Figure 4.

Quantification of myelin and mitochondrial changes in reconstructed axons in 3D following injury. (a) Correlation between axon diameter and myelin thickness generated from values in 2D from sections 5 µm apart along the axons; normal optic nerves R² = 0.449, whilst after injury R² = 0.249. Linear regression line for normal fits the equation: y = 0.20 × +0.13, and injured: y = 0.21 × +0.15; n = 30 axons in each group. (b) Myelin decompaction was quantified for normal and injured axons at 5 µm intervals along the length of each reconstructed axon. Individual reconstructed axons classified as having >40% myelin decompaction in single sections at 5 µm intervals were given a ‘point’ and total points of decompaction along the axon were expressed as a proportion of the measured length of the axon. (c) Representative image of a myelin segment <40% decompaction (green, arrow) and >40% decompaction (orange, double arrow) from transverse sections. Myelin was considered decompacted by the presence of light grey regions located between the concentric darker, compact myelin lamellae. (d) Paranodal gap was increased following injury to the optic nerve. (e) Representative image of paranode before (green) and after (orange) injury; there is an elongation of the paranodal gap, shown visualizing the 3D dataset longitudinally. The (f) number and (g) length of mitochondria within all nodes of Ranvier in the 30 axons examined per experimental group were quantified for normal and injured axons. Figures are expressed as scatter plots with the line representing the mean ± SEM. Differences between experimental groups are shown by *(p ≤ 0.05). Representative images of mitochondria from normal (h) and injured optic nerve (i) are shown. Scale bar for c and e = 1.5 µm; for h and i = 1 µm.