Tiam Mana Saffari

153 FUNCTIONAL MOTOR RECOVERY OF ANGIOGENESIS OF NERVE ALLOGRAFTS 7 APPENDIX - MATERIALS AND METHODS All outcomes were performed bilaterally and compared to the non-operated, contralateral side. Ultrasonography - was performed using a GE Vivid 7 Ultrasound system (General Electric, Fairfield, CT, USA) and the cross-sectional area (X-C area) was determined bilaterally using Adobe Photoshop CC 2018 (Adobe Systems Incorporated, San Jose, CA, USA) 1 . Maximum ITF measurements - were performed bilaterally as previously described with optimization of preload 2 . In short, the tendon of the tibial anterior muscle was exposed via a second skin incision and incised at the insertion. The leg was fixed to a platform with two Kirschner wires and the tendon was attached to the force transducer. A bipolar stimulator was used to generate the stimulus to the peroneal nerve branch and acquire a signal which was processed using LabVIEW (National Instruments, Austin, Texas). The muscle was kept hydrated with warm 0.9% NaCl during the experiment. Histological outcomes - A 3-mm segment of both peroneal nerves of all rats (N=10/ group/time point), 5-mm distal to the graft, was harvested and immediately stored in Trumps solution. Samples were processed with 0.1M Phosphate Buffer, 1%Osmium tetroxide in buffer, graded series of alcohols and acetone. Samples were subsequently infiltrated in a 50%, 75% and finally 100% epoxy resin and polymerized at 65°C for 12-18 hours. Samples were cut into 1-µm sections, placed on slides, stained with toluidine blue (Fisher Scientific, Pittsburgh, Pennsylvania, USA) for 2-2.5 minutes and coverslipped. Immunofluorescent outcomes - Sciatic nerve graft samples and the contralateral sciatic nerves were harvested, fixed in 10% formalin for 48 hours and stored in 70% ethanol, until embedding in paraffin. Samples were sectioned transversely to a thickness of 5-µm and retrieved for 20 minutes using Epitope Retrieval2 (EDTA based; Leica). Samples were incubated in 10% Normal Goat Block (Fisher Scientific, Pittsburgh, Pennsylvania, USA) for 30minutes and reacted with immunofluorescent markers CD34 3 and protein gene product 9.5 (PGP 9.5) 4 , to quantify vascularity and nerve fibers, respectively. The CD34 primary antibody (rabbit monoclonal; Abcam, UK) was diluted