FIGURE 5.

E6020 accelerates macrophage activation and myelin debris clearance after lysolecithin-induced demyelination. (a, b) Demyeli-nated lesions were revealed by myelin stain with EC (blue) and axon immunolabeling with anti-NF at day 3 (a, b), day 7 (a’, b’), and day 14 (a”, b”) in animals co-injected with vehicle (a) or E6020 (b). Spinal cord schematic (a) outlines area of demyelination in lateral white matter (blue oval). Acutely after lysolecithin, both groups show demyelination (lack of EC stain) and slight edema. Over the course of 2 weeks, edema resolves and lesions shrink. E6020 did not alter lesion size at any time. (c, d) CD11b immunoreactivity at the same time points and from the same representative animals in a and b (area outlined with black box) demonstrates increased macrophage activation between 3 and 14 days postinjection in vehicle (c, c’, c”) and E6020 (d, d’, d”) groups. E6020 treatment induced greater macrophage reactivity at day 7 (d’) and day 14 (d”), while vehicletreated animals had significantly greater macrophage activation at day 14 (c”) compared to E6020. (e, f) Myelin debris phagocytosis measured with oil red O stain from the same representative animals in a and b (area outlined with black box) shows that E6020 accelerates myelin debris clearance. Oil red O is significantly greater at day 7 after E6020 treatment (f) compared to vehicle (e’) and is cleared from the lesion by day 14 (f”) at the time when staining is the highest with vehicle (e”). Quantification of (g) lesion size (EC/NF), (h) macrophage activation (CD11b), and (i) myelin debris clearance (oil red O stain) at 3, 7, 14, and 21 days post-lysolecithin plus vehicle (black bars) or E6020 (striped bars) co-injection. *p < .05, **p < .01, ***p < .001 versus vehicle. Scale bars = 100 µm (a, b), 50 µm (c–f). Data are mean ±SEM. [Color figure can be viewed at wileyonlinelibrary.com]