General introduction

Multiple sclerosis (MS), also known as disseminated sclerosis or encephalomyelitis disseminata, is a chronic, inflammatory, and demyelinating disease that affects the central nervous system (CNS). It is the most common neurological disorder in young adults in the Western hemisphere, being more common in females. MS appears in several courses with new symptoms either in discrete episodes (relapsing–remitting form) or slowly accumulating over time (progressive form). Most patients are first diagnosed with relapsing–remitting MS but enter at later stages the secondary-progressive stage. Between such episodes, symptoms may disappear completely. However, neurological problems often persist, especially as disease progresses. The most widely held hypothesis is that MS reflects a primary T cell-controlled autoimmune reaction against myelin-related proteins which predominantly affects the white matter. Activation of auto-reactive T cells subsequently results in the recruitment of brain resident and peripheral inflammatory cells. Even if the autoimmune pathogenesis of MS is a highly productive concept, its formal proof remains elusive [53, 74]. This scheme is oversimplified and cannot explain the entire spectrum of lesion formation in MS cohorts [9, 55]. Although inflammatory lesions typically appear in plaques within the white matter [9, 55], recent histopathological studies have convincingly shown that grey matter regions are also massively affected [32, 55]. In addition, diffuse white matter inflammation can be observed in the so-called normal appearing white matter [2, 74]. Beyond myelin destruction, acute axonal injury is frequently observed within actively demyelinating lesions [50]. In consequence, chronic plaques display a pronounced decrease in neurite density [55]. Although still a matter of discussion, it is assumed that inflammatory demyelination is not central to the pathogenesis of MS [13]. Since the pattern of demyelination differs between subgroups of patients, it is suggested that MS follows, at least to some extent, an individual and heterogeneous course [55]. The mechanism of this disease is largely unknown and there is no cure for it. However, there are several well-characterized experimental animal models that may help to understand pathological processes that may cause or affect this disease. Experimental therapies designed for MS rely on the testing of drugs in appropriate animal models before using it in clinical trials. Experimental allergic encephalomyelitis (EAE) is one of the most frequently used model which mimics MS pathology to study therapeutic intervention strategies. The cuprizone model represents a toxic experimental model that is frequently used. (For reviews of different MS animal models see [11, 52, 75, 98]).

The present article reviews recent data concerning demyelination and remyelination in the cuprizone model. Particular focus is given to similarities between human MS lesions and the cuprizone-induced pathology. The importance of gender and strain is highlighted. “Acute and chronic demyelination” is explained in detail and the differences are discussed. Finally, the roles of particular growth factors and neurotrophic cytokines, i.e. leukemia inhibitory factor (LIF), insulin-like growth factor-1 (IGF-1), and platelet-derived growth factor (PDGF) under different treatment paradigms are discussed.

Histopathological subtypes of MS: relevance of the cuprizone model

A detailed histopathological study conducted by Lucchinetti et al. has highlighted the profound heterogeneity in the pattern of demyelination among MS patients [60, 61]. Despite the similarities in the inflammatory reaction (inflammatory infiltrates composed mainly of T cells and macrophages), the investigated lesions can be segregated into four distinct patterns with the common hallmark of myelin destruction. Pattern I lesions can be considered as sites of active demyelination associated with a T cell- and macrophage-dominated inflammation [60]. Pattern II lesions additionally display deposition of immunoglobulin and activated complement at sites of active myelin destruction. Pattern I and II lesions can be described as macrophage- and antibody-mediated demyelinating events, respectively. Both lesion types are typically centered by small veins or venules and show sharply demarcated edges. Loss of different myelin proteins, such as myelin associated glycoprotein (MAG), myelin oligodendrocyte protein (MOG), proteo-lipid protein (PLP), or CNPase occurs to the same extent. Pattern III lesions also contain an inflammatory infiltrate, composed mainly of T cells and macrophages/microglia, but there is no deposition of immunoglobulin or complement. Furthermore, lesions are not centered by veins but preservation of a rim of myelin is frequently observed around an inflamed vessel within the demyelinated plaque. While type I and II lesions display a sharp lesion edge, the borders of pattern III lesions are ill-defined. Another striking difference is that pattern III lesions display a preferential loss of MAG. Within these areas, myelin-forming oligodendrocytes show nuclear condensation and fragmentation which are typical signs of apoptosis. Consequently, these cells are also stained by in situ tailing for DNA fragmentation. The active plaque border displays a pronounced loss of oligodendrocytes; the inactive center is almost completely devoid of oligodendrocytes. Pattern IV lesions contain microglia/macrophages and T cells (as pattern I lesions); however, due to the significant cell death in the periplaque white matter they resemble pattern III lesions. In summary, pattern III and IV lesions can be subsumed as lesions with signs of oligodendrocyte dystrophy/apoptosis. Pattern II is most frequently observed in MS cases, followed in the order of magnitude by pattern III, pattern I, and pattern IV. Interestingly, pattern III lesions were mainly found in patients with less than 2 months duration of disease before biopsy or autopsy [60].

In summary, pattern III lesions are fundamentally different in several important aspects compared to type I and II lesions. Such histopathological features have never been described in any model of autoimmune encephalomyelitis. These data suggest that in MS patients with pattern III lesions, demyelination is induced by a functional disturbance of oligodendrocytes, possibly as a result of infection or damage mediated by some unknown toxin.

Some authors state that the histopathological pattern of cuprizone-induced demyelination resembles type III lesions [92]. Indeed, there are some similarities between both forms. MAG mRNA levels seem to be more down-regulated during early demyelination compared to other myelin protein markers [71], demyelination does not present in a perivenous distribution, borders are ill-defined, and pronounced oligodendrocyte apoptosis can be found in active demyelinating lesions. However, other features of pattern III lesions, such as inflamed blood vessels and the presence of CD3+ T cells, are not observed in the cuprizone model. The later might be due to a direct suppression of cuprizone on T cell function [26, 65].

The biochemical events leading to oligodendrocyte damage in response to cuprizone treatment are cryptical [96, 100]. Although cuprizone is a copper chelator, there is a controversial discussion of changes in CNS copper levels under the toxic challenge [96, 100]. Furthermore, the mechanisms responsible for oligodendroglial death in MS lesions are not clear. It is questionable whether similar pathomechanisms are responsible for oligodendroglial loss in MS lesions and in the cuprizone model. The question arises, whether cuprizone-induced demyelination models myelin loss observed in human MS patients. This assumption is often challenged by experts in the field. However, we want to stress that MS is presently regarded as a disorder with many different features and facets. No current experimental animal model covers the entire complexity of MS in humans. Instead, the various animal models and intoxications can be applied to selectively investigate and uncover distinct pathological processes and repair mechanisms of MS.

MS animal models

EAE is the most frequently used animal model for human MS. This form of encephalomyelitis pictures an acute, chronic-relapsing, acquired, inflammatory demyelinating autoimmune disease (for reviews see [30, 52, 70, 98]). Animals are injected with proteins that are part of the myelin sheath surrounding axons. MBP, PLP, and MOG are frequently used antigens. These proteins, when systemically introduced, induce an autoimmune response which, in consequence, leads to an attack on the myelin. EAE can be induced in different animal species including mice, rats, guinea pigs, rabbits, macaques, rhesus monkeys, and marmosets [52, 99]. For various reasons such as the number of immunological tools, availability, lifespan, fecundity, and similarity of the resulting pathology to human MS appearance, mice and rats are the most frequently used species. Other experimental systems for MS studies encompass cuprizone, lysolecithin, or ethidium bromide-induced demyelination and are often pooled together as “toxic demyelination models” [11]. Another animal model used for more than 30 years to investigate MS-related symptoms is Theiler’s murine encephalomyelitis (TMEV). This model is based on virus-induced demyelination [75]. The cuprizone model is characteristic for toxic demyelination and represents a reversible demyelination and remyelination system [86, 89, 92]. In the following chapters, we will highlight the benefits and shortcomings of this particular animal model.

The cuprizone animal model

Cuprizone ingestion in mice induces a highly reproducible demyelination of distinct brain regions, among them the corpus callosum (CC) which represents the most frequently investigated white matter tract in this animal model [47, 87]. After 5–6 weeks of cuprizone treatment, the CC is almost completely demyelinated, a process called “acute demyelination”. Acute demyelination is followed by spontaneous remyelination during subsequent weeks when mice are fed normal chow. In contrast, remyelination is highly restricted when cuprizone administration is prolonged (12 weeks or longer), a process called “chronic demyelination”. During late stages of acute demyelination, spontaneous remyelination occurs partially but fails under a continued cuprizone challenge [4, 37, 89, 92].

First experiments using cuprizone as a toxic compound date back to the late 1960s and were conducted by Carlton [16, 17]. He reported that oxalic biscyclohexylidenehydrazide, a chelator used as a reagent for copper analysis, induces microscopic lesions in the brain accompanied by edema, hydrocephalus, demyelination, and astrogliosis [17]. He administered different doses of cuprizone mixed in basic chow ranging from 0.2 to 0.5%. Mice fed cuprizone showed signs of growth retardation in a dose-dependent manner. At higher concentrations (0.5%), cuprizone caused high mortality especially when introduced in the chow early during development. The mortality in 4-week-old mice (average weight 11.4 g) was 100%, whereas mortality declined to 20% in 8-week-old mice (average weight 26.0 g). In parallel, development of a severe hydrocephalus was more frequent in younger animals [17]. Based on these early findings, most laboratories use 6- to 9-week-old mice feeding a diet containing 0.2–0.3% cuprizone [1, 40, 73, 86]. To assure proper demyelination in aged animals, the cuprizone concentration needs to be increased [40, 62, 73, 80]. Furthermore, a strain-dependent susceptibility to cuprizone has to be considered [90]. For example, it has been shown that SJL mice display a unique pattern of demyelination that does not follow the profile that is described in C57BL/6 mice. SJL mice do not readily demyelinate at the midline within the CC but show greater demyelination directly lateral to midline [90].

There exists controversy about the site of lesion in cuprizone-induced demyelination. Demyelination was previously believed to predominantly affect white matter tracts (CC and the superior cerebellar peduncles) [92]. Consequently, the CC was in the focus of most of the studies. However, Carlton [17] already noted in his pioneering work myelin loss in other brain regions such as the cerebellar medulla. This was recently confirmed by our own studies revealing marked demyelination in deep cerebellar nuclei [35]. Cuprizone induces distinct demyelination in several brain regions such as the hippocampus [38, 73], the cerebellum [35, 84], the caudate-putamen, and the ventral part of the caudate nucleus (submitted). Interestingly, distinct grey matter regions are also affected in the cortex [36] and hippocampus formation [73]. Cuprizone-induced demyelination results from degeneration of supporting oligodendrocytes rather than a direct attack on myelin sheaths [16, 17, 89]. As pointed out later, distal parts of oligodendrocytes appear to be primarily vulnerable before cytopathological changes in the oligodendrocyte cell body can be observed.

The extent of demyelination can be assessed by various laboratory methods including histological procedures (luxol-fast blue staining, LFB), immunohistochemistry (IHC) against the myelin components MBP and PLP, electron microscopy, and gene/protein expression studies [1, 37, 73]. LFB is an alcohol-soluble counterpart of water-soluble Alcian Blue. Staining is due to the presence of lipoproteins and an acid–base reaction with salt formation [85]. This method shows only limited sensitivity and is, therefore, not suited to uncover grey matter pathologies. IHC is a better choice and allows visualization of the exact myelin status [73, 84]. Gene expression studies cover a wide scale of myelin and interacting proteins as well as other markers of mature and premature oligodendrocyte. Para-clinical methods such as MRT [1] or diffusion tensor magnetic resonance imaging [37] are also usable to assess ongoing pathology or recovery in longitudinal studies.

Mechanisms of cuprizone-induced demyelination

Cuprizone [oxalic acid bis(cyclohexylidene hydrazide)] is a well-known copper-chelating agent, discovered and described in the early 1950s [68]. The resulting copper–cuprizone complex is characterized by a very intense and unusual absorption band in the visible region, centered at 595 nm. Due to the highly chromogenic reaction of cuprizone with copper (II) ions, cuprizone was largely exploited for the quantitative determination of copper in various samples. Starting from the early 1970s, a lot of interest focused again on cuprizone, mainly within the biomedical scientific community, as this compound was reported to possess unique neurotoxic properties and, therefore, to serve as a valuable pharmacological tool for CNS demyelination and spongiosis in various laboratory animals [16, 17, 68]. The underlying mechanisms of cuprizone-induced oligodendrocyte cell death are not fully understood. It is well known that feeding of cuprizone causes the formation of mega-mitochondria in the liver. Liver tissue from cuprizone-fed animals was studied with respect to mitochondrial dysfunction [22, 39, 91]. The formation of mega-mitochondria also termed “giant mitochondria” is linked to metabolic injury [39]. Mega-mitochondria reveal short cristae that are restricted to the organelle periphery. The matrix is expanded and lacks membrane-like structures but is of normal density. Similar mitochondrial alterations are found when mice are fed nialamide, ethidium bromide, or are injected with triamcinolone. Such mega-mitochondria can emerge by two different mechanisms: Normal sized mitochondria are enlarged or mitochondria fuse [39]. Early observations indicate that both mechanisms, mitochondrial fusion and fission, are involved in mega-mitochondria formation in this animal model [39, 97]. However, also the inhibition of mitochondrial division has been postulated as a putative mechanism [28]. Since cuprizone induces alterations of mitochondrial morphology, it is speculated that the neurotoxic properties of this copper-chelating compound are due to a disturbance of cellular respiration, a key function of mitochondria [6]. Besides structural changes in mitochondrial morphology, it appears that the activity of a set of enzymes is disturbed prior to myelin loss during the first days or weeks of cuprizone exposure. Already in the early 1970s, Venturini [96] reported a decrease in monoamine oxidase and cytochrome oxidase activities in the brain and liver following cuprizone treatment. Cytochrome oxidase is a complex enzyme consisting of 13 subunits, 3 of which are encoded by the mitochondria genome and fulfill the catalytic function, whereas 10 additional nuclear-encoded subunits provide a regulatory potential to the enzyme [6]. Carbonic anhydrase II (CA II) activity declines in the brain of cuprizone-exposed animals well before demyelination develops [15, 49]. This enzyme plays an important role in buffering acute changes in pH in the brain. Disturbance of the intracerebral pH (acidosis) might contribute to oligodendrocyte stress and, in consequence, to cell death [45]. In the myelin sheath, CA II supports compaction of myelin by stimulating the co-transport and efflux of ions and water from myelin membranes [16, 17]. Interestingly, enzymatic activities were unaffected in copper-chelated cuprizone-treated animals [96]. In contrast, administration of copper to cuprizone-fed animals failed to reduce toxicity in other studies [16, 17]. It is still not clear whether the copper-chelating nature of cuprizone is implicated in its neurotoxic properties. It is also possible that cuprizone is actually chelating other heavy metals, such as Fe2+ and Mn2+ or Zn2+ [39, 96, 100]. The fact that a simple deficiency of metals such as copper, iron, or manganese does not produce morphological or biochemical changes similar to those produced by cuprizone militates against any mechanism that solely involves chelation. It may be speculated that a metabolite of cuprizone is in fact responsible for the observed effects. However, there is no evidence for a metabolic transformation of cuprizone. Additional mechanisms such as inhibition of oligodendrocyte precursor (OPC) differentiation [14] and secretion of pro-inflammatory cytokines from activated microglia/macrophages are also discussed [76]. In the next chapters, we aim to define and characterize acute and chronic demyelination by highlighting their cellular reactions.

Acute demyelination

Morell et al. [71] described the myelin status of the CC during a 6 weeks cuprizone (0.2%) exposure period using C57BL/6J mice. In LFB-stained sections, significant demyelination was evident after 3 weeks and approached a maximum until week 5. Thereafter, no further significant increase of demyelination was observed. Similar results have been reported by other groups [1, 57, 58, 73]. Expression of genes encoding proteins considered to be expressed by mature oligodendrocytes using Northern hybridization showed that the levels of MAG, ceramide galactosyltransferase (CGT), and MBP mRNAs were already diminished after 1 week cuprizone and further declined by 90% compared to control animals after 3 weeks. The time course of down-regulation of these mRNA species clearly precedes the onset of significant demyelination [71]. This observation was confirmed several times on the mRNA and protein level [1, 44, 73]. The expression of MBP, MAG, and CGT returned to normal values during the following 3 weeks, even when cuprizone exposure was continued. The reappearance of myelin protein expression during late stages of acute demyelination has also been reported by Lindner et al. [57] using C57BL/6J mice.

This clearly suggests that 6 weeks of cuprizone exposure cannot be regarded as pure “acute demyelination”. It is well known that during acute demyelination partial remyelination occurs between week 3 and 6. The depletion of oligodendrocytes, moreover, stimulates OPC proliferation, migration towards the demyelinated area, and differentiation as well as remyelination [1, 5, 37]. This complex process is initiated already during the acute demyelinating event. Our studies as well as data from other groups document that the expression of pro-myelination factors such as IGF-1 and FGF-2 is up-regulated very early during acute demyelination [48]. It is obvious that spontaneous remyelination or, at least, the approach occurs during the 6-week treatment interval and, therefore, the term “acute demyelination” is misleading.

However, it should be noticed that the magnitude of remyelination during the acute demyelinating event critically depends on several variables. Figure 1 summarizes results from four different groups. As referred above to the study of Morell et al. [71], expression levels of MBP, MAG, and CGT mRNAs returned to normal levels between week 3 and 6 despite continuous cuprizone exposure. The described recovery of myelin protein transcripts or proteins cannot be observed, or at least to a lesser extent, if cuprizone concentration is increased [57], or the weight of animals used at the beginning of cuprizone exposure decreased (see Fig. 1) [1]. Conflicting results might also be due to different regenerative properties of different brain regions. In the study of Morell et al. [71], total RNA from half of a mid-sagittally sectioned brain was used for gene expression study. In contrast, we and others used isolated RNA from specific brain regions, such as the CC or cerebral cortex. Different brain regions might display different remyelinating capacities as recently demonstrated by Gudi et al.: the time course and dynamics of demyelination and remyelination vary between the CC and cortex, suggesting different underlying pathomechanisms [36]. In consequence, results of gene expression studies during a 6-week treatment interval might be different, depending on which brain region is in the focus of the study. In summary, spontaneous remyelination, or at least the attempt, occurs during the 6-week treatment interval. As pointed out later, remyelinating processes during acute demyelination should be considered in data interpretation.

Fig. 1
figure 1

The figure summarizes data from four different studies using the cuprizone mouse model and studying myelination parameters during a 5–6 weeks exposure period. Age, weight, genetic background, animal provider, and cuprizone concentration in the chow are given for each study. The respective experimental approach to determine the myelination index (expressed in % of control) is additionally presented. Note the variations in the myelination index and the heterogeneity of the study design

Full size image

Recent studies have selected the CC or the rostral cerebellar peduncle as the site for the analysis of post-cuprizone demyelination and remyelination. However, the CC is not uniformly affected by the cuprizone challenge. As shown by Stidworthy et al. [87], the splenium of the CC and the dorsal hippocampal commissure regions were most severely affected. In contrast, large areas of the central, rostral, and ventral genu of the CC were only slightly affected. Changes in the proportion of myelinated and non-myelinated axons might additionally contribute to these disparities. This study clearly highlights the importance comparing the same regions between study groups in this animal model.

In summary, individual and strain differences need to be considered. Additionally, the weight of the animal in experiment appears to be critical for the outcome. To achieve both, homogenous weight and age distribution between experimental groups seems to be very important. A direct comparison of animals at the same age but different weights would be required to support these preliminary observations.

Chronic demyelination

The prolonged administration of cuprizone is usually called “chronic demyelination”. Most investigators claim that after chronic demyelination, remyelination fails [5, 37, 92]. This was first shown by Ludwin et al. [63]. Armstrong et al. [5] were able to demonstrate that the myelin status of the CC significantly improves after acute demyelination (6 weeks cuprizone) during a 6-week follow-up period, whereas endogenous remyelination failed after chronic cuprizone (12 weeks) exposure using mice on the 129 Sv-Ev:Black-Swiss genetic background. However, a repopulation by mature oligodendrocytes in the demyelinated CC was found during a 6-week recovery period. A delay of remyelination after chronic demyelination was also shown by other groups. Tansey et al. [89] reported that CA II protein increases during a 2-day normal chow period after acute demyelination for 4 weeks, whereas no such effect was seen when cuprizone administration is prolonged. Male swiss mice from the Charles River Laboratory were used. In the same study, the myelin status of the corpus striatum was assessed using anti-2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) IHC and a persisting severe demyelination in the center of corpus striatum was reported after 9 weeks cuprizone followed by 10 weeks without cuprizone [89]. This is in good agreement with another report studying the myelin status of the CC in sagittally orientated sections after chronic demyelination by cuprizone (0.2%) for 12 weeks and a follow-up of 12 weeks without treatment using anti-MBP IHC, the number of APC-positive mature oligodendrocytes, and the number of myelinated axons as parameters [37]. None of them improved during the 12 weeks remyelination period.

In a very recent study, chronic demyelination was induced by feeding cuprizone (0.2%) for 12 weeks, followed by a 12-week recovery period [58]. The endogenous remyelination capacity of the CC was compared with an acute demyelinated group (6 weeks cuprizone, 0.2%) in male C57BL/6 mice. Withdrawal of the toxin resulted in spontaneous remyelination. Although remyelination was extensive even after chronic cuprizone treatment, the rate of remyelination was considerably reduced. Accordingly, Nogo-A+-mature oligodendrocytes displayed a significantly slower recovery rate after chronic demyelination [58].

A closer look at the experimental framework of these studies reveals profound heterogeneity between the groups. Armstrong et al. [5] used 8-week-old male mice with a 129 Sv-Ev:Black-Swiss background and put them on a 0.2% cuprizone diet, whereas Tansey et al. [89] used 6-week-old male swiss mice and applied 0.5% cuprizone. There were also different experimental conditions between the studies. It seems that the genetic background of the study animals could be one explanation for the controversial findings. Additionally, the sex of the study animals might influence the outcome. In the study of Harsan et al., female animals were used, whereas male animals where used by Lindner et al. [37, 58]. Gender-based differences or gender-related hormonal influences are well accepted to influence remyelination efficacy [1, 90]. We will come back to this point below. Concerning the endogenous remyelination capacity after chronic demyelination, it becomes evident that remyelination is, at least, delayed compared to spontaneous remyelination after acute demyelination. Whether this applies to all brain regions and whether regional differences in the remyelination capacity exist have to be clarified in future studies.

In MS, brain plaques partially remyelinate by endogenous mechanisms. The reappearance of oligodendrocytes in demyelinated lesions is frequently seen in MS plaques [77]. Patrikios et al. [77] systematically analyzed the incidence and distribution of completely remyelinated lesions (so-called shadow plaques) or partially remyelinated lesions (shadow plaque areas) in 51 autopsies of patients with different clinical courses and disease durations. The extent of remyelination was extremely variable between cases. In 20% of the patients, the extent of remyelination was extensive with 60–96% of the global lesion area remyelinated. Remyelination was found not only in patients with relapsing MS, but also in a subset of patients with progressive disease. In most of the chronic lesions, however, oligodendrocytes with multiple extended processes that are associated with demyelinated axons can be found. These pre-myelinating oligodendrocytes failed to myelinate. Therefore, remyelination seems not to be limited by an absence of OPC or their failure to generate oligodendrocytes [18, 78].

Remyelination protects against progressive axonal injury and reduces progression of long-term disability. It is not clear why remyelination occurs only in a subset of patients, whereas it fails in others. Remyelination might be diminished due to precursor cell depletion and/or a non-permissive local environment. Our recent studies and results from other groups [37] deny the concept of a depletion of OPC in the demyelinated white and grey matter. Olig2+ cells are frequently found within the CC and the adjacent neocortex after 13 weeks cuprizone exposure. The same applies for another toxic demyelination model [76]. Therefore, it is likely, that a non-permissive local environment is mainly responsible for the failure of adequate remyelination in this animal model. This is also supported by the observation that abundant PLP mRNA-expressing cells are present in the CC after chronic demyelination despite remyelination failure [5]. Furthermore, remyelination can be induced after chronic demyelination by different strategies such as thyroid hormone or progesterone therapy (unpublished, [37]). We assume that positive effects of both therapeutic strategies are due to indirect effects on astrocyte/microglia stimulation, growth factor release and, in consequence, OPC proliferation, migration, and differentiation.

The well-described diminishing remyelination capacity of the brain due to aging is another approach to study underlying mechanisms of remyelination failure [80, 81]. Shen et al. [80] used the cuprizone model to investigate the molecular mechanisms underlying remyelination failure in old animals. Although the pattern and degree of demyelination after cuprizone feeding is the same in young and old mice, there is a clear consistent reduction in remyelination capacity in the old mice. Shen et al. demonstrated that this impaired remyelination in older animal was not the result of an altered inflammatory response, an abnormal increase in the number of ‘reactive’ astrocytes, or axonal degeneration at the lesion site but rather the result of loss of ‘epigenetic memory’ in the OPCs. The epigenetic changes detected in the OPCs of old mice resulted in abnormal gene transcription patterns during remyelination. Besides epigenetic memory loss, other potential mechanisms behind the poor remyelination capacity in older animals can be adequately studied using the cuprizone model.

Gender aspect in the cuprizone model

As pointed out above, gender differences, i.e. plasma levels of protective hormones can certainly influence cuprizone-induced CNS pathology. Clinical and experimental observations describe sexual dimorphisms in MS with a higher prevalence in females of childbearing age and different clinical courses in the female and male population. These data point at a potential role for gonadal steroids and other sex-specific hormones such as prolactin, oxytocin, etc. for the predisposition and severity of MS in humans [1, 3, 20, 25, 46]. The idea that sex steroids are implicated in the susceptibility and course of MS and that they might be a therapeutic option also originates from studies revealing that pregnancy affects MS disease activity [20]. A reduction of relapse frequency is observed during pregnancy, in particular during the last trimester when estrogen and progesterone plasma levels are at their maximum [1, 20]. Postpartum, MS symptoms exacerbate again and the incidence rates of MS are reduced in multipara compared to nullipara. Furthermore, is has been shown that females remyelinate more efficiently than males following demyelination in the aged adult CNS [56]. Removal of gonads did not affect remyelination of either gender. These results indicate a sex-associated divergence in remyelination efficiency that occurs with aging that is unaffected by the removal of gonadal sources of sex steroid hormones. After having shown in the EAE model that hormonal substitution can ameliorate symptomatology, non-pregnant female patients were treated with estriol in an attempt to reflect pregnancy [33, 83]. This strategy of therapy underscored the importance of estrogens as therapeutics. All these data suggest that sex steroids, gender, and myelin are important dependent variables.

This conclusion is also documented by our recent findings. We have tested the hypothesis whether sex steroids, 17β-estradiol and progesterone alone or in a combined treatment, influence the myelin status during acute demyelination. Steroids were injected into male mice at the age of 6 weeks for a 5-week period into the neck region twice a week at concentrations yielding steroid plasma levels which conform to plasma concentrations measured during the last trimester of rodent pregnancy. Our data show that both hormones possess the capacity to reduce demyelination effectively but only the combined approach resulted in highly significant restoration of myelination in the CC [1]. The improvement of the myelination index was paralleled by enhancement of OPC proliferation, PDGFα-receptor expression, and precursor migration from the CC midline to the lateral parts followed by an induction of the expression of myelin proteins. In addition, we observed an early astrogliosis in the demyelinated areas and a moderate stimulation of IGF-1 expression. This is of particular interest, since IGF-1 synergizes with FGF-2 to stimulate oligodendrocyte progenitor entry into the cell cycle [41, 42]. This effect was potentiated by the application of hormones. Moreover, the number of microglial cells in the affected areas was significantly increased in hormone-treated animals compared to controls (see Fig. 2).

Fig. 2
figure 2

Effect of cuprizone (cup, 5 weeks) and estrogen (E) and progesterone (P) on myelination of the corpus callosum (CC) in male mice. a Luxol-fast blue/cresyl violet stainings. Note that cuprizone administration causes a massive demyelination of the CC (arrowheads) which is partly restored by E and P treatment and almost completely prevented by a combined hormone treatment. b Immunocytochemical staining for the mature oligodendrocyte protein “adenomatous polyposis coli gene clone CC1 (APC-CC1)” in the midline of the CC as denoted by the quare in a (cup + E/P). It is evident that cuprizone-induced disappearance of oligodendrocytes is diminished by E/P. c T2-weighted magnetic resonance imaging (MRI). Myelin signal intensity in the CC is reflected by a black hypo-intense (strong myelination) and white hyper-intense signal (low myelination). Data are modified from Acs et al. [1]

Full size image

These data clearly point at the interference of gender, sex hormones, and cuprizone-induced demyelination. Individual cellular responses and the cell–cell communication between astroglia, microglia, and oligodendroglia might be targeted by sex hormones.

Therapeutic intervention in the cuprizone model

In this chapter, we present novel findings in the cuprizone model with respect to novel therapeutic strategies in human MS. Although extensive remyelination may occur in some 20% of MS cases, repair processes fail frequently despite the presence of OPC in the MS plaque that are in principle capable of remyelinating axons [18]. The reasons for this remyelination failure may be manifold [29, 86] and the knowledge of the molecular mechanisms responsible for remyelination is only limited. A large number of factors are known to influence the complex process of remyelination including cytokines and growth factors. In the following, we describe and discuss the effects of three different cytokines on cuprizone-induced demyelination and remyelination after an acute or chronic demyelinating event. We will emphasize the protective role of LIF, IGF-1, and PDGF, notwithstanding that there are many other factors that have been investigated in this model in the last few years that also would need consideration [10].

LIF

Neurotrophic cytokines such as LIF play a key role in neuronal and oligodendrocyte survival [59]. The relevance of LIF for MS was recently reviewed by Linker et al. In vitro and in vivo LIF supports the differentiation and survival of OPC and prevents oligodendrocyte apoptosis in response to serum withdrawal or cytokine challenge. LIF also supports motoneuron survival under different experimental conditions [59]. Besides its effects on neuronal and glial cells, LIF possesses pleiotropic functions in different cell types and organs [66]. T cells and monocytes in vitro and in inflammatory MS lesions produce LIF [94, 95]. Furthermore, LIF mRNA expression is induced in the CC during early acute demyelination in the cuprizone model [27]. After a 4-week cuprizone challenge, mice were given LIF. These animals displayed an improved myelin status as well as a reduced loss of GST-pi+ mature oligodendrocyte in the midline of CC compared to placebo-treated animals [66]. Concordantly, LIF knock-out animals revealed aggravated demyelination after 3 weeks cuprizone exposure compared to wild-type (wt) animals [66]. Protective effects of LIF might be achieved by two different mechanisms. LIF could prevent mature oligodendrocyte cell loss and, in consequence, diminish the loss of myelin sheaths. LIF could also induce precursor cell proliferation and differentiation in the injured CC and, thus, promote remyelination. It appears that the first assumption is mainly responsible for the observed effects, since the quantification of NG2+ cells (OPC) was not influenced by LIF [66]. Furthermore, LIF knock-out mice exhibit intensified demyelination and increased numbers of caspase-3-positive oligodendrocytes compared to wt littermates after a 3-week cuprizone challenge [27, 66]. Taken together, these data indicate that LIF prevents cuprizone-induced oligodendrocyte loss and does not stimulate remyelination during the acute demyelination.

In a next step, the effect of LIF during spontaneous remyelination after a 5-week cuprizone intoxication period was investigated. Exogenous LIF failed to promote remyelination after acute demyelination. In contrast, mice deficient for endogenous LIF production showed delayed remyelination after acute demyelination. This discrepancy might be due to abundant LIF expression during early remyelination. Additional exogenous LIF administration, therefore, might fail to be beneficial. Interestingly, numbers of GST-pi+ (mature) oligodendrocytes and NG2+ cells (progeni) were similar in wt and LIF −/− animals. This discrepancy of myelin status and cell types involved in remyelination implicates that the proliferation, migration, and differentiation of OPC is not under the direct control of LIF. Nevertheless, final steps of remyelination, namely axon sheathing and myelin production, might be positively influenced.

These studies demonstrate the complexity of what is called “acute demyelination”. Cuprizone administration induces oligodendrocyte apoptosis, microglia cell activation, phagocytosis of myelin sheaths and, in consequence, demyelination. At the same time, demyelination induces an activation of neural stem/progenitor cells (NSPCs). This occurs in so-called neurogenic regions of the adult brain, such as the subventricular zone (SVZ) of the lateral ventricles. Unpublished data from our group support this idea and demonstrate an increase of proliferating cells (BrdU incorporation) within the SVZ around the lateral ventricles (Fig. 3c). In parallel, the number of nestin+ NSPCs is elevated after 3 weeks cuprizone exposure in the SVZ (Fig. 3d). Activated NSPCs probably migrate towards the demyelinated CC, differentiate into OPC and finally into mature oligodendrocytes, contributing to the formation of new myelin sheets. Interestingly, this activation also occurs in NSPCs located in nonconventional neurogenic regions [42, 43] affected by cuprizone, such as the thalamus or corpus striatum (Fig. 3e, f). Since nestin has also been found in reactive astrocytes in the cuprizone model, it has to be clarified to what extent these nestin+ cells participate in remyelination.

Fig. 3
figure 3

Schematic illustration of the complexity of cellular reactions during “acute demyelination”. a Summary of histopathological findings in the acute (3–6 weeks) demyelinated brain. Besides myelinated axons, some axons are completely demyelinated. Yellow cells indicate phagocytosis of the myelin sheet by microglia/macrophages, a typical sign of successive demyelination. Oligodendrocyte precursor cells proliferate, migrate towards the injured lesion (green arrowhead), and form new myelin sheets. b shows well-known aspects during acute demyelination. Oligodendrocytes undergo apoptosis (caspase-3), microglia cells are attracted (Iba-1), and, finally, axons are demyelinated (PLP). c–f Summarizes regenerative aspects during acute cuprizone-induced demyelination. High proliferation activity within the subventricular zone (SVZ) of the lateral ventricles is indicated by BrdU incorporation (c). Besides proliferation, nestin-positive neural stem/progenitor cells increase in number in the neurogenic area (SVZ) (d). However, an increase in the number of nestin-positive neural stem/progenitor cells can also be found in nonconventional neurogenic regions in the brain, such as the thalamus (e) or the corpus striatum (f). Both mechanisms might contribute to remyelinating processes during an acute demyelinating event

Full size image

It is not clear at the moment at what particular stage first signs of remyelination start during early demyelination. A previously conducted Affymetrix gene expression analysis conducted in our lab suggests that remyelinating pathways are activated very early during acute demyelination. In male mice fed cuprizone for 2 weeks, gene expression in the CC revealed that genes known to be implicated in remyelination, such as IGF-1 [48], epidermal growth factor [34], transforming growth factor-beta-1 [24], and FGF-2 [4] were up-regulated, whereas anti-myelination factors, such as semaphorin-4D [88], were down-regulated. Therefore, protective effects of any therapeutic intervention during cuprizone-induced demyelination might be due to (1) the prevention of oligodendrocyte apoptosis or (2) induction of remyelination at any step described above.

IGF-1

IGF-1 has been shown to induce myelination in vitro and in vivo, while also protecting mature oligodendrocytes from a pathological insult [23, 31, 67]. Furthermore, IGF-1 promotes the long-term survival of mature oligodendrocytes in culture and inhibits mature oligodendrocyte apoptosis in vitro [19, 31]. As already shown for LIF and FGF-2, IGF-1 transcripts are robustly up-regulated during acute demyelination [1, 48, 69]. In cuprizone-treated animals, there is a strong IGF-1 mRNA hybridization signal detectable in the CC, caudate-putamen, diagonal bands, anterior commissure, superior cerebellar peduncles, and tegmental tracts. White matter tracts which are not affected by demyelination do not display increased IGF-1 transcript levels. It was further shown that IGF-1 is expressed by hypertrophic astrocytes during cuprizone-induced demyelination [48].

Transgenic IGF-1 (IGF-1tg) animals express IGF-1 under the mouse metallothionein-I promoter [67]. To investigate the impact of IGF-1 over-expression on myelin status during cuprizone-provoked demyelination, electron microscopy studies and quantification of the percentage of myelinated axons during a 5-week cuprizone exposure period were assessed in wt and IGF-1tg animals. Furthermore, mature oligodendrocytes, apoptosis of mature oligodendrocytes, and microglia accumulation were studied. After 3 weeks of cuprizone, the medial region of the CC was significantly demyelinated in both, wt and IGF-1tg animals [67]. In contrast, the number of GST-pi+ mature oligodendrocytes within the medial CC was significantly higher in IGF-1tg animals compared to wt. It is surprising and unexpected that cuprizone-provoked loss of mature oligodendrocytes was less pronounced in IGF-1tg animals at week 3, whereas the number of myelinated axons did not differ between both groups at this particular time point. The authors suggested in this study that high levels of IGF-1 in IGF-1tg mice can protect cell bodies of mature oligodendrocytes from apoptosis, whereas myelin sheets are not protected [67]. This phenomenon, i.e. discrepancy between oligodendrocyte cell loss and effects on myelin, might relate to a process called “dying-back oligodendrogliopathy” [54, 64]. This concept was first introduced as a mechanism of cell damage in experimental models of axonal degeneration [79]. It can be induced by a variety of toxic or metabolic insults, responsible for different forms of axonal neuropathy [54], and is thought to be the consequence of a chronic metabolic disturbance of the neuron which is manifested primarily at the most distal cell processes. Morphologically, it is characterized by the accumulation of neurofilaments, mitochondria, and lysosomes in affected axons. In spite of extensive degeneration of distal axons, the neuronal perikarya may survive the insult for extended periods. In 1981, Ludwin and Johnson [64] suggested that a similar dying-back process may not only affect nerve cells but also oligodendrocytes. In cuprizone-induced demyelination, they observed primary degenerative changes in the peri-axonal collar of oligodendrocyte processes which was followed by demyelination and to some degree oligodendrocyte destruction [64]. Similar alterations of oligodendrocytes have been described in brain biopsies from MS plaques [60, 61]. The findings of Mason and colleagues suggest that IGF-1 over-expression protects against “dying-back oligodendrogliopathy” during the 3 weeks cuprizone exposure period. However, since remyelination processes might start very early during cuprizone-induced demyelination, this discrepancy between myelin status and the number of mature oligodendrocyte cells might also be due to an early recruitment of oligodendrocyte precursor cells in the demyelinated midline and differentiation into GST-pi+ cells. One might argue that this is unlikely since NG2+ cell numbers were not increased at week 3, 4, or 5 of cuprizone exposure in IGF-1tg animals. However, it can be assumed that repopulation occurs between week 0 and 3 and, therefore, was not detectable at late stages of demyelination. The importance of IGF-1 signaling for the recruitment of mature oligodendrocytes during remyelination has been shown by the same group in a later study.

PDGF

OPC in MS lesions and in experimental demyelination have been identified by expression of NG2 proteoglycan and platelet-derived growth factor-α receptor (PDGF-α receptor) [72, 93, 99]. PDGF-α ligand activation of PDGF-α receptor signaling can stimulate the proliferation of OPC in response to acute experimental demyelination [72]. To investigate the role of PDGF-α during demyelination or remyelination, Woodruff et al. used human PDGF-α transgenic (hPDGF-αtg) mice with the human PDGF-α gene under control of the GFAP promoter (astrocytes are a major source of endogenous PDGF-α in demyelinating lesions). After 4 weeks cuprizone exposure (0.2%), OPC increased by four times in the CC of PDGFα over-expressing animals compared to controls. This study clearly shows that PDGF-α can act as a mitogen on OPC or neural stem/progenitor cells and, therefore, increases the number of OPC in a demyelinated lesion [99]. In another study, it was investigated whether PDGF-α over-expression influences remyelination after chronic cuprizone exposure (12 weeks cuprizone followed by up to 6 weeks of remyelination). The same model system, i.e. human PDGFα protein over-expression under the control of GFAP promoter, was applied. During the cuprizone treatment period, mice of both genotypes exhibited a similar disease progression with a similar extent of demyelination at 12 weeks of cuprizone ingestion determined by anti-MOG IHC and PLP mRNA in situ hybridization [93]. During the recovery period with normal chow for 6 weeks, myelination of the CC in hPDGF-αtg mice improved significantly compared to 12-week cuprizone values, whereas extensive demyelination persisted in wt mice during the recovery period. When investigating the SVZ of the lateral ventricles for proliferation activity and OPC, surprisingly no differences between groups were seen. TUNEL staining during the 6-week recovery period revealed that there is ongoing apoptosis after chronic demyelination. Apoptosis was significantly diminished in PDGFα over-expressing animals. The authors conclude that protective effects of hPDGF-αtg over-expression are due to an attenuation of oligodendrocyte apoptosis in the remyelination phase after chronic demyelination. In contrast, cuprizone induced mature oligodendrocyte apoptosis during the demyelination period was not different in hPDGF-αtg over-expressing and wt animals. Thus, it is possible that cuprizone-induced oligodendrocyte apoptosis is triggered by another mechanism compared to oligodendrocyte apoptosis occurring during remyelination and, therefore, might be prevented to a different extent by PDGFα.

As a limitation of this study, it is not entirely clear whether the lower number of apoptotic cells during remyelination in the CC of hPDGF-αtg over-expression contributes to changes in OPC, mature oligodendrocytes, or other cell types such as microglia or astrocytes. Our recent studies indicate that numbers of astroglia and microglia are lower after 12 weeks cuprizone administration compared to 5 weeks intoxication. The loss of astrocytes and/or microglia during the late phase of chronic demyelination might be, in part, responsible for the failure/delay of remyelination after prolonged cuprizone exposure and, in consequence, lower levels of pro-myelination factors, such as IGF-1 or FGF-2, are available at the site of lesion [12, 48]. Therefore, prevention of astrocyte/microglia apoptosis by PDGFα is a visible protective/regenerative mechanism [7, 8, 51].

Summary

The cuprizone model is increasingly used to study processes related to de- and re-myelination in the CNS and to address questions concerning the potential of compounds to regulate myelination of axons. It is certainly debatable to what extent cuprizone-induced demyelination reflects human MS pathology. Primary oligodendrocyte apoptosis in connection with an activation of microglia are two major histopathological hallmarks of the cuprizone animal model. These pathological features are also characteristic for human MS lesion formation [9]. Thus, cuprizone-induced oligodendrocyte apoptosis may reflect pathological steps of newly forming MS lesion in humans. In addition, human type III and IV MS lesions are partially mimicked by the cuprizone model [60].

The most convincing advantage of this animal model is that demyelination can be induced in a highly reproducible manner and lesion formation can be predicted under temporal and spatial aspects. Furthermore, remyelination processes as well as remyelination failure is experimentally approachable. Since the knowledge about cellular mechanisms leading to MS pathology is, despite many efforts, still sparse, this animal model together with the other existing models mentioned at the beginning of this article might be valuable tools to uncover these pathological processes step by step. At least, mechanisms not involving primary inflammatory events can be investigated in the cuprizone model. Experiments need to be planned precisely about which animal model is the best-suited for a particular question. Another important advantage of this animal model over others is the possibility to selectively study factors and molecules that might positively affect myelination processes in the brain either by recruiting or activating OPC by preventing the death of mature oligodendrocytes, as well as for studying the remyelination potential of exogenous, implanted OPC [21, 82]. Before implantation of exogenous remyelinating cells, cuprizone diet should be stopped to prevent the implanted cells from being affected by cuprizone. From that moment, there will be a competition in the CC on remyelination between exogenous and endogenous OPC migrating in from the environment, but with proper labeling the remyelination activity of the exogenous cells can be adequately studied. Moreover, if implantations are performed unilaterally, the contralateral side of the CC can be used as a perfect control. Lastly, axonal damage and protection of axonal degeneration following demyelination is likewise accessible if aged animals are used [40] (not discussed in this review). Another strategy for this animal model is also the use of selective knock-out and transgenic mouse models to be exposed to cuprizone. This allows investigating particular loss-of-function or gain-of-function models in the context of myelination and axonal pathology.