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Objective: Cold-inducible RNA-binding protein (CIRBP) has been shown to be involved not only in cooling-induced cellular protection but also as a mediator of sterile inflammation, a critical mechanism of the innate immune response in ischemia/reperfusion (I/R) injury. The role of microglia and its activation in cerebral I/R injury warrants further investigation as both detrimental and regenerative properties have been described. Therefore, we investigated the effects of cooling, specifically viability, activation, and release of damage associated molecular patterns (DAMPs) on oxygen glucose deprivation/reperfusion- (OGD/R-) induced injury in murine BV-2 microglial cells., Methods: Murine BV-2 microglial cells were exposed to 2 to 6 h OGD (0.2% O 2 in glucose- and serum-free medium) followed by up to 19 h of reperfusion, simulated by restoration of oxygen (21% O 2 ) and nutrients. Cells were maintained at either normothermia (37°C) or cooled to 33.5°C, 1 h after experimental start. Cultured supernatants were harvested after exposure to OGD for analysis of DAMP secretions, including high-mobility group box 1 (HMGB1), heat shock protein 70 (HSP70), and CIRBP, and cytotoxicity was assessed by lactate dehydrogenase releases after exposure to OGD and reperfusion. Intracellular cold-shock proteins CIRBP and RNA-binding motif 3 (RBM3) as well as caspases 9, 8, and 3 were also analyzed via Western blot analysis. Furthermore, inducible nitric oxide synthase (iNOS), ionized calcium-binding adaptor molecule 1 (Iba1), tumor necrosis factor- α (TNF- α ), interleukin-6 (IL-6), interleukin-1 β (IL-1 β ), interleukin-1 α (IL-1 α ), monocyte chemotactic protein 1 (MCP-1), transforming growth factor β (TGF β ), CIRBP, and RBM3 gene expressions were assessed via reverse transcription polymerase chain reaction, and TNF- α , IL-6, and IL-1 β releases into the cultured supernatants were assessed via enzyme-linked immunosorbent assays (ELISA)., Results: Prolonged exposure to OGD resulted in increased BV-2 necrotic cell death, which was attenuated by cooling. Cooling also significantly induced cold-shock proteins CIRBP and RBM3 gene expressions, with CIRBP expression more rapidly regulated than RBM3 and translatable to significantly increased protein expression. DAMPs including HMGB-1, HSP70, and CIRBP could be detected in cultured supernatants after 6 h of OGD with CIRBP release being significantly attenuated by cooling. Exposure to OGD suppressed cytokine gene expressions of IL-1 β , TNF- α , MCP-1, and TGF β independently of temperature management, whereas cooling led to a significant increase in IL-1 α gene expression after 6 h of OGD. In the reperfusion phase, TNF- α and MCP-1 gene expressions were increased, and cooling was associated with significantly lower TGF β gene expression. Interestingly, cooled Normoxia groups had significant upregulations of microglial activation marker, Iba1, IL-1 β , and TNF- α gene expressions., Conclusion: BV-2 microglial cells undergo necrotic cell death resulting in DAMP release due to OGD/R-induced injury. Cooling conveyed neuroprotection in OGD/R-injury as observable in increased cell viability as well as induced gene expressions of cold shock proteins. As cooling alone resulted in both upregulation of microglial activation, expression of proinflammatory cytokines, and cold shock protein transcript and protein expression, temperature management might have ambiguous effects in sterile inflammation. However, cooling resulted in a significant decrease of extracellular CIRBP, which has recently been characterized as a novel DAMP and a potent initiator and mediator of inflammation., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Jana Lücht et al.)

Introduction: Fever is frequently observed after acute ischemic events and is associated with poor outcome and higher mortality. Targeted temperature management (TTM) is recommended for neuroprotection in comatose cardiac arrest survivors, but pyrexia after rewarming is proven to be detrimental in clinical trials. However, the cellular mechanisms and kinetics of post-TTM rebound pyrexia remain to be elucidated. Therefore, we investigated the effects of cooling and post-TTM pyrexia on the inflammatory response and apoptosis in a cardiomyocyte ischemia-reperfusion (IR) injury model., Methods: HL-1 cardiomyocytes were divided into the following groups to investigate the effect of oxygen-glucose deprivation/reperfusion (OGD/R), hypothermia (33.5°C), and pyrexia (40°C): normoxia controls maintained at 37°C and warmed to 40°C, OGD/R groups maintained at 37°C and cooled to 33.5°C for 24 h with rewarming to 37°C, and OGD/R pyrexia groups further warmed from 37 to 40°C. Caspase-3 and RBM3 were assessed by Western blot and TNF- α , IL-6, IL-1 β , SOCS3, iNOS, and RBM3 transcriptions by RT-qPCR., Results: OGD-induced oxidative stress (iNOS) in cardiomyocytes was attenuated post-TTM by cooling. Cytokine transcriptions were suppressed by OGD, while reperfusion induced significant TNF- α transcription that was exacerbated by cooling. Significant inductions of TNF- α , IL-6, IL-1 β , and SOCS3 were observed in noncooled, but not in cooled and rewarmed, OGD/R-injured cardiomyocytes. Further warming to pyrexia induced a sterile inflammatory response in OGD/R-injured groups that was attenuated by previous cooling, but no inflammation was observed in pyrexic normoxia groups. Moreover, cytoprotective RBM3 expression was induced by cooling but suppressed by pyrexia, correlating with apoptotic caspase-3 activation., Conclusion: Our findings show that maintaining a period of post-TTM "therapeutic normothermia" is effective in preventing secondary apoptosis-driven myocardial cell death, thus minimizing the infarct area and further release of mediators of the innate sterile inflammatory response after acute IR injury., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2019 Giang Tong et al.)

Introduction. Ischemia/Reperfusion (I/R) is a primary cause of myocardial injury after acute myocardial infarction resulting in the release of damage-associated molecular patterns (DAMPs), which can induce a sterile inflammatory response in the myocardial penumbra. Targeted temperature management (TTM) after I/R has been established for neuroprotection, but the cardioprotective effect remains to be elucidated. Therefore, we investigated the effect of TTM on cell viability, immune response, and DAMP release during oxygen-glucose deprivation/reperfusion (OGD/R) in murine primary cardiomyocytes. Methods. Primary cardiomyocytes from P1-3 mice were exposed to 2, 4, or 6 hours OGD (0.2% oxygen in medium without glucose and serum) followed by 6, 12, or 24 hours simulated reperfusion (21% oxygen in complete medium). TTM at 33.5°C was initiated intra-OGD, and a control group was maintained at 37°C normoxia. Necrosis was assessed by lactate dehydrogenase (LDH) release and apoptosis by caspase-3 activation. OGD-induced DAMP secretions were assessed by Western blotting. Inducible nitric oxide synthase (iNOS), cytokines, and antiapoptotic RBM3 and CIRBP gene expressions were measured by quantitative polymerase chain reaction. Results. Increasing duration of OGD resulted in a transition from apoptotic programmed cell death to necrosis, as observed by decreasing caspase-3 cleavage and increasing LDH release. DAMP release and iNOS expression correlated with increasing necrosis and were effectively attenuated by TTM initiated during OGD. Moreover, TTM induced expression of antiapoptotic RBM3 and CIRBP. Conclusion. TTM protects the myocardium by attenuating cardiomyocyte necrosis induced by OGD and caspase-3 activation, possibly via induction of antiapoptotic RBM3 and CIRBP expressions, during reperfusion. OGD induces increased Hsp70 and CIRBP releases, but HMGB-1 is the dominant mediator of inflammation secreted by cardiomyocytes after prolonged exposure. TTM has the potential to attenuate DAMP release. [ABSTRACT FROM AUTHOR]