Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Abstract

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Figure 1:. Preparation of heart graft for transplantation.

(A) The heart is excised from the donor mouse. (B,C) The pulmonary artery trunk is exposed and pulled through a 20 G cuff, folded back, and secured with a 10–0 nylon suture. (D) A 10–0 nylon suture is placed through the edge of the recipient’s external jugular vein and fixed to underlying tissue. (E) A 10–0 nylon suture is placed through the edge of the donor aorta and secured to the underlying tissue adjacent to the recipient carotid artery. (F) The recipient’s common carotid artery cuff is inserted into the donor aorta and secured with an 8–0 silk suture. (G) The donor pulmonary artery cuff is inserted into the recipient’s external jugular vein and secured with an 8–0 silk suture. (H) Proximal slipknot on the recipient’s external jugular vein is released, followed by the release of the common carotid artery slipknot.

Figure 2:. Intra-operative view of cardiac graft.

A 1 mm 20 G cuff is pulled over the donor’s pulmonary artery and secured with a 10–0 nylon tie. A 0.6 mm 24 G cuff is pulled over the recipient’s right common carotid artery and secured with a 10–0 nylon tie. Anchor sutures (10–0 nylon) are placed in the wall of the donor aorta and the recipient’s right external jugular vein and secured to underlying tissue to prevent movement during cuff insertion. (AO = aorta, PA = pulmonary artery, CCA = common carotid artery, EJV = external jugular vein).

Figure 3:. Intravital two-photon imaging of leukocyte dynamics in the heart graft.

Intravital two-photon imaging of beating heart transplanted from B6 mouse to B6 LysM-GFP recipient demonstrates trafficking of recipient neutrophils into the cardiac graft tissues between 2–3 h postoperatively. (Green = neutrophils, red = vessels labeled with quantum dots). Scale bar = 20 μm.