Figure 5.

Inhibition of ROCK signaling facilitates axonal sorting in ILK mutant mice. ILK conditional control and mutant mice were injected i.p. with either PBS or 40 mg/kg/d fasudil over six consecutive days from P8 to P13. SNs were collected at P14. (A, a–d) Toluidine blue–stained 0.5-µm semithin sections of control and mutant animals injected with either fasudil or with PBS only. (a and b) There were no visible differences in myelination between fasudil- and PBS-injected control nerves. Bars, 10 µm. (B, a–d) Electron micrographs of mutant animals injected with either fasudil or with PBS only. PBS-injected mutant mice display large unsorted axonal bundles (a and c) as seen before, whereas bundles in fasudil-injected mutant mice are smaller (b and d; n = 3; P_0–5 = 0.012; P_6–10 = 0.0064; P_51–100 = 0.011; P_101–250 = 0.027). (e) Quantification of the unsorted bundle area reveals a higher frequency of small bundles in the fasudil-injected mice (6–25 µm²) and a higher frequency of large bundles in PBS-injected mice (51 to >250 µm²). Crosses indicate that no bundles within the size category were found in any of the animals analyzed. In the axonal bundle depicted in d, asterisks mark axons that have presumably been recently sorted and are larger than those remaining in the bundle. (f and g) There is a significant increase in the number of 1:1 relations in fasudil- versus PBS-injected mutant animals (f; n = 3, P = 0.0052); however, the total number of myelinated axons in the nerves analyzed does not differ between PBS- and fasudil-injected mice (g). Error bars indicate ± SEM. *, P < 0.05; **, P < 0.01. Boxes in a and b indicate regions that are shown at a higher magnification in c and d, respectively. Bars: (a and b) 5 µm; (c) 2 µm; (d) 1 µm.