Traumatic Brain Injury (TBI) is caused by a mechanical insult to the head and often results in prolonged or permanent brain dysfunction. TBI represents a major health and socioeconomic problem; in the U.S. over 2.8 million are affected annually. Even so-called “mild” TBI – otherwise known as concussion – may induce neurophysiological deficits affecting learning, memory, and concentration that do not resolve in up to 15-20% of patients. There are currently no approved treatments to improve recovery from TBI. Through previous extensive neuropathological examinations, we have shown that mild TBI is associated with subtle changes in synaptic loss or gain and neuronal hypertrophy occurring concomitantly with migration and activation of microglia – the resident immune cell of the central nervous system. We have also detailed persistent changes to microglia morphology in distinct white matter and gray matter regions after a single mild TBI. The objective of this proposal is to establish the function of microglia and the overall neuroinflammatory response in secondary pathophysiology for a range of TBI severities, with a particular focus on the role of hyper-ramified microglia on responses to repeat TBI. To accomplish these objectives, we will utilize our porcine model of closed-head rotational-acceleration-induced TBI, which closely replicates human head injury biomechanics that cannot be reproduced in small animals. In Aim 1, we will characterize the effect of mild or moderate injury severity on TBI pathology through transcriptomic profiling and immunohistochemical protein expression. In particular, RNA sequencing will be performed to examine neurodegenerative and neuroinflammatory pathways followed by immunohistochemistry to provide regional context to neuronal loss and immune cell activation, amongst other pathologies. In Aim 2, animals will perform a battery of neurobehavioral tasks to assess spatial memory and working memory. We will then create a statistical model to correlate the extent of behavioral and cognitive deficits with transcriptomic and neuropathological changes post-TBI. Finally, in Aim 3, we will test the effect of repeat TBI on neuroinflammatory states. We will subject pigs to a second TBI at either a timepoint shown to present maximal elevations in hyper-ramified microglia or a timepoint where the density of this microglial phenotype has returned to baseline. Changes in gene expression, neuropathology, and behavior will be assessed. Our hypothesis is that repeat TBI during a period with elevated levels of hyper-ramified microglia will result in excessive synaptic pruning/loss, exacerbated neuronal hypertrophy, and elevated deficits in hippocampal- dependent cognitive tasks. Across these aims, we will use established methodologies to generate comprehensive genomic, pathological, and cognitive profiling of clinically relevant TBI and create open access datasets that other TBI researchers can interrogate against rodent and human model ...