2.6. Counting of Myelinated Axons and Morphometric Analysis

The sciatic nerves were processed for myelinated axon counting and morphometric evaluation by transverse semithin sections (0.5 μm thick), stained in Sudan black (0.7% in 70% alcohol) solution, and examined using a bright field microscope Leica CTR 6500® (Leica Microsystems CMS GmbH) and Adobe Photoshop CS5® (Adobe Systems) software. The analyses were performed by sampling at least 30% of each nerve cross section (magnification of 1,000x), generating approximately 12 images per animal. Sampling bias was avoided by spreading the micrographs systematically over the entire cross section, according to the procedure proposed by Mayhew and Sharma [36]. The images were used for counting the total number of myelinated axons in each specimen. For the morphometric analysis, two sampled fields in each nerve (magnification of 1,000x) were utilized. The images were converted to black and white, so that any background artefacts would not be measured, including fibers that were partly captured. The myelinated axons were identified and the measurements were carried out automatically, after calibration. The measurements provided were myelin sheath's area, perimeter, and smaller diameter of each myelinated fiber. These values were used to calculate the myelinated axons diameter, myelin thickness (fiber diameter − axon diameter/2) and “g” ratio (axon diameter/fiber diameter). The data are represented as the mean ± standard error (SE) for each group.