Dolph Houben

162 CHAPTER 8 Post-operative management All animals received a 2-week immunosuppressive triple therapy consisting of Tacrolimus 0.6- 1.5mg/kg (Sandoz Inc. Princeton, NJ), Mycophenolate Mofetil 30-60mg/kg (Mylan Institutional Inc., Rockford, IL) and Methylprednisolone sodium succinate (Pfizer Inc., NY, NY) 500mg start dose followed by 125mg the first post-operative day. 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 adjustments were made for the immunosuppression to be in therapeutic range (Tacrolimus: 5.0-15.0ng/ml, Mycophenolate 1.0-3.5 ng/ml). The methylprednisolone was tapered over the immunosuppression period (-12.5mg/day). The animals received two weeks of prophylactic antibiotic therapy with Enrofloxacin (Baytril, 7.5mg/kg, Bayer Healthcare LLC, Shawnee Mission, KS) and Ceftiofur (Excede, 100mg/kg, Pfizer Inc, New York, NY). Direct weight-bearing was allowed after recovering fromanesthesia. Daily observation permitted administration of appropriate analgesics, used until no evidence of pain was observed. The animals were individually housed and received standard feed and water ad libitum. Although we had planned to briefly anesthetize all animals at 2, 4, 6, 10 weeks for radiographic views, ultrasound evaluation of the vascular pedicle, and periodic blood draws. The need to terminate the experiment due to unacceptable complications did not permit the collection of any of this data. Sacrifice procedure All animals were anesthetized (Telazol + Xylazine IM) and euthanized with intravenous administration of Pentobarbital Sodium (Vortech Dearborn MI, 0.22 ml/kg) as recommended by the Panel on Euthanasia of the American Veterinary Medical Association and performed according to NIH guidelines under the direction of the Institutional Animal Care and Use Committee. The femoral artery of the operated side was dissected, cannulated and flushed with heparinized saline and then injected with the contrast agent Microfil (MV-122, Flow Tech, Carver, MA). With the use of Micro-Computed Tomography (Inveon PET CT, Siemens Medical Solutions USA, Inc., Malvern, PA) at a voltage of 80 kV and 500uA with a current version of imaging software (PMOD Technologies, Zurich, Switzerland) a micro-angiography of the knee was made in a medium magnification resolution. The complete allotransplant was dissected, fixed in 10% buffered formalin for 48 hours, embedded in methyl methacrylate, sectioned in two 15um-thick sections using a diamond band saw (Exakt Technologies Inc., Oklahoma City, OK) and stained with hematoxylin/eosin (H&E). Results Anatomic study The blood supply to the hind limb is provided by the superficial femoral artery and its popliteal and saphenous artery branches. The saphenous artery and vein are of great importance in our hind limb model since it provides continuous blood flow to the entire hind limb if left undisturbed. In case the femoral artery thromboses after anastomoses or manipulation, the hind limb continues to survive on it blood supply from the saphenous artery [16] . We were able to raise a pedicled gracilis muscle flap from its insertion and found the dominant