The aim of this procedure is to transplant a donor mouse abdominal aorta into an allogeneic recipient mouse. This is accomplished by first obtaining the donor aorta. Next, the graft site is constructed in the recipient animal.
The native aorta of the recipient animal is then dissected and divided. The final step is to use two end-to-end vascular anastomosis to implant the donor aorta. Ultimately, this procedure can be used to study the development of post-transplant vascular disease in allogeneic strain combinations.
The advantage of this method over, for example, heterotopic heart transplantation, is that it's easier to do morphometric analysis because the vessels are of uniform size. In addition, the vessel is not surrounded by parenchymal tissue, which can be surrounded by cellular infiltrates and other things that can complicate the analysis The data generated. Using this technique can be particularly useful in illustrating the mechanisms involved in post transplant vascular disease, which means an intractable problem in human clinical transplantation.
To minimize the effects of stress and to maximize their energy reserves, maintain the animals in the vivarium for at least 72 hours before surgery. On the day of surgery, place a metal operating board on top of a heating pad set to 35 to 38 degrees Celsius. Then after anesthetization, remove the hair on the ventral abdomen of the animal.
Use laboratory tape to mount the animal in a spread eagled position on the operating table, and then sanitize the entire shaved area of the animals three times with a disinfectant followed by ethanol using sterile scissors. Next, make an incision through the body wall to expose the abdominal viscera. Then use a sterile cotton swab to push the viscera off to the side onto a piece of sterile gauze wetted with warm saline, exposing the great vessels of the abdomen.
Now, very carefully blunt, dissect the abdominal aorta away from the surrounding tissue, including the inferior vena caver, and then carefully lift and cauterize the lumbar artery. The section of the aorta for dissection should be below the renal vessels and above the bifurcation of the aorta into the femoral arteries. After identifying the area tire suture just below the renal arteries and another just above the bifurcation.
Next, use scissors to divide the aorta and then very carefully hold one end of the vessel and drip heparinized saline so that it runs through the vessel by gravity. Then handling the ends of the vessel as little as possible. Remove the graft and immediately place it in a container of ice cold saline.
After administering anesthesia and analgesia to the recipient animal, shave the ventral abdomen as just shown. Apply gentamicin ophthalmic ointment to the eyes to prevent them from drying out, and then sanitize the abdomen as just demonstrated to ensure adequate disinfection. Now make a midline incision through the skin and body wall into stages.
Then insert a spreader to hold the abdominal incision open, pointing the screw assembly towards the hind end of the animal to keep the bulk of the clamp out of the way. Next place a saline soaked sterile gauze over the intestines. Then holding the gauze gently in place with a finger in.
Insert a sterile cotton swab under the intestines, and gently reflect them so that the intestines are sitting on top of the gauze. Place another piece of sterile gauze onto the intestines and wet it with saline. After gently removing any fatty tissue covering their ATA and the inferior vena caver, use forceps to carefully dissect the infrarenal aorta from the inferior vena caver.
Now clear an area large enough to provide room for two clamps, and with enough vessels such that when divided, there will be enough vessel to which the graft can be sutured. Note that the black suture can be used to feel your weight along the vessel. When dissecting look for vessel branches, cauterizing the branches with a low temperature cauterizer as necessary.
Next, insert one vascular clamp just below the renal artery and another vascular clamp just above the bifurcation. Then cut the aorta. In this instance, the cut is made closer to the bifurcation because a branch vessel was cauterized near the middle of the two clamps, making that a pore site for the anastomosis, the ends of the aorta will usually retract, leaving a space of about five millimeters.
Check for hemostasis. If the clamps are working correctly, only a small amount of blood should escape after the cut. Next, rinse the cut ends of the aorta with heparinized saline and then swab away the extra solution.
Now tack the graft into place using three discontinuous sutures at each end. Then suture one side of the anastomosis at each end and fill in the rest with seven to eight discontinuous sutures. If a running suture is preferred, be careful that the vessel walls remain relaxed and that stenosis at the anastomosis is not caused.
Now carefully remove the clamp at the cranial end of the aorta and look for leaks at the suture lines. Next, gently press on the aorta above the anastomosis site with a wetted cotton applicator and remove the second clamp Lightly press and release the aorta a couple of times to check that the vessel is patent. The graft will be perfused immediately and a pulse should be visible.
Now, remove the gauze covering the intestines and move them back into place, maintaining their normal anatomical orientation and avoiding twisting. Use a five zero Vicryl suture to close the muscle layer and then close the skin with a five zero or six zero proline suture. Treat the mouse subcutaneously with carprofen to augment the analgesia before terminating the anesthesia, and then give the mouse 0.5 to 0.8 milliliters of saline by the same root.
Finally, place the mouse in a heated cage for recovery and monitor the animal carefully. At 12 and 24 hours after the operation, administer buprenorphine and carprofen subcutaneously to the animal respectively. This first figure shows an aortic graft.
Note the suture lines as denoted by the white arrows here. A typical experiment in which recipient survival was followed for a period of 56 days is depicted. The control group consisted of wild type C 57 BL six by FVB recipient mice transplanted with bowel C aortas.
The experimental group consisted of C 57 BL six by FVB recipients, deficient in expression of heme oxygenase one. This knockout results in thrombosis of the grafts within 24 hours. Notably, this resulted in the death of all the recipients.
This last figure shows the echo measurements of the inferior vena, caver and abdominal aorta in a normal animal and in a transplant recipient. Note that the graft is patent and similar in appearance to the non-transplant aorta. As you've seen, the tissues are very delicate and small, so you need to bear in mind that this procedure is definitely an acquired skill and so therefore it will take a good bit of practice before the procedure can be done successfully, routinely, Once mastered.
This technique can be done within one hour if it's performed properly.
