FIG. 9.

Thy1-YFP-16 mice show axonal pathology in the cortex and corpus callosum after r-mTBI. (A and B) Thy1-YFP (green) shows labeling of neurons throughout the cortex along with axons projecting through the corpus callosum in mice perfused 3 days after r-sham (A) or r-mTBI (B) procedure. Nissl labeling (red) of the cytoplasmic rough endoplasmic reticulum along with DAPI nuclei counterstaining (blue) shows the overall cortical cytoarchitecture, which is not markedly disrupted after r-mTBI. Arrows (B) point to examples of swollen segments of damaged axons. (C and D) At higher magnification, Thy1-YFP (green) labeled axons in the corpus callosum exhibit a generally uniform longitudinal profile in r-sham mice (C) in contrast to the numerous swollen axonal segments observed in r-mTBI mice (D). (E and F) High-magnification Thy1-YFP (green) cortical regions analyzed in r-sham mice (E) and r-mTBI mice (F). Large swellings and thickened Thy1-YFP axonal profiles are evident in the cortex of r-mTBI mice (F, arrows) compared to the thin processes of Thy1-YFP axons in r-sham mice (E, arrow). Lack of DAPI nuclear labeling (blue) shows that enlarged Thy1-YFP regions (F) are not cell bodies. (G) Quantification of damaged axonal profiles (swollen, axonal bulbs, and varicosities) labeled with Thy1-YFP in the cingulate and M2 motor cortices that comprise the region of interest in the medial cortex for DTI/DKI analyses. A significant increase of damaged axons was observed in r-mTBI mice compared to r-sham. Values are mean ± standard error of the mean; n = 3; three to nine sections per animal. **p < 0.01; ****p < 0.0001. Scale bars = 200 μm (A and B) and 20 μm (C–F). Avg, average; d, days; DAPI, 4′,6-diamidino-2-phenylindole; DKI, diffusion kurtosis imaging; DTI, diffusion tensor imaging; r-mTBI, repetitive mild traumatic brain injury; r-sham, repetitive sham.