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 at 1 (n=6), 3 (n=5), 5 (n=5), 7 (n=6), 10 (n=4), and 14 dpl (n=4), and in NAWM (n=3). 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 5 dpl in untreated (n=5) and platelet depleted mice (n=3). 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 in untreated (n=3) and platelet depleted mice (n=5). G) Representative images of toluidine blue staining of remyelination in untreated (n=5) and platelet depleted mice (n=3) 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 OPCs co-cultured with 1 (n=6), 5 (n=6), and 10% (n=6) washed platelets (WP) for 3 days in vitro (DIV), followed by WP removal for an additional 3 DIV (Pulse). Additionally, OPCs were co-cultured in the presence of 10% WP for 6DIV (n=5) (Sustained). Vehicle treated OPCs represents the control condition (n=6). B) Graph represents the percentage of Olig2+MBP+ oligodendrocytes within the total Olig2 population (quantitative analysis of OPC differentiation). C) Representative images of OPCs exposed to 1% platelet lysate (PL) (n=5) for 6 DIV. Vehicle treated OPCs represents the control condition (n=5). D) Graph represents the quantitative analysis of OPC differentiation as in B. E) Representative images of OPCs exposed to either PL for 9 DIV (Sustained) (n=5) or 6DIV with PL followed by its removal for an additional 3 more DIV (Withdrawn) (n=5). Vehicle treated OPCs represents the control condition (n=5). F) Graph shows the quantitative analysis of OPC differentiation as in B and D. Data were analyzed using One-way Anova followed by Tukey’s post-hoc test or an un-paired t-test. Data represent the mean ± SD. *** p ≤0.001; **** p ≤0.0001; ns (not significant), p >0.05. Scale bars, 50 mm.

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 (n=4 & n=5, respectively), 10 ((n=5 & n=6, respectively), and 14 dpl ((n=4 & n=6, respectively). C) Quantification of CD41+ signal in demyelinated lesions of WT vs CALRHET mice at 5 dpl (n=5 & n=5, respectively) and 10 dpl (n=4 & n=4, respectively). 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) (n=4). 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.

Platelet depletion does not alter BBB permeability.

A) Representative immunofluorescence images of neutrophils (NIMP-R14+) and B) number of neutrophils in LPC-induced white matter spinal cord lesion at 7dp in untreated (n=3) and platelet depleted mice (n=5). C) Representative immunofluorescence images of Fibrinogen and D) quantification of Fibrinogen signal within the demyelinated lesion at 7 dpl in untreated and platelet depleted mice (n=4). Scale bar 50 μm. Data were analyzed using an unpaired t-test. Data represent the mean ± SD. ns (not significant), p >0.05.

Changes in circulating platelet numbers does not alter the macrophage/microglia population during remyelination.

A) Platelets (CD41+) are located in close proximity to the macrophage/microglia population (IBA1+) at 5 dpl. Scale bar 50 um. B) Total macrophage/microglia population (IBA-1+), M1 (CD16/32+) and M2 (Arg-1+) macrophage/microglia populations at 10 dpl and (C-E) their respective quantitative analysis in untreated (n=6), platelet depleted (n=3), and CALRHET mice (n=4). F) Representative oil-red staining of myelin debris at 10 dpl. G) Quantification of Oil-red staining in untreated (n=6) and platelet depleted mice (n=3). Scale bar 100μm. Data were analyzed using an unpaired t-test. Data represent the mean ± SD. ns (not significant), p >0.05.