D-Aspartate treatment attenuates myelin damage and stimulates myelin repair (2018)
Figure 2

Figure 2. Effect of D-Asp treatment on myelination and remyelination in cerebellar organotypic slices.

A. Schematic diagram showing D-Asp exposure protocol in organotypic slices.

B. Maximum intensity projection of z-stack confocal images displaying MBP (red) and NF200 (green) immunoreactivities in 7 DIV cerebellar organotypic slices cultured in absence (b-d) or in presence of 100 μM D-Asp (f-h). Panels d and h display colocalized points (white). Panels a and e show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence (a) or in presence of 100 μM D-Asp (e). Scale bars in a and e: 200 μm; in b-d and f-h: 50 μm.

C. Scatter plot histogram analysis of the myelination index in 7 DIV cerebellar slices cultured in absence or in presence of 100 μΜ D-Asp.

D. Western blotting analysis of MBP protein levels from homogenates of organotypic cerebellar slices cultured in absence or in presence of 100 μΜ D-Asp. Data were normalized on the basis of β-actin and expressed as percentage of controls.

E. Schematic diagram showing D-Asp exposure protocol in organotypic slices after LPC exposure. F. Maximum intensity projection of z-stack confocal images displaying MBP (red) and NF200 (green) immunoreactivities in control cerebellar organotypic slices (b-d), and in cerebellar slices at 6 days after LPC exposure (dpl) in absence (f-h) or in presence 100 μM D-Asp (j-l). Panels d, h, and l display colocalized points (white). Panels a, e, and i show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence (a) or in presence of 100 μM D-Asp (e). Scale bars in a, e ,i: 200 μm; in b-d, f-h, and j-l: 50 μm;

G. Western blotting and quantitative analysis of MBP protein levels from homogenates of organotypic cerebellar slices at 6 dpl cultured in absence or in presence of 100 μΜ D-Asp.

H. Scatter plot histogram analysis of the remyelination index in cerebellar explants at 6dpl after LPC exposure cultured in absence or in presence of 100 μΜ D-Asp. Data were normalized to vehicle control.

I. Maximum intensity projection of z-stack confocal images displaying MBP (red) and NF200 (green) immunoreactivities in control cerebellar organotypic slices (b-d), and in cerebellar slices at 10 dpl in absence (f-h) or in presence of 100 μM D-Asp (j-l). Panels d, h, and l display colocalized points (white). Panels a, e, and i show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence (a) or in presence of 100 μM D-Asp (e). Scale bars in a, e ,i: 200 μm; in b-d, f-h,and j-l: 50 μm.

J. Scatter plot histogram analysis of the remyelination index in cerebellar explants at 10 dpl cultured in absence or in presence of 100 μΜ D-Asp. Data were normalized to vehicle control.

Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in C, two-tailed Student's t test, *P<0.05 versus control (n=4 animals, 3-4 slices per group); D, two-tailed Student's t test, *P<0.05 versus controls (n=3); G, one-way ANOVA P=0.003 followed by Bonferroni post hoc test, *P<0.05 versus control, ˄P< 0.05 versus LPC (n=3); H, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus controls, ˄P< 0.05 versus LPC (n=4 animals, 3-4 slices per group); J, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus controls, ˄P< 0.05 versus LPC (n=4 animals, 3-4 slices per group). See the exact P‐values from comparisons tests in Appendix Table Figure 2.

Source data are available online for this figure