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Galina Dvoriantchikova, Andrea Rachelle Santos, Xenia Dvoriantchikova, Eleut Hernandez, Dmitry Ivanov; Extracellular Hsp70 Mediates Ischemia-Induced Retinal Damage Through A Tlr4-Dependent Pathway. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6358.
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Changes in the levels of intracellular and extracellular Hsp70 produce different responses after ischemia-reperfusion (IR) injury: intracellular Hsp70 mediates neuroprotection after IR, while extracellular Hsp70 can induce an immune response that can promote tissue damage. Since the role of extracellular Hsp70 in IR injury of the retina remains unclear, we investigated whether Hsp70 contributes to retinal damage after IR.
Retinal IR injury was induced by unilateral elevation of intraocular pressure and the level of Hsp70 in vitreous humor was analyzed 24 hours after reperfusion. To test the functional significance of Hsp70 release, retinas were treated with the recombinant Hsp70 protein and retinal damage was evaluated by direct counting of neurons in the ganglion cell layer of flat-mounted retinas 7 days after IR. Mouse macrophage RAW-blue reporter cell line was used to monitor the activation of NFκB after Hsp70 treatment. To investigate the mechanism and establish the cell type specific Hsp70-mediated pro-inflammatory response, primary cultures of glial cells isolated from Tlr4, Myd88, and Trif knockout animals were used. We generated retinal ganglion cell (RGC)/glial co-cultures to test whether Hsp70 induces RGC death via pro-inflammatory activity of glial cells.
Hsp70 is accumulated in the vitreous humor 24 hours after IR. Treatment of retinas with Hsp70 induced a loss of RGCs. Stimulation of RAW-blue reporter cell line with Hsp70 led to an increase in NFκB activation. We observed increased levels of cytokine, chemokine NADPH oxidase and nitric oxide synthase activity in glial cells treated with Hsp70. This activity was significantly reduced in glial cells isolated from Tlr4 and Myd88 knockout animals. Genetic ablation of Tlr4 and Myd88 in glial cells suppressed RGC loss in co-cultures with glia challenged by treatment with Hsp70.
Our data strongly support the hypothesis that extracellular release of Hsp70 contributes to retinal IR injury. The effects of Hsp70 in retinal ischemia could be explained by engagement of glial Tlr4, resulting in the Myd88-dependent activation of pro-inflammatory NFκB signaling cascades, which mediate RGC death.
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