Brain mapping and spatial protein profiling reveal functional connectivity deficits and molecular changes following repetitive mild traumatic brain injury in wild-type mice.

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 rmTBI 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 sought to define the extent of altered brain functional connectivity (FC) and expression of neuropathological markers after rmTBI. We performed two rmTBI (2x 0.6{square}J impacts 24{square}h apart) in male and female C57BL/6J wild-type (WT) (~2.5-3mo) mice using closed head injury model of engineered rotational acceleration (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 to determine the extent of gliosis. The expression of disease-associated protein markers in grey and white matter regions were evaluated using the NanoString-GeoMx digital spatial protein profiling (DSP) platform. 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. In addition, DTI revealed significantly decreased fractional anisotropy (FA) and axial diffusivity in the optic tract. 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 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.