Tiam Mana Saffari

137 FUNCTIONAL MOTOR RECOVERY OF ANGIOGENESIS OF NERVE ALLOGRAFTS 7 After rats were euthanized with Pentobarbital Sodium (Fatal Plus, 390 mg/mL, Vortech, MI, USA), tissues were collected. Tibial muscles were carefully dissected and weighed to evaluate muscle mass. Sciatic nerves and peroneal branches were harvested bilaterally and immediately stored in fixative. Histology - Peroneal nerve samples were processed and subsequently infiltrated in 50%, 75% and finally 100% epoxy resin and polymerized at 65°C for 12-18 hours to allow cutting 1-µm transverse sections. Samples were stained on a warming plate with toluidine blue (Fisher Scientific, Pennsylvania, USA) for 2-2.5 minutes to evaluate histological outcomes. Nerve area, myelin thickness, axon count, total axon area were determined using NIS-Elements software (NIS-Elements BR 4.51.01), and the N-ratio (ratio between the myelinated fiber area and tissue cable area), indicating the number of axonal sprouting and maturation of the regenerating nerve 30-32 , was calculated. Immunofluorescence - Sciatic nerves were embedded in paraffin, sectioned transversely to a thickness of 5-µm and reacted with immunofluorescent markers CD34, staining vascular endothelial cells and extensively expressed on blood vessels 33 , and protein gene product 9.5 (PGP 9.5), a pan-neuronal marker, staining both myelinated and unmyelinated nerve fibers 34 , to quantify vascularity and axons, respectively. The CD34 primary antibody (1:2000, rabbit monoclonal, Abcam, UK) was diluted in background reducing diluent (Dako, Agilent Technologies Inc., CA, USA) and incubated for 60 minutes. PGP9.5-Alexa 568 conjugate (1:50, rabbit polyclonal, Dako), using the Zenon-Alexa 568-Rabbit IgG kit (Fisher Scientific) was incubated for 60 minutes, prior to staining with the appropriate secondary antibody (Alexa Goat-Anti-Rabbit 488, 1:200, Fisher Scientific). Counterstain was performed using Hoechst 33342 (Fisher Scientific) for 10 minutes, to stain cell nuclei. Slides were cover slipped and images were obtained using confocal microscope (Zeiss LSM 780, Carl Zeiss Surgical GmbH, Oberkochen, Germany). The total nerve area was defined by the combined area of tissues positively stained for any of PGP 9.5, CD34 and Hoechst. Density of axons and vascularity were normalized to the total nerve area and expressed in percentages. A detailed description of evaluation measurements is provided (See Appendix).