Orthotopic Hind-Limb Transplantation in Rats

Abstract

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies. 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. Protocol Donor Operation and1. 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 ...

Orthotopic Hind Limb Transplantation in the Mouse

“…Although there have been various small 13,16,18,20,25,26 and large animal 14,27-29 models described for VCA as recently reviewed by Brandacher et al 30 only very few are actually applicable for basic mechanistic immunological research 16,24,30 . Due to the small vessel diameter of the mouse femoral artery and vein, it requires advanced surgical and super-microsurgical training and skills to perform successful anastomosis as the key component of orthotopic hind limb transplantation.…”

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

“…19 In fact, the cuff technique has been implemented in multiple other animal transplant models. [37][38][39][40][41][42][43][44] Heterotopic sternal transplantation in rats was introduced in 1999 by Santiago et al as an alternative method to study vascularized bone marrow transplantation. 1 They were able to show long term peripheral chimerism, tolerance, and survival following cessation of immunosuppression on postoperative day 30.…”

Role of the plasma cascade systems in ischemia/reperfusion injury of bone

“…Analgesia was administered with 0.05 mg/kg of buprenorphine (Temgesic, Reckitt Benckiser, Switzerland AG), subcutaneously injected 30 min before induction of the surgical procedure. Our rat model was established according to our previous work [29][30][31][32] and rat vascular anatomy [33].…”

“…Oxygen saturation, heart and breath rate, breath distention (for depths of anesthesia) and body temperature were monitored throughout the anesthesia using a Mouse-Ox plus System (Starr Life Sciences, Oakmont, PA, USA). The used rat model of crush I/R injury was established based on our previous work [29][30][31][32] and a study published by Hsieh et al [4]. Following incision in the left groin, femoral artery, vein, and nerve were isolated from the surrounding tissue.…”