A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity.

The mouse model of spleen transplantation offers a great opportunity for uncovering the role of spleen cells in the regulation of local and systemic immune responses in inflammatory disease. The main advantage of this protocol is that by using congenic mice, spleen transplantation makes it possible to check the fate, longevity, and function of spleen cells. Before beginning the procedure, place a sterile disposable drape over the surgical platform and confirm the lack of response to toe pinch in the anesthetized donor mouse.

Shave the hair from the entire abdominal area and place the mouse onto a sterile surgical platform under an operating microscope at a 6 to 10X magnification. Sterilize the exposed skin with an alcohol prep pad and secure the limbs with surgical tape. Enter the abdomen via a three to four centimeter midline vertical skin incision from the pubis to the xyphoid process.

Cauterize the skin vessels and extend the incision to the lateral abdomen on both sides to achieve better exposure. Using a sterile cotton swab, move the intestines to the right flank of the abdomen to expose the spleen and use a sterile low temperature cautery to cauterize the short gastric vein attached to the spleen. Place a piece of sterile gauze soaked with 37 degree Celsius saline over the spleen to keep it moist and separate and mobilize the portal vein from the pancreatic tissue.

Ligate the branches of the portal vein as demonstrated. Place a suture around the portal vein distal from the splenic vein and flip the spleen to the right side to expose the aorta and the splenic artery on the celiac trunk. Dissect and mobilize the aortic celiac splenic arteries and place a suture around the aorta proximal to the celiac artery.

Inject 100 international units of heparin into the inferior vena cava or IVC to heparinize the whole body. After three minutes, ligate the hepatic and gastric arteries in the aorta proximal to the celiac artery. Transect the portal vein and perfuse the whole body with 10 milliliters of four degree Celsius heparinized saline from the abdominal aorta distal to celiac trunk.

Collect the spleen graft and block with the associated aortic celiac splenic segment in the portal vein along with the segment of splenic vein and a small portion of pancreatic tissue. Then preserve the graft in five milliliters of four degree Celsius saline. 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 a 37 degree 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 four 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 microscissors.

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 four degree Celsius saline soaked gauze. Using two 11-0 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 four to five 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 and close 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 a 30 degree 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.

Hematoxylin and eosin of spleen isografts days one and seven after transplantation reveals that the architecture of the spleen isografts remains intact during the first post operative week. Flow cytometric analysis to investigate donor spleen cell migration after transplantation demonstrates that approximately 50%of the spleen cells are donor derived at one day after transplantation and approximately 46%are recipient derived. Seven days after transplantation, the donor derived leukocytes account for approximately 32%of the total spleen cells while the recipient derived cells increased up to approximately 53%Donor splenic leukocytes also migrated into the lymph nodes, blood, and bone marrow as early as day one and are maintained to at least day seven generating a unique Chimera valuable for splenocyte trafficking research.

Using genetically modified knock-in mice as spleen donors will allow the investigation of the roles of spleen cell derived mediators or key signaling pathways in disease processes. This model could be a powerful tool for exploring the mechanisms of spleen cell populations in response to pathogens, injury, inflammation, or transplant rejection.