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Cited by 119Neonatal and fetal brain pathologyNeurogenesis and neuroplasticity mechanismsCircadian rhythm and melatonin

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Melatonin Promotes Oligodendroglial Maturation of Injured White Matter in Neonatal Rats

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Olivier, Paul, Fontaine, Romain H., Loron, Gauthier, Van Steenwinckel, Juliette, Biran, Valérie, Massonneau, Véronique, Kaindl, Angela, Dalous, Jeremie, Charriaut-Marlangue, Christiane, Aigrot, Marie-Stéphane, Pansiot, Julien, Verney, Catherine, Gressens, Pierre, Baud, Olivier·2009·PLoS ONE
DOI 10.1371/journal.pone.0007128PMC PMC2742165
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Abstract

OBJECTIVE: To investigate the effects of melatonin treatment in a rat model of white matter damage (WMD) in the developing brain. Additionally, we aim to delineate the cellular mechanisms of melatonin effect on the oligodendroglial cell lineage. METHODS: A unilateral ligation of the uterine artery in pregnant rat at the embryonic day 17 induces fetal hypoxia and subsequent growth restriction (GR) in neonatal pups. GR and control pups received a daily intra-peritoneal injection of melatonin from birth to post-natal day (P) 3. RESULTS: Melatonin administration was associated with a dramatic decrease in microglial activation and astroglial reaction compared to untreated GR pups. At P14, melatonin prevented white matter myelination defects with an increased number of mature oligodendrocytes (APC-immunoreactive) in treated GR pups. Conversely, melatonin was not found to be associated with an increased density of total oligodendrocytes (Olig2-immunoreactive), suggesting that melatonin is able to promote oligodendrocyte maturation but not proliferation. These effects appear to be melatonin-receptor dependent and were reproduced in vitro. INTERPRETATION: These data suggest that melatonin has a strong protective effect on developing damaged white matter through decreased microglial activation and oligodendroglial maturation leading to a normalization of the myelination process. Consequently, melatonin should be a considered as an effective neuroprotective candidate not only in perinatal brain damage but also in inflammatory and demyelinating diseases observed in adults.

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Figure 1 Melatonin attenuates myelination defect in the cingulum. (A–D) Cingulate white matter MBP-immunoreactivity in P14 rat pups demonstrating that myelin content was defective in PBS-treated GR pups (B) compared to PBS- (A) and melatonin-treated controls (C). In contrast, melatonin-treated animals (D) showed an MBP-immunoreactivity density similar to controls. Bars = 200 µm. (E) Quantitative analysis of the MBP-positive fibers optical density in the cingulate white matter of controls and GR pups treated with PBS or with melatonin 20 mg/kg. The area of optic density measurement is indicated by an asterisk in (A–D). Statistical analysis compared either control and GR pups or untreated and treated GR pups. (**p<0.01 and ***p<0.001 using one-way ANOVA with the Newman-Keuls correction). (F) Scattered plot representation of MBP-positive fibers immunoreactivity density in the cingulate white matter of PBS- and melatonin-treated controls and GR pups at P14 according to their birth weight. Squares (in red) and line represent PBS-treated animals, triangles (in blue) and line represent melatonin-treated animals. Dotted-lines represents weight of 5.50 g and 5.87 g, respectively the upper limit (−2 SD) of GR animals birth weight and the lower limit (−1 SD) of control animals birth weight.
Figure 1 Melatonin attenuates myelination defect in the cingulum. (A–D) Cingulate white matter MBP-immunoreactivity in P14 rat pups demonstrating that myelin content was defective in PBS-treated GR pups (B) compared to PBS- (A) and melatonin-treated controls (C). In contrast, melatonin-treated animals (D) showed an MBP-immunoreactivity density similar to controls. Bars = 200 µm. (E) Quantitative analysis of the MBP-positive fibers optical density in the cingulate white matter of controls and GR pups treated with PBS or with melatonin 20 mg/kg. The area of optic density measurement is indicated by an asterisk in (A–D). Statistical analysis compared either control and GR pups or untreated and treated GR pups. (**p<0.01 and ***p<0.001 using one-way ANOVA with the Newman-Keuls correction). (F) Scattered plot representation of MBP-positive fibers immunoreactivity density in the cingulate white matter of PBS- and melatonin-treated controls and GR pups at P14 according to their birth weight. Squares (in red) and line represent PBS-treated animals, triangles (in blue) and line represent melatonin-treated animals. Dotted-lines represents weight of 5.50 g and 5.87 g, respectively the upper limit (−2 SD) of GR animals birth weight and the lower limit (−1 SD) of control animals birth weight.CC BY · PubMed Central
Figure 2 Melatonin attenuates myelination defect in the corpus callosum. (A–D) White matter MBP-immunoreactivity in P14 rat pups demonstrating that myelin content was defective in the genu of the corpus callosum in PBS-treated GR pups (B) compared to PBS- (A) and melatonin-treated controls (C). In contrast, melatonin-treated animals (D) showed an MBP-immunoreactivity density similar to controls. Bars = 200 µm. (E) Quantitative analysis of the MBP-positive fibers optical density in the corpus callosum white matter of controls and GR pups treated with PBS or with melatonin 20 mg/kg. The area of optic density measurement is indicated by a square in (A–D). Statistical analysis compared either control and GR pups or untreated and treated GR pups. (***p<0.001 using one-way ANOVA with the Newman-Keuls correction).
Figure 2 Melatonin attenuates myelination defect in the corpus callosum. (A–D) White matter MBP-immunoreactivity in P14 rat pups demonstrating that myelin content was defective in the genu of the corpus callosum in PBS-treated GR pups (B) compared to PBS- (A) and melatonin-treated controls (C). In contrast, melatonin-treated animals (D) showed an MBP-immunoreactivity density similar to controls. Bars = 200 µm. (E) Quantitative analysis of the MBP-positive fibers optical density in the corpus callosum white matter of controls and GR pups treated with PBS or with melatonin 20 mg/kg. The area of optic density measurement is indicated by a square in (A–D). Statistical analysis compared either control and GR pups or untreated and treated GR pups. (***p<0.001 using one-way ANOVA with the Newman-Keuls correction).CC BY · PubMed Central
Figure 3 Melatonin effect is dose-dependent and mediated by its specific receptors. (A) Cingulate white matter MBP-immunoreactivity optical density in P14 GR pups compared to controls demonstrating that melatonin was able to restore normal MBP content with a minimal dose of 0.2 mg/kg. Controls and treated GR animals were compared to untreated GR pups. *p<0.05 and ***p<0.001 using one-way ANOVA with the Newman-Keuls correction. (B) The melatonin effect on myelination was reversed when melatonin was co-injected with luzindole, a specific MT1/MT2 antagonist. **p<0.01, NS: not significant using one-way ANOVA with the Newman-Keuls correction.
Figure 3 Melatonin effect is dose-dependent and mediated by its specific receptors. (A) Cingulate white matter MBP-immunoreactivity optical density in P14 GR pups compared to controls demonstrating that melatonin was able to restore normal MBP content with a minimal dose of 0.2 mg/kg. Controls and treated GR animals were compared to untreated GR pups. *p<0.05 and ***p<0.001 using one-way ANOVA with the Newman-Keuls correction. (B) The melatonin effect on myelination was reversed when melatonin was co-injected with luzindole, a specific MT1/MT2 antagonist. **p<0.01, NS: not significant using one-way ANOVA with the Newman-Keuls correction.CC BY · PubMed Central
Figure 4 Melatonin improves the ultrastructural myelin aspect in GR pups. (A–F) Cingulate white matter ultrastructural aspect observed using electron microscopy. PBS-treated GR pups (B, C) exhibited swollen axons (marked with an asterisk) and dramatically low cellular, axonal and myelinated axons density compared to PBS- (A) and melatonin-treated controls (D). Note the large proportion of myelinated axons surrounded with uncompacted myelin sheets (arrows) in PBS-treated GR-pups (B, C). In contrast, melatonin-treated GR animals exhibited a high proportion of well-compacted myelination processes, similar to controls (E–F). Interestingly, melatonin treatment appeared to not be able to restore a normal axonal aspect within the developing white matter (E). Swollen axons were marked with an asterisk. Quantitative analysis of myelin ultrastructural aspect was reported in table 2.
Figure 4 Melatonin improves the ultrastructural myelin aspect in GR pups. (A–F) Cingulate white matter ultrastructural aspect observed using electron microscopy. PBS-treated GR pups (B, C) exhibited swollen axons (marked with an asterisk) and dramatically low cellular, axonal and myelinated axons density compared to PBS- (A) and melatonin-treated controls (D). Note the large proportion of myelinated axons surrounded with uncompacted myelin sheets (arrows) in PBS-treated GR-pups (B, C). In contrast, melatonin-treated GR animals exhibited a high proportion of well-compacted myelination processes, similar to controls (E–F). Interestingly, melatonin treatment appeared to not be able to restore a normal axonal aspect within the developing white matter (E). Swollen axons were marked with an asterisk. Quantitative analysis of myelin ultrastructural aspect was reported in table 2.CC BY · PubMed Central
Figure 5 Melatonin promotes oligodendroglial maturation. (A) Quantitative analysis of Olig2-immunoreactive cells in the cingulate white matter at P14. Melatonin treatment was found to not be able to reverse the loss of total oligodendrocytes observed in GR pups. (B) Quantitative analysis of APC-immunoreactive cells in the cingulate white matter at P14. Melatonin treatment was associated with a significant reduction in mature oligodendrocytes deficit observed in GR pups. *p<0.05 and ***p<0.001 using one-way ANOVA with the Newman-Keuls correction. (C–F) PBS-treated GR pups exhibited a defective mature (APC-immunoreative) oligodendrocyte density (D) compared to PBS- and melatonin-treated controls (C, E). Melatonin treatment partially restored mature oligodendrocytes density (F). Microphotographes show cingulate white matter with the standardized counting area indicated by dotted lines. V: ventricule. cc = corpus callosum underlying the cingulum. Bars = 200 µm.
Figure 5 Melatonin promotes oligodendroglial maturation. (A) Quantitative analysis of Olig2-immunoreactive cells in the cingulate white matter at P14. Melatonin treatment was found to not be able to reverse the loss of total oligodendrocytes observed in GR pups. (B) Quantitative analysis of APC-immunoreactive cells in the cingulate white matter at P14. Melatonin treatment was associated with a significant reduction in mature oligodendrocytes deficit observed in GR pups. *p<0.05 and ***p<0.001 using one-way ANOVA with the Newman-Keuls correction. (C–F) PBS-treated GR pups exhibited a defective mature (APC-immunoreative) oligodendrocyte density (D) compared to PBS- and melatonin-treated controls (C, E). Melatonin treatment partially restored mature oligodendrocytes density (F). Microphotographes show cingulate white matter with the standardized counting area indicated by dotted lines. V: ventricule. cc = corpus callosum underlying the cingulum. Bars = 200 µm.CC BY · PubMed Central
Figure 6 Melatonin is associated with a decreased glial activation. (A) Quantitative analysis of amyboid tomato-lectin-immunoreactive cells in the cingulate white matter in P3 and P14 rat pups. PBS-treated GR pups exhibited a significant increase in density of reactive microglia compared to PBS- and melatonin-treated controls. In contrast, microglial activation in melatonin-treated GR animals was similar to controls. (B) Quantitative analysis of GFAP-immunoreactive cells in the cingulate white matter in P14 rat pups. PBS-treated GR pups exhibited a significant increase in reactive astrocyte density compared to controls. Melatonin-treatment was associated with a normal astroglial density in treated-GR pups compared to controls. Analysis was performed using two-way ANOVA with the Newman-Keuls correction *p<0.05, **p<0.01 and ***p<0.001 indicating significant differences between GR and control rat pups. #p<0.05, ##p<0.01 and ###p<0.001 indicating significant differences between PBS- and melatonin-treated GR animals.
Figure 6 Melatonin is associated with a decreased glial activation. (A) Quantitative analysis of amyboid tomato-lectin-immunoreactive cells in the cingulate white matter in P3 and P14 rat pups. PBS-treated GR pups exhibited a significant increase in density of reactive microglia compared to PBS- and melatonin-treated controls. In contrast, microglial activation in melatonin-treated GR animals was similar to controls. (B) Quantitative analysis of GFAP-immunoreactive cells in the cingulate white matter in P14 rat pups. PBS-treated GR pups exhibited a significant increase in reactive astrocyte density compared to controls. Melatonin-treatment was associated with a normal astroglial density in treated-GR pups compared to controls. Analysis was performed using two-way ANOVA with the Newman-Keuls correction *p<0.05, **p<0.01 and ***p<0.001 indicating significant differences between GR and control rat pups. #p<0.05, ##p<0.01 and ###p<0.001 indicating significant differences between PBS- and melatonin-treated GR animals.CC BY · PubMed Central
Figure 7 Melatonin receptors are differentially expressed in white matter glial cells in vitro. (A) Relative expression of MT1 (amplicon: 109 bp) and MT2 (amplicon: 75 bp) mRNA in various cell cultures including astrocytes, neurons, immature oligodendrocytes and microglia.(B) Immuno-labelling of various primary cell cultures (microglia, neuron, oligodendrocytes, astrocytes) using either MT1 or MT2 markers. Arrows show small fraction of astrocytes contaminating oligodendrocyte cell cultures.
Figure 7 Melatonin receptors are differentially expressed in white matter glial cells in vitro. (A) Relative expression of MT1 (amplicon: 109 bp) and MT2 (amplicon: 75 bp) mRNA in various cell cultures including astrocytes, neurons, immature oligodendrocytes and microglia.(B) Immuno-labelling of various primary cell cultures (microglia, neuron, oligodendrocytes, astrocytes) using either MT1 or MT2 markers. Arrows show small fraction of astrocytes contaminating oligodendrocyte cell cultures.CC BY · PubMed Central
Figure 8 Melatonin promotes myelin production and prevents LPS-induced microglial activation in vitro. (A) Immuno-labelling of primary oligodendroglial cell cultures using either O4 or MBP at DIV6 or DIV10 with or without treatment with 1 µmol melatonin. (B) Quantitative analysis of the density of O4 or MBP immunoreactive cells at DIV6 and DIV10 with or without treatment with 1 µmol melatonin. **p<0.01, using one-way ANOVA with the Newman-Keuls correction. (D) Immuno-labelling of microglial cell cultures using tomato-lectin in presence of LPS with and without 1 µmol melatonin. Note the marked increase in tomato-lectin immunoreactivity in microglia subjected to LPS. (C) Quantitative analysis of the density of tomato-lectin positive cells in presence of LPS with and without 1 µmol melatonin. ***p<0.001, using two-way ANOVA with the Newman-Keuls correction.
Figure 8 Melatonin promotes myelin production and prevents LPS-induced microglial activation in vitro. (A) Immuno-labelling of primary oligodendroglial cell cultures using either O4 or MBP at DIV6 or DIV10 with or without treatment with 1 µmol melatonin. (B) Quantitative analysis of the density of O4 or MBP immunoreactive cells at DIV6 and DIV10 with or without treatment with 1 µmol melatonin. **p<0.01, using one-way ANOVA with the Newman-Keuls correction. (D) Immuno-labelling of microglial cell cultures using tomato-lectin in presence of LPS with and without 1 µmol melatonin. Note the marked increase in tomato-lectin immunoreactivity in microglia subjected to LPS. (C) Quantitative analysis of the density of tomato-lectin positive cells in presence of LPS with and without 1 µmol melatonin. ***p<0.001, using two-way ANOVA with the Newman-Keuls correction.CC BY · PubMed Central

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Metadata

Authors
Olivier, P., Fontaine, R. H., Loron, G., Van Steenwinckel, J., Biran, V., Massonneau, V., Kaindl, A., Dalous, J., Charriaut-Marlangue, C., Aigrot, M.-S., Pansiot, J., Verney, C., Gressens, P., & Baud, O.
Affiliations
  • Inserm (FR)
  • Centre National de la Recherche Scientifique (FR)
Corresponding author
Baud, Olivier
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2009-09-22
Volume
4
Issue
9
Pages
e7128
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0007128
PMC
PMC2742165
OpenAlex
W1966357480
Cited by
119
References
37
Citations by year
2025: 3 · 2024: 7 · 2023: 5 · 2022: 11 · 2021: 17 · 2020: 6 · 2019: 8 · 2018: 4 · 2017: 7 · 2016: 6 · 2015: 7 · 2014: 9 · 2013: 9 · 2012: 9
FWCI
5.64
Type
journal article
Language
en
Update notices
correction (2009-10-15)
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Research Portal (King's College London) (publishedVersion, other-oa)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Ikaria
  • Institut National de la Santé et de la Recherche Médicale
  • Centre National de la Recherche Scientifique (UMR 7101)
Topics
  • Health Sciences › Medicine › Pediatrics, Perinatology and Child Health › Neonatal and fetal brain pathology
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
  • Life Sciences › Neuroscience › Endocrine and Autonomic Systems › Circadian rhythm and melatonin
Keywords
melatonin, oligodendrocyte maturation, white matter injury, fetal hypoxia, growth restriction, uterine artery ligation, myelination, microglial activation, astroglial reaction, APC, Olig2, neuroprotection, perinatal brain injury, melatonin receptors
MeSH terms
Animals, Animals, Newborn, Brain, Cells, Cultured, Immunohistochemistry, Melatonin, Microscopy, Fluorescence, Nerve Fibers, Myelinated, Oligodendroglia, RNA, Messenger, Time Factors, Rats, Sprague-Dawley, Gene Expression Regulation, Developmental, Cell Lineage, Rats

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