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Izzy Livne-Bar, Samih Alqawlaq, Jeremy M Sivak; A Screen for Astrocyte-Secreted Activity Which Protects Ganglion Cells from Metabolic Stress. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4829.
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© ARVO (1962-2015); The Authors (2016-present)
Astrocytes and related Müller glia play critical homeostatic roles in the inner retina, and rapidly activate with disease or injury. However, the positive and/or negative effects of activation on retinal ganglion cells (RGCs) are not fully understood. We have previously demonstrated that retinal astrocytes protect RGCs when quiescent. However, glial activation reduces this protective effect on RGCs in vivo. Here, we describe a cell-based screen to characterize and identify the astrocyte-derived protective activity.
For in vivo experiments, fresh media or astrocyte conditioned media were injected intravitreally into recipient mice prior to kainic acid induced excitotoxic RGC injury. The in vivo protective effect was quantified by apoptosis assay (TUNEL). For in vitro assays we exposed a neuronal cell line (Ht22) to glutamate neurotoxic treatment. Prior to glutamate treatment, Ht22 cells were incubated with fresh media or astrocyte conditioned media. The protective effect from glutamate-induced cell death, was measured by a mitochondrial activity (XTT) assay.
We established that astrocyte-conditioned medium is strongly neuroprotective both to RGCs in vivo and to the Ht22 cell line. This protection was sensitive to trypsin treatment. Similar protective activity was not produced by scleral fibroblasts or by immortalized astrocyte cell lines. We developed a large scale screening strategy to identify the pathways and molecules mediating this protection. We will present preliminary results on the pathways involved in this neuronal protection.
We consistently demonstrated that retinal astrocytes secrete neuroprotective factors that reduce glutamate toxicity both in vivo and in vitro.<br /> We have now developed a cell-based screen to identify the astrocyte-derived protective factors. This neuroprotective activity appears to be mediated, in part, by the PI3k pathway.
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