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Cited by 8Multiple Sclerosis Research StudiesNeurogenesis and neuroplasticity mechanismsNeuroinflammation and Neurodegeneration Mechanisms

Cuprizone-Dependent De/Remyelination Responses and Functional Correlates in Mouse Strains Adopted to Model Relapsing, Chronic and Progressive Experimental Autoimmune Encephalomyelitis

Open access
Buonvicino, Daniela, Ranieri, Giuseppe, Chiarugi, Alberto·2021·Neurotoxicity Research
DOI 10.1007/s12640-021-00331-3PMC PMC8096768
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Abstract

NOD mice represent a unique strain that recapitulates some aspects of progressive MS when subjected to experimental autoimmune encephalomyelitis (EAE). It is unknown, however, whether a proneness to demyelination and/or defect in remyelination contribute to disease progression in NOD mice. Answering to this question might help deciphering the molecular and cellular events underpinning disease evolution in progressive MS. Here, we compared the cuprizone-dependent demyelination and remyelination responses, as well as their functional correlates, in NOD, C57BL/6, and SJL mice typically adopted to model progressive, chronic or relapsing EAE. We report that demyelination occurred to a similar extent in the three mice strains, and that in none of them there was evidence of axonal degeneration during prolonged demyelination. Moreover, immunostaining for GFAP + astrocytes, Iba1 + microglia, and NG2 + oligodendrocyte precursor cells similarly increased in the 3 mouse strains after cuprizone exposure. The mice underwent concomitant and complete remyelination 2 weeks after cuprizone withdrawal. On a functional level, NOD mice showed the earliest reduction of spontaneous motility and full recovery, but no impairment of motor skill. Conversely, C57BL/6 animals showed phasic reduction of both spontaneous motility and motor skill. Lastly, SJL mice presented the most severe neurological impairment with long-lasting reduction of spontaneous motility and motor skill. Overall, data suggest that the unique feature of EAE progression in NOD mice is not due to proneness to demyelination or intrinsic defects in myelin formation. Findings also unravel important functional differences in the response of the three mouse stains to cuprizone that can be harnessed to design and interpret future experiments.

AI snapshot

Written from the paper’s full text.

TL;DR

In female NOD, C57BL/6, and SJL mice, 6-week cuprizone caused similar callosal demyelination, glial/OPC activation, and complete remyelination 2 weeks after withdrawal, with no axonal degeneration. Despite similar pathology, strains differed functionally: NOD had earliest spontaneous motility loss and no motor-skill deficit, C57BL/6 had phasic motor-skill loss after withdrawal, and SJL had the most severe and long-lasting deficits.

Key findings

  • Six weeks of 0.4% cuprizone produced massive corpus callosum demyelination that did not differ among NOD, C57BL/6, and SJL mice; Bielschowsky staining showed no evidence of abnormal neurofilament staining, and myelin completely recovered in all three strains 2 weeks after cuprizone withdrawal.
  • Immunostaining for GFAP+ astrocytes, Iba1+ microglia, and NG2+ oligodendrocyte precursor cells increased after cuprizone exposure with no difference among strains (n = 4 per strain, 3 sections per animal).
  • Spontaneous motility declined in all strains, but NOD mice showed reduction at day 10 versus day 30 for C57BL/6 and SJL; NOD and C57BL/6 recovered 10 days after withdrawal, whereas SJL recovered only at day 70. Motor skill was unaffected in NOD, transiently reduced in C57BL/6 at days 10 and 20 after withdrawal, and most severely impaired in SJL during cuprizone treatment.
  • NOD glycemia measured weekly from week 12 never exceeded control values (< 250 mg/dL), arguing against diabetes as the cause of motor impairment.

Methods

  • Five-week-old female NOD/ShiLtj, SJL, and C57BL/6 mice were fed 0.4% cuprizone for 6 weeks, then standard chow; total cuprizone-exposed mice were 20 per strain. Histology used 10 mice per strain (5 at 6 weeks, 5 at 2 weeks after withdrawal), behavior used 10 mice per strain, and immunohistochemistry used 4 mice per strain.
  • Corpus callosum was assessed by Luxol Fast Blue for myelin and Bielschowsky silver staining for axons; GFAP, Iba1, and NG2 immunofluorescence was quantified with ImageJ by a blinded operator.
  • Spontaneous motility was measured by board test every 10 days; motor skill was measured by rota rod at 30 RPM with a 300 s cutoff. Statistics used ANOVA plus Tukey’s or Dunnett’s tests, or Kruskal–Wallis plus Dunn’s post test, with alpha 0.05.

Limitations

  • The paper states that cuprizone-dependent demyelination is massive and acute, so the data do not rule out a subtle remyelination defect in NOD mice under a less acute but more prolonged autoimmune demyelination such as EAE.
  • The authors state they do not have an explanation for the apparent inconsistency in NOD mice, namely earlier reduction of spontaneous motility despite complete maintenance of motor skill.
  • Evident from the text: only female mice were studied; the study uses cuprizone rather than direct EAE comparison; histological analysis focuses on corpus callosum; and functional assessment is limited to board test and rota rod.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1007/s12640-021-00331-3

Cites

27 of 27 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

11 of 19 cited sources checked · 6 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    NOD mice represent a unique strain that recapitulates some aspects of progressive MS when subjected to EAE.

    “NOD mice represent a unique strain that recapitulates some aspects of progressive MS when subjected to experimental autoimmune encephalomyelitis (EAE).”
    • Also stated in: § Introduction · boosted
      “we recently characterized the EAE NOD mouse model and provided neurological, neuropathological, and immune evidence that it recapitulates key features of PMS (Buonvicino et al. 2019).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Buonvicino et al. 2019 source
  • Premisecitation

    SJL mice are differently affected by cuprizone compared to C57BL/6 mice.

    “Data are also in keeping with prior work showing that SJL mice are differently affected by cuprizone compared to those of the C57BL/6 strain (Taylor et al. 2009).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Taylor et al. 2009 source
  • Premisecitation

    The neuropathological correlate of EAE NOD progression consists in widespread spinal cord demyelination invariably accompanied by axonal loss heralded by impairment of mitochondrial structure and function.

    “the neuropathological correlate consists in widespread spinal cord demyelination that is invariably accompanied by axonal loss heralded by impairment of mitochondrial structure and function.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Buonvicino et al. 2019 source
  • Premisecitation

    Progression of EAE in NOD mice can be counteracted by the bioenergetic-boosting drug dexpramipexole.

    “it can be counteracted by the bioenergetic-boosting drug dexpramipexole (Buonvicino et al. 2020).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Buonvicino et al. 2020 source
  • Premisecitation

    Reduction of cytotoxic CD8+ lymphocytes by dexamethasone does not provide protection from disease progression in EAE NOD mice.

    “reduction of this neuroimmune response by dexamethasone does not provide protection from disease progression (Buonvicino et al. 2019).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Buonvicino et al. 2019 source

      Section 3.6 reports that dexamethasone did not reduce disease progression in PPEAE NOD mice despite reducing neuroinflammation. It specifically reports a significant reduction in spinal-cord CD4+ lymphocytes, but does not establish a reduction in CD8+ lymphocytes; that cell-specific part of the claim is unsupported by the results passage. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • May combine two claims. 11
  • Classified as probably not a substantial claim. 4
  • The quote only partly states this claim. 1

  • May combine two claims.

    Premisecitation

    Activation of innate immunity within demyelinated regions during cuprizone treatment lasts about 1 month, which is shorter than the 2-4 months necessary for progression onset in NOD animals.

    “It is worth noting, indeed, that activation of innate immunity within demyelinated regions during cuprizone treatment (Fig. 2 and (Skripuletz et al. 2013)) lasts for an extent of time of about 1 month that is shorter than that of 2–4 months necessary for progression onset in NOD animals (Buonvicino et al. 2019).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Skripuletz et al. 2013 source
    • cites Buonvicino et al. 2019 source

      The citation is attached to the claim that progression in NOD mice takes 2–4 months to begin. Section 3.1 instead reports disease onset around day 20 after immunization in the 10-week-old mice used for the study and describes their disability as gradually worsening from onset. It reports 12–122 days to reach score 4 *after* onset, not a 2–4-month wait for progression to begin; the cuprizone duration is attributed to a different citation. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Cuprizone-dependent demyelination is massive within the corpus callosum but also occurs within additional brain regions such as the cortex and hippocampus.

    “cuprizone-dependent demyelination is massive within the corpus callosum but also occurs within additional brain regions such as the cortex and hippocampus (Koutsoudaki et al. 2009; Skripuletz et al. 2008; Steelman et al. 2012).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Koutsoudaki et al. 2009 source
    • cites Skripuletz et al. 2008 source

      The section “De-and Remyelination in the Cortex Are Similar to Changes in the Corpus Callosum” reports severe demyelination in the corpus callosum and complete demyelination in the cortex after cuprizone treatment. Our copy does not report hippocampal demyelination, so it does not support that part of the claim. (LLM, conf=0.89)

      Experimental — accuracy not yet measured
    • cites Steelman et al. 2012 source

      The Results report prominent demyelination in the cerebral cortex, and the Discussion reports nearly complete demyelination in caudal regions of the corpus callosum. But the paper says rostral callosal regions are less affected, so it does not support massive demyelination throughout the corpus callosum. It examines the dorsal hippocampal commissure, not demyelination of the hippocampus itself (Results; Discussion; page numbers unavailable). (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Histological sections were analyzed by a blinded operator.

    “sections were analyzed by a blinded operator by using ImageJ software (Buonvicino et al. 2019).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Buonvicino et al. 2019 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Exposure to a cuprizone-containing diet is a prototypical model of demyelination probably due to copper-chelating effects in oligodendrocytes.

    “Exposure to a cuprizone-containing diet is a prototypical model of demyelination probably due to the copper-chelating effects of the compound in oligodendrocytes (Vega-Riquer et al. 2019).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Vega-Riquer et al. 2019 source

  • May combine two claims.

    Findingown data

    C57BL/6 mice showed unaffected motor skill during cuprizone treatment but a delayed and phasic motor skill reduction at day 10 and 20 after cuprizone withdrawal.

    “C57BL/6 mice also showed unaffected motor skill during cuprizone treatment but, oddly, underwent a delayed and phasic motor skill reduction at day 10 and 20 after cuprizone withdrawal (Fig. 3e).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    Recovery of motor skill in SJL mice was more rapid than that of spontaneous motility but showed the highest degree of reduction.

    “Recovery of motor skill in SJL mice was more rapid than that of spontaneous motility but showed the highest degree of reduction.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    NOD mice showed reduced spontaneous motility at day 10 after cuprizone exposure, whereas C57BL/6 and SJL mice showed reduction at day 30.

    “NOD mice showed reduced motility at day 10 after cuprizone exposure, whereas C57BL/6 and SJL mice showed a reduction in spontaneous motility at day 30.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    There was no evidence of axonal degeneration or abnormal neurofilament staining during prolonged demyelination in NOD, C57BL/6, and SJL mice.

    “we found no evidence of abnormal neurofilament staining even in those regions undergoing complete demyelination.”
    • Also stated in: § Abstract · plain
      “in none of them there was evidence of axonal degeneration during prolonged demyelination.”
    • Also stated in: § Discussion · plain
      “none of the mouse strains showed sign of neurodegeneration during demyelination.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Peculiarities of the autoimmune attack during EAE and/or primary proneness to axonopathy in NOD mice are implicated in the cause of disease progression.

    “Rather, the present study, along with data showing an impairment of the T regulatory response in NOD mice (Tang et al. 2008), point to peculiarities of the autoimmune attack during EAE and/or primary proneness to axonopathy in this mouse strain.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Tang et al. 2008 source

  • May combine two claims.

    Interpretationreasoning

    Neither proneness to demyelination nor impaired remyelination processes underpin disease progression in EAE NOD mice.

    “As a whole, data suggest that neither proneness to demyelination nor impaired remyelination processes underpin disease progression in EAE NOD mice.”
    • Also stated in: § Abstract · plain
      “the unique feature of EAE progression in NOD mice is not due to proneness to demyelination or intrinsic defects in myelin formation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In EAE NOD mice, disease slowly but inexorably progresses to severe neurological impairment that always leads to death without signs of disease stabilization, at variance with RR-EAE in SJL or chronic EAE in C57BL/6.

    “in EAE NOD mice slowly but inexorably progresses to a severe neurological impairment that always leads to death without signs of disease stabilization (Buonvicino et al. 2019, 2020).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Buonvicino et al. 2019, 2020

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Premisenone given

    A prompt remyelination response typically follows cuprizone withdrawal and is sustained by OPC proliferation.

    “A prompt remyelination response typically follows cuprizone withdrawal and is sustained by olygodendrocyte precursor cell proliferation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All animal care and experimental procedures were performed according to European Community guidelines and were approved by the Committee for Animal Care and Experimental Use of the University of Florence.

    “All animal care and experimental procedures were performed according to the European Community guidelines for animal care (European Communities Council Directive 2010/63/EU) and were approved by the Committee for Animal Care and Experimental Use of the University of Florence.”
    Quote in the paperNot a substantial claim
13 of 13

  • The authors state this as an assumption.

    It is unknown whether a proneness to demyelination and/or defect in remyelination contribute to disease progression in NOD mice.

    “It is unknown, however, whether a proneness to demyelination and/or defect in remyelination contribute to disease progression in NOD mice.”

  • The authors state this as an assumption.

    The neuropathological basis of disease progression in EAE NOD mice and why other mouse strains do not show the same feature of disease evolution are still unknown.

    “In spite of these achievements, however, the neuropathological basis of disease progression in EAE NOD mice as well as why other mouse strains do not show the same feature of disease evolution are still unknown.”
2 of 2

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • If animals are provided standard chow after acute cuprizone-induced demyelination, endogenous remyelination occurs, which is on the histological level complete after 3-4 weeks.

    “If the animals are provided standard chow after acute cuprizone-induced demyelination, endogenous remyelination occurs, which is on the histological level complete after 3–4 weeks [25].”

    From How to Use the Cuprizone Model to Study De- and Remyelination (2024) · cites this paper as [25]

    The Experimental Procedures say mice were returned to standard chow after cuprizone feeding. The Results report complete recovery of callosal demyelination when assessed two weeks after withdrawal, but do not establish the claimed 3–4-week timeframe. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 3 panel b is assigned to two different outcomes

    Minor

    The Results identify panels a-c as spontaneous motility, which makes panel b a motility panel. The Figure 3 caption instead identifies panel b as motor skill.

1 of 1

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Related

Authors
Buonvicino, D., Ranieri, G., & Chiarugi, A.
Affiliations
University of Florence (IT)
Corresponding author
Buonvicino, Daniela
ORCID
Venue
Neurotoxicity Research
Publisher
Springer Science and Business Media LLC
Published
2021-01-21
Volume
39
Issue
3
Pages
658-666
ISSN
  • 1029-8428 (ISSN-L)
  • 1476-3524
DOI
10.1007/s12640-021-00331-3
PMC
PMC8096768
OpenAlex
W3126017551
Cited by
8
References
27
Citations by year
2026: 2 · 2025: 4 · 2024: 2
FWCI
0.29
Type
journal article
Language
en
Open access
Licence
Locations
  • Neurotoxicity Research (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Florence Research (University of Florence) (submittedVersion, other-oa)
  • PubMed Central (submittedVersion, cc-by)
Funders
  • Fondazione Italiana Sclerosi Multipla (2014/R/6)
  • Università degli Studi di Firenze
Topics
  • Health Sciences › Medicine › Pathology and Forensic Medicine › Multiple Sclerosis Research Studies
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
cuprizone, demyelination, remyelination, experimental autoimmune encephalomyelitis, multiple sclerosis, NOD mice, C57BL/6 mice, SJL mice, mouse strain differences, astrocytes, microglia, oligodendrocyte precursor cells, axonal degeneration, motor function, spontaneous motility, disease progression
MeSH terms
Remyelination, Animals, Chelating Agents, Chronic Disease, Cuprizone, Demyelinating Diseases, Disease Models, Animal, Encephalomyelitis, Autoimmune, Experimental, Female, Mice, Inbred C57BL, Myelin Sheath, Mice, Inbred NOD, Disease Progression, Mice

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