Dolph Houben

78 CHAPTER 4 Short-term immunosuppression All animals received a 2-week immunosuppressive triple therapy consisting of Tacrolimus (Sandoz Inc. Princeton, NJ), Mycophenolate Mofetil (Mylan Institutional Inc., Rockford, IL) and Methylprednisolone sodium succinate (Pfizer Inc., NY, NY). Tacrolimus and Mycophenolate were administrated orally and Methylprednisolone intravenously. Immunosuppression levels were monitored by blood draws taken every other day from a central venous catheter. Dose adjustment was made to maintain a therapeutic level (Tacrolimus: between 5.0-15.0 ng/ml, Mycophenolate, between 1.0-3.5 mcg/ml). The methylprednisolone was tapered over the immunosuppression period. Surgical Procedure [23] Donor VCA harvest The transplants were performed with two surgical teams, first harvesting two tibia segments from the left and right hindlimb of a single donor, then immediately transplanting the bone into matched tibial defects in one hindlimb of two recipient animals. In a series of prior hindlimb dissections, we determined that prolonging the vascular pedicle to include the femoral artery and vein greatly facilitated tibial VCA transplantation, permitting end-to-side arterial and end-to-end venous anastomoses of large vessels in the recipient hindlimb. This precluded using a single animal as both donor and recipient. Instead, in a non-survival surgery, one male Yucatan donor provided two tibial segments. This is a modification of our previously described method [23] . After initial induction of anesthesia with Tiletamine HCL + Zolazepam HCL 5 mg/kg IM (Telazol, Zoetis Inc, Kalamazoo, MI.), Xylazine 2 mg/kg IM (Xylamed, Bimeda-MTC, Cambridge ON, Canada), the animal was euthanized with Pentobarbital Sodium 0.22ml/kg (Vortech Dearborn MI). Using the previously-described method, a 3.5 cm tibial segment was harvested from each hindlimb. The segment harvested included the major nutrient pedicle of the tibia, located on the posterior- lateral border of the tibia immediately distal to the location of the tibial tubercle anteriorly and supplied by the caudal interosseous artery. An extensive dissection of its more proximal inflow to include to the femoral artery and vein provided large caliber vessels to facilitate its immediate transplantation. VCA Transplantation On the same operative day, two female Yucatan recipients were simultaneously anesthetized, then intubated and maintained with inhalation anesthesia (Isoflurane 1-3%). Prior to the incision, 1 gram of cefazolin (Hospira, Lake Forest, IL) was administered intravenously for infection prophylaxis. Tacrolimus and mycophenolate were administered by a gastric tube and methylprednisone, intravenously at this time. Exposure of the tibia and cranial tibial AV-bundle was as described previously [18] , but modified with a second incision to expose the femoral vessels proximal to the knee. Exposure of the tibia and cranial tibial vessels was made through an antero-lateral incision. The cranial autogenous tibial AV-bundle was ligated distally and mobilized proximally from the surface of the interosseous membrane. With the use of our custom cutting jig, a 3.5cm tibial segment, identical in location to the donor VCA was removed. The donor segment was transplanted, and the vascular pedicle subcutaneously tunneled to the femoral artery and vein, where microsurgical