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D. Wang, W. Huang, A. Ray, C. Ergorul, K. Rodgers, Y. Ben, C. L. Grosskreutz; Downregulation of Neuregulin 1 in Retinal Ganglion Cells in Experimental Glaucoma. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2075.
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We recently performed microarray analysis of mRNA from retinal ganglion cells (RGC) isolated by laser capture microdissection (LCM) and found many differentially expressed genes in glaucomatous RGC. One gene is the neurogenesis and survival gene Neuregulin (Nrg-1). We sought to further investigate Nrg-1 in RGC and its decrease in experimental glaucoma.
RGCs were bilaterally back-labeled by stereotaxic injection of Fluorogold into the superior colliculus. Sustained unilateral intraocular pressure (IOP) elevation was produced by hypertonic saline episcleral vein injection in Brown Norway rats with the fellow eye serving as a control. IOP was measured in conscious rats every other day and the area under the pressure time curve was calculated. When the IOP had been elevated for 10 days and the AUC reached 250 mmHg-days, the rats were sacrificed and their retinas were removed and processed for tissue sectioning, RNA extraction or protein isolation. In the first group (n = 5), an equal number of RGCs were isolated by LCM from retinal tissue sections of rat eyes with and without high IOP. qRT-PCR was used to validate the micrcoarray results of decreased mRNA expression of Nrg-1 in the RGCs. Retinal tissue sections were also used to perform immunohistochemical localization of Nrg-1. In the second group (n = 6), Western blotting was performed on whole retinal lysates to examine whole retinal Nrg-1 protein levels. In the third group (n = 6), qRT-PCR was performed to examine the expression of Nrg-1 in whole retina.
qRT-PCR of mRNA from RGC isolated by LCM confirmed our microarray analysis result that the level of Nrg1 was significantly down-regulated in RGCs from eyes with high IOP when compared to control eyes (glaucoma:control mean±SD: -6.56±0.47, P < 0.05, n = 5). Immunohistochemistry showed that Nrg1 protein was also downregulated in the ganglion cell layer of the glaucomatous retinas. Nrg1 mRNA was abundant in RGC and was 11.61 fold (P < 0.001, n = 6) enriched in normal RGC vs. normal whole retina. However, neither the whole retinal mRNA level (glaucoma:control 0.90±1.42, P = 0.19, n = 6) nor the whole retinal protein level of Nrg-1 (glaucoma:control 1.84±1.18, P = 0.14, n = 6) were significantly changed.
Our results show that Nrg1 message and protein are selectively decreased in RGC in experimental glaucoma. Nrg-1 has been shown to be a survival factor for RGC in culture, for spiral ganglion cells in the inner ear, and is a potent neuroprotective factor in stroke models. We speculate that the decreased expression of Nrg1 may contribute to RGC death in glaucoma.
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