Electron microscopic analysis and axonal tracing shows no axonal damage after LPC induced demyelinating lesions. (A) In a demyelinated optic nerve (left), unmyelinated axons of various axonal diameters are seen compared to a crushed optic nerve (right) where axonal diameters are smaller. (B) To quantify axon diameter, 200 axons of each of n = 3 fish were measured per condition and are shown as frequency of axons per axon diameter bin. This is also shown as a cumulative frequency, and a box and whisker plot (showing median, interquartile range and maximum and minimum values). These representations all illustrate that small diameter axons are more frequent after nerve crush than in unlesioned controls or after LPC treatment. Furthermore, large diameter axons are more frequent after LPC treatment. (C) Tracer was applied to optic nerve (cut proximal to lesion) 8 days after treatment with LPC (or PBS for control) so that with no axon damage, the tracer labels the entire contralateral optic tectum (top picture), but with axon damage, partial tectal labelling is observed (bottom picture). (D) There are no differences in tracer labelling in brains after treatment with LPC or PBS-control, whereas a partial crush results in distinct areas of lower labelling intensity (black arrows), (dorsal is up), also seen by quantification of tectal labelling compared to the contralateral unlabelled control side (E) (p < 0.05, n = 3 fish, Kruskal Wallis test, Dunn’s post-test). Mean ± SEM. Scale bars: A = 1 μm, D = 200 μm (top), 100 μm (bottom).