Orthotopic Hind-Limb Transplantation in Rats

I am Robert Suha from the Daniels Roski Research Laboratory of the Department of Visceral Transplant and Thoracic Surgery from Innsbrook Medical University. Today we are going to show you the procedure of he limb transplantation in the red, introducing a cuff technique for revascularization. This he limb transplantation surgery is performed by our surgical residents, including myself.

We use this animal model to study ischemia and reperfusion associated injuries in a composite tissue allograft. Furthermore, we use this procedure to study immunological events of composite tissue allograft rejection, as well as strategies to prevent rejection from occurring. These strategies include the minimization or even avoidance of maintenance immunosuppression through the use of targeted immunomodulatory regimens.

The red hind limb transplantation procedure begins with the harvest of the donor composite tissue graft in which the right hind limb is removed from the donor rat and stored on ice. Next, the right hind limp is removed from the recipient rat to make room for the donor limp. The donor limp is then removed from ice and transplanted to the recipient rat.

In this model, vascular anastomosis are performed using a cuff technique. For our experiments, male Louis Reds weighing 250 grams were used. Anesthesia was induced using 50 milligrams per kilogram intraperitoneal injection of pentobarbital.

The anesthetized red is shaved and sanitized over the groin and limp for the procedure. A complete set of microsurgical instruments is required including straight microsurgical, scissors, vessel dilators, and two curved forceps. Furthermore, the most important instruments are the microsurgical vessel clamps shown here.

To test depth of anesthesia, the toe pinch reflex is utilized. The rat is placed on a sterile platform and secured with all four Limbs in full extension prior to incision. The limp is whipped free of Any axis here to prevent contamination.

An incision is made across the right groin approximately at the level of the inguinal ligament. The femoral vessels appear immediately under the skin and they're dissected free with bipolar tery and blunt dissection. The epigastric vessels are then dissected free and cauterized.

A Single loop as holding suture is placed around the vein and artery to allow traction without directly handling the vessels with instruments after separation. The deep branches of the artery and vein are ligated with eight oh silk suture proximally and cauterized distally Before harvest of the donor limp. 50 units of heparin are administered via the penal Vein.

After one minute, Attention is returned to the femoral vessels. The femoral artery is ligated as approximately as possible using eight o silk, and the same procedure is repeated for the Fal. Both vessels are then cut distally from the ligature and the hind limb is flashed through the artery with heparinized ice cold HDK solution over five Minutes.

Next, all muscle groups are carefully divided with scissors. The osteotomy is performed using a rotating electrical saw at the level of the distal third of the femur. The harvested limp is then submerged in 20 milliliters of ice cold HDK solution for preservation and cold storage until transplantation.

The recipient operation begins similarly to the donor operation with isolation and dissection of the femoral vessels. Following careful dissection, the vessels are ligated using eight o silk as distal as possible, but proximal to the epigastric vessels Takeoff. We then proceed with the cuff mounting of the recipient vein by pulling the polyline cuff over the vessel.

A single vein Clamp is applied proximal to the cuff over the vein and cuff handle to ensure complete hemostasis. The ligature at the distal end of the vein is cut and the vein is then averted Over the cuff. An eight O Ligature is tied circumferentially around the vessel and the cuff securing the recipient vein in Place.

The exact same procedure is performed for the artery with the smaller diameter cuff. The recipient's limp Is dissected similarly to the donor limp with sharp dissection of muscle groups and osteotomy performed in the mid femur. We are now ready to Transplant the hind limp.

The donor limp is removed from the HDK solution. Transplantation begins with osteo synesis of the femur with an 18 gauge needle as an intramedullary rod. The tip is cut off and the remaining needle is cut to one and a half to two centimeters.

Ventral Muscle groups are closely approximated with four oh Vicryl or proline interrupted sutures to stabilize the limb and prepare for vascular Anastomosis. For Cuff R anastomosis, the donor vein is dilated, pulled over the recipient's vein and cuff and secured with another circumferential eight o ligature. The same procedure is performed for the donor artery.

Finally, Once both vessels are estamos, the venous clamp is released, followed by the arterial Clamp. Reperfusion Is immediate and easily visualized by return of color of the distal limb and foot, as well as pulsitile flow through the artery. The remaining dorsal muscle groups are re approximated.

Skin is closed with four oh Vicryl or proline interrupted sutures, and transplantation Is complete Postoperatively. The red is warmed on a heating pad and analgesics and antibiotics are administered. The red is monitored closely for 48 to 72 hours for signs of distress or surgical complications.

The cuff technique of re anastomosis has been widely performed in different surgical research areas over the last decades from marine kidney transplant models to heterotopic heart transplantation models. It allows for studying various aspects of his chemo, reperfusion associated injuries, as well as immunological phenomena. Following our transplantation.

Here we show the first application of this unique cuff technique to he limb composite tissue transplantation. In a small animal model, the cuff technique significantly reduces operative time, microsurgical expertise required by the operator and intraoperative blood loss when compared to conventional suture techniques for anastomosis.