Therapeutic hypothermia is an effective cytoprotectant and promising intervention shown to improve outcome in patients following cardiac arrest and neonatal hypoxia-ischemia. However, despite our clinical and experimental experiences, the protective molecular mechanisms of therapeutic hypothermia remain to be elucidated. Therefore, in this brief overview we discuss both the clinical evidence and molecular mechanisms of therapeutic hypothermia in order to provide further insights into this promising intervention.

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.

Therapeutic hypothermia during cardiac surgery has been widely used for neuroprotection and to attenuate the systemic inflammatory response due to cardiopulmonary bypass (CPB). Experimental data suggest that cold-shock protein RNA-binding motif 3 (RBM3), which is induced in response to hypothermia, plays a key role in hypothermia-induced organ protection. To date, investigation on RBM3 has been performed exclusively in vitro or in animal models, and the detection and regulation of RBM3 in human blood has not been investigated until now. The aim of this study was to investigate the level of RBM3 protein and cytokine expression profile involved in the inflammatory response in patients with congenital heart disease undergoing cardiac surgery involving CPB and therapeutic hypothermia. A single-center prospective trial with 23 patients undergoing cardiac surgery with CPB was performed. RBM3 protein was quantified in blood serum samples collected from patients and healthy individuals employing a new developed enzyme-linked immunosorbent assay. Cytokine levels were analyzed from dry blood spot samples using a Quanterix Simoa Immunoassay. For the first time, RBM3 protein was detected in blood samples of patients with congenital heart disease undergoing cardiac surgery. Hereby, RBM3 protein concentrations were significantly elevated in patients after cardiac surgery with CPB and mild hypothermia as compared with pre-surgery levels. Moreover, a complex immune reaction with significant induction of pro-inflammatory cytokines (interleukin [IL]-1 beta, IL-6, IL-8, IL-16, IL-18, monocyte chemotactic protein 1, CC-chemokine ligand [CCL]3, CCL4, intercellular adhesion molecule-1) in response to CPB was detected. Significantly elevated vascular endothelial growth factor and matrix metallopeptidase 3 concentrations reflecting ischemia/reperfusion-induced injury were observed 24 hours after weaning from CPB. The use of CPB is still associated with a complex inflammatory response. RBM3 protein is measurable in blood samples of patients with significantly higher concentrations after cardiac surgery with CPB and mild-to-moderate hypothermia. RBM3 is a new candidate as a biomarker for therapeutic hypothermia and a possible new therapeutic target for organ protection.