and Miura et al. [8] indicatedthat the fresh bitter-gourd juice caused a significant reduction

Received 12th of August 2016; Accepted 10th of September 2016. Address for correspondence: Komolafe, Omobola Aderibigbe, PhD, Department of Anatomy
and Cell Biology, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria; phone: +2348034498833; e-mail: bkomolaf@yahoo.com

8

Fiziologia - Physiology • 2016.26.4(92)in plasma glucose concentration, and an improvement in the
response to an oral glucose load. Bitter melon contains anarray of biologically active plant chemicals including
triterpenes, proteins and steroids. In addition, a protein foundin bitter melon, momordin, has clinically demonstrated
anticancerous activity against Hodgkin's lymphoma in animals.
Other proteins in the plant, alpha- and beta-momorcharin andcucurbitacin B have been tested for possible anticancerous
effects [9]. In numerous studies, at least three different groupsof constituents found in all parts of bitter melon have clinicallydemonstrated hypoglycemic (blood sugar lowering) properties
or other actions of potential benefit against diabetes mellitus[10]. These chemicals that lower blood sugar include a mixtureof steroidal saponins known as charantins, insulin-like peptides,
and alkaloids. The hypoglycemic effect is more pronounced in
the fruit of bitter melon where these chemicals are found ingreater abundance.
The present study investigated the restorative effects of
M. charantia on haematological parameters in streptozotocin-induced diabetic Wistar rats.

MATERIALS AND METHODS
Animal care:
Forty healthy adult Wistar rats of both sexes, withaverage weight of 134.4 g were used for this experiment.
The rats were bred in the animal holding of College of Health
Sciences, Obafemi Awolowo University, Ile-Ife. They weremaintained on standard rat pellet (Capsfeed, Ibadan,
Nigeria) and water was provided ad libitum.
The animals were randomly assigned into five groups A,
B, C, D and E of eight rats each.
 group A were the control (normal rats)
 group B were the experimentally-induced diabetic
rats administered with 10% tween 80
 group C were the experimentally-induced diabetic
rats treated with methanolic extracts of Momordica
charantia dissolved in 10% tween 80 for two weeks(withdrawal group) group D were the experimentally-induced diabetic
rats treated with methanolic extracts of Momordica
charantia dissolved in 10% tween 80 for four weeks
 group E were the diabetic rats treated with astandard diabetic drug (2 mg/kg of glimepiride)
dissolved in 10% tween 80 for four weeks
The animals received humane treatment as outlined in
the “Care and Management of Laboratory Animals”
published by the National Institute of Health [11].
Plant material:
Matured leaves of Momordica charantia (Cucurbitaceae)
were collected during the raining season from suburban
villages of Ile-Ife metropolis in Osun State of Nigeria. The
leaves were taken to the Herbarium in the Department ofFiziologia - Physiology • 2016.26.4(92)Botany, Obafemi Awolowo University, Ile-Ife to confirm
identification and a voucher specimen number (UHI 16510)
was placed in the Herbarium.
Preparation of methanolic extract of M. charantia:
Leaves of Mormodica charantia (MC) were air dried andpowdered in a warring blender. A 765 g of the powdered
leaves were extracted in 1,950 mls of absolute methanol for
72 hours with intermittent shaking and filtered. The filtrate
were concentrated in vacuo at 350C using a vacuum rotary
evaporator (Bṻchi Rotavapor R110, Schweiz). The extract
was partitioned between water and dichloromethane, thedichloromethane fraction (5.94%) was oven-dried at 37oC
and stored until it is ready to be used. The aqueous portion
obtained was very little. Aliquot portions of the extract were
weighed and dissolved in 10% tween 80 for use on each dayof the experiment.
Induction of diabetes:
Diabetes mellitus was experimentally-induced in groups
B, C, D and E by a single intraperitoneal injection of 65
mg/kg body weight of streptozotocin (Tocris Bioscience, UK)dissolved in 0.1M sodium citrate buffer (pH 6.3). Diabetes
was confirmed in animals 48 hours after induction, bydetermining fasting blood glucose level using a digital
glucometer (Accu-chek® Advantage, Roche Diagnostic,
Germany) consisting of a digital meter and the test strips
using blood samples obtained from the tail vein of the rats.
Animals in group A were given equal volume of citrate bufferused in dissolving streptozotocin intraperitoneally.
Administration of extract and anti-diabetic drug:
Methanolic extracts of the leaves of M. charantia (100mg/kg) was dissolved in10% tween 80 and administereddaily (orally) by gastric intubation to the rats in groups C and
D for 2 and 4 weeks respectively. The standard antidiabetic
drug (glimepiride, 2 mg/kg) was administered to group E rats
for four weeks [12] while those in group B were left
untreated.
Haematological parameters:
Blood samples were by cardiac puncture from the animals
under slight chloroform anesthesia using syringes containing
the 10%-EDTA. This procedure was carried out within 0.8-1.2
minutes to minimize stress. The blood was later transferred
into EDTA K2 vacuum tube (GD040EK). Haematologicalparameters were performed using automated heamatology
analyzer (Sysmex: KX-21N). The following heamatologicalparameters were determined: white blood cell (WBC), redblood cell (RBC), heamoglomin concentration (HGB), meancell volume (MCV), Mean corpuscular heamoglobin (MCH),
Mean heamoglobim concentration (MCHC), platelet (PLT),
lymphocytes (LYM), neutrophil (NEUT), red blood cell
distribution weight (RDW_SD), red blood cell distribution
weight (RDW_CV), Platelet distribution weight (PDW), mean
platelet volume (MPV), P_LCR.
9

Statistical analysis:
All values were presented as means ± SEM. Data wereanalyzed using one way analysis of variance (ANOVA) with
Duncan multiple range test (DMRT) using Statistical
Package for Social Science (SPSS 17).

RESULTS
WBC profile (WBC, LYMP & NEUT):White Blood Cell Count
There was a significant (p>0.05) decrease in the WBC
count in the diabetic group (group B, 6.60±0.20) whencompared with the control (12.15±2.85). Administration of
MC extract for only two weeks to the animals in group C(withdrawal group) reduced the WBC count to 3.70±0.90.
The continuation of the extract administration for four weeks(group D), increased the WBC count to 7.95±1.25. The
group administered with gimepiride (group E) showed
unprecedented significant (p<0.05) increase in the WBC
count when compared with all other animal groups (Table I).
Lymphocytes Count
There was no significant (p>0.05) difference in thelymphocyte count of the diabetic (79.35 ± 3.65) group whencompared with the control group (61.95±3.25). The
lymphocyte count of group C (withdrawal group) was
reduced to 41.90±18.20 when compared with group D.
However, the rats treated with extract for four weeks (groupD) showed an increase in the lymphocyte count by 63.13%
when compared with the group C. The lymphocyte count ingroup D (68.35±13.45) was not significantly different(p>0.05) when compared with the group treated withglimepiride (group E, 75.90±7.50) (Table I).
Neutrophil count
There was a significant (p<0.05) decrease in theneutrophil count of the diabetic rats (group B, 20.65±3.65)
when compared with the control group (group A,
38.05±3.25). The animals in group C (withdrawal group)
shows an increase in the neutrophil count (58.10±18.20) as
compared with group D. Continuation of the MC extract
administration for four weeks reduces the neutropil count by
45.52%. There was no significant (p>0.05) difference in theeffect of glimepiride on neutropil count (24.10±7.50) whencompared with group D (31.65±13.45) (Table I).
RBC Indices (RBC, HGB, HCT, MCV, MCH, MCHC,RDW_SD, RDW_CV):Red Blood Cell (RBC) Count
There was no significant (p>0.05) increase in RBC
count in the diabetic group (7.94±0.04) when compared withthe control group (7.64±0.63). There was a slight decrease(p>0.05) in the RBC count of the animals administered withextracts for two weeks (withdrawal group C) when compared

10

with group D rats that increased with 38.77%. The group
administered with glimepiride (group E, 8.33±0.05)
compared non significantly with group D (8.59±0.30) ratsthat were administered with extract for four weeks (Table I).
HGB
The HGB concentration increases significantly (p<0.05)
in the diabetic group (group B) when compared with thecontrol group (group A).With the administration of MC extract
for four weeks (group D), HGB concentration increased to15.60± 0.20 when compared with the control (13.05±0.75)
rats (group A). Following withdrawal of the MC extract for two
weeks (group C), the HGB concentration reduced to 10.60±2.00. The group administered with glimepiride (group E)
shows no significant difference (p>0.05) with group D animals
treated with MC extract for four weeks after the initial fourweeks of STZ induction (Table I).
HCT
There was a significant (p<0.05) increase in HCT in the
diabetic group (group B) when compared with the control(group A) as shown in Table 4.9. HCT count shows asignificant (50.90 +_1.80 p<0.05) increase in the grouptreated with MC extract for four weeks (group D) whencompared with the control group. Withdrawal of MC extract
adminisration for two weeks (group C) lowered the HCT to41.05±4.95 when compared with the diabetic group (group
B, 55.40±1.60). There was no significant (p>0.05) difference
between the group administered with glimepiride (group E)
and the group that was treated with MC extract for fourweeks after the induction of STZ (Table I).
MCV, MCH and MCHC
There was an increase in the MCV and MCH in the
diabetic group (69.70±1.70 and 19.75±0.55 respectively)
when compared with the control group (55.50±1.30,
17.10±0.40, respectively). However, MCHC shows asignificant decrease (p<0.05, 28.35±0.05) in the diabetic
group when compared with the control group (30.85 ±0.05).
Administration of MC extract for four weeks (group D) further
reduced the MCV and MCH count to 59.15 ±0.05 and 18.15±0.15 respectively while MCHC increased to 30.60 ±0.30.
Withdrawal of the extract administration in (group C) rats
significantly increased the MCV count but shows a decreasein the MCH and MCHC count.The group administered withglimepiride (group E) shows a non significant difference(p>0.05) when compared with the group treated with MC
extract for four weeks (group D) (Table I).
RDW_SD, RDW_CV
The RDW_SD and RDW_CV shows an increase in the
diabetic group (group B) when compared with the control
group (Table 1). Administration of extract for four weeks
further increased the RDW _SD but reduced the RDW _CV.
Withdrawal of the extract administration in group C
significantly (p<0.05) increased the RDW_SD to 48.80±1.50
Fiziologia - Physiology • 2016.26.4(92)when compared with the group D treated with extract for fourweeks (37.85±0.95), while the RDW _CV was increased to
21.46±4.45 when compared with the group D treated withextract for four weeks (19.65±0.25). The group E treated withglimepiride presented a non significant difference (p>0.05)
when compared with the group treated with MC extract for
four weeks (group D) (Table I).

respectively) as compared with the control (group A)(770.50±32.50, 10.75 ±0.05 and 8.45 ±0.05 respectively).
Administration of MC extract for four weeks reduced the
PLT, PDW and MPV to 828.00 ±29.00, 10.95±0.65 and8.55±0.35 respectively. Withdrawal of extract administration
for two weeks further reduced the PLT and MPV to544.00±371 and 8.00 ±2.40 respectively while PDW was
increased to 12.60±5.10. Group E animals treated withglimepiride showed a decrease of PLT to 725.00 ± 23.00
and increased the PDW and MPV to 13.50 ± 0.40 and 9.55
± 0.05, respectively (Table I).

Platelet (PLT, PDW and MPV)
The PLT, PDW and MPV were increased in the diabetic
rats (group B) (1033.00±73.00, 11.85±1-25 and 9.25±0.85

Table I. The effect of M. Charantia on Heamatological ParamatersWBCx10³/µl

LYM
%

NEUT
%

Group

12.15 ±

61.95 ±

38.05 ±

RBC
x10⁶/
µl
7.64 ±

HGB
g/dl

HCT
%

MCV
Fl

MCH
Pg

MCHC
g/dl

13.05 ±

42.30 ±

55.50 ±

17.10 ±

30.85 ±

RDW_S
D
fl
33.85 ±

RDW_
CV %

PLT
x10³/µl

PDW
fl

MPV
fl

17.15 ±

770.50 ±10.75 ±

8.45 ±

A

2.85a

3.25abc

3.25a

0.63b

0.75ab

2.50a

1.30a

0.40a

0.05b

0.85a

1.75a

32.50ab

0.05a

0.05a

Group

6.60 ±

79.35 ±

20.65 ±

7.94 ±

15.70 ±

55.40 ±

69.70 ±

19.75 ±

28.35 ±

45.00 ±

18.10 ±

1033.00

11.85 ±

9.25 ±

B

0.20ab

3.65ab

3.65ab

0.04b

0.50b

1.60b

1.70c

0.55a

0.05ab

1.50bc0.50a± 73.00b

1.25a

0.85a

Group

3.70 ±

41.90 ±

58.10 ±

6.19 ±

10.60±

41.05 ±

66.05 ±

17.00 ±

25.60 ±

48.80 ±

21.45 ±

544.00 ±12.60 ±

8.00 ±

C

0.90c

18.20a18.20abc

0.38a

2.00a

4.95a

3.95bc

2.20a

1.80a

5.30c

4.45a

371.00a

5.10a

2.40a

Group

7.95 ±

68.35 ±

31.65 ±

8.59 ±

15.60 ±

50.90 ±

59.15 ±

18.15 ±

30.60 ±

37.85 ±

19.65 ±

828.00 ±10.95 ±

8.55 ±

D

1.25ab

13.45a13.45a

0.30b

0.70b

1.80ab

0.05ab

0.15a

0.30b

0.95ab0.25a29.00ab

0.65a

0.35a

Group

17.50 ±

75.90 ±

24.10 ±

8.33 ±

14.75 ±

48.80 ±

58.55 ±

17.70 ±

30.25 ±

34.85 ±

17.25 ±

725.00 ±13.50 ±

9.55 ±

E

8.10a

7.50a

7.50ab

0.05b

0.05b

0.60ab

0.35ab

0.20a

0.45b

0.85a

0.65a

23.00ab

0.40a

0.05a

Values are given as Mean ± SEM for each haematological parameters WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, LYM, NEUT,
RDW_SD, RDW_CV, PDW and MPV respectively in each group. a, b, c, d, ab, bc, cd, abc within column signifies that means with different
letters differs significantly at p < 0.05 while means with the same letters does not differ significantly at p < 0.05 (using one way ANOVA with
Duncan multiple range test)

DISCUSSION
Reactive oxygen species has also been implicated in themechanism of red cell damage [13]. In diabetes, the excess
glucose present in the blood reacts with the haemoglobin toform glycosylated haemoglobin which leads to a decrease
in total haemoglobin level [14]. The present result showed
an increase in RBC indices in diabetic rats which whentreated with M. charantia and glimepiride for four weeks
significantly increase it except RBC, MCH and RDW-CV ascompared with the control group. The reason for thisobservation calls for further investigation but the duration ofthis study may not be enough to have allowed pronounced
pathogenesis required for full expression of anaemia.
Withdrawal of the M. charantia decreased the RBC indiceswith the exception of MCV and RDW_SD. The reason forthis occurrence is not yet known. It has also been reported
that occurrence of anemia in DM is more common and ismostly due to an increase in non-enzymatic glycosylation oferythrocyte membrane glycoproteins, which correlates withhyperglycemia [15]. These increases in oxidation of
glycoproteins induce an increase in the production of lipid
peroxides causing hemolysis of erythrocytes [16].
Fiziologia - Physiology • 2016.26.4(92)The WBC and NEUT were significantly reduced in the
diabetic animals except the lymphocyte count which wassignificantly increased. Treatment with M. charantia andglimepiride for four weeks increased the WBC and NEUT
while the lymphocyte count was not affected significantly.
Following the withdrawal of the M. charantia, the NEUT
counts showed further increase when compared with thediabetic rats, the reason for the decrease in WBC and
NEUT counts in diabetic rats may be due to reduction in the
immunity in corroboration with other investigation [17]. The
increase noticed in lymphocyte count in diabetic rats may
be a strategy to cope with infection associated with diabetes
mellitus [ 16].
Platelet count, PDW and MPV was significantly
increased in the diabetic rats when compared with thecontrol group. Increase in PLT and MPV has been reported
to be implicated in diabetes [18]. This increase may be due
to dehydration which often characterizes diabetic animals as
result of polyuria [19]. Zuberi et al [20] also reported anincrease in MPV to be due platelet hyperactivity. Treatment
with M. charantia and glimepiride restored the Platelet
count, PDW and MPV to normal.

11This study has revealed that methanolic M. charantia
could restore some haematological parameters in diabetic
rats in a manner similar to glimepiride. Though the present
study showed that M. charantia restored somecompromised haematological parameters in DM, there isneed for further study to fully understand the mechanisms ofadverse haematological changes associated with diabetic
rat models and the effects of useful therapies on thesechanges.

CONFLICT OF INTEREST
There was no conflict of interest among the authors andevery necessary detail was agreed upon during the
preparation of the work.

REFERENCES
1. Vats V, Yadav SP. and Grover JK. (2004) Ethanolic extract ofOcimum sanctum leaves partially attenuates streptozotocininduced alterations in glycogen content and carbohydrate
metabolism in rats. J. Ethnopharmacol., 90: 155-160.
2. Nicholas LB, Kolb Y, Prinssen EP. (2006) A combined marble
burying ocomotor activity test in mice: a practical screening
test with sensitivity to different classes of anxiolytics andantidepressants. Eur J Pharmacol. 547: 106 – 115.
3. Teoh SL, Latiff AA and Das S. (2009b) The effect of topicalextract of Momordica charantia (bitter gourd) on woundhealing in nondaibetic rats and in rats with diabetes induced
by streptozotozin. Clin Exp Dermatol 34(7):815 -22.
4. Platel K and Srinivasan K. (1997) Plant foods in the
management of diabetes mellitus: vegetables as potential
hypoglycaemic agents. Nahrung, 41(2):68-74.
5. Raman A And Lau C (1996) Anti-diabetic properties andphytochemistryofMomordicacharantia
L.
(Cucurbitaceae).Phytomedicine 2; 349-662.
6. Akhtar, M. S., Athar, M.A. and Yaqub, M. (1981): Effect ofMomordica charantia on blood glucose level of normal andalloxan-diabetic rabbits. Planta Med. 42:205-212.
7. Sarkar S, Pranava M and Marita R. (1996) Demonstration of
the hypoglycemic action of Momordica charantia in a validate
animal model of diabetes. Pharmacol. Res. 33: 1-4.
8. Miura T, Ichiki H, Hashimoto I, Iwamoto N, Kato M, Kubo M,
et al. (2001) Impairment of insulin-stimulated GLUT4translocation in skeletal muscle and adipose tissue in the12

Tsumura Suzuki obese diabetic mouse: a new genetic animalmodel of type 2 diabetes. Eur J Endocrinol; 45: 785-790.
9. Nagasawa H, Watanabe K and Inatomi H (2002) Effects ofbitter melon (Momordica charantia) or ginger rhizome(Zingiber offifinale Rosc.) on spontaneous mammary
tumorigenesis in SHN mice. Am. J. Clin. Me.; 30(2–3): 195–
205.
10. Tan FJ,
Fire AZ. and Hill RB. (2007) Regulation of
apoptosis by C. elegans CED-9 in the absence of the Cterminal transmembrane domain. Cell Death Differ. 14, 19251935.
11. National Institute of Health Guide for the Care and Use ofLaboratory Animals. DHEW
12. Publication (NIH) 1985, revised, Office of Science and
Health Reports, DRR/NIH, Bethesda, USA.
13. Mir SH, Darzi MM, Ahmad F, Chishti MZ and Mir MS (2008)
The Influence of Glimepiride on the Biochemical andHistomorphological Features of Streptozotocin-Induced
Diabetic Rabbits. Pakistan Journal of Nutrition 7(3): 404-407.
14. Tanko Y, Mabrouk MA,
Adelaiye AB, Fatihu MY, andMusa KY. (2011) Anti-diabetic and some haematologicaleffects of ethylacetate and n-butanol fractions of Indigofera
pulchra extract on alloxan-induced diabetic Wistar rats.
Journal of Diabetes and Endocrinology Vol. 2(1) 1-7.
15. Rao BK, Sudarshan PR, Rajasekhar MD, Nagaraju N and
Rao CA. (2003) Antidiabetic activity of Terminalia pallida
fruit in alloxan induced diabetic rats. J. Ethnopharmacol., 85:
169-172.
16. Sheela CG and Augusti KT. (1992) Antidiabetic effects of Sallyl cysteine sulphoxide isolated from garlic Allium sativum
Linn. Indian J. Exp. Biol., 30: 523-526.
17. Ceriello A. (2003) New insights on oxidative stress anddiabetic complications may lead to a “casual” antioxidant
therapy. Diabetes Care; 53:701–710.Kennedy L and Baynes
JW. (1984) Non-enzymatic glycosylation and the chronic
complications of diabetes: An overview. Diabetologia, 26:9398.
18. Irshaid F, and Mansi K. (2009) The effects of methanol extract
derived from Urtica pililufera leaves on some haematologicaland biochemical parameters of Diabetic Rats .Res J. Biol. Sc.
4 (6) 675-681.
19. Malone JI, Schocken DD, Morrison AD, and Gilbert-Barness
E. (1999) Diabetic cardiomyopathy and carnitine deficiency.
Jour Diab Comp.); 13: 86–89.Diabetic explain.com (DE)(2012)
http://www.diabetesexplained.com/the-plateletcount.html#top.Retrieved on 21/01/12.
20. Zuberi BF, Akhtar N and Afsars S. (2008) Comparison of mean
platelet volume in patients with diabetes mellitus,impaired
fasting glucose and non-diabetic subjects. S. Med J. 49(2):114-116.

Fiziologia - Physiology • 2016.26.4(92)QUALITY OF LIFE IN HAEMOPHILIA PATIENTSWITH KNEE HAEMARTHROSISBOGDAN HOGEA1*,LAURA NUSSBAUM2*, MIHAELA AUNGURENCE3,
LAVINIA HOGEA2*
1Orthopedics and Trauma Department, „Victor Babes“ University of Medicine and Pharmacy, Timisoara, Romania
2Neuroscience Department, „Victor Babes“ University of Medicine and Pharmacy, Timisoara, Romania
3Psychologist, Psychotherapist* These authors have equal contribution and thus share first authorship.

ABSTRACT
Purpose. The study brings to your attention a rare pathology whose treatment is expensive but with its help encouraging results
may be obtained, with consecutive growth of the quality of life of hemophilic patients and their social reintegration.
Material and methods. The study included a number of 83 patients with hemarthrosis with ages between 6 and 40 years. Data
collecting was made by using the EQ-5D quality of life questionnaire, HAL and the VAS visual scale.
Results. The quality of life questionnaires show a significant difference with improvements to the quality of life between thetime period previous to the recovery and the after recovery time period (p=0.002).
Conclusions. Patients suffering from hemarthrosis tend to report that the ability to perform daily activities, social and physicalactivities is severely affected. Furthermore, the ones with the severe illness have the lowest quality of life score in all areas.
Keywords: haemophilia, quality of life, haemarthrose

INTRODUCTION
Haemarthrosis represent one of the hemorrhagic
manifestations with a high incidence at hemophiliacs,
considering that 3 out of 4 patients suffer from ahemarthrosis.
Hemarthrosis generally appears with the begging ofwalking, between the ages 1 and 5 and represents the firstmanifestation of the illness. After its apparition, after 10
years, as a unique hemorrhage it may indicate an
attenuated form of illness.
The triggering factor is always traumatizing, and theaffected articulations with the highest predilection are the knees,ankles and elbows. The articulations of the shoulders, hands,
thighs and fingers are affected less often [1, 2, 3].
The hemarthorsis triggering is insidious, being precededby sensations of intraarticular tension, accompanied byarticular instability, after which the very live pain appears
accompanied by the tumefaction and functional deficiencyof the articulation. The evolution towards hemarthorsis may
be rapid, in a few minutes or hours, according to theimportance of the suffered traumatism [1, 4, 5].
The increased frequency of hemarthrosis to patientswith a severe form of illness, the apparition and evolution ofchronic hemophiliac arthropathy, which produces disability

and modifies the body image may lead to a decrease in thequality of life of the hemophilia patients.
The purpose of the evaluations of the patient’s quality oflife consists of underlying the effect which different
pathological events and therapeutic methods have on thepatient’s life taking into consideration aspects regarding thepersonal satisfaction, regarding social life. The evaluation of
the treatment quality of a chronic affection implies theimperious reference to the quality of life of the patient who
benefits from that treatment [6, 7].

MATERIAL AND METHOD
The present study was conducted on a group formed by
83 subjects diagnosed with severe hemophilia and who alsopresented hemarthrosis. The patients presented
themselves for the comprehensive evaluation and for the
locomotory recovery treatment. The patients had ages
between 6 and 40 years.
Their quality of life has been evaluated with the help of
the EQ-5D and HAL questionnaires, and the VAS scale. The
EQ-5D questionnaire contains five questions, eachmeasuring a health domain: mobility, self-care, usual
activities, pain/discomfort, restlessness/depression. Based

Received 10th of September 2016. Accepted 15th of October 2016. Address for correspondence: Lavinia Hogea, Department of Psychology, “Victor Babes”
University of Medicine and Pharmacy, Eftimie Murgu Square No. 2A, Timisoara, Romania; phone: +40745042035; e-mail: laviniahogea@yahoo.com

Fiziologia - Physiology • 2016.26.4(92)13on the given answers a profile was calculated that indicatesthe affected level in each health domain as well as anaverage score of each domain. Furthermore, a healthevaluation of the patients was done using the VAS visual
scale (the biggest value is 100 and the smallest is 0).
With the help of the HAL questionnaire the patientscompleted their daily activities that they can participate toand the way the muscle-skeletal functioning influences it.
Aspects related to the following domains were pursued: arm
functions, leg functions, self-caring, home activities, leisureactivities, using the public means of transport, others meansof transport.
Global and domain centered scores were calculated forevery questionnaire before and after the recovery process.
The statistical processing of data was made using the
SPSS program that allowed the calculation of the frequency,
mean, standard deviation, the appraisal of the p and r
probability and the Pearson coefficient.

RESULTS
After you apply the EQ-5 d questionnaire, the proportionof patients with and without problems on the five areas
before and after recovery was appreciated, it has thus been
observed an increased proportion of patients with or without
problems, namely the decrease in the number of patientswith large problem after recovery. The average scores wereassessed to the 83 patients of the group. Thus, before
recovery, the average scores obtained varied between DS =0.46 in the area of self-care and DS = 0.54 in the area ofpain/discomfort. In the field of common activities, mobility
and restlessness/depression scores do not vary greatly,
being roughly equal, DS = 0.52 [Figure 1].
We may conclude that patients with hemarthrosis
appreciate the quality of life as negative on all five areas:
mobility, usual activities, pain, anxiety/depression and selfcare [8, 9, 10].
The field of pain and discomfort measures theunpleasant physical sensations which a person felt and thedegree of influence over their life. At the same time, itmeasures the person's control that it has on the pain and theease with which he frees himself by the sensation of pain.
Being a subjective sensation, response to pain differsfrom person to person, depending on the degree oftolerance, acceptance, but it is certain that it has an impact
on the quality of life [11, 12].
Studies have shown that pain and discomfort have a strong
impact on quality of life, and the presence of pain influences theperception of other areas of the quality of life [13].
After the recovery intervention it was found a decreasein the average score across all fields, values vary between
DS = 0.52 in the self-care domain and DS = 0.50 in the areaof pain/discomfort. In the areas of common activities,
mobility, and anxiety/depression the scores are DS = 0.51
[Figure 1].
14

The results found that the areas of self-care and pain
are more affected in patients with a severe form of illness.

Fig. 1. The graphical representation of quality of the life before andafter recovery

The VAS score appreciated depending on the severity,
proved a significant increase after recovery in the case ofpatients with a severe form, the significant difference being
p = 0,002.
The evaluation of the quality of life with the aid of the
HAL list of activities has shown that patient had increased
scores, the initial self-evaluation reveals the affecting of all
functional abilities. The highest score was in the area of ofdaily living activity, DS = 24.1.
After the recovery, a significant decrease in score wasreported from fields related to arms functions - p=0.003,
posture - p=0.003, p=0.005- domestic activities.
The study carried out by Prof. Schramm et al. over thequality of life of patients with hemarthrosis from the countries
where the prophylactic treatment is being provided
compared to those who don't receive treatment have abetter quality of life.

CONCLUSION
Patient assessment has revealed that in the absence ofa replacement treatment, from a tender age the chronic
suffering and impairment within a joint arises. The
progresive affected joints will result in disability, handicap
and a decrease of the quality of life. Most patients had theirknee joint damaged , 96,6%, 46% had a damaged elbow, ,23.2% ankle 6.15%, the scapula-humerus joint and 1%, thecoxo-femoral joint [14, 15].
Questionnaires measuring the quality of life show asignificant difference with the improvement of the quality oflife between the period prior to the recovery and after
recovery (p = 0,002).
The implementation of a prophylaxis program and hometreatment is imperative, starting at an early age in order toprevent the appearance of hemarthrosis and arthropathy
with incapacitating potential [16].
Fiziologia - Physiology • 2016.26.4(92)REFERENCES
1. Poenaru, D.V., et al. (2005). Artropatiilehemofilice. Editura
Academiei Romane, 53.
2. Alcalay, M., Deplas, A. (2002). Rheumatologicalmanagement of patients with hemophilia. Part II: Muscle
hematomas and pseudotumors. Joint Bone Spine 2002;
69(6):556-559.
3. Berntorp, E. (2004). Hemophilia. Octapharma. Stockholm.
4. Alcalay, M., Deplas, A. (2002). Rheumatologicalmanagement of patients with hemophilia. Part 1: Joint
ifestations. Joint Bone Spine 2002; 69(5):442-449.
5. Aledort, L.M. (2003). Orthopedic Outcome Studies andCost Issues. Semin Thromb Hemost, 29(1): 55-60.
6. Allison, P., Locker, D., Feine, J. (1997). Quality of life: adynamic construct. Soc Sci Med 45:221-230.
7. Hogea L.M., Nussmaum L., Evaluation of life quality inpatients with haemophilia, RevistamedicalaRomana,
București 2016, Vol. LXIII, Nr. 1, Pag. 31, ISSN: 12205478;
8. Bullinger, M., Von Mackensen, S., The Haemo- QoLGroup. (2003). Quality of life in children and families withbleeding disorders. J. PediatrHematolOncol, Vol. 25,
Suppl. 1.
9. Bullinger, M., Von Mackensen, S. (2004). Quality of life
assessment in haemophilia.Haemophilia, 10 Suppl 1,
9-16.
10. Hogea L.M., Hogea G.B., Oancea C., Ilie A.C.,

Psychological and clinical aproach of hemophilicpatients, Medicine in Evolution, Volume XIX, 2013, No.
3, 6 pag, 2065-376X;
11. Carol, S., Burckhardt, L., Anderson, B.A.,Hägg,O.
(2003). Health and Quality of Life. Outcomes.
12. Hogea L.M., Nussmaum L., Dumache R., Assessmentof obsessive-compulsive behavior in patients withhaemophilia, Revista Physiology, Timisoara 2016,
26.1(89), Pag. 31, ISSN: 1223-2076;
13. Skevington, S.M. (2001).Validation of the WHOQOL100: pain management improves quality of life forchronic pain patients, 267-275.
14. W, Schramm et al. (2012). Haemophilia Care in Europe:
the ESCHQoL study. 10.1111/j.1365-2516.2012.02847.x
15. Jones, P. (2002). Living with haemophilia. Oxford
University Press.
16. Rodriguez-Merchan, E.C., (1996). Effects of
haemophilia on articulation of children and adults. Clin
Orthopedics Related Res, 328:7-13.
17. Tedgard, U., Ljung, R., Bullinger, M., Von Mackensen,
S. (2008). Quality of life in haemophilia carriers –preliminary results of the ESCHQoL study. Session
B-2.5: Multidisciplinary free papers 01. 29 FP 05.
Haemophilia.
18. Hogea L.M., Nussmaum L., Alexithymia in patients withhaemophilia. Comparative study with the healthy
population, RevistamedicalaRomana, Bucuresti 2016,
Vol. LXIII, Nr. 2, Pag. 136, ISSN: 1220-5478.

CALITATEA VIEȚII LA PACIENȚII HEMOFILICICU HEMARTROZĂ LA GENUNCHIREZUMAT
Scop. Studiul aduce in atentie o patologie rara al carei tratament este costisitor, dar cu ajutorul caruia se pot obtine rezultate
incurajatoare, cu cresterea consecutive a calitatii vietii pacientilor hemofilici si reintegrarea lor sociala.
Material si metode. Studiul a inclus un numar de 83 de pacienti cu hemartroza, cu vârsta cuprinsă între 6-40. Colectarea
datelor s-a realizată folosind chestionarul de masurare a calitatii vietii EQ-5D, HAL si scala vizuala VAS.
Rezultate. Chestionarele de masurare a calitatii vietii arata o diferenta semnificativa cu imbunatatirea calitatii vietii intre perioada
anterioara recuprarii si dupa recuperare (p=0,002).
Concluzii. Pacientii cu hemartoza tind să raporteze că le sunt afectate sever în capacitatea de a efectua activitatile de zi cu zi,
activitățile lor fizice și sociale. Deasemenea, cei cu boala severa au cel mai scazut scor al calitatii vietii pe toate domeniile.
Cuvinte cheie: hemofilie, calitatea vietii, hemartroza

Fiziologia - Physiology • 2016.26.4(92)15BODY MASS DEPENDENCE OF REACTIVE OXYGENSPECIES COMPARTMENTAL DISTRIBUTION
IN THE RAT DIABETIC KIDNEYSADRIAN VADUVA1, ADRIAN STURZA*2,3, DIANA ANDREEA TIUGAN2,4,
DANINA MUNTEAN*2,3, ALIS DEMA1
1Department of Morphological Sciences - Morphopathology, “Victor Babes” University of Medicine and Pharmacy of Timisoara, Romania
2Department of Functional Sciences - Pathophysiology, “Victor Babes” University of Medicine and Pharmacy of Timisoara, Romania
3Center for Translational Research and Systems Medicine, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania
4Department of Morphological Sciences – Medical Genetics, “Victor Babes” University of Medicine and Pharmacy of Timisoara, Romania

ABSTRACT
Background: Oxidative stress is widely recognized as a major contributor to kidney damage in the setting of diabetes. We
aimed to characterize reactive oxygen species (ROS) production in the different kidney compartments in rats.
Material and methods: Diabetes was induced in Wistar rats (n=12) by a single IP injection (50 mg/kg) of streptozotocin. After
2 months, the rats were separated in two groups, under and above 400g body weight, respectively. The kidneys were harvested
and dihidroethidium (DHE) stained frozen sections were examined under an Olympus Fluoview FV1000 laser scanning confocal
microscope. Fluorescence intensity was measured using a custom designed protocol in Icy bioimage analysis software.
Results: In animals weighing less than 400g, the average fluorescence intensity was over 900 AU for 2/6 rats (33%), while in
the over 400g group all rats (6/6) had higher levels. Interestingly, in the > 400g group (at variance from the < 400g group) a
major ROS production occurred in the vascular compartment (followed by the glomeruli) that displayed the highest DHE
intensities. No changes were evident between the groups for the tubular ROS production.
Conclusion: Our results strongly suggest that an increased body mass influences the ROS generation, especially in bloodvessels and glomeruli, an observation that clearly warrants further investigations with the appropriate data reporting.
Keywords: kidney ROS, confocal DHE, weight adjusted ROS levels, diabetic kidney disease

INTRODUCTION
Diabetic nephropathy (DN) is a distinct renal disease
incurred by long time exposure to high glycaemic levels in
diabetic patients. The spectrum of lesions is wide, as DN
affects the glomeruli (mesangial expansion, podocyte
damage, thickening of the basement membranes andeventually obliteration of the capillary tuft), the tubular andinterstitial compartment (thickening of basement
membranes, tubular atrophy, interstitial fibrosis), and thevascular structures, respectively (hyaline change) (17).
In the past decades, oxidative stress (OS) has emerged
both as a central pathomechanism for tissue damage
triggered by persistent high glucose levels in diabetic
patients as well as potential a therapeutic target (7,12). OS
is defined by the imbalance between the increase in reactive
oxygen species (ROS) generation and the decrease innative antioxidant mechanisms (3). ROS comprise a familyof oxygen metabolism-derived intermediates, superoxidebeing the foremost element, which can be further converted
in hydrogen peroxide and hydroxyl radical. Superoxide isalso responsible for the generation of peroxynitrite, which
results from its reaction with nitric oxide(13).
In living cells, several sources for superoxide were
described. Nicotinamide adenine dinucleotide phosphate(NADPH) oxidases and mitochondria are the major sources
for superoxide generation (15). Interestingly, Brodsky
demonstrated that under hyperglycaemic stress, in vitro,endothelial cells supress nitric oxide production andsuperoxide is generated at higher rates, via themitochondrial nitric oxide synthase (4).
Over the years, several fluorescent probes weredeveloped to adequately monitor ROS production, like
dihidroethidium (DHE) and mitochondrial targeted DHE –MitoSOX for superoxide, dichlorodihydrofluoresceindiacetate (DCF) for H202, dihydrorhodamine for
peroxynitrite (9). DHE is a freely permeable fluorescent dyethat can easily cross membranes and upon oxidation by

Received 2nd of September 2016. Accepted 3rd of October 2016. Address for correspondence: Danina Muntean, MD, PhD, Department of Pathophysiology,“Victor Babeș” University of Medicine and Pharmacy of Timişoara, 14, Tudor Vladimirescu Street, 300173 Timisoara, RO; tel/fax: +40-256-493085; e-mail:daninamuntean@umft.ro16

Fiziologia - Physiology • 2016.26.4(92)ROS forms ethidine and 2-hydroxyethidium. It wasdemonstrated that out of these two, 2-hydroxyethidium is a
specific product resulting only through superoxide oxidationof DHE; accordingly, the detection of the latter compoundappears to be a superior marker for the superoxide
quantification (20). Since the oxidized forms of DHE haveoverlapping emission spectra and, in order to separate thefluorescent signal, a specific setting for excitation was
documented: the first of the two excitation peaks of the DHE
seems to be specific for superoxide generated 2hydroxyethidium (396nm) (16).
We aimed to quantify the levels of ROS generated in the
diabetic kidneys, using the well-established model of
streptozotocin (STZ)-induced experimental diabetes in rat(2). Our main outcome was to characterize thecompartmentalization of ROS production in the different
structures of the kidney: glomeruli, blood vessels andtubules, using the confocal microscopy technique in order toquantify DHE fluorescence.

MATERIAL AND METHODSAll experimental procedures used in this research wereconducted according to the Romanian Law no.43/2014 andthe 2010/63/EU Directive regarding protection of theanimals used in scientific experiments, upon approval of theexperimental protocol by the Committee for Research Ethicsof the University of Medicine and Pharmacy “Victor Babes”
Timisoara.
Diabetes induction and evolution
We used the previously described streptozotocin
method to induce diabetes in adult male Wistar rats (n =12). A single IP injection (50mg/kg) of STZ wasadministered and glycemia values of > 200mg/dL detected
after 2 days in the blood sample harvested from the tailvein was considered a positive test for successful
induction of diabetes. The animals were monitored over a
period of 2 months, being housed in a controlledenvironment (22.5 +/- 2oC, alternating 12h light/dark cycles,
food and water ad libitum). On the day prior to organ
harvesting, total food restriction was instated.
Organ harvesting and imaging
Kidneys were removed under general anaesthesia andimmediately cryo-embedded in OCT (TissueTek). 20um
thick sections were cut using a cryostat (SLEE MTC) andincubated for 30 minutes with DHE (Sigma-Aldrich, D7008).
Control slides were created by incubating them with PBS
instead of DHE. The resulting slides were coverslippedusing Vectashield (Vector Labs) mounting medium andimmediately examined on an Olympus Fluoview FV1000
laser scanning confocal microscope (UPLSAPO 20x
objective (NA=0.75), 405nm laser excitation, barrier filter
position at 500nm, barrier filter range 100nm and 4us/pixel
Fiziologia - Physiology • 2016.26.4(92)sampling speed). Randomly selected images were acquired
from the cortical area of every kidney (Fig. 1a).

Fig. 1. a. Sample confocal image of DHE stained kidney. b. Sample
PAS stained kidney section showing no histological changes.

Image analysis
The first step in image analysis was backgroundcorrection to remove auto fluorescent signal. This wasdone in Olympus Fluoview F10-ASW 4.0 software, bysubtraction of the unstained images from the stained ones.
From every acquired image, sample areas representing
glomeruli, tubulo-interstitial and vascular compartments
were manually cropped. The images thus obtained werethen fed through a custom designed batch processing
algorithm in Icy bioimage analysis software (Fig.2). Briefly,
nuclei were segmented as regions of interest (ROI) andROI statistics were computed. We used the average
fluorescence intensity values to determine the meanvalues for nuclear staining intensity in each compartmentof the kidney cortex from every rat. Fluorescence intensity
was expressed in arbitrary units (AU).

Fig. 2. Custom designed protocol for batch analysis of DHE stained
kidney sections.

Statistical analysis included two-way ANOVA tests
performed in GraphPad Prism. A p value less than 0.05 wasconsidered significant. All values were expressed as means+/-SEM.
17

RESULTS
Diabetic state and body weight
All animals developed diabetes and the glycaemia levels
at the end of the experiment were  285mg/dl. The average
weight of the rats at the beginning of the experiment was
520  28g. At the end of the experiment, 6 rats had weights
up to 400g, while the remaining 6 were over 400g. Therefore,
we decided to separate them in 2 groups, under and above
400g, respectively.
DHE staining
The average fluorescence intensity was over 900 AU for
2/6 (33%) in the under 400g group, while in the over 400g
group all rats (6/6) had higher than 900 AU levels (Fig.3).

Fig. 3. ROS levels in different kidney compartments, expressed as
average values per rat. ANOVA test results are shown underneaththe rat’s weight, while significant Tukey’s multiple comparison test
results are displayed over the bars.

The analysis of variance for the three kidneycompartments (glomerular, vascular and tubulo-interstitial)
showed significance in 2 out of 6 rats under 400g (p=0.01,
respectively p=0.04), while for the over 400g group, 2 out of
6 rats presented higher significance values (p<0.001) (Fig.3)
The ANOVA analysis was completed with Tukey’s multiple
comparison test for individual variances in the kidneycompartments and it yielded significant differences between
the vascular and tubulo-interstitial compartment in only 1 of
the 6 rats under 400g group. In the over 400g group, 2 outof 6 rats presented very significant p values when comparing
the glomerular with the vascular compartment, respectively
glomerular versus tubulo-interstitial compartment.
Of note, in the <400g group, 4 out of 6 rats presented
higher ROS levels in glomeruli than in blood vessels, whilein the other group, vascular levels were higher thanglomerular in 4 out 6 rats and higher than tubular level in 5
out six rats.
The two-way ANOVA analysis of the 2 groups (below
and above 400g) showed very significant effect for the18

weight factor (p=0.006), while the kidney compartment
factor had no effect overall (p=0.8123).
Weight adjusted ROS levels
We computed weight adjusted values by dividing thedetected DHE fluorescence levels to the body mass of theanimal. The resulting values showed similar ROS levels forthe tubular compartment in both groups, while theglomerular and vascular DHE values were higher inthe >400g group. We noticed that the vascular compartment
had the most striking change, as it appear to produce thehighest levels of ROS (followed by glomeruli and then by thetubules in the >400g group. Interestingly, in the <400g group,
blood vessels displayed the lowest values for DHE
fluorescence no significant difference between the twogroups was recorded for the ROS generation in the tubulointerstitial compartment.

Fig. 4. Weight adjusted ROS levels in the different kidneycompartments

Optical microscopy
We detected no chronic changes in the kidneyparenchyma: absent glomerular nodular sclerosis, no
thickening of the glomerular and tubular basement
membranes, while the interstitium appeared to be within
normal limits, with no added inflammatory changes (Fig.1b).

DISCUSSION
The present study was purported to characterize thecompartmental distribution of ROS levels in the kidneys of
diabetic rats, using confocal microscopy to quantify
florescence intensity of DHE, a marker for superoxide.
Our major finding is that in STZ-induced diabetes in
Wistar rats, the weight of the animals at the end of theexperiment influenced the ROS levels detected in the
diabetic kidneys. In the presence of diabetes, all rats
became leaner over the two months period of diabetes
follow-up and this weight loss is in concordance with trends
previously described in literature (2). Interestingly, in ourhands, the rats with final body weight >400g had overall
Fiziologia - Physiology • 2016.26.4(92)higher ROS values in all compartments in comparison to
the <400g group. The compartmental distribution of ROS
was also different between the groups. We identified thatthe vascular compartment presented a lower contribution
to the overall ROS levels in the kidneys of the <400ggroup.
A fairly recent Cochrane meta-analysis, which took into
consideration studies on the impact of administering
antioxidants to chronic kidney disease patients (8),suggested that this therapy might slow the progression
towards end-stage renal disease in patients notundergoing dialysis. As medication dosage is generally
calculated in concordance to body mass, we normalized
the ROS levels to the body weight of an individual animal(wROS). The compartmental distribution analysis on thisnew parameter showed that there was little difference attubular level between the two groups, whereas in vascularand glomerular compartments higher levels of superoxide
occurred in the >400g group. Of note, the vascularcompartment, displayed the highest values of DHE
fluorescence in the "fat" rats and the lowest in the "lean"rats.
Quigley et al. have shown that in the setting of obesity,
the increased OS is a factor that elicits kidney injury (14).
Interestingly, in obesity, the lesions in the kidney covered adifferent spectrum as compared to diabetic kidney disease,glomerulomegaly
and
focal
and
segmental
glomerulosclerosis being the major components (5,10,17). It
was documented that weight loss improves kidney function
by decreasing total proteinuria and albuminuria (1,11).
Moreover, higher ROS levels have been proven to modify
the glomerular permeability (18). Our weight normalized
parameter - wROS is in line with the previous observations
and appears to indicate that weight reduction in diabetes is
associated with improved levels of superoxide in vascularand glomerular compartments of the kidney. In this respect,
it would be tempting to speculate that in the setting of type
II obese diabetics, weight loss could also elicit acompartment dependent-mitigation of OS.
Interestingly, a recent study published by Dugan et al.
(6), shows that in the setting of diabetes, the ROS levels in
the kidney are actually decreased, contrary to previous
literature findings. Corroborating these findings with our
results, we conclude that body weight plays a definite role inincreasing ROS levels in the diabetic rats, and this increase
particularly occurs in the vascular compartments, regardlesstheir location (interstitial or intra-glomerular – inside thecapillary tuft).
Of note, the histological examination of the formalin fixed,
paraffin embedded kidney samples showed no inflammatory
cell infiltrates in the interstitium. It has been postulated that the
infiltrating immune cells increase the local cellular ROS
generation (19). In this setting, we conclude that in our
experiment, the changes in ROS levels occurred, mostprobably, in response to the chronic hyperglycaemia.

Fiziologia - Physiology • 2016.26.4(92)CONCLUSIONSIn the STZ-induced diabetes in rats, ROS levels largely
varied across the different compartments of the kidney.
These levels showed a body mass - dependence, especiallyin glomeruli and blood vessels. In studies dealing withoxidative stress, we propose the normalization of ROS
values to body weight in order to provide a more accurateinterpretation of the results.
Acknowledgement. The confocal microscope used in this study
was purchased within the framework of the Hungary-Romania
Cross-Border Cooperation project HURO/0802/011_AF (HUROCARDIOPOL).

REFERENCES
1.

2.
3.
4.

5.
6.
7.
8.
9.

10.
11.

12.
13.

Afshinnia F, Wilt TJ, Duval S, et al.: Weight loss andproteinuria: systematic review of clinical trials andcomparative cohorts. Nephrol Dial Transplant.
2010;25(4):1173–83.
Akbarzadeh A, Norouzian D, Mehrabi MR, et al.: Inductionof diabetes by Streptozotocin in rats. Indian J Clin Biochem.
2007;22(2):60–4.
Betteridge DJ: What is oxidative stress? Metabolism.
2000;49(2 Suppl 1):3–8.
Brodsky S V., Gao S, Li H, et al.: Hyperglycemic switch frommitochondrial nitric oxide to superoxide production inendothelial cells. Am J Physiol Heart Circ Physiol.
2002;283(5):H2130-9.
D’Agati VD, Chagnac A, de Vries APJ, et al.: Obesity-related
glomerulopathy: clinical and pathologic characteristics andpathogenesis. Nat Rev Nephrol. 2016;12(8):453–71.
Dugan LL, You YH, Ali SS, et al.: AMPK dysregulation
promotes diabetes-related reduction of superoxide andmitochondrial function. J Clin Invest. 2013;123(11):4888–99.
Forbes JM, Coughlan MT, Cooper ME: Oxidative stress asa major culprit in kidney disease in diabetes. Vol. 57,
Diabetes. 2008. p. 1446–54.
Jun M, Venkataraman V, Razavian M, et al.: Antioxidants forchronic kidney disease. Cochrane database Syst Rev.
2012;10(10):CD008176.
Kalyanaraman B, Darley-Usmar V, Davies KJA, et al.:Measuring reactive oxygen and nitrogen species withfluorescent probes: Challenges and limitations. Free Radic
Biol Med. 2012;52(1):1–6.
Kambham N, Markowitz GS, Valeri AM, et al.: Obesityrelated glomerulopathy: An emerging epidemic. Kidney Int.
2001;59(4):1498–509.
Navaneethan SD, Yehnert H, Moustarah F, et al.: Weightloss interventions in chronic kidney disease: a systematic
review and meta-analysis. Clin J Am Soc Nephrol.
2009;4(10):1565–74.
Okamura DM, Pennathur S: The balance of powers: Redox
regulation of fibrogenic pathways in kidney injury. Redox Biol.
2015;6:495–504.
Poyton RO, Ball KA, Castello PR: Mitochondrial generationof free radicals and hypoxic signaling. Trends Endocrinol
Metab. 2009;20(7):332–40.

19

14.

15.
16.

17.

Quigley JE, Elmarakby AA, Knight SF, et al.: Obesity
induced renal oxidative stress contributes to renal injury insalt-sensitive hypertension. Clin Exp Pharmacol Physiol.
2009;36(7):724–8.
Ratliff BB, Abdulmahdi W, Pawar R, et al.: Oxidant
Mechanisms in Renal Injury and Disease. Antioxid Redox
Signal. 2016;25(3):119–46.
Robinson KM, Janes MS, Pehar M, et al.: Selective
fluorescent imaging of superoxide in vivo using ethidiumbased probes. Proc Natl Acad Sci U S A.
2006;103(41):15038–43.
Sharma SG, Bomback AS, Radhakrishnan J, et al.: The
Modern Spectrum of Renal Biopsy Findings in Patients with
Diabetes. Clin J Am Soc Nephrol. 2013;8(10):1718–24.

18.

19.

20.

Sverrisson K, Axelsson J, Rippe A, et al.: Acute reactive
oxygen species (ROS)-dependent effects of IL-1β, TNF-α,
and IL-6 on the glomerular filtration barrier (GFB) in vivo. Am
J Physiol Renal Physiol. 2015;309(9):F800-6.
Turkmen K: Inflammation, oxidative stress, apoptosis, andautophagy in diabetes mellitus and diabetic kidney disease:
the Four Horsemen of the Apocalypse. Int Urol Nephrol.
2016 Dec 29
Zielonka J, Kalyanaraman B: Hydroethidine- and
MitoSOX-derived red fluorescence is not a reliableindicator of intracellular superoxide formation: Anotherinconvenient truth. Free Radic Biol Med. 2010;
48(8):983–1001.

MASA CORPORALĂ INFLUENȚEAZĂ DISTRIBUȚIA
SPECIILOR REACTIVE DE OXIGEN ÎN DIFERITECOMPARTIMENTE ALE RINICHIULUI DE ȘOBOLAN
DIABETICREZUMAT
Introducere: Stresul oxidativ este larg acceptat ca fiind un important factor implicat in lezarea rinichiului in diabet. Noi am dorit
sa cuantificam productia de specii reactive de oxigen (ROS) in diferitele compartimente ale rinichiului de sobolan.
Material si metode: Diabetul zaharat a fost indus in sobolani Wistar (n=12) printr-o singura injectie intraperitoneala (50 mg/kg)
de streptozotocin. Dupa 2 luni, sobolanii au fost separati in doua grupuri, cu greutate sub, respectiv peste 400g. Rinichii au fostrecoltati, iar sectiunile facute la gheata au fost incubate cu dihidroethidium (DHE) si examinate cu un microscop confocal
Olympus Fluoview FV1000. Intensitatea fluorescentei a fost masurata folosind un protocol personalizat dezvoltat in programul
de analiza de imagini Icy.
Rezultate: La animalele cu greutate mai mica de 400g, 2/6 (33%) din sobolani au prezentat nivele medii de intensitate mai maride 900 AU, in timp ce in grupul celor cu greutate peste 400g, toti au prezentat nivele mai mari. In mod interesant, in grupul celorde peste 400g (spre deosebire de cei <400g), s-a identificat o productie mult crescuta de ROS la nivel vascular (urmat de celglomerular), ce au prezentat cele mai mari intensitati ale DHE. Nu am identificat diferente intre nivelele ROS la nivel tubular.
Concluzie: Rezultatele noastre sugereaza cu tarie faptul ca o masa corporala crescuta influenteaza productia renala de ROS
in diabet, in special in vase si glomeruli, observatie ce necesita studii suplimentare.
Cuvinte cheie: specii reactive de oxigen in rinichi, DHE confocal, nivele ROS ajustate la greutate, rinichi diabetic

20

Fiziologia - Physiology • 2016.26.4(92)BIOPRINTING: CURRENT TECHNIQUESAND APPLICATIONSHURMUZ MIHAI1, BOJIN FLORINA.2, MIU CATALIN.1, IONAC MIHAI. 3, TATU FABIAN1
1“Victor Babes” University of Medicine and Pharmacy Timisoara, XVth Department
2“Victor Babes” University of Medicine and Pharmacy Timisoara, IIIrd Department
3“Victor Babes” University of Medicine and Pharmacy Timisoara, Xth Department

ABSTRACT
Bioprinting is an evolving technology that uses 3d printing in order to create complex macro and micro structures capable ofreplicating and enhancing the healing capacity of the native body. Through the printing process different materials and cells canbe mixed in order to obtain artificial tissues and organs which in the future can represent an alternative to tissue transplant or
implants. We have reviewed the main techniques of cell bioprinting options and presented the advantages and drawbacks ofeach method.
Key words: biofabrication, 3d printing, tissue engineering

INTRODUCTION
Tissue engineering is a combination of different
scientific branches that combines engineer’s principles withmedical knowledge in order to repair damaged tissues, torestores specific functions of an organ or to replace it entirely[1-3]. The classical method implies the combination ofdifferent cells types with a scaffold to obtain an implantable
construct. The overall result assures mechanical features
similar to the native tissue, promote cell adhesions andproliferation [4]. Although in most of the cases the macro
structures resembles to the anatomical shape most of thetimes the internal architecture lacks the complexity of natural
tissues or organs. Common techniques for fabricating
tissue engineering scaffolds such as gas foaming, solventcasting, fiber bonding, phase separation, particulateleaching, and freeze drying provide macroscale scaffold but
provide limited accuracy in cell placement, cell density,
enable proper cell connectivity. The micro and nanostructure of a design is equally important as the large scale.
All these inconveniences have determined the scientists tosearch for other solutions.
Bioprinting has been intensely studied after the
successful transformation in the early 2000s of a traditional
ink jet printer when cells replaced the traditional ink from the
cartridges [5]. In the recent years many publications havedocumented research in the 3d printing field (Fig.1).
Bioprinting can be defined as an “innovative technology thatallows for the generation of organized 3D tissue constructs

via a layer-by-layer deposition process that combines cells
and biomaterials in an ordered and predetermined way” [6].
One of the major advantages of 3D bioprinting is thatpatient-specific information can be directly incorporated into
the biofabrication process in order to generate anatomicallycorrect shapes [7].

Fig. 1. Number of publications related to 3D printing according to
PubmedBIOPRINTING TECHNIQUESThere are many bioprinting techniques available. We
have tried to classify them and present there advantages
and disadvantages in order to better understand the current
status of knowledge. During the bioprinting process care

Received 22nd of September 2016. Accepted 13th of October 2016. Address for correspondence: Mihai Hurmuz, MD, PhD student, , Department XV, “VictorBabeș” University of Medicine and Pharmacy of Timişoara, Eftimie Murgu No. 2A, 300041-RO, Timisoara; tel/fax: +40-256-490507; e-mail:hurmuz_mihai@yahoo.com

Fiziologia - Physiology • 2016.26.4(92)21

must be taken in order to assure cell viability and cellfunction after the printing process has ended.

Fig. 2. Classification of bioprinting techniques

INKJET Bioprinting

type of printer uses crystal that under an electric charge
generate a vibration that will determine the ink to bediscarded from the nozzle [16].
The heating process presumably affects the quality andviability of the cells [15]. However in 2014 Lorber et al proved
using adult retinal ganglion cells and glia rat cells in apiezoelectric inkjet that the process has no side effects oncell characteristics [17].
Boland et al transformed a normal inkjet printer into
bioprinter and managed to print multiple layers of cells andpolymers [18]. Other groups of researchers have managed
to adapt multiple printing heads to one printer combining
several types of cells and polymers thus obtaining complex
structures [19-21]. Watanabe et al, Cambell et al, Roda et al
printed growth factors, proteins and biological cells using
inkjet machines [22-24].
In order to be able to be printed the viscosity of thesubstances used needs to be below 0,1 Pa s-1 [25]. The sizeof the droplet can vary from 1 to 300 picoliters and theamount of droplets per second can be up to 10000 [8].
LASER bioprinting

Fig. 3. Schematic representation of inkjet bioprinting

The bioprinting process with ink consists of adding cells
to different biomaterials and through a nozzle discarded inlayers to form the final construct. The printing processcontrols very accurate the amount of substances released
and so very sophisticated forms can be created [8,9]. Oneof the reasons for the extensive us of this technique is the
speed of the printing, the non-contact procedure, theresolution obtain and the reduced cost [10,11]. The
increased speed poses though some problems regarding
the material that can be used. The polymers used need totransform form liquid to solid, to gelatinate, very quickly, inorder to be able to maintain the shape of printing [12]. During
the gelatination phase chemical crosslinking is used and it
can be physical, chemical or through ultraviolet methods
obtained.
The thermal inkjet system contains the ink reservoir anda heating plate. The raising of the heat in the metal plate
generates bubbles which will determine the drop of thesubstances from the reservoir [13-15]. The piezoelectric22

Fig. 4. Schematic representation of laser bioprinting

Laser technology for bioprinting was used by Odde et alin 1999 for the first time [26]. Laser-based direct writing(LDW) is one of the techniques on which a laser pulse
determines the movement of the cells, each at a time, fromthe reservoir to a collector base. The laser pulse provokes
the generation of a bubble which forces the exit of the cells
from the donor slide to the collector slide. The LDW
techniques can be divided supplementary in laser-induced
forward transfer (LIFT) and matrix-assisted pulsed laser
evaporation direct writing (MAPLE DW). All those laser
procedures allow an accurate distribution of cells and so
Lothar et al, Wang et al and Riggs et al managed to print
skin cell(fibroblasts), human mesenchymal stem cells andFiziologia - Physiology • 2016.26.4(92)biopolymers [27-29]. The viability of skin cells after printing
was 98% while for mesenchymal stem cells was 90%.
Nathan et al. studied different types of cells (dermal, neural,pulmonary, cancer) from different sources (humans, rats,
mice, bovine) and concluded that laser printing is an efficientand consistent technique in observation collagen formation,
advancement of cancer and growth of neural cell [30].
The technique has no limitations regarding the viscosityof the gels unlike inkjet printing. So using high viscosity
bioink can also be employed [31,32]. The precision of thebioprinting method has permitted Gaebel et al. toincorporate human umbilical vein endothelial cells on apolyester urethane cardiac patch and proved cell efficiency
after several weeks [33]. By printing mammalian cells someauthors have proved that stacks of cells of 50 microns canbe obtained [34]. Guillotin et al. proved also that highdensity microscale construct can be printed with the laser
practice [32].
Like every method employed the laser technology has
its own side effects. The heat and pulse generated by thelaser can alter the functionality and the outcome of theconstruct [32,35]. Laser printing techniques provide themost accurate and reliable cell placement but is limited in
producing height for the scaffolds.
Extrusion bioprinting

Fig. 5. Schematic representation of extrusion bioprinting

Extrusion bioprinting uses force, pneumatic or mechanic,
to liberate the bioink from a dispenser. The piston
mechanism allows a better control over the amount of liquid
been dispensed. The bioink used for this process needs tohave minimal resistance under flow. After dispense of the
ink the composite is crosslinked by light thus allowing for 3d
constructs with a continuous deposition of ink.
In a 2005 study Yan et al. printed artificial liver tissue
using hepatocytes and adipose derived cells [36]. Sun andLee proved that cell viability is directly correlated by theconcentration of the ink, the pressure of the piston andnozzle geometry [37]. In latest studies researchers havereached angiogenesis by incorporating endothelial cells intoa fibrin network filled with support cells [38]. Billiet et al. in a
recent study ended up with an artificial liver made up out ofhepatocytes and gelatin methacrylamide hydrogel and with
Fiziologia - Physiology • 2016.26.4(92)a more than 95% cell viability after the printing process [39].
Horvath et al. realized a very accurate blood-air barrier in a
printed lung tissue [40].
Pati et al. printed human adipose derived mesenchymal
stem cells onto a decellularized matrix components ofadipose tissue, being able to create various shape ofstructures and moreover proved that bioprinted cells havemore important adipogenic differentiation capacity [41].
The extrusion printing process has great speed and the
hardware required in not as expensive as in other methods.
Through this techniques a wide variety of bioinks, including
cell aggregates, cell-laden hydrogels, micro-carriers anddecellularized matrix components can be utilized [42-47].
Although is a very convenient method, easy to use andadaptable, the extrusion process has some disadvantages
also. The minimum resolution is over 100 microns and socells can be grossly printed thus not allowing very finedeposition of cells. Hydrogel cannot be used due togelatination and solidification process.

FUTURE DIRECTIONS
We are facing a rapid development of tissue engineering
techniques and processes and 3d printing is among them.
The new research has led to creation of tissue grafts andwhole organs. The scientist managed to replace parts ofintestines, to replace bladder or to help drug delivery [48-50].
There are still things that need to be sorted out such as
quality and resolution of cell deposition, obtain both nanostructure and macro structure that would resemble thenative tissue, improve the speed of creation of structures.
The current material used for scaffolds such as collagen,gelatin, hyaluronic acid, alginate should be improved and beaccompanied be new ones which respect some rules like:biocompatibility, promote cell differentiation and cell
multiplication.
The new lines to be investigated could include in situ
printing to allow quicker and more reliable healing andrehabilitation outcomes.

REFERENCES
1.
2.
3.
4.

Langer R, Vacanti P: Tissue Engineering. Science
1993; 260(5110): 920–926.
Bonassar J, Vacanti A: Tissue Engineering: The First
Decade and Beyond. J. Cell. Biochem. Suppl 1998;
72(30): 297–303.
Griffith G, Naughton G: Tissue Engineering—Current
Challenges and Expanding Opportunities. Science
2002; 295(5557):1009–1014.
Dietmar H, Michael S, Makarand R: Scaffold-BasedTissue Engineering: Rationale for Computer-Aided

23

5.
6.
7.
8.
9.
10.

11.

12.

13.
14.

15.

16.

17.

18.
19.

20.

24

Design and Solid Free-Form Fabrication Systems.
Trends Biotechnol 2004; 22(70):354–362.
Wilson W, Boland T: Cell and Organ Printing 1: Protein
and Cell Printers. Anat. Rec. 2003; 272A(2):491–496.
Claudia Di B, Amanda F, Davide M, et al.: Front Surg.
2015; 2: 39.
Ibrahim O, Hemanth G: A review on design forbioprinting. Bioprinting 2016; (3) 1–14.
Murphy SV, Atala A: 3D bioprinting of tissues andorgans. Nature Biotechnology 2014; 32, 773.
Xu T, Jin J, Gregory C, Hickman JJ, Boland T: Inkjet
printing of viable mammalian cells. Biomaterials 2005;
26, 93.
Schuurman W, Levett P, Pot M, van Weeren P, et al :Gelatin-Methacrylamide Hydrogels as Potential
Biomaterials for fabrication of TissueEngineered
Cartilage Constructs. Macromolecular Biosciences
2013; 13, 56.
Billiet T, Gevaert E, De Schryvwer T, Cornerlson M:
The 3D printing of gelatin methacrylamide cell-laden
tissue-engineered constructs with high cell viability.
Biomaterials 2014; 35, 49.
Kolesky D, Truby R, Gladman S, Busbee T, Homan K,
Lewis JA: 3D Bioprinting of Vascularized,
Heterogeneous Cell-Laden Tissue Constructs.
Advanced Materials 2014; 26, 3124.
Le Hue P: Progress and Trends in Inkjet Printing
Technology. J. Imaging Sci. Technol. 1998;
42(1):49–62
Dababneh A B, Ozbolat I T: Bioprinting Technology: A
Current State-of-the-Art Review. Journal of
Manufacturing Science and Engineering 2014; 136,
061016,
Kesti M, Muller M, Becher J, Schnabelrauch, M, et al:
A versatile bioink for three-dimensional printing ofcellular scaffolds based on thermally andphototriggered tandem gelation. Acta Biomater 2015;
11, 162.
Stachowiak J, Richmond D, Li T, Brochard F, Fletcher
A: Inkjet Formation of Unilamellar Lipid Vesicles for
Cell-Like Encapsulation. Lab Chip 2009 9(14): 2003–2009
Barbara L, Wen-Kai H, Ian M, Keith R: Adult Rat
Retinal Ganglion Cells and Glia Can Be Printed by
Piezoelectric Inkjet Printing. Biofabrication 2014; 6(1):015001.
Wilson W, Boland T: Cell and Organ Printing 1: Protein
and Cell Printers. Anat. Rec. 2003; 272A(2): 491–496.
Phillippi JA, Miller E, Weiss L, et al:
Microenvironments Engineered by Inkjet Bioprinting
Spatially Direct Adult Stem Cells Toward Muscle- andBone-Like Subpopulations. Stem Cells 2008; 26, 127.
Ker D F E, Chu B, Phillippi J A, et al: Engineering
Spatial Control of Multiple Differentiation Fates withina Stem Cell Population. Biomaterials 2011; 32, 3413.

21.

22.
23.
24.

25.
26.
27.
28.

29.
30.

31.
32.

33.

34.
35.
36.
37.

Cooper G, Miller E D, Decesare G E, et al: Inkjetbased biopatterning of bone morphogenetic protein-2
to spatially control calvarial bone formation. Tissue
engineering Part A 2010; 16, 1749.
Roda A, Guardigli M, Russo C, Pasini P, Baraldini M:
Protein Microdeposition Using a Conventional Ink-Jet
Printer. Biotechniques 2000; 28(3): 492–496.
Watanabe K, Miyazaki T, Matsuda R: Fabrication of
Growth Factor Array by Color Inkjet Printer. Zool. Sci.
2003; 20(4): 429–434.
Campbell P, Miller E, Fisher G, et al: Engineered
Spatial Patterns of FGF-2 Immobilized on Fibrin Direct
Cell Organization. Biomaterials 2005; 26(33): 6762–
6770.
Calvert P: Inkjet Printing for Materials and Devices.
Mater. Sci. Eng. 2001;13(10): 3299–3305.
Odde D J, Renn M J: Laser-guided direct writing for
applications in biotechnology. Nanotechnology 1999;
17, 385.
Lothar K, Stefanie K, Heiko S, et al: Laser Printing ofSkin Cells and Human Stem Cells. Tissue Eng. 2010;16(5): 847–854.
Wang W, Li G, Huang Y: Modeling of Bubble
Expansion-Induced Cell Mechanical Profile in LaserAssisted Cell Direct Writing. Journal of Manufacturing
Science and Engineering 2010; 1312009.
Riggs BC, Dias AD, Schiele NR, et al: Matrixassisted
pulsed laser methods for biofabrication. MRS Bulletin
2011; 36, 1043
Nathan R, Ryan A, David T, et al: Laser Direct Writingof Combinatorial Libraries of Idealized Cellular
Constructs: Biomedical Applications. Appl. Surf. Sci.
2009; 255(10): 5444–5447
Devillard R, Pages E, Correa MM, et al: Patterning by
Laser-Assisted Bioprinting. Methods in Cell Biology
2014; 119, 159.
Guillotin B, Souquet A, Catros S, et al: Laser assisted
bioprinting of engineered tissue with high cell density
and microscale organization. Biomaterials 2010; 31,
7250.
Gaebel R, Mn N, Liu J, Guan J,et al: Patterning
Human Stem Cells and Endothelial Cells With Laser
Printing for Cardiac Regeneration. Biomaterials 2011;
32(35): 9218–9230.
Barron J, Ringeisen B, Kim H, et al: Application of
Laser Printing to Mammalian Cells. Thin Solid Films.
2004; 453(383): 383–387
Guillemot F, Souquet A, Catros S, et al: Highthroughput laser printing of cells and biomaterials fortissue engineering. Acta Biomaterialia. 2010; 6, 2494.
Yan Y, Wang X, Pan Y, et al: Fabrication of Viable
Tissue-Engineered Constructs With 3D Cell-AssemblyTechnique. Biomaterials 2005 26(29): 5864–5871.
Nair K, Yan K, Sun W: A Multi-Level Numerical Modelfor Qunatifying Cell Deformation in Encapsulated

Fiziologia - Physiology • 2016.26.4(92)

38.

39.

40.
41.
42.
43.

Alginate Structures. J. Mech. Mater. Struct. 2007; 2(6):1121–1139.
Lee V, Lanzi A, Ngo H, Yoo SS, et al: Generation ofMulti-Scale Vascular Network System Within 3D
Hydrogel Using 3D Bio-Printing Technology. Cell. Mol.
Bioeng. 2014; 7(3): 1–13.
T Billiet, E Gevaert, T De Schryver, M Cornelissen, P.
Dubruel: The 3D printing of gelatin methacrylamidecell-laden tissue-engineered constructs with high cell
viability. Biomaterials 2014; 35 49e62.
L Horv ath, Y Umehara, C Jud, et al: Engineering an
in vitro air-blood barrier by 3D bioprinting. Sci. Rep.
2015; 5 7974.
F Pati, D Ha, J Jang, HH Han, J Rhie, DW Cho:
Biomimetic 3D tissue printing for soft tissue
regeneration. Biomaterials 2015; 62: 164-175.
JHY Chung, S Naficy, Z Yue, et al: Bio-ink properties
and printability for extrusion printing living cells.
Biomater. Sci. 2013; 1 763.
S Khalil, W Sun: Bioprinting endothelial cells withalginate for 3D tissue constructs. J. Biomech. Eng.
2009; 131 111002.

44.
45.
46.
47.
48.

49.
50.

J Malda, J Visser, FP Melchels, et al: 25th anniversary
article: engineering hydrogels for biofabrication. Adv.
Mater. 2015; 25: 5011-5028.
SV Murphy, A Skardal, A Atala: Evaluation ofhydrogels for bio-printing applications. J. Biomed.
Mater. Res. Part A. 2013; 101A: 272-284.
R Levato, J Visser, J Planell, et al: Biofabrication oftissue constructs by 3D bioprinting of cell-laden
microcarriers, Biofabrication 2014; 6 35020.
F Pati, J Jang, DH Ha, et al: Printing three-dimensional
tissue analogues with decellularized extracellularmatrix bioink. Nat. Commun 2014; 5 3935.
Choi RS, Vacanti JP: Preliminary studies of tissueengineered intestine using isolated epithelial organoid
units on tubular synthetic biodegradable scaffolds.
Transplantation proceedings 1997; 29, 848.
Atala A: Tissue engineering of human bladder. British
Medical Bulletin 2011; 97, 81.
Figallo E, Cannizzaro C, Gerecht S, et al: Microbioreactor
array
for
controlling
cellularmicroenvironments. Lab on a chip 2007; 7, 710.

BIOIMPRIMAREA: TEHNOLOGII ACTUALE ȘI APLICAȚIIREZUMAT
Bioprintarea este o ramura tehonologica in plina dezvoltare care utilizeaza imprimarea tridimensionala pentru a crea structurecomplexe micro si macroscopice cu rol in replicarea si sporirea capacitatilor de vindecare ale organismului. Prin procesul deprintare diferite material si celule sunt amestecate in vederea obtinerii tesuturilor si organelor artificiale care in viitor vor
reprezenta o alternativa la transplantul tisular sau la implanturile existente. Ne-am aplecat atentia asupra tehnicilor principalede bioimprimare celulara prezentandu-le avantajele si dezavantajele.
Cuvinte cheie: inginerie tisulara, imprimare 3d, bioimprimare

Fiziologia - Physiology • 2016.26.4(92)25HISTOCHEMICAL AND HEAMATOLOGICAL STUDY
OF THE EFFECTS OF ETHANOLIC LEAF EXTRACT C.
ZAMBESICUS ON THE STOMACH IN DIABETIC
RATS
D. A OFUSORI*, O. A KOMOLAFE, O. S ADEWOLE
Department of Anatomy and Cell Biology, Faculty of Basic Medical Sciences, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria.

ABSTRACTThe aim of this study was to verify the histochemical and haematological effects of C. zambesicus on the stomach in STZinduced wistar rats.
Forty adult wistar rats were divided into four groups n=10. Group A serves as negative control animals receiving equivalentamount of citrate buffer, group B animals were induced with 65mg/kg of STZ, group C were pretreated with C. zambesicus leaf
extract two weeks before STZ induction and the treatment continued throughout the study while group D was administered with
C. zambesicus leaf extract four weeks after STZ induction. At the expiration of the study, the animals were sacrificed by cervicaldislocation. The blood samples were collected for haematological analysis while the stomachs were excised and fixed in 10%
buffered formaline for the demonstration of connective tissue fibres (collagen and elastic) using Verhoeff’s vanGieson staining
and histochemical analysis for detection of glycogen deposit using PAS staining with diastase control.
The result showed that the ethanolic leaf extract of C. zambesicus was able to prevent hyperglycemia and also restore
connective tissue fibres, glycogen deposit and haematological parameters to near normal in adult wistar rats.
We therefore concluded that C. zambesicus exhibited antidiabetic potentials and thus recommend it for further studies aimed
at corroborating these findings in the quest to finding a safer alternative product in the management gastric disorders resulting
from diabetes.
Key words: C. zambesicus, Diabetes, Stomach, Histochemistry, Haematology, Connective tissue fibers

INTRODUCTION
Diabetes is the failure of the body to metabolize blood
sugar properly; it is now progressing very rapidly among theAmericans and other developed countries [1]. In developing
nations, diabetes is gradually becoming a deadly disease as
they switch to westernized lifestyles [1]. Gastric tissuecompromise has been implicated in diabetes [2]. At least 75%
of diabetic patients have significant gastrointestinal
symptoms [3,4]. It therefore becomes very important tostudy gastric complications resulting from diabetes and also
try to unearth the possible remedy to these pathologic
conditions. Some of the solutions of diabetic-mediatedcomplications come with the treatment of diabetes itself
while in some cases; diabetes becomes alleviated living
behind the complications such as gastric disorders [5]. Mostof the gastrointestinal manifestations in patients have been
attributed to disordered motor function [2]. Streptozotocin(STZ) induction of diabetes is an experimental model widelyused to study glycemic and lipidemic changes in plasma [6].

In mammalian cells, the mechanism of action of STZ that
results in cell death has not been fully identified, but isthought to be a result of DNA and chromosomal damage
brought forth by mechanisms involving free radical
generation during STZ metabolism [1].
The extract of C. zambesicus has been documented to
produce a significant reduction in blood glucose level after asingle dose of the extract (150mg/kg) and in prolonged
treatment for 7 days [7]. Also Ofusori et al., [8] has reported
an improvement in gastric emptying following theadministration of C. zambesicus in adult wistar rats. Ngadjui
et al., [9] has successfully identified some very importantcompounds in C. zambesicus which include abiatane,
diterpenoids, quinines, triterpenoids, flavonoid, Labdane,clerodane and trachylobane.
In recent times, traditional therapy has been employedby different researchers with a view to finding lasting andsafer solutions not only to diabetes but also thecomplications that come along with it. It is in this view we set

Received 26th of October 2016. Accepted 3rd of November 2016. Address for correspondence: David Ofusori, Department of Anatomy and Cell Biology,
Faculty of Basic Medical Sciences, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria; phone: +234-803-445-5715; e-mail: davidofus234@yahoo.com

26

Fiziologia - Physiology • 2016.26.4(92)out to assessing the histochemical and heamatologicaleffects of C. zambesicus in the prevention and restoration ofgastric integrity in diabetic rats.

Group A: Control rats administered intraperitoneally with
0.1 M sodium citrate buffer (pH 4.5)

MATERIALS AND METHODSGroup C: Diabetic rats but in which C. zambesicus leaf
extract (200 mg/kg body weight/day/rat) in 10%
tween 80 therapy started 2 weeks prior toinduction and continued throughout the period
the experiment lasted (8 weeks).

Materials
Streptozotocin (STZ) was purchased from Tocris
Bioscience, UK.. tween 80 was purchased from sigma
chemical company, St Louis, Missouri. USA.
Animal Care
Forty adult male albino rats of the Wistar strain were
procured and acclimatized for two weeks at the Animal
Holdings of the College of Health Sciences, Obafemi
Awolowo University, Ile-Ife, Nigeria before thecommencement of the research work. Animals were fed withstandard rat feed (Capfeeds, Ibadan) and given water
liberally.
All the animal experiments were conducted inaccordance with the “Guide for the Care and Use ofLaboratory Animals” prepared by the National Academy of
Sciences and published by the National Institutes of Health,USA [10].
Preparation of Plant Extract
The leaves of C. zambesius Müll. Arg. (Euphorbiaceae)
were collected and authenticated at the Botany Department,
Obafemi Awolowo University where specimen sample withnumber (UHI 16511) was deposited. The fresh leaves of theplant were air dried and grinded using squeezing andcrushing machine (Daiki Rika Kogyo Co-ltd, Japan).
Thereafter, the ethanolic extract was obtained. The extract
obtained was partitioned between dichloromethane andwater. The dichloromethane fraction was dissolved in 10%
tween 80 and administered orally at a dose of 200 mg/kg as
the plant extract.
Induction of experimental diabetes
All the animals were fasted for 16-h, but still allowed freeaccess to water throughout. At the end of the fasting period– taken as 0h, initial glycemia of all animals were determined
and recorded. The animals in groups B, C and D were
induced with streptozotocin (65mg/kg body weight)
dissolved in 0.1M sodium citrate buffer (pH 4.5). These
animals were stabilized for four weeks after which the leaf
extract in aqueous solution was administered orally throughgavages at a concentration of 200 mg/kg bodyweight/rat/day to group D for another 4 weeks. Animals ingroup C were pretreated with C. zambesicus therapy 2
weeks prior to induction of diabetes.
Experimental Design
The animals were divided into four groups as follows,with ten animals in each group.
Fiziologia - Physiology • 2016.26.4(92)Group B: Diabetic rats administered orally with 10% tween
80 for 4 weeks after the initial four weeks ofdiabetic induction.

Group D: Diabetic rats administered orally with C.
zambesicus leaf extract (200 mg/kg bodyweight/day/rat) in 10% tween 80 for 4 weeks
after the initial four weeks of diabetic induction.
Animal sacrifice
Twenty-four hours after the last administration, all animals
were sacrificed by cervical dislocation.
Elastic and collagen fibers analysis
Stomach from each of the animals were excised andfixed in 10% Neutral buffered formalin and Verhoeff vanGieson stain was used for demonstration of collagen andelastic fibres.
Histochemical procedure
A periodic Acid Schiff (PAS) with diastase control
method by Gomori (1946) was used to demonstrate
Glycogen. This included fixation in 10% Neutral bufferedformalin; thereafter, the specimen were deparaffinized andhydrated to water, treated with periodic acid, aqueous for 5
minutes, washed in running water for 5 min, treated with
Schiff reagent for 10 min, transfered through sulfite solutions
three changes for 1.5-2 minutes each, washed in runningwater for 5 min, counterstained, dehydrated, cleared andmounted with DPX for permanent storage.
Haematological procedure
Haematological Analysis was performed using
automated heamatology analyzer (Sysmex KX-21N, USA).
Blood samples were drawn using syringes containing the
10%-EDTA from the heart of the animals placed under slightchloroform anesthesia. Average period of 1min was adopted
in this procedure to reduce stress. Blood samples were
thereafter transferred into EDTA K2 vacuum tube
(GD040EK). The following parameters were analysed: whiteblood cell (WBC), red blood cell (RBC), heamoglomin
concentration (HGB), mean cell volume (MCV), Mean
corpuscular heamoglobin (MCH), Mean heamoglobim
concentration (MCHC), platelet (PLT), lymphocytes (LYM),
neutrophil (NEUT), red blood cell distribution weight(RDW_SD), red blood cell distribution weight (RDW_CV),
Platelet distribution weight (PDW), mean platelet volume(MPV).
27

Photomicrography
All the prepared slides were examined and archived at
different magnification under a LEICA research microscope(LEICA DM750, Switzerland) with a digital camera attached(LEICA ICC50).

the untreated diabetic group (Figure 2). The histologicaloutline is comparable with the groups pretreated with
200mg/kg of C. Zambesicus.

Statistical analysis
Data were expressed as Mean ± Standard Error of
Mean (S.E.M). The statistical significance was evaluated by
one-way analysis of variance (ANOVA) using SPSS version
17.0 (SPSS, Cary, NC, USA) with Duncan's Multiple Range
Test (DMRT) option. A value of p<0.05 was considered toindicate a significant difference between groups.

RESULTS
Blood Glucose Level (BGL)
The result from this study showed that there was asignificant increase in the BGL in the untreated diabetic
group when compared with the negative control (Fig. 1).
Group C that was pretreated with C. zambesicus prevented
the elevation of BGL while the group treated with C.
zambesicus for 4 weeks reduced BGL to near normal as
shown in Figure 1.

Fig. 1. Showing the antihyperglycemic effect of C. Zambesicus indiabetic rats.

Collagen and Elastic Fibers
There was general reduction in the elastic and collagenfibers in the untreated diabetic group when compared withthe control animals. With early commencement of extract
administration two weeks prior to STZ induction, there were
evidences of increased concentration of elastic fibers in themuscularis externa as well as mucosa and increased
concentration of collagen fibers in the submucosa andmucosa when compared with the untreated diabetic groupand control (Figure 2). Administration of the extract for fourweeks after four weeks of diabetic stabilization (group D)
also showed an increase in the concentration of elastic
fibers in the muscularis externa and mucosa as well as
increased concentration of collagen fibers in the submucosa
and mucosa when compared with histological sections of28

Fig. 2. Photomicrograph of the transverse section of the stomachof (a) Control group. Note the high deposits of collagen fibers in the
submucosa (SM) and elastic fibers in the muscularis externa (ME).
(b) Untreated diabetic group. Note the depletion of collagen fibers
in the submucosa (SM) and elastic fibers in the muscularis externa(ME). BLUE ARROW-Collagen fibers, RED ARROW-Elastic fibers(C) Note the preservation of collagen fibers in the submucosa (SM)and elastic fibers in the muscularis externa (ME) (d) Note the few
strands of collagen fibers in the submucosa (SM). BLUE ARROWCollagen fibers, RED ARROW-Elastic fibers VVG x100.

Histochemical Analysis
The examination of the stomach for PAS staining in all
animal groups suggest that the stomach of untreateddiabetic group showed an increase in the glycogen deposit
when compared with the control animals (Figures 3 & 4).
With early commencement of extract administration two
weeks prior to STZ induction, there was an evidence of
reduction in glycogen deposit as marked by decreased PAS
positive staining characterized by purple granules whencompared with the untreated diabetic group and control(Figures 4 & 5). Administration of the extract for four weeks
after four weeks of diabetic stabilization (Figure 6) also
showed a reduction in glycogen deposit in a similar fashion
with the group with early commencement of extract
administration (Figures 5 & 6).

Fig. 3. Photomicrograph of the transverse section of the stomachof Negative control animals showing (a) Diastase control. (b)Normal staining. PAS x100
Fiziologia - Physiology • 2016.26.4(92)Fig. 4. Photomicrograph of the transverse section of the stomachof untreated diabetic animals showing (a) Diastase control. Notethe depletion of glycogen (b) Normal staining. Note theaccumulation of glycogen deposits as evident by the high staining
intensity. PAS x100

Fig. 5. Photomicrograph of the transverse section of the stomachof animals pretreated with C. zambesicus before STZ induction (a)Diastase control. (b) Normal staining. PAS x100

Fig. 6. Photomicrograph of the transverse section of the stomachof animals treated with C. zambesicus four weeks after STZ
induction (a) Diastase control. (b) Normal staining. PAS x100

Heamatological Analysis
White blood cell profiles
WBC
The WBC in the untreated diabetic animals (group B)
was significantly lowered by 44.31% when compared withthe control group (group A). Early commencement of extract
administration two weeks prior to STZ induction significantly(p<0.05) raised the WBC by 43.50% (Table I).
Treatment of diabetic animals with extract for four weeks(group D) significantly (p<0.05) raised the WBC count by
96.35% when compared with untreated diabetic group(group B).

Table I. Effects of C. Zambesicus on the Heamatological Parameters in STZ induced Diabetic rats

Values are given as mean ± SEM for each haematological parameters in each group. a, b, c, d, ab, bc, cd, abc, bcd within column signifies
that means with different letters differs significantly at p < 0.05 while means with the same letters does not differ significantly at p < 0.05
(using one way ANOVA with Duncan multiple range test).

LYM and NEUT
The LYM in the untreated diabetic animals (group B) was
significantly (p<0.05) increased by 29.12% while the NEUT
was lowered by 46.81% when compared with the control
group. Early commencement of extract administration two
weeks prior to STZ induction slightly (p>0.05) lowered the
LYM count by 3.39% while the NEUT count was slightly(p>0.05) raised by 13.24% (Table I).

Fiziologia - Physiology • 2016.26.4(92)Treatment of diabetic animals with extract for four weeks(group D), raised (p>0.05) the LYM count by 7.35% andreduced the NEUT count by 28.68% when compared withuntreated diabetic group (group B).
Red Blood Cell Indices
RBC, HGB and HCT
RBC, HGB and HCT counts were significantly (p<0.05)
increased in untreated diabetic group (group B) when29compared with the control (Table 16). These values wereincreased by 19.67%, 5.08% and 4.96% respectively withearly commencement of extract treatment two weeks priorto STZ-induction. Treatment of diabetic animals with extract
for four weeks after four weeks of diabetic stabilization(group D) presented 11.55% increament in RBC as well as
1.59% and 2.94% decreament in HGB and HCT
respeactively as compared with the untreated diabetic group(group B).
MCV, MCH and MCHC
The MCV and MCH count was significantly (p<0.05)
increased in untreated diabetic group (group B) while MCHC
was significantly decreased (p<0.05) when compared withthe control (Table I). MCV and MCH values were decreasedby 12.38% and 12.03% respectively with earlycommencement of extract treatment two weeks prior to STZ
induction while MCHC was increased by 0.03% whencompared with the untreated diabetic group. Treatment ofdiabetic animals with extract for four weeks after four weeks
of diabetic stabilization also presented a 11.55% and11.78% decreament in MCV and MCH respectively while
MCHC presented a decement as compared with theuntreated diabetic group (group B).
RDW_SD and RDW_CV
The RDW_SD and RDW_CV count was significantly(p<0.05) increased in untreated diabetic group (group B)
when compared with the control (Table I). RDW_SD value
was decreased by 15.29% while RDW_CV was increased
by 4.72% with early commencement of extract treatment two
weeks prior to STZ induction. Treatment of diabetic animals
with extract for four weeks after four weeks of diabetic
stabilization also presented a 16.29% decreament in
RDW_SD and 5.00% increament in RDW_CV as comparedwith the untreated diabetic group (Group B).
Platelet profile
PLT
The PLT count was significantly (p<0.05) increased in
untreated diabetic group (group B) when compared with thecontrol (Table I). This was reduced by 31.38% with earlycommencement of extract treatment two weeks prior to STZ
induction. Treatment of diabetic animals with extract for fourweeks after four weeks of diabetic stabilization alsopresented 11.55% decreament in PLT as compared with theuntreated diabetic group (group B).
PDW and MPV
The PDW and MPV count was increased in untreateddiabetic group (group B) when compared with the control(Table I). These values were reduced by 19.67% and 5.08%
respectively with early commencement of extract treatment
two weeks prior to STZ induction. Treatment of diabetic
animals with extract for four weeks after four weeks ofdiabetic stabilization also presented a reduction in PDW and30

MPV as compared with the untreated diabetic group (groupB) (Table I).

DISCUSSION
Our finding showed that C. zambesicus reduced the
BGL significantly and also prevented the elevation in thegroup pretreated before diabetic induction. This observation
is in inline with previously documentations [11,12] Thisfurther established that C. zambesicus, if properly adopted
in our diet can serve as preventive measure for
hyperglycemia.
Positive staining indicating the presence of collagen andelastic tissue fibers were observed in varying degrees ofintensity in the stomach of the negative control. These fibers
were drastically depleted in the untreated diabetic group.
The tunica mucosa is composed of epithelium, a lamina
propria (of collagen, elastic and reticular fibers) and a
muscularis mucosae. The submucosa contains collagenfibers, fat and the submucosal nerve plexuses [13]. The
tunica muscularis externa has two coats: an inner circularand an outer longitudinal layer. The reduction in the staining
intensity of collagen fibers in the submucosa of untreateddiabetic animals is indicative of reduction in collagen fibers.
This condition will subsequently lead to a reduction in thetensile strength which is required to support the mechanicalactivity of the stomach. This may have been responsible forthe poor gastric emptying earlier noticed by Ofusori et al.,[8]. A reduction in the elastic tissue fibers may compromise
the churning process in the stomach. The elastic fibers are
responsible for the stretching and recoiling property needed
for the proper churning of the ingesta. Pretreatment andtreatment with C. zambesicus moderately restored theconnective tissue fibers to near normal in a similar fashion
as glimepiride.
The histochemical results showed an increase inglycogen deposits in the entire gastric outline of untreateddiabetic rats. Gastric pathology is a common complication in
DM. Glycogen deposit is a reference point for probablecompromise in carbohydrate metabolism in alloxan induced
diabetes thus resulting in hyperglycaemia [14]. It is therefore
presumed that depletion of insulin secretion,
hyperglycaemia and altered metabolism of lipids,
carbohydrates and proteins, in addition to damaged β cellsof pancreas are factors which contributed to the increase inglycogen deposit in the untreated diabetic group. In thisstudy, the restoration of the glycogen deposits towards
normal levels when treated with C. zambesicus presented
similar pattern with that of glimepiride treated group, thus
unearthing the gastroprotective effect of C. zambesicus.
Some WBC profiles (WBC and NEUT) were significantly
reduced in the diabetic animals except the lymphocyte countwhich was increased. Preatment with C. zambesicus two
weeks prio to STZ induction and treatment with glimepiride
for four weeks provide better remediation vis-à-vis groups
Fiziologia - Physiology • 2016.26.4(92)treated with C. zambesicus for four weeks after four weeks
of diabetes stabilization. The lymphocyte count was only
moderately affected. This suggests that pretreatment with C.
zambesicus could reasonably boost the immunity response
in diabetes as evident in this study. The increament oflymphocytes in the diabetic state may be an adaptive meansof coping with series of infections that are diabetic related[15]. Lymphocytes are the main constituents of the immune
system which is a defense against the attack of pathogenic
micro-organisms such as viruses, bacteria, fungi andprotista [16].
Platelet count, PDW and MPV were significantly
increased in the untreated diabetic rats when compared withthe control group. Previous study has shown that increasein PLT and MPV are evident in diabetic state 17. This
increase may be connected with dehydration implicated indiabetes as a result of polyuria [18]. Evidence from previous
study has also shown that platelet hyperactivity could leadto an increase in MPV [17]. Pretreatment and treatment with
C. zambesicus restored the platelet count, PDW and MPV
to near normal in a similar fashion as glimepiride.
In conclusion, this study has shown that C. zambesicus
is capable of restoring connective tissue fibres, glycogendeposit and also boost the immunity in the treated groups.
We therefore, advocate for further studies to corroborate thisstudy.

5.
6.
7.

8.

9.
10.

11.
12.

13.
Acknowledgement
We acknowledged the technical input of A. Abbas, T. Onayade,and Y. Ayanleye of Molecular Biology Laboratory, Obafemi
Awolowo University Teaching Hospital Complex, Ile-Ife, Osun State,
Nigeria during the haematomogical analysis.

14.
15.

REFERENCES
1.
2.

3.
4.

Bolzan AD, Bianchi MS. Genotoxicity of Streptozotocin. Mutat
Res. 2002; 512: 121-134.
Bjelakovic G, Nagorni A, Stamenkovic I, Benedeto-Stojanov
D, Bjelakovic M, Petrovic B, Antic S. Diabetes mellitus anddigestive disorders. Acta fac. med. NAISS. 2005; 22 (1): 4350.
Piyachaturawat P, Poprasit J, Glinsukon T. Gastricmucosal secretions and lesions by different doses of
streptozotocin in rats. Toxicol. Letter. 1991; 55: 21-29.
Wolosin JD, Edelman SV. Diabetes and the Gastrointestinal
Tract. Clinical Diabetes. 2000; 18(4): 148-151.

Fiziologia - Physiology • 2016.26.4(92)

16.
17.

18.

Locke GR III. Epidemiology of gastrointestinal complicationsof diabetes mellitus. Eur J Gastroenterol Hepatol. 1995; 7:
711-716.
Bailey CJ, Day C. Traditional plants medicines as treatment
for diabetes. Diabetes Care. 1989; 12: 553-564
Okokon JE, Bassey AL, Obot J. Antidiabetic activity of
ethanolic leaf extract of croton zambesicus muell.(thunderplant) in alloxan diabetic rats. Afri. J Trad. Comp. Alt. Med.
2006; 3(2): 21-26.
Ofusori DA, Komolafe OA, Adewole OS. Ethanolic Leaf
Extract of Croton zambesicus (MÜll. Arg.) Improves Gastric
Emptying Capacity and Gastric Mucosa Integrity in
Streptozotocin-induced Diabetic Rats. International Journalof Diabetes Research 2012a; 1(4): 58-67
Ngadjui BT, Abegaz BM, Keumedjio F, Folefoc GN, Kapche
GWF. Diterpenoids from the stem bark of Croton zambesicus.
Phytochemistry. 2000; 60: 345–349.
National Institute of Health Guide for the Care and Use ofLaboratory Animals. DHEW Publication (NIH). Revised,
Office of Science and Health Reports, DRR/NIH, 1985;
Bethesda, USA.
Okokon JE, Umoh UF, Udobang JA, Etim EI. Antiulcerogenic
Activity of Ethanolic Leaf Extract of Croton Zambesicus Muell.
Arg. Afr. J. Biomed. Res. 2011; 14: 43 -47
Ofusori DA, Komolafe OA, Adewole OS, Abiodun AA, Ayoka
AO. Ethanolic leaf extract of Croton zambesicus inhibits
Gastric Lesion in Streptozotocin induced diabetic rats.
Journal of Pharmeceutical and scientific innovation. 2012b;
1(3): 32-36
Williams DRR, Baxter HS, Airey CM, Ali S, Turner B. Diabetes
UK funded surveys of the structural provision of primary carediabetes services in the UK. Diabetic Medicine 2002; 19(Supplements 4): 21–26
Newell FW, Kurimoto S. Histochemistry of the retina in thealloxan-diabetic rat.Br J Opthamol. 1963; 47: 596-600
Malone J, Schwartz S, Quattrin T, Maclaren NK. Age andfamily relationship accentuate the risk of IDDM in relatives ofpatients with insulin-dependent diabetes. J Clin. Endocrinol.
Metab. 1995; 80: 3739-3743
Ceriello A. New insights on oxidative stress and diabetic
complications may lead to a “causal” antioxidant therapy.
Diabetes Care. 2003; 26: 1589-1596
Zuberi BF, Akhtar N, Afsar S. Comparison of mean plateletvolume in patients with diabetes mellitus, impaired fasting
glucose and nondiabetic subjects. Singapore Med J. 2008;49(2): 114-116
Davis TM, Millns H, Stratton IM, Holman RR, Turner RC. Risk
factors for stroke in type 2 DM: United Kingdom Prospective
Diabetes Study (UKPDS) 29. Arch Intern Med 1999; 159:
1097–1103

31

USEFULNESS OF REAL-TIME SHEAR WAVE
ELASTOGRAPHY IN THE DIAGNOSISOF MALIGNANT THYROID NODULES IN PATIENTSWITH AUTOIMMUNE THYROIDITISMARIA-CRISTINA OPREA1, LAZAR FULGER2
1Victor Babes University of Medicine and Pharmacy Timisoara, Endocrinology Department
2Pius Brinzeu County Clinical Emergency Hospital Timisoara, 2nd Surgery Clinic

ABSTRACT
Thyroid disorders are common and both low and high iodine intake are responsible for a wide range of health consequences.
Thyroid nodules and autoimmune thyroiditis are also associated with changes in iodine intake. Diagnosis of both diffuse thyroid
disease and thyroid nodules is often challenging. Elastography, a new imaging modality, has recently emerged and has beenshown to be useful in the assessment of thyroid nodules, with good inter- and intra-observer reproducibility and accuracy indetecting thyroid malignant disease.
We have analysed 19 thyroid nodules (13 benign, 6 malignant) assessed by means of conventional and Doppler ultrasound, as
well as by shear wave elastography. The ultrasound and shear wave elastography studies were performed during a single
procedure on the same equipment. At conventional ultrasound, we found only one feature associated with malignancy (transonicareas) was statistical significant. The colour-flow Doppler correctly detected blood flow patterns correlated with benign andmalignant disease. SWE produced statistically significant results for all the four parameters analysed (mean and maximum
elasticities, standard deviation in the elasticity index and the ratio between elasticities inside and outside the nodule). However,in our group, the elasticity scores did not help us discriminate between benign and malignant lesions.
SWE proves to be a helpful diagnostic tool, but further studies are needed to confirm its potential.
Keywords: shear wave elastography, thyroid nodule, thyroid cancer, autoimmune thyroiditis

INTRODUCTION
Thyroid disorders are common, particularly in areas ofiodine deficiency, with high prevalence of goitre, reaching up
to 80% in areas of severe iodine deficiency [1]. The healthconsequences of iodine deficiency also include infantmortality, as well as altered growth and development due toimpaired thyroid hormone production [2] Conversely, highiodine intake may lead to autoimmune thyroid disorders, withestimated incidences of hypothyroidism and hyperthyroidism
reaching
350/100,000/year
and80/100,000/year,
respectively, in women, while the figures are much lower inmen (80/100,000/year and 8/100,000/year) [3]. Changes in
iodine intake from low to high elicit effects on thyroid function,
resulting in increased prevalence of thyroiditis [4]. Thyroid
dysfunctions are associated with levels of thyroid-stimulating
hormone (TSH) so that high TSH levels are seen in
hypothyroidism and low levels in hyperthyroidism. Low serum
TSH in iodine-deficient areas leads to formation of thyroid
nodules, which is a common pathology (50%-60% of healthy

subjects) [5]. These nodules are either unable to synthesize
hormones or, inversely, have the capacity to autonomously
synthesize and release thyroid hormones independently of
the TSH level. Finally, some nodules are seen in the settingof simple or multinodular goitre. Elevated serum TSH in
iodine-replete areas, on the other hand, results in increased
hypothyroidism prevalence due to thyroid hypofunction
secondary to autoimmune thyroiditis, and inhibition of thyroid
hormone synthesis and release caused by excessive iodine[4]. Patients with chronic autoimmune thyroiditis are exposed
to an increased risk of overt hypothyroidism with increasing
serum TSH levels above 2 mlU/L, and in the presence of bothanti-thyroid microsomal and anti-thyroglobulin antibodies [6].
Diagnosis of both diffuse thyroid disease and thyroid
nodules is often challenging and includes history, clinicalexamination, laboratory tests, fine-needle aspiration biopsy(FNAB), conventional ultrasound (US) and other imaging
techniques. Assessment of various thyroid hormones andantibodies may guide further investigation in patients with

Received 2nd of September 2016. Accepted 16th of October 2016. Address for correspondence: Oprea Maria-Cristina, MD, PhD student, Department ofEndocrinology, University of Medicine and Pharmacy of Timişoara, Eftimie Murgu No. 2A, 300041-RO, Timisoara; tel/fax: +40356433366

32

Fiziologia - Physiology • 2016.26.4(92)nodular and/or diffuse thyroid disease [7]. Conventional US
allows for the identification of both pathologies, while FNAB
is recommended for nodules larger than 1 cm withsuspicious features at ultrasound examination [8].
Elastography, a new imaging modality, has recently
emerged and has been shown to be useful in theassessment of thyroid nodules, with good inter- and intraobserver reproducibility and accuracy in detecting thyroid
malignant disease [9,10]. Various elastographic techniquescurrently used in clinical practice are able to assess tissue
elasticity using a compression force to measure the degreeof tissue displacement [11]. Tissue stiffness is associatedwith the risk of malignancy [12], although thyroid tissue isalso stiffer in patients with thyroiditis. However, datareported so far suggest elastography may become a useful
tool in discriminating benign from malignant nodules in thepresence of autoimmune thyroid disorders [13,14].
We have analysed 19 thyroid nodules by means ofconventional and Doppler ultrasound, as well as by shear
wave elastography (SWE) in order to assess the diagnostic
ability of the latter technique to discriminate between benign
and malignant nodules in the presence of autoimmune
thyroiditis.

MATERIALS AND METHODS
We conducted this preliminary retrospective study inorder to evaluate the ability of SWE to correctly diagnosebenign and malignant thyroid nodules in patients with
autoimmune thyroiditis. We have thus extracted only thosenodules found in patients with autoimmune thyroiditis asconfirmed by histopathological diagnosis.
The study was conducted from October 2015 toFebruary 2016 in the Endocrinology Department of theVictor Babes University of Medicine and Pharmacy
Timisoara (UMFVBT) and the 2nd Surgery Clinic within the
Pius Brinzeu County Clinical Emergency Hospital Timisoara(SCJUT). Out of the 43 thyroid nodules assessed, we
extracted the 19 thyroid nodules found in 11 patients with
autoimmune thyroiditis, all women, mean age 58.36 years(min. 39 years, max. 76 years). The nodules were split in
two subgroups, i.e. benign (n=13) and malignant (n=16). All
nodules were surgically removed in the 2nd Surgery Clinic of
SCJUT, and blinded histopathologic diagnosis served bothas inclusion criterion and reference standard. All patientsgave their informed consent prior to inclusion in the study.
We have performed our ultrasound and shear wave
elastography studies during a single procedure on thesame equipment, an Aixplorer TM ultrasound system(SuperSonic Imagine, Aix-en-Provence, France), using a
SC5-1 linear transducer. First, we have assessed thesonographic features of the nodules by conventional
ultrasound, namely nodule number, sizes and volume,
aspect of margins, echogenicity, homogeneity, halo sign,
microcalcifications and transonic areas inside nodules.
Fiziologia - Physiology • 2016.26.4(92)Afterwards, the blood flow in the nodule and thesurrounding area was evaluated by means of colour-flow
Doppler and classified into intranodular, perinodular orabsent blood flow.
Lastly, we performed real-time SWE, with theelastographic and B-mode images simultaneously shown on
two separate screens. Shear wave elastography relies onan acoustic radiation force that creates quasiplane shear
waves, with the shear source being moved at supersonic
speed [15]. During the procedure, we have recorded thequantitative parameters displayed by the SWE system:
mean elasticity index of the nodule (SWE-mean), maximum
elasticity index of the nodule (SWE-max), standard deviationin the elasticity index (SWE-SD), and the ratio between
elasticity of the nodule and that of the normal surrounding
thyroid tissue (SWE-ratio), all expressed in kilo-Pascals(kPa).
Statistical Analysis
We used the Statistical Package for the Social Sciences(SPSS) programme (version 17.0 for Windows, SPSS Inc.,
Chicago, Illinois, USA) to conduct statistical analysis. The
19 nodules were assigned to benign (13 nodules, 68.4%) or
malignant (6 nodules, 31.6%) subgroups. Student’s t-test
was used to compare data and p-values <0.05 wereconsidered statistically significant.

RESULTS
All patients included underwent either lobectomy or total
thyroidectomy in the 2nd Surgery Clinic of SCJUT. The
blinded histopathologic diagnosis was performed according
to the World Health Organization guidelines [16], revealing
6 papillary carcinomas, out of which 1 was classical variant,
1 microcarcinoma and 4 - follicular variant. Among the 13
benign nodules, 2 were follicular and 1 oxyphilic adenomas,
2 hyperplasic nodules, and 8 cystic colloid nodules.
All findings at conventional and Doppler ultrasound, as
well as shear wave elastography described below werecompared with the histopathologic diagnosis to assess theaccuracy of imaging techniques used in this study.
Conventional ultrasound is routinely used to assess
thyroid nodules, being able to identify suspicious nodules
that need further evaluation by means of FNAB and/or
histology, as recommended by guidelines [5, 17, 18].
Several features of thyroid nodules are considered to bepredictive for malignancy, such as irregular margins,
hypoechogenicity, inhomogeneity, absence of hallo sign,
presence of microcalcifications and transonic areas, while
US features of thyroiditis can vary according to the disease
phase and severity, usually presenting as an array of
hypoechoic nodules and echogenic fibrous bands [13].
In our group, we had 10 patients with solitary nodules,
one with 3 nodules and other 3 with 2 nodules. The
average nodule volume was 2.2550 ml in the benign
33

subgroup and 3.0017 ml in the malignant nodules
subgroup. Measurements of length, width, thickness andmaximum diameter showed no statistical differences
between the benign and malignant nodules. Among the USfeatures associated with malignancy, the presence oftransonic areas was the only parameter to reach statisticalsignificance, at p = 0.033. In our series, only 7 nodules
presented with irregular margins, most of which (n = 5)
were diagnosed as benign conditions. Because most of thenodules (n = 16) in our group were hypoechoic, including
all malignant nodules, this feature was not useful indiscriminating between benign and malignant lesions.
Similarly, more than half of the nodules (n = 12, out of
which 5 were malignant) had a non-homogenous
appearance. The halo was absent in 12 nodules (2malignant) in our series, while 17, including all malignant,nodules, did not had microcalcifications.
Doppler ultrasound is reliably detecting the blood flow inthyroid nodules, with marked intranodular hypervascularity
being a distinctive pattern of malignancy, and perinodular
blood flow more often seen in benign thyroid nodules, whileavascular thyroid nodules are almost always benign. In ourgroup, 4 of the 6 malignant nodules had intravascular blood
flow, 2 had perivascular blood flow, while all avascular
nodules (n = 5) were benign. These results proved to bestatistically significant, at a p-value of 0.043.
Shear wave elastography, a recent imaging techniquedemonstrating good diagnostic potential for thyroid
pathologies, enables the quantitative assessment of tissue
elasticity by measuring the shear wave propagation speed,
owing to the high sensitivity of shear modulus (i.e., elasticity
coefficient for shear force) to physiological and pathologicalchanges in tissues under investigation.
The transducer covered with US gel was placed on
patient’s skin without applying tissue compression. With
SWE, images are acquired within seconds and then a regionof interest is placed on the frozen image to perform themeasurements.
We have recorded four quantitative parameters (SWEmean, SWE-max, SWE-SD and SWE-ratio), the meanvalues of which are shown in Table 1 for both subgroups,
with statistically significant (p-value <0.05) higher values
recorded in the malignant nodules subgroup, consistent with
reports in literature. Nevertheless, elastographic findings in
diffuse thyroid diseases, such as autoimmune thyroiditis,
indicate higher tissue stiffness as well, correlated with theseverity of disease [19]. Despite this, we found that tissue
stiffness in malignant nodules in patients with concurrent
autoimmune thyroiditis is significantly higher for all SWE
parameters, with great differences between the highest
values recorded in the two subgroups, as follows: SWE
mean 59.1 kPa in a malignant nodule and 33.2 kPa in abenign one; SWE max 96.06 kPa/46.5 kPa; SWE SD 14.4

34

kPa/4.9 kPa; SWE ratio 2.03 kPa and 1.53 kPa,
respectively.
Table I. Mean values of SWE parameters in the two subgroups
Mean value

Parameter
SWE mean
SWE max
SWE SDSWE ratio

Standard

p-value

Deviation
benign19.33 kPa

9.09

malignant

28.41 kPa

15.52

benign36.11 kPa

10.71

malignant56.65 kPa

27.43

benign5.92 kPa

3.18

malignant

9.42 kPa

5.31

benign0.93 kPa

0.38

malignant1.63 kPa

0.56

0.005
0.010
0.020
0.010

Tissue elasticity can also be assessed qualitatively, theelastographic image showing a range of colours, from blue
for maximum tissue elasticity to dark red for marked
stiffness. We have assigned scores from 1 to 5 according to
Ueno scale [20], where score 1 is assigned to normal tissue
elasticity and score 5 to stiffness in the entire nodule and thesurrounding area. In our study, the results were notstatistically significant, as almost all nodules had elasticity
scores of 2, with only one elasticity score of 3 recorded on amalignant nodule.
The US and SWE images of a benign and a malignant
nodule in our study group are shown in Figures 1 and 2,
revealing differences in appearance and particularly in therecorded SWE parameters.

Fig. 1. SWE and US images of a benign nodule. A 53-year old
woman with a follicular adenoma and chronic autoimmune
thyroiditis, ES 2 on SWE. US shows a homogeneous hypoechoic
nodule, with regular margins, no microcalcifications, absence of
halo sign and cystic content.

Fiziologia - Physiology • 2016.26.4(92)Fig. 2. SWE and US images of a malignant nodule. A 57-year-old
woman with papillary carcinoma, follicular variant, and chronic
autoimmune thyroiditis, ES 3 on SWE. US shows anonhomogeneous hypoechoic nodule, taller than wide, with anincomplete hypoechoic halo and an area of microcalcification.

DISCUSSIONS
Conventional ultrasound of thyroid nodules is useful in
identifying suspicious masses, but further investigation isneeded to confirm the diagnosis, particularly in patients withconcurrent autoimmune thyroiditis, as many of the featuresconsidered to be predictive of malignancy are also seen ina large number of benign nodules. On the other hand,
Bonavita et al. [21] identified four US patterns associatedwith benign nodules: spongiform configuration, cyst withcolloid clot, giraffe pattern, and diffuse hyperechogenicity.
The US findings in the setting of thyroiditis typically shows
enlarged thyroid gland with one of the following patterns:
several diffuse small hypoechoic nodules interspersed withechogenic bands; round hyperechoic areas surrounded bylinear hypoechoic areas; or a hyperechoic nodule [19].Error!
Bookmark not defined. Therefore, the hypoechoic pattern in ourgroup was not useful in discriminating between between
benign and malignant lesions, as hypoechogenicity is not
only a distinctive US feature for malignancy, but is alsocharacteristic to thyroiditis as a result of marked
inflammatory process following infiltration of thyroid withlymphocytes and plasma cells [22,23]. The nonhomogenous appearance is also a common finding in
nodular or adenomatous goiters and a typical aspect of
thyroid parenchyma seen in patients with thyroiditis [21,24].
Absence of a hypoechoic rim around the nodule is due tolymphocytic infiltration, development of fibrous connective
tissue and compression of thyroid parenchyma [25], and thismay explain the absence of halo sign in both cancerous andbenign nodules in our group. It is therefore not surprising
Fiziologia - Physiology • 2016.26.4(92)that a halo was absent in 12 nodules (2 malignant) in ourseries. Similarly, microcalcifications, which are considered ahallmark of thyroid malignancy, are also found in
autoimmune thyroiditis and follicular adenomas [26]. In ourstudy, we only found two nodules with microcalcifications.
Marked intranodular blood flow identified at colour-flow
Doppler is considered a distinctive pattern of malignancy,
although it is also present in more than half of benign solid
thyroid nodules [27]. Perinodular blood flow is more often
seen in benign thyroid nodules, but is not uncommon inmalignant thyroid conditions [28], while avascular thyroid
nodules are almost always benign. The patterns of bloodflow seen in our study allowed for the fair discrimination
between benign and malignant nodules.
SWE has emerged as an imaging technique with great
potential in the diagnosis of thyroid diseases and provides
both qualitative and quantitative data regarding tissue
elasticity. Various studies in literature [9,29-31] have proved
the value of SWE in diagnosing thyroid malignancies, whileothers [13,14,24] have shown it can be useful in detecting
thyroid cancer even in the setting of autoimmune thyroiditis,which is also characterized by an increased tissue stiffness.
Though, the higher stiffness due to autoimmune thyroiditis
seems not to tackle the ability of SWE to differentiate
between benign and malignant conditions, as values of
SWE parameters in autoimmune thyroiditis appear to becloser to those recorded in benign thyroid lesions.
Szczepanek-Parulska et al.[32], for instance, compared thestiffness of benign thyroid nodules in patients with andwithout autoimmune thyroiditis and found no significant
differences between groups. Nodules in our study were all
found in patients with autoimmune thyroiditis, so that acomparison with benign lesions in absence of thyroiditis is
not possible. However, we have shown that malignant
nodules exhibit significantly higher stiffness and that
concurrent thyroiditis is not affecting the results. SWE
allowed for the correct diagnosis of nodules and, more
important, was able to detect all malignancies. Conversely,
US produced significant results for only one parameter,while colour-flow Doppler added in the characterisation ofblood flow within nodules and the surrounding region,
confirming the presence of vascularity inside malignant
nodules. A limitation of our study is the small number of
nodules included. Because of this, we could not establish an
optimal cut-off, nor sensitivities and specificities.

CONCLUSIONS
Shear wave elastography is consistently proving its
diagnostic value in thyroid disorders, being able to identify
malignancies with high accuracy. Being a recent technique,studies are still needed to confirm its precision. For now, it
is being used as an adjunct to conventional ultrasound, aiding
in patient guidance to FNAB or surgery. If its value anddiagnostic potential will be confirmed in the future, SWE will35

help reduce the number of unnecessary FNAB procedures
and surgeries, to the best benefit and care of patient.

REFERENCES
1. Vanderpump MPJ. The epidemiology of thyroid disease. British
Medical Bulletin. 2011; 99:39-51.
2. Zimmermann MB, Jooste PL, Pandav CS. Iodine-deficiency
disorders. The Lancet. 2008; 372(9645):1251-1262.
3. McGrogan A, Seaman HE, Wright JW et al. The incidence ofautoimmune thyroid disease: a systematic review of theliterature. Clin Endocrinol (Oxf). 2008;69:687-96.
4. Laurberg P, Bulow Pedersen I, Knudsen N et al. Environmental
iodine intake affects the type of non-malignant thyroid disease.
Thyroid. 2001;11:457–69.
5. Gharib H, Papini E, Garber JR, Duick DS, Harrell RM, Hegedüs
L, Paschke R, Valcavi R, Vitti P. American Association of Clinical
Endocrinologists, American College of Endocrinology, andAssociazione Medici Endocrinologi Medical Guidelines forClinical Practice for The Diagnosis and Management of Thyroid
Nodules-2016 Update. Endocrine Practice. 2016;22(Suppl 1).
6. Vanderpump MPJ, Tunbridge WMG, French JM et al. The
incidence of thyroid disorders in the community: a twenty-year
follow-up of the Whickham survey. Clin Endocrinol (Oxf).
1995;43:55-69.
7. Tamhane S, Gharib H. Thyroid nodule update on diagnosis andmanagement. Clinical Diabetes and Endocrinology.
2016;2(1):17.
8. Cantisani V, Lodise P, Grazhdani H, ... & Catalano C. Ultrasoundelastography in the evaluation of thyroid pathology. Current
status. European Journal of Radiology. 2014;83(3):420-428.
9. Rago T, Santini F, Scutari M, Pinchera A, Vitti P. Elastography:
new developments in ultrasound for predicting malignancy inthyroid nodules. J Clin Endocrinol Metab. 2007;92(8):2917-22.
10. Lim DJ, Luo S, Kim MH, Ko SH, Kim Y. Interobserver agreement
and intraobserver reproducibility in thyroid ultrasoundelastography. American Journal of Roentgenology.
2012:198(4):896-901.
11. Kwak JY, Kim EK. Ultrasound elastography for thyroid nodules:
recent advances. Ultrasonography. 2014;33(2):75-82.
12. Gharib H, Papini E. Thyroid nodules: clinical importance,
assessment, and treatment. Endocrinology and metabolism
clinics of North America. 2007 Sep 30;36(3):707-35.
13. Magri F, Chytiris S, Capelli V, Alessi S, Nalon E, Rotondi M,
Cassibba S, Calliada F, Chiovato L. Shear wave elastography inthe diagnosis of thyroid nodules: feasibility in the case ofcoexistent chronic autoimmune Hashimoto’s thyroiditis. Clin
Endocrinol Oxf. 2012;76:137-41.
14. Hekimoglu K, Donmez FY, Arslan S, Ozdemir A, Demir C, Yazici
C. The role of shear wave elastography in the diagnosis ofchronic autoimmune thyroiditis. Medical Ultrasonography.
2015;17(3):322-326.
15. Bercoff J, Tanter M, Fink M. Supersonic shear imaging: a newtechnique for soft tissue elasticity mapping. IEEE transactions
on ultrasonics, ferroelectrics, and frequency control.
2004;51(4):396-409.
16. Heidinger CH, Sobin LH. Histological typing of thyroid tumors.
International histological classification of tumors. 1974;11:17-27.
17. Pacini F, Castagna MG, Brilli L, Pentheroudakis G, ESMO
Guidelines Working Group. Thyroid cancer: ESMO Clinical

36

Practice Guidelines for diagnosis, treatment and follow-up.
Annals of Oncology. 2012;23(suppl 7):vii110-9.
18. Cooper DS, Doherty GM, Haugen BR, Kloos RT, Lee SL,
Mandel SJ, Mazzaferri EL, McIver B, Pacini F, Schlumberger M,
Sherman SI. Revised American Thyroid Association
management guidelines for patients with thyroid nodules anddifferentiated thyroid cancer: the American Thyroid Association(ATA) guidelines taskforce on thyroid nodules and differentiatedthyroid cancer. Thyroid. 2009 Nov 1;19(11):1167-214.
19. Menzilcioglu MS, Duymus M, Avcu S. Sonographic elastographyof the thyroid gland. Polish Journal of Radiology. 2016;81:152.
20. Itoh A, Ueno E, Tohno E, Kamma H, Takahashi H, Shiina T,
Yamakawa M, Matsumura T. Breast disease: clinical applicationof us elastography for diagnosis 1. Radiology. 2006;239(2):34150.
21. Bonavita JA, Mayo J, Babb J, Bennett G, Oweity T, Macari M,
Yee J. Pattern recognition of benign nodules at ultrasound of the
thyroid: which nodules can be left alone? American Journal of
Roentgenology. 2009;193(1): 207-213.
22. Pedersen OM, Aardal NP, Larssen TB, Varhaug JE, Myking O,
Vik-Mo H. The value of ultrasonography in predicting
autoimmune thyroid disease. Thyroid. 2000;10(3):251-259.
23. Yeh HC, Futterweit W, Gilbert P. Micronodulation:
ultrasonographic sign of Hashimoto thyroiditis. Journal of
Ultrasound in Medicine. 1996;15(12):813-819.
24. Fink H., Hintze G. Autoimmune thyroiditis (Hashimoto's
thyroiditis): current diagnostics and therapy. Medizinische Klinik(Munich, Germany: 1983);2010;105(7): 485-493.
25. Hoang JK, Lee WK, Lee M, Johnson D, Farrell S. US Featuresof thyroid malignancy: pearls and pitfalls. Radiographics.
2007;27(3):847-860.
26. Taki S, Terahata S, Yamashita R, et al. Thyroid calcifications:sonographic patterns and incidence of cancer. Clin Imaging.
2004; 28(5):368–371.
27. Frates MC, Benson CB, Doubilet PM, Cibas ES, Marqusee E.
Can color Doppler sonography aid in the prediction ofmalignancy of thyroid nodules? J Ultrasound Med.
2003;22(2):127–131.
28. Chan BK, Desser TS, McDougall IR, Weigel RJ, Jeffrey RB Jr.
Common and uncommon sonographic features of papillary
thyroid carcinoma. J Ultrasound Med. 2003;22(10):1083–
1090.
29. Sebag F, Vaillant-Lombard J, Berbis J, Griset V, Henry JF,
Petit P, Oliver C. Shear wave elastography: a new ultrasoundimaging mode for the differential diagnosis of benign andmalignant thyroid nodules. The Journal of Clinical
Endocrinology & Metabolism. 2010;95(12):5281-8.
30. Hong Y, Liu X, Li Z, Zhang X, Chen M, Luo Z. Real‐timeultrasound elastography in the differential diagnosis of benign
and malignant thyroid nodules. Journal of Ultrasound in
Medicine. 2009;28(7):861-867.
31. Szczepanek-Parulska E, Woliński K, Stangierski A, Gurgul E,
Biczysko M, Majewski P, Rewaj-Łosyk M, Ruchała M.
Comparison of diagnostic value of conventional
ultrasonography and shear wave elastography in theprediction of thyroid lesions malignancy. PLoS One.
2013;8(11):e81532.
32. Szczepanek-Parulska E, Woliński K, Stangierski A, Gurgul E,
Ruchała M. Biochemical and ultrasonographic parameters
influencing thyroid nodules elasticity. Endocrine.
2014;47(2):519-527.

Fiziologia - Physiology • 2016.26.4(92)RISK FACTORS ASSOCIATED WITH CHRONICVENOUS INSUFFICIENCY IN CENTRAL INDIA
SMITA R SORTE, SACHIN B RATHOD
Department of Physiology, Shri Shankaracharya Institute of Medical Sciences, Bhilai, Chhattisgarh, India.
Department of Physiology in coordination with Department of Tifac - Core of Interventional Radiology, of Datta Meghe Institute ofMedical Sciences, Jawaharlal Nehru Medical College, Sawangi (Meghe), Wardha, Maharashtra, India.

ABSTRACT
Chronic venous insufficiency is not only an individual problem but also a national problem as it had increased economic burden
due to recurrence, treatment, disability and decreasing quality of life. Incidence of chronic venous insufficiency varies in different
populations ranges from 10% to 65% which may be due to varied risk factors affecting diverse population. We want to find risk
factor in central India rural population who lack awareness of this condition. A case control study was done, on 57 cases and29 age-matched healthy controls. Various risk factors were studied – sex, age, height, weight, parity, history of prolong standing,prolong sitting, any family history, constipation, OCP, Post thrombotic and any prior invasive surgery of vein, smoking, cancer,
polycythemia, hypertension and diabetes. Factors like prolong standing, prolong sitting, family history, prior invasive venous
surgery, post thrombotic was higher in cases than controls which was found to be statistically significant (P<0.05).
Keywords: varicose veins, risk factors, chronic venous insufficiency

INTRODUCTION
Venous disorders of the legs occur frequently whichrange in severity from minor asymptomatic incompetence ofvenous valves to chronic leg ulceration. Varicose veins are
a common manifestation of venous incompetence in thelower limb, and they appear as dilated, elongated, tortuous,
pouched, thickened, inelastic and friable veins which havepermanently lost its valvular efficiency. Incompetence of thedeep, superficial and/or perforating veins leads to raised
venous pressure in the lower leg, which can result in skinchanges such as hyperpigmentation and indurations with
eventual ulceration. These changes in the skin andsubcutaneous tissues of the lower leg are often referred toclinically as chronic venous insufficiency (CVI) (13).
Incidence of varicose veins and their complication is not
only an individual, but also a national problem of great
importance especially in western countries due to its highprevalence, cost of investigations and treatment, and loss ofworking days (1,20). Varicose veins are present in 25% to
33% of female and 10% to 20% of male adults.
(2,3,8.9,14,25,28,38,39,42,44) Prevalence estimates vary
widely by geographic location, with the highest reported
rates in Western countries. . Data from the Brazilian Security
System show that CVI is the 14th most-frequently quoted
disease leading to temporary work absenteeism and the32nd most frequent reason for permanent disability andpublic financial assistance.(11) The annual cost of venous
ulcers has been estimated to be £400 to 600 million for theUnited Kingdom (6,16) and >$1billion for the United States(15). The total direct and indirect cost of CVI to society islikely to be $1 billion US dollars in each of 3 European
countries (the United kingdom, France, and Germany) (21,22). The reported ranges in prevalence estimations
presumably reflect differences in the population distributionof risk factors, accuracy in application of diagnostic criteria,
the quality and availability of medical diagnostic resources.
Wardha is District in Maharashtra which lies in central
India. The district has 6,309 sq km and population of
1,300,774 out of which 67.46 % live in rural areas according
to the 2011 Census. The percentage of population living inrural Wardha is far greater than in the state as a whole. If
not treated then, nearly 50% of patients of superficial venous
insufficiency will eventually experience chronic venous
insufficiency which is characterized by swelling of lower
extremity, pigmentation, hemorrhage, eczema andulceration (43). Chronic venous insufficiency (CVI) can be
regarded as the common clinical endpoint of various venous
diseases.
Till now, no study has been conducted to see theseverity of the problem. People are not aware of the hazards
of prolong standing. Chronic venous insufficiency, like a
glacier, moves slowly but forcefully over time. It hasenormous clinical and socioeconomic consequences,

Received 12th of September 2016. Accepted 10th of October 2016. Address for correspondence: Samita R Sorte, Assistant Professor, Department of
Physiology, Shri Shankaracharya Institute of Medical Sciences, Junwani, Bhilai, India; phone: +91-9407796105 / +91-9423409931; e-mail:drsmyta@gmail.com

Fiziologia - Physiology • 2016.26.4(92)37because the severity and chronicity of the CVI associatedsigns and symptoms can profoundly interfere with thepatients’ quality of life and work capacity on the one side,and the financial resources of the health care system on theother(12,31,32,41).
Scarcity of information regarding health and safety ofrural population provides strong rationale for rural healthprofessionals to initiate research and educational programs
focusing on health promotion/safety needs of rural
population. Our study aims to find the risk factors for chronic
venous insufficiency in central India.

METHOD AND MATERIAL
A case control study was done. Clearance from
Institutional review board was obtained. Informed writtenconsent was taken from the patient. 57 patients wereenrolled for the study. Patients attending Acharya Vinoba
Bhave Rural Hospital for management of Chronic Venous
insufficiency were subjected to standard questionnaire.
The following data was collected: age, height, weight, parity,history of prolong standing, prolong sitting, any family
history, constipation, OCP, Post thrombotic and any priorinvasive surgery of vein, , smoking, cancer, polycythemia,
hypertension and diabetes.
Clinical examination of leg was done in following heads-Venous dilatation, edema, Skin pigmentation and venous
ulceration. A classification and grading of chronic venous
disease was done according to the American Venous
Forum is CEAP classification. It was followed by duplex
scanning examination with colour Doppler unit. All tests
were performed in morning hours between 0900Hrs to
1200Hrs. The diagnosis of varicose veins was done bycombination of clinical and duplex ultrasonographic
examination. All patients in the study had normal arterial
examination on duplex scanning. Inclusion criteria werevaricose veins of grade C3-C6. Exclusion criteria were
Occlusive arterial diseases of leg, obliterativearteriosclerosis, atherosclerosis of leg veins; varicose
veins grade C1, C2 .Twenty nine normal, healthy age
matched volunteer were selected as controls. They had nosign or symptom of venous or arterial diseases in any limb.
Colour Doppler findings were normal.
Statistical analysis
All parameters in the study were assumed
nonparametric as the control group has less than 30 patientsand nonparametric tests was used for statistical analyses.
Comparison between groups was tested by Mann-Whitney
U at 95% confidence interval (CI; 𝛼 = 0, 05 level). Allanalyses were performed using SPSS 16.0

38

RESULTS

Table I and II shows Classification of chronic venous
disease.
Table I. Classification of chronic venous disease (Porter andMoneta 1995) Mark Definition
C

Clinical signs (grade 0-6), supplemented by(S) for
symptomatic and (A) for asymptomatic presentation

E

Etiologic Classification (Congenital, Primary, Secondary)A

Anatomic distribution (Superficial, Deep, or Perforator, alone
or in combination)

P

Pathophysiologic dysfunction (reflux or obstruction, alone orin combination)
Table II. CEAP Clinical classification of chronic lower extremity
venous disease (Porter and Moneta 1995)
Class

Clinical signs

C0

No visible or palpable signs of venous disease

C1

Superficial spider veins, Telangiectasias, reticularveins, malleolar flare

C2

Simple varicose veins only

C3

Ankle edema of venous origin (not foot edema)

C4

Skin changes ascribed to venous disease(pigmentation,
venouseczema,Lipodermatosclerosis)

C5

Skin changes (as defined above) in conjunction
with healed ulceration

C6

Skin changes (as defined above) in conjunction
with active ulceration

Table III shows clinical data of cases and control group.
Mean age of the cases and control was 43.9 + 12.2 yrs and42.94 ± 13.6yrs respectively. There was no statisticaldifference between ages of two groups. The sex ratio (M: F)in cases and control was 4.7:1 and 1.9:1. BMI, Height, OCP(in females), smokers, hypertensive, diabetics andConstipation history in cases was greater than control groupbut not statistically significant (P>0.05). Other factors like
Prolong Standing, Prolong Sitting, Family History, Prior
surgery, Post thrombotic was higher in cases than controls
which was found statistically significant (P<0.05).

Fiziologia - Physiology • 2016.26.4(92)Table III. Demographic and clinical data of cases and control

SrnoParameters

Cases

Controls

P
Value

1

Numberpatients n

57

29

-

2

Age (years;
mean ± SD)

43.9 +1242.94 ±
13.60

0.56

3

Sex
ratio
(Male:
Females)

47/10

19/10

-

BMI
(kg/m2,mean
± SD)

21.31
3.608

5

Height(m,mean ± SD)

6
7

of

20.96
4.5

+

1.68+0.09

1.65
0.06

+

Parity (birth no>1) (%)

10 (100%)

10(100%)-

ProlongStanding
(%)

n,

42
(73.68%)6(20.68%)0.01

8Prolong
Sitting n, (%)

10(17.54%)2(6.89%)0.01

9FamilyHistory (%)

28(49.12%)4(13.79%)0.05

3 (30%)

2 (20%)

0.2

4

(in

±

0.75

Age
Group
(yrs)

Male

Female

Total

11-20

1(1.75%)1(1.75%)2(3.51%)21-30

7(12.2%)1(1.75%)8(14.04%)31-40

10(17.54%)

1(1.75%)11(19.30%)

41-50

13(22.81%)

5(8.77%)18(31.58%)>50

16(28.07%)2(3.51%)18(31.58%)

Total

47(82.46%)

10(17.54%)

57(100%)

Table V. Distribution of cases according to occupation

0.66

10

OCP
females)

11

Constipation

8(14.03%)6(20.68%)0.59

12Prior surgery

10(17.54%)1 (3.44%)

0.05

13

Post
thrombotic

7(12.28%)0 (0%)

0.01

14

Smokinghistory

18

15

0.82

15

Hypertension

27

15

0.67

16

Diabetes

15

9

0.74

Table IV shows age and Gender wise distribution ofcases and Table V shows distribution of cases according to
occupation.
CEAP classification of chronic venous
insufficiency was shown in Table VI.

Fiziologia - Physiology • 2016.26.4(92)Table IV. Age wise and Gender wise distribution of cases

Occupation

No.of
patients

Percentage (%)

Prolonged standing

42

73.68421

Prolonged sitting

10

17.54386

Weight pullers

58.77193

Total

57

100.00

Table VI. CEAP Classification of patients
SR
NO

CEAP

Cases

Control

Males

Females

Males

Females

1

C0

-

-

-

-

2

C1

-

-

-

-

3

C2

-

-

-

-

4

C3

10 (21.27%)

3 (30%)

-

-

5

C4

12 (25.53%)

3 (30%)

-

-

6

C5

13(27.65%)

2 (20%)

-

-

7

C6

12 (25.53%)

2 (20%)DISCUSSION
Incidence of chronic venous insufficiency varies indifferent populations ranges from 10% to 65% which may be
due to varied risk factors affecting diverse population. We
had tried to find risk factor in central India rural population
who lack awareness of this condition.
The sex ratio in the cases was male: female 4.7:1. We
may interpret that chronic venous insufficiency is more39

common in males as compared to female in rural Wardha
population of central India. It might be due to; firstly males
are occupied more in occupation requiring prolonged
standing. Secondly women bother less about the medical
problem and do not seek medical advice till the condition
becomes severe and unbearable. Being a rural area female
consume less oral contraceptive pills and so are at less riskof deep venous thrombosis and Chronic venous
insufficiency. These findings are in accordance with Nobl(27) 1910 and Nicholson (26) 1927 who found male
predominance. Stanhope J M (36) 1975 showed modest
prevalence in men and very low prevalence in women. Most
authors (Briscoe G 1918, Gibson 1937, Hooker 1914,
Leconte 1922, Lyons 1922, Villaret 1921, Winsor 1946)
found lower values for women as cited by Ochsner A (29)(1951). Callman M J (7) cited following table showing female
predominance in the prevalence of varicose veins and themale: female ratio was between 1: 0.6 to 1: 4.0 depending
upon the definition used.
Reference
Nobl

Year
1910

Sex Ratio M:F
1:0.6

Nicholson

1927

1:0.6

Widmer

1978

1:1.0

Brand et al

1988

1:1.3

De Takats

1929

1:1.5

Dodd and Cockett

1956

1:1.5

Prior et al

1970

1:1.7

Lartson and smith

1943

1:2.0

Pratt

1950

1:2.0

Dodd

1964

1:2.0

Drury

1965

1:2.0

Lake et al

1942

1:2.0

Babek et al

1962

1:2.1

Arnoldi

1958

1:2.1

Lofgren

1966

1:2.1

Logan and Cushion

1958

1:2.5

Logan and Brooke

1957

1:2.6

Harding Le Riche et al

1962

1:2.7

Abramson

1981

1:2.8

Danish National Morbidity Survey

1954

1:2.9

Haakenaasen

1963

1:3.0

Dick

1966

1:3.5

Weddell

1969

1:3.5

Payne

1936

1:4.0

Phillips

1963

1:4.0

US National Health Survey

1938

1:4.0

40

According to Callman M J (7) (1994), use of a wide
variety of sampling techniques, definition of varicose veins(VV) and regardless of whether they were looking at
incidence, point or period prevalence, various studies haveshown a female predominance with a female: male ratio up
to 4: 1. Two factors may account at least in part for the wide
variation in results. First, the studies that gave a very highfemale predominance were almost exclusively
questionnaire based and this method tends to be lessaccurate Dale JJ 1983 (10), Weddell JM 1969 (42). Second,the data from Bernlsens (5) 1927 and the Basle study by
Widmer LK (44) 1978 among others, suggest that thefemale: male ratio increases with age. The varying ages ofthe samples in different studies may have contributed to thewide range of results.
In our study, 18 (31.58%) cases were from the agegroup 41 to 50yrs and 18 (31.58%) cases were in the agegroup of >51. Mean age of the cases was 43.9 yrs + 12.2
yrs. The present study shows that chronic venous
insufficiency is more common in more than 40 yrs of age in
population of rural Wardha. Our finding are in accordancewith Mekky S et al (25) in 1969, Beaglehole R (4) in 1986,
Staffa R (35) 2002 and Widmer LK (44) 1978 who foundthat varicose veins increase with increasing age. Data fromother studies (Abramson JH (12) 1978, Coon W (8) W1973,
US Department, Hirai M, Lorenzi G) Show similar trends,
although the absolute percentages varied, because different
definition of varicose veins and population samples wasused as cited by Callman M J in 1994.
The cause for increase varicosity with increase age canbe explained on the basis of Structural alterations of thevenous wall. There is reduction of smooth muscle fibers ofthe venous wall and qualitative and quantitative alterationsin the wall of the varicose vein. Fibers are deformed with
reduced collagen and disorderly disposed, with an excess
of ‘proteoglycans.’ In addition, there is an increase in all theactivities of lysosomal enzymes (hyaluronidases,
glucosaminidases, and phosphatases) Sciannameo (33)1993. According to Silveira (34) 1993, varicose saphenous
veins presents significant structural modifications in its wall,
occurring, in addition to a greater intimal thickening, deep
modifications in the structure of the tunica media, withinterposition of elastic fibers to smooth muscle clusters,
consequently altering the resistance of the damaged venous
wall. The loss of elasticity in the venous wall leads to agerelated increases of venous diameter in the venous wall andmay predispose for varicose veins (17).
BMI, Height, OCP (in females), history of constipation,
hypertension, diabetics and smoking, in cases was greaterthan control group but not statistically significant (p>0.05).
Other factors like prolong standing, prolong sitting, family
history, prior venous surgery, post thrombotic was higher incases than controls which was found statistically significant(P<0.05).
In our study, out of 57 patients, 46 were occupied in
occupation requiring prolonged standing. This study
Fiziologia - Physiology • 2016.26.4(92)elucidates that, patients whose occupation involves
prolonged standing, are at greater risk for developing
chronic venous insufficiency. These findings are in line withLake (19) et al 1942 and Kontosic (23) 2000 study showingrole of posture in causing varicose veins. According to
Stanhope J M100 in 1975 sitting habits may be important in
the etiology of varicose veins. According to Malhotra S L (24)1972 and Beaglehole (3) 1975 and Weddell J M (35) 1969
posture does not appear to contribute to the causation ofvaricose veins. Weddell J M5 postulated men who wereinvolved in heavy lifting work did have a significant
influence. Following table shows the association between
occupation and varicose veins, as cited by Callman M J (7).
ReferenceYear

Occupation

Cause

POSITIVE ASSOCIATION
Mekky et al

1969

Cotton workers

Standing

Weddell

1969

Community
samplesHeavy lifting

Abramson

1981

Community
samplesStanding

Lorenzi et al

1986

Metal workers

Standing

constantly raised. The veins became locally dilated distal toa valve and that there is media hypertrophy, which later
progresses to atrophy and sacculation (King (18) 1950).
Disorganization of the circular muscle in the media occurs,
and that the collagen contents decreases and the watercontent increases (Prerovsky et al. (20) 1962).
Veins lose their elasticity and its diameter increases toaccommodate the increased venous volume. Due towhich the venous valve does not approximate and become
functionless in long run. They can no longer prevent theback flow of the blood in the leg. Once the venous valves
are incompetent, then even walking may actually increase
venous pressure in the lower extremities because of areversal in blood flow. Increase in leg veins blood volume
and increase in the hydrostatic pressure produce highpressure zone in deep veins. This high pressure thendamages the perforator valves and leaks into the superficial
veins. Impeded blood flow leads to stasis in veins of thelower extremities. Stasis in the venous system is a key
mechanism in venous vascular disease. Stasis increases
the risk for coagulation and thrombus formation. (FinnTuchsen (40) 2000).
People occupied in occupation which requires
prolonged standing should be advised to walking at workand alternate with other positions such as sitting, preferablywith the legs in an elevated position. They should do“vajrasan” which helps to make calf muscle stronger and use
compression stockings as a preventive measure.

NO ASSOCIATION
Guberan et al

1973

Store employee

Standing

CONCLUSION

Maffei1986

Out patients

Standing

Weddell

1969

Community
sampleStanding

Our case control study of small sample size found thefollowing risk factors were associated with chronic venous
insufficiency: male sex, increasing age, prolong standing,prolong sitting, family history, prior venous surgery and post
thrombotic conditions. Further studies with larger sample
size and cohort study may show significance of parameter
which we found to be insignificant in our study like parity,
OCP and constipation.

These finding can be explained as venous pressure in
the healthy human leg is determined by the rate of arterial
inflow and venous outflow. In supine position, venous
pressure in foot vein is 10 mmHg and about 90 mm Hg
approximately in standing position which is equal to thepressure of a column of blood extending vertically from thatvein to the level of the heart Christian Kügler. With walking,lower extremity venous pressure is reduced fromapproximately 100 mm Hg (depending on height) to a meanof 22 mm Hg within 7 to 12 steps (44).
The regular contraction and relaxation of the deep leg
muscles is largely responsible for the upward blood flow.
Prolonged standing, on the other hand, is not a natural habit
in man, and it certainly hampers the blood flow in the lower
half of the body A. M. Stewart (37). The biological basis forthe standing hypothesis is, the muscle pump is less utilized
in people who stand continuously during work. The blood is
not pumped towards heart, the venous pressure remains
Fiziologia - Physiology • 2016.26.4(92)REFERENCES
1. Abenhaim L, Kurz X, Norgren L, et al. The management ofchronic venous disorders of the leg: an evidence-based reportof an international task force. Phlebology. 1999;14(suppl1):1–126.
2. Abramson JH, Hopp C, Epstein LM. The epidemiology ofvaricose veins: a survey of Western Jerusalem. J Epidemiol
Commun Health. 1981;35:213–217.
3. Beaglehole R, Prior IAM, Salmond CE, et al. Varicose veins in
the South Pacific. Int J Epidemiol. 1975;4:295–299.
4. Beaglehole R. Epidemiology of varicose veins. World Journalof Surgery 1986; 10(6): 898-902.

41

5. Bernlsen A. Des varices du membre inferieur, spccialemcnt au
point de vue de I'etiologie et du traitement chirurgical. Acla
Chirurgica ScandÍllarica 1927; 62: 61-85.
6. Callam M. Prevalence of chronic leg ulceration and severe
chronic venous disease in Western Countries. Phlebology.
1992;7(suppl 1):6s–12s.
7. Callman M J. Epidemiology of varicose veins. British Journalof Surgery 1994; 81: 167-173.
8. Coon MM, Willis PW, Keller JB. Venous thromboembolism andother venous disease in the Tecumseh Community HealthStudy. Circulation. 1973;48:839–846.
9. Da Silva A, Widmer LK, Martin H, et al. Varicose veins andchronic venous insufficiency: prevalence and risk factors in
4376 subjects in the Basle Study II. Vasa. 1974;3:118 –125.
As cited by Nicolaides A N. Investigation of Chronic Venous
Insufficiency: A Consensus Statement. Circulation 2000,
102:126-163.
10. Dale JJ, Callam MJ, Ruckley CV, Harper DR, Berrey PN.
Chronic ulcers of the leg: a study of prevalence in a Scottish
commurutv. Health Bull (Edinb) 1983; 41: 310-14.
11. De Castro-Silva M. Chronic venous insufficiency of the lower
limbs and its socioeconomic significance. Int Angiol.
1991;10:152–157.
12. DeBacker G. Epidemiology of chronic venous insufficiency.
Angiology 1997;48:569–576.
13. Evans C, Fowkes F, Ruckley C and Lee A. Prevalence ofvaricose veins and chronic venous insufficiency in men andwomen in the general population: Edinburgh Vein Study. J
Epidemiology Community Health 1999;53:149-153.
14. Guberan W, Widmer LK, Glaus L, et al. Causative factors ofvaricose veins: myths and facts. Vasa. 1973;2:115–120.
15. Hume M. Presidential address: a venous renaissance? J Vasc
Surg.1992;15:947–951.
16. Jantet G. Impact socio-e´conomique de la pathologie veineuseen Grande sBretagne. Phle´bologie. 1992;45:433– 437.
17. K Kröger, C Ose, G Rudofsky, J Roesener, D Weiland and H
Hirche. Peripheral veins: influence of gender, body massindex, age and varicose veins on cross-sectional area. Vasc
Med 2003;8:249-225.
18. KING, E. S. J. The genesis of varicose veins. Aust. N. Z. J.
Surg 1950; 20:126. As cited by Weddell JM. Varicose Veins
Pilot Study,1966. Br J Prev Soc Med. 1969;23: 179–186
19. Kontosic I et al. Work conditions as risk factors for varicose
veins of the lower extremities in certain professions of theworking population of Rijeka. Acta Med Okayama 2000; 54:
33-38.
20. Kurz X, Kahn SR, Abenhaim L, et al. Chronic venous disordersof the leg: epidemiology, outcomes, diagnosis andmanagement: summary of an evidence-based report of the
VEINES Task Force. Int Angiol. 1999;18:83–102.
21. Lafuma A, Fagnani F, Peltier-Pujol F, et al. La maladieveineuse en France: un proble`mede sante´ publiqueme`connu. J Mal Vasc. 1994;19: 185–189.
22. Laing W. Chronic Venous Diseases of the Leg. London, UK:
Office of Health Economics; 1992:1– 44.
23. Lake M, Pratt G H and Wright I S. Journal of the American
Medical Association 1942; 119:696.
24. Malhotra S L. An Epidemiological Study of Varicose Veins inIndian Railroad Workers from the South and North of India,

42

with Special Reference to the Causation and Prevention of
Varicose Veins. Int. J. Epidemiol. 1972; 1(2):177-183.
25. Mekky S, Schilling RSF, Walford J. Varicose veins in womencotton workers: an epidemiological study in England andEgypt. BMJ. 1969; 2:591-595.
26. Nicholson BB. Varicose veins: etiology and treatment. Arch
Surg 1927; 15: 351-72.
27. NoblG.
Der raricose Symptomencomplex. Seine
Grund/agen und Behandlung. 2nd ed. Berlin, 1910.
28. Novo S, Avellone G, Pinto A, et al. Prevalence of primitive
varicose veins of lower limbs in a random population sample
of western Sicily. Int Angiol. 1988; 7: 176-81.
29. Ochsner A, Colp R, Burch G E. Normal Blood Pressure in the
Superficial Venous System of Man at Rest in the Supine
Position. Circulation 1951, 3:674-680.
30. Paerovskv, I, Linhart, J, Dejdar, R, Svejcar, J, Kruml, J, And
Vavrejn, B. Research on the primary varicose veins andchronic venous insufficiency. Rev. Czech. Med. 1962; 8: 171.
31. Ruckley CV. Socioeconomic impact of chronic venous
insufficiency and leg ulcers. Angiology 1997;48:67–69.
32. Rudofsky G. Untersuchungen zur Epidemiologie,
Pathophysiologie, Diagnostik und Therapie venöser
Erkrankungen. Vasa 1980;suppl. 7:1-48.
33. Sciannameo F, Ronca P, Alberti D and Madami C. Varicose
veins in pregnancy: physiopathology and therapeutic
approach. Minerva Ginecol. 1993;45:539-43.
34. Silveira P R M. Estudo estrutural da veia safena magna normal
e varicosa. Rev Angiol Cir Vasc. 1993;2:116-33.
35. Staffa R. Chronic venous insufficiency. Epidemiology. Bratisl
Lek Listy 2002; 103 (4.5): 166.168
36. Stanhope J M. Varicose Veins in a Population of Lowland New
Guinea. Int. J. Epidemiol 1975; 4 (3):221-225.
37. Stewart A, Webb J and Hewitt D. Social medicine studies
based on civilian medical board records, Physical andoccupational characteristics of men with varicose conditions.
Brit. J. Prev. Soc. Med. 1955;9: 26-32.
38. Strano A, Novo S, Avellone G, et al. Pre´valence des varices
primitives des membres infe´rieurs dans une population
randomise´e de la Sicile occidentale. Art Veines. 1984;2:167–
171.
39. Stvrtinova V, Kolesar J, Wimmer G. Prevalence of varicose
veins of the lower limbs in the women working at a department
store. Int Angiol. 1991;10:2–5.
40. Tuchsen F, Krause N, Hannerz H, Burr H and Kristensen T,
Standing at work and varicose veins. Scand J Work Environ
Health 2000;26(5):4 14-420.
41. Venenerkrankungen D, Geschehen E K. Phlebologie
1997;26:164-168.
42. Weddell J M. Varicose Veins Pilot Survey, 1966. Brit. J. Prev.
Soc. Med. 1969; 23: 179-186.
43. Widmer LK, Mall TH, Martin H. Epidemiology and social
medical importance of diseases of the veins. Munch Med
Wochenschr 1974; 116:1421–1426.
44. Widmer LK. Peripheral Venous Disorders: Prevalence andSocio- Medical Importance: Observations in 4529 Apparently
Healthy Persons: Basle III Study. Bern, Switzerland: Hans
Huber; 1978:1–90.

Fiziologia - Physiology • 2016.26.4(92)Emotional regulation of children diagnosed
with Autistic Spectrum Disorder
Cristina Gianina Anghel1,2, Liana Kobylinska3,4, Mihaela Stancu1, Iuliana Dobrescu1,2
1. Child and Adolescent Psychiatry Department, “Prof. Dr. Al. Obregia” Clinical Psychiatry Hospital, Bucharest, Romania
2. Department of Child and Adolescent Psychiatry, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
3. Department of Physiology and Fundamental Neuroscience, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
4. Child and Adolescent Psychiatry Department, “Dr. Constantin Gorgos” Psychiatry Hospital, Bucharest, Romania

Abstract: Emotional regulation refers to the evaluation and appropriate modulation of emotional responses according to thesocial context or for achieving a goal. Emotional regulation is a spontaneous process which starts from an early age and is
influenced by family interactions, observational learning and modeling and represents one of the pillars of social skills. A
deficiency in ER could explain the emotional and behavioral problems in ASD, as well as the impaired social functioning ofthese individuals. This article tries to point out the particularities of ER in ASD and underline the need for further research inthis area, as well as for the development of therapeutic programs focusing on improving ER.
Abstract: Reglarea emotionala se refera la evaluarea si modularea adecvata a raspunsurilor emotionale in functie de contextul
social sau pentru atingerea unui scop. Reglarea emotionala este un proces spontan care incepe de la varsta mica si este
influentat de interactiunile familiale, invatarea observationala si sta la baza interactiunii sociale. Un deficit al reglarii emotionalear putea explica problemele emotionale si comportamentale din TSA, precum si deficitul in interactiunea sociala. Articolul defata incearca sa sublinieze particularitatile reglarii emotionale in TSA si importanta continuarii studiilor in acest domeniu, precum
si dezvoltarea unor programe de terapie bazate pe imbunatatirea RE.
Key-words: emotional regulation, autism, alexithymia, facial processing
Key-words: reglare emotionala, autism, alexitimie, procesare faciala

INTRODUCTION
The Autistic Spectrum Disorder (ASD) diagnosis criteria
include marked difficulties in social interaction, using nonverbal behaviors and the presence of repetitive andstereotype behaviors [1]. Children with ASD have frequently
been described as having difficulties in all emotional
aspects; deficiencies in understanding and expression ofemotions, in decoding facial expressions, and appropriate
management of emotions. [2] There is currently a growinginterest in emotional regulation (RE) literature in TSA due tobehavioral consequences that affect quality of life andrequire appropriate therapeutic measures [3].
Emotional regulation (ER) refers to the complex anddynamic processes involved in evaluating and appropriately
modulating emotional responses according to the social
context or for achieving a goal [4]. ER is a multifactorial
process with biological, cognitive, behavioral andobservational components [2]. In ASD, emotional regulation
could explain some of the behavioral and emotional issues
that affect functioning in all areas of child activity.
Studies have shown that there is a lack of emotional
regulation in children with ASD, which leads to increased
use of psychiatric services [5]. 74% of children with ASD

have difficulty of ER compared to only 18% in children withtypical development and these deficits occur early andcould be observed starting with the age of 12 months [6].
Emotional dysregulation is associated with majordifficulties especially in school environment, and often
children with ASD fail to graduate school or experiencefrequent class changes due to atypical RE strategies [7].
At the age of adolescence these difficulties are associatedwith depression, anxiety and behavioral problems.
Depression has recently been recognized as an important
contributor to suicidal thoughts experienced by adults
diagnosed with ADS [2].
Currently, efforts are made for trying to conceptualize
poor ER specific to ASD. Because these deficits are notfound in the diagnostic criteria, there is a growing tendency
to diagnose an associated condition in TSA thatencompasses consecutive behavioral manifestations
caused by the deficit of ER.
Thus, emotional dysregulation in ASD could no longer
be considered as a mark of ADS. In support of this statement
come the literature that shows that emotion dysregulation is
not always present in ASD [3].

Autor corespondent: Mihaela Stancu, ms.stancu@yahoo.ro

Fiziologia - Physiology • 2016.26.4(92)43In addition, there is evidence of a possible biologicalvulnerability that affects ER in ASD, including the variabilityof cardiac frequency and neuronal activity. [3]and immature theory-of-mind abilities, characteristic ofpreschoolers with ASD, further complicate their ability toregulate social stress. [10]Neural mechanisms involved in ER
Studies show that in ASD there is an up-regulative
failure in the brain regions involved in adaptive RE strategies
such as cognitive re-evaluation that involves changing
cognition about a situation. ER activates areas of prefrontal
cortex (ventrolateral, medial and dorsolateral) involved incognitive control and subcortical areas involved in arousal
and motivation (Critchley et al, 2007). The prefrontal cortex
modulates the activity of the limbic regions that alter theexperiential, behavioral and neurobiological aspects ofemotional responses. Connectivity abnormalities between
these areas have been described in ASD, some studies
showing that there is a hypo-connectivity, while others havedescribed cerebral hyper-connectivity [3][8]. It is currently
believed that in ASD there is a hyper-connectivity of brain
areas that are close to each other and a decrease inconnectivity in long brain circuits that ensure large-scale
integration [9]Maladaptive strategies used in ASD: ¼ of restrictive andstereotype behaviors appear in response to emotional
triggering as a ER trial; this strategy can be effective in the
short term but maladaptive over time. Studies that examined
ER strategies and their effectiveness in ASD for 3 different
emotions (anger, anxiety, and amusement) showed that thosewith ASD experienced more negative emotions and less fun.
Expression of anger and anxiety through tantrum crises,
accompanied by low levels of positive emotions, most affects
the daily functioning and well-being of children with ASD [12].
Other maladaptive behaviors used are avoidance,
suppression of emotions, self-talk, remaining focused on the
stressor which are often ineffective in lowering negative
activation [2,11]. Studies have shown that in ASD there is a
trial-error approach because children with ASD aged 3-10
years use a wider range of strategies than TD [13].

But there are other brain structures involved inmodulation of ER from ASD. Recent neuro-imaging studies
have shown the insulae involvement in experimenting andanticipating the negative consequences and striated cortex
in the rewards component of RE and the abnormal
impairment or functioning of these brain areas in ASD. [5]Behavioral Strategies for ER
Children with typical development use adaptive
strategies for ER: goal-oriented behaviors, social support,
cognitive re-evaluation. Cognitive revaluation involves
reassigning the meaning of a stimulus to changing the
trajectory of the emotional response [2]. Adaptive strategies
are context-dependent and selectively applied depending on
the situation. In contrast, maladaptive tends to be universally
applied. Compared to TD, ASDs use maladaptive strategies
due to cognitive inflexibility, deficit in perspective taking, anddifficulty in modulating behaviors [5].
Individuals with ASD use multiple maladaptive strategies
that are associated with a wide range of adverse
consequences such as impairment of social functioning andthe more frequent occurrence of symptoms of anxiety anddepression [3]. Because they fail to properly manage theiremotions, they often act impulsively to emotional stimuli
through crises of anger, self-harm and aggression, and thesebehaviors are interpreted as deliberate and defiant. Preschool children with ASD often use simpler tactics such as
self-soothing or proximity-seeking, and do not resort tocomplex strategies involving cognitive and attention-based
processes such as attention diversion or substitutive play. [10]Difficulties in affection sharing , limited understanding of
emotional messages, reduced eye-to-eye communication,

44

In ASD, the ER adaptive strategies are used less andrepetitive behaviors occur more frequently. These behaviors
can be used to control the environment and deal withoverwhelming emotions, but could not explain the presenceof behavioral speculations in general. However, they can beeffective in helping the child with ASD to cope with
undesirable emotions and could be a way of accepted by the
RE when they are not potentially dangerous for the child orothers and the cognitive modalities of ER could not be
accessed. Behavioral stereotypes may, however, also
reflect a state of distress, so caution is recommended ininterpreting them in the conducted studies [5].
Among the possible reasons why preschoolers with TSA
have difficulty with RE, there may be an increased frequencyof anxiety and irritability, abnormal brain response, difficultyin understanding the mental status of others [2].
Negative emotions and Anxiety in ASD
Impaired ER has been associated with several disorders
including anxiety and mood disorders. Another possible
explanation for the ER deficiency in ASD is the presence of
these associated conditions that may be responsible forthese emotional difficulties. On the other hand, the DE
deficiency can also be intrinsic to the underlying pervasive
disorder and then it becomes difficult to identify the sourceof consecutive behavioral problems [3].
.

The tripartite model of anxiety and depression claims
that general distress is a common factor but that thephysiological hyper-arousal is specific to anxiety andanhedony is specific to depression. In ASD we encounterincreased levels of irritability since small ages and hyperarousal physiology in response to emotional distress. Thesefindings explain the excess presence of ASD anxiety and theefficacy of arousal management therapy. [5]Fiziologia - Physiology • 2016.26.4(92)Increased levels of anxiety, anger, and negative
reactions such as tantrum and aggression crises arecommon in children and adolescents with ASD and are arisk factor for the development of other psychiatric disorders.
Moreover, increased negative emotional affects social
functioning leading to difficulties in interpreting clues socialand consequent inappropriate behavior in these situations[3]. In order to objectively determine the symptoms ofanxiety in children, we used measurements of thephysiological response in response to stress and especiallythe autonomic nervous system response: heart rate,electrodermal activity and body temperature. In the studies
performed, children with ASD had an increased response toelectrodermal activity (dampened EDA response to faces ascompared to objects (Kushki et al, 2013).
Recognizing emotions in facial expressions
Developing ER skills at young ages is a spontaneous
process, due to family interactions, observational learning,modeling, and social reference. Social deficiencies in the
ASD and especially the reduced preference for human faces
and the difficulties in recognizing emotions interfere with thelearning process through observation and social experience.
[14]
Neuroimaging and behavioral evidences indicate to thefact that people with ASD show important anomalies in the
facial processing. These differences are often explained asa result of a innate deficiency of neural circuits or as asecondary consequence of low social interest levels.
Literature reviews that analyze typical and atypical facial
processing show that this represents a developmental ability
which is mainly mediated by early experiences. People with
ASD may experience cerebral defficiencies which hinder
their ability to atribute statuses to faces and expressions,
therefore narrowing down the number of visual stimuli andlimiting the development of specialized neural areas for
facial processing. Recognizing facial emotions represents
the base of emotion regulation because these offer us
information regarding the mental and emotional state ofothers. The difficulties in facial emotion recognition areheavily associated with ASD, although there arecontradictions of study results.
Eye-tracking and electrophysiology studies show thatfrom early childhood people with ASD experience adiminishing interest for faces and a lack of social motivation.
[15] These features are maintained by avoiding eye contact
which can occur as early as 6 months. [16] At the age of
15 months, children with ASD focus more on the mouth
region of the face, which negatively impacts face
expressions recognition and causes a loss of social stimuli.
Therefore, there is established an early deficient base of ER
development and social interaction.
Studies that analyzed the deficit of emotion recognition
in individuals with ASD show a great variability with results
Fiziologia - Physiology • 2016.26.4(92)between 5-70%. These differences made the authors
conclude that there can't be said that emotion recognition
deficit is a universal trait of ASD phenotype. [17]A 43 transversal studies meta-analysis of facial emotion
recognition (FER) in people with ASD have shown that acommon difficulty is especially indentifying fear and anger
and other subtle emotions expressions. These studies saythat a lack of facial emotion recognition in different social
contexts contribute to the social communication deficit. FER
is one of the main problems in ASD and represent a main
target in therapeutic intervention. Improvement of FER
through therapy leads to an improvement in social
interaction. [18]It is still unclear if FER deficit is applicable for the wholeemotional range or is limited to a division of basic emotions,or just for complex or subtle emotions. The processes
responsible for FER variability are not yet well known. [19]Furthermore, children with ASD also have difficulties inadequately expressing emotions. Even if they have a richemotional experience, often times their emotional
expression is inadequate to the context or being described
as "plain".
Alexithymia has been proposed as a factor which can
explain the FER difficulties and inadequate emotion
expression, met in certain children with ASD. The main
characteristics of alexithymia consist in difficulties in
identifying and describing emotions, which affects the abilityof understanding the emotional state of oneself andexpressing an emotional adequate response in a situation.
It is associated with social-affective deficits as well as lackof empathy. Studies have proven that alexithymia is more
frequently met in children with ASD compared to typicalchildren. Moreover, the facial expressions of those with highlevels of alexithymia are much more limited. Therefore, theoverlap of alexthymia and neurodevelopmental disorders
could explain difficulties in identifying and expressing
emotions. [20]ASD individuals also experience a deficiency in thetheory of mind (ToM). TOM refers to the ability to determine
the internal status of others, their thoughts, beliefs andmotivation of the persons we came in contact with, all thesewith the purpose of foreseeing their behavior. Studies
have shown that from a very early age, children react
depending on the emotions of others. Once a childunderstands that facial expression reveals something about
the internal status of a person, this knowledge represents
the pillar of "mind-reading" development, which reflects inthe ability to attribute a mental status to other people. ToM
underlines the importance of emotional information andtherefore the attention for facial expressions is being
stimulated. Baron-Cohen et al, 1986) A deficiency in ToM
development contributes to the difficulties regarding
emotional expression and regulation [21]Although emotional regulation (ER) is not included in
ASD criteria, it is frequently observed in this pathology. ER
45studies are still in an early stage, but it is important that themechanisms of ER deficiency in ASD be elucidated.
Emotional and behavioral problems in ASD have been
associated with a low level of independence and a severe
deficit in self-care as an adult. [3] Studies observing the
behavior in emotional contexts are rare in ASD and theauthors pointed out the necessity of further research in ER
behaviors in these children for pinpointing the social
stressing moments. The development of therapeutic
programs focusing on stimulation of using adaptive
strategies are bound to increase the coping in stressful
situations while also optimizing long term results for young
people with ASD.

REFERENCES
1. American Psychiatric Association, Diagnostic and statisticalmanual of mental disorders, (4th ed., text revision),
Washington, DC: American Psychiatric Association, 2000
2. Hirschler-Guttenberg Y, Goland O, Ostfeld-Etzion S, et al.
Mothering, fathering, and the regulation of negative andpositive emotions in high-functioning preschoolers with autism
spectrum disorder. J. Child Psychol Psychiatry.
2015
May;56(5):530-9
3. Mazefsky CA, Emotion Regulation and Emotional Distressin Autism
Spectrum
Disorder:Foundations
andConsiderations for Future Research, J Autism Dev. Discord,
2015 Nov; 45(11): 3405-8
4. Thompson, R.A, Emotion regulation: A theme in search ofdefinition. Monographs of the Society for Research in Child
Development, 1994, 59, 25-52.
5. Mazefsky CA, Herrington J, Siegel M, Scarpa A, et al, The roleof emotion regulation in autism spectrum disorder, J Am Acad
Child Adolesc Psychiatry. 2013 Jul;52(7):679-88
6. Totsika, V., Hastings, R.P., Emerson, E. et al. A populationbased investigation of behavioural and emotional problems
and maternal mental health: Associations with autism
spectrum disorder and intellectual disability, Journal of Child
Psychology and Psychiatry,2011, 52, 91-99
7. Samson, A.C., Hardan, A.Y., Lee, I.A. et al. Maladaptive
behavior in autism spectrum disorder: The role of emotion
experience and emotion regulation. Journal of Autism andDevelopmental Disorders,2015, 45, 3424–3432.
8. Anthony Richey J, Damiano CR, Sabatino A et al. Neural
Mechanisms of Emotion Regulation in Autism Spectrum
Disorder, J Autism Dev Disord. 2015 Nov; 45(11): 3409–3423.

46

9. Tager-Flusberg H, The origins of social impairments in autism
spectrum disorder: studies of infants at risk, NeuralNetw. 2010 Oct-Nov;23(8-9):1072-6
10. Jahromi LB, Meek SE, Ober-Reynolds S. Emotion regulationin the context of frustration in children with high functioning
autism and their typical peers. J Child Psychol
Psychiatry. 2012;53:1250–8
11. Mazefsky, C.A., Borue, X., Day, T.N et al. Emotion regulation
patterns in adolescents with highfunctioning autism spectrum
disorder: Comparison to typically developing adolescents andassociation with psychiatric symptoms. Autism Research,
2014, 7, 344–354.
12. Samson AC, Wells WM, Phillips JM et al. Emotion regulation
in autism spectrum disorder: evidence from parent interviews
and children's daily diaries. J Child Psychol Psychiatry. 2015
Aug;56(8):903-13
13. Konstantareas, M.M., & Stewart, K. Affect regulation andtemperament in children with autism spectrum disorder.
Journal of Autism and Developmental Disorders, 2006, 36,
143–154
14. Morris AS, Silk JS, Steinberg L, Myers SS, et al. The role ofthe family context in the development of emotion regulation,
Social Development, 2007, 16 (2): 361-388
15. Dawson G, Webb SJ, McPartland J. Understanding the natureof face processing impairment in autism: Insights frombehavioral and electrophysiological studies, Developmental
Neuropsychology. 2005;27:403–424
16. Jones W, Klin A, Attention to eyes is present but in decline in
2-6-month-old infants later diagnosed with autism. Nature.
2013 Dec 19; 504(7480):427-31.
17. Taylor LJ, Maybery MT, Grayndler L, et al. Evidence for shared
deficits in identifying emotions from faces and from voices in
autism spectrum disorders and specific language impairment.
International journal of language & communication disorders /Royal College of Speech & Language Therapists.2015,
50.10.1111/1460-6984.12146.
18. MB Harms, A. Martin, GL Wallace. Facial emotion recognition
in autism spectrum disorders: a review of behavioral andneuroimaging studies. Neuropsychol Rev. 2010 Sep;
20(3):290-322.
19. Berggren S, Engström AC, Bölte S. Facial affect recognition in
autism, ADHD and typical development. Cogn
Neuropsychiatry. 2016 May;21(3):213-27.
20. Costa AP, Steffgen G, Samson AC. Expressive Incoherence
and Alexithymia in Autism Spectrum Disorder. J Autism Dev
Disord, 2017,Jun;47(6):1659-1672.
21. Eack SM, Mazefsky CA, Minshew NJ. Misinterpretation of
facial expressions of emotion in verbal adults with autism
spectrum disorder. Autism, 2015 Apr;19(3):308-15

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