Moderate Hypothermia after Hypoxia Is Neuroprotective Possibly via Upregulation of Cold Shock Protein RBM3

Objective: Hypoxic brain injury during cardiac arrest or resuscitation is a significant cause of long-term neurodevelopmental disability. Mild to moderate therapeutic hypothermia is known to be an effective neuroprotectant, possibly improving the neurological outcome of these patients. Even though many clinical trials have demonstrated a beneficial effect of mild to moderate hypothermia after hypoxic injury, the underlying cellular mechanisms of hypothermia-induced neuroprotection remain unclear. Though global cellular protein synthesis is attenuated during hypothermia, a small group of RNA-binding proteins including the RNA-binding motif 3 (RBM3) is up-regulated in response to cooling. Therefore, the aim of this study is to establish a cell-based model to investigate the effects of hypoxia and hypothermia on neuronal cell survival, as well as to examine the kinetics of RBM3 expression.

Experimental procedure: Experiments were performed using a human SK-N-SH neuroblastoma cell line exposed to either severe (0.2% O₂) or mild hypoxia (8% O₂) for 24 hours, followed by maintenance at moderate hypothermia (33.5°C) or normothermia (37°C) for 24, 48, and 72 hours. Cell death was examined by quantification of LDH and NSE releases into the cell culture medium. Neuronal cell morphology was examined by immunofluorescence staining. The regulation of the cold shock protein RBM3 expression was assessed by RT-qPCR and western blot analysis.

Results: Exposure to severe hypoxia (0.2% O₂) for 24 hours significantly increased cell death in the SK-N-SH neurons, whereas mild hypoxia (8% O₂) had no significant impact on cell death. Treatment with moderate hypothermia for 48 or 72 hours rescued the neurons from severe hypoxia-induced cell death. Moreover, severe hypoxia resulted in observable cell swelling, which was attenuated by moderate hypothermia. Moderate hypothermia also significantly induced RBM3 mRNA and protein expressions.

Conclusion: Moderate hypothermia protects neurons from severe hypoxia-induced cell injury. Moreover, the expression of the cold-shock protein RBM3 is induced by hypothermia and is a promising possible mediator of hypothermia-induced neuroprotection.