Dolph Houben

138 CHAPTER 7 Systemic inflammatory response To evaluate the systemic immune response, we obtained blood at baseline (before surgery) and at 1, 2, 4, 6, 10 and 20 weeks after surgery. A total of 10ml blood was obtained with heparin (50USP per 10ml of blood). Five milliliters were used for hematology tests, including differential leucocyte counts. The remainder was centrifuged for 10 minutes at 3000 RPM at 5⁰C. The supernatant was transferred into a clean 1.5mml microtube and stored at -80⁰C. Cytokine analyses were then performed using a Milliplex porcine cytokine/chemokine magnetic bead panel (Millipore Sigma, Cat. No PCYTMG-23K-13 PX, Darmstadt, Germany). This panel enabled quantification of thirteen different cytokines. The panel allowed measurement of: IL-1A, IL-1B, TNF-A, INF-G, IL- 2, IL-4, IL-6, IL-8, IL-10, IL12, IL-18 and GM-CSF, especially designed for porcine cytokines. This multiplex analysis was performed by the Immunochemical Core laboratory at our institution following the manufacturer’s protocol. Sacrifice procedure At 20 weeks, all animals were anesthetized with Tiletamine HCL + Zolazepam HCL. Thereafter, the animals were euthanized as recommended by the Panel on Euthanasia of the American Veterinary Medical Association with Pentobarbital Sodium (Vortech Dearborn MI, 0.22 ml/kg IV). The entire tibia including the allotransplant was harvested under sterile conditions, removing the internal fixation. The allotransplant was divided using a cooled oscillating saw, including a 2mm section snap-frozen in liquid nitrogen and stored at -80⁰C reserved for PCR analyses, and a 5mm bone section reserved for histology, fixed in 10% buffered formalin for 48h. The 5mm bone section was embedded in methyl methacrylate and sectioned into 15µm-thick sections using a diamond band saw (Exakt Technologies Inc., Oklahoma City, OK) and stained with hematoxylin/ eosin(H&E). Transplant Pedicle The allogenic vascular pedicle and the site of the microsurgical arterial and venous anastomoses were identified, removed, fixed in 10% buffered formalin for 48h. The vascular pedicle was embedded in paraffin, sectioned in 5µm-thick sections, deparaffinized and stained with an Elastica-van Gieson stain.