Figure 2.
Radial contraction and dilation jointly drive the rapid beading in stressed axons. (A) Structure of an AoC with rat hippocampal neurons seeded in opposing soma chambers (red) and axons extending to the central injury chamber (blue). Bar = 0.5 cm. (B) On DIV7–8, axons expressing Lifeact-RFP underwent mechanical stress with culture medium flux into the central injury chamber. Red circles denote growth cones, and the white box marks the live-imaging window, magnified on the right, showing axon shafts are the predominantly stressed axonal region. Bar = 200 µm (left), 100 µm (right). (C) Schematic timeline of the flux-induced axon stress assay with a 180-s flux and ∼10-min recovery for live imaging. Fixation for staining occurred at 2 h (for SCG10 or Cleaved Caspase 3 staining) or 24 h (for in vitro fragmentation analysis) after flux. (D) Representative fields showing the axons of the central injury chamber before, during, and after flux with automatically detected beading regions (magenta). The red arrow indicates the flux direction. Bar = 20 µm (top) and 10 µm (bottom). (E) Quantification of the bead number in response to different flow-rated flux. (F) Quantification of the Ir (N = 12, 70, 22, 46). (G) Mild mechanical stress (20 µl/min flux, 180 s) induced beading in axons. Flux onset was set as 0 s. Beading regions are magenta, and non-beading between segments are cyan. Bar = 10 µm (left) and 5 µm (right). (H) Curves showing instant area changes in beading (red), between (blue), and total (black) regions during flux of G. (I) Quantification of the total axon area changes during the flux of G (N = 30). (J) The z-stack raw images show morphological changes in the same axon before and after a 50 µl/min flux, with 3D Imaris renderings at the bottom. Axon diameter is color-coded. Bar = 30 μm (top) and 2 μm (bottom). (K–M) Paired analysis of the Imaris-rendered 3D surfaces within the same axon, with quantifications of (K) the median axon diameter, (L) the distribution of axon diameter, and (M) the axon volume (N = 10). (N) Quantification of volume change ratio contributed by beading (red), between (blue), and total (black) regions of stressed axons (N = 10). (O) Model for axon beading formation. Left: axonal regions between beads contract to form the string (blue). Right: beaded regions dilate to form beads (red). Rapid axon beading formation is a mixture of the two models. Data represent mean ± SEM; in F and I, unpaired two-tailed Student’s t test; in K and L, paired two-tailed Student’s t test; in M, Wilcoxon matched-pairs signed rank test; *P < 0.05, **P < 0.01, ***P < 0.001; n.s., non-significant.

Radial contraction and dilation jointly drive the rapid beading in stressed axons. (A) Structure of an AoC with rat hippocampal neurons seeded in opposing soma chambers (red) and axons extending to the central injury chamber (blue). Bar = 0.5 cm. (B) On DIV7–8, axons expressing Lifeact-RFP underwent mechanical stress with culture medium flux into the central injury chamber. Red circles denote growth cones, and the white box marks the live-imaging window, magnified on the right, showing axon shafts are the predominantly stressed axonal region. Bar = 200 µm (left), 100 µm (right). (C) Schematic timeline of the flux-induced axon stress assay with a 180-s flux and ∼10-min recovery for live imaging. Fixation for staining occurred at 2 h (for SCG10 or Cleaved Caspase 3 staining) or 24 h (for in vitro fragmentation analysis) after flux. (D) Representative fields showing the axons of the central injury chamber before, during, and after flux with automatically detected beading regions (magenta). The red arrow indicates the flux direction. Bar = 20 µm (top) and 10 µm (bottom). (E) Quantification of the bead number in response to different flow-rated flux. (F) Quantification of the Ir (N = 12, 70, 22, 46). (G) Mild mechanical stress (20 µl/min flux, 180 s) induced beading in axons. Flux onset was set as 0 s. Beading regions are magenta, and non-beading between segments are cyan. Bar = 10 µm (left) and 5 µm (right). (H) Curves showing instant area changes in beading (red), between (blue), and total (black) regions during flux of G. (I) Quantification of the total axon area changes during the flux of G (N = 30). (J) The z-stack raw images show morphological changes in the same axon before and after a 50 µl/min flux, with 3D Imaris renderings at the bottom. Axon diameter is color-coded. Bar = 30 μm (top) and 2 μm (bottom). (K–M) Paired analysis of the Imaris-rendered 3D surfaces within the same axon, with quantifications of (K) the median axon diameter, (L) the distribution of axon diameter, and (M) the axon volume (N = 10). (N) Quantification of volume change ratio contributed by beading (red), between (blue), and total (black) regions of stressed axons (N = 10). (O) Model for axon beading formation. Left: axonal regions between beads contract to form the string (blue). Right: beaded regions dilate to form beads (red). Rapid axon beading formation is a mixture of the two models. Data represent mean ± SEM; in F and I, unpaired two-tailed Student’s t test; in K and L, paired two-tailed Student’s t test; in M, Wilcoxon matched-pairs signed rank test; *P < 0.05, **P < 0.01, ***P < 0.001; n.s., non-significant.

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