Fig. 7: FDA-approved small molecules induce sustained and long-distance axon regeneration after optic nerve crush (ONC).

a Schematic diagram illustrating the glycopyrrolate (1 µg) and mexiletine (1 µg) treatment paradigm for ONC. Axon regeneration was analyzed by cholera toxin subunit B-555 (CTB-555) tracing. b Robust axon regeneration was observed in glycopyrrolate-treated and mexiletine-treated mice at 2 weeks after ONC. c Glycopyrrolate and mexiletine induced more than 17-fold and 6-fold increase in the number of CTB-labeled regenerating axons extending 1.0 mm from the site of injury. d Serial transverse cryosections (20µm-thick) of retinae were immunostained with anti-RBPMS antibodies for retinal ganglion cell (RGC) survival assay. RBPMS-positive RGCs were counted in every fifth section per retina (3–5 sections). RGC survival rate of small-molecule treatment groups nearly doubled after injury, when compared with vehicle control group. e Sustained long-distance axon regeneration was observed in glycopyrrolate-treated mice at 4 weeks after ONC, and some CTB-labeled axons regenerated along the whole length of the optic nerve to reach the optic chiasm in glycopyrrolate-treated mice [yellow insets in (e)]. f Arrowheads indicated CTB-labeled regenerating axons projecting into the optic chiasm in glycopyrrolate-treated mice at 4 weeks after ONC. g At 2 mm distal to the lesion site, glycopyrrolate treatment resulted in more than 70-fold increase in the number of CTB-labeled regenerating axons, while up to 24-fold more regenerating axons were seen in mexiletine treatment group, compared with vehicle controls. h At 4 weeks after ONC, both glycopyrrolate and mexiletine induced a similar increase in RGC survival promoting effects, when compared with vehicle-treated controls. Scale bars: 200 µm in (b, e), 100 µm in (f), 20 µm in (d, h). Mean ± SEM (n = 6–8 per group); *P < 0.05; one-way ANOVA followed by post hoc Bonferroni test in (c, d, g, h).