An astrocyte-specific proteomic approach to inflammatory responses in experimental rat glaucoma

To delineate astrocyte-mediated inflammatory processes in glaucoma, we analyzed proteomic responses of retinal astrocytes in an experimental rat model using a cell-specific approach.

IOP elevation was induced in rats by hypertonic saline injections into episcleral veins. Enriched samples of astrocytes were isolated through the immunomagnetic cell selection process established originally for retinal ganglion cell (RGC) sampling. Ocular hypertensive and control samples were collected by pooling from rat eyes matched for the cumulative IOP exposure. Protein expression was analyzed complementarily by quantitative two-dimensional capillary liquid chromatography and linear ion trap mass spectrometry (LC-MS/MS) followed by quantitative Western blot analysis and retinal tissue immunolabeling using specific antibodies to selected proteins.

Following validation of enriched astrocyte samples, LC-MS/MS analysis resulted in the identification of over 2000 proteins with high confidence. Bioinformatic comparison analysis of the high-throughput MS/MS data along with the findings of immunoblotting and immunohistochemistry supported distinct responses of ocular hypertensive astrocytes during the experimental paradigm, which exhibited predominantly cellular activation and immune/inflammatory responses as opposed to activation of cell death signaling in ocular hypertensive RGCs. Inflammatory responses of astrocytes in experimental glaucoma included up-regulation of a number of immune mediators/regulators linked to TNF-α/TNFR signaling, nuclear factor kappa-B (NF-κB) activation, autophagy regulation, and inflammasome assembly.

These findings validate an astrocyte-specific approach to quantitatively identify proteomic alterations in experimental glaucoma, and highlight many immune mediators/regulators characteristic of the inflammatory responses of ocular hypertensive astrocytes. By dissecting the complexity of prior data obtained from whole tissue, this pioneering approach should enable astrocyte responses to be defined and new treatments targeting astrocytes to be developed.