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We developed a new analytic tool based on visible-light optical coherence tomography fibergraphy (vis-OCTF) to longitudinally track individual axon bundle transformation following acute optic nerve crush injury (ONC) in mice. We analyzed four parameters: lateral bundle width, axial bundle height, cross-sectional area, and the shape of individual bundles. We showed that axon bundles became wider and thicker at 3-days post ONC. The bundle swelling at 3-days post-ONC has correlated with about 15% retinal ganglion cell (RGC) soma loss. At 6-days post-ONC, axon bundles showed a significant reduction in lateral width and cross-sectional area, followed by a reduction in bundle height at 9-days post-ONC. Bundle shrinking at 9-days post-ONC has correlated with about 68% RGC soma loss. Both experimental and simulated results suggested that the cross-sectional area of individual RGC axon bundles is more sensitive than the bundle width and height to indicate RGC soma loss. This study is the first to track and quantify individual RGC axon bundles in vivo following ONC injury and establish the correlation between the morphological changes of RGC axon bundles and RGC soma loss.

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We seek to develop techniques for high-resolution imaging of the tree shrew retina for visualizing and parameterizing retinal ganglion cell (RGC) axon bundles in vivo. We applied visible-light optical coherence tomography fibergraphy (vis-OCTF) and temporal speckle averaging (TSA) to visualize individual RGC axon bundles in the tree shrew retina. For the first time, we quantified individual RGC bundle width, height, and cross-sectional area and applied vis-OCT angiography (vis-OCTA) to visualize the retinal microvasculature in tree shrews. Throughout the retina, as the distance from the optic nerve head (ONH) increased from 0.5 mm to 2.5 mm, bundle width increased by 30%, height decreased by 67%, and cross-sectional area decreased by 36%. We also showed that axon bundles become vertically elongated as they converge toward the ONH. Ex vivo confocal microscopy of retinal flat-mounts immunostained with Tuj1 confirmed our in vivo vis-OCTF findings.

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