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Background/purpose: Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide, with more than 6 million TBIs sustained annually in the United States alone. Human TBIs are heterogeneous; penetrating injuries result in focal necrosis and immune cell infiltration, while diffuse injuries cause more widespread pathology with minimal cell death. The absence of overt gross pathology causes diffuse TBI to seem milder; however, these patients experience myriad complications including brain swelling and seizures and increased risk for later development of depression and Alzheimer’s disease. Microglia, the innate immune cells of the brain, become acutely activated after TBI and release inflammatory cytokines, phagocytize debris, and take on altered morphologies. One such morphology is rod-shaped microglia, previously shown to align with neuronal structures. Rod microglia have been observed in human brains after chronic infection such as syphilis and typhus, in addition to animal models of stroke and Alzheimer’s disease. The role of rod microglia in TBI is currently unknown, thus we were interested in further characterization to determine if they contribute to neuropathology. Our objectives were (1) to determine if rod microglia align with blood vessels in the injured cortex, and (2) to identify whether their source of origin is from peripherally derived macrophages. Research method: A diffuse TBI was induced in mice using a midline fluid percussion injury. This models diffuse TBI in humans, evidenced by the absence of gross pathology, neuronal death, or long-term motor dysfunction. After 7d, brain tissue was fixed, sliced, and labeled for structural and activation markers including Ly6C, Iba1, and CD45. To assess peripheral trafficking, green fluorescent protein (GFP) chimeras were created. In these mice, all peripherally derived immune cells, including monocytes and macrophages, are GFP+ and readily identified by microscopy. Results: We show novel data indicating that Iba1+ rod-shaped myeloid cells form long trains perpendicular to the cortex in mice. Additionally, rod microglia aligned with Ly6C+ blood vessels, and these cells were thicker and in proximity to deramified highly activated microglia. Furthermore, both rod-shaped microglia and adjacent deramified microglia showed increased labeling of CD45, a marker associated with peripheral monocytes. Notably, thin rod microglia, in cortical areas with less microglial reactivity overall, did not associate with blood vessels. Using GFP chimeras, we found that TBI does result in trafficking of GFP+ monocytes to the brain. These peripheral myeloid cells differentiate into ramified macrophages and, after TBI, can take on a rod-shaped morphology; however, the vast majority of rod-shaped myeloid cells are resident microglia. Implications: We have confirmed that rod microglia are resident microglia, and not peripheral macrophages masquerading as microglia. Furthermore, our findings show that highly activated CD45+ rod-microglia align with blood vessels. In contrast, CD45- thin rod-microglia may align with neuronal structures. Our findings suggest that there may be two subsets of rod-microglia that may have different functions in the injured brain; the former may affect blood-brain barrier integrity while the latter may be involved in axonal repair and regeneration.