Figure 1.
Mechanical stress induces reversible axonal beading process in vitro and in vivo. (A) Schematic representation of the axonal resistance hypothesis (middle), the mechanical stress brain (left), and the AoC device (right). The axonal resistance hypothesis suggests that mild mechanical impacts to the head can activate intrinsic resistance mechanisms in axons, promoting recovery. The AoC device applies transverse stress to axons by injecting culture medium at varying flow rates, mimicking different levels of head impact stress. (B) Modified AoC device for stress on the somatodendritic region. In the microfluidic device with three chambers attached to a glass-bottom dish, neurons were seeded into the central injury chamber (blue), instead of the two reservoirs (red). Bar = 0.5 cm. (C) Representative images of neurons within the central injury chamber, with axons (blue) and dendrites (red) marked with brackets. The right panels provide magnified views of the bracketed ROIs, revealing beading (top) and the intensity of Ca2+ sensor GCaMP-6f (bottom) dynamics before (0 s), during (35.6 s), and after (783 s) the 180-s flux. The arrowheads highlight the beading regions, which are further magnified in the lower-right insets. Bar = 20 μm. (D) Paired analysis of beading density on axons (N = 44) and dendrites (N = 45). (E) Quantification of Ca2+ fluctuation in beads of stressed axon (blue) and dendrites (red). The flux span is indicated with shades. (F and G)Ir quantification for (F) beading process (N = 39, 44) and (G) Ca2+ intensity (N = 53, 59). (H) Photograph of the immobilized Thy1-YFP transgenic mouse head during mild mechanical stress, with imaging window (blue) and impacted site (red) marked on the skull. Bar = 5 mm. (I) Representative two-photon images showing axon beading before and 5 or 150 min after impact. Boxed ROIs are amplified in bottom panels. Bar = 20 μm (top) and 5 μm (bottom). (J) Paired analysis of beading density in different axon-like processes before and 5 or 150 min after impact (N = 34, 45, 40 axons from four mice). (K) Quantification of the Ir for axon-like processes (N = 30). Data represent mean ± SEM; in D and J, paired two-tailed Student’s t test; in F and G, Mann-Whitney test; ***P < 0.001.

Mechanical stress induces reversible axonal beading process in vitro and in vivo. (A) Schematic representation of the axonal resistance hypothesis (middle), the mechanical stress brain (left), and the AoC device (right). The axonal resistance hypothesis suggests that mild mechanical impacts to the head can activate intrinsic resistance mechanisms in axons, promoting recovery. The AoC device applies transverse stress to axons by injecting culture medium at varying flow rates, mimicking different levels of head impact stress. (B) Modified AoC device for stress on the somatodendritic region. In the microfluidic device with three chambers attached to a glass-bottom dish, neurons were seeded into the central injury chamber (blue), instead of the two reservoirs (red). Bar = 0.5 cm. (C) Representative images of neurons within the central injury chamber, with axons (blue) and dendrites (red) marked with brackets. The right panels provide magnified views of the bracketed ROIs, revealing beading (top) and the intensity of Ca2+ sensor GCaMP-6f (bottom) dynamics before (0 s), during (35.6 s), and after (783 s) the 180-s flux. The arrowheads highlight the beading regions, which are further magnified in the lower-right insets. Bar = 20 μm. (D) Paired analysis of beading density on axons (N = 44) and dendrites (N = 45). (E) Quantification of Ca2+ fluctuation in beads of stressed axon (blue) and dendrites (red). The flux span is indicated with shades. (F and G)Ir quantification for (F) beading process (N = 39, 44) and (G) Ca2+ intensity (N = 53, 59). (H) Photograph of the immobilized Thy1-YFP transgenic mouse head during mild mechanical stress, with imaging window (blue) and impacted site (red) marked on the skull. Bar = 5 mm. (I) Representative two-photon images showing axon beading before and 5 or 150 min after impact. Boxed ROIs are amplified in bottom panels. Bar = 20 μm (top) and 5 μm (bottom). (J) Paired analysis of beading density in different axon-like processes before and 5 or 150 min after impact (N = 34, 45, 40 axons from four mice). (K) Quantification of the Ir for axon-like processes (N = 30). Data represent mean ± SEM; in D and J, paired two-tailed Student’s t test; in F and G, Mann-Whitney test; ***P < 0.001.

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