Tiam Mana Saffari

194 CHAPTER 9 at 65°C for 12-18 hours. Samples were cut in sections at 0.6microns, placed on slides and stained on a warming plate with Toluidine blue for 2-2.5 minutes. The total tissue cable area (nerve area), axon area, axon count and myelin area were obtained using a Nikon Eclipse 50i microscope and Image Pro Plus Software. The N-ratio was calculated by dividing themyelinated fiber area (axon area andmyelin area) by the tissue cable area 36 . Immunofluorescence - Both sciatic nerves of five randomly selected rats per group were dissected and fixed in 10% formalin for 48 hours. Nerves samples were vertically embedded in paraffin and sections from the exact middle were stained for Schwann cell marker S100 and protein gene product 9.5 (PGP9.5), a pan neuronal marker. Immunohistochemical staining was performed at the Pathology Research Core (Mayo Clinic, Rochester, MN, USA) using the Leica Bond RX stainer (Leica, Buffalo Grove, IL, USA). The S100 (rabbit polyclonal; Dako, Agilent Technologies Inc., Carpinteria, CA, USA) and PGP9.5 primary antibody (rabbit polyclonal; Dako, Agilent Technologies Inc.) were diluted to 1:5000 in Background Reducing Diluent (Dako, Agilent Technologies Inc.) and incubated for 60 minutes with the samples, prior to staining with the appropriate secondary antibody (Alexa Goat-Anti-Rabbit 488, 1:300, for S100 and Alexa Goat-Anti- Rabbit 568, 1:200 for PGP9.5) and counterstainedwith Hoechst 33342 (all ThermoFisher Scientific, MA, USA). Images of the stained slides were obtainedwith a fluorescence laser confocal microscope (Zeiss LSM 780, Carl Zeiss Surgical GmbH, Oberkochen, Germany). The mean fluorescent density of both stains was measured using ImageJ software. Statistical analysis All obtained images were blinded and all outcomes were expressed as a percentage of the contralateral side to correct for biological variability between rats. Non- physiologic outcomes were excluded from analysis after review by a statistician and an independent researcher. Two-way analysis of variance (ANOVA) was used for the cross-sectional tibial area measurements. One-way ANOVA was used to compare all other outcome measures between groups. Post-hoc Bonferroni was used to correct for multiple comparisons. Outcomes were expressed as the mean and the standard error of the mean (SEM). Outcomes of cross-sectional tibial muscle area were expressed as mean difference and the standard error of the mean difference. The level of significance was set at α≤0.05.