The cuprizone model for demyelination

Background –  Important advances in multiple sclerosis (MS) research have been made as a direct or indirect result of experiments in animal models for the disease, although MS is a disease only affecting humans. The cuprizone model is a model for toxic demyelination. In this model, young mice are fed with the copper chelator cuprizone, leading to oligodendrocyte death and a subsequent reversible demyelination. Spontaneous remyelination can be seen as early as 4 days after withdrawal of cuprizone.
Materials and methods –  This article reviews previous research on this model and discusses the potential of the model for future application in MS research.
Discussion –  The cuprizone model correlates with newer histopathological data in MS and is a valuable tool for studies on de- and remyelination. The use of the C57BL/6 strain offers the potential for future studies on transgene and knockout mice.     

Psychological stress effects on myelin degradation in the cuprizone‐induced model of demyelination

Multiple sclerosis (MS) is known as the most common demyelinating disease worldwide in which previous studies have shown that stress is a risk factor for the disease's onset and progression. Nevertheless, further studies are needed to investigate the consequences of stress in MS pathology. In this study, after 5 days of exposure to psychological and physical stress as a repetitive distress modality, rats were treated with cuprizone. The demyelination degree was compared in animal groups using Luxol fast blue staining, immunohistochemical staining for myelin basic protein and transmission electron microscopy. Outcomes revealed that animals exposed to stress prior to cuprizone ingestion, elicit more intense demyelination. Continuous psychological distress has more severe effects on myelin sheath destruction in the preclinical stage.     

Cuprizone‐induced demyelination in CNP::GFP transgenic mice

Cuprizone (bis‐cyclohexanone oxaldihydrazone) was previously shown to induce demyelination in white matter enriched brain structures. In the present study we used the cuprizone demyelination model in transgenic mice expressing the enhanced green fluorescent protein (GFP) under the 2′‐3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) promoter. The use of these particular transgenic mice allows easy detection of cells belonging to the entire oligodendroglial (OLG) lineage, ranging from OLG precursors to mature myelinating OLGs. We were able to evaluate the precise extent of oligodendroglial cell damage and recovery within the murine adult central nervous system (CNS) after inducing demyelination by acute cuprizone intoxication. A generalized loss of GFP+ cells was observed after cuprizone exposure and correlated with a decline in myelin basic protein (MBP) expression. OLGs were depleted in many brain areas that were previously thought to be unaffected by cuprizone treatment. Thus, in addition to the well‐known cuprizone effects on the medial corpus callosum, we also found a loss of GFP+ cells in most brain structures, particularly in the caudatus putamen, cortex, anterior commissure, olfactory bulb, hippocampus, optic chiasm, brainstem, and cingulum. Loss of GFP+ cells was accompanied by extensive astrogliosis and microglial activation, although neurons were not affected. Interestingly, cuprizone‐treated animals showed both activation of GFAP expression and a higher proliferation rate in subventricular zone cells. A week after cuprizone removal from the diet, GFP+ oligodendroglial cells began repopulating the damaged structures. GFP expression precedes that of MBP and allows OLG detection before myelin restoration. J. Comp. Neurol. 518:2261–2283, 2010. © 2010 Wiley‐Liss, Inc.     

The function of contactin‐2/TAG‐1 in oligodendrocytes in health and demyelinating pathology

The oligodendrocyte maturation process and the transition from the pre‐myelinating to the myelinating state are extremely important during development and in pathology. In the present study, we have investigated the role of the cell adhesion molecule CNTN2/TAG‐1 on oligodendrocyte proliferation, differentiation, myelination, and function during development and under pathological conditions. With the combination of in vivo, in vitro, ultrastructural, and electrophysiological methods, we have mapped the expression of CNTN2 protein in the oligodendrocyte lineage during the different stages of myelination and its involvement on oligodendrocyte maturation, branching, myelin‐gene expression, myelination, and axonal function. The cuprizone model of central nervous system demyelination was further used to assess CNTN2 in pathology. During development, CNTN2 can transiently affect the expression levels of myelin and myelin‐regulating genes, while its absence results in reduced oligodendrocyte branching, hypomyelination of fiber tracts and impaired axonal conduction. In pathology, CNTN2 absence does not affect the extent of de‐ and remyelination. However during remyelination, a novel, CNTN2‐independent mechanism is revealed that is able to recluster voltage gated potassium channels (VGKCs) resulting in the improvement of fiber conduction.     

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     

Altered transition metal homeostasis in the cuprizone model of demyelination

In the cuprizone model of demyelination, the neurotoxin cuprizone is fed to mice to induce a reproducible pattern of demyelination in the brain. Cuprizone is a copper chelator and it has been hypothesized that it induces a copper deficiency in the brain, which leads to demyelination. To test this hypothesis and investigate the possible role of other transition metals in the model, we fed C57Bl/6 mice a standard dose of cuprizone (0.2% dry chemical to dry food weight) for 6 weeks then measured levels of copper, manganese, iron, and zinc in regions of the brain and visceral organs. As expected, this treatment induced demyelination in the mice. We found, however, that while the treatment significantly reduced copper concentrations in the blood and liver in treated animals, there was no significant difference in concentrations in brain regions relative to control. Interestingly, cuprizone disrupted concentrations of the other transition metals in the visceral organs, with the most notable changes being decreased manganese and increased iron in the liver. In the brain, manganese concentrations were also significantly reduced in the cerebellum and striatum. These data suggest a possible role of manganese deficiency in the brain in the cuprizone model.     

Ultrastructural abnormalities and loss of myelinated fibers in the corpus callosum of demyelinated mice induced by cuprizone

It is now accepted that white matter abnormalities play an important role in demyelinating diseases and a wide range of psychiatric disorders. Experimental demyelination (especially induced by cuprizone) has been investigated extensively. However, details regarding demyelination and ultrastructural changes of myelinated fibers have not been previously reported. Therefore, we determined the extent of demyelination using quantitative stereology. Mice exposed to cuprizone in the current study showed abnormal anxiety‐like behavior without impaired spatial learning or memory. The myelinated fibers in whole corpus callosum of mice exposed to cuprizone showed extensive myelin deficiencies and occasional axonal injuries. The total length of the myelinated fibers in whole corpus callosum of mice exposed to cuprizone was significantly decreased by 45% compared with control mice. The loss of myelinated fibers was mainly due to the marked loss of the fibers with a diameter of 0.4 to 0.8 μm. The g‐ratio of the myelinated fibers in the corpus callosum of mice exposed to cuprizone (0.69 ± 0.02) was significantly decreased compared with control mice (0.76 ± 0.02). These results might help us to further understand the role of white matter abnormalities in demyelinating diseases or a wide range of psychiatric disorders. © 2016 Wiley Periodicals, Inc.     

Amyloid precursor protein and amyloid precursor-like protein 2 have distinct roles in modulating myelination,demyelination, and remyelination of axons

The identification of factors that regulate myelination provides important insight into the molecular mechanisms that coordinate nervous system development and myelin regeneration after injury. In this study, we investigated the role of amyloid precursor protein (APP) and its paralogue amyloid precursor-like protein 2 (APLP2) in myelination using APP and APLP2 knockout (KO) mice. Given that BACE1 regulates myelination and myelin sheath thickness in both the peripheral and central nervous systems, we sought to determine if APP and APLP2, as alternate BACE1 substrates, also modulate myelination, and therefore provide a better understanding of the events regulating axonal myelination. In the peripheral nervous system, we identified that adult, but not juvenile KO mice, have lower densities of myelinated axons in their sciatic nerves while in the central nervous system, axons within both the optic nerves and corpus callosum of both KO mice were significantly hypomyelinated compared to wild-type (WT) controls. Biochemical analysis demonstrated significant increases in BACE1 and myelin oligodendrocyte glycoprotein and decreased NRG1 and proteolipid protein levels in both KO brain tissue. The acute cuprizone model of demyelination/remyelination revealed that whereas axons in the corpus callosum of WT and APLP2-KO mice underwent similar degrees of demyelination and subsequent remyelination, the myelinated callosal axons in APP-KO mice were less susceptible to cuprizone-induced demyelination and showed a failure in remyelination after cuprizone withdrawal. These data identified APP and APLP2 as modulators of normal myelination and demyelination/remyelination conditions. Deletion of APP and APLP2 identifies novel interplays between the BACE1 substrates in the regulation of myelination.     

Episodic demyelination and subsequent remyelination within the murine central nervous system: changes in axonal calibre

Exposure of young adult C57BL/6 mice to cuprizone in the diet initiated profound and synchronous demyelination of the corpus callosum, which was virtually complete by 4 weeks of exposure. Interestingly, even in the face of a continued exposure to cuprizone, there was spontaneous remyelination 2 weeks later. This remyelination preferentially involved smaller calibre axons. There was a suggestion of yet another cycle of demyelination (at 10 weeks) and remyelination (at 12 weeks), but by 16 weeks of exposure, the regenerative capacity was exhausted and the animals were near death. The relapsing-remitting pattern suggests this may be a useful model for certain human demyelinating disorders. In contrast to the above chronic model, the corpus callosum from mice exposed to cuprizone for only 6 weeks continued to remyelinate, with 67% of the axons being myelinated or remyelinated at 10 weeks. Interestingly, a significant reduction in the mean value for axonal diameter was observed during acute demyelination. Upon remyelination, however, the axonal calibre distribution returned to near-normal. In contrast, when mice were maintained on a cuprizone diet for 16 weeks, the mean value for axonal diameter was reduced to 60% of normal. These results provide further evidence that the interactions between oligodendrocytes and axons alter axonal calibre.     

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.     

Mechanisms of lysophosphatidylcholine‐induced demyelination: A primary lipid disrupting myelinopathy

For decades lysophosphatidylcholine (LPC, lysolecithin) has been used to induce demyelination, without a clear understanding of its mechanisms. LPC is an endogenous lysophospholipid so it may cause demyelination in certain diseases. We investigated whether known receptor systems, inflammation or nonspecific lipid disruption mediates LPC‐demyelination in mice. We found that LPC nonspecifically disrupted myelin lipids. LPC integrated into cellular membranes and rapidly induced cell membrane permeability; in mice, LPC injury was phenocopied by other lipid disrupting agents. Interestingly, following its injection into white matter, LPC was cleared within 24 hr but by five days there was an elevation of endogenous LPC that was not associated with damage. This elevation of LPC in the absence of injury raises the possibility that the brain has mechanisms to buffer LPC. In support, LPC injury in culture was significantly ameliorated by albumin buffering. These results shed light on the mechanisms of LPC injury and homeostasis.     

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     

Effects of dietary intervention on MRI activity, de- and remyelination in the cuprizone model for demyelination

Whether differences in diet composition may influence demyelinating diseases remains controversial. The aim of this study was to analyse if diets with a different composition of polyunsaturated fatty acids (PUFAs) could influence demyelination and remyelination in cuprizone fed mice, a widely used animal model for de- and remyelination. C57Bl/6 mice were fed with 0.2% cuprizone on three different diets. The diets consisted of the same ingredients, except the lipid source, which came from 1) salmon fillets rich in marine n − 3 polyunsaturated fatty acids (PUFAs), 2) cod liver oil rich in marine n − 3 PUFAs, or 3) a control diet containing soybean oil rich in n − 6 PUFAs. After 5 weeks of cuprizone treatment, the mice given salmon-cuprizone had significantly less hyperintense lesion volume on brain magnetic resonance imaging (MRI) than the two other groups (P < 0.0005). After 6 weeks of cuprizone treatment, the salmon-cuprizone group had less demyelination in the corpus callosum, as measured with luxol fast blue (LFB) (P < 0.0005) and anti-proteolipid protein (PLP) (P = 0.014). The salmon-cuprizone group also had enhanced remyelination compared to the cod liver oil-cuprizone group (LFB; P = 0.003, PLP; P = 0.018). This study indicates that a fish rich diet may offer a protective role in demyelination. The source of N − 3 PUFAs, or other components in the fish, may be important, as no effect of a cod liver oil based diet was observed. This may be of importance related to the discrepant results in dietary intervention studies for demyelinating diseases.     

PLP/DM20 Expression and turnover in a transgenic mouse model of pelizaeus‐merzbacher disease

The most common cause of Pelizaeus‐Merzbacher (PMD) is due to duplication of the PLP1 gene but it is unclear how increased gene dosage affects PLP turnover and causes dysmyelination. We have studied the dynamics of PLP/DM20 in a transgenic mouse model of PMD with increased gene dosage of the proteolipid protein gene (Plp1). The turnover of PLP/DM20 were investigated using an ex‐vivo brain slice system and cultured oligodendrocytes. Homozygous mice have reduced PLP translation, markedly enhanced PLP degradation, and markedly reduced incorporation of PLP into myelin. Proteasome inhibition (MG132) prevented the enhanced degradation. Numerous autophagic vesicles are present in homozygous transgenic mice that may influence protein dynamics. Surprisingly, promoting autophagy with rapamycin decreases the degradation of nascent PLP suggesting autophagic vacuoles serve as a cellular storage compartment. We suggest that there are multiple subcellular fates of PLP/DM20 when overexpressed: the vast majority being degraded by the proteasome, a proportion sequestered into autophagic vacuoles, probably fused with endolysosomes, and only a small proportion entering the myelin sheath, where its association with lipid rafts is perturbed. Transgenic oligodendrocytes have fewer membrane sheets and this phenotype is improved with siRNA‐mediated knockdown of PLP expression that promotes the formation of MBP+ myelin‐like sheets. This finding suggests that RNAi technology is in principle applicable to improve CNS myelination when compromised by PLP/DM20 overexpression. © 2010 Wiley‐Liss, Inc.     

Three‐dimensional morphometric analysis of microglial changes in a mouse model of virus encephalitis: age and environmental influences

Many RNA virus CNS infections cause neurological disease. Because Piry virus has a limited human pathogenicity and exercise reduces activation of microglia in aged mice, possible influences of environment and aging on microglial morphology and behavior in mice sublethal encephalitis were investigated. Female albino Swiss mice were raised either in standard (S) or in enriched (EE) cages from age 2 to 6 months (young – Y), or from 2 to 16 months (aged – A). After behavioral tests, mice nostrils were instilled with Piry‐virus‐infected or with normal brain homogenates. Brain sections were immunolabeled for virus antigens or microglia at 8 days post‐infection (dpi), when behavioral changes became apparent, and at 20 and 40 dpi, after additional behavioral testing. Young infected mice from standard (SYPy) and enriched (EYPy) groups showed similar transient impairment in burrowing activity and olfactory discrimination, whereas aged infected mice from both environments (EAPy, SAPy) showed permanent reduction in both tasks. The beneficial effects of an enriched environment were smaller in aged than in young mice. Six‐hundred and forty microglial cells, 80 from each group were reconstructed. An unbiased, stereological sampling approach and multivariate statistical analysis were used to search for microglial morphological families. This procedure allowed distinguishing between microglial morphology of infected and control subjects. More severe virus‐associated microglial changes were observed in young than in aged mice, and EYPy seem to recover microglial homeostatic morphology earlier than SYPy . Because Piry‐virus encephalitis outcomes were more severe in aged mice, it is suggested that the reduced inflammatory response in those individuals may aggravate encephalitis outcomes.