Ia32 image analysis system

Eight weeks following grafting, grafts underwent histomorphometric analysis.^{24 (link)} Grafts were harvested and stored in 3% glutaraldehyde (Polysciences Inc., Warrington, PA). The grafts were post-fixed in 1% osmium tetroxide, dehydrated, embedded in epoxy resin (Polysciences), and cross-sectioned at 1μm. Slides were counter-stained with 1% toluidine blue dye and analyzed at 1000× using a Leco IA32 Image Analysis System (Leco, St.Joseph, MI) to quantify myelinated axon counts, width, density, and percent neural tissue by a blinded observer. For electron microscopy, graft samples 1cm from the proximal coaptation and immediately adjacent to the distal coaptations were collected and processed as just described. These samples were sectioned at 90 nm and stained with uranyl acetate and lead citrate. Ultramicrographs were taken with a Joel 1200EX electron microscope at 10,000× magnification.

At 6 weeks post-operatively, animals were re-anesthetized and prepped to expose the right sciatic nerve and graft. Following tissue harvest, animals were euthanized. En bloc specimens of the graft and distal sciatic nerve underwent histomorphometric analysis as previously described (Hunter et al. 2007 (link)). Briefly, nerve was harvested and stored in 3% glutaraldehyde (Polysciences Inc., Warrington, PA). The nerves were post-fixed in 1% osmium tetroxide and serially dehydrated in ethanol and toluene. The nerves were 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 1000× on a Leitz Laborlux S microscope. The Leco IA32 Image Analysis System (Leco, St. Joseph, MI) was utilized to quantify nerve fiber counts and percent neural tissue. All analysis was done by an observer blinded to the experimental groups.

Nerves from Lewis rats, including the regenerating tissue from the proximal nerve end or ANA, were harvested and marked with a proximal suture, then stored in 3% glutaraldehyde (Electron Microscopy Sciences; Hatfield, PA) in 0.1 M pH 7.2 phosphate buffer (Fisher Scientific; Fair Lawn, NJ) at 4° C until processing. The explanted tissues were processed and analyzed as described previously^{32 (link)}. Briefly, the tissues were post-fixed in 1% osmium tetroxide (Polysciences, Inc., Warrington, PA), and serially dehydrated in 90% ethanol (Thermo Fischer Scientific, Winmill Hill, UK). The tissues were then embedded in grade 502 epoxy (Polysciences, Inc., Warrington, PA) and sectioned to 1 μm with an ultramicrotome. The slides were stained with 1% toluidine blue and analyzed under light microscopy at 1000x (Leitz Laborlux S, Leica; Buffalo Grove, IL) using the IA32 Image Analysis System (Leco; St. Joseph, MI) in order to quantify nerve fiber counts, fiber width, fiber density, and percent neural tissue. One hundred twenty (120) days after the initial nerve injury, the number of myelinated axons within the proximal, middle and distal segment of regenerated tissue, as well as proximal to the initial injury were examined. All analysis was conducted by a blinded observer to the experimental groups.