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

Abstract

Diffuse traumatic brain injury (TBI) leads to complex pathophysiological processes that result in clinical symptoms. Neuroinflammation and associated microglia activation are hallmark pathophysiological processes of diffuse TBI and contribute significantly to both damage and ensuing repair. Microglia are the resident innate immune cells in the brain and microglia function is linked to cellular morphology. Inflammatory signaling after diffuse TBI initiates microglia activation where microglia go from a ramified morphology (small soma with long, highly branched processes) to an activated morphology (swollen soma with enlarged, retracted processes). We also report an abundance of rod microglia (elongated soma with polarized processes) in the cortex after diffuse TBI. Rod microglia were first described in the early 1900’s and have since been sporadically reported in neurological conditions such as chemical exposure, viral infection, Alzheimer’s disease, Lewy Body dementia, ischemia, and TBI. Despite their occurrence across injury and disease, very little is known about rod microglia. Investigations have been limited to post-mortem histology using general microglia markers. The objective of this dissertation was to identify markers and develop tools to differentiate rod microglia from other microglia morphologies and confirm rod microglia mechanisms after TBI with the central hypothesis that rod microglia have a unique molecular profile compared to other microglia morphologies. First, we applied phage display biopanning to isolate antigen-binding domains specific to rod microglia compared to other microglia morphologies. We modified a novel discovery pipeline to develop antibody-mimetics from antigen-binding domains that can be validated for rod microglia specificity and used for downstream applications such as immunohistochemistry. Next, we investigated gene expression of rod microglia pathology with single nucleus RNA sequencing to provide insight into rod microglia function. We identified three distinct subclusters of microglia within the somatosensory cortex and inferred that those with inflammatory and neurological disease pathways may represent rod microglia. Finally, we used diffusion magnetic resonance imaging (MRI) to refine a non-invasive imaging signature of rod microglia. Water restriction was visible by diffusion MRI in the somatosensory cortex and corresponding histology reported activated microglia in regions where water restriction was observed. While not a marker of rod microglia specifically, novel post-image processing was sensitive to detect changes in microglia. This dissertation is the first step in developing and validating tools to target rod microglia. Until rod microglia mechanisms can be investigated with new tools, their role in the evolution, progression, and resolution of diffuse TBI pathophysiology remains unknown.