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Cited by 599Multiple Sclerosis Research StudiesNeuroinflammation and Neurodegeneration MechanismsNeurogenesis and neuroplasticity mechanisms

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Inefficient clearance of myelin debris by microglia impairs remyelinating processes

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Lampron, Antoine, Larochelle, Antoine, Laflamme, Nathalie, Préfontaine, Paul, Plante, Marie-Michèle, Sánchez, Maria Gabriela, Yong, V. Wee, Stys, Peter K., Tremblay, Marie-Ève, Rivest, Serge·2015·Journal of Experimental Medicine
DOI 10.1084/jem.20141656PMC PMC4387282
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Abstract

An imbalance between remyelinating and demyelinating rates underlies degenerative processes in demyelinating diseases such as multiple sclerosis. An optimal therapeutic strategy would be to stimulate remyelination while limiting demyelination. Although accumulation of myelin debris impairs remyelination, the mechanisms regulating the clearance of such debris by mononuclear phagocytic cells are poorly understood. We demonstrate that after cuprizone intoxication, CCR2-dependent infiltration of mouse bone marrow-derived cells is abundant in demyelinating areas, but that these cells do not impact demyelination. However, in CX3CR1-deficient mice, the clearance of myelin debris by microglia was blocked greatly, affecting the integrity of the axon and myelin sheaths and thus preventing proper remyelination. These results highlight the crucial role played by CX3CR1 in myelin removal and show that there can be no efficient remyelination after a primary demyelinating insult if myelin clearance by microglia is impaired.

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Figure 1. BM-derived cells are recruited in a CCR2-dependent manner into demyelinating sites of the CNS in the cuprizone model. (A) A Busulfan/Cyclophosphamide chemotherapy regimen was used to prepare WT mice to receive the injection of BM cells from GFP+/− mice. 6 wk after transplantation, 0.2% Cuprizone was added to the diet for up to 6 wk. Mice were sacrificed after 2, 3, 4, 5, and 6 wk on a cuprizone-supplemented diet. Another group was sacrificed 2 wk after removing cuprizone from the diet to allow remyelination. (B) Flow cytometry analysis of GFP expression in circulating monocytes of WT mice, GFP+/− mice, chimeric GFP → WT mice, and and CCR2−/− → WT mice. (C) GFP+ cells were counted with a stereologic apparatus. Reported is the total number of GFP+ cells per slice counted per animal. (D-F) Representative confocal images of GFP+ cells (green) and immunoreactive Iba1+ cells (red) in the hippocampus and corpus callosum of chimeric mice either untreated (D) or after 5 wk of treatment with a cuprizone diet in GFP (E) and CCR2−/− (F) chimeric mice. One representative experiment out of two is shown. n = 3–9 mice/group. ****, P < 0.0001. Bars, 100 µm.
Figure 1. BM-derived cells are recruited in a CCR2-dependent manner into demyelinating sites of the CNS in the cuprizone model. (A) A Busulfan/Cyclophosphamide chemotherapy regimen was used to prepare WT mice to receive the injection of BM cells from GFP+/− mice. 6 wk after transplantation, 0.2% Cuprizone was added to the diet for up to 6 wk. Mice were sacrificed after 2, 3, 4, 5, and 6 wk on a cuprizone-supplemented diet. Another group was sacrificed 2 wk after removing cuprizone from the diet to allow remyelination. (B) Flow cytometry analysis of GFP expression in circulating monocytes of WT mice, GFP+/− mice, chimeric GFP → WT mice, and and CCR2−/− → WT mice. (C) GFP+ cells were counted with a stereologic apparatus. Reported is the total number of GFP+ cells per slice counted per animal. (D-F) Representative confocal images of GFP+ cells (green) and immunoreactive Iba1+ cells (red) in the hippocampus and corpus callosum of chimeric mice either untreated (D) or after 5 wk of treatment with a cuprizone diet in GFP (E) and CCR2−/− (F) chimeric mice. One representative experiment out of two is shown. n = 3–9 mice/group. ****, P < 0.0001. Bars, 100 µm.CC BY-NC-SA · PubMed Central
Figure 2. Infiltrated cells are immunoreactive for macrophage markers. (A) The diagram represents a coronal section where the pictures in B–E were taken (red box). Iba1 immunostaining was performed on brain sections from GFP → WT and CCR2−/− → WT chimeric mice. Proportion of GFP+ cells that were also Iba1+ after 4 wk of treatment as counted on the stereological apparatus. (B–E) Representative confocal images reveal apparent co-localization of GFP (green) with Iba1 (red; B and C) and F4/80 (red; D and E) in the corpus callosum of GFP → WT (B and D) and CCR2−/− → WT chimeric mice (C and E). Merged images are also shown. One representative experiment out of two is shown. n = 4–5 mice/group. ****, P < 0.0001 from an unpaired two-tailed Student’s t test. Bars, 20 µm.
Figure 2. Infiltrated cells are immunoreactive for macrophage markers. (A) The diagram represents a coronal section where the pictures in B–E were taken (red box). Iba1 immunostaining was performed on brain sections from GFP → WT and CCR2−/− → WT chimeric mice. Proportion of GFP+ cells that were also Iba1+ after 4 wk of treatment as counted on the stereological apparatus. (B–E) Representative confocal images reveal apparent co-localization of GFP (green) with Iba1 (red; B and C) and F4/80 (red; D and E) in the corpus callosum of GFP → WT (B and D) and CCR2−/− → WT chimeric mice (C and E). Merged images are also shown. One representative experiment out of two is shown. n = 4–5 mice/group. ****, P < 0.0001 from an unpaired two-tailed Student’s t test. Bars, 20 µm.CC BY-NC-SA · PubMed Central
Figure 3. Infiltration of BMDCs into the CNS in the cuprizone model does not impact demyelination or remyelination. (A) Staining of myelin with Black Gold II was performed on brain sections from GFP → WT and CCR2−/− → WT chimeric mice. Representative micrograph images show Black Gold staining of untreated (left), GFP → WT (middle), and CCR2 → WT (right) chimeric mice after 6 wk of treatment with cuprizone or normal chow. (B and C) Demyelination (B) and remyelination (C) were measured in the corpus callosum of either GFP → WT or CCR2−/− → WT after 6 wk of treatment with cuprizone (B) or 2 wk of normal chow after 6 wk of cuprizone (C). Differences between the GFP and CCR2−/− groups were found not significant, with adjusted p-values over 0.8 in both cases in a one-way ANOVA followed by Turkey post-hoc test for multiple comparisons. One representative experiment out of two is shown. n = 3–10 mice/group. Bars, 100 µm.
Figure 3. Infiltration of BMDCs into the CNS in the cuprizone model does not impact demyelination or remyelination. (A) Staining of myelin with Black Gold II was performed on brain sections from GFP → WT and CCR2−/− → WT chimeric mice. Representative micrograph images show Black Gold staining of untreated (left), GFP → WT (middle), and CCR2 → WT (right) chimeric mice after 6 wk of treatment with cuprizone or normal chow. (B and C) Demyelination (B) and remyelination (C) were measured in the corpus callosum of either GFP → WT or CCR2−/− → WT after 6 wk of treatment with cuprizone (B) or 2 wk of normal chow after 6 wk of cuprizone (C). Differences between the GFP and CCR2−/− groups were found not significant, with adjusted p-values over 0.8 in both cases in a one-way ANOVA followed by Turkey post-hoc test for multiple comparisons. One representative experiment out of two is shown. n = 3–10 mice/group. Bars, 100 µm.CC BY-NC-SA · PubMed Central
Figure 4. CX3CR1 knockout severely impacts cuprizone-induced demyelination. (A) Staining of myelin with Black Gold II was performed on brain sections from WT and CX3CR1−/− mice 3 and 5 wk after the cuprizone treatment or normal chow. Representative photomicrographs are shown. (B) Quantification of demyelination was measured in the corpus callosum using a stereological procedure 5 wk after normal chow or cuprizone treatment in WT and CX3CR1−/− mice. (C) Time spent in the open arm of the elevated plus maze behavioral test, efficient to assess behavioral deficits induced by cuprizone (Lister, 1987). (D) Stereologic assessment of APC+ cells in the corpus callosum of WT and CX3CR1−/− mice after 5 wk of a cuprizone-supplemented diet or normal chow. APC is a specific marker for mature myelinating oligodendrocytes. (E) Representative electron microscopy images of oligodendrocytes in the corpus callosum of CX3CR1−/− mice, either untreated, or after 6 wk of a cuprizone-supplemented diet. One representative experiment out of two is shown. n = 4–12 mice/group. (B) **, P = 0.002; ****, P < 0.0001; (C and D) **, P = 0.0093; ****, P < 0.0001. Bars: (A) 100 µm; (E) 2 µm.
Figure 4. CX3CR1 knockout severely impacts cuprizone-induced demyelination. (A) Staining of myelin with Black Gold II was performed on brain sections from WT and CX3CR1−/− mice 3 and 5 wk after the cuprizone treatment or normal chow. Representative photomicrographs are shown. (B) Quantification of demyelination was measured in the corpus callosum using a stereological procedure 5 wk after normal chow or cuprizone treatment in WT and CX3CR1−/− mice. (C) Time spent in the open arm of the elevated plus maze behavioral test, efficient to assess behavioral deficits induced by cuprizone (Lister, 1987). (D) Stereologic assessment of APC+ cells in the corpus callosum of WT and CX3CR1−/− mice after 5 wk of a cuprizone-supplemented diet or normal chow. APC is a specific marker for mature myelinating oligodendrocytes. (E) Representative electron microscopy images of oligodendrocytes in the corpus callosum of CX3CR1−/− mice, either untreated, or after 6 wk of a cuprizone-supplemented diet. One representative experiment out of two is shown. n = 4–12 mice/group. (B) **, P = 0.002; ****, P < 0.0001; (C and D) **, P = 0.0093; ****, P < 0.0001. Bars: (A) 100 µm; (E) 2 µm.CC BY-NC-SA · PubMed Central
Figure 5. Cuprizone treatment triggered microgliosis and inflammation in the corpus callosum of mice in a CX3CR1-independent manner. (A) Iba1 was immunostained on brain sections from WT and CX3CR1−/− mice after 4 wk of normal show or a cuprizone-supplemented diet, the peak time point for microglial accumulation in the corpus callosum. Representative Iba1 immunostaining photomicrographs in the corpus callosum. The area covered by Iba1+ staining was measured in the corpus callosum using a stereological procedure (right). (B and C) TLR2 and C1qα mRNA was hybridized on brain sections from WT and CX3CR1−/− mice after 4 wk of treatment with cuprizone or normal show. Semiquantitative expression levels of TLR2 (B) and C1q-α (C) mRNA signal were measured in the corpus callosum using ImageJ. The coronal sections at the bottom depict representative images of TLR2 (B) and C1q-α (C) in situ hybridization signal. One representative experiment out of two is shown. n = 3–6 mice/group. **, P = 0.01; ****, P < 0.001. Bars: (A) 100 µm.
Figure 5. Cuprizone treatment triggered microgliosis and inflammation in the corpus callosum of mice in a CX3CR1-independent manner. (A) Iba1 was immunostained on brain sections from WT and CX3CR1−/− mice after 4 wk of normal show or a cuprizone-supplemented diet, the peak time point for microglial accumulation in the corpus callosum. Representative Iba1 immunostaining photomicrographs in the corpus callosum. The area covered by Iba1+ staining was measured in the corpus callosum using a stereological procedure (right). (B and C) TLR2 and C1qα mRNA was hybridized on brain sections from WT and CX3CR1−/− mice after 4 wk of treatment with cuprizone or normal show. Semiquantitative expression levels of TLR2 (B) and C1q-α (C) mRNA signal were measured in the corpus callosum using ImageJ. The coronal sections at the bottom depict representative images of TLR2 (B) and C1q-α (C) in situ hybridization signal. One representative experiment out of two is shown. n = 3–6 mice/group. **, P = 0.01; ****, P < 0.001. Bars: (A) 100 µm.CC BY-NC-SA · PubMed Central
Figure 6. The phagocytic response of microglia is impaired in CX3CR1−/− mice after cuprizone treatment. TREM2 and CD11c expression levels were used as indicators of microglial phagocytic activity. (A) In situ hybridization signal for trem2 mRNA in the mouse brain 4 wk after the cuprizone treatment or nomal chow. (B) Flow cytometry analysis of CD11c surface protein expression in CD45+ CD11b+ cells in the CNS. (C) CD45+ cells were selected among live cells, which were then gated against CD11b+ and CD11c+, cells positive for both markers are reported. (D and E) Iba1-immunoreactive signal in the brain of WT and CX3CR1−/− mice 5 wk after normal show or cuprizone supplemented diet. Representative electron microscopy images of microglia are shown in WT (D) and CX3CR1−/− (E) mice. Unstained endosomes (red arrow), partially digested myelin products localized within the endosomes (green arrow), and cholesterol crystals inside of their cytoplasm (blue arrow) were abundant in WT (D) but not in CX3CR1−/− microglia (E). One representative experiment out of two is shown. n = 3–6 mice/group. ****, P < 0.0001. Bars, 2 µm.
Figure 6. The phagocytic response of microglia is impaired in CX3CR1−/− mice after cuprizone treatment. TREM2 and CD11c expression levels were used as indicators of microglial phagocytic activity. (A) In situ hybridization signal for trem2 mRNA in the mouse brain 4 wk after the cuprizone treatment or nomal chow. (B) Flow cytometry analysis of CD11c surface protein expression in CD45+ CD11b+ cells in the CNS. (C) CD45+ cells were selected among live cells, which were then gated against CD11b+ and CD11c+, cells positive for both markers are reported. (D and E) Iba1-immunoreactive signal in the brain of WT and CX3CR1−/− mice 5 wk after normal show or cuprizone supplemented diet. Representative electron microscopy images of microglia are shown in WT (D) and CX3CR1−/− (E) mice. Unstained endosomes (red arrow), partially digested myelin products localized within the endosomes (green arrow), and cholesterol crystals inside of their cytoplasm (blue arrow) were abundant in WT (D) but not in CX3CR1−/− microglia (E). One representative experiment out of two is shown. n = 3–6 mice/group. ****, P < 0.0001. Bars, 2 µm.CC BY-NC-SA · PubMed Central
Figure 7. Myelin staining and electron microscopy reveal the presence of persistent aberrant myelin patterns in the brain of CX3CR1−/− mice after cuprizone feeding. (A–D) Myelin staining with 0.5 mM 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (red) and Iba1-immunoreactive staining (green). (E–J) Imaging at (E, F, and G) low or (H, I, and J) high magnifications of myelin state in representative sagittal microsections of the corpus callosum of either (E and H) untreated WT or (F and I) treated WT and (G and J) CX3CR1−/− with a cuprizone diet for 5 wk. (K–M) High powered magnification of aberrant myelin patterns frequently seen in the corpus callosum of CX3CR1−/− mice. Such patterns included disorganization or separation of their myelin sheath (K and L) and spheroid-like myelin-enclosed structures (M). Images were taken from mice sacrificed after 5 wk of cuprizone diet. One representative experiment out of two is shown. n = 3–6 mice/group. Bars: (A–D) 20 µm; (E–G) 5 µm; (H-M) 2 µm.
Figure 7. Myelin staining and electron microscopy reveal the presence of persistent aberrant myelin patterns in the brain of CX3CR1−/− mice after cuprizone feeding. (A–D) Myelin staining with 0.5 mM 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (red) and Iba1-immunoreactive staining (green). (E–J) Imaging at (E, F, and G) low or (H, I, and J) high magnifications of myelin state in representative sagittal microsections of the corpus callosum of either (E and H) untreated WT or (F and I) treated WT and (G and J) CX3CR1−/− with a cuprizone diet for 5 wk. (K–M) High powered magnification of aberrant myelin patterns frequently seen in the corpus callosum of CX3CR1−/− mice. Such patterns included disorganization or separation of their myelin sheath (K and L) and spheroid-like myelin-enclosed structures (M). Images were taken from mice sacrificed after 5 wk of cuprizone diet. One representative experiment out of two is shown. n = 3–6 mice/group. Bars: (A–D) 20 µm; (E–G) 5 µm; (H-M) 2 µm.CC BY-NC-SA · PubMed Central
Figure 8. Demyelination-induced recruitment of oligodendrocyte precursor cells (OPCs) is impaired in CX3CR1−/− mice. Complementary histological markers were used to measure OPC proliferation and recruitment in the corpus callosum. (A and B) Platelet-derived growth factor receptor α (PDGFR-α) mRNA hybridization signal in the corpus callosum of mice killed 4 wk after being fed with a cuprizone-supplemented diet. (C) Representative Olig2-immunoreactive staining in the corpus callosum of WT and CX3CR1−/− mice 5 wk after a cuprizone treatment or normal show. (D) Stereologic quantification of Olig2-immunoreactive cells in the corpus callosum of WT and CX3CR1−/− after 5 wk of cuprizone or normal feeding. High expression of this protein is specific to OPCs and preoligodendrocytes (Levine et al., 2001). (E) Immunoreactive signal for Olig2 (red)/Ki67 (green), PDGFRα (red), and IGF-1 (red). One representative experiment out of two is shown. n = 3–6 mice/group. (B) **, P = 0.0083; ****, P < 0.0001; **, P = 0.0022; (D) ***, P = 0.0009; ***, P = 0.0003. Bars: (C) 100 µm; (E) 20 µm.
Figure 8. Demyelination-induced recruitment of oligodendrocyte precursor cells (OPCs) is impaired in CX3CR1−/− mice. Complementary histological markers were used to measure OPC proliferation and recruitment in the corpus callosum. (A and B) Platelet-derived growth factor receptor α (PDGFR-α) mRNA hybridization signal in the corpus callosum of mice killed 4 wk after being fed with a cuprizone-supplemented diet. (C) Representative Olig2-immunoreactive staining in the corpus callosum of WT and CX3CR1−/− mice 5 wk after a cuprizone treatment or normal show. (D) Stereologic quantification of Olig2-immunoreactive cells in the corpus callosum of WT and CX3CR1−/− after 5 wk of cuprizone or normal feeding. High expression of this protein is specific to OPCs and preoligodendrocytes (Levine et al., 2001). (E) Immunoreactive signal for Olig2 (red)/Ki67 (green), PDGFRα (red), and IGF-1 (red). One representative experiment out of two is shown. n = 3–6 mice/group. (B) **, P = 0.0083; ****, P < 0.0001; **, P = 0.0022; (D) ***, P = 0.0009; ***, P = 0.0003. Bars: (C) 100 µm; (E) 20 µm.CC BY-NC-SA · PubMed Central
Figure 9. Persistence of myelin aberrations in CX3CR1−/− mice after remyelination. (A) Remyelination was assessed by the histological Black Gold staining in the corpus callosum 1 wk after cuprizone was removed from the diet. These photomicrographs are representative examples of such histological preparations for each group. Black Gold area was quantified in the corpus callosum with a stereological procedure. (B) Representative electron microscopy imaging of myelin aberrations in the corpus callosum of WT and CX3CR1−/− mice after remyelination. 5 wk after a cuprizone-supplemented diet, mice were killed 1 wk after normal feeding. (C) Stereologic assessment of the number of axons with aberrant myelin patterns. Presented are the means of at least three fields of view at 1900X per each animal. On average, 152 axons were counted per field of view. One representative experiment out of two is shown. n = 4–6 mice/group. *, P = 0.027; ***, P = 0.0010; ****, P < 0.0001. Bars: (A) 100 µm; (B) 500 nm (left); 2 µm (right).
Figure 9. Persistence of myelin aberrations in CX3CR1−/− mice after remyelination. (A) Remyelination was assessed by the histological Black Gold staining in the corpus callosum 1 wk after cuprizone was removed from the diet. These photomicrographs are representative examples of such histological preparations for each group. Black Gold area was quantified in the corpus callosum with a stereological procedure. (B) Representative electron microscopy imaging of myelin aberrations in the corpus callosum of WT and CX3CR1−/− mice after remyelination. 5 wk after a cuprizone-supplemented diet, mice were killed 1 wk after normal feeding. (C) Stereologic assessment of the number of axons with aberrant myelin patterns. Presented are the means of at least three fields of view at 1900X per each animal. On average, 152 axons were counted per field of view. One representative experiment out of two is shown. n = 4–6 mice/group. *, P = 0.027; ***, P = 0.0010; ****, P < 0.0001. Bars: (A) 100 µm; (B) 500 nm (left); 2 µm (right).CC BY-NC-SA · PubMed Central

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Metadata

Authors
Lampron, A., Larochelle, A., Laflamme, N., Préfontaine, P., Plante, M.-M., Sánchez, M. G., Yong, V. W., Stys, P. K., Tremblay, M.-È., & Rivest, S.
Affiliations
  • Université Laval (CA)
  • University of Calgary (CA)
Corresponding author
Rivest, Serge
ORCID
Venue
Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
2015-03-16
Volume
212
Issue
4
Pages
481-495
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.20141656
PMC
PMC4387282
OpenAlex
W2096423240
Cited by
595
References
77
Citations by year
2026: 45 · 2025: 42 · 2024: 46 · 2023: 78 · 2022: 86 · 2021: 55 · 2020: 60 · 2019: 61 · 2018: 44 · 2017: 38 · 2016: 29 · 2015: 11
FWCI
32.27
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Experimental Medicine (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
Canadian Institutes of Health Research
Topics
  • Health Sciences › Medicine › Clinical Neurology › Multiple Sclerosis Research Studies
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
Keywords
remyelination, myelin debris clearance, microglia, CX3CR1, CCR2, demyelination, multiple sclerosis, cuprizone, bone marrow-derived cells, axon integrity
MeSH terms
CX3C Chemokine Receptor 1, Animals, Axons, Bone Marrow Cells, Demyelinating Diseases, Myelin Sheath, Phagocytes, Microglia, Mice, Knockout, Receptors, Chemokine, Mice, Receptors, CCR2

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