Precision Identification and Targeting of Rod Microglia in Diffuse Brain-Injured Cortex

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PROJECT SUMMARY: After experimental diffuse traumatic brain injury (TBI), we rediscovered the rod microglia – a morphological variant of activated microglia in the pathology or repair of the brain. Rod microglia are depicted in the drawings of Nissl, Cajal, and del Rio Hortega in cases of general paresis, but were largely ignored for around 100 years. Rod microglia have recently been visualized in human Alzheimer’s disease, Autism spectrum disorder and experimental models of TBI, bacterial infection, and seizure, but no function has been reported. We reproducibly observe rod microglia that align in trains across cortical layers adjacent to neuronal processes in foci of neuropathology after diffuse TBI and they form and recede in a time course consistent with neuropathology. Yet, advanced molecular tools are absent from the arsenal necessary to understand rod microglia structure and function. We hypothesize that rod microglia have a unique molecular profile compared to other microglia morphologies, which provide tools to confirm rod microglia mechanisms in diffuse TBI. To investigate the significance and impact of rod microglia in diffuse TBI, the current proposal will provide critical data to distinguish rod microglia from activated and ramified microglia by molecular profile using phage display biopanning and RNA sequencing. We will induce diffuse TBI by midline fluid percussion injury in adult male and female CX3Cr1-eGFP mice and identify rod microglia based on morphology. Laser capture microdissection will isolate rod microglia and non-rod microglia (activated and ramified) from cortical regions. Isolated cells in aim 1 will be used for phage display biopanning to develop and validate a cell surface marker unique to rod microglia. Isolated cells in aim 2 will be used for next-generation sequencing (RNAseq) to reveal upregulated gene expression in rod microglia compared to activated and ramified morphologies. Pathway analysis will be incorporated to identify unique expression domains associated with the function and origin of rod microglia. The sponsor and mentoring team provide collective experience and expertise to provide advanced training for the applicant to achieve the aims. Data from this proposal will define the molecular profile of rod microglia and result in developed tools for quantitative and isolation protocols of rod microglia. With refined molecular tools, rod microglia can be investigated across neurological conditions (e.g. Alzheimer’s disease, Autism spectrum disorder, seizure) and activated or inhibited to drive treatment of neurological symptoms.