Tiam Mana Saffari

92 CHAPTER 5 USA). The skin was closed subcutaneously, using the same suture. Postoperatively, the rats were kept warm with towels. The rats were observed until completion of the experiment. Nonsurvival procedure After completion of the designated survival period, rats were sacrificed and neoangiogenesis was measured using two measures; the vascular surface area and the vascular volume. Anesthesia At survival time points (two, 12 and 16 weeks), rats were anesthetized and euthanized with 1 mL intraperitoneal injection of Pentobarbital Sodium. Vascular preservation Both thighs as well as the abdomen of the rat were shaved. On both sides, the sciatic nerve was exposed carefully. The vasculature of the lower extremity was preserved by aortic infusion 19 . A long longitudinal cut was made medially to expose the vena cava and aorta and these were cleaned from debris. These vessels were ligated as proximal as possible using a 5-0 Vicryl suture (Vicryl Rapide, Ethicon Inc., Sommerville, NJ, USA). Distal to the ligation, a catheter was inserted in the aorta. A yellow Microfil® compound (MV 8ml, diluent 15 ml, and curing agent 1.2 ml, Flow Tech, Inc., Carver, MA, USA) was infused into the aorta. After the contrast agents had cured, bilateral sciatic nerves were harvested. Nerves were temporarily stored in PBS and cleared in graded series of ethyl alcohol (25%, 50%, 75%, 95%, 100%) and placed in methyl salicylate. Clearing the nerve tissue while preserving the injected Microfil® allowed for measurement of the vascularity of the nerve segments. Outcome measurements Preserved vasculature in the nerve segments was quantified using a SkyScan 1276 micro computed tomography (micro CT, Bruker Corporation, Billerica, MA, USA) to calculate the vascular volume (three dimensional) and a Canon 5D Mark IV camera, (Manual Mode, ISO 200, 1/200th of a sec, f/16), a Canon MP-E 65mm Macro lens and