Cervical Heterotopic Mouse Heart Transplantation using End-to-side Anastomosis Technique to Study Transplant Immunology

We study transplant immunology using refined mice cervical heart transplant model with end-to-side anastomosis. Compared to traditional cuff method, our end-to-side anastomosis avoids foreign material, preserves green indocyanine, and improves long-term graft survival. To begin, expose the peritoneal cavity of the anesthetized donor mouse covered with a sterile surgical gauze.

After retracting the small intestine, expose the abdominal aorta and the inferior vena cava. Inject 0.3 milliliters of heparinized saline into the inferior vena cava, and wait for one minute to allow systemic heparinization. Use scissors to transect the abdominal aorta and inferior vena cava to exsanguinate the donor.

Use scissors to incise the diaphragm, along with the anterior chest wall, to create a U-shaped incision through the ribcage and expose the thoracic cavity. Inject two milliliters of cold saline at four degrees Celsius into the heart through the intrathoracic inferior vena cava to flush residual blood. Cover the heart with crushed ice and gauze soaked in ice-cold saline, and keep the gauze moist with additional ice-cold saline to maintain hypothermia.

Identify and isolate the intrathoracic inferior vena cava. Ligate it using a 6-0 silk suture. Using microscissors, transect the vessel distal to the ligation.

Identify and remove the thymus to expose the aortic arch and pulmonary artery. Identify and isolate the superior vena cava. Ligate it using a 6-0 silk suture, and transect distal to the ligation.

Next, identify and isolate the ascending aorta. Transect the aorta proximal to the innominate artery to maximize vessel length. Identify and isolate the pulmonary artery.

Transect it just proximal to its bifurcation. Ligate all vessels posterior to the heart, including the pulmonary veins, with a 6-0 silk suture. Use microscissors to carefully excise the heart.

Remove the attached lung tissue and any excess surrounding tissue. Gently transfer the heart into cold saline placed on ice for storage until transplantation. Through the longitudinal neck incision on the anesthetized recipient mouse, carefully excise the right submandibular gland using forceps and a microcautery pen.

Next, mobilize the external jugular vein by cauterizing its branches. Transect the right sternocleidomastoid muscle to create a sufficient pocket for donor heart implantation. Isolate the common carotid artery while avoiding damage to the vagus nerve.

Use two microvascular clamps to first clamp the cranial ends of the vessels, then the caudal ends, to simultaneously occlude the external jugular vein and common carotid artery for anastomosis and reduce tracheal compression. After rotating the mouse 90 degrees clockwise, use an 11-0 suture to pass the needle through the vessel wall, and lift it at a distance roughly equal to the donor vessel's diameter. Use microscissors to excise a small portion of the vessel wall to create an anastomotic window.

Flush the lumen with heparinized saline to remove residual blood clots. Rotate the mouse again to place the donor heart in the cervical pocket. Anchor the donor ascending aorta to the recipient common carotid artery using two 11-0 sutures at proximal and distal ends.

Position the heart on the right side and cover it with wet sterile gauze. Using the distal suture, perform a continuous running suture on the posterior walls in a distal-to-proximal direction with six to eight stitches. Rotate the mouse 180 degrees.

Reflect the donor heart to the left and cover it with wet sterile gauze. Flush the lumen with saline. Perform the anterior wall anastomosis in a proximal-to-distal direction with six to eight stitches.

Make a longitudinal incision on the external jugular vein matching the diameter of the donor pulmonary artery. Flush the lumen with heparinized saline to thoroughly remove residual blood. Anchor the donor pulmonary artery to the recipient external jugular vein using two 11-0 sutures at proximal and distal ends.

Using the proximal suture, continuously suture the posterior walls in a proximal-to-distal direction with five to seven stitches. Continue suturing the anterior walls from outside in a distal-to-proximal direction, and after flushing the lumen with saline, tie at the proximal apex. After covering the sutures with sterile cotton, rotate the mouse and release the distal clamp first, followed by the proximal clamp, to initiate reperfusion.

Observe the cardiac graft turning red and regaining normal rhythm and contraction within one to two minutes. Confirm hemostasis and position the cardiac graft within the subcutaneous pocket. Close the skin incision using 5-0 sutures.

Survival analysis of 10 syngeneic and 10 allogenic recipients showed that recipients of syngeneic grafts survived for over 100 days with normal contraction rhythm and strength. In contrast, recipients with allogeneic grafts showed typical rejection within six to eight days post-transplantation. Histological evaluation revealed that syngeneic grafts maintained intact myocardial architecture without inflammation or graft vasculopathy at postoperative days seven and 100.

Conversely, allogeneic grafts at postoperative day seven exhibited severe acute cellular rejection, marked my massive lymphocyte infiltration, cardiomyocyte edema, and necrosis. This protocol is particularly suitable for genetically or drug-induced FRET mice and secondary or combined transplantations. Manual suture of tiny cervical vessels is technically challenging.

This method's so certain with clear abdominal heart transplant appearance. Future studies can explore tolerance mechanisms, secondary transplantation, combined organ transplantation models, and novel immunosuppressive strategies.