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Dmitry V Ivanov, Isabel Perea-Martinez, Dagmara Danek, Jasmine R Naik, Steve Pappas, Xenia Dvoriantchikova, Galina Dvoriantchikova; The Molecular Mechanisms Of Sterile Inflammation In Ischemic Retina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):6168.
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© ARVO (1962-2015); The Authors (2016-present)
The role of sterile inflammation (innate immune response that occurs in the absence of live pathogens) was proposed in many retinal disorders including glaucoma, ischemic optic neuropathy and diabetic retinopathy. Thus, understanding the mechanisms of sterile inflammation will provide substantial insight and novel therapeutic potential to regulate disease pathogenesis in the retina. The purpose of this study was to investigate signaling cascades that regulate sterile inflammation in the retina after ischemia-reperfusion (IR) injury.
TLR4, PKCα, MYDd88- and TRIF-deficient animals and C57BL/6J mice as the wild type control were obtained from the Jackson Laboratory. The mouse model of retinal IR injury was employed for in vivo experiments. Whole retina flatmounts were stained for the neuronal marker NeuN to quantify the number of surviving neurons in the ganglion cell layer of ischemic and control retinas. Primary retinal ganglion cells (RGCs) isolated from retinas according to the two-step immunopanning protocol and glial cells obtained by the “shaking method” were used for in vitro experiments. Quantitative RT-PCR, ELISA and western blot analysis were used to study production of pro-inflammatory factors in ischemic retinas and in primary RGC and glial cultures.
We found that the initial ischemic stress leads to cell injury (necrosis) and release of HSP70. Our in vitro and in vivo studies indicate that upon HSP70 ligation to TLR4, glial cells produce neurotoxic levels of TNFα in a TRIF-and MYD88-dependent manner. Our data also indicate that PKC isoform α (PKCα) is critical in mediating the HSP70/TLR4 -dependent pro-inflammatory response, which is sufficient to mediate RGC death. We also found that TNFα mediates NF-κB activation in glial cells, which facilitates survival of these cells but mediates production of TNFα in the retina. Meanwhile, the lack of NF-κB activity in RGCs in the presence of TNFα, as well as sustained JNK activation, both contribute to RGC death, mostly by necrosis.
Our findings suggest that HSP70, released by necrotic RGCs, induces glial cells to produce TNFα in a TLR4/ PKCα/ TRIF/MYD88-dependent manner. In turn, TNFα triggers RGC necrosis that can lead to increased levels of extracellular HSP70. This cycle can be repeated many times and can result in significant retinal damage after IR.
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