Tiam Mana Saffari

134 CHAPTER 7 MATERIALS AND METHODS Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC A3348-18). A total of 60 male Lewis rats, weighing 200-300 grams (Envigo, USA), were randomly divided into three groups. All rats underwent excision of a 10-mm portion of the sciatic nerve. In group I (nerve autograft, gold standard), this nerve defect was repaired with an ipsilateral reversed autologous graft. In group II, the same gap was reconstructed with a 10-mm processed nerve allograft. In group III, the allograft was wrapped with a SIEF flap 19 . All groups had survival periods of 12 and 16 weeks (N=10 per time point/group) for evaluation of outcomes. Time points were chosen based on previous research and the neuroregenerative capacity of rats 5 . Muscle atrophy and reinnervation was noninvasively measured over time using ultrasonography. Nerve allograft harvest and processing Twenty male Sprague-Dawley rats, weighing 250-300 grams, (Envigo, USA) served as donors for harvesting 15-mm segments of the sciatic nerve bilaterally. This species of rats were specifically chosen to create a histocompatibility mismatch to the Lewis rats 24,25 . The sciatic nerves were cleaned from external debris and processed into acellular allografts using a nerve decellularization protocol 26 . Nerves were harvested under sterile conditions and underwent a series of washing steps, were sterilized using g-irradiation and stored at 4°C until use 26 . Surgical procedure Rats were anesthetized in an isoflurane chamber, shaved and positioned in the nose cone to maintain anesthesia throughout the procedure. Preoperatively Enrofloxacin (Infection prophylaxis, Baytril, Germany, 10mg/kg), 5 mL of NaCl 0.9%, and buprenorphine SR (pain control, Buprenorphine SR-LAB, ZooPharm pharmacy, 0.6mg/ kg) were injected subcutaneously. Body temperature was maintained at 37°C with a heating pad. The sciatic nerve was fully exposed proximally from the inferior margin of the piriformis muscle to approximately 5-mm distal to the bifurcation, under an operating microscope (Zeiss OpMi 6, Carl Zeiss Surgica, Oberkochen, Germany). A 10-mm segment