The overall goal of the following experiment is to transplant a donor vessel into the abdominal aorta of a recipient. The first step is to dissect the thoracic aorta from the donor mouse. Next anastomotic sites are created in the abdominal aorta of the recipient mouse.
The donor vessel is then implanted into the recipient abdominal aorta. Ultimately, successful transplantation can be accessed by the intact hind limb motor function of the recipient mouse. So this model is gonna help us understand the pathogenesis of graft arterial sclerosis.
And it is technically challenge, which requires a lot of prac practice and the patient to be perfect. So demonstrating the procedure will be Dr.Chen and assistant by Louanne. To begin confirm the proper depth of anesthesia in the donor mouse.
Once anesthetized, place the mouse in the supine position on a tray under an operating microscope. Clean the chest with repeated scrubbings of iodine and alcohol after exposing the heart and flushing it through with saline, remove the heart and lungs to expose the entire length of the thoracic aorta. Once visualized, begin carefully dissecting the thoracic aorta away from the surrounding tissue.
When lumbar branches are reached, ligate them near the aorta with silk suture before cutting them away. When freed from the surrounding tissue, excise the whole thoracic aorta. Once isolated, flush the donor aorta with heparinized saline and store it in this solution on ice until transplantation.
After ensuring adequate anesthesia in the recipient mouse, shave the fur from the anterior abdominal region. Cover the eyes with ophthalmic ointment and clean the area as seen before. When ready, place the mouse in the supine position on a tray under an operating microscope.
Incise the abdominal wall at the midline from xiphoid to pubis, and then use a retractor to spread the wall apart and expose the abdominal cavity Once exposed, wrap the intestines in saline moistened gauze, and push them up and away from the abdominal cavity. Use more saline moistened gauze to wrap the remaining abdominal areas and use this to retract the rectum to the right side of the abdomen. Next, move the reproductive organs inferiorly.
Remember to keep the exposed tissues moist by applying saline solution. Regularly identify the infrarenal aorta located proximal to the renal vessel and distal to the aortic bifurcation blunt. Dissect it carefully to separate it from the inferior vena cva.
Identify ligate and cut free all of the small branches originating from this segment of aorta. Once the aortic segment is isolated, place a vascular clamp at each end approximately five millimeters apart. Next, create the anastomotic sites by transecting the abdominal aorta between the clamps.
Using sharp micro scissors resect a small segment of the aorta from one cut end to accommodate the donor aortic graft. Flush the aortic segments between the clamps using heparinized saline to remove the residual blood. Remember to periodically irrigate both aortic samples with heparinized saline solution throughout the procedure.
Next, transect both sides of the donor aorta to create a segment graft of 2.5 millimeters in length for transplantation. Place the donor aortic graft in position lining up the proximal and distal ends for continuous sutures. Place stay sutures at the three and nine o'clock positions of both ends first.
Next, join the cut edges with three to four running sutures and tie the running sutures to the stay sutures with a double knot for interrupted sutures. Construct four anastomosis sites at the three and nine o'clock positions of both sides of the graft.First. Next, join the cut edges with three to four stitches placed between each of these sites.
Once the sutures are in place, release the distal clamp to allow retrograde flow into the graft. Identify any bleeding sites and make additional stitches within three minutes. When satisfactory hemostasis is achieved, release the proximal clamp amp.
The grafts patency can be confirmed by the presence of vigorous pulsation. If the pulsation diminishes, inject heparin into the vessel to prevent thrombosis. When ready, return the intestines into the abdominal cavity and suture the muscle and skin layers closed.
Place the recipient mouse into a warm, clean cage and monitor for one to two hours until consciousness returns. Assess the motor function of the mouse after recovery and again, on the second day, a transplantation surgery is successful if no dysfunction of the hind limbs is observed. Histological analysis of the artery grafts taken four weeks after surgery revealed an increased thickness in the vessel wall in female recipients of a male donor.
However, no graft arteriosclerosis was observed in the male to male group. In this example, the thoracic artery from a male donor was transplanted into an interferon gamma knockout mouse interferon gamma expression was then induced in the recipient mouse. Before samples were taken.
A few weeks later, histological analysis of artery grafts show that no graft arteriosclerosis was observed in the treated group. Now you know how to do this surgery. Keep trying.
