Activations of actomyosin-II protect mechanically stressed axons from AAD. (A) Procedure timeline of the closed-skull mTBI mouse model. (B) Schematic illustrating the relative locations of stereotaxic cortical injection of AAV and impact sites in the brain of the mTBI mouse model. (C) Brain map showing AAV-hSyn-GFP injection and impact locations. (D) A representative tiled image shows axon tracts in the corpus callosum and impacted sites. Bar = 2 mm. (E) Confocal images of the corpus callosum and impacted sites, with boxed ROIs magnified in lower panels to reveal individual axon morphology. Bar = 100 μm (top) and 20 μm (bottom). (F) Z-stack raw images of axon tracts with 3D Imaris renderings of selected axonal segments shown on the right. White boxed outlines are magnified below. The volume of the axonal segments is color-coded. Bar = 100 μm (top) and 20 μm (bottom). (G) Quantification of the proportion of fragmented axon (sphericity >0.8; length <10 μm) in F (N = 18, 26, 24, 25). (H) Representative images show the axons of neurons expressing either MRLC-WT, MRLC-SA, MRLC-SD or empty vector (GFP) in commissural axons crossing the corpus callosum (top) or projecting to the impacted site (bottom). Axons in sparse (1#) or dense (2#) labeled regions are shown in lower panels. The volume of axonal fragments is color-coded. Bar = 20 μm. (I) Percentage of fragmented axons (sphericity >0.8; length <10 μm) for the corpus callosum (top) and impacted site (bottom) in H (top: N = 73, 34, 37, 31; bottom: N = 70, 35, 40, 33). Data represent mean ± SEM; unpaired two-tailed Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001.