Partial Heterotopic Hindlimb Transplantation Model in Rats

We present a reliable and reproducible surgical model of heterotopic hindlimb transplantation in rats. This technique is ideal for short-term studies on preservation or immune rejection in VCA. The step-by-step description will allow trainees to rapidly master the surgery.

To begin, make a circumferential incision on the skin above the ankle at the distal third of the leg. Skeletonize and cauterize the saphenous artery and the terminal branch of the popliteal artery using bipolar forceps. Cauterize and cut the gastrocnemius, soleus, tibialis anterior, and biceps femoris muscles until the tibial bone is exposed.

Make a 2.5 centimeter incision at the right inguinal crease and dissect the inguinal fat pad. Use a fish hook retractor to grasp the inguinal ligament. Hold the inguinal fat pad distally with clamping forceps and retract the fat pad to expose the femoral vessels.

Dissect the femoral vessels, individualize the Murphy branches, and ligate with 8/0 nylon ties. Heparinize the donor rat by injecting the heparin in the penile dorsal vein using a 27.5 gauge needle. Complete the skin incision around the hip.

Use the bipolar forceps to cauterize the biceps femoris and gluteus superficialis muscles, then cauterize and cut the sciatic nerve at mid-femur length. Expose the femur proximally at the level of the posterior femoral crest and ligate the femoral vessels with nylon ties at the level of the inguinal ligament. Perform an arteriotomy on the femoral artery just below the ligature and dilate to insert the 24 gauge angiocatheter.

Cauterize and cut the remaining muscle underneath the pedicle to expose the anterior side of the femur. Use the bone cutter to cut the tibia proximally and cut the femur as distally as possible. Flush the partial hindlimb with two milliliters of heparin saline to obtain a clear venous outflow.

Place the hindlimb on ice in a sterile gauze until the microvascular transfer. Dissect the inguinal fat pad and recline the inguinal fat pad distally to expose the femoral vessels. Use a hook to retract the inguinal ligament and clamping forceps to hold the inguinal fat pad distally.

Dissect the femoral vessels, individualize the Murphy branches, and ligate with nylon ties as demonstrated. Ligate both vessels above the epigastric vessels using nylon ties. Place the approximator clamps proximally, dilate the vessel lens, and rinse with heparin saline.

Make an incision on the left flank above the hip and create a subcutaneous pocket with a subcutaneous tunnel to the inguinal crease. Place the proximal part of the partial limb and the inguinal fat pad through the subcutaneous tunnel for microvascular transfer. Perform the venous and arterial anastomoses using nylon sutures.

Remove both approximator clamps and observe the revascularization of the limb. Perform a milking test on both vessels to assess the patency of each anastomosis. Make a longitudinal skin incision on the medial side of the transplanted limb and insert the graft.

Remove the excess skin of the graft and close the wound with separate sutures and a running suture using absorbable 4/0 sutures. Suture together the inguinal fat pads of the transplanted limb and the recipient using two separate absorbable sutures and close the inguinal crease after the last checkup of the microvascular anastomosis. The evolution of the heterotopic hindlimb model was monitored until the end of the study.

The hair regrowth was observed during the first postoperative week, and the cutaneous retraction appeared after two weeks. Vascularized composite allotransplantation failure can occur during the first postoperative week due to microvascular thrombosis. Venous thrombosis was the cause of early euthanasia in 20%of the cases, all of which occurred before postoperative day five.

The skin appeared blue and became darker each day. Self-mutilation is a serious concern in nonsensate grafts and often occurs between postoperative day two and seven. If limited to less than a third of the graft surface and concerns only skin, surgical debridement and suture using non-absorbable sutures can be discussed with the staff veterinarian.

Prevention of self-mutilation relies on using an e-collar stitched to the neck until postoperative day seven and cleaning any blood or crust on the animal's surgical wounds. Severe autophagia of multiple layers of the graft leads to euthanasia of the animal. Dermal cysts appeared after postoperative day 14, sometimes with a cutaneous necrotic center before the fistula.

It is crucial to ensure that the graft placement will not prevent the animal from being ambulatory, and that the pedicle is not kinked or tight during the graft inset. Our team performed preservation studies in the procured limbs before transplantation. The effects of different durations of static cold storage have been evaluated as well as different limb perfusion protocols.