Optic Nerve Axon Quantification

One-μm-thick cross-sections of the optic nerve embedded in epoxy resin were used for imaging-based axon quantification in a masked fashion similar to our previous studies [25 (link)]. Images of the toluidine blue-stained sections were collected as non-overlapping tile images using Zeiss AxioObserver.Z1 microscope for wide-field fluorescence microscopy (Carl Zeiss, Thornwood, NY) and the Zen software (Blue Edition; Carl Zeiss) to allow axon counts representing the entire surface area of cross-sections as previously described [25 (link)]. Axon counting was performed by a researcher masked for the experimental group. After image acquisition, nerve outlines were manually traced on mosaics of images. Image processing determined the size and shape parameters to exclude intervening glia, myelin debris, and highly degenerated axons. The axon loss was determined by the ratio of axon counts in ocular hypertensive eye to normotensive fellow eye.