Repetitive mild traumatic brain injuries (rmTBIs) are serious trauma events responsible for the development of numerous neurodegenerative disorders. A major challenge in developing diagnostics and treatments for the consequences of rmTBI is the fundamental knowledge gaps of the molecular mechanisms responsible for neurodegeneration. It is both critical and urgent to understand the neuropathological and functional consequences of rmTBI to develop effective therapeutic strategies. Using the Closed-Head Impact Model of Engineered Rotational Acceleration, or CHIMERA, we measured neural changes following injury, including brain volume, diffusion tensor imaging, and resting-state functional magnetic resonance imaging coupled with graph theory and functional connectivity analyses. We determined the effect of rmTBI on markers of gliosis and used NanoString-GeoMx to add a digital-spatial protein profiling analysis of neurodegenerative disease-associated proteins in gray and white matter regions. Our analyses revealed aberrant connectivity changes in the thalamus, independent of microstructural damage or neuroinflammation. We also identified distinct changes in the levels of proteins linked to various neurodegenerative processes including total and phospho-tau species and cell proliferation markers. Together, our data show that rmTBI significantly alters brain functional connectivity and causes distinct protein changes in morphologically intact brain areas.

Background: Repetitive mild traumatic brain injury (rmTBI) is a leading and severe threat to cognition that often goes undiagnosed. A major challenge in developing diagnostics and treatments for the consequences of rmTBI is the fundamental knowledge gaps that explain how TBI promotes brain dysfunction. It is both critical and urgent to understand the neuropathological and functional consequences of rmTBI to develop effective therapeutic strategies. In this study, we defined the extent of altered brain functional connectivity (FC) and expression of neuropathological markers after rmTBI. Methods: We performed two rmTBI (2x 0.6 J impacts 24 h apart) in male and female C57BL/6J wild-type (WT) (~2.5-3mo) mice using CHIMERA or sham procedures. At 5-6 days post-injury (dpi), we measured changes in brain volume and FC using T2-weighted images, resting-state functional MRI (rsfMRI) and graph theory analyses. We used diffusion tensor imaging (DTI) to assess microstructural changes in white matter tracts. In addition, at 7dpi, we measured changes in Iba1 and GFAP in specific brain regions to determine the extent of gliosis. The expression of disease-associated protein markers in grey and white matter (WM) regions were evaluated using the NanoString-GeoMx digital spatial protein profiling spatial profiling (DSP) platform. Results: The rsfMRI data revealed aberrant changes in connectivity such as node clustering coefficient, global and local efficiency, participation coefficient, eigenvector centrality, and betweenness centrality in thalamus and other key brain regions that process visual, auditory, and somatosensory information. Using DTI, we found that fractional anisotropy (FA) and axial diffusivity in the optic tract was significantly decreased. Also, mean, radial, and axial diffusivity (L1) were significantly increased in the hippocampus. DSP revealed that phospho-serine 199 tau (pS199) as well as glial markers such as GFAP, cathepsin-D, and Iba1 were significantly increased in the optic tract. In thalamic nuclei, the neuroinflammatory marker GPNMB was increased significantly, and the cell proliferation marker Ki-67 was significantly decreased in the rmTBI group. Our data suggest that rmTBI significantly alters brain functional connectivity and causes a profound inflammatory response in gray matter regions, beyond chronic white matter damage.