anesthetize male rats weighing 20020 grams with isoflurane on cone mask ( 3% for the induction , 2% during the operation , 1 l / min air flow , fio2 70% ) .  
perform a large median and transverse laparotomy and cauterize the epigastric vessels on both sides .  
cut the falciform ligament , and separate the left diaphragmatic vein from the suprehepatic vena cava .
divide the hepato - esophageal ligament and artery between 7 - 0 ligatures ( coagulation is a valid alternative ) .  
separate the right renal vein from the surrounding tissues and divide it between a 10 - 0 and a 7 - 0 ligature , on its proximal and distal ends , respectively .  
divide the right suprarenal vein between 7 - 0 ligatures and free the liver from its posterior ligaments by cutting under gentle traction .  
isolate the gastrosplenic vein and divide it between a 10 - 0 and a 7 - 0 ligature , on its proximal and distal ends , respectively .  
isolate the duodeno - pancreatic vein and divided it between a 10 - 0 and a 7 - 0 ligature , on its proximal and distal end , respectively .  
insert a 22 g , 3.5 mm stent into the common bile duct by practicing a small incision at 1 cm distance from the hepatic hilum .
secure the stent in position with a 7 - 0 ligature .   inject 10 ui of heparin , diluted into 1 ml of normal saline , through the dorsal vein of the penis .  
cannulate the vena porta ( as far as possible from the hilum ) with a 21 g needle and gently flush the liver with 20 ml of cold ringer lactate ( or other preservation solutions ) . at the same time , cut the vena cava below the renal veins to allow an adequate outflow .
place the liver into a basin filled with cold ringer lactate and lay the basin on an ice pad .  
insert the vena porta into its cuff , then evert the vessel around the edge to expose the intima . secure the everted segment on the cuff with a 7 - 0 ligature .  
position a micro - clamp ( 4 - 6 mm length ) on the proximal part of the infrahepatic vena cava .
overturn the liver and insert two prolene 8 - 0 sutures at the opposite lateral edges of the suprahepatic vena cava , from outside to inside .  
store the graft ( immersed into ringer lactate solution ) in the refrigerator at 0 - 4 c .   anesthetize male rats weighing 20020 grams with isoflurane on cone mask ( 3% for the induction , 2% during the operation , 1 l / min air flow , fio2 70% ) and keep on a warm pad . of note ,
inject 10 ml of normal saline subcutaneously and 0.03 g of piperacillin / tazobactam intramuscular before laparotomy .  
perform a midline xipho - pubic laparotomy .   reverse the xiphoid process with an auto - static forceps in order to better expose the liver .   cut the anterior ligaments and divide the left diaphragmatic vein between two 7 - 0 ties .  
divide the hepato - esophageal ligament and artery between 7 - 0 ligatures ( coagulation is a valid alternative ) .
divide the right suprarenal vein between 7 - 0 ligatures and free the liver from its posterior ligaments by cutting under gentle traction ( not shown in the video ( identical to donor operation ) ) .  
place a 7 - 0 ligature around the common bile duct , just below its division .
isolate the hepatic artery at the hilum and divide it between 7 - 0 ligatures .  
pay attention not to damage the common hepatic artery or the gastro - splenic vein .  
position the caval ring around the vena cava , cranially to the right renal vein . then secure the vessel wall to the ring by placing four cardinal 9 - 0 sutures .  
repeat the same operation with the portal ring on the vena porta .   inject 10 ui of heparin , diluted into 1 ml of normal saline , through the dorsal vein of the penis .   set the anesthesia on 0.25% and the fio2 on 100% .  
clamp the infrahepatic vena cava just above the right renal vein , then clamp the vena porta just above the gastro - splenic vein . finally clamp the suprahepatic vena cava being sure to include at least 2 mm of diaphragm .  
cut the left and the right portal branches and divide the septum in between .  
cut the infrahepatic vena cava close to the parenchyma , paying attention not to incise any of the sutures previously positioned .
perform an end - to - end anastomosis between the suprahepatic vena cava of the graft and the recipient .
start with the running suture of the posterior wall from left to right , then proceed on the anterior wall from right to left .
keep the anterior suture loose until the end in order to allow a generous flushing of the interior lumen before tightening the ends .  
insert the caudal branch of the approximator into the portal ring handle ; then insert the vena porta of the graft into the slit of the cranial branch of the approximator , with the portal cuff finding its position into the slot .  
close the approximator while flushing the recipient 's vena porta with normal saline and allow the cuff to insert into the vessel .
secure the recipient 's vena porta around its cuff with a 7 - 0 circumferential tie .   remove the approximator .
open the clamp on the suprahepatic vena cava and the vena porta in order : portal flow is now reestablished .  
remove the portal ring by cutting the 9 - 0 ligatures with extra - fine scissors .
insert the caudal branch of the approximator into the caval ring handle ; then insert the infrahepatic vena cava of the graft into the slit of the cranial branch of the approximator , with the caval cuff finding its position into the slot .  
close the approximator while flushing the recipient 's infrahepatic vena cava with normal saline and allow the cuff to insert into the vessel .
secure the recipient 's vena cava around its cuff with a 7 - 0 circumferential tie .   remove the approximator .
open the microclamps on the infrahepatic vena cava of the graft and the recipient in order : venous flow is now reestablished .  
perform a partial incision of the recipient 's common bile duct , apply a gentle traction towards the head , and insert the free side of the stent previously inserted into the bile duct of the graft .
is recommended .   of note : end - to - end hepatic artery anastomosis can be performed , when required by the experimental design ( the artery is not required for long - term survival after rat liver transplantation ) .  
the described technique could be successfully mastered by our team after approximately 10 training surgeries .
it allowed 100% long - term ( > 21 days ) survival on 36 consecutive lewis - to - lewis and dark agouti - to - lewis transplantations , after the short training period ( a number of syngeneic liver graft recipients have been kept alive for over 12 months now ) .
post- syngeneic transplant day 1 liver function tests showed median aspartate transaminase ( ast ) : 84
u / l ( 51.7 - 92.7 ) and median alanine transaminase ( alt ) : 172.5 u / l ( 127.5 - 240.2 ) .
the mean anhepatic phase ( from vena porta clamping to graft re - perfusion ) was 14 2 min , with the longest step being the suprahepatic vena cava anastomosis ( running suture ) time 92 min .
the quick - linker system allowed the positioning of 1.55 mm - bore cuffs on vena porta and 2.40 mm - bore cuffs on vena cava ( recipients weighing 200 20 g ) .
by contrast , when we tried transplanting using kamada 's technique , they could not exceed 1.40 mm and 2.16 mm , respectively ( rats matching for strain and weight ) .
as shown in figure 7 , quick - linker rings are designed to keep recipient 's vessels on optimal stretching , allowing minimum caliper and length wasting , which results in closer - to - physiological hemodynamic results .  
anesthetize male rats weighing 20020 grams with isoflurane on cone mask ( 3% for the induction , 2% during the operation , 1 l / min air flow , fio2 70% ) .  
perform a large median and transverse laparotomy and cauterize the epigastric vessels on both sides .  
cut the falciform ligament , and separate the left diaphragmatic vein from the suprehepatic vena cava .
divide the hepato - esophageal ligament and artery between 7 - 0 ligatures ( coagulation is a valid alternative ) .  
separate the right renal vein from the surrounding tissues and divide it between a 10 - 0 and a 7 - 0 ligature , on its proximal and distal ends , respectively .  
divide the right suprarenal vein between 7 - 0 ligatures and free the liver from its posterior ligaments by cutting under gentle traction .  
isolate the gastrosplenic vein and divide it between a 10 - 0 and a 7 - 0 ligature , on its proximal and distal ends , respectively .  
isolate the duodeno - pancreatic vein and divided it between a 10 - 0 and a 7 - 0 ligature , on its proximal and distal end , respectively .  
insert a 22 g , 3.5 mm stent into the common bile duct by practicing a small incision at 1 cm distance from the hepatic hilum .
secure the stent in position with a 7 - 0 ligature .   inject 10 ui of heparin , diluted into 1 ml of normal saline , through the dorsal vein of the penis .  
cannulate the vena porta ( as far as possible from the hilum ) with a 21 g needle and gently flush the liver with 20 ml of cold ringer lactate ( or other preservation solutions ) . at the same time , cut the vena cava below the renal veins to allow an adequate outflow .
place the liver into a basin filled with cold ringer lactate and lay the basin on an ice pad .
insert the vena porta into its cuff , then evert the vessel around the edge to expose the intima . secure the everted segment on the cuff with a 7 - 0 ligature .  
position a micro - clamp ( 4 - 6 mm length ) on the proximal part of the infrahepatic vena cava .
overturn the liver and insert two prolene 8 - 0 sutures at the opposite lateral edges of the suprahepatic vena cava , from outside to inside .  
store the graft ( immersed into ringer lactate solution ) in the refrigerator at 0 - 4 c .
anesthetize male rats weighing 20020 grams with isoflurane on cone mask ( 3% for the induction , 2% during the operation , 1 l / min air flow , fio2 70% ) and keep on a warm pad . of note , recipient and
inject 10 ml of normal saline subcutaneously and 0.03 g of piperacillin / tazobactam intramuscular before laparotomy .  
perform a midline xipho - pubic laparotomy .   reverse the xiphoid process with an auto - static forceps in order to better expose the liver .   cut the anterior ligaments and divide the left diaphragmatic vein between two 7 - 0 ties .  
divide the hepato - esophageal ligament and artery between 7 - 0 ligatures ( coagulation is a valid alternative ) .
divide the right suprarenal vein between 7 - 0 ligatures and free the liver from its posterior ligaments by cutting under gentle traction ( not shown in the video ( identical to donor operation ) ) .  
place a 7 - 0 ligature around the common bile duct , just below its division .
isolate the hepatic artery at the hilum and divide it between 7 - 0 ligatures .  
pay attention not to damage the common hepatic artery or the gastro - splenic vein .  
. then secure the vessel wall to the ring by placing four cardinal 9 - 0 sutures .  
repeat the same operation with the portal ring on the vena porta .   inject 10 ui of heparin , diluted into 1 ml of normal saline , through the dorsal vein of the penis .   set the anesthesia on 0.25% and the fio2 on 100% .  
clamp the infrahepatic vena cava just above the right renal vein , then clamp the vena porta just above the gastro - splenic vein .
finally clamp the suprahepatic vena cava being sure to include at least 2 mm of diaphragm .  
cut the left and the right portal branches and divide the septum in between .  
cut the infrahepatic vena cava close to the parenchyma , paying attention not to incise any of the sutures previously positioned .
perform an end - to - end anastomosis between the suprahepatic vena cava of the graft and the recipient .
start with the running suture of the posterior wall from left to right , then proceed on the anterior wall from right to left .
keep the anterior suture loose until the end in order to allow a generous flushing of the interior lumen before tightening the ends .  
insert the caudal branch of the approximator into the portal ring handle ; then insert the vena porta of the graft into the slit of the cranial branch of the approximator , with the portal cuff finding its position into the slot .  
close the approximator while flushing the recipient 's vena porta with normal saline and allow the cuff to insert into the vessel .
secure the recipient 's vena porta around its cuff with a 7 - 0 circumferential tie .   remove the approximator .
open the clamp on the suprahepatic vena cava and the vena porta in order : portal flow is now reestablished .  
remove the portal ring by cutting the 9 - 0 ligatures with extra - fine scissors .
insert the caudal branch of the approximator into the caval ring handle ; then insert the infrahepatic vena cava of the graft into the slit of the cranial branch of the approximator , with the caval cuff finding its position into the slot .  
close the approximator while flushing the recipient 's infrahepatic vena cava with normal saline and allow the cuff to insert into the vessel .
secure the recipient 's vena cava around its cuff with a 7 - 0 circumferential tie .   remove the approximator .
open the microclamps on the infrahepatic vena cava of the graft and the recipient in order : venous flow is now reestablished .  
perform a partial incision of the recipient 's common bile duct , apply a gentle traction towards the head , and insert the free side of the stent previously inserted into the bile duct of the graft .
administer effective analgesia according to local institutional protocols . a minimum duration of 24 h is recommended .   of note
: end - to - end hepatic artery anastomosis can be performed , when required by the experimental design ( the artery is not required for long - term survival after rat liver transplantation ) .
the described technique could be successfully mastered by our team after approximately 10 training surgeries .
it allowed 100% long - term ( > 21 days ) survival on 36 consecutive lewis - to - lewis and dark agouti - to - lewis transplantations , after the short training period ( a number of syngeneic liver graft recipients have been kept alive for over 12 months now ) .
post- syngeneic transplant day 1 liver function tests showed median aspartate transaminase ( ast ) : 84
u / l ( 51.7 - 92.7 ) and median alanine transaminase ( alt ) : 172.5 u / l ( 127.5 - 240.2 ) .
the mean anhepatic phase ( from vena porta clamping to graft re - perfusion ) was 14 2 min , with the longest step being the suprahepatic vena cava anastomosis ( running suture ) time 92 min .
the quick - linker system allowed the positioning of 1.55 mm - bore cuffs on vena porta and 2.40 mm - bore cuffs on vena cava ( recipients weighing 200 20 g ) .
by contrast , when we tried transplanting using kamada 's technique , they could not exceed 1.40 mm and 2.16 mm , respectively ( rats matching for strain and weight ) .
as shown in figure 7 , quick - linker rings are designed to keep recipient 's vessels on optimal stretching , allowing minimum caliper and length wasting , which results in closer - to - physiological hemodynamic results .  
early deaths , occurring within the first three hours from reperfusion , are most often due to a bowel infarction , as a consequence of a long portal clamping .
it is therefore recommended to keep the anhepatic phase within the ideal limit of 15 minutes ( 20 minutes still acceptable ) .
central air embolism can also be a cause of sudden death and it is compulsory to flush all veins prior to anastomosis .  
deaths occurring between postoperative day one and three are often due to liver failure or thrombosis of the vena cava .
one should therefore avoid any aggressive graft flushing before the explantation in the donor ( we flush with 20 ml over approximately 60 seconds ) and ensure an appropriate cold preservation ( full immersion into 0 - 4 c ringer lactate solution ) .
the risk of thrombosis can be decreased by minimizing the manipulation of the intima and preserving its integrity .
deaths between postoperative day four and nine are often related to bacterial cholangitis . based on our experience , a single dose of piperacilline / tazobactam ( 0.03 g ) given before laparotomy is enough to minimize the risk of common bile duct infection .
however , due to absence of arterial flow , some bile duct problems can be observed with cholastasis .
of note , a cold ischemia time up to 24 hours can be considered as acceptable .
the quick - linker system was originally designed to perform liver transplantation with three cuff anastomoses ( very short anhepatic phase ) .
however , the three - cuff technique requires the use of very small calipers for the suprahepatic vena cava ( shvc ) cuff and it has not been widely accepted due to the risk of liver outflow problems .
however , implantation phases up to 15 - 18 minutes are acceptable , allowing the use of the two - cuff technique , and a more physiological shvc drainage . as shown in this video paper , we routinely use the quick - linker system in the second part of the implantation only .
while some extra surgical time is required to position the quick - linker handles , this part of the procedure is associated with minimal risks and allows for a shortening of the critical anhepatic phase thanks to easier cuffs insertions .
other advantages of the quick - linker system are a better graft - recipient vessel alignment and the use of wider cuffs with better hemodynamic results compared to the previously described techniques ( including the one described by kamada et al.)(fig 7 ) .
 clinical progress in the field of liver transplantation has been largely supported by animal models1,2 . since the publication of the first orthotopic rat liver transplantation in 1979 by kamada et al.3 , this model has remained the gold standard despite various proposed alternative techniques4 . 
 nevertheless , its broader use is limited by its steep learning curve5.in this video paper , we show a simple and easy - to - establish revision of kamada 's two - cuff technique . 
 the suprahepatic vena cava anastomosis is performed manually with a running suture , and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick - linker cuff system6 . manufacturing the quick - linker kit is shown in a separate video paper . 
Protocol
1. Donor Operation
2. Graft Preparation
3. Recipient Operation
4. Representative Results
Discussion
Disclosures