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S.L. Bernstein, Y. Guo, Z. Mehrabyan; Whole Mount Retinal TUNEL Assay Reveals Delayed Apoptosis in Rodent AION . Invest. Ophthalmol. Vis. Sci. 2005;46(13):156.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Anterior ischemic optic neuropathy (AION) is an optic nerve stroke. We recently described a new rodent model of AION (rAION) in rats (Bernstein et al, IOVS 2003) and mice Goldenberg–Cohen et al, IOVS, 2004). We wanted to determine whether retinal ganglion cells (RGCs) die by apoptosis or necrosis following optic nerve stroke in rats. We refined the TUNEL assay method to enable whole retina analysis of apoptosis. Using this new assay system, we were able to determine that RGCs die by apoptosis following AION.Methods: AION was induced in one eye of male Wistar rats (120–150g), using the published technique (Bernstein et al, 2003). One eye was left untreated as a control. Following induction, animals were euthanized at 1–21 days. Retinae were fixed in 4% paraformaldehyde–PBS. Post–fixation, retinae were removed in one piece, permeabilized with cytonin (Trevigen), freezing, and treated with a number of other permeabilization steps. Retinae were then incubated with the enzyme TdT and FITC–dUTP (Boerhinger–Mannheim), and flat mounted with glycerin. Positive controls were produced by incubating whole retinae with diluted DNAse I. Tissues were examined using an Olympus confocal microscope at 488nm. Results: Retinal structure was grossly intact after the modified assay. TUNEL negative cells were apparent as ‘holes’ in the RGC layer. TUNEL positive cells were apparent as brightly fluorescent cells against a darker background at the same tissue level as the large retinal vessels. The positive control showed strong signal in all cells in the RGC layer. rAION resulted in TUNEL positive nuclei only in the RGC layer. Few positive cells were seen prior to 1 week. The number of TUNEL positive cells increased dramatically after 9 days post–induction, but many TUNEL positive cells were still present 15 days post–induction. Conclusions: The retina flat mount TUNEL assay is a useful tool for evaluating patterns of RGC death in models involving optic nerve damage. rAION results in RGC death by apoptosis. The major loss of RGCs occurring from AION is considerably later than the wave of RGC death following either optic nerve crush or cut. This suggests that the potential therapeutic window of opportunity following optic nerve stroke may be longer than previously thought.
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