Cuff-Free Suture-Based Cervical Model for Secondary Heterotopic Heart Transplantation to Evaluate Donor-Specific Immune Tolerance in Mice

Our lab develops a cuff-free, suture-based cervical secondary transplant model to study donor-specific immune tolerance. This protocol addresses the need for a reproducible cuff-free cervical technique that enables reliable secondary grafting in tolerance experiments. To begin, place an anesthetized donor mouse in the supine position on the surgical surface.

Secure the limbs with tape to prevent movement. Remove the hair from the surgical area and disinfect the skin three times using 70%isopropyl alcohol swabs. After confirming anesthetic depth by toe pinch reflex, use fine scissors to perform a midline laparotomy, followed by a midline sternotomy, and expose the abdominal and thoracic cavity.

With moistened swab, gently mobilize the intestines to the left side, then place saline-soaked gauze over the intestines to keep them moist. To begin perfusion preparation, gently cannulate the inferior vena cava using a 26 to 30-gauge needle or catheter. Then, slowly perfuse 0.2 to 0.3 milliliters of cold heparinized saline over 30 seconds, and wait one to two minutes for optimum heparinization.

Then, perform a bilateral thoracotomy to expose the heart with maximum visualization. Separate the anterior thoracic cage from the dorsal side and reflect it upward, securing it with a needle or clamp. Using blunt dissection, dissect the connective tissue between the ascending aorta and the pulmonary trunk.

Ligate the inferior and superior vena cava close to the heart using 8-0 nylon sutures. Transect the ligated vessels distal to the ligatures. Dissect the pulmonary trunk as distally as possible.

Next, ligate the left and right pulmonary veins together using a 6-0 nylon suture. Transect the ligated vessels distal to the ligature. Excise the heart while preserving adequate length of the ascending aorta by transecting it at the origin of the brachiocephalic artery.

Preserve the anterior wall of the pulmonary trunk as long as possible. Then, trim the ascending aorta and pulmonary trunk to ensure a secure anastomosis. Place the heart into a 60-millimeter cell culture dish filled with chilled normal saline.

Gently reperfuse the heart with one milliliter of cold heparinized saline through the ascending aorta or pulmonary trunk. Trim the vessels on the chilled plate as needed. Place the heart in cold ringers lactate solution on ice until implantation.

Begin recipient preparation by injecting 0.5 milliliters of warm normal saline subcutaneously into the dorsal region of an anesthetized mouse on a heating pad. Position the mouse supine with the tail facing the operator, and secure the head, limbs, and tail with tape. After skin disinfection, don fresh sterile gloves and make a longitudinal skin incision in a reverse T shape over the neck from the sternum to the right mandibular angle.

Using blunt dissection, mobilize the right external jugular vein from the right medial clavicle to its major confluence. Cauterize and transect the small branches of the vein. Next, dissect and remove the submandibular salivary gland using cautery.

Transect the sternum mastoid muscle to expose the right carotid artery. Using blunt dissection, mobilize the right carotid artery as far as possible above the carotid bifurcation. Apply a straight glover atraumatic vascular bulldog clamp to the proximal portion of the carotid artery.

Then, place a 10-0 nylon ligature around the distal portion at the carotid bifurcation. After ligating the external jugular vein at the proximal end and applying a clamp at the confluence of the retro mandibular and posterior auricular veins, transect the external jugular vein and carotid artery between the ligature and clamp using fine scissors. Then, flush the vessel openings with normal saline and keep the field moist.

Place the donor heart graft upside down in the cervical region, with the pulmonary trunk oriented laterally and the ascending aorta oriented medially. Cover the graft with moistened gauze while leaving the pulmonary trunk and descending aorta exposed. Anastomose the donor ascending aorta to the recipient carotid artery using 11-0 nylon sutures with a sleeve technique.

Now, anastomose the donor pulmonary trunk to the recipient external jugular vein. After release of vascular clamps, the graft artery filled with blood produced a palpable pulse, and the graft became visibly red within 10 seconds. Kaplan-Meier survival curves demonstrated prolonged graft survival in intolerant mice compared with rapid rejection of third-party grafts.

Gross examination at endpoint showed visible differences between healthy and necrotic grafts. Histopathology showed preserved myocardial architecture in syngeneic intolerant grafts, but interstitial hemorrhage, necrosis, and infiltrates in rejected third-party grafts. Our cuff-free sleeve anastomosis of intraluminal stents works well in small caliber vessels and is supposed consistent to secondary change testing.

Key challenges includes microsurgical handling of small vessels, parenting symbiosis, and achieving consistent success across operators. This method standardizes secondary challenge experiments and improves rigor and reproducibility for mechanistic studies of donor-specific transplanters.