Cuprizone-induced Demyelination In Mice Research Articles

5Z-7-Oxozaenol attenuates cuprizone-induced demyelination in mice through microglia polarization regulation.

Demyelination is a key factor in axonal degeneration and neural loss, leading to disability in multiple sclerosis (MS) patients. Transforming growth factor beta activated kinase 1 (TAK1) is a critical molecule involved in immune and inflammatory signaling pathways. Knockout of microglia TAK1 can inhibit autoimmune inflammation of the brain and spinal cord and improve the outcome of MS. However, it is unclear whether inhibiting TAK1 can alleviate demyelination. Eight-week-old male c57bl/6j mice were randomly divided into five groups: (a) the control group, (b) the group treated with cuprizone (CPZ) only, (c) the group treated with 5Z-7-Oxozaenol (OZ) only, and (d) the group treated with both cuprizone and 15 μg/30 μg OZ. Demyelination in the mice of this study was induced by administration of CPZ (ig) at a daily dose of 400mg/kg for consecutive 5weeks. OZ was intraperitoneally administered at mentioned doses twice a week, starting from week 3 after beginning cuprizone treatment. Histology, rotarod test, grasping test, pole test, Western blot, RT-PCR, and ELISA were used to evaluate corpus callosum demyelination, behavioral impairment, oligodendrocyte differentiation, TAK1 signaling pathway expression, microglia, and related cytokines. Our results demonstrated that OZ protected against myelin loss and behavior impairment caused by CPZ. Additionally, OZ rescued the loss of oligodendrocytes in CPZ-induced mice. OZ inhibited the activation of JNK, p65, and p38 pathways, transformed M1 polarized microglia into M2 phenotype, and increased brain-derived neurotrophic factor (BDNF) expression to attenuate demyelination in CPZ-treated mice. Furthermore, OZ reduced the expression of proinflammatory cytokines and increases anti-inflammatory cytokines in CPZ-treated mice. These findings suggest that inhibiting TAK1 may be an effective approach for treating demyelinating diseases.

Neurobehavioral, biochemical and histological assessment of the effects of resveratrol on cuprizone-induced demyelination in mice: role of autophagy modulation

Resveratrol is known to exhibit neuroprotective effects in many neurological disorders via autophagy modulation. However, controversial results have been reported about the therapeutic potential of resveratrol and the implication of autophagy in demyelinating diseases. This study aimed to evaluate the autophagic changes in cuprizone-intoxicated C57Bl/6 mice and explore the effect of autophagy activation by resveratrol on the demyelination and remyelination processes. Mice were fed with chow containing 0.2% cuprizone for 5 weeks, followed by a cuprizone-free diet for 2 weeks. Resveratrol (250 mg/kg/day) and/or chloroquine (an autophagy inhibitor; 10 mg/kg/day) were given for 5 weeks starting from the third week. At the end of the experiment, animals were tested on rotarod and then sacrificed for biochemical assessment, luxol fast blue (LFB) staining, and transmission electron microscopy (TEM) imaging of the corpus callosum. We observed that cuprizone-induced demyelination was associated with impaired degradation of autophagic cargo, induction of apoptosis, and manifest neurobehavioral disturbances. Oral treatment with resveratrol promoted motor coordination and improved remyelination with regular compacted myelin in most axons without a significant impact on myelin basic protein (MBP) mRNA expression. These effects are mediated, at least in part, via activating autophagic pathways that may involve SIRT1/FoxO1 activation. This study verified that resveratrol dampens cuprizone-induced demyelination, and partially enhances myelin repair through modulation of the autophagic flux, since interruption of the autophagic machinery by chloroquine reversed the therapeutic potential of resveratrol.

Neuroprotective effect of newly synthesized 4-aminopyridine derivatives on cuprizone-induced demyelination in mice-a behavioral and immunohistochemical study.

The aim of this study was to assess the effect of newly synthesized derivatives of 4-aminopyridine (4-AP) on cuprizone-induced model of brain demyelination in mice. 4-AP is already approved for the treatment of walking difficulties in patients with multiple sclerosis. The model of demyelination was carried out by the administration of cuprizone to the drinking water of the experimental mice. Besides cuprizone, 4-AP derivatives and 4-AP were administered to the groups in order to assess their protective effect on the demyelination. We used immunohistochemistry for visualization of changes in corpus callosum. Memory storage processes were also assessed with the passive avoidance test on the last two days of the experiment. The experimental mice treated with compounds 4b and 4c increased significantly their latency time on the second day in comparison to the control group which indicated an improved memory process. The number of mature oligodendrocytes in the groups treated with compounds 4b, 4c and 4-AP is closer to those in the control group. The results of our studies showed that the newly synthesized compounds 4b and 4c reverse the effect of cuprizone. These groups also showed increased latency time in the passive avoidance test in comparison to the control group.

Neuroprotective Effects of Telmisartan and Nifedipine Against Cuprizone-Induced Demyelination and Behavioral Dysfunction in Mice: Roles of NF-κB and Nrf2

Multiple sclerosis is a chronic inflammatory neurodegenerative disease of the central nervous system which injures the myelin sheath. Telmisartan and nifedipine are antihypertensive drugs that recently showed neuroprotective properties against neurodegenerative diseases. This study evaluated the neuroprotective effect of telmisartan or nifedipine in cuprizone-induced demyelination in mice by examining the underlying mechanisms. C57BL/6 mice received a diet containing 0.7% (w/w) cuprizone for 7 days followed by 3 weeks on a 0.2% cuprizone diet. Telmisartan (5 mg/kg/day, p.o.) or nifedipine (5 mg/kg/day, p.o.) was administered for 3 weeks starting from the second week. Telmisartan or nifedipine improved locomotor activity and enhanced motor coordination as demonstrated by open field, rotarod, and grip strength tests. Furthermore, telmisartan or nifedipine restored myelin basic protein mRNA and protein expression and increased luxol fast blue-staining intensity. Telmisartan or nifedipine attenuated cuprizone-induced oxidative stress and apoptosis by decreasing brain malondialdehyde and caspase-3 along with restoring reduced glutathione and brain-derived neurotrophic factor levels. Telmisartan or nifedipine exerted an anti-inflammatory effect by reducing the expression of nuclear factor kappa B (NF-κB p65) as well as pro-inflammatory cytokines and elevating the expression of IκB-α. In parallel, telmisartan or nifedipine upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and the levels of heme oxygenase-1 and NADPH quinone oxidoreductase 1 enzymes. In conclusion, the current study provides evidence for the protective effect of telmisartan and nifedipine in cuprizone-induced demyelination and behavioral dysfunction in mice possibly by modulating NF-κB and Nrf2 signaling pathways.

Myelin Protection by Ursolic Acid in Cuprizone-Induced Demyelination in Mice.

Neuronal survival in multiple sclerosis (MS) and other demyelinating diseases depends on the preservation of myelin and remyelination of axons. Myelin protection is the main purpose to decrease myelin damage in the central nervous system (CNS). Ursolic acid (UA) as a natural product in apple is suggested to protect neural cells. This study is the first to demonstrate an effect for UA on CNS myelin loss induced by cuprizone toxin. In the current study, we hypothesized that daily treatment with UA in drinking water (1 mg/mL) prevents myelin damage by 6 weeks administration of CPZ in mice pellet which lead to corpus callosum axonal demyelination. We assessed the myelin content and the number of myelinating cells in corpus callosum by FluoroMyelin and luxol fast blue staining as well as by immunostaining against MBP and Olig2. Our finding indicated that UA could decrease the extent of demyelination area and enhanced myelin stain intensity within CC and protected oligodendrocyte lineage cells against cuprizone toxin. We could conclude that myelinated structures could be protected by UA in corpus callosum, which provide favorable evidence for the possibility of application of UA in demyelinating diseases and traumatic injuries.

Neuroprotective effect of linagliptin against cuprizone-induced demyelination and behavioural dysfunction in mice: A pivotal role of AMPK/SIRT1 and JAK2/STAT3/NF-κB signalling pathway modulation

Multiple sclerosis is a chronic inflammatory demyelinating central nervous system disorder leading to serious neurological deficits. Linagliptin, a dipeptidyl peptidase-4 inhibitor, recently showed neuroprotective properties against neurodegenerative diseases. This study investigated the possible neuroprotective effect of linagliptin against cuprizone-induced demyelination in mice and its potential early-remyelinating properties. C57Bl/6 mice were fed chow containing 0.7% cuprizone for 1 week, followed by 3 weeks of a 0.2% cuprizone diet. Linagliptin (10 mg/kg/day, p.o.) was given for 3 weeks starting from the second week. Linagliptin treatment improved behavioural and motor abnormalities induced by cuprizone, as demonstrated by open field, rotarod and grip strength tests. In parallel, linagliptin lessened the demyelination through enhancing Olig2 gene expression, as shown by increased myelin basic protein, myelin proteolipid protein levels and Luxol fast blue-staining intensity. Linagliptin attenuated cuprizone-induced oxidative stress by decreasing brain thiobarbituric acid reactive substances along with restoring reduced glutathione levels. Linagliptin exerted an anti-inflammatory effect by reducing brain tumor necrosis factor-alpha. Interestingly, linagliptin diminished phosphorylated JAK2, phosphorylated STAT3 and NF-κB p65 protein expression while up-regulating phosphorylated AMP-activated protein kinase (p-AMPK) protein and SIRT1 gene expression levels. In conclusion, linagliptin exerted a neuroprotective effect in mice with cuprizone-induced demyelination possibly by modulating AMPK/SIRT1 and JAK2/STAT3/NF-κB signalling pathways.

Histological validation of fast macromolecular proton fraction mapping as a quantitative myelin imaging method in the cuprizone demyelination model

Cuprizone-induced demyelination in mice is a frequently used model in preclinical multiple sclerosis research. A recent quantitative clinically-targeted MRI method, fast macromolecular proton fraction (MPF) mapping demonstrated a promise as a myelin biomarker in human and animal studies with a particular advantage of sensitivity to both white matter (WM) and gray matter (GM) demyelination. This study aimed to histologically validate the capability of MPF mapping to quantify myelin loss in brain tissues using the cuprizone demyelination model. Whole-brain MPF maps were obtained in vivo on an 11.7T animal MRI scanner from 7 cuprizone-treated and 7 control С57BL/6 mice using the fast single-point synthetic-reference method. Brain sections were histologically stained with Luxol Fast Blue (LFB) for myelin quantification. Significant (p < 0.05) demyelination in cuprizone-treated animals was found according to both LFB staining and MPF in all anatomical structures (corpus callosum, anterior commissure, internal capsule, thalamus, caudoputamen, and cortex). MPF strongly correlated with quantitative histology in all animals (r = 0.95, p < 0.001) as well as in treatment and control groups taken separately (r = 0.96, p = 0.002 and r = 0.93, p = 0.007, respectively). Close agreement between histological myelin staining and MPF suggests that fast MPF mapping enables robust and accurate quantitative assessment of demyelination in both WM and GM.

Regulatory effect of triiodothyronine on brain myelination and astrogliosis after cuprizone-induced demyelination in mice.

Chronic demyelination and plaque formation in multiple sclerosis is accompanied by persisting astrogliosis, negatively influencing central nervous system recovery and remyelination. Triiodothyronin (T3) is thought to enhance remyelination in the adult brain by the induction of oligodendrocyte maturation. We investigated additional astrocyte-mediated mechanisms by which T3 might promote remyelination in chronically demyelinated lesions using the cuprizone mouse model. C57BL/6 mice were fed cuprizone for 12 weeks to induce lesions with an impaired remyelination capacity. While the expression of oligodenrocyte progenitor markers, i.e., platelet derived growth factor-α receptor was not affected by T3 administration, myelination status, myelin protein expression as well as total and adult oligodendrocyte numbers were markedly increased compared to cuprizone treated controls. In addition to these effects on oligodendrocyte numbers and function, astrogliosis but not microgliosis was ameliorated by T3 administration. Intermediate filament proteins vimentin and nestin as well as the extracellular matrix component tenascin C were significantly reduced after T3 exposure, indicating additional effects of T3 on astrocytes and astrogliosis. Our data clearly indicate that T3 promotes remyelination in chronic lesions by both enhancing oligodendrocyte maturation and attenuating astrogliosis.

MRI study of the cuprizone-induced mouse model of multiple sclerosis: demyelination is not found after co-treatment with polyprenols (long-chain isoprenoid alcohols)

Multiple sclerosis is a neurological disorder with poorly understood pathogenic mechanisms and a lack of effective therapies. Therefore, the search for new MS treatments remains very important. This study was performed on a commonly used cuprizone animal model of multiple sclerosis. It evaluated the effect of a plant-derived substance called Ropren® (containing approximately 95% polyprenols or long-chain isoprenoid alcohols) on cuprizone- induced demyelination. The study was performed on 27 eight-week old male CD-1 mice. To induce demyelination mice were fed 0.5% cuprizone in the standard diet for 10 weeks. Ropren® was administered in one daily intraperitoneal injection (12mg/kg), beginning on the 6th week of the experiment. On the 11th week, the corpus callosum in the brain was evaluated in all animals using magnetic resonance imaging with an 11.7 T animal scanner using T2- weighted sequence. Cuprizone treatment successfully induced the model of demyelination with a significant decrease in the size of the corpus callosum compared with the control group (p<0.01). Mice treated with both cuprizone and Ropren® did not exhibit demyelination in the corpus callosum (p<0.01). This shows the positive effect of polyprenols on cuprizone-induced demyelination in mice.

Activation of Nuclear Receptors RAR, RXR, and LXR Does Not Reduce Cuprizone-Induced Demyelination in Mice

Experiments with animal models of multiple sclerosis have shown that the expression of retinoid X receptors (RXR) increases during demyelination and that RXR is involved in the regulation of remyelination. After ligand binding RXRs form heterodimeric transcription factors with other nuclear receptor (NR) families including the retinoic acid receptors (RAR) and liver X receptors (LXR). We tested whether activation of these nuclear receptor complexes reduces pathological demyelination using the cuprizone mouse model. Cuprizone, which causes oligodendrocyte degeneration, was given for three weeks as a food additive. For the activation of nuclear receptors mice were treated with daily i.p. injections of agonists for RXR (9-cis RA), RAR (all-trans RA), and LXR (T0901317). Myelin status, oligodendrocyte survival, astrogliosis, microglial activation, and axon density were monitored with immunohistochemistry and evaluated quantitatively. Three weeks of cuprizone feeding caused severe demyelination and significantly raised the number of Iba1 immunoreactive microglia cells in the caudal corpus callosum. This increase of microglia activity was reduced with 9-cis RA treatment but was enhanced with all-trans RA and was not affected by T0901317. Nuclear receptor activation did not influence the degree of demyelination, oligodendrocyte survival, astrogliosis, or axonal preservation. We conclude that RXR activation, although affecting Iba1-positive microglia, does not protect oligodendrocytes from cuprizone toxicity and does not induce compensatory mechanisms in the initial phase of demyelination.

Cuprizone-induced demyelination in mice: age-related vulnerability and exploratory behavior deficit

Schizophrenia is a mental disease that mainly affects young individuals (15 to 35 years old) but its etiology remains largely undefined. Recently, accumulating evidence indicated that demyelination and/or dysfunction of oligodendrocytes is an important feature of its pathogenesis. We hypothesized that the vulnerability of young individuals to demyelination may contribute to the onset of schizophrenia. In the present study, three different age cohorts of mice, i.e. juvenile (3 weeks), young-adult (6 weeks) and middle-aged (8 months), were subjected to a 6-week diet containing 0.2% cuprizone (CPZ) to create an animal model of acute demyelination. Then, age-related vulnerability to CPZ-induced demyelination, behavioral outcomes, and myelination-related molecular biological changes were assessed. We demonstrated: (1) CPZ treatment led to more severe demyelination in juvenile and young-adult mice than in middle-aged mice in the corpus callosum, a region closely associated with the pathophysiology of schizophrenia; (2) the higher levels of demyelination in juvenile and young-adult mice were correlated with a greater reduction of myelin basic protein, more loss of CC-1-positive mature oligodendrocytes, and higher levels of astrocyte activation; and (3) CPZ treatment resulted in a more prominent exploratory behavior deficit in juvenile and young-adult mice than in middle-aged mice. Together, our data demonstrate an age-related vulnerability to demyelination with a concurrent behavioral deficit, providing supporting evidence for better understanding the susceptibility of the young to the onset of schizophrenia.

The neural androgen receptor: a therapeutic target for myelin repair in chronic demyelination

Myelin regeneration is a major therapeutic goal in demyelinating diseases, and the failure to remyelinate rapidly has profound consequences for the health of axons and for brain function. However, there is no efficient treatment for stimulating myelin repair, and current therapies are limited to anti-inflammatory agents. Males are less likely to develop multiple sclerosis than females, but often have a more severe disease course and reach disability milestones at an earlier age than females, and these observations have spurred interest in the potential protective effects of androgens. Here, we demonstrate that testosterone treatment efficiently stimulates the formation of new myelin and reverses myelin damage in chronic demyelinated brain lesions, resulting from the long-term administration of cuprizone, which is toxic for oligodendrocytes. In addition to the strong effect of testosterone on myelin repair, the number of activated astrocytes and microglial cells returned to low control levels, indicating a reduction of neuroinflammatory responses. We also identify the neural androgen receptor as a novel therapeutic target for myelin recovery. After the acute demyelination of cerebellar slices in organotypic culture, the remyelinating actions of testosterone could be mimicked by 5α-dihydrotestosterone, a metabolite that is not converted to oestrogens, and blocked by the androgen receptor antagonist flutamide. Testosterone treatment also failed to promote remyelination after chronic cuprizone-induced demyelination in mice with a non-functional androgen receptor. Importantly, testosterone did not stimulate the formation of new myelin sheaths after specific knockout of the androgen receptor in neurons and macroglial cells. Thus, the neural brain androgen receptor is required for the remyelination effect of testosterone, whereas the presence of the receptor in microglia and in peripheral tissues is not sufficient to enhance remyelination. The potent synthetic testosterone analogue 7α-methyl-19-nortestosterone, which has been developed for long-term male contraception and androgen replacement therapy in hypogonadal males and does not stimulate prostate growth, also efficiently promoted myelin repair. These data establish the efficacy of androgens as remyelinating agents and qualify the brain androgen receptor as a promising drug target for remyelination therapy, thus providing the preclinical rationale for a novel therapeutic use of androgens in males with multiple sclerosis.

Effects of intraventricular methotrexate administration on Cuprizone-induced demyelination in mice

We previously showed that intrathecal administration of methotrexate slowed disability progression in multiple sclerosis (MS) patients with progressive disease. In general MS patients with progressive disease respond poorly to anti-inflammatory therapies. In order to better understand the mechanism by which methotrexate is protective in progressive MS, we analyzed its impact on the non-inflammatory cuprizone-induced demyelination model. When low-dose methotrexate was administered intracerebroventricularly it reduced demyelination and accumulation of GFAP+ reactive astrocytes in the corpus callosum. Administration of methotrexate after the withdrawal of cuprizone neither delayed remyelination nor influenced the number of astrocytes in the corpus callosum suggesting that methotrexate does not interfere with repair processes in the CNS. Moreover, methotrexate increased the expression of IGF1 in vitro and in vivo, a factor known to protect oligodendrocytes and limit the activation of astrocytes. Our studies show that methotrexate has an impact on pathogenic process in a demyelination model whose pathophysiological basis is not primarily related to inflammatory mechanisms, similar to neurodegenerative mechanisms associated with progressive MS. The pronounced inhibitory influence of methotrexate on the accumulation of astrocytes in the corpus callosum suggests that intrathecal methotrexate modulates astroglial activation in progressive MS possibly by promoting CNS production of IGF1.

Reduced astrocytic NF-κB activation by laquinimod protects from cuprizone-induced demyelination

Laquinimod (LAQ) is a new oral immunomodulatory compound that reduces relapse rate, brain atrophy and disability progression in multiple sclerosis (MS). LAQ has well-documented effects on inflammation in the periphery, but little is known about its direct activity within the central nervous system (CNS). To elucidate the impact of LAQ on CNS-intrinsic inflammation, we investigated the effects of LAQ on cuprizone-induced demyelination in mice in vivo and on primary CNS cells in vitro. Demyelination, inflammation, axonal damage and glial pathology were evaluated in LAQ-treated wild type and Rag-1-deficient mice after cuprizone challenge. Using primary cells we tested for effects of LAQ on oligodendroglial survival as well as on cytokine secretion and NF-κB activation in astrocytes and microglia. LAQ prevented cuprizone-induced demyelination, microglial activation, axonal transections, reactive gliosis and oligodendroglial apoptoses in wild type and Rag-1-deficient mice. LAQ significantly decreased pro-inflammatory factors in stimulated astrocytes, but not in microglia. Oligodendroglial survival was not affected by LAQ in vitro. Astrocytic, but not microglial, NF-κB activation was markedly reduced by LAQ as evidenced by NF-κB reporter assay. LAQ also significantly decreased astrocytic NF-κB activation in cuprizone-treated mice. Our data indicate that LAQ prevents cuprizone-induced demyelination by attenuating astrocytic NF-κB activation. These effects are CNS-intrinsic and not mediated by peripheral immune cells. Therefore, LAQ downregulation of the astrocytic pro-inflammatory response may be an important mechanism underlying its protective effects on myelin, oligodendrocytes and axons. Modulation of astrocyte activation may be an attractive therapeutic target to prevent tissue damage in MS.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-012-1009-1) contains supplementary material, which is available to authorized users.

Myelin debris regulates inflammatory responses in an experimental demyelination animal model and multiple sclerosis lesions

In multiple sclerosis (MS), gray matter pathology is characterized by less pronounced inflammation when compared with white matter lesions. Although regional differences in the cytoarchitecture may account for these differences, the amount of myelin debris in the cortex during a demyelinating event might also be contributory. To analyze the association between myelin debris levels and inflammatory responses, cortical areas with distinct and sparse myelination were analyzed for micro- and astrogliosis before and after cuprizone-induced demyelination in mice. In postmortem tissue of MS patients, leucocortical lesions were assessed for the type and level of inflammation in the cortical and white matter regions of the lesion. Furthermore, mice were injected intracerebrally with myelin-enriched debris, and the inflammatory response analyzed in white and grey matter areas. Our studies show that the magnitude of myelin loss positively correlates with microgliosis in the cuprizone model. In MS, the number of MHC class II expressing cells is higher in the white compared with the grey matter part of leucocortical lesions. Finally, direct application of myelin debris into the corpus callosum or cortex of mice induces profound and comparable inflammation in both regions. Our data suggest that myelin debris is an important variable in the inflammatory response during demyelinating events. Whether myelin-driven inflammation affects neuronal integrity remains to be clarified.

Combination of Growth Factors Enhances Remyelination in a Cuprizone-induced Demyelination Mouse Model

Loss of oligodendrocytes (OLs) is often associated with demyelination. PDGF-AA, bFGF, NT3 and IGF-1 are known to regulate OL proliferation, survival and/or differentiation. Following cuprizone-induced demyelination in mice a combination of above four growth factors (GF) was intracranially injected to stimulate remyelination in vivo. Activation of cell signaling and transcription factors involved in cell proliferation, survival and differentiation was observed in response to GF. Increased cell proliferation and migration occurred in corpus callosum, lateral ventricles, rostral migratory stream and cerebri at 2-5 days post injection (dpi) of GF cocktail. The fate of these newly formed nestin or bromodeoxyuridine (BrdU) positive progenitors was traced to proteoglycan NG2 and glutathione transferase (GST) pi positive cells, early and mature OL lineage markers, respectively. Immunostaining for myelin showed the presence of more myelinated fibers in GF-injected brains at 21 dpi. Remyelination in response to GF was confirmed by electron microscopy. In conclusion, this combination of GF is a promising tool to consider for remyelination strategies.