We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.
In this procedure, the donor's aorta is placed in the orthopneic position and superficial bites taken in the feeding vessel. Anesthetize the mice with a mixture of 1.5 volume percent isoflurane and 100%oxygen through a face mask. Lay the mouse on a platform in the supine position and tape all the legs to the operating table.
Place ophthalmic ointment on the eyes to prevent dryness during the procedure. Check reflexes, pinching the hind feet to be sure the mouse is sufficiently anesthetized. Remove all hair from the abdomen.
Apply a depilatory cream or use a shaver. Perform the operation under sterile conditions. Disinfect the abdomen.
To remove the donor aorta, make a midline abdominal incision and retract the bowel manually to the right. Gently manually reflect the intestines over to the side using powder-free gloves. Place the bowels on a piece of gauze wetted with saline to keep them moist.
Dissect away the abdominal aorta very carefully from the surrounding tissue using blunt dissection with tweezers. Dissect the segment of the aorta between the renal artery and the bifurcation from the vena cava with the tweezers. Secure all the small branches of this segment using 11-0 monofilament single suture.
Before removing the aorta, inject 0.5 milliliter of saline solution containing 50 units of heparin into the inferior vena cava. Let the donor animal exsanguinate before the segment of the aorta has been removed. Rinse the graft fully with saline and place it after that immediately in a container of ice cold saline.
To prepare the recipient, anesthetize the animal through a face mask, remove the hair, and disinfect as above, and make a midline incision from the xiphoid to the pelvis with a scalpel. Retract the abdominal walls with retractors. Wrap the bowels in saline solution-moistened gauze and displace them very gently to the animal's right.
Dissect the infrarenal aorta free between the renal arteries proximally and the bifurcation distally with the tweezers. Secure all the small branches of this segment using 11-0 monofilament single suture. Clamp the proximal and distal portions of the aorta via a 6-0 single silk suture.
Divide the aorta in the middle between the clamps and irrigate the cut ends with heparinized saline to flush the lumen open. Place the graft in the orthopneic position. Perform the anastomosis using the sleeve technique with sutures using 11-0 monofilament.
With this method, the end of the feeding vessel is placed into the receiving vessel. Place the end of the infrarenal recipient aorta within the proximal end of the donor aorta. Perform the distal anastomosis in the same way.
Place the distal end of the donor aorta within the distal end of the recipient aorta. Take care to avoid any torsion of the aorta by ensuring the perfect alignment of donor and recipient aorta. The moment of the clamp's release is a very important step of the procedure and must be performed very carefully.
After checking that the anastomosis is in order, release the clamps. First release the distal clamp gently, followed by the proximal clamp. In this way there is low pressure to hold the walls together before the high-pressure side is released.
The graft is perfused immediately and a pulse is visible. Gently remove the remnants of the silk. Put back the abdominal contents into the abdominal cavity and close the wound using a running 3-0 polyglycolic acid suture.
Administer buprenorphine 0.05 milligram per kilogram to the mouse before terminating anesthesia. We present the examples of the necropsy pictures of the graft six weeks after transplantation and its ultrasound three-dimensional and B-mode view. The pictures show the patency of the graft.
There are no changes in lumen dimensions shown by the graft. In addition, we have found no stenosis or aneurysmal formations. We did not observe any significant lesions in transplanted aortas by histological and by immunohistochemical staining.
Nuclei were counter-stained by DAPI. In conclusion, this very rapid anastomosis method could be used to study vascular disease in genetically-engineered mice.
