Therapeutic hypothermia (TH) is an attractive target for mild traumatic brain injury (mTBI) treatment, yet significant gaps in our mechanistic understanding of TH, especially at the cellular level, remain and need to be addressed for significant forward progress to be made. Using a recently-established 3D in-vitro neural hydrogel model for mTBI we investigated the efficacy of TH after compressive impact injury and established critical treatment parameters including target cooling temperature, and time windows for application and maintenance of TH. Across four temperatures evaluated (31.5, 33, 35, and 37°C), 33°C was found to be most neuroprotective after 24 and 48 hours post-injury. Assessment of TH administration onset time and duration showed that TH should be administered within 4 hours post-injury and be maintained for at least 6 hours for achieving maximum viability. Cellular imaging showed TH reduced the percentage of cells positive for caspases 3/7 and increased the expression of calpastatin, an endogenous neuroprotectant. These findings provide significant new insight into the biological parameter space that renders TH effective in mitigating the deleterious effects of cellular mTBI and provides a quantitative foundation for the future development of animal and preclinical treatment protocols.
