Liver Cold Storage and Transplantation in the Cold-Adaptive Daurian Ground Squirrels

We study cold-adapted mammals, like DGS, to create better organ preservation methods. We goal is to understand their natural cold storage and improve human organ preservation. Our future work will explore how hibernation level, extreme cold, and stress and use that knowledge to improve organ preservation, extend storage time, and make transplants more successful.

To begin, make the required incision extending from the xiphoid process to the pubic symphysis on the anesthetized animal. Use cotton swabs to carefully free the infrahepatic inferior vena cava above the right renal vein from the surrounding tissues of the animal. Then, ligate the right adrenal and lumbar veins draining into the inferior vena cava near the liver using 7-0 sutures.

Isolate the proper hepatic artery, gastroduodenal artery, and common hepatic artery from the surrounding tissues using microforceps. Insert a seven-gauge intravenous needle into the aorta and secure it with a clamp. Then, incise the diaphragm to expose the thoracic aorta and place a vascular clamp on the thoracic aorta.

After perfusing the liver, incise the intrathoracic vena cava and the infrahepatic inferior vena cava above the right renal vein to allow the perfusate to drain out. During the perfusion, excise the gallbladder using surgical scissors. Make a small incision on the anterior wall of the common bile duct and insert the bile duct stent into the duct while securing it with a 7-0 silk suture.

Now, using microforceps, isolate the portal vein from the surrounding connective tissue. Ligate and transect the pyloric vein with a 7-0 suture and transect the portal vein trunk at the level of the splenic vein. Ligate and transect the gastroduodenal artery distally and create a small incision on the anterior wall of the common hepatic artery.

Insert the hepatic artery stent into the common hepatic artery and secure it with a 7-0 silk suture. Isolate the left diaphragmatic vein from the suprahepatic inferior vena cava and ligate it with a 7-0 suture. Dissect the suprahepatic inferior vena cava as close to the diaphragm as possible using fine surgical instruments.

Ligate the hepatosplenic ligament using a 7-0 suture. Secure the handle of the portal vein cuff with a vascular clamp and stabilize it using mosquito forceps. Carefully thread the portal vein through the cuff, ensuring it is not twisted, and fold the portal vein over the cuff and secure it in place with a 7-0 suture.

Ligate the cystic duct securely. Place a single 7-0 silk suture around the infrahepatic inferior vena cava between the attached cuff and the right inferior lobe of the liver. Insert 8-0 stay sutures at both lateral corners of the suprahepatic inferior vena cava from the exterior to the interior.

Carefully pass blunt forceps behind the suprahepatic inferior vena cava and place a 7-0 silk suture around it. Ligate the common bile duct proximally with a 7-0 silk suture and transect it above the ligature. After separating the bile duct, transect the gastroduodenal artery and proper hepatic artery between the ligations.

At the end of the common hepatic artery, form a Y-shaped bifurcation and leave a two-centimeter length of suture on the proper hepatic artery ligature for later use. Free the right and left branches of the portal vein. Then, place five-centimeter 7-0 stay sutures on each branch to aid in later handling.

Now, clamp the infrahepatic inferior vena cava above the right renal vein, followed by clamping the portal vein above the pyloric vein. Inject 1.5 milliliters of saline into the right portal vein within 10 seconds using a two-milliliter syringe to flush the blood from the liver. To retract the diaphragm, gently pull the 7-0 silk suture placed around the suprahepatic inferior vena cava.

Then, apply a bulldog clamp to the suprahepatic inferior vena cava, including the portion of the diaphragm in the clamp. Next, transect the suprahepatic inferior vena cava just above the liver. Secure the 7-0 stay sutures on both portal vein branches and transect the portal vein above these ligatures.

Then, transect the infrahepatic inferior vena cava close to the liver parenchyma and promptly remove the liver by cutting any remaining ligamentous attachments. Suture the posterior wall with eight to 10 continuous stitches beginning at the left corner. Continue with the anterior wall closure using 10 to 12 stitches moving from right to left with the same suture.

For portal vein reconstruction, use the previously placed five-centimeter stay sutures to gently elevate both portal vein bifurcations by applying upward traction with vessel clamps. Flush the portal vein lumen with heparinized saline at a concentration of 300 international units per milliliter. Insert the cuff into the portal vein and secure it using a 7-0 silk suture.

Sequentially release the clamps on the portal vein and the suprahepatic inferior vena cava to reestablish graft perfusion and conclude the anhepatic phase. Then, make a small incision at the bifurcation of the Y configuration at the terminus of the recipient's common hepatic artery. Flush the common hepatic artery lumen with heparinized saline at 300 international units per milliliter.

Insert the arterial stent into the common hepatic artery and secure it with a 7-0 silk suture. Tie the stay sutures on both the donor and recipient sides to stabilize the stent and prevent its displacement. Release the microvascular clip to restore arterial blood flow.

Then, make a small incision on the anterior wall of the recipient's common bile duct. Insert the bile duct stent into the lumen and secure it using a 7-0 silk suture. Finally, tie the stay sutures on both donor and recipient bile ducts to hold the stent securely in place and prevent displacement.

Recipients of liver grafts preserved in cold UW solution for 24 hours exhibited a 100%survival rate at seven days post-transplantation, regardless of whether they were in a hibernation state prior to transplantation or not. In contrast, all SD rats receiving liver grafts from other SD rats also preserved for 24 hours in cold UW solution succumbed within 12 hours post-transplantation.