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Dmitry V. Ivanov, Galina Dvoriantchikova, David J. Barakat, Eleut Hernandez, Valery I. Shestopalov; The Role of HMGB1 Protein In Retinal Ischemia. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6059.
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HMGB1 is a ubiquitous and abundant nuclear protein that is released from necrotic cells. It induces an inflammatory response through TLR4 and RAGE and its levels are often increased in areas of ischemic insult. At the same time, suppression of HMGB1 signaling significantly improves outcome in the brain, heart, liver, and the kidney after ischemia-reperfusion (IR) injury. Since mechanisms of cell death after ischemia share similarity in many tissues, we hypothesize that HMGB1 plays a significant role in retinal IR injury.
IR injury was induced by unilateral elevation of intraocular pressure via direct corneal canulation. Samples of vitreous humor were collected 24 hours after reperfusion and analyzed by western blot using an antibody against HMGB1. To test the functional significance of HMGB1 release, the neutralizing anti-HMGB1 antibody and control IgG were injected intraperitoneally 15 min before ischemia. Cell death was evaluated by direct counting of NeuN labeled ganglion cell layer (GCL) neurons of flat-mounted retinas 7 days post-reperfusion. To elucidate the downstream signaling pathway involved in HMGB1 induced effects in the ischemic retina, we used TLR4-deficient mice. The changes in expression of pro-inflammatory genes 24 h postreperfusion by quantitative PCR and survival of GCL neurons 7 days after ischemic injury were assessed in TLR4 knockout and wild type (control) mice.
Our data indicate thatHMGB1 is released into the extracellular space and accumulates in the vitreous humor 24 hours after retinal IR injury. Following ischemic injury, mice treated with neutralizing anti-HMGB1 antibody showed significant increase in survival of neurons in the GCL relative to the controls. The analysis of TLR4-deficient vs. wild type mice showed significantly reduced expression of pro-inflammatory genes 24 hours after reperfusion and significantly increased survival of GCL neurons 7 days after ischemic injury.
Our results suggest that an increased level of HMGB1 and signaling via the TLR4 receptor contribute to inflammatory response and neurotoxicity following retinal IR injury.
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