PROJECT SUMMARY Patients aged 65 and older account for an increasing portion of traumatic brain injury (TBI) patients. TBI contributes to over one third of trauma related deaths and aged TBI patients suffer increased morbidity and mortality compared to young TBI patients. The impact of TBI is also highlighted through its significant long-term cognitive and psychiatric complications suffered by survivors. After TBI, microglia adopt a constitutively active state leading to an increased inflammatory response. Our previous studies show that TBI results in an influx of monocytes, monocyte derived leukocytes, and CD8+ T-cells into the brain. Additionally, we demonstrated that infiltrating inflammatory immune cells mediate injury and neurocognitive outcomes after TBI, and reduced infiltration attenuates poor neurologic outcomes and improves behavioral outcomes. But the interplay between microglia, immune cells, and the gut is still poorly understood. To assess recruitment of monocytes and T-cells by activated microglia, we will deplete microglia in young and aged mice and observe immune cell infiltration after TBI. Microglial depletion will occur through a colony-stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622 formulated into rodent chow. After microglia depletion, mice will undergo TBI or sham injury. Activation, proliferation, and differentiation of monocyte and T-cell subtypes will be assessed through flow cytometry to determine whether microglial depletion attenuates immune cell accumulation. To investigate the impact of the gut microbial community on immune infiltration and microglial activation we will utilize a murine FMT model. We will prepare fecal slurry from young and aged mice to administer to aged mice either before or after TBI with PBS as a vehicle control. Mice will undergo neurocognitive testing to assess phenotypic alterations due to FMT. We will assess neuroinflammation in these mice by flow cytometry, microglial gene expression through scRNAseq, and clonal expansion of T-cells through scTCRseq. Overall, this project will assess the interplay of microglia, infiltrating immune cells, and the gut in the aged brain after TBI, potentially determining routes for therapeutic development. This project will expand my capabilities through advanced training in neuroimmunology, RNA sequencing, and neurocognitive testing while utilizing my expertise in immunology and advanced flow cytometry. I will present my findings in numerous professional settings for peer review including national conferences and I will publish my findings in peer-reviewed journals in preparation for a future MOSAIC K99/R00 award.