A functional role of NMDA receptor in regulating the differentiation of oligodendrocyte precursor cells and remyelination

Differentiation of oligodendrocyte precursor cells (OPCs) is the most important event for the myelination of central nervous system (CNS) axons during development and remyelination in demyelinating diseases, while the underlying molecular mechanisms remain largely unknown. Here we show that NMDA receptor (NMDAR) is a functional regulator of OPCs differentiation and remyelination. First, GluN1, GluN2A, and GluN2B subunits are expressed in oligodendrocyte lineage cells (OLs) in vitro and in vivo by immunostaining and Western blot analysis. Second, in a purified rat OPC culture system, NMDARs specially mediate OPCs differentiation by enhancing myelin proteins expression and the processes branching at the immature to mature oligodendrocyte transition analyzed by a serial of developmental stage‐specific antigens. Moreover, pharmacological NMDAR antagonists or specific knockdown of GluN1 by RNA interference in OPCs prevents the differentiation induced by NMDA. NMDA can activate the mammalian target of rapamycin (mTOR) signal in OPCs and the pro‐differentiation effect of NMDA is obstructed by the mTOR inhibitor rapamycin, suggesting NMDAR exerts its effect through mTOR‐dependent mechanism. Furthermore, NMDA increases numbers of myelin segments in DRG‐OPC cocultures. Finally, NMDAR specific antagonist MK801 delays remyelination in the cuprizone model examined by LFB‐PAS, immunofluorescence and electron microscopy. This effect appears to result from inhibiting OPCs differentiation as more NG2⁺ OPCs but less GST‐π⁺ mature oligodendrocytes are observed. Together, these results indicate that NMDAR plays a critical role in the regulation of OPCs differentiation in vitro and remyelination in cuprizone model which may provide potential target for the treatment of demyelination disease.     

Functional antagonism of sphingosine‐1‐phosphate receptor 1 prevents cuprizone‐induced demyelination

Recent evidence suggests that the oral drug Fingolimod (FTY720) for relapsing‐remitting multiple sclerosis (MS) may act directly on the central nervous system (CNS) and modulate disease pathogenesis and progression in experimental models of MS. However, the specific subtype of sphingosine‐1‐phosphate (S1P) receptors that mediates the effect of FTY720 on the CNS cells has not been fully elucidated. Here, we report that S1P receptor 1 (S1PR1) is elevated in reactive astrocytes in an autoimmunity independent mouse model of MS and that selective S1PR1 modulation is sufficient to ameliorate the loss of oligodendrocytes and demyelination. The non‐selective S1PR modulator, FTY720, or a short‐lived S1PR1‐specific modulator, CYM5442, was administered daily to mice while on cuprizone diet. Both FTY720‐ and CYM5422‐treated mice displayed a significant reduction in oligodendrocyte apoptosis and astrocyte and microglial activation in comparison to vehicle‐treated groups, which was associated with decreased production of proinflammatory mediators and down‐regulation of astrocytic S1PR1 protein. Interestingly, S1PR1 modulation during the early phase of cuprizone intoxication was required to suppress oligodendrocyte death and consequent demyelination as drug treatment from 10 days after the initiation of cuprizone feeding was no longer effective. CYM5442 treatment during the brief cuprizone exposure significantly prevented Il‐1β, Il‐6, Cxcl10, and Cxcl3 induction, resulting in suppression of subsequent reactive gliosis and demyelination. Our study identifies functional antagonism of S1PR1 as a major mechanism for the protective effect of FTY720 in the cuprizone model and suggests pathogenic contributions of astrocyte S1PR1 signaling in primary demyelination and its potential as a therapeutic target for CNS inflammation.     

Fibroblast growth factor 1 (FGFR1) modulation regulates repair capacity of oligodendrocyte progenitor cells following chronic demyelination

The adult mammalian brain contains multiple populations of endogenous progenitor cell types. However, following CNS trauma or disease, the regenerative capacity of progenitor populations is typically insufficient and may actually be limited by non-permissive or inhibitory signals in the damaged parenchyma. Remyelination is the most effective and simplest regenerative process in the adult CNS yet is still insufficient following repeated or chronic demyelination. Our previous in vitro studies demonstrated that fibroblast growth factor receptor 1 (FGFR1) signaling inhibited oligodendrocyte progenitor (OP) differentiation into mature oligodendrocytes. Therefore, we questioned whether FGFR1 signaling may inhibit the capacity of OP cells to generate oligodendrocytes in a demyelinating disease model and whether genetically reducing FGFR1 signaling in oligodendrocyte lineage cells could enhance the capacity for remyelination. FGFR1 was found to be upregulated in the corpus callosum during cuprizone mediated demyelination and expressed on OP cells just prior to remyelination. Plp/CreER^T:Fgfr1^fl/fl mice were administered tamoxifen to induce conditional Fgfr1 deletion in oligodendrocyte lineage cells. Tamoxifen administration during chronic demyelination resulted in reduced FGFR1 expression in OP cells. OP proliferation and population size were not altered one week after tamoxifen treatment. Tamoxifen was then administered during chronic demyelination and mice were given a six week recovery period without cuprizone in the chow. After the recovery period, OP numbers were reduced and the number of mature oligodendrocytes was increased, indicating an effect of FGFR1 reduction on OP differentiation. Importantly, tamoxifen administration in Plp/CreER^T:Fgfr1^fl/fl mice significantly promoted remyelination and axon integrity. These results demonstrate a direct effect of FGFR1 signaling in oligodendrocyte lineage cells as inhibiting the repair capacity of OP cells following chronic demyelination in the adult CNS.     

Cuprizone [Bis(Cyclohexylidenehydrazide)] is Selectively Toxic for Mature Oligodendrocytes

Cuprizone [bis(cyclohexylidenehydrazide)]-induced toxic demyelination is an experimental animal model commonly used to study de- and remyelination in the central nervous system. In this model, mice are fed with the copper chelator cuprizone which leads to oligodendrocyte death with subsequent demyelination. The underlying mechanisms of cuprizone-induced oligodendrocyte death are still unknown, and appropriate in vitro investigations to study these mechanisms are not available. Thus, we studied cuprizone effects on rat primary glial cell cultures and on the neuroblastoma cell line SH-SY5Y. Treatment of cells with different concentrations of cuprizone failed to show effects on the proliferation and survival of SH-SY5Y cells, microglia, astrocytes, and oligodendrocyte precursor cells (OPC). In contrast, differentiated mature oligodendrocytes (OL) were found to be significantly affected by cuprizone treatment. This was accompanied by a reduced mitochondrial potential in cuprizone-treated OL. These results demonstrate that the main toxic target for cuprizone is mature OL, whilst other glial cells including OPC are not or only marginally affected. This explains the selective demyelination induced by cuprizone in vivo.     

Differential local tissue permissiveness influences the final fate of GPR17‐expressing oligodendrocyte precursors in two distinct models of demyelination

Promoting remyelination is recognized as a novel strategy to foster repair in neurodegenerative demyelinating diseases, such as multiple sclerosis. In this respect, the receptor GPR17, recently emerged as a new target for remyelination, is expressed by early oligodendrocyte precursors (OPCs) and after a certain differentiation stage it has to be downregulated to allow progression to mature myelinating oligodendrocytes. Here, we took advantage of the first inducible GPR17 reporter mouse line (GPR17‐iCreER^T2xCAG‐eGFP mice) allowing to follow the final fate of GPR17⁺ cells by tamoxifen‐induced GFP‐labeling to unveil the destiny of these cells in two demyelination models: experimental autoimmune encephalomyelitis (EAE), characterized by marked immune cell activation and inflammation, and cuprizone induced demyelination, where myelin dysfunction is achieved by a toxic insult. In both models, demyelination induced a strong increase of fluorescent GFP⁺ cells at damaged areas. However, only in the cuprizone model reacting GFP⁺ cells terminally differentiated to mature oligodendrocytes, thus contributing to remyelination. In EAE, GFP⁺ cells were blocked at immature stages and never became myelinating oligodendrocytes. We suggest these strikingly distinct fates be due to different permissiveness of the local CNS environment. Based on previously reported GPR17 activation by emergency signals (e.g., Stromal Derived Factor‐1), we propose that a marked inflammatory milieu, such as that reproduced in EAE, induces GPR17 overactivation resulting in impaired downregulation, untimely and prolonged permanence in OPCs, leading, in turn, to differentiation blockade. Combined treatments with remyelinating agents and anti‐inflammatory drugs may represent new potential adequate strategies to halt neurodegeneration and foster recovery.     

Progesterone and nestorone promote myelin regeneration in chronic demyelinating lesions of corpus callosum and cerebral cortex

Multiple Sclerosis affects mainly women and consists in intermittent or chronic damages to the myelin sheaths, focal inflammation, and axonal degeneration. Current therapies are limited to immunomodulators and antiinflammatory drugs, but there is no efficient treatment for stimulating the endogenous capacity of myelin repair. Progesterone and synthetic progestins have been shown in animal models of demyelination to attenuate myelin loss, reduce clinical symptoms severity, modulate inflammatory responses and partially reverse the age‐dependent decline in remyelination. Moreover, progesterone has been demonstrated to promote myelin formation in organotypic cultures of cerebellar slices. In the present study, we show that progesterone and the synthetic 19‐nor‐progesterone derivative Nestorone® promote the repair of severe chronic demyelinating lesions induced by feeding cuprizone to female mice for up to 12 weeks. Progesterone and Nestorone increase the density of NG2⁺ oligodendrocyte progenitor cells and CA II⁺ mature oligodendrocytes and enhance the formation of myelin basic protein (MBP)‐ and proteolipid protein (PLP)‐immunoreactive myelin. However, while demyelination in response to cuprizone was less marked in corpus callosum than in cerebral cortex, remyelination appeared earlier in the former. The remyelinating effect of progesterone was progesterone receptor (PR)‐dependent, as it was absent in PR‐knockout mice. Progesterone and Nestorone also decreased (but did not suppress) neuroinflammatory responses, specifically astrocyte and microglial cell activation. Therefore, some progestogens are promising therapeutic candidates for promoting the regeneration of myelin. GLIA 2015;63:104–117     

Galanin is an autocrine myelin and oligodendrocyte trophic signal induced by leukemia inhibitory factor

In order to further investigate the molecular mechanisms that regulate oligodendrocyte (OC) survival, we utilized microarrays to characterize changes in OC gene expression after exposure to the cytokines neurotrophin3, insulin, or leukemia inhibitory factor (LIF) in vitro. We identified and validated the induction and secretion of the neuropeptide galanin in OCs, specifically in response to LIF. We next established that galanin is an OC survival factor and showed that autocrine or paracrine galanin secretion mediates LIF‐induced OC survival in vitro. We also revealed that galanin is up‐regulated in OCs in the cuprizone model of central demyelination, and that oligodendroglial galanin expression is significantly regulated by endogenous LIF in this context. We also showed that knock‐out of galanin reduces OC survival and exacerbates callosal demyelination in the cuprizone model. These findings suggest a potential role for the use of galanin agonists in the treatment of human demyelinating diseases. GLIA 2015;63:1005–1020     

Intravenous administration of human embryonic stem cell-derived neural precursor cells attenuates cuprizone-induced central nervous system (CNS) demyelination

S. J. Crocker, R. Bajpai, C. S. Moore, R. F. Frausto, G. D. Brown, R. R. Pagarigan, J. L. Whitton and A. V. Terskikh (2011) Neuropathology and Applied Neurobiology 37, 643–653 Intravenous administration of human embryonic stem cell‐derived neural precursor cells attenuates cuprizone‐induced central nervous system (CNS) demyelination Aims: Previous studies have demonstrated the therapeutic potential for human embryonic stem cell‐derived neural precursor cells (hES‐NPCs) in autoimmune and genetic animal models of demyelinating diseases. Herein, we tested whether intravenous (i.v.) administration of hES‐NPCs would impact central nervous system (CNS) demyelination in a cuprizone model of demyelination. Methods: C57Bl/6 mice were fed cuprizone (0.2%) for 2 weeks and then separated into two groups that either received an i.v. injection of hES‐NPCs or i.v. administration of media without these cells. After an additional 2 weeks of dietary cuprizone treatment, CNS tissues were analysed for detection of transplanted cells and differences in myelination in the region of the corpus callosum (CC). Results: Cuprizone‐induced demyelination in the CC was significantly reduced in mice treated with hES‐NPCs compared with cuprizone‐treated controls that did not receive stem cells. hES‐NPCs were identified within the brain tissues of treated mice and revealed migration of transplanted cells into the CNS. A limited number of human cells were found to express the mature oligodendrocyte marker, O1, or the astrocyte marker, glial fibrillary acidic protein. Reduced apoptosis and attenuated microglial and astrocytic responses were also observed in the CC of hES‐NPC‐treated mice. Conclusions: These findings indicated that systemically administered hES‐NPCs migrated from circulation into a demyelinated lesion within the CNS and effectively reduced demyelination. Observed reductions in astrocyte and microglial responses, and the benefit of hES‐NPC treatment in this model of myelin injury was not obviously accountable to tissue replacement by exogenously administered cells.     

G protein‐coupled receptor 30 contributes to improved remyelination after cuprizone‐induced demyelination

Estrogen exerts neuroprotective and promyelinating actions. The therapeutic effect has been shown in animal models of multiple sclerosis, in which the myelin sheath is specifically destroyed in the central nervous system. However, it remains unproven whether estrogen is directly involved in remyelination via the myelin producing cells, oligodendrocytes, or which estrogen receptors are involved. In this study, we found that the membrane‐associated estrogen receptor, the G protein‐coupled receptor 30 (GPR30), also known as GPER, was expressed in oligodendrocytes in rat spinal cord and corpus callosum. Moreover, GPR30 was expressed throughout oligodendrocyte differentiation and promyelinating stages in primary oligodendrocyte cultures derived from rat spinal cords and brains. To evaluate the role of signaling via GPR30 in promyelination, a specific agonist for GPR30, G1, was administered to a rat model of demyelination induced by cuprizone treatment. Histological examination of the corpus callosum with oligodendrocyte differentiation stage‐specific markers showed that G1 enhanced oligodendrocyte maturation in corpus callosum of cuprizone‐treated animals. It also enhanced oligodendrocyte ensheathment of dorsal root ganglion (DRG) neurons in co‐culture and myelination in cuprizone‐treated animals. This study is the first evidence that GPR30 signaling promotes remyelination by oligodendrocytes after demyelination. GPR30 ligands may provide a novel therapy for the treatment of multiple sclerosis. © 2012 Wiley Periodicals, Inc.     

Deficiency of NG2+ cells contributes to the susceptibility of stroke-prone spontaneously hypertensive rats

Aims: The purpose of this study is to investigate whether the NG2⁺ cells, a class of oligodendrocyte progenitor cells, is involved in the pathophysiology of stroke in stroke‐prone spontaneously hypertensive rat (SHR‐SP). Methods: SHR‐SP, SHR, Wistar‐Kyoto rats (WKY), and C57BJ/6 mice were used. Immunohistochemistry was conducted to evaluate the number of NG2⁺ cells in frozen brain sections. Demyelination was evaluated by Sudan black staining and serum level of myelin basic protein. Middle cerebral artery occlusion (MCAO) was performed to prepare experimental stroke model. Results: The number of NG2⁺ cells was significantly decreased in infarct core and increased in penumbra in WKY rats after MCAO. In brain sections of 6‐month‐old SHR‐SP, the number of NG2⁺ cells was significantly (P < 0.01) less than that in age‐matched SHR and WKY rats. However, this phenomenon was not observed in 3‐month‐old rats. Demyelination was found in 6‐month‐old SHR‐SP but not in 3‐month‐old SHR‐SP. Pharmacological treatment of cuprizone in mice induced demyelination and enlargement of cerebral infarction after MCAO. Conclusion: The decline of NG2⁺ cells may cause demyelination and contribute to the susceptibility of SHR‐SP to ischemic brain injury.     

Astrocyte-specific expression of a soluble form of the murine complement control protein Crry confers demyelination protection in the cuprizone model

Complement has been implicated as a potential effector mechanism in neurodegeneration; yet the precise role of complement in this process remains elusive. In this report, we have utilized the cuprizone model of demyelination-remyelination to examine the contribution of complement to disease. C1q deposition was observed in the corpus callosum of C57BL/6 mice during demyelination, suggesting complement activation by apoptotic oligodendrocyte debris. Simultaneously, these mice lost expression of the rodent complement regulatory protein, Crry. A soluble CNS-specific form of the Crry protein (sCrry) expressed in a transgenic mouse under the control of an astrocyte-specific promoter was induced in the corpus callosum during cuprizone treatment. Expression of this protein completely protected the mice from demyelination. Interestingly, sCrry mice had low levels of demyelination at later times when control mice were remyelinating. Although the sCrry transgenic mice had lower levels of demyelination, there was no decrease in overall cellularity, however there were decreased numbers of microglia in the sCrry mice relative to controls. Strikingly, sCrry mice had early recovery of mature oligodendrocytes, although they later disappeared. TUNEL staining suggested that production of the sCrry protein in the transgenic mice protected from a late apoptosis event at 3 weeks of cuprizone treatment. Our data suggest complement provides some protection of mature oligodendrocytes during cuprizone treatment but may be critical for subsequent remyelination events. These data suggest that temporal restriction of complement inhibition may be required in some disease settings.     

Long‐term impact of neonatal inflammation on demyelination and remyelination in the central nervous system

Perinatal inflammation causes immediate changes of the blood‐brain barrier (BBB) and thus may have different consequences in adult life including an impact on neurological diseases such as demyelinating disorders. In order to determine if such a perinatal insult affects the course of demyelination in adulthood as “second hit,” we simulated perinatal bacterial inflammation by systemic administration of lipopolysaccharide (LPS) to either pregnant mice or newborn animals. Demyelination was later induced in adult animals by cuprizone [bis(cyclohexylidenehydrazide)], which causes oligodendrocyte death with subsequent demyelination accompanied by strong microgliosis and astrogliosis. A single LPS injection at embryonic day 13.5 did not have an impact on demyelination in adulthood. In contrast, serial postnatal LPS injections (P0‐P8) caused an early delay of myelin removal in the corpus callosum, which was paralleled by reduced numbers of activated microglia. During remyelination, postnatal LPS treatment enhanced early remyelination with a concomitant increase of mature oligodendrocytes. Furthermore, the postnatal LPS challenge impacts the phenotype of microglia since an elevated mRNA expression of microglia related genes such as TREM 2, CD11b, TNF‐α, TGF‐β1, HGF, FGF‐2, and IGF‐1 was found in these preconditioned mice during early demyelination. These data demonstrate that postnatal inflammation has long‐lasting effects on microglia functions and modifies the course of demyelination and remyelination in adulthood. GLIA 2014;62:1659–1670     

Astrocyte‐targeted production of interleukin‐6 reduces astroglial and microglial activation in the cuprizone demyelination model: Implications for myelin clearance and oligodendrocyte maturation

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system. Interleukin (IL)−6 is a pleiotropic cytokine with a potential role in MS. Here we used transgenic mice with astrocyte‐targeted production of IL‐6 (GFAP‐IL6Tg) to study the effect of IL‐6 in the cuprizone‐induced demyelination paradigm, which is an experimental model of de‐ and re‐myelination, both hallmarks of MS. Our results demonstrated that cuprizone‐treated GFAP‐IL6Tg mice showed a significant reduction in astroglial and especially microglial activation/accumulation in the corpus callosum in comparison with the corresponding cuprizone‐treated wild type (WT). Production of a key microglial attracting chemokine CXCL10, as well as CXCL1 and CCL4 was lower in cuprizone‐treated GFAP‐IL6Tg mice compared with cuprizone‐treated WT. Reduced microglial cell accumulation was associated with inefficient removal of degraded myelin and axonal protection in cuprizone‐treated GFAP‐IL6Tg mice, compared with WT mice at the peak of demyelination. In addition, transgenic production of IL‐6 did not alter initial oligodendrocyte (OL) apoptosis and oligodendrocyte precursor cell recruitment to the lesion site, but it impaired early OL differentiation, possibly due to impaired removal of degraded myelin. Indeed, a microglial receptor involved in myelin phagocytosis, TREM2, as well as the phagolysosomal protein CD68 were lower in cuprizone‐treated GFAP‐IL6Tg compared with WT mice. Our results show for the first time that astrocyte‐targeted production of IL‐6 may play a role in modulating experimental demyelination induced by cuprizone. Further understanding of the IL‐6‐mediated molecular mechanisms involved in the regulation of demyelination is needed, and may have implications for the development of future therapeutic strategies for the treatment of MS. GLIA 2016;64:2104–2119     

Cuprizone induces similar demyelination in male and female C57BL/6 mice and results in disruption of the estrous cycle

Multiple sclerosis is a demyelinating neurological disease that is influenced by gender, primarily reflected in greater susceptibility to disease development in women than in men. Cuprizone intoxication, an animal model that is used to study demyelination and remyelination, has been extensively characterized in male C57BL/6 mice. Here, we have undertaken a comprehensive characterization of the morphological and cellular processes that occur in female C57BL/6J mice during cuprizone‐induced demyelination and subsequent remyelination and compared them with age‐matched male mice. We find that the pattern of demyelination and remyelination is similar between genders and that there is little or no difference in the loss or repopulation of mature oligodendrocytes or accumulation of reactive glia. Furthermore, examination of αERKO and βERKO mice suggests that estrogen receptors do not affect the outcome for demyelination or remyelination. Interestingly, we found that cuprizone treatment disrupts estrous cyclicity in female mice, possibly interfering with potential hormone influences on demyelination and remyelination. Therefore, cuprizone‐induced demyelination in C57BL/6J mice may have limitations as a model for the study of sex differences. © 2009 Wiley‐Liss, Inc.     

Lineage tracing reveals dynamic changes in oligodendrocyte precursor cells following cuprizone‐induced demyelination

The regeneration of oligodendrocytes is a crucial step in recovery from demyelination, as surviving oligodendrocytes exhibit limited structural plasticity and rarely form additional myelin sheaths. New oligodendrocytes arise through the differentiation of platelet‐derived growth factor receptor α (PDGFRα) expressing oligodendrocyte progenitor cells (OPCs) that are widely distributed throughout the CNS. Although there has been detailed investigation of the behavior of these progenitors in white matter, recent studies suggest that disease burden in multiple sclerosis (MS) is more strongly correlated with gray matter atrophy. The timing and efficiency of remyelination in gray matter is distinct from white matter, but the dynamics of OPCs that contribute to these differences have not been defined. Here, we used in vivo genetic fate tracing to determine the behavior of OPCs in gray and white matter regions in response to cuprizone‐induced demyelination. Our studies indicate that the temporal dynamics of OPC differentiation varies significantly between white and gray matter. While OPCs rapidly repopulate the corpus callosum and mature into CC1 expressing mature oligodendrocytes, OPC differentiation in the cingulate cortex and hippocampus occurs much more slowly, resulting in a delay in remyelination relative to the corpus callosum. The protracted maturation of OPCs in gray matter may contribute to greater axonal pathology and disease burden in MS.     

SOX17 is expressed in regenerating oligodendrocytes in experimental models of demyelination and in multiple sclerosis

We have previously demonstrated that Sox17 expression is prominent at developmental stages corresponding to oligodendrocyte progenitor cell (OPC) cycle exit and onset of differentiation, and that Sox17 promotes initiation of OPC differentiation. In this study, we examined Sox17 expression and regulation under pathological conditions, particularly in two animal models of demyelination/remyelination and in post‐mortem multiple sclerosis (MS) brain lesions. We found that the number of Sox17 expressing cells was significantly increased in lysolecithin (LPC)‐induced lesions of the mouse spinal cord between 7 and 30 days post‐injection, as compared with controls. Sox17 immunoreactivity was predominantly detected in Olig2⁺ and CC1⁺ oligodendrocytes and rarely in NG2⁺ OPCs. The highest density of Sox17⁺ oligodendrocytes was observed at 2 weeks after LPC injection, coinciding with OPC differentiation. Consistent with these findings, in cuprizone‐treated mice, Sox17 expression was highest in newly generated and in maturing CC1⁺ oligodendrocytes, but low in NG2⁺ OPCs during the demyelination and remyelination phases. In MS tissue, Sox17 was primarily detected in actively demyelinating lesions and periplaque white matter. Sox17 immunoreactivity was co‐localized with NOGO‐A+ post‐mitotic oligodendrocytes both in active MS lesions and periplaque white matter. Taken together, our data: (i) demonstrate that Sox17 expression is highest in newly generated oligodendrocytes under pathological conditions and could be used as a marker of oligodendrocyte regeneration, and (ii) are suggestive of Sox17 playing a critical role in oligodendrocyte differentiation and lesion repair. GLIA 2013;61:1659–1672     

Abbreviated exposure to cuprizone is sufficient to induce demyelination and oligodendrocyte loss

Cuprizone intoxication is one of several animal models used to study demyelination and remyelination. Early treatment protocols exposed mice to cuprizone for 6 weeks to induce demyelination; however, more recent reports have varied exposure times from 4 to 5 weeks. The goal of this study was to determine the minimal exposure of cuprizone in C57BL/6 mice that would induce a pathology of robust demyelination and gliosis similar to that described for a 5‐ or 6‐week treatment. We found that an abbreviated insult of only 2 weeks of exposure to cuprizone induced significant demyelination 3 weeks later (5‐week time point) but was somewhat variable. Three weeks of exposure to cuprizone produced extensive demyelination by week 5, equivalent to that observed with 5 weeks of exposure. The depletion of mature oligodendrocytes, as well as microglia and astrocyte accumulation, showed trends similar to those with 5‐week exposure to cuprizone. Once mature oligodendrocytes are perturbed after a 3‐week treatment, the progression to demyelination occurs without requiring further exposure. Furthermore, the early removal of cuprizone did not accelerate remyelination, suggesting that other sequences of events must follow before repair can occur. Thus, a short, “hit and run” CNS insult triggers a cascade of events leading to demyelination 2–3 weeks later. © 2012 Wiley Periodicals, Inc.