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Stewart A Bloomfield, Sandeep Kumar, Abram Akopian; Blockade of Retinal Gap Junctions Offers Significant Neuroprotection in an Experimental Mouse Model of Glaucoma. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2589.
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© ARVO (1962-2015); The Authors (2016-present)
Secondary cell death via gap junctions (GJs) plays a crucial role in the progression of retinal ganglion cell (RGC) and amacrine cell (AC) loss under a number of neurodegenerative insults to the retina. Here we studied the role of secondary cell death in the loss of RGCs and ACs in a mouse model of glaucoma and determined whether pharmacologic or genetic blockade of GJs forms a novel approach for protection of neurons in glaucomatous retinas.
Experimental glaucoma was induced by intracameral injections of polystyrene microbeads in mice eyes. IOP measurements were performed weekly and animals were sacrificed at 1, 4, or 8 weeks after initial bead injection. To access cell loss, RGCs and coupled ACs were retrogradely labeled with Neurobiotin and retinal sections were counterstained with DAPI. RGCs were identified by Brn3a antibody. Retinal damage was assessed by GFAP expression. In some experiments GJs were blocked with meclofenamic acid (MFA) or genetically ablated in connexin KO (knockout) mice. Data are presented as mean ± SEM per vertical section length.
Injection of microbeads raised IOP from 11.4 ± 0.3 to 21.9 ± 0.5 mm Hg for at least 8 weeks. There was a significant decrease in RGC count 4 weeks (40 ± 3; p < 0.05) and 8 weeks after bead injection (33 ± 1; p < 0.001), a 20% and 33% population decrease, respectively. Retinal injury was evidenced by an upregulation of GFAP in Muller cells. We next blocked GJs before and after bead injection with MFA (50 μM). MFA prevented the loss of RGCs by bead injection as evidenced by cell counts of 47 ± 4 (p > 0.1) at 8 weeks, comparable to control data. We next induced experimental glaucoma in connexin KO mice. At 8 weeks after bead injections the RGC count was 40 ± 1 in Cx36 KOs, 43 ± 1 for Cx45 KOs, and 49 ± 3 for Cx36/45 dKO, indicating a 41%, 59%, and 94% increase in survivability, respectively. We also observed a 20-30% loss of ACs in bead-injected WT mouse retinas, which was significantly prevented in Cx36 KO mice.
Our results indicate that secondary cell death via GJs plays a critical role in the progressive loss of neurons in experimental glaucoma. Both Cx36- and Cx45-expressing GJs are involved and their effects on cell death are additive. Overall, our results suggest that targeting retinal GJs forms a novel therapeutic approach to protect retinal neurons in glaucomatous retinas.
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