Platelets accumulate in response to demyelination.

A) LPC induced demyelinating lesions in spinal cord white matter of WT mice at 1, 3, 7, and 14 dpl, stained for platelets (CD41+). Scale bar 100 μm. B) Quantification of CD41+ signal within the demyelinated lesion. Scale bar 50 μm. C) Platelet staining (CD41+) in spinal cord white matter injected with PBS/DAPI. D) Upper left panel: localization of platelets within blood vessels (ColIV+) and in close proximity with OPCs (Olig2+) at 5 dpl. Upper right panel: IMARIS 3D projection shows the spatial distribution of platelets. Lower panels: magnification of the IMARIS projection showing platelet aggregation within the blood (left panel) and penetration into the parenchyma (right panel). Data were analysed using one-way ANOVA. Data represent the mean ± SD. *p<0.05.

Platelet depletion impairs remyelination in vivo.

A) Schematic representation of the LPC-induced demyelination model coupled with platelet depletion using anti-CD42b. B) Quantitative analysis of CD41+ signal at 5dpl in untreated and platelet depleted mice. C) Representative images of immunofluorescence staining of oligodendroglial lineage cells in untreated and platelet depleted mice at 7 dpl using Olig2+ (upper panels) and mature oligodendrocytes using Olig2+/CC1+ (lower panels). Boxed areas represent high magnification images. (D-F) Quantitative analysis of oligodendroglia at 7 dpl. G) Representative images of toluidine blue staining of remyelination in untreated and platelet depleted mice at 14 dpl and (H-I) its quantification by relative ranking analysis. Data were analyzed using an Unpaired Student’s t-test or Mann-Whitney U test. Data represent mean ± SD. *p ≤0.05; ns (not significant), p >0.05. Scale bars, 100 μm.

Prolonged exposure to platelets suppresses their ability to enhance OPC differentiation.

A) Representative fluorescence images of OPC differentiation (Olig2+/MBP+) 6 DIV and B) its quantification. Data were analyzed using One-way Anova followed by Tukey’s post-hoc test. Data represent the mean ± SD. *** p ≤0.001; **** p ≤0.0001; ns (not significant), p >0.05. Scale bars, 50 μm.

A sustained increase in circulating platelets impairs remyelination in-vivo.

A) Representative fluorescence images of platelets (CD41+) in LPC induced demyelinating lesions of spinal cord white matter of WT and CALRHET mice at 5 and 10 dpl B) Quantification of circulating platelets in WT vs CALRHET mice at 5 & 10 dpl. C) Quantification of CD41+ signal in demyelinated lesions of WT vs CALRHET mice D) Representative immunofluorescence staining of oligodendroglial lineage cells in untreated and platelet depleted mice at 10 dpl using Olig2+ (upper panels) and mature oligodendrocytes using Olig2+/CC1+ (lower panels). Scale bar 100 μm. (E-G) Quantitative analysis of oligodendroglia at 10 dpl. H) Correlation between the circulating platelet number with the number of Olig2+/CC1+ cells within the demyelinated lesion. Data were analyzed using an unpaired t-test or Spearman’s rank correlation analysis. Data represent the mean ± SD. *p ≤0.05; ** p ≤0.001; ns (not significant), p >0.05.