The overall goal of this procedure is to perform an orthotopic intestinal transplant in the rat. This is accomplished by first procuring the small intestine from the donor animal. The second step of the procedure is the so-called back table preparation of the intestinal graft prior to transplantation.
The third step of the procedure is the engraftment by performing the arterial and venous anastomosis, followed by graft reperfusion. The final step of the procedure is the reestablishment of enteral continuity by completing the intestinal anastomosis in a fashion of end-to-end due to geogen ostomy and ileo ileostomy respectively. Ultimately, results can be obtained that show long-term survival of iso genetically transplanted animals through excellent graft function and sufficient oral nutrient absorption in allergenic combinations.
However, different aspects of acute and chronic intestinal rejection can be studied. This method can help to answer key questions in the field of intestinal transplantation, such as the mechanisms of acute and chronic rejection that may lead to graft failure and impair the clinical outcome. Visual demonstration of this method is critical.
The crucial steps are difficult to learn, mainly because of small vessel size and the necessity to employer fast and accurate microsurgical technique To minimize warm ischemia time Before surgery on the donor rat, keep it fasting with free access to water and glucose solution for 24 hours. After anesthetizing the animal with 2%isof fluorine inhalation. Check for sedation by performing a toe.
Pinch, shave the abdomen and disinfect skin three times with skin prep. Then perform a median incision after subcutaneous administration of the analgesic using a scalpel. Perform a median incision under a surgical microscope.
Wrap the small bowel in saline soaked gauze and place it on the left side of the abdomen with a sterile cos and swab. Gently separate the physiological adhesions between the pancreas and the ascending colon using micros if necessary, using seven zero silk like eight, and divide the ileocecal and right a middle colic vessels after spreading the ascending colon to the right side of the superior mesenteric vein or SMV with seven zero ties like eight and divide the right and middle colic vessels using a mosquito clamp held in place with plaster scene formed into shape. Retract the stomach upward so that the entire SMV is straightened out and exposed.
Next ligate and divide the pancreatic duodenal veins coming from the SMV. Then turn the graft over to the right side of the abdomen with the graft still on the right side of the abdomen, the loose connective tissue including all lymphatics between the SMV and the abdominal, A altar becomes accessible. Use seven zero silk to ligate the tissue and divide it to avoid postoperative lymph re from the intestinal graft.
Alter the connective tissue is divided ligate and divide the right renal artery. Systemic heparinized the rat using 200 units of heparin IV via the penile vein. Ligate the marginal arteries and divide the small bowel of the duino deju junction and of the terminal ileum.
Ligate the aorta proximally to the origin of the SMA transect the portal vein at the confluence with the splenic vein. Harvest the graft with its vascular pedicle consisting of the SMA with an aortic segment. Using a 20 gauge IV catheter on a 10 milliliter syringe via the aortic conduit.
Perfuse the graft using three milliliters of chilled University of Wisconsin solution or uw. Then with a 50 milliliter syringe, irrigate the intestinal lumen from the AL end with 30 milliliters of cold cetin and solution during the preparation of the recipient. Store the graft in cold UW solution before surgery on the recipient rat.
Keep it fasting with free access to water glucose solution for 24 hours after the animal is anesthetized with 2%isof fluorine. Administer carprofen for intraoperative analgesia. Shave the abdomen and disinfect skin three times with skin prep.
Then perform a median incision. Wrap the recipient bowel in normal saline soaked GREs and place it on the recipient's chest. Use sterile cos and swabs to bluntly open the retroperitoneum and expose the abdominal aorta and inferior vena caver just below the renal vessels, down to the level of the iliac vessels to avoid blood loss like eight.
The small lumbar and spinal tributaries from the aorta and vena caver with micro vessels.Clips. Cross clamp the aorta and the IVC below the left renal vessels, proximally and above the iliac bifurcation. Distally then incise the anterior wall of both vessels and wash out any remaining blood.
Move the graft to the field to perform an end-to-end anastomosis between the graft aortic segment and the recipient's infrarenal aorta. Using a continuous 10 zero proline suture. Place the graft in the right side of the abdomen and perform the back wall stitches of the arterial anastomosis and tie the lower stay suture.
Then turn over the graft to the left side of the abdomen to expose the front wall of the anastomosis with the graft still on the left side of the abdomen. Start the porter cable anastomosis by placing a lower stay suture. Then with 10 zero proline, perform an end-to-end anastomosis between the graft portal vein and the recipients IVC.
Then from the outside, secure the front wall stitches when all the sutures are in place. Remove the distal clamps followed by the upper clamps. Use direct pressure with cotton swabs to control any anastomotic bleeding.
Check the graft for equal and quick reperfusion after ligation of the mesenteric vessels, resect the recipient's entire small intestine, preserving two to three centimeters of proximal jejunum and one centimeter of distal ileum using approximately 16 uninterrupted six zero Monocryl sutures. Restore enteric continuity by proximal and distal end-to-end intestinal anas. Irrigate the peritoneal cavity with normal saline until clean.
Administer two milliliters of normal saline intraperitoneal for fluid replacement. Then use three zero Vicryl for the muscle layer, plus a continuous skin suture to close the abdomen after surgery. For a quick recovery, place the rat under a heat lamp.
Keep the animal fasting with access to water and glucose solution and administer subcutaneous injections of normal saline and glucose every eight hours for the first 24 hours on day two, restart them on rathel and water ad libitum following the transplantation procedure. General appearance, fur condition, and mucosal appearance should be normal after postoperative day one. The activity level should return to preoperative status.
However, apathic or abnormal behavior suggests early surgical complications. This figure shows the average postoperative weight after orthotopic small bowel transplantation. After an initial loss of up to 20%of body weight, the rats will start to gain weight again around postoperative day six to eight, and will have reached around 90%of their preoperative weight normally around postoperative day 14.
While attempting this procedure, it's important to focus on minimal blood loss and short warm ischemia. Time to be successful After its development. This technique paved the way for researchers in the field of intestinal transplantation to explore acute and chronic rejection in a rodent model.
