title: An astrocyte-specific proteomic approach to inflammatory responses in experimental rat glaucoma.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Ophthalmology & Visual Sciences, University of Louisville School of Medicine, Louisville, KY 40202, USA. gulgun.tezel@louisville.edu
authors.forename: Gülgün
authors.initials: G
authors.lastname: Tezel
authors.forename: Xiangjun
authors.initials: X
authors.lastname: Yang
authors.forename: Cheng
authors.initials: C
authors.lastname: Luo
authors.forename: Jian
authors.initials: J
authors.lastname: Cai
authors.forename: David W
authors.initials: DW
authors.lastname: Powell
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism* / Q000473:pathology; D015153:Blotting, Western; D004195:Disease Models, Animal; D005901:Glaucoma / Q000378:metabolism* / Q000473:pathology; D007249:Inflammation / Q000378:metabolism* / Q000473:pathology; D040901:Proteomics / Q000379:methods*; D051381:Rats; D011914:Rats, Inbred BN; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D015854:Up-Regulation