Quantitative Nerve Histomorphometry

The axial 1 cm of the nerve distal to the injured site was isolated and fixed at 4°C with 3% glutaraldehyde (Polysciences Inc., Warrington, PA), washed in 0.1 M phosphate buffer (pH 7.2), post-fixed with 1% osmium tetroxide (Fisher Scientific, Pittsburgh, PA), dehydrated in graded ethanol solutions, and embedded in Araldite 502 (Polysciences Inc.). Axial semi-thin sections, with 1-μm-thick nerve specimens, taken at a 5-mm distance from the injured site, were stained with 1% toluidine blue for histomorphometric analysis. Binary image analysis was used for semi-automated quantitative analysis of multiple components of nerve histomorphometry; this was performed by an observer blinded to experimental conditions [33 (link), 63 (link)]. Total myelinated fiber counts were measured based on 6 representative fields at 1000 × magnification. Fiber width, axon width, total fiber area, fiber area, myelin area, axon area, and fiber debris area was calculated and compared.