Tiam Mana Saffari

110 CHAPTER 6 MATERIALS AND METHODS Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC A3348-18). All animals were individually housed with ad libitum access to food and water, with a twelve-hour light-dark cycle until sacrifice. Experimental design Thirty-nine male Lewis rats, weighing between 200 to 300 grams (Envigo, Madison, WI, USA), were randomly divided into three groups. All rats underwent excision of a 10-mm portion of the sciatic nerve that was reconstructed with (i) autografts, (ii) decellularized processed nerve allografts, or (iii) allografts wrapped around with a superficial inferior epigastric fascial (SIEF) flap to provide vascularization to the wound bed (SIEF group). At one and two weeks, CD4 and CD8 levels were measured using flow cytometry. Four extra unoperated rats served for baseline control levels. Animals were sacrificed at two weeks (N=5/group) or 12 weeks and 16 weeks (N=4/ group/time point) to evaluate gene expression profiles and immunohistochemistry staining in the nerve graft samples, respectively (Table 1). Table 1. Experimental design. SIEF flap: Superficial inferior epigastric artery fascial (SIEF) flap, qPCR: quantitative polymerase chain reaction Groups Outcome measurements Time points and N I Autograft • qPCR analysis • T cell measurement in blood samples • Immunohistochemistry analysis • At 2 weeks • 5 samples/group II Allograft III Allograft + SIEF flap • At 12 and 16 weeks • 4 samples/group Nerve allograft harvest and processing A total of thirteen male Sprague-Dawley rats, weighing 250-300 grams, (Envigo, Madison, WI, USA) served as donors for the bilateral harvest of 15-mm segments of the sciatic nerve. These rats were used to create a histocompatibility barrier 11,12 . The sciatic nerves were cleaned from external tissue debris and treated using a five-day decellularization protocol to generate acellular allografts as described previously 13 . Briefly, the nerves underwent multiple sequential washing steps, were incubated in