変更されたカフを用いた膵臓移植マウス モデル

My name is Benno Cardini, from the Daniel Swarovski Research Laboratory of the Department of Visceral, Transplant, and Thoracic Surgery of the Medical University Innsbruck. Today, we are going to show you a mouse model for pancreas transplantation using a modified cuff technique. Anesthetize the donor animal with an intraperitoneal injection of ketamine and xylazine, and shave the abdominal region.

Then place the recipient on an operative field in a supine position, and scrub the surgical region three times with chlorhexidine. Now, perform a midline laparotomy from the pubic region to the xiphoid and enlarge the incision subcostally to both sides to gain a better view on the operative field. Then exteriorize the viscera to the left side, and inject heparin into the infrarenal vena cava.

In the next step, identify the abdominal aorta proximal to the crossing of the left renal vein, undermine the aorta, and tie it at that point. Now, move to the liver hilum, identify and dissect the common bile duct close to the liver, and ligate it close to the pancreas. Finally, transect the common bile duct above the ligature.

Next, dissect the portal vein close to the liver to achieve enough length for a venous anastomosis. As a tip, leave a small piece of liver on the portal vein for later easier identification. Next, undermine the left renal vein in the periaortic tissue and ligate it.

Then transect the renal vein and the periaortic tissue, staying on the right side of the ligature. Next, bluntly dissect the abdominal aorta from the celiac trunk to the diaphragm, undermine the aorta close to the diaphragm and transect it at that point. Next, identify and coagulate all lumbar branches and transect them.

Now transect the aorta under the previously set ligature. Now perfuse the pancreas via the aortic patch with ice cold perfusion solution until you see effluent coming out of the portal vein. On the top you can see the perfused portal vein and below the perfused aorta.

Now replace the viscera into the peritoneal cavity and separate step-wise the pancreas from the intestine using 8-0 silk ligatures. Starting from the postpyloric region undermine the duodenum twice and pull 8-0 silk ligature through it. Then ligate it and sharply separate the pancreas from the duodenum.

This approach is repeatedly performed until reaching the ligament of Treitz. It is essential not to damage the duodenum since it would exclude the graft from the experiments due to possible contamination. After the pancreas has completely been separated from the duodenum, move onto the mesenterium, undermine it and ligate it and then transect it.

In analogy to the previous steps, separate the pancreas from the transverse colon by undermining it, ligating it and finally transecting it. Now using the same approach, separate the pancreas from the biliary region by sharply dissecting it after ligation. Subsequently, bluntly dissect the pancreas from the stomach.

Identify the left gastric artery, ligate it and transect it. At the end, remove the pancreas with the attached spleen and put it in ice cold reperfusion solution until further implantation. Use the spleen as a handle.

In analogy to the donor, anesthetize the recipient animal with an intraperitoneal injection of ketamine and xylazine and shave the cervical region. Afterward put the donor animal on the operative field in a supine position and scrub the cervical region three times with chlorhexidine. Next, perform a skin incision from the jugular incision to the right mandibular angle.

Next, identify all lateral branches of the right external jugular vein, mobilize them, undermine them and divide them after bipolar coagulation. Afterwards, grasp the lower pole of the right submandibular gland, lift it cranially and dissect it after bipolar coagulation of its feeding vessels. Now transect all medial branches of the right external jugular vein after bipolar coagulation.

Ligate the external jugular vein twice as cranially as possible and divide it between ligatures. Pass the proximal end of the external jugular vein through a polyethylene cuff. Here it is important that the handle of the cuff faces the operating surgeon.

As next step, place the microvascular venous clamp coming from the lateral side. Now moisten the vein with heparin, remove the ligature and gently pull the vein over the polyethylene cuff. At the end, fix the vein over the cuff with a circular 8-0 silk ligature with one knot on the back and three in front.

To gain access to the right common carotid artery, mobilize and transect the right sternocleidomastoid muscle after proximal and distal coagulation. Subsequently, gently mobilize the common carotid artery, taking care not to harm the adjacent structures such as the vagal nerve. In analogy to the vein, ligate the common carotid artery twice, as cranially as possible, and divide it between ligatures.

Now pass the vessel through the arterial cuff. Finally, fix the arterial cuff with the microvascular clamp, coming from the medial side. Next, remove the ligature and widen the lumen of the artery using vessel dilators.

After sufficient widening of the lumen, pull the artery over the arterial cuff. At the end, in analogy to the vein, fix the artery with the 8-0 silk ligature. Here you can see the vein and the artery repaired for arterial venous anastomosis.

For implantation, place the graft on a moistened Ace in the cervical region. Then, pull the portal vein through an 8-0 silk ligature loop, shorten the vein for a tension-free anastomosis and next pull the portal vein over the venous cuff. Finally, fix the vein with a circular 8-0 silk ligature.

In analogy to the vein, pass the aortic stump through a ligature loop and shorten the aorta. Then, pull the aorta over the arterial cuff and finally fix it with a circular 8-0 silk ligature. Before reperfusion, identify the splenic vessels and ligate them and finally dissect them sharply and remove the spleen.

For reperfusion, first remove the venous clamp, followed by the arterial clamp. An adequate reperfusion of the graft shows a visible pulsation of the artery and a pinkish color of the whole graft. Moisten the graft with normothermic saline solution.

Before wound closure, remove the handle of the venous cuff to avoid a kinking of the vessel. At the end, close the wound by 6-0 running suture and inject 0.5 milliliters of normothermic saline subcutaneously. To validate the model, we tested the endocrine function of the transplanted pancreas.

Mice were rendered hypoglycemic by pretreatment with streptozotocin four days before surgery and were considered hypoglycemic if blood glucose levels were above 300 milligram percent. This line shot shows blood glucose levels of live non-transplanted control mice in red, as well as of recipients of grafts not exposed to prolonged cold ischemia time in blue. In addition, blood glucose levels of recipients of pancreatic grafts exposed to 16 hours cold ischemia time are shown in green.

Live mice remained hypoglycemic over the entire observation period. In contrast, transplantation of pancreatic grafts not exposed to prolonged cold ischemia time resulted in normal glycemia within 24 hours, confirming the endocrine function of the graft. Transplantation of grafts exposed to 16 hours cold ischemia time did not restore normal glycemia to recipients.

Even more, in this group all mice had to be sacrificed within 48 hours due to ischemia reperfusion injury associated early graft damage. In this video we presented a method for pancreas transplantation in mice using a non-suture cuff technique. This is technically and microsurgically feasible with excellent success rates.

However, given the progredient fibrosis of the pancreas, due to the duct occlusion, it is our opinion that this model is suited best for research areas focusing on the earlier ischemia reperfusion injury associated graft damage.