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M. Duong, K. Hockmann, A.K. Ball; Retinal Microglial Responses to Cellular Injury Following ET–1 Mediated RGC Death . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2985.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Retinal ganglion cell (RGC) loss due to injury is accompanied by glial responses. We have previously shown that injection of 5µL (500µM) endothelin–1 (ET–1) into the vitreous of rat eyes results in the death of 40% of RGCs within 4 weeks and increases GFAP expression by astrocytes and Muller cells. These responses are not accompanied by an obvious extrinsic inflammatory immune response. In the present study, we describe the activation and re–organization of microglia (MG) after ET–1 induced RGC death. Methods:ET–1 (5µL; 500µM) was injected into the vitreous chamber of adult rat eyes. Animals were killed at 2, 4, 7, 14 and 21 days post–ET–1 injection, eyes were fixed in phosophate buffered 4% formaldehyde and processed for double label immunohistochemistry. MG were identified using antisera directed against Iba–1 (NIH; 1:500) and the lysosomal membrane antigen, ED–1 (Serotec; 1:250). Activated MG were differentiated from resting MG when Iba–1–immunoractive (–IR) microglia also expressed ED–1. MG organization was analysed at each time point by nearest neighbor analysis at the 3 layers where MG normally reside (proximal inner plexiform layer (IPL), distal IPL and the outer plexiform layer (OPL)). Number of cells were quantified in flat mounts and frozen sections cut perpendicular to the retinal layers. Changes in the cytoplasmic volume were measured in confocal micrographs using ImageJ software. Results:In normal, untreated retinas, nearest neighbor analysis revealed that the Iba–1–IR MG were uniformaly arranged and none expressed ED–1. Expressoin of Iba–1 and ED–1 increased at 2 days post–ET–1 injection, peaked at 4 days and decreased to control levels by 21 days post–ET–1 injection. The increase in immunoreactivity was due to changes in cellular expression because cell numbers remained unchanged. Nearest neighbor analysis revealed that the distribution of retinal MG became random between 2 and 7 days post–ET–1 injection, suggesting that MG were migrating through the retinal layers during this time. The distribution of MG returned to the uniform arrangement observed in control retinas by 21 days post–ET–1 injection. Conclusions:These results demonstrate that MG become activated within days of ET–1 mediated injury and precedes the gradual sustained loss of RGCs that occurs over 4 weeks. Rather than being recruited to the retina upon injury, our results suggest that MG exist in the retina in a resting state with a uniform distribution. Immediately following injury, they become activated and migrate. Surprisingly, the activation and redistribution peaked at 4 days and returned to control levels despite the continued loss of RGCs.
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