README File for Title: "Application of mild hypothermia successfully mitigates neural injury in a 3D in-vitro model of traumatic brain injury" Authors: Mark T. Scimone Ph.D(1,2), Harry C. Cramer III(1,2), Paul Hopkins(1,2), Jonathan B. Estrada Ph.D(3), and Christian Franck Ph.D(4) Affiliations: 1. Center for Biomedical Engineering, Brown University 2. School of Engineering, Brown University 3. Mechanical Engineering, University of Michigan - Ann Arbor 4. Mechanical Engineering, University of Wisconsin - Madison Abstract: 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. Funding: Office of Naval Research, Grant Numbers: N00014-132-1-0708, N00014-16-1-2869, and N00014-17-1-2644. Permanent Identifier Address: http://digital.library.wisc.edu/1793/79929 DOI: 10.21231/m4kj-ns45 File Descriptions: Note: Please see the associated publication for imaging parameters and methods. Filename: LiveDeadImpact_Tiffs.zip Size: 7.900 Gb Description: Contains all data required to replicated findings pertaining to figures 2 and 3. Each channel is separated, such that each raw data file is split into 2 channels (Calcein AM - Live, and Ethidium Homodimer-1 - Dead). Filenames describe the experimental group and time points. Filename: Delayed_hypothermia_Tiffs.zip Size: 2.475 Gb Description: Contains all data required to replicated findings pertaining to figure 4a. Each channel is separated, such that each raw data file is split into 2 channels (Calcein AM - Live, and Ethidium Homodimer-1 - Dead). Filenames describe the experimental group and time points. Filename: Abbreviated_hypothermia_Tiffs.zip Size: 4.231 Gb Description: Contains all data required to replicated findings pertaining to figure 4b. Each channel is separated, such that each raw data file is split into 2 channels (Calcein AM - Live, and Ethidium Homodimer-1 - Dead). Filenames describe the experimental group and time points. Filename: Caspase_Tiffs.zip Size: 397.6 Mb Description: Contains all data required to replicated findings pertaining to figure 5. Each channel is separated, such that each raw data file is split into 3 channels (Hoechst, Caspase CellEvent, and Ethidium Homodimer-1). Filenames describe the experimental group and time points. Filename: Calpastatin_Tiffs.zip Size: 895.5 Mb Description: Contains all data required to replicated findings pertaining to the results shown in figure 6. Each channel is separated, such that each raw data file is split into 3 channels (Dapi, Calpastatin, brightfield). Filenames describe the experimental group and time points. Filename: CellROX_Tiffs.zip Size: 954.2 Mb Description: Contains all data required to replicated findings pertaining to figure S2. Each channel is separated, such that each raw data file is split into 3 channels (Hoechst, ROS CellROX, and Ethidium Homodimer-1). Filenames describe the experimental group and time points.