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

Murine heterotopic cardiac transplantation is a well-established model for investigating allograft rejection and immune tolerance. In tolerance studies, the placement of a second heterotopic cardiac graft is often required to assess donor specificity, with the cervical region providing an ideal anatomical site. While cuff-based techniques simplify vascular anastomoses, these methods introduce artificial materials that can promote peri-anastomotic inflammation, alter hemodynamics, and confound immunological analyses. Additionally, vessel narrowing resulting from genetic modifications, immunosuppressive treatment, advanced recipient age, or chronic experimental conditions can make cuff placement technically challenging in small-caliber vessels. Here, we present a cuff-free, suture-based technique for cervical heterotopic heart transplantation in mice. This method avoids the use of intraluminal polymers, preserves physiological blood flow, and reduces complications associated with altered flow dynamics. The protocol includes meticulous preparation of both the donor and recipient vessels, end-to-end micro-anastomoses of the carotid artery and external jugular vein, intraoperative patency assessments, and postoperative monitoring strategies. By minimizing the use of artificial materials and adapting to small-caliber vessels, this cuff-free technique establishes a physiological and reproducible platform for secondary heart transplantation. This approach enables rigorous mechanistic studies of donor-specific tolerance in murine cardiac transplantation models.