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Gustavo Munguba, Daisy Lam, Ling Ge, Sanja Galeb, Sinthia Samad, Mary Tapia, Andrew Camp, Xiaoli Xing, Richard Lee; Longitudinal Assessment of Retinal Ganglion Cell Degeneration In Vivo. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2245.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the progressive nature of injury-induced neurodegenerative structural changes occurring following injury to retinal ganglion cell (RGC) axons, including spatial information by taking advantage of recent advances in in-vivo imaging techniques and assess whether structural changes to the RGC+IP layers correlate well with direct assessment of RGC density.
All animals used in this study were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the UM-IACUC. To track degenerative progress in retinas following optic nerve crush (ONC) injury, we used Bioptigen’s SD-OCT system and manual segmentation of OCT images. RGC density was measured by confocal laser scanning ophthalmoscope (CSLO) (HRA2; Heidelberg Engineering) and counts were performed using blood vessels as landmarks with which to track anatomically defined progressive RGC loss.
Approximately 50% of RGCs homogeneously distributed throughout the retina of our transgenic line express Thy-1 promoter driven EYFP, labeling RGC dendrites, soma and axons. Static histology studies 3 weeks after optic nerve crush injury show a >70% reduction in the number of Brn3a and GFP positive RGCs present in injured retinas as compared to sham injury control. Unlike histology findings, in vivo density of RGCs expressing EYFP, as tracked by CSLO, progressively decreases as a consequence of ONC injury and can be tracked in a dynamic manner providing both spatial information and nature of progression. RGC+IP layer thickness decreases in response to ONC injury serve as a reliable surrogate structural marker for RGC death. Changes in RGL+IPL thickness heat-maps provide spatial information regarding progressive RGC axonal loss.
In vivo CSLO imaging can track EYFP expression by RGCs, providing dynamic assessment of CNS neuron survival following injury. The use of RGC+IP layer thickness as a surrogate marker for RGC survival is an effective method to follow progressive loss of RGC soma and axons after axonal injury. In vivo visualization of neuron specific gene expression to follow cell survival in a progressive manner may provide a more dynamic view of animal model disease pathophysiology than conventionally used histologic methods. Assessment of RGC density by CSLO is a potentially useful clinical application.
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