Abstract TP271: Time-Dependent Neuroprotection of Pre-reperfusion Hypothermia: CREB1/GATA3-Driven RBM3 Transcription Cooperates with Mitochondrial Preservation to Block Endoplasmic Reticulum (ER) Stress-Induced Pyroptosis

Background: The neuroprotective efficacy of therapeutic hypothermia is highly time-dependent, with pre-reperfusion hypothermia offering superior benefit. The cold shock protein RBM3 is upregulated during hypothermia, while the specific mechanisms on temporal advantage by which pre-reperfusion cooling enhances RBM3 accumulation remain to be determined. Methods: Rat middle cerebral artery occlusion/reperfusion (MCAO/R) and SH-SY5Y cell oxygen-glucose deprivation/reoxygenation (OGD/R) models were treated with pre-reperfusion hypothermia (1-h pre-reperfusion) or during-reperfusion hypothermia. Pharmacological cooling (chlorpromazine+promethazine) was applied in vivo and physical hypothermia (32°C) were applied in vitro . Infarct volume (TTC), apoptosis/pyroptosis (TUNEL, flow cytometry), mitochondrial function (ATP, respiratory complex I/II, ROS)(ELISA), RBM3 expression, inflammasome (NLRP3, TXNIP, GSDMD-N), and ER stress (p-PERK, p-eIF2α, CHOP) were assessed using qPCR and Western Blot (WB). TXNIP-CHOP interaction was verified by co-immunoprecipitation (co-IP) and immunofluorescence. RBM3 was knockdown by AAV in vivo . In vitro, CREB1/GATA3 regulation of RBM3 transcription was examined via siRNA silencing, and by WB, qPCR, Co-IP, and ChIP. Mitochondrial function was monitored (JC-1, MitoSOX, mPTP). Oxidative damage was modeled by hydrogen peroxide exposure. Results: Both cooling regimens activated CREB1/GATA3 and increased RBM3 transcription. Only pre-reperfusion hypothermia preserved mitochondrial function and suppressed the reperfusion ROS bursts ( in vivo and in vitro ), thereby sustaining RBM3 protein levels, as evidenced by direct ROS suppression in hydrogen peroxide-treated cells. Animal experiments proved that elevated RBM3 protein attenuated ER stress and disrupted TXNIP-CHOP binding, inhibited NLRP3 inflammasome activation, and thus reduced pyroptosis and apoptosis. During-reperfusion hypothermia failed to protect mitochondria, resulting in ROS accumulation, RBM3 loss, and thus infarction. Conclusion: Pre-reperfusion hypothermia uniquely enables RBM3 accumulation by combining CREB1/GATA3-driven transcription with mitochondrial preservation to avert ROS-mediated suppression. RBM3 acts as a pivotal determinant of neuroprotection, providing strategic targets for stroke therapy.