Histomorphometric Analysis of Tibial Nerve

At 56 days post-operatively, Lewis rats were re-anesthetized, and the right tibial nerve was exposed. Following tissue harvest, animals were euthanized. En bloc specimens of the tibial nerve 5mm distal to the repair site underwent histomorphometric analysis as previously described^{24 (link),25 (link)}. Nerve was harvested and stored in 3% glutaraldehyde (Polysciences Inc., Warrington, PA, USA). The nerves were post-fixed in 1% osmium tetroxide, serially dehydrated in ethanol and toluene, then embedded in epoxy (Polysciences) and sectioned on an ultramicrotome into 1μm cross sections. Slides were counter-stained with 1% toluidine blue dye. The slides were then analyzed at 1000x on a Leitz Laborlux S microscope. The Leco IA32 Image Analysis System (Leco, St. Joseph, MI, USA) was utilized to quantify nerve fiber counts, percent neural tissue, fiber sizes, and myelin thickness. All analyses were done by an observer blinded to the experimental groups.