Zigmond, J. W.; Lamberty, B. G.; Simon, G. J.; Wood, J. M.; Elliott, N. M.; Miller, D. W.; Stauch, K. L.

Despite traumatic brain injury (TBI) being a leading cause of death and disability in the United States, there is currently no FDA-approved medication that can prevent, heal, or reverse brain damage caused by TBI. TBI is caused by primary and secondary brain damage resulting in loss of neurons, astrocytic gliosis, and microglial activation which culminate in complex neurological disorders. The goal of the current study was to investigate the neuroprotective potential for Creatine Hydrochloride (C-HCl) supplementation following TBI in wild-type mice. Four C-HCl supplementation paradigms were examined including both 7 day pre-injury and 14-day post-injury treatments consisting of the following: 1) water/water, 2) water/C-HCl (100 mg/kg), 3) C-HCl (100 mg/kg)/C-HCl (100 mg/kg), 4) C-HCl (100 mg/kg)/C-HCl (450 mg/kg). Following controlled cortical impact (CCI) injury at a parietal location, measures of tissue and cellular (neuronal and glial) damage were assessed at 14 days post-injury (dpi). At 14 dpi, there were no significant effects on gross anatomical cavitation, tissue loss, third ventricle expansion, or systemic fluid biomarkers with any treatment group, though female mice exhibited larger baseline lesion boundaries overall compared to male mice. However, microstructural evaluation revealed a profound and sex-dependent effect of C-HCl supplementation in the hippocampus with female mice showing reduced neuroinflammation and male mice displaying reduced neurodegeneration (Fluoro-Jade C staining of neurons) across the CA1 and dentate gyrus (DG) compared to placebo control. Furthermore, mice receiving C-HCl pre-injury followed by high-dose C-HCl post-injury demonstrated distinct remodeling of inhibitory circuits to restore network balance with males significantly modifying presynaptic machinery via vesicular GABA transport (VGAT puncta) in the DG, whereas females reinforced postsynaptic architectural framing through scaffolding elements (gephyrin puncta) in both the CA1 and DG. These results suggest C-HCl supplementation holds promise as a neuroprotective therapeutic strategy for TBI. Crucially, the molecular mechanisms utilized to achieve this brain cell homeostasis are radically distinct between the sexes, females benefit primarily via upstream glial stabilization, while males derive protection via direct downstream neuronal survival. These outcomes highlight a critical requirement for sex-stratified profiling in translational pipelines for neurometabolic interventions for TBI. Ultimately, these data indicate that C-HCl successfully counteracts chronic secondary injury cascades, mitigating neurodegeneration, dampening neuroinflammation, and remodeling inhibitory circuitry to prevent post-traumatic network hyperexcitability.