This article details a surgical procedure for heterotopic spleen transplantation in mice. It outlines the steps necessary to successfully transplant a donor spleen into a recipient mouse while minimizing blood loss and ensuring proper vascular connections.
Take an anesthetized recipient mouse for spleen transplantation.
Make a midline incision, exposing the abdominal cavity, and displace the intestine to locate the spleen.
Ligate the splenic artery and vein to minimize blood loss and remove the spleen.
Externalize the intestine and apply saline-soaked gauze to keep the exposed tissue moistened. Locate the inferior vena cava, or IVC, and the adjacent infrarenal abdominal aorta, or IAA.
Ligate the branches of the IVC and IAA. Clamp the vessels to interrupt blood flow and create openings for connecting the donor's spleen.
Flush the vessels with heparinized saline to remove blood and prevent clot formation.
Heterotopically position the donor's spleen and connect its artery to the IAA opening and the vein to the IVC opening.
Remove the clamps to allow blood flow to the spleen and close the incision.
Subcutaneously inject warm saline to rehydrate the mouse and monitor its recovery. Identify living donor spleen immune cells in the mouse to confirm successful transplantation.
Enter the recipient abdomen via midline incision after the surgical area is prepared, as it is for the donor, and remove recipient native spleen by ligating the splenic vein and artery. Cover the intestine tissues with the 37 degrees Celsius soaked gauze, and carefully move the intestine to the left side.
Dissect and ligate the lumbar branches of the infrarenal aorta in the IVC, and cross-clamp the infrarenal aorta in the IVC, with two 4-milliliter microvascular clamps. Place an 11-0 nylon suture through the infrarenal aorta, and retract the vessel to allow the creation of an elliptical aortotomy with micro-scissors.
Use a 30-gauge needle to pierce the IVC to create an elliptical venotomy and extend the opening to the length of the donor portal vein. Flush the aorta and IVC with 500 microliters of heparinized saline to clear the intraluminal blood or blood clots from the vessels, and place the donor spleen graft into the right flank of the recipient abdomen.
Carefully identify the donor aortic cuff and portal vein. After confirming that the vessels are not twisted, cover the spleen graft with 4 degrees Celsius saline-soaked gauze. Using two 11-0 nylon stay sutures, connect the donor aortic cuff to the proximal and distal apex of the recipient aortotomy, and make an anastomosis with two to three bites of continuous 11-0 nylon sutures between the donor aortic cuff and the anterior wall of the recipient aortotomy. Turn the spleen graft to the left side of the recipient, and make the anastomosis between the donor aortic cuff and the posterior wall of the recipient aortotomy.
Perform the venous anastomosis between the donor portal vein and the recipient IVC with 4 to 5 bites of continuous sutures on each side. Suture the posterior wall within the vessel lumen first, and then close the anterior wall using the same suture. Complete the anastomosis with a knot placed on the lower corner outside of the IVC.
Now, release the vessel clamps, and use a sterile cotton swab to tamponade the bleeding until the spleen color is recovered. Close the abdomen with a 5-0 synthetic absorbable vicryl suture in a continuous pattern. Enclose the skin layer with a 5-0 nylon suture in an interrupted pattern.
Then, inject 250 microliters of warm saline subcutaneously into four separate locations, and warm the mouse in the 30 degrees Celsius temperature-controlled incubator for the first few hours, post-operation, with monitoring until the animal has regained sufficient consciousness for transfer to the home cage.
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