Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

The small number of vascularized composite allotransplants performed clinically necessitate a reliable, reproducible animal model in order to study the underlying causes of graft rejection. Clinically-relevant tissues such as skin, muscle, and bone are all present in the model. Additionally, the non-functional nature of our model allow for faster surgery, less ischemic time for the flap, and lower morbidity compared to orthotopic models of VCA.

In hand transplant recipients, vasculopathy of donor vessels can lead to a complete closure of the vessels, and ischemic damage to the grafted hand. This model is a tool for studying the development of these vasculopathic events. Before attempting this surgical model, the primary surgeon should be familiar with and proficient in macrovascular surgical techniques.

This is a complicated and long procedure. The visual demonstration will help other labs to prepare accordingly, and understand the steps of the surgery. To begin, first harvest the hind limb of a donor rat by making a skin incision in the groin concavity, exposing the femoral vessels.

Make a ventral skin incision, which will assist in dissection of the pedicle. Following disarticulation of the knee and the ankle, make dorsal skin cuts, transect the pedicle to free the graft, and transport the graft immediately to the recipient surgical table, where the surgical site of the recipient animal is prepared in advance to minimize the graft’s ischemic time. On the recipient animal, expose and dissect the femoral vessels as before, and then anastomose the graft, vein end-to-side, artery end-to-end.

Inset the graft into the inguinal pocket and orient it upside-down. Finally, suture the skin. To begin the procedure, shave an anesthetized donor rat, and then use depilatory cream.

Inject 30 units of heparin subcutaneously in the scruff area. Place the animal supine on a heating pad and produce a sterile surgical field by prepping, scrubbing, and draping the surgical area, including the dorsal aspect of the leg. Use scalpel blade number 15 to make a 3-centimeter skin incision in the groin concavity of the donor rat.

Use Iris scissors to laterally reflect the inguinal fat pad. Using blunt dissection with a cotton swab, expose the common femoral vessels, and then place a wire hook with an elastic band to retract the abdominal muscles. Under a dissecting microscope with up to 40X magnification, use micro-scissors, number three straight jeweler forceps, and number five 45-degree-angled forceps to dissect the pedicle proximally from the emergence of the common femoral vessels under the inguinal ligament, and distally to the confluence of popliteal vessels into the graft.

Using micro-clips and bipolar jewelers forceps, ligate and divide the large arterial and venous branches to mobilize the main femoral vessels. Use fine bipolar forceps to cauterize any small branches. Use Iris scissors to make a skin incision from the center of the previous skin cut along the ventral side of the hindlimb to the ankle area.

Cut the gracilis muscle and the other adductor muscles underneath it in a vertical fashion to expose and ligate the medial proximal genicular vessels, deep-branching small vessels, and the sciatic nerve. At this point, on a separate surgical table, the other surgeon should intubate and anesthetize the recipient animal to prepare the recipient surgical site in time for graft placement, and minimize the graft’s ischemic time. Make circumferential skin incisions at the level of the knee and ankle, disarticulate the knee and ankle, and remove extraneous muscle and tissue.

Make a vertical skin incision on the dorsal side of the hindlimb to free the graft, composed of fibula and tibia, and now connected only by the pedicle. Place small clamps as proximally as possible on the femoral artery and vein and cut the pedicle as close as possible to the inguinal ligament. Use a 27-gauge flushing blunt cannula to inject heparinized saline into the femoral artery to flush the graft of blood.

Wrap the isolated graft in warm saline-soaked gauze and transport it immediately to the table with the recipient animal, with the surgical site already prepared for vascular anastomosis. Intubate the recipient rat, and deeply anesthetize with 2.5 to 3%vaporized isoflurane, balanced with one liter per minute of oxygen. Place the animal supine on a heating pad, and scrub and drape the surgical site.

While the first surgeon is working on freeing the graft from the donor animal, use scalpel blade number 15 to make a three-centimeter skin incision in the groin concavity of the anesthetized recipient rat. Use Iris scissors to laterally reflect the inguinal fat pad. After exposing the common femoral vessels in the same fashion as with the donor animal, place a wire hook with an elastic band to retract the abdominal muscles.

Then ligate and divide Murphy branches. Using 10-0 nylon interrupted sutures, anastomose donor vessels. To recipient vessels via venous end-to-side technique.

Then perform arterial anastomosis via end-to-end technique. After gradually releasing the clamps, monitor the anastomotic sites for bleeding and add additional sutures if needed. In order to assure effective graft reperfusion, visually assess the vascular anastomosis.

Then inset the graft into the inguinal pocket, and orient it upside-down, with the ankle joint superior, and the knee joint inferior. Use tucking sutures to secure the graft to adjacent muscles. Finally, close the skin via interrupted horizontal mattress skin-absorbable 4-0 sutures, and continue with post-operative care as described in the manuscript.

The rat VCA heterotopic hindlimb osteomyocutaneous flap model allows for long-term allograft survival under immunosuppression. The flap is well-hidden in the groin area, and constitutes minimal morbidity and discomfort to the animal. When immunosuppression treatment was stopped, the graft was rejected.

Doppler images of the allograft show a shift from minimal blood profusion immediately post-VCA, to full revascularization of the flap on day 64. Skin biopsy of a syngeneic allograft on day 45 shows normal morphology of the skin components, whereas there is atrophy and mononuclear cell infiltration in an allogeneic allograft in rejection on day 75 treated daily with a lower dose of an immunosuppressant. Muscle biopsy of a syngeneic allograft on day 45 shows normal morphology, whereas muscle atrophy and mononuclear cell infiltration are apparent in an allogeneic allograft in rejection on day 98, treated daily with a lower dose of an immunosuppressant.

Femoral artery biopsy of a syngeneic allograft on day 45 shows normal morphology of the artery, in contrast to femoral artery of an allogeneic allograft in rejection on day 98, treated daily with a lower dose of an immunosuppressant. This model provides a tool to test the effects of acute rejection, chronic rejection, and environmental factors such as infection and surgical insults on the development of vasculopathy. Immunosuppressive drugs are required for long-term survival of the graft.

These drugs are chemically hazardous, and should be handled with caution and proper personnel protective equipment.