The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver's common hepatic artery to the recipient liver's proper hepatic artery.
Rat orthotopic liver transplantation model is a powerful tool for studying multiple aspects of liver transplantation. From an immune response on the infection, to technical aspects of the procedure. The main advantages of this technique are that it can reduce complications, better representing comely liver transplantation, and improve recipient survival.
This modified rat orthotopic liver transplantation protocol closely mimics aspects of human liver transplantation and will serve as variable and clinically relevant research model. It is our hope that through the visual demonstration, viewers can learn all the details and shorten the learning curve for mastering the microsurgery skill required for this technique. After injecting 300 international units of heparin sodium into the intrahepatic inferior vena cava of an anesthetized 12 to 14 week old rat, make a five millimeter incision below the bile duct bifurcation, and insert the bile duct stent into the common bile duct.
Secure the stent with one 7-0 silk ligature, one millimeter above the incision, and one ligature 10 millimeters below the bifurcation. Cut the bile duct between the two ties and expose the proper hepatic artery. Divide the gastroduodenal artery between two 7-0 silk ligatures, and expose the left gastric artery, splenic artery and celiac trunk.
Then cut the left gastric artery, splenic artery, and celiac trunk between the artery ties. Use a 20 milliliter syringe equipped with a 21.5 gauge needle to slowly inject 20 milliliters of four degrees Celsius Ringer's lactate solution into the portal vein. To prepare the donor liver, cut open the bifurcation of the celiac trunk, splenic artery and left gastric artery to form a larger arterial sleeve cuff and insert the 1.5 millimeter length 24 gauge arterial stent into the donor common hepatic artery via the cuff.
Then secure the stent with an 8-0 polypropylene ligature and flush the stent with Ringer's lactate solution. After exposing the portal vein of a 12 to 14 week old rat recipient, divide the recipient bile duct at 0.5 centimeters below a tiler bifurcation, and insert a bile duct stent into the distal common bile duct. Expose the left, middle, and right hepatic arteries and tie the vessels distal to the hepatic artery bifurcation.
Cut the arteries close to the liver above the ties and place a long thin piece of gauze behind the super hepatic inferior vena cava. Place a 3D printed intrahepatic vena cava holder behind the intrahepatic vena cava and use a Tenno non-absorbable monofilament suture to sew the ends of the 3D printed handle together. Loosely tie a 7-0 silk ligature below both the 3D printed holder between the intrahepatic vena cava and the portal vein, and clamp the intrahepatic vena cava just above the right renal vein below the 3D printed cava holder.
Clamp the portal vein just above the pyloric vein and below the 3D printed PV holder. Using a three milliliter syringe with a 27 gauge needle attached, flush two milliliters of 37 degrees Celsius Ringer's lactate solution via the bifurcation of the portal vein, and clamp the super hepatic vena cava above the liver with a Kitzmiller clamp. Then cut below the clamp as close to the liver as possible and cut above the 3D printed holders for both the portal vein and the inter hepatic vena cava to remove the recipient liver.
For donor liver transplantation, carefully orient the donor liver within the recipient's body cavity in such a way that an upper cobble anastomosis can be created. Insert the portal vein cuff from the donor into the recipient portal vein and tighten the 7-0 silk tie. Remove the atraumatic clamp from the super hepatic vena cava and remove the microvascular clip for the portal vein.
After re-perfusing the liver with warm blood, insert the donor intrahepatic vena cava cuff into the recipient intrahepatic vena cava and secure the vessel with the 7-0 silk tie. Then remove the donor intrahepatic vena cava clip before removing the recipient clip. To perform an arterial anastomosis, cut off the portion of the celiac trunk from the donor that extends beyond the stent, and clamp the proper hepatic artery of the recipient.
Cut off the tie at the end and any extra tissue surrounding the vessel. And use Ringer's lactate solution to flush the lumens of both the donor and recipient vessel ends. Using a curved needle, place a 10-0 ethilon suture through the left aspect of the donor hepatic artery, 2.5 millimeters above the distal orifice of the stent, and out through the end of the stent.
Transfix the recipient proper hepatic artery 0.5 millimeters below the vessel orifice from the left side of the vessel, to the right side of the artery. Place the next suture through the right wall of the donor hepatic artery from the inside to the outside, at a distance from the stent orifice identical to the original stitch, and pull up on the two ends of the 10-0 nonabsorbable monofilament to slip the recipient proper hepatic artery up, and into the hepatic artery stent. Then tie the 10-0 nonabsorbable monofilament with itself over the donor hepatic artery.
Flush both the recipient and donor bile ducts, and insert the donor bile ducts stent into the recipient bile duct. Then tighten the tie that was previously placed around the recipient bile duct. In this representative experiment, the non hepatic artery anastomosis rat orthotopic liver transplant model demonstrated 50 and 37.5%survival rates, at 21 days and 60 days, post-operation respectively.
By contrast, the optimized hepatic artery reconnection procedure significantly increased the long term survival. Histological analysis of a representative subset of transplanted animals without hepatic artery reconnection revealed signs of hypoxic liver injury with central lobular necrosis, at day six and thirteen post-transplant. Extensive liver necrosis was associated with tremendously elevated levels of alanine immuno transferees and aspartate amino transferase in these animals.
In contrast, transplanted rats with hepatic artery reconnection exhibited no signs of liver injury and histological analysis revealed a normal liver parenchyma structure, with organized acini, lobules, arteries and bile ducts. In addition, histological analysis of the livers from non hepatic artery reconnection model rats revealed reactive changes after hypoxic liver injury. Including market bile duct proliferation, peri portal fibrosis and inflammation, and distorted liver parenchyma.
This protocol can be adapted to study many immunological and surgical aspects of liver transplantation and can serve as a model for testing novel therapeutic interventions relevant to transplantation.
