induction of anesthesia of the donor mouse is initiated with 5% isoflurane . the mouse is orotracheally intubated with a 20-gauge intravenous catheter and then placed on a rodent ventilator , using 100% oxygen at a rate of 125 breaths / minute and approximately 0.5 ml tidal volume ( 2% of its body weight ) .
heparin at 100 u / kg is injected into the inferior vena cava ( ivc ) , from just below the liver .
the diaphragm is cut along the ventral costal attachment toward the spine , and the thoracic cavity is exposed by cutting both sides of the chest wall to the neck .
after incising the ivc at the level of diaphragm , the right atrial appendage is cut and the lungs flushed with 2 ml of cooled ( 4 c ) lactated ringer 's injection and 0.1 ml of heparin via a transverse incision at the root of the pulmonary artery ( pa ) trunk . arresting ventilation at two thirds of end - tidal inflation , the heart - lung block is excised and stored on ice ( 4 c ) .
identify the pa , located at the most cranial aspect of the hilum , as well as the attached main bronchus ( br ) . carefully dissect the pa from the bronchus .
the cuff for the bronchus is derived from a 20-gauge iv catheter , and cut in length of 1.0 mm with an extension of 0.7 mm . using the same materials , the pv cuff varies with the weight of the donor mouse . specifically , for mice 24 - 27 grams the cuff size is 22-gauge , 0.7 mm in length with a 0.7 mm extension . for mice weighing 27 - 32 grams ,
the studies utilized a 20-gauge catheter that is 0.7 mm in length with a 0.7 mm extension .
the cuffs are inserted into the distal ends of the pa , pv and br and secured with a 9 - 0 suture .
the donor lung is flushed and washed with sterile saline with sterile heparin before storage .
the donor lung is then wrapped with sterile gauze soaked in sterile saline on ice ( 4 c ) , which keeps the lung very clean and sterile .  
a microvessel clamp is placed on the bronchus to prevent lactated ringer entry into the airway .
induction of anesthesia and mechanical ventilation are the same as described for the donor above .  
the left chest wall is shaved and prepped with 70% alcohol and the surgical field is draped .
a thoracotomy incision is made in the left third intercostal space , extending the incision dorsally close to the spine , and a microvessel clamp placed on the left pulmonary vessels and bronchus adjacent to the heart .
you can see pa at the cranial aspect , pv at the caudal end of hilum and br between them . using gentle traction on the hemostat to cause mild tension on the pa , br and pv ,
the left lung is pulled out from the thoracic cavity while leaving the central hilar structures clamped . the pa , pv and br are isolated by blunt dissection followed by placing a 9 - 0 suture positioned loosely around the pa , pv and br . after dissecting the pa completely from its adventitial sheath , a small transverse incision of approximately one fourth of the vessel 's circumference
is made into the anterior wall , leaving the continuation of the dorsal part of the artery intact .
the donor lung , wrapped in cold , lactated ringer - soaked cotton gauze and prepared as described above , is then positioned into the thoracic cavity , and cuffs inserted into the recipient pa , pv and br and secured with 9 - 0 suture .
after positioning the transplanted lung back into the recipient thorax , the thoracotomy incision is closed using a 5 - 0 suture .
buprenorphine ( 0.05 - 0.15 mg / kg ) is administered immediately after surgery , and for every 8 hours for 2 - 3 days post - surgery . our experience has taught us that it requires several months of repeated practice to become proficient in the mouse lung transplant model .
after proficiency is attained , we achieved a 96% ( 96/100 consecutive surgeries ) perioperative survival rate with deaths occurring within seven days post operatively .
two deaths were due to bleeding that began intraoperatively , and pneumothraces were the cause of death in the other two mice .
for all procedures , the warm ischemia time was 14.32  3.14 minutes , and cold ischemia time was 58.51 18.06 minutes .
three orthotopic lung transplant groups were studied : isograft : c57bl/6c57bl/6 , allograft : c57bl/10c57bl/6 and c57bl/6c57bl/10 .
we used only male mice but our technique can be also applied to female mice , because there is no significant anatomical difference between the sexes .
grading of rejection pathology was conducted in a blinded fashion utilizing standard criteria for clinical lung transplantation(table 1 ) . whereas we observed mild or no rejection in isograft ( c57bl/6c57bl/6 ) , both allograft combinations developed comparable acute or chronic rejection ( figure 1 ) .
in contrast , ob was significantly more frequent in the c57bl/10c57bl/6 than c57bl/6c57bl/10 group by day28 ( table 1 ) .
scoring of acute rejection ( " a " scores ) by standard criteria as described in representative results .
data represents the mean  sd of " a " scores at days 28 post transplantation .
data represents the quantity and percentage of mice in each group that developed ob at days 21 and 28 post transplantation .
panel 1b and 1c represents h&e and masson 's trichrome stained bl/10 lung allograft transplanted into bl/6 mouse recipient , which developed ob and non ob , respectively .
main trouble shooting during the procedure were as follows . blood flow disorder : flush donor lung until the color becomes white in color .
difficulty of cuff insertion into the donor : ascertain that vasculature is of sufficient length and free of attached fat and connective tissues .
bleeding at anastomotic site : use q tips to apply pressure at affected site for approximately 5 minutes .
recipient pa blood flow obstruction : mainly caused by torsion of the during cuff insertion . if air is observed within the pa then re - do of the anastomosis is necessary .
recipient pv blood flow obstruction : make sure the position and direction of the cuff . release the compression from the bronchial cuff .
pneumothorax : this may be caused by either bronchial perforation due to manipulation of the airway , or perforation of lung surface due to trauma associated with the transplant procedure .
the former is treated with surgical closure of the hole within the airway wall ; and the latter is treated by oversewing the leak on the lung surface using 10 - 0 nylon suture .
induction of anesthesia of the donor mouse is initiated with 5% isoflurane . the mouse is orotracheally intubated with a 20-gauge intravenous catheter and then placed on a rodent ventilator , using 100% oxygen at a rate of 125 breaths / minute and approximately 0.5 ml tidal volume ( 2% of its body weight ) .
heparin at 100 u / kg is injected into the inferior vena cava ( ivc ) , from just below the liver .
the diaphragm is cut along the ventral costal attachment toward the spine , and the thoracic cavity is exposed by cutting both sides of the chest wall to the neck .
after incising the ivc at the level of diaphragm , the right atrial appendage is cut and the lungs flushed with 2 ml of cooled ( 4 c ) lactated ringer 's injection and 0.1 ml of heparin via a transverse incision at the root of the pulmonary artery ( pa ) trunk . arresting ventilation at two thirds of end - tidal inflation , the heart - lung block is excised and stored on ice ( 4 c ) .
identify the pa , located at the most cranial aspect of the hilum , as well as the attached main bronchus ( br ) . carefully dissect the pa from the bronchus .
the cuff for the bronchus is derived from a 20-gauge iv catheter , and cut in length of 1.0 mm with an extension of 0.7 mm . using the same materials , the pv cuff varies with the weight of the donor mouse . specifically , for mice 24 - 27 grams the cuff size is 22-gauge , 0.7 mm in length with a 0.7 mm extension . for mice weighing 27 - 32 grams ,
the studies utilized a 20-gauge catheter that is 0.7 mm in length with a 0.7 mm extension .
the cuffs are inserted into the distal ends of the pa , pv and br and secured with a 9 - 0 suture .
the donor lung is flushed and washed with sterile saline with sterile heparin before storage .
the donor lung is then wrapped with sterile gauze soaked in sterile saline on ice ( 4 c ) , which keeps the lung very clean and sterile .  
a microvessel clamp is placed on the bronchus to prevent lactated ringer entry into the airway .
induction of anesthesia and mechanical ventilation are the same as described for the donor above .  
the left chest wall is shaved and prepped with 70% alcohol and the surgical field is draped .
a thoracotomy incision is made in the left third intercostal space , extending the incision dorsally close to the spine , and a microvessel clamp placed on the left pulmonary vessels and bronchus adjacent to the heart .
you can see pa at the cranial aspect , pv at the caudal end of hilum and br between them . using gentle traction on the hemostat to cause mild tension on the pa , br and pv , the left lung is pulled out from the thoracic cavity while leaving the central hilar structures clamped . the pa , pv and br are isolated by blunt dissection followed by placing a 9 - 0 suture positioned loosely around the pa , pv and br . after dissecting the pa completely from its adventitial sheath , a small transverse incision of approximately one fourth of the vessel 's circumference is made into the anterior wall , leaving the continuation of the dorsal part of the artery intact .
the donor lung , wrapped in cold , lactated ringer - soaked cotton gauze and prepared as described above , is then positioned into the thoracic cavity , and cuffs inserted into the recipient pa , pv and br and secured with 9 - 0 suture .
after positioning the transplanted lung back into the recipient thorax , the thoracotomy incision is closed using a 5 - 0 suture .
buprenorphine ( 0.05 - 0.15 mg / kg ) is administered immediately after surgery , and for every 8 hours for 2 - 3 days post - surgery .
our experience has taught us that it requires several months of repeated practice to become proficient in the mouse lung transplant model .
after proficiency is attained , we achieved a 96% ( 96/100 consecutive surgeries ) perioperative survival rate with deaths occurring within seven days post operatively .
two deaths were due to bleeding that began intraoperatively , and pneumothraces were the cause of death in the other two mice . for all procedures ,
the warm ischemia time was 14.32  3.14 minutes , and cold ischemia time was 58.51 18.06 minutes .
three orthotopic lung transplant groups were studied : isograft : c57bl/6c57bl/6 , allograft : c57bl/10c57bl/6 and c57bl/6c57bl/10 .
we used only male mice but our technique can be also applied to female mice , because there is no significant anatomical difference between the sexes .
grading of rejection pathology was conducted in a blinded fashion utilizing standard criteria for clinical lung transplantation(table 1 ) . whereas we observed mild or no rejection in isograft ( c57bl/6c57bl/6 ) , both allograft combinations developed comparable acute or chronic rejection ( figure 1 ) .
in contrast , ob was significantly more frequent in the c57bl/10c57bl/6 than c57bl/6c57bl/10 group by day28 ( table 1 ) .
scoring of acute rejection ( " a " scores ) by standard criteria as described in representative results .
data represents the mean  sd of " a " scores at days 28 post transplantation .
data represents the quantity and percentage of mice in each group that developed ob at days 21 and 28 post transplantation .
panel 1b and 1c represents h&e and masson 's trichrome stained bl/10 lung allograft transplanted into bl/6 mouse recipient , which developed ob and non ob , respectively .
main trouble shooting during the procedure were as follows . blood flow disorder : flush donor lung until the color becomes white in color .
difficulty of cuff insertion into the donor : ascertain that vasculature is of sufficient length and free of attached fat and connective tissues .
bleeding at anastomotic site : use q tips to apply pressure at affected site for approximately 5 minutes . recipient pa blood flow obstruction : mainly caused by torsion of the during cuff insertion .
if air is observed within the pa then re - do of the anastomosis is necessary .
recipient pv blood flow obstruction : make sure the position and direction of the cuff .
pneumothorax : this may be caused by either bronchial perforation due to manipulation of the airway , or perforation of lung surface due to trauma associated with the transplant procedure .
the former is treated with surgical closure of the hole within the airway wall ; and the latter is treated by oversewing the leak on the lung surface using 10 - 0 nylon suture .
orthotopic lung transplantation in mice is challenging due to microsurgical demands and extreme fragility of tissues .
introduction of the cuff technique has allowed for the widespread use of the orthotopic lung transplantation in rats .
this became the basis for the development of the orthotopic lung transplant model in mice in our lab . in mice and rats , unlike humans , the left lung contains only one lobe and makes up only 25% of the total lung mass .
this makes left - single lung transplantation feasible in the murine model without the need for a circulatory support system .
our preliminary surgeries revealed that ventilation and perfusion of the transplanted lung was highly dependent on the size of pv anastomosis .
inappropriate cuff sizing resulted in either atelectasis of the transplanted lung or dehiscence of the pv anastomosis .
okazaki et al reported the use of combined vessel ligation and clipping the bronchus . in the current study
we use an aneurysm clip for all hilar structures , which we suggest may shorten warm ischemic time .
one limitation of the orthotopic left lung transplant in the murine model is that recipient animals can survive after alloimmune - mediated necrosis of their allografts .
therefore , survival studies are not feasible and graft assessment depends on histological examination of the transplanted lung . using this technique
this suggests the role of minor , and not major , histocompatibility antigens in obliterative bronchiolitis pathogenesis .
this model represents a novel research tool for the examination of lung transplantation and the advancement of clinical lung transplants .
 orthotopic lung transplantation in rats was first reported by asimacopoulos and colleagues in 1971 1 . 
 currently , this method is well accepted and standardized not only for the study of allo - rejection but also between syngeneic strains for examining mechanisms of ischemia - reperfusion injury after lung transplantation . 
 although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies , the scope of those investigations is limited by the scarcity of knockout and transgenic rats . due to no effective therapies for obliterative bronchiolitis , the leading cause of death in lung transplant patients , there has been an intensive search for pre - clinical models that replicate obliterative bronchiolitis . 
 the tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3 . however , the lack of an intact vasculature with no connection to the recipient 's conducting airways , and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4 . unlike transplantation of other solid organs , vascularized mouse lung transplants have only recently been reported by okazaki and colleagues for the first time in 2007 5 . applying the basic principles of the rat lung transplant 
 , our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single - left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6 . 