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Xiangjun Yang, Cheng Luo, Jian Cai, David W. Powell, Gulgun Tezel; Immune/Inflammatory Responses of Retinal Astrocytes in Experimental Glaucoma. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3833.
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With the advantage of cell-specific sampling, this proteomic study aimed to analyze retinal astrocyte-specific responses in a chronic pressure induced rat model of glaucoma.
Intraocular pressure elevation was induced in rats by hypertonic saline injections into episcleral veins. Enriched samples of retinal astrocytes were isolated through the two-step immunomagnetic cell selection process originally established to enrich retinal ganglion cell samples. Ocular hypertensive and control samples were collected by pooling from rat eyes matched for the cumulative intraocular pressure exposure. Protein expression was complementarily analyzed by quantitative LC-MS/MS followed by quantitative Western blot analysis and immunohistochemical analysis using cleavage or phosphorylation site-specific antibodies to selected proteins.
Western blots verified GFAP expression in enriched astrocyte samples; however, neuronal markers, including NeuN and Brn-3, were not detectable in these samples. After validation of astrocyte-specific protein sampling, analysis of cell-specific protein expression identified hundreds of proteins with high confidence (by two peptides or more at the 0.2% peptide and 0.1% protein false discovery rates). The MS/MS data also included GFAP expression in astrocyte samples. Bioinformatic comparison analysis of the cell-specific high-throughput data by the Ingenuity Pathways Analysis supported distinct responses of astrocytes during the experimental paradigm, which predominantly exhibited cellular activation and immune/inflammatory responses as opposed to cell death signaling in retinal ganglion cells. As also validated by Western blot analysis and tissue immunolabeling using specific antibodies, a number of proteins mediating inflammatory processes exhibited significant up-regulation in ocular hypertensive astrocyte samples (Mann-Whitney Rank Sum test; p<0.05). These proteins included a number of low-abundant downstream proteins linked to TNF-α/TNFR1 signaling (TRADD, TNFAIP2, and an astrocytic phosphoprotein, PEA-15), NF-ΚB activation (RIPK2, IΚKB, p65, p100/52, COP9-signalosome), and inflammasome assembly (NLRP1, NLRP3, caspase-1).
Findings of this study indicated various molecules characterizing retinal astrocyte responses during the experimental paradigm. By highlighting various co-players of inflammatory responses mediated by ocular hypertensive astrocytes, these findings support that dissection of astrocyte-specific responses can benefit the development of new treatment strategies for glaucoma targeting not only retinal ganglion cells but also astrocytes.
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