Figure 4. Microtubule dynamics after acute and chronic injury.

(a,b) Acute axotomy by two-photon laser (axotomy site outside of the frame; arrow in a) induces a rapid increase in comet density at the proximal axon-end (asterisk; Supplementary Movie 6). Comet density in the lower axon is unchanged, because the cut is>60 μm outside of the frame. Quantification (b) shows that this effect is confined to within less than 60 μm from the axon-end and comet orientation is largely unaltered (horizontal line shows mean±s.e.m.; polar plot of the 0–10 μm segment shown as insert; the dashed green line indicates control values; n≥7 neurites from ≥5 mice for each group; polar plot from four axons from four mice). (c–e) In contrast to the local increase in microtubule dynamics seen acutely after axotomy, global changes in comet density are found within 12 h after nerve cut (schematic of experiment and measurement sites in c). The ‘neighbour’ is an uncut adjacent nerve that can sprout into the denervated territory (green sprout symbol on the right in c). Proximal of the cut (~1 mm away) comet density steadily increases, while distally an initial spike in dynamics is followed by a decline before fragmentation (d,e, left). (e) Right shows that axons in the neighbouring nerve that are not cut, but receive a growth stimulus (‘Nbr’), also show increased microtubule dynamics after 48 h (n≥10 neurites from ≥3 mice for each condition; bar showing ‘cut, 48 h’ is a re-plot from time course on left; horizontal line and data symbols show mean±s.e.m., respectively). (f,g) Changes in microtubule dynamics in the distal axotomized stump are abolished by overexpressing a fusion protein that blocks Wallerian degeneration (‘ΔNLS’), while the proximal changes are delayed but still present (n≥11 neurites from ≥2 mice for each group; data symbols indicate mean±s.e.m.). In panels e,g some error bars are hidden by the data symbol. Scale bar in all panels 5 μm. ***P<0.001, analysis of variance.