Abstract Text: Despite increased survival rates, traumatic brain injuries (TBI) are still a leading cause of death and disability worldwide. Varying injury mechanisms, patient demographics, and the development of secondary injuries are all factors which contribute to the heterogeneous nature of TBI. Current diagnostics include CT, MRI, and the somewhat subjective Glasgow Coma Scale (GCS). Yet new tools are sought to add precision, inform on underlying pathobiology, and better predict patient outcomes. Recently the brain injury peptidome has been recognized as an underutilized source of molecular biomarkers. Large macromolecule protein markers often investigated are more size restricted than smaller peptide fragments, with the latter’s size permitting ready diffusion and brain efflux through normal waste clearance. TBI pathobiology involves dynamic activation of proteases within cells and the extracellular space, releasing peptides to the periphery as indicators of those remodeling processes in the brain. In our previous work, we interrogated the urinary TBI peptidome during inpatient rehabilitation to identify relevant biomarker peptides that track with greater patient functional improvement. Biomarker models were built against standard functional independence measure (FIM) and disability rating scale (DRS) assessments, with correlation assessed across time between facility admission and discharge. Two key peptides were identified as fragments of hnRNPM and CSF1R, warranting the follow-up studies to investigate their relevance to TBI pathobiology. In the present study, a controlled cortical impact (CCI) injury model was used to investigate the cellular localization, changes of these proteins over time, and how this relates to the observed changes in the urine of human subjects. Sprague Dawley rats were injured to model a moderate injury severity with brains collected prior to and at 1-, 4-, 7- and 14-days post injury. Brains were snap frozen and sectioned (20 µm) through the site of injury. Sections were probed with antibodies to hnRNPM, CSF1R, IBA1, and GFAP and imaged using immunofluorescence microscopy. During TBI rehabilitation, hnRNPM and CSF1R levels demonstrated an inverse correlation with greater functional recovery. We thus hypothesized that in modeled TBI, both proteins would track downward later in recovery, between 1 and 2 weeks in rats. Staining for hnRNPM and CSF1R peaked between 4- and 7- days post TBI, with a reversal to lower levels for hnRNPM by 14-days in surviving tissues below the contusion. Conversely, after a sharp decrease at 7-days, CSF1R increased and remained elevated at 14-days post injury. Within the lesion itself, both hnRNPM and CSF1R levels first decline acutely after TBI, with a rebound by 4-days post and then a rapid decline as the tissue died off. Differentiation between surviving and dying tissue was further evidenced through the post-TBI cellular localization of hnRNPM. In surviving tissues, the acute increase in hnRNPM associated with a proliferation of astrocytes, observed to proliferate around the lesion and migrate to form a glial scar by 14-days. However, within the lesion, there was no such glial localization, as hnRNPM expression declined with dying cells. CSF1R was primarily associated with IBA1+ microglia rather than astrocytes. hnRNPM and CSF1R peptide formation is predicted through cathepsin (n-term) and calpain (c-term) cleavage, both known to increase in activity acutely following TBI. We surmise thus that these biomarkers reflect on the loss in hnRNPM and CSF1R coincident to a loss in lesioned / dying tissue and formation of a glial scar as found here after modeled TBI. Thus, logically, a greater loss of tissue would track with worse initial functional performance, allowing for greater recovery following rehabilitation. Ultimately, our findings support the relevance of biomarker peptides that track patient functional improvement during inpatient rehabilitation per their association with recovery from contusional burden after TBI. Further research is expected to identify additional connections to the TBI peptidome with post injury neurobiology, supporting further development of these biomarkers for injury assessment as well as therapeutic intervention and recovery monitoring.