The overall goal of this procedure is to perform and assess the health of a murine heterotopic heart transplant. This is accomplished by first carefully harvesting the heart of the donor. In the second step, the graft site is surgically crafted within the recipient to allow sufficient space to implant the donor tissue.
Next, the donor heart is transplanted through the formation of arterial and venous anastomosis. In the final step, hemostasis of the vascular suture lines is verified before closing the recipient. Ultimately, the health of the graft can be monitored by daily palpation to evaluate the effects of various factors on donor graft survival.
Visual demonstration of this method is critical as the anastomotic steps are difficult to learn. The tension of the sutures must be just right to avoid leakage and blood flow constriction. To harvest the donor heart first immobilize the anesthetized donor animal with four-way restraints.
After clipping the fur, wipe the skin with alcohol. After dissecting away the skin, make a transverse incision just inferior to the diaphragm. To open the abdominal cavity, cut the diaphragm posterior to the costal insertion.
Extend a full thickness, cut up the posterior lateral thoracic cavity on the left and right sides of the chest ice, isolate the inferior vena cava or IVC, and then place a loose five aught silk suture around the IVC adjacent to its insertion. Into the right atrium, inject one cc of four degrees Celsius. Heparinized SA saline into the IVC and ligate the vessel with the five T silk suture and divide.
Then after ligating and suturing the right superior vena CVA in a similar fashion, gently roll the heart towards the animal's right side and isolate and ligate the left superior vena cva exposing the left pulmonary artery. Now secure the heart under a wet gauze and blunt, dissect the thymus away from the main pulmonary artery, the left and right pulmonary artery branches, and the ascending aortic arch blunt. Dissect the aortic arch free of the surrounding tissues, and then use micro scissors to divide the aorta proximal to the right brachial cephalic artery until there are no branches between the heart and the division point of the aorta.
This section of the aortic arch forms the arterial cuff for the implant process. Next, reflect the aortic cuff inferiorly to expose the pulmonary artery trunk and the left and right branches of the pulmonary artery. Then blunt, dissect the left and right pulmonary artery branches away from the surrounding tissues as far from the heart as possible.
Divide the pulmonary artery trunk as distally as possible just proximal to its bifurcation to form the venous cuff for the implant process. Then tie a five aught silk suture around the base of the heart. Cut it free at the base and transfer the excised tissue into four degrees Celsius saline.
Total harvest time is approximately 10 to 15 minutes to implant the donor heart. After anesthetizing and immobilizing the recipient animal, wipe the skin with povidone iodine and rinse in between with alcohol or sterile water. Repeat this three times and then drape the surgical site in a sterile fashion.
Then begin the procedure by making a two centimeter midline vertical abdominal incision. To enter the abdominal cavity, retract the bowel superiorly, and externalize the tissue onto the chest, wrapping it in sterile gauze moistened with sterile saline. Next, isolate the abdominal aorta and inferior vena cava below the renal vessels and place four aught cotton ties around the tissue's superior, then inferior to the anastomosis site.
Identify any lumbar vessels within the field and ligate them with 10 T nylon. Suture, then not the inferior cotton tie, followed by the superior one to retain some blood in the aorta to make the A autotomy easier to perform. After securing the knots, enter the lumen of the aorta with a 30 gauge needle.
Then use fine micro scissors to extend the incision to a length of approximately two millimeters in a straight line along the longitudinal axis of the vessel. Now place a 10 aut nylon suture. Stay stitch in the donor aorta and to the inferior angle of the incision in the recipient aorta to make an end to side anastomosis of the donor aorta to the recipient aorta.
After tying the suture, place a second 10 T nylon opposite the first in the donor aorta and in the superior corner of the incision and the abdominal aorta, and tie this suture as well. When the second suture is in place, make a running suture line from the superior to the inferior in the lateral wall of the aorta, taking care to bring the intima together and tie it against the previously placed stay stitch. Next, suture the medial side in a running fashion and tie it placing the first and last stitches on each side as close to the stay stitches as possible.
Aim to have three evenly spaced stitches between these two, making a total of five stitches. Then to make an end to side anastomosis of the donor pulmonary artery to the recipient IVC first puncture the IVC with a 30 gauge needle, and then use the fine micro scissors to extend the incision for about two millimeters in a straight line along the longitudinal axis of the vessel. Next, tie the donor pulmonary artery to the inferior corner of the incision in the IVC with 10 knot nylon.
Place a second 10 oh nylon opposite the first in the donor artery and the superior corner of the incision in the abdominal IVC and tie. Then make a running suture line between the pulmonary artery and the IVC and tie it, placing the first and last stitches on each side as close to the stay stitches as possible. Aiming to have five evenly spaced stitches between these two, making a total of seven stitches.
Now release the distal four OTT cotton tie reestablishing venous flow. Once hemostasis of the venous anastomosis has been observed, gradually loosen the proximal four OTT cotton tie and observe the arterial anastomosis for hemostasis. When both anastomosis are considered secure, remove the cotton ties from the mouse, return the bowel to the abdomen and use five aught silk to close the abdominal wall in two layers.
In a running fashion, administer a one milliliter bolus of sterile, warm normal saline into the abdomen as fluid resuscitation upon closing and inject 0.8 milliliters of normal saline subcutaneously. Finally, to assess the graft function by daily transabdominal palpation, restrain the mouse as for an intraperitoneal injection. Then gently press the tip of a forefinger against the abdominal wall to ascertain the beating strength and regularity of the graft.
Give the palpation quality a score from four for a normal amplitude and frequency to zero for a non beating rejected graft. This surgical technique facilitates both simple graft survival studies as well as complex experimental protocols. For example, in this representative experiment, the involvement of fast and or perforin as mechanisms of CD four positive T-cell mediated cardiac rejection was investigated.
The results demonstrate that the direct rejection of the cardiac allografts by CD four positive effector T cells requires the alternative contribution of graft fast expression and T-cell perran expression indicating for the first time that cytolytic activity by CD four positive T cells can play an obligate role in primary acute allograft rejection in vivo. After watching this video, you should have a good understanding of how to place the sutures in your anastomosis to result in a healthy beating cardiac graft.
