Circulating platelets modulate oligodendrocyte progenitor cell differentiation during remyelination
- Laboratory of Stem Cells and Neuroregeneration, Institute of Anatomy, Histology and Pathology, Faculty of Medicine, Universidad Austral de Chile, Chile
- Center for Interdisciplinary Studies on the Nervous System (CISNe), Universidad Austral de Chile, Chile
- Wellcome-MRC Cambridge Stem Cell Institute & Department of Clinical Neurosciences, University of Cambridge, United Kingdom
- Translational Regenerative Neurobiology Group (TReN), Molecular and Integrative Biosciences Research Programme (MIBS), Faculty of Biological and Environmental Sciences, University of Helsinki, Finland
- Escuela de Ciencias Agrícolas y Veterinarias, Universidad Viña del Mar, Chile
- Department of Haematology and NHS Blood and Transplant, University of Cambridge, United Kingdom
- Institute of Molecular Regenerative Medicine, Paracelsus Medical University, Austria
- Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Chile
- Laboratory of Cellular Pathology, Institute of Anatomy, Histology & Pathology, Faculty of Medicine, Universidad Austral de Chile, Chile
- School of Life Sciences, University of Westminster, United Kingdom
Figures
Figure 1
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). (C) Platelet staining (CD41+) in spinal cord white matter injected with PBS/DAPI. Scale bar 50 μm. (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. Scale bar 10 μm. Lower panels: magnification of the IMARIS projection showing platelet aggregation within the blood (left panel) and penetration into the parenchyma (right panel). Scale bars: 5 μm (left panel) and 7 μm (right panel). Data were analysed using a Kruskal Wallis test. Data represent the mean ± SD. ** p<0.01; ns (not significant), p>0.05.
Figure 2 with 2 supplements
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=6) and platelet depleted mice (n=3) at 14 dpl and (H–I) its quantification by relative ranking analysis. Data were analysed using an Unpaired Student’s t-test or Mann-Whitney U test. Data represent mean ± SD. * p<0.05; ** p<0.01; ns (not significant), p>0.05. Scale bars, 100 μm.
Figure 2—figure supplement 1
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 7 dpl 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 analysed using an unpaired t-test. Data represent the mean ± SD. ns (not significant), p>0.05.
Figure 2—figure supplement 2
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 (IBA-1+) at 5 dpl. Scale bar 10 μm. (B) Total macrophage/microglia population (IBA-1+), M1 (CD16/32+) and M2 (Arg-1+) cell subpopulations at 10 dpl. Scale bar 50 μm. (C–E) Quantitative analysis of total, M1 and M2 cell subpopulations in untreated (n=6), platelet depleted (n=3), and Calr+/- mice (n=4). (F) Representative Oil-Red O staining of myelin debris at 10 dpl. (G) Quantification of Oil-Red O (ORO) staining in untreated (n=6) and platelet depleted mice (n=3). Scale bar 100 μm. Data were analysed using an ordinary one-way ANOVA and or an unpaired t-test. Data represent the mean ± SD. ns (not significant), p>0.05.
Figure 3
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 6 DIV (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 6 DIV 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 analysed using one-way ANOVA followed by Tukey’s post-hoc test, a Mann-Whitney U test, or Kruskal-Wallis test. Data represent the mean ± SD. * p<0.05; *** p<0.001; **** p<0.0001; ns (not significant), p>0.05. Scale bars, 50 μm.
Figure 4
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 Calr +/-mice at 5 and 10 dpl. Scale bar 50 μm. (B) Quantification of circulating platelets in WT vs Calr +/-mice at 5 (n=4 and n=5, respectively) and 10 dpl (n=5 and n=6, respectively). (C) Quantification of CD41+ signal in demyelinated lesions of WT vs Calr +/-mice at 5 dpl (n=5 and n=5, respectively) and 10 dpl (n=4 and 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 analysed using a two-way ANOVA followed by Bonferroni’s post-hoc test, an unpaired t-test, Welch’s t-test, a Mann-Whitney U test, or Pearson’s correlation coefficient analysis. Data represent the mean ± SD. * p<0.05; ** p<0.01; ns (not significant), p>0.05.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | CD41 rat monoclonal | Abcam | Cat# ab33661; RRID:AB_726487 | Working dilution (1:200) |
| Antibody | CD16/32 rat monoclonal | BD Biosciences | Cat # BD 553142 RRID:AB_394656 | Working dilution (1:200) |
| Antibody | Iba-1 rabbit polyclonal | WAKO | Cat# 019–19741; RRID:AB_839504 | Working dilution (1:500) |
| Antibody | Arg-1 goat polyclonal | Santa Cruz | Cat# sc-18351; RRID:AB_2258542 | Working dilution (1:200) |
| Antibody | NIMP-R14 rat monoclonal | Abcam | Cat# ab2557; RRID:AB_303154 | Working dilution (1:200) |
| Antibody | Olig2 rabbit monoclonal | Abcam | Cat# Ab109186; RRID:AB_10861310 | Working dilution (1:200 in vivo) (1:500 in vitro) |
| Antibody | CC1 mouse monoclonal | Millipore | Cat# OP80; RRID:AB_2057371 | Working dilution (1:1000) |
| Antibody | MBP rat monoclonal | Bio-rad | Cat# MCA409S; RRID:AB_325004 | Working dilution (1:500) |
| Antibody | Collagen IV (ColIV) goat polyclonal | Millipore | CAT# AB769; RRID:AB_92262 | Working dilution (1:100) |
| Antibody | Fibrinogen rabbit polyclonal | Abcam | Cat # ab34269 RRID:AB_732367 | Working dilution (1:200) |
| Chemical compound, drug | L-α-lysophosphatidylcholine | Sigma-Aldrich | Cat # L1381 | Demyelinating agent, Working concentration 1% |
| Chemical compound, drug | CD42b (mixture of rat monoclonal antibodies) | Emfret Analytics; Evans et al., 2021 | Cat #R300 RRID:AB_2721041 | Platelet depletion antibody, Working concentration 0.6 μg/g |
| Strain, strain background (Mus musculus) | Mouse: C7BL/6 | Charles River Laboratories | RRID:SCR_003792 | |
| Strain, strain background (Mus musculus) | Mouse: Calrfl/+:Vav1-Cre mice | Li et al., 2018 | ||
| Strain, strain background (Rattus norvegicus) | Rat: Sprague Dawley | Charles River Laboratories | RRID:SCR_003792 |
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Circulating platelets modulate oligodendrocyte progenitor cell differentiation during remyelination
eLife 12:RP91757.
https://doi.org/10.7554/eLife.91757.3
