Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

The overall goal of this procedure is to perform an end-to-end aortic interposition grafting in mice to study vascular rejection and transplant arteriosclerosis. This is accomplished by first harvesting a segment of the abdominal aorta from the donor mouse. The segment is then ortho topically grafted into the intrarenal aorta of an allogeneic recipient that is genetically different from the donor.

In the final step, the transplanted vessel segment is excised and analyzed for the development of transplant arteriosclerosis. Ultimately, the extent of transplant arteriosclerosis and immune mediated allograft. Vascular rejection can be assessed through the measurement of the intimal thickening and luminal narrowing, as well as by the immunohistochemical analysis of the tissue for the accumulation of immune cells.

Generally, individuals new to this procedure need time to become comfortable with microsurgical technique because it takes a high level of dexterity and practice the master. However, the method should be attainable to most researchers with practice diligence in patients. So demonstrating the procedure with Ms.Winnie ends a talented neurosurgeon and Adam Roso, a talented PhD student in my lab Begin by placing the donor mouse in the supine position on a clean, thin plexiglass board wrapped in a sterile drape under the operating microscope at an eight 30 x magnification.

After confirming adequate surgical anesthesia by toe pinch, use sterile scissors to make a single midline incision from the pubis to the ZipID 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 five forceps to separate the aorta from the IVC at the left renal artery near the bifurcation. Then use 10 zero 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 is just demonstrated. Then place two four millimeter microvascular clamps, proximal and distal to the segment of interest, approximately five millimeters apart.

Next, using vanous tube engine micro scissors make a single horizontal aorta otomy 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 three to four millimeter graft segment as just demonstrated. Then use a 25 gauge five eight 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 heparin ice 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 five forceps to avert it slightly.

Now use the forceps to drive a needle attached to a 10 zero 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 nine o'clock in both the upper and lower ends of the graft. Then starting at the upper end of the graft from three o'clock anastomosis.

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 grof forceps to close the abdominal wall with five zero 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. The amount of immune mediated allograft vascular damage is examined, quantifying the intimal thickening and luminal narrowing in grafted aortic segments at day 30. Post transplantation.

Indeed as observed, the allograft artery segments develop significant intimal thickening and luminal occlusion, whereas no changes are observed in the ISO graft control artery segments. Further, the recruitment of leukocytes into the allograft artery can be evaluated by examining the accumulation of CD four and CD eight positive T cells and macrophages by immunohistochemistry. As expected, all three types of immune cells were detected within the intima of the donor arterial tissues After development.

This technique paved the way for researchers in the field of transportation to explore alloimmune mediated vascular reduction and transplant arterial sclerosis.