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M.S. Pezda, M. Tapia, A.S. Hackam, R.K. Lee; Brn–3b Is Selectively Down–Regulated in a Mouse Model of Glaucoma . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1327.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Identification of molecules specifically down–regulated in the retina of glaucomatous mice. DBA2/J mice develop chronic, progressive glaucoma and are a useful model for exploring basic signaling mechanisms contributing to retinal ganglion cell (RGC) death. Our initial approach to gain insight into the biological pathways contributing to glaucomatous changes was to identify patterns of gene expression changes during glaucoma progression in DBA2/J mice. Methods: Pooled whole retinas from young, non–glaucomatous (younger than 3 months of age) and from aged, glaucomatous (12 months and older) DBA2/J mice were homogenized and total RNA was extracted using Trizol and purified with Qiagen RNeasy separation columns. First strand cDNA was synthesized and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 gene expression microarrays to determine transcriptional levels of brn–3b expression. Retinal cDNA was also used for real–time PCR with brn–3 a, b, and c primers to quantitatively confirm the microarray gene expression data. In addition, immunohistochemistry using brn–3b antibodies was used to determine the protein expression levels of brn–3b Results: The gene array data was analyzed with Affymetrix Microarray Suite 5.0 software and brn–3b was identified as one of the top ten most down–regulated genes expressed in aged, glaucomatous DBA2/J retinas compared to young, non–glaucomatous DBA2/J retinas. The differential brn–3b expression in glaucomatous retinas compared to non–glaucomatous retinas was confirmed by real–time PCR and by immunohistochemistry. The decrease in brn–3b expression levels correlated with the histological loss of RGC in glaucomatous retinas. Conclusions: In a genome wide screen of genes expressed in the mouse retina, we found significantly decreased levels of brn–3b expression in glaucomatous aged DBA2/J retinas from 12 to 17 month old mice that correlates with age and severity of RGC loss. Even though there are fewer RGC than other cell types in the retina, the level of decrease in brn–3b expression was one of the most significant compared to all retinal genes. Brn–3b, a member of the brn–3 family of POU–IV domain transcription factors, is critically important for retinal ganglion cell development. Our data suggests that brn–3b plays a role not only as a marker for RGC loss, but that brn–3b may play a functional role in keeping RGC alive. One explanation is that brn–3b down–regulation is a proximal event in susceptibility to RGC death in response to glaucomatous stress and that decreased levels of brn–3b leads to a cascade of changes in the RGC that eventually trigger apoptosis in susceptible glaucomatous RGC.
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