Dual-clamp technique facilitating end-to-side suture anastomosis in murine cervical heart transplantation

Heterotopic cervical heart transplantation (HT) in mouse is a widely used preclinical model in transplant immunology and chronic allograft vasculopathy research 1,2 . Although the cuff technique is popular for its simplicity, it compromises unilateral cerebral blood flow, requires custom-made cuffs, and carries higher risks of thrombosis and vessel twisting. In our center, we favored manual end-to-side suture anastomosis, as it preserves the continuity of the common carotid artery (CCA) and external jugular vein (EJV), thereby preventing neurological complications and adhering to the 3Rs principle. Moreover, it avoids the high-velocity arterial jets in the end-to-end grafts and ensures maximum comparability between studies using abdominal HT model 3 .A major technical hurdle of this manual approach is that the CCA lies deep and is widely separated from the EJV, making precise suturing highly demanding, especially in the confined cervical space. To overcome this, we applied a dual-clamp occlusion technique 4 (Figure 1A). Two micro-clamps are used: one placed at the proximal end and one at the distal end, each simultaneously clamping both the CCA and EJV, physically bringing the two vessels into close proximity and effectively eliminating height mismatch (Figure 1B-E). Additionally, sterile nonwoven pads moistened with cold saline are placed over the clamp handles. The gentle downward pressure not only keeps the graft cool but also elevates the deeper CCA up to the superficial plane. This simple modification makes the artery-first anastomosis and the entire everting suture feasible.Since implementing this refined technique in our established HT program (>1000 cases since 2010), we achieved a technical success rate of 83/87 (95.4%) in recent consecutive procedures. Technical success was defined as graft survival with maintained palpable contractility throughout the 100-day observation period (assessed daily by palpation). All 83 successful grafts remained functional over this period without thrombosis or other vascular complications. Although manual suturing is generally considered technically more demanding than cuff method, the total recipient operative time (~25-30 min) was not prolonged. In fact, the anastomosis itself (15-20 min) was faster than cuff installation (20-30 min) 5