Figure 3.

ET-2 but not CXCL13 promotes remyelination in cerebellar slice cultures. (A) Experimental schematic for remyelinating slice cultures. Following exposure to lysolecithin (LPC), fully myelinated cultures are demyelinated without damage to axons. After removal of lysolecithin, cultures are maintained for 14 additional days and remyelinate (MBP, green; NFH, red). (B) Quantification demonstrates a loss of myelin after exposure to lysolecithin. Control slices not exposed to lysolecithin (myelination) are compared with those exposed to lysolecithin (demyelination) at 15 days in vitro. The data are analysed by t-test and the significant difference (***P < 0.001) is shown. (C) Confocal z-stacks of cerebellar slice cultures (labelled and analysed as in Fig. 2) demonstrate an increase in remyelination following the addition of ET-2. (D) Quantification demonstrates an increase in remyelination with 10 ng/ml ET-2. Values shown are mean + SD (n = 3). The data are analysed by one-way ANOVA with Dunnett’s multiple comparison test, and the significant difference (***P < 0.001) is shown. (E) Confirmation of remyelination in the slices as revealed by formation of nodes of Ranvier, labelled as in Fig. 2, and by (F) electron microscopy showing the loss of myelin following lysolecithin (left) and the presence of compact myelin following remyelination (right). In the demyelination panel, asterisks denote demyelinated axons. Note the high neurofilament density. M = reactive microglial cell. In the remyelination panel, asterisks denote remyelinated axons, and the arrow denotes a demyelinated axon surrounded by oligodendrocyte processes, likely in the process of being remyelinated. Scale bars: C = 30 µm; E = 10 µm; F = 2 µm.