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Pharmacologically increasing Na+ channel activation exacerbates myelin swelling in zebrafish (A) Schematic of experimental set-up for veratridine treatment in the Tg(mbp:mCherry-NTR) model. (B) Confocal images of all myelin sheaths in the ventral spinal cord labelled by the transgenic reporter Tg(mbp:EGFP-CAAX) from Tg(mbp:mCherry-NTR)-expressing animals treated with or without mtz for 1 day, with our without treatment with veratridine for 1 day. Asterisks = swellings. Scale bar = 10 µm. (C) Quantification of disorder in the ventral spinal cord post-treatment in controls + vehicle (n = 8 animals), controls + veratridine (n = 8 animals), mtz + vehicle (n = 12 animals), or 5 mM mtz + veratridine (n = 11 animals). Two Way ANOVA with Šídák’s multiple comparisons test. Error bars are mean ± 95% CI. (D) Quantification of the number of swellings in the ventral spinal cord post-treatment in controls + vehicle (n =8 animals), controls + veratridine (n =8 animals), mtz + vehicle (n = 12 animals), or mtz + veratridine (n = 11 animals). Data are normalised to 100 µm length of spinal cord. Unpaired t-test with Welch’s correction (as values for DMSO-treated groups all = 0). (E) Schematic of experimental set-up for veratridine treatment in the Tg(mbp:mCherry-NTR) model for following fate of single oligodendrocytes 5 days post treatment (dpt). Ver = veratridine. dpf = days post fertilisation. (F) Confocal images of myelin sheaths in single oligodendrocytes in the ventral spinal cord mosaically labelled with (mbp:EGFP-CAAX) from Tg(mbp:mCherry-NTR)-expressing animals treated with mtz + vehicle or mtz + veratridine immediately post treatment (0 dpt). Scale bar = 5µm. (G) Quantification of number of swellings per cell immediately post treatment with mtz + vehicle (n=33) or mtz + veratridine (n=33). One cell per animal. Unpaired two-tailed t-test with Welch’s correction. Error bars are mean ± 95% CI. (H) Quantification of number of unaffected myelin sheaths (myelin sheaths with no swellings) per cell immediately post treatment with mtz + vehicle (n=33) or mtz + veratridine (n=33). Unpaired two-tailed t-test with Welch’s correction. Error bars are mean ± 95% CI. (I) Quantification of average size of swellings per cell immediately post treatment with mtz + vehicle (n=13) or mtz + veratridine (n=14). Unpaired two-tailed t-test with Welch’s correction. Error bars are mean ± 95% CI. (J) Quantification of total swelling area per cell immediately post treatment with mtz + vehicle (n=13) or mtz + veratridine (n=14). Unpaired two-tailed t-test with Welch’s correction. Error bars are mean ± 95% CI. (K) Quantification of number of oligodendrocytes that degenerated or survived following treatment with mtz + vehicle (n=23) or mtz + veratridine (n=36). Chi-square test P value = 0.0430. (L) Confocal images of myelin sheaths in single oligodendrocytes in the ventral spinal cord followed over time with treatment of mtz + veratridine. Scale bar = 5µm. (M) Quantification of total swelling area per cell immediately post treatment with mtz + vehicle (clear datapoints) or mtz + veratridine (blue datapoints), separated into oligodendrocytes that survived (n=19) vs. degenerated (n=8). Unpaired two-tailed t-test with Welch’s correction. Error bars are mean ± 95% CI.
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