Our vascular cuff placement allows for laminar blood flow and we utilize a larger cuff for the donor pulmonary artery to ensure ample blood return to the recipients. These modifications used in this protocol decrease the likelihood of turbulent blood flow and thrombus formation and result in improved survival rates. This method provides valuable insight for studying immune responses after transplantation such as ischemia reperfusion injury and graft rejection.
Additionally, our technique allows for easier post-transplant monitoring or re-transplantation. Demonstrating this procedure will be Dr.Wen Jun Li, an expert microsurgeon in our laboratory who developed this technique. Prepare the surgical area by shaving the hair from the chest and abdomen using an electric razor.
Administer 100 units of heparin intravenously into the penile vein for males or external jugular vein for males and females. Place the mice in a supine position with forelimbs overhead. Secure the forelimbs and hindlimbs with surgical tape and disinfect the skin with 0.75%iodine and 70%ethanol.
Perform an incision for the median laparosternotomy from the umbilicus to sternal angle, followed by a bilateral thoracotomy along each costal margin. Fold the anterior chest wall over the neck for full exposure of the mediastinum. Excise the thymus and expose the intrathoracic inferior vena cava.
For retrograde perfusion, inject 1.5 milliliters of four degrees Celsius saline into the intrathoracic inferior vena cava with the needle oriented superiorly toward the graft. Ligate the superior vena cava using an 8-0 silk suture and divide distally. Repeat the retrograde perfusion by injecting another 1.5 milliliters of four degrees Celsius saline via the inferior vena cava.
Ligate the inferior vena cava using an 8-0 silk suture and divide distally. Dissect the aortic arch and pulmonary artery trunk for graft harvesting and transect both distally. Ligate the pulmonary veins on the posterior surface of the heart using a 6-0 silk suture and divide distally.
Perform graft preparation by removing the donor heart from the chest cavity. Place the excised heart in a plastic container filled with four degrees Celsius heparinized saline for one to two minutes. Transfer the graft onto a sterile plastic flask filled with ice for cuff placement.
Place a one millimeter long 20 gauge angiocatheter cuff over the pulmonary artery for the donor cuff. Using fine forceps, gently fold the edges of the artery back over the cuff. Secure the folded vessel to the cuff using a 10-0 nylon tie.
Store the donor heart in heparinized saline or another preservation solution at four degrees Celsius. Prepare the surgical area by shaving the hair from the cervical area using an electric razor. Apply sterile non-medicated ophthalmic ointment to the eyes to prevent corneal drying.
Place the animal in a supine position with forelimbs adjacent to the body and the head turned slightly to the left. Secure the forelimbs and hindlimbs with surgical tape. Disinfect the skin with 0.75%iodine and 70%ethanol.
After making a midline cervical incision from the lower mandible to the sternum, transect the right sternocleidomastoid muscle. Excise the right lobe of the inframandibular gland to create space for graft implantation. Tie a slipknot over the proximal external jugular vein using a 6-0 silk suture.
Ligate the distal external jugular vein and adjacent branches using an 8-0 silk suture. Suture under the vein and cover with gauze. Make a transverse incision across the anterior wall of the external jugular vein.
Place a 10-0 nylon suture through the edge of the proximal external jugular vein and the underlying tissue to secure the vein during cuff insertion. Ligate the distal right common carotid artery using an 8-0 silk suture just inferior to the carotid bifurcation. Tie a slipknot over the proximal common carotid artery using a 6-0 silk suture.
Transect the artery distally between the sutures. Then place a 0.6 milliliter long 24 gauge angiocatheter cuff over the recipient's right common carotid artery. Using fine forceps, gently fold the edges of the artery back over the cuff.
Secure the folded vessel to the cuff using a 10-0 nylon tie. Place the donor heart superior to the right cervical area. Drip cold saline onto the heart graft every few minutes during implantation.
Place a 10-0 nylon suture through the edge of the donor aorta and through a superficial bite of the underlying tissue to secure the graft in place. Flush the donor aorta with 0.5 milliliters of 0.9%heparinized saline. Insert the recipient's common carotid artery cuff into the donor aorta.
Secure the anastomosis with an 8-0 silk tie. Remove the aortic anchor suture. De-air the external jugular vein by flushing the recipient's external jugular vein with 0.5 milliliters of 0.9%heparinized saline.
Perform pulmonary artery anastomosis by inserting the donor pulmonary artery cuff into the recipient's external jugular vein and secure with an 8-0 silk tie. Remove the external jugular vein anchor suture and transect the remaining posterior wall of the external jugular vein to free the graft from the underlying tissue. Ensure the graft is properly oriented without kinking or twisting of the anastomosis.
Release the slipknots on the recipient's external jugular vein, followed by the common carotid artery to initiate heart graft reperfusion. The technique demonstrated in this protocol showed a survival rate of approximately 97%This could potentially be attributed to the larger 20 gauge cuff placed on the donor pulmonary artery to ensure ample return of blood flow to the recipient. Additionally, the alignment of blood flow with cuff placement in the present technique minimizes the risk of thrombosis and anastomotic turbulence.
The mean recipient operation time was approximately 37 minutes with an average cold ischemia time of 20 minutes. For survival studies, cardiac grafts were assessed daily by direct visualization and digital palpation of the heartbeat. Intravital two-photon imaging was performed early after transplantation to evaluate leukocyte trafficking.
It is important to check that the graft is properly positioned and oriented prior to skin closure to prevent twisting. This method can be used in re-transplantation models to identify resident immune cells in transplanted grafts that mediate alloimmune responses.
