Orthotopic Hind-Limb Transplantation in Rats

Donor Operation and Hind-Limb Harvest

1. For the operative procedure a sterile set of microsurgical instruments is utilized. The surgeon should wear proper attire including sterile gloves, which have to be changed prior to the start of the surgical procedure.
2. The rat is anesthetized with an intraperitoneal injection of pentobarbital (50mg/kg IP) and placed supine on a heated operation field, which facilitates a stable animal body temperature throughout the entire surgical procedure.
3. Prior to skin incision the filed of operation is sanitized three times each with both, a disinfectant and alcohol. To prevent the animal’s corneas from drying out, eyes are lubricated with an ophthalmic ointment (Vidisic) prior to surgery.
4. A skin incision is made across the right groin approximately at the level of the inguinal ligament. The femoral vessels are dissected free with bipolar cautery and blunt dissection; deep branches are ligated with 8-0 silk.
5. 300μl (50 IU) of heparin are injected via the penile vein for anti-coagulation.
6. Femoral vessels are ligated at the level of the inguinal ligament and cut distal to the ligature.
7. The hind limb is flushed with ice-cold (4°C) HTK solution (5ml) via the femoral artery over 5 minutes.
8. All muscle groups are sharply divided with scissors and osteotomy is performed with a rotating electrical saw at the level of the distal third of the femur.
9. The harvested limb is then submerged in 20mL of ice-cold HTK solution for preservation and cold storage until transplantation.

Recipient Operation and Transplantation

1. The rat is anesthetized with an intraperitoneal injection of pentobarbital and placed supine on the operation field.
2. A skin incision is made across the right groin approximately at the level of the inguinal ligament. The femoral vessels are dissected free with bipolar cautery and blunt dissection.
3. Femoral vessels are ligated as distal as possible, but proximal to the epigastric vessel takeoff. Vascular clamps are placed at the level of the inguinal ligament on artery and vein.
4. A polyethylene cuff is pulled over the artery (inside cuff diameter: 0.912 mm, cuff-wall thickness: 0.010 mm, cuff length: 2.0 mm) and vein (inside cuff diameter: 1.151 mm; cuff-wall thickness: 0.010 mm, cuff length: 2.0 mm). The edge of the vessel lumen is then pulled with a vessel dilator and the femoral artery and vein are everted over the whole body of the cuff (approximately 2mm) and fixed with a circumferential 8-0 silk ligature.
5. To make room for the donor limb the recipient limb is removed with sharp dissection of muscle groups and osteotomy is performed at the mid-femur.
6. The donor limb is removed from the HTK solution and transplantation begins with osteosynthesis of the femur using an 18-gauge needle (length: 1.5-2.0 cm) as an intramedullary rod.
7. Ventral muscle groups are closely approximated with 4-0 Vicryl or Prolene interrupted sutures.
8. Vascular anastomoses are performed by pulling the donor vein and artery over the recipient’s vein and cuff complex. A circumferential 8-0 silk ligature is again placed to secure the anastomosis.
9. The venous clamp is released followed by the arterial clamp ,and reperfusion is visualized by return of colour to the distal limb and foot, as well as pulsatile flow through the artery.
10. The remaining dorsal muscle groups are re-approximated with 4-0 Vicryl or Prolene interrupted sutures.
11. The skin is closed with 4-0 vicryl or prolene interrupted sutures.
12. The recipient rat is placed on a heating pad and monitored closely until recovery.
13. Recipient animals receive prophylactic antibiotics (Cefazolin, 20 mg/day, IM) and Buprenex (0.1 mg/kg, SC) for three posttransplant days.