A Mouse Aortic Interposition Model to Study Alloimmune-Induced Vascular Rejection
Transcrição
Begin by placing the donor mouse in the supine position on a clean, thin Plexiglas board wrapped in a sterile drape under the operating microscope at an 8-30X magnification.
After confirming adequate surgical anesthesia by toe pinch, use sterile scissors to make a single midline incision from the pubis to the xiphoid process. Next, use a small retractor to open the abdominal walls, exposing the abdominal cavity. Then, use sterile cotton-tipped applicators to gently retract the intestines superiorly to the animal's left. Cover the tissue with gauze moistened with saline, and move the reproductive organs inferiorly to locate the infrarenal aorta and the inferior vena cava or IVC.
Now, use medical number 5 forceps to separate the aorta from the IVC at the left renal artery near the bifurcation. Then, use 10-0 polyamide monofilament sutures to ligate the small branches near the aorta. When the donor aorta and IVC have been separated, saturate the vessel with saline. Cover the exposed cavity with moistened gauze and set the donor aside in a sterile area.
Now, place the recipient mouse under the microscope, and separate the recipient aorta from the IVC as just demonstrated. Then, place two 4-millimeter microvascular clamps proximal and distal to the segment of interest approximately 5 millimeters apart. Next, using Vannas-Tübingen microscissors, make a single horizontal aortotomy, and resect no more than 0.5 millimeters of the abdominal aorta to accommodate the donor aortic graft.
Return the donor to the microscope and transect a 3 to 4-millimeter graft segment as just demonstrated. Then. use a 25-gauge 5/8 needle to flush the excised aorta with heparinized saline, taking care that the tip of the needle does not come into contact with the vessel, and place the vessel in a container of heparinized saline on ice.
Within 30 minutes of the excision, place the donor aortic graft in the orthotopic position in the recipient, matching the respective graft anatomical orientation with that of the recipient. Then, gently grasp the tunica externa of the vessel and use the number 5 forceps to invert it slightly. Now, use the forceps to drive a needle attached to a 10-0 polyamide monofilament suture through the full thickness of the vessel wall to secure the donor aortic graft to the recipient's resected vessel.
For the placement of continuous stitches, position stay sutures at 9 o'clock in both the upper and lower ends of the graft. Then, starting at the upper end of the graft from 3 o'clock, anastomose the resected ends with two running sutures and secure the suture to the stay suture. Then, flip the graft over and continue the running suture to the dorsal part of the vessel, meeting the origin stay suture. Secure the suture without applying much pressure on the vessel, and repeat the suturing for the lower anastomosis.
Once the anastomosis is complete, release the distal microvascular clamp to allow retrograde blood flow through the vessel. If no bleeding is observed, release the proximal clamp. If no blood obstructions are observed, use a pair of forceps to gently grasp one end of the stay sutures and slightly avert the vessel to inspect the back wall of the vessel. No puckering should be observed. If a vigorous pulse pattern is observed in both the donor and recipient vessels, use cotton-tipped applicators to return the intestines to the abdominal cavity.
Finally, use a needle holder and Graefe forceps to close the abdominal wall with 5-0 polypropylene sutures and continuous stitching. Using a subcuticular closure, close the skin layer with the same sutures. The motor function of the hind limbs can be observed to assess the success of the surgery.
