Post-TTM Rebound Pyrexia after Ischemia-Reperfusion Injury Results in Sterile Inflammation and Apoptosis in Cardiomyocytes.

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-1ß, 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-1ß, 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.

RBM3 and CIRP expressions in targeted temperature management treated cardiac arrest patients-A prospective single center study.

BACKGROUND: Management of cardiac arrest patients includes active body temperature control and strict prevention of fever to avoid further neurological damage. Cold-shock proteins RNA-binding motif 3 (RBM3) and cold inducible RNA-binding protein (CIRP) expressions are induced in vitro in response to hypothermia and play a key role in hypothermia-induced neuroprotection. OBJECTIVE: To measure gene expressions of RBM3, CIRP, and inflammatory biomarkers in whole blood samples from targeted temperature management (TTM)-treated post-cardiac arrest patients for the potential application as clinical biomarkers for the efficacy of TTM treatment. METHODS: A prospective single center trial with the inclusion of 22 cardiac arrest patients who were treated with TTM (33°C for 24 hours) after ROSC was performed. RBM3, CIRP, interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and inducible nitric oxide synthase (iNOS) mRNA expressions were quantified by RT-qPCR. Serum RBM3 protein concentration was quantified using an enzyme-linked immunosorbent assay (ELISA). RESULTS: RBM3 mRNA expression was significantly induced in post-cardiac arrest patients in response to TTM. RBM3 mRNA was increased 2.2-fold compared to before TTM. A similar expression kinetic of 1.4-fold increase was observed for CIRP mRNA, but did not reached significancy. Serum RBM3 protein was not increased in response to TTM. IL-6 and MCP-1 expression peaked after ROSC and then significantly decreased. iNOS expression was significantly increased 24h after return of spontaneous circulation (ROSC) and TTM. CONCLUSIONS: RBM3 is temperature regulated in patients treated with TTM after CA and ROSC. RBM3 is a possible biomarker candidate to ensure the efficacy of TTM treatment in post-cardiac arrest patients and its pharmacological induction could be a potential future intervention strategy that warrants further research.

Combined Cyclosporin A and Hypothermia Treatment Inhibits Activation of BV-2 Microglia but Induces an Inflammatory Response in an Ischemia/Reperfusion Hippocampal Slice Culture Model.

INTRODUCTION: Hypothermia attenuates cerebral ischemia-induced neuronal cell death associated with neuroinflammation. The calcineurin inhibitor cyclosporin A (CsA) has been shown to be neuroprotective by minimizing activation of inflammatory pathways. Therefore, we investigated whether the combination of hypothermia and treatment with CsA has neuroprotective effects in an oxygen-glucose deprivation/reperfusion (OGD/R) injury model in neuronal and BV-2 microglia monocultures, as well as in an organotypic hippocampal slice culture (OHSC). METHODS: Murine primary neurons, BV-2 microglia, and OHSC were pretreated with CsA and exposed to 1 h OGD (0.2% O2) followed by reperfusion at normothermia (37°C) or hypothermia (33.5°C). Cytotoxicity was measured by lactate dehydrogenase and glutamate releases. Damage-associated molecular patterns (DAMPs) high mobility group box 1 (HMGB1), heat shock protein 70 (Hsp70), and cold-inducible RNA-binding protein (CIRBP) were detected in cultured supernatant by western blot analysis. Interleukin-6 (IL-6), Interleukin-1α and -1ß (IL-1α/IL1-ß), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein 1 (MCP1), inducible nitric oxide synthase (iNOS), glia activation factors ionized calcium-binding adapter molecule 1 (Iba1), and transforming growth factor ß1 (TGF-ß1) gene expressions were analyzed by RT-qPCR. RESULTS: Exposure to OGD plus 10 µM CsA was sufficient to induce necrotic cell death and subsequent release of DAMPs in neurons but not BV-2 microglia. Moreover, OGD/R-induced secondary injury was also observed only in the neurons, which was not attenuated by cooling and no increased toxicity by CsA was observed. BV-2 microglia were not sensitive to OGD/R-induced injury but were susceptible to CsA-induced toxicity in a dose dependent manner, which was minimized by hypothermia. CsA attenuated IL-1ß and Iba1 expressions in BV-2 microglia exposed to OGD/R. Hypothermia reduced IL-1ß and iNOS expressions but induced TNF-α and Iba1 expressions in the microglia. However, these observations did not translate to the ex vivo OHCS model, as general high expressions of most cytokines investigated were observed. CONCLUSION: Treatment with CsA has neurotoxic effects on primary neurons exposed to OGD but could inhibit BV-2 microglia activation. However, CsA and hypothermia treatment after ischemia/reperfusion injury results in cytotoxic neuroinflammation in the complex ex vivo OHSC.

Moderate therapeutic hypothermia induces multimodal protective effects in oxygen-glucose deprivation/reperfusion injured cardiomyocytes.

OBJECTIVE: Therapeutic hypothermia has been shown to attenuate myocardial cell death due to ischemia/reperfusion injury. However, cellular mechanisms of cooling remain to be elucidated. Especially during reperfusion, mitochondrial dysfunction contributes to cell death by releasing apoptosis inductors. The aim of the present study was to investigate the effects of moderate therapeutic hypothermia (33.5°C) on mitochondrial mediated apoptosis in ischemia/reperfusion-injured cardiomyocytes. METHODS: Ischemic injury was simulated by oxygen-glucose deprivation for 6h in glucose/serum-free medium at 0.2% O2 in mouse atrial HL-1 cardiomyocytes. Simulation of reperfusion was achieved by restoration of nutrients in complete supplemented medium and incubation at 21% O2. Early application of therapeutic hypothermia, cooling during the oxygen-glucose deprivation phase, was initiated after 3h of oxygen-glucose deprivation and maintained for 24h. Mitochondrial membrane integrity was assessed by cytochrome c and AIF protein releases. Furthermore, mitochondria were stained with MitoTracker Red and intra-cellular cytochrome c localization was visualized by immunofluorescence staining. Moreover, anti-apoptotic Bcl-2 and Hsp70 as well as phagophore promoting LC3-II protein expressions were analyzed by Western-blot analysis. RESULTS: Therapeutic hypothermia initiated during oxygen-glucose deprivation significantly reduced mitochondrial release of cytochrome c and AIF in cardiomyocytes during reperfusion. Secondly, anti-apoptotic Bcl-2/Bax ratio and Hsp70 protein expressions were significantly upregulated due to hypothermia, indicating an inhibition of both caspase-dependent and -independent apoptosis. Furthermore, cardiomyocytes treated with therapeutic hypothermia showed increased LC3-II protein levels associated with the mitochondria during the first 3h of reperfusion, indicating the initiation of phagophores formation and sequestration of presumably damaged mitochondrion. CONCLUSION: Early application of therapeutic hypothermia effectively inhibited cardiomyocyte cell death due to oxygen-glucose deprivation/reperfusion-induced injury via multiple pathways. As hypothermia preserved mitochondrial membrane integrity, which resulted in reduced cytochrome c and AIF releases, induction of both caspase-dependent and -independent apoptosis was minimized. Secondly, cooling attenuated intrinsic apoptosis via Hsp70 upregulation and increasing anti-apoptotic Bcl-2/Bax ratio. Moreover, therapeutic hypothermia promoted mitochondrial associated LC3-II during the early phase of reperfusion, possibly leading to the sequestration and degradation of damaged mitochondrion to attenuate the activation of cell death.

Neuroprotection via RNA-binding protein RBM3 expression is regulated by hypothermia but not by hypoxia in human SK-N-SH neurons.

OBJECTIVE: Therapeutic hypothermia is an established treatment for perinatal asphyxia. Yet, many term infants continue to die or suffer from neurodevelopmental disability. Several experimental studies have demonstrated a beneficial effect of mild-to-moderate hypothermia after hypoxic injury, but the understanding of hypothermia-induced neuroprotection remains incomplete. In general, global protein synthesis is attenuated by hypothermia, but a small group of RNA-binding proteins including the RNA-binding motif 3 (RBM3) is upregulated in response to cooling. The aim of this study was to establish an in vitro model to investigate the effects of hypoxia and hypothermia on neuronal cell survival, as well as to examine the kinetics of concurrent cold-shock protein RBM3 gene expression. METHODS: Experiments were performed by using human SK-N-SH neurons exposed to different oxygen concentrations (21%, 8%, or 0.2% O2) for 24 hours followed by moderate hypothermia (33.5°C) or normothermia for 24, 48, or 72 hours. Cell death was determined by quantification of lactate dehydrogenase and neuron-specific enolase releases into the cell cultured medium, and cell morphology was assessed by using immunofluorescence staining. The regulation of RBM3 gene expression was assessed by reverse transcriptase-quantitative polymerase chain reaction and Western blot analysis. RESULTS: Exposure to hypoxia (0.2% O2) for 24 hours resulted in significantly increased cell death in SK-N-SH neurons, whereas exposure to 8% O2 had no significant impact on cell viability. Post-hypoxia treatment with moderate hypothermia for 48 or 72 hours rescued the neurons from hypoxia-induced cell death. Moreover, exposure to severe hypoxia led to observable cell swelling, which was also attenuated by moderate hypothermia. Finally, moderate hypothermia but not hypoxia led to the induction of RBM3 expression on both transcriptional and translational levels. CONCLUSION: Moderate hypothermia protects neurons from hypoxia-induced cell death. The expression of the cold-shock protein RBM3 is induced by moderate hypothermia and could be one possible mediator of hypothermia-induced neuroprotection.