Single Cell RNA Sequencing after Moderate Traumatic Brain Injury: Effects of Therapeutic Hypothermia
Traumatic brain injury (TBI) initiates a cascade of cellular and molecular events that promote acute and long-term patterns of neuronal, glial, vascular, and synaptic vulnerability leading to lasting neurological deficits.  These complex responses lead to patterns of programmed cell death, diffuse axonal injury, increased blood-brain barrier disruption, neuroinflammation, and reactive gliosis, each a potential target for therapeutic interventions. Posttraumatic therapeutic hypothermia (TH) has been reported to be highly protective after brain and spinal cord injury and studies have investigated molecular mechanisms underlying mild hypothermic protection while commonly assessing heterogenous cell populations. In this study we conducted single-cell RNA sequencing (scRNAseq) on cerebral cortical tissues after experimental TBI followed by a period of normothermia or hypothermia to clarify multiple cell type-specific transcriptional responses. C57BL/6 mice underwent moderate controlled cortical impact (CCI) injury or sham surgery and then placed under sustained normothermia (37°C) or hypothermia (33°C) for 2 hrs. After 24 hours, cortical tissues including peri-contused regions were processed for scRNAseq. Unbiased clustering and differential expression tests revealed cellular heterogeneity, reactive astrocytes, microglia, and leukocyte infiltration at this subacute posttraumatic time point. The analysis also revealed vascular and immune subtypes associated with neovascularization and debris clearance, respectively. Compared to normothermic conditions, TH treatment altered the abundance of specific cell subtypes and induced reactive astrocyte-specific modulation of neurotropic factor gene expression. In addition, an increase in the proportion of endothelial tip cells in the hypothermic TBI group was documented compared to normothermia. These data emphasize the importance of early temperature-sensitive glial and vascular cell processes in producing potentially neuroprotective downstream signaling cascades in a cell-type-dependent manner. The use of single cell sequencing to address cell-specific mechanisms underlying therapeutic treatments provides a valuable resource for identifying targetable biological pathways for the development of neuroprotective and reparative interventions. Overall design: Twelve male C57BL/6 mice (2–3 months old) were randomly assigned to four groups (n=3 per group): 1. TBI–Normothermia 2. Sham–Normothermia 3. TBI–Hypothermia  4. Sham–Hypothermia A moderate controlled cortical impact (CCI) traumatic brain injury (TBI) was administered, followed by a 2-hour selective brain hypothermia (33°C) or normothermia (37°C) protocol starting 30 minutes post-injury. Mice were sacrificed at 24 hours post-injury or sham surgery, and the ipsilateral cortex was dissected for single-cell RNA-sequencing. One sample corresponds to one individual animal, for a total of 12 samples.