An Astrocyte-Specific Proteomic Approach to Inflammatory Responses in Experimental Rat Glaucoma

Abstract

PURPOSE. To delineate astrocyte-mediated inflammatory processes in glaucoma, we analyzed proteomic responses of retinal astrocytes in an experimental rat model using a cellspecific 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. 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. (Invest Ophthalmol Vis Sci. 2012;53:4220-4233)

Axonal Protection via Modulation of the Amyloidogenic Pathway in Tumor Necrosis Factor–Induced Optic Neuropathy

“…Most recently, TNF/TNFR signaling has been reported to mediate immune/inflammatory responses in astrocytes during glaucomatous neurodegeneration. 30 In the present study, activation of astrocytes was observed in the optic nerve after TNF injection. The TNF injection model has a limitation in mimicking the glaucoma model as it shows axonal degeneration in 2 weeks without further degeneration over longer periods of time, 23 while the hypertensive glaucoma model shows progressive axonal degeneration.…”

The Complex Role of Neuroinflammation in Glaucoma

“…Several studies using transcriptional profiling have identified early up-regulation of genes associated with inflammatory pathways in the retina and ONH in experimental models of glaucoma (Ahmed et al 2004;Johnson et al 2007Johnson et al , 2011Yang et al 2007;Kompass et al 2008;Nikolskaya et al 2009;Panagis et al 2010;Howell et al 2011aHowell et al , 2012. Collectively, these studies showed an up-regulation of inflammatory inducers/sensors (PRRs, e.g., TLRs), transducers (e.g., Mapk, Trif, MyD88) and amplifiers (e.g., Il1, Il6, Tnf, Ccl2) in human glaucoma Yang et al 2011;Tezel et al 2012) and in early stages of experimental glaucoma (Howell et al 2011a;Johnson et al 2011). In our study using DBA/2J mice, an inherited model of glaucoma, we were able to define early molecular changes that occur before detectable loss of RGCs and axons (Howell et al 2011a).…”

“…Not surprisingly, proteomic analysis of glaucomatous retinas from humans and rats found increased expression of kinases such as RIPK, NIK, and IkK that are involved in the activation of the NF-kB pathway (Yang et al 2011;Tezel et al 2012). Activation of NF-kB results in the transcription of genes from the IL-1 cytokine family.…”

Autophagy dysregulation and the fate of retinal ganglion cells in glaucomatous optic neuropathy

“…Retina and optic nerve astrocytes exert both neurosupportive and detrimental effects on RGCs over the course of glaucoma; proteomic analysis of astrocytesenriched samples from hypertensive rats (hypertonic saline injection) showed upregulation of mTOR, Atg3, and Atg7 (Tezel et al, 2012).…”