Current issues in liver transplantation require the implementation of large animal models. This protocol describes the technique of porcine orthotopic liver transplantation without the use of a veno-venous bypass. This technique significantly reduces the logistical and technical complexity of the procedure.
Furthermore, by engrafting livers after 20 hours of static cold storage, our model simulates extended criteria donor conditions. Begin by placing the anesthetized donor pig in a supine position and fix the limbs at the base of the operation table with elastic bands. Scrub the skin with an antiseptic agent and cover the animal with sterile drapes.
After confirming the loss of petal reflex, perform a midline laparotomy beginning at the xiphoid process by using monopolar cautery and sever the falciform and the triangular ligaments using scissors and bipolar cautery. After sufficient liver dissection, incise the left portion of the diaphragm over a distance of 5 to 10 centimeters using scissors to locate the thoracic segment of the descending aorta. Encircle and place a 3-0 polyfilament ligature without tightening.
Incise the right portion of the diaphragm over a distance of 5 to 10 centimeters using scissors and identify the suprahepatic vena cava inferior. Relocate the intestine to the upper left of the donor and enter the retroperitoneal space by transverse incision of the peritoneum over a distance of 5 to 10 centimeters using scissors. Place two 3-0 polyfilament ligatures around the abdominal aorta.
One cranial of the iliac bifurcation and one approximately 3 centimeters cranially without tightening. Place another ligature around the infrahepatic vena cava inferior without tightening. Tighten the caudally-located first ligature around the abdominal aorta.
After occluding the abdominal aorta cranially of the second ligature, make a transverse incision between both the ligatures using scissors. Insert the cannula into the incision and secure it with the remaining ligature. Using scissors, sever the suprahepatic inferior vena cava far cranially close to the right atrium.
After blood loss of approximately 1, 500 to 2, 000 milliliters, cross-clamp the thoracic segment of the descending aorta by tying the ligature and start to antegrade perfusion. Tighten the ligature placed around the infrahepatic vena cava inferior. Incise the vessel cranially of the ligature and insert the surgical aspirator.
After perfusion with 3, 500 milliliters of preservation solution over the course of approximately 10 to 15 minutes, sever the inside supra hepatic vena cava inferior of the donor pig, then sever the infrahepatic vena cava inferior at the level of the left renal vein. Sever the bile duct cranial of the pancreatic tissue between two 3-0 polyfilament ligatures to avoid bile spillage, then sever the portal vein cranial of the pancreas. Locate the celiac artery after blunt preparation and follow dorsally to the abdominal aorta.
Excise the respective aortic segment to create a patch for the engraftment. Excise the diaphragm around the super hepatic vena cava inferior and sever the remaining adhesions using scissors. Extract the liver and place on ice.
Perform a cholecystectomy or tighten a ligature around the cystic duct, then flush the common bile duct with at least 20 milliliters of preservation solution. Place the perfusion cannula into the portal vein and flush the graft with 500 milliliters of preservation solution. Place the graft in a sterile bowl placed on ice.
Remove the lymphatic tissue of the aortic segment and the hepatic artery, thereby identifying and occluding the arterial side branches and lymphatic vessels with either clips, ligatures, or sutures. Proceed similarly with the portal vein. Perform shortening of the vessels and preparation of the aortic patch only upon and engraftment to take into account the individual anatomic circumstances.
Identify the super hepatic vena cava inferior and place sutures around both diaphragmatic veins after removing surrounding diaphragmatic tissue. Place the anesthetized recipient pig in a supine position. Enter the abdominal cavity and sever the falciform and triangular ligaments of the anesthetized animal using scissors and bipolar cautery as demonstrated earlier.
After sufficient liver dissection, encircle both the supra and infrahepatic vena cavae inferior close to the liver parenchyma. Incise the superficial peritoneal layer covering the hepatoduodenal ligament and identify the hepatic arteries before entering the liver parenchyma. Dissect using bipolar cautery or placing the clips, ligatures, or sutures.
Dissect and sever the common bile duct below the cystic duct junction between two ligatures. Identify the portal vein and encircle close to the liver parenchyma. Identify the abdominal aorta after incising the avascular layer between the right and left diaphragmatic muscles.
Prepare the aorta for aortic anastomosis by removal of the surrounding tissue. Perform recipient hepatectomy by placing in a traumatic vascular clamp on the portal vein and sever the portal vein close to the liver parenchyma followed by the infrahepatic vena cava. Place an atraumatic vascular clamp on the suprahepatic vena cava including the surrounding diaphragm while caudally retracting the liver and sever the vessel close to the liver parenchyma.
Remove the recipient liver from the abdominal cavity. Place the donor liver into the abdominal cavity and shorten the donor or recipient suprahepatic vena cava inferior to an adequate length while avoiding kinking or too much tension on the anastomosis. Place a single 5-0 monofilament suture as a supporting thread adapting the right corner of the donor and recipient supra hepatic vena cavae inferior.
Begin the dorsal side of the anastomosis from the left corner of the vessels with a 5-0 monofilament double-armed running suture. Remove the supporting thread when reaching the right corner and secure the running suture with a clamp, then continue with the ventral side of the anastomosis, again, beginning from the left corner of the vessels. After completing the ventral side of the anastomosis, tighten the suture with multiple knots without constricting the vessel diameter to avoid stenosis.
Perform a vascular anastomosis of the donor and recipient portal vein as demonstrated earlier using a 6-0 monofilament double-armed suture. Begin the portal venous reperfusion by removing the vascular clamp, occluding the recipient portal vein and occlude the donor infrahepatic vena cava inferior with a vascular clamp after draining approximately 200 to 400 milliliters of blood. Slowly remove the vascular clamp occluding the recipient supra hepatic vena cava inferior and search for active bleeding.
Shorten the donor and/or recipient infrahepatic vena cava inferior. then perform a vascular anastomosis of the donor and recipient infrahepatic vena cavae inferior. After clamping the abdominal aorta and making an incision to fit the patch, begin the aortic anastomosis with a 6-0 monofilament double-armed running suture at the cranial corner of the incision or patch.
When reaching the caudal corner, secure the running suture with a clamp and complete the anastomosis again beginning at the cranial corner. Tighten the suture with multiple knots and slowly remove the vascular clamp after checking all vascular anastomoses. Place a catheter in the common bile duct and secure it with a single ligature.
Close the abdomen temporarily by adapting the muscular fascia and the skin with a running suture and cover the abdomen with clinging film or drapes to avoid thermal loss. The mean operation time was 103.50 minutes including a mean and hepatic phase of 27.13 minutes. Only two recipients underwent an anhepatic phase of more than 30 minutes while all recipients showed declining lactate serum concentrations 4 hours after portal venous reperfusion.
Recipients required increased concentrations of norepinephrine immediately before and throughout the anhepatic phase to stabilize the mean arterial pressure at 60 millimeters of mercury or higher. Low epinephrine concentrations were simultaneously used to increase cardiac output in this vulnerable time period. After engraftment, the mean arterial pressure and required doses of vasopressors remained stable.
The LiMAx values obtained 6 hours after portal venous reperfusion were comparable to the values measured in the liver donors before organ procurement in all but one recipient with an anhepatic phase of 34 minutes. This model can be of special interest as a readout for workups focusing on mechanisms of ischemia and reperfusion injury and the reconditioning of extended criteria donor organs.
