Investigating the fundamental mechanisms of transplant immunology relies on reproducible animal models. The orthotopic hindlimb model we propose here using a refined technique for vascular anastomosis allows for more reliable anastomosis and better success rates than with previously described techniques, even at a vessel diameter as small as 200¯o;m.

Methods: The model was designed for mice weighing 20-35g (6-20 weeks). C57BL/6 (n=20) were used as recipients, Balb/c (allogeneic) and C57BL/6 (syngeneic) as donors. The donor graft was harvested after dissecting the femoral artery and vein, and muscle cuts were made at the midfemoral level. The recipient animal was prepared in a similar fashion, and the graft was attached in an orthotopic position, performing osteosythesis of both femoral bones with an intramedullary rod. Following the muscle attachment, the vein was anastomosed using a non-suture cuff technique, while the artery was sutured with 6 stitches using 11-0 Nylon (Figure) with the special aid of intravital staining. After reperfusion, the skin was closed with 6-0 Vicryl to complete the surgery.

Results: Our orthotopic hindlimb model in the mouse allowed for success rates of 80%. The major complication vas venous thrombosis. Discovery of early rejection signs and application of the standard Banff grading criteria was possible. Long-term follow-up in syngeneic grafts at 50 days showed normal histological appearance.

The here proposed model will serve as an important tool in the future basic research of reconstructive transplantation.

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