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J. L. Wheat, L. D. Carter-Dawson, N. B. Patel, R. S. Harwerth; Changes in the Axonal Component of the Retinal Nerve Fiber Layer in Experimental Glaucoma. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2104.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this experiment was to quantify and compare the axonal component of the retinal nerve fiber layer (RNFL) in the peripapillary region using confocal microscopy images of immunostained experimental glaucoma and control eyes.
Tissue from glaucomatous retinas and the fellow control retinas of macaque monkeys were imaged using confocal microscopy after staining with DAPI, Alexa-phalloidin, glutamine synthetase, and glial fibrilary acidic protein (GFAP). A customized MATLAB program was used to evaluate the number and intensity of pixels in a given image. A paired t-test was used to compare the amount of each stain in terms of pixels per total pixel area in the RNFL and in terms of intensity per total pixel area of RNFL between corresponding regions.
Phalloidin showed a high prevalence for axonal staining in the RNFL. Pixel/area staining revealed statistically significant differences in the temporal region (7-20% reduction), with nasal, inferior, and superior differences varying between subjects. Intensity/area of phalloidin staining in the RNFL was found to be statistically significant for all regions imaged. GFAP and GS showed smaller increases in glaucomatous retinas, with temporal regions showing the largest increases in glial staining per pixel area.
Experimental glaucomatous atrophy causes a decrease in the density and quality of phalloidin staining in the RNFL that appears to be regionally selective. These changes are likely representative of a change in axon density in diseased RNFL. Glial content changes in experimental glaucoma are less uniform, but appear to increase overall.
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