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PubMed1997

Rapid upregulation of the Pi isoform of glutathione-S-transferase in mouse brains after withdrawal of the neurotoxicant, cuprizone.

Cuprizone intoxication has been used as a model for reversible demyelination in the CNS. During the course of cuprizone intoxication, the glutathione-S-transferase isoform, Pi, normally and oligodendrocytic marker, appears in reactive astrocytes (Cammer ad Zhang, 1993). The present experiments address the changes in expression of Pi after removal of cuprizone from the diet of the affected mice. In order to localize Pi message, a riboprobe was prepared and in situ hybridization (ISH) performed. Western blots and immunocytochemistry were used to examine Pi protein and other glial cell markers. The data indicated that Pi protein increased during the first 2 d after withdrawal of the toxicant, when the level of the myelin marker, 2',3'-cyclic nucleotide-3'phosphohydrolase, remained minimal. Results of ISH suggested that levels of Pi message in the corpus striatum decreased during cuprizone feeding and began to recover within 2d after withdrawal of the toxicant. Both microglia and astrocytes appeared during the first week of cuprizone administration and persisted during two to three additional weeks on cuprizone. Reactive astrocytes remained in the tissue for at least 6 wk after cuprizone was withdrawn, while microglia receded within days. The findings suggest that astrocytes continue to express Pi after withdrawal of cuprizone.

Animals↗

PubMed2006

Effects of commissural de- and remyelination on motor skill behaviour in the cuprizone mouse model of multiple sclerosis.

Feeding of copper chelator cuprizone induces reversible demyelination, predominantly of the corpus callosum in C57/Bl6 mice. With the availability of knockout and transgenic mice, this animal model of multiple sclerosis has increasingly attracted scientists to study the roles of various factors involved in de- and remyelination. However, central motor deficits have not been reported in this model so far. In the present study, we introduce a novel murine motor test, the motor skill sequence (MOSS). This test is designed to detect latent deficits in motor performance. In a first step, we habituated mice to training wheels composed of regularly spaced crossbars till maximal wheel-running performance was achieved. Subsequently, the animals were exposed to wheels with irregularly spaced crossbars demanding high-level motor coordination. This two-step approach minimized a contribution of cardiopulmonary and musculoskeletal training to any improvement of motor performance on the complex wheels. We applied the MOSS test under acute cuprizone-induced demyelination as well as in remyelinated mice after cuprizone withdrawal. Demyelinated animals on a cuprizone diet already showed reduced running performance on the training wheels as compared to control animals. This was even more pronounced when these mice were subsequently exposed to the complex wheels. In contrast, remyelinated animals after cuprizone withdrawal did not exhibit any functional impairment on the training wheels. Latent motor skill deficits were however revealed on the complex wheels, although clearly ameliorated as compared to acutely demyelinated mice. Our results show that latent motor deficits of cuprizone-induced demyelination and after remyelination can be quantified by MOSS. This motor test thus expands the usability of the cuprizone model to a functional level and might also be applicable to other animal models of human CNS diseases associated with subtle motor deficits of central origin.

Analysis of Variance↗

PubMed1975

The failure of supplemental dietary copper to prevent cuprizone-induced alterations in mouse hepatocytes.

Weaning mice were fed a powdered complete diet containing either 0.5% cuprizone, 0.5% cuprizone + 0.01% copper sulfate, or 0.01% copper sulfate. With cuprizone as the sole additive, hepatic mitochondria became greatly enlarged. When the diet contained cuprizone + copper sulfate, giant mitochondria were still present, and, in addition, numerous lysosome-like structures became evident. When only copper sulfate was added to the normal diet, the mitochondria were of normal size, but the hepatocytes contained abundant lysosomes. Dietary supplementation with cuprizone or with cuprizone + copper sulfate resulted in considerably depressed (20-50%) rates of mitochondrial oxidation. Supplementation solely with copper sulfate produced virtually no changes in oxidative activity. It may be concluded that the subcellular effects of cuprizone are not based on its ability to produce copper deficiency by chelation of copper, but on other properties of this drug.

Animal Nutritional Physiological Phenomena↗

PubMed2006

The neurotoxic effect of cuprizone on oligodendrocytes depends on the presence of pro-inflammatory cytokines secreted by microglia.

In order to further characterize the still unknown mechanism of cuprizone-induced demyelination, we investigated its effect on rat primary oligodendroglial cell cultures. Cell viability was not significantly affected by this treatment. However, when concentrations of IFNgamma and/or TNFalpha having no deleterious effects per se on cell viability were added together with cuprizone, cell viability decreased significantly. In mitochondria isolated from cuprizone-treated glial cells, we observed a marked decrease in the activities of the various complexes of the respiratory chain, indicating a disruption of mitochondrial function. An enhancement in oxidant production was also observed in cuprizone and/or TNFalpha-treated oligodendroglial cells. In in vivo experiments, inhibition of microglial activation with minocycline prevented cuprizone-induced demyelination. Based on the above-mentioned results we suggest that these microglial cells appear to have a very active role in cuprizone-induced oligodendroglial cell death and demyelination, through the production and secretion of pro-inflammatory cytokines.

Animals↗

PubMed2006

Noninvasive detection of cuprizone induced axonal damage and demyelination in the mouse corpus callosum.

Previously, we tested the prediction that axonal damage results in decreased axial diffusivity (lambda(parallel)) while demyelination leads to increased radial diffusivity (lambda(perpendicular)). Cuprizone treatment of C57BL/6 mice was a highly reproducible model of CNS white matter demyelination and remyelination affecting the corpus callosum (CC). In the present study, six C57BL/6 male mice were fed 0.2% cuprizone for 12 weeks followed by 12 weeks of recovery on normal chow. The control mice were fed normal chow and imaged in parallel. Biweekly in vivo DTI examinations showed transient decrease of lambda(parallel) in CC at 2-6 weeks of cuprizone treatment. Immunostaining for nonphosphorylated neurofilaments demonstrated corresponding axonal damage at 4 weeks of treatment. Significant demyelination was evident from loss of Luxol fast blue staining at 6-12 weeks of cuprizone ingestion and was paralleled by increased lambda(perpendicular) values, followed by partial normalization during the remyelination phase. The sensitivity of lambda(perpendicular) to detect demyelination may be modulated in the presence of axonal damage during the early stage of demyelination at 4 weeks of cuprizone treatment. Our results suggest that lambda(parallel) and lambda(perpendicular) may be useful in vivo surrogate markers of axonal and myelin damage in mouse CNS white matter.

Animals↗

PubMed2006

Remyelination after cuprizone-induced demyelination in the rat is stimulated by apotransferrin.

Twenty-one-day-old Wistar rats were fed a diet containing 0.6% cuprizone for 2 weeks. Studies carried out after withdrawal of cuprizone showed histological evidences of marked demyelination in the corpus callosum. Biochemical studies of isolated myelin showed a marked decrease in myelin proteins, phospholipids, and galactocerebrosides as well as a marked decrease in myelin yield. Treatment of these animals with a single intracranial injection of 350 ng of apotransferrin at the time of withdrawal of cuprizone induced a marked increase in myelin deposition resulting in a significantly improved remyelination, evaluated by histological, immunocytochemical, and biochemical parameters, in comparison to what was observed in spontaneous recovery. Immunocytochemical studies of cryotome sections to analyze developmental parameters of the oligodendroglial cell population at the time of termination of cuprizone and at different times thereafter showed that in the untreated animals, there was a marked increase in the number of NG2-BrdU-positive precursor cells together with a marked decrease in MBP expression at the peak of cuprizone-induced demyelination. As expected, the amount of precursor cells decreased markedly during spontaneous remyelination and was accompanied by an increase in MBP reactivity. In the apotransferrin-treated animals, these phenomena occurred much faster, and remyelination was much more efficient than in the untreated controls. The results of this study suggest that apotransferrin is a very active promyelinating agent which could be important for the treatment of certain demyelinating conditions.

Analysis of Variance↗

PubMed2003

Quantifying the early stages of remyelination following cuprizone-induced demyelination.

The demyelinating toxin cuprizone is used increasingly in mouse studies of central nervous system remyelination. The value of this model for such studies depends on an accurate description of its quantifiable features. We therefore investigated histology and ultrastructure during the early oligodendrocyte differentiation phase of remyelination in mice given cuprizone and allowed to recover for 2 weeks. Limiting the dose of cuprizone to 0.2% overcame significant mouse morbidity and weight loss seen with a 0.4% dose, but the distribution of cuprizone-induced demyelination was anatomically variable. The caudal corpus callosum and dorsal hippocampal commissure mostly demyelinated at this dose, but the rostral corpus callosum and rostral cerebellar peduncles did not. This variable response, together with small axon diameters and hence thin myelin sheaths, hindered analysis of the progress of early remyelination. The proportion of myelinated and unmyelinated axons in defined regions followed expected trends, but there was pronounced variation between animals. Furthermore, group mean G ratios did not change as expected during the early stages of remyelination, and regression analysis revealed a complex relationship between axon diameter and myelin sheath thickness during this period. We also noted axonal pathology that persisted for at least 2 weeks after cuprizone withdrawal.

Animals↗

PubMed1998

Gene expression in brain during cuprizone-induced demyelination and remyelination.

When C57BL/6J mice, 8 weeks of age, received 0.2% Cuprizone in their diet, extensive demyelination in corpus callosum was detectable after 3 weeks, and there was massive demyelination by 4 weeks. As expected, the accumulation of phagocytically active microglia/macrophages correlated closely with demyelination. When Cuprizone was removed from the diet, remyelination was soon initiated; after 6 weeks of recovery, myelin levels were near-normal and phagocytic cells were no longer prominent. Steady-state levels of mRNA for myelin-associated glycoprotein, myelin basic protein, and ceramide galactosyltransferase were already profoundly depressed after 1 week of Cuprizone exposure and were only 10-20% of control values after 2 weeks. Unexpectedly, upregulation of mRNA for these myelin genes did not correlate with initiation of remyelination but rather with accumulation of microglia/macrophages. After 6 weeks of exposure to Cuprizone, mRNA levels were at control levels or higher-in the face of massive demyelination. This suggests that in addition to effecting myelin removal, microglia/macrophages may simultaneously push surviving oligodendroglia or their progenitors toward myelination.

Animals↗

PubMed1988

Cuprizone neurotoxicity in the rat: morphologic observations.

Weanling male Wistar rats fed a diet containing 0.5-2% cuprizone developed intramyelinic edema of the cerebellar white matter, hilum of the dentate nucleus and superior cerebellar peduncle. Oligodendrocytes in these regions showed hyperchromatic nuclei and abnormally dense cytoplasm, with an increase in the number of free ribosomes and enlargement of mitochondria. Unlike the lesions produced by cuprizone in weanling mice, those in the rat did not lead to demyelination. Cuprizone caused a distal peripheral axonopathy, with degeneration of myelinated axons in the sciatic nerve but preservation of the spinal nerve roots, dorsal root ganglia, posterior spinal funiculi and anterior horn cells. Unmyelinated fibers were largely spared. Some axonal regeneration occurred despite the continued administration of cuprizone. The intramyelinic edema and the nuclear and cytoplasmic abnormalities of the oligodendrocytes disappeared after the resumption of a normal diet.

Animals↗

PubMed1999

The neurotoxicant, cuprizone, retards the differentiation of oligodendrocytes in vitro.

The effects of oxalyldihydrazone (cuprizone) on weanling rodents provided an early protocol for toxic demyelination in vivo, in which degeneration of oligodendrocytes preceded disruption of the myelin sheath, and in which remyelination could take place. We administered cuprizone to oligodendrocyte-enriched glial-cell cultures and to mixed glial-cell cultures from neonatal rat brains. The cultures were treated with cuprizone for 1 h and allowed to continue differentiating on subsequent days. Treated cultures and respective control cultures were fixed with 4% paraformaldehyde (w/v) and immunostained with double immunofluorescence. MAbO4 was used to mark precursors and mature oligodendrocytes, and anti-myelin basic protein (MBP) to mark mature oligodendrocytes (O4+/MBP+), as distinguished from precursors, which were O4+/MBP-. Cell counts suggested that cuprizone inhibited the maturation of oligodendrocytes without diminishing the numbers of precursors, and appeared to affect the mitochondria in those cells.

Acridine Orange↗

PubMed1998

Microglial/macrophage accumulation during cuprizone-induced demyelination in C57BL/6 mice.

To study microglial/macrophage infiltration, a cuprizone-induced model for demyelination in C57BL/6 mice was established. Cuprizone is known to cause demyelination in Swiss mice, however, cuprizone-induced demyelination in C57BL/6 mice has not been previously described. Induction of demyelination in C57BL/6 mice enables examination of the function of microglia/macrophage through comparative analyses of syngeneic mice with various targeted genetic mutations. In this report, cuprizone-induced demyelination is easily inducible, localized, and predictable. Concurrent with the initiation of demyelination, we noted microglial/macrophage accumulation and changes in astrocyte morphology. Astrogliosis promptly followed microglia/macrophage recruitment. These observations suggested that microglia/macrophage actively contribute to the demyelination process.

Animals↗

PubMed2001

The neurotoxicant, cuprizone, as a model to study demyelination and remyelination in the central nervous system.

Myelin of the adult CNS is vulnerable to a variety of metabolic, toxic, and autoimmune insults. That remyelination can ensue, following demyelinating insult, has been well demonstrated. Details of the process of remyelination are, however difficult to ascertain since in most experimental models of demyelination/remyelination the severity, localization of lesion site, or time course of the pathophysiology is variable from animal to animal. In contrast, an experimental model in which massive demyelination can be reproducibly induced in large areas of mouse brain is exposure to the copper chelator, cuprizone, in the diet. We review work from several laboratories over the past 3 decades, with emphasis on our own recent studies, which suggest an overall picture of cellular events involved in demyelination/remyelination. When 8 week old C57BL/6 mice are fed 0.2% cuprizone in the diet, mature olidgodendroglia are specifically insulted (cannot fulfill the metabolic demand of support of vast amounts of myelin) and go through apoptosis. This is closely followed by recruitment of microglia and phagoctytosis of myelin. Studies of myelin gene expression, coordinated with morphological studies, indicate that even in the face of continued metabolic challenge, oligodendroglial progenitor cells proliferate and invade demyelinated areas. If the cuprizone challenge is terminated, an almost complete remyelination takes place in a matter of weeks. Communication between different cell types by soluble factors may be inferred. This material is presented in the context of a model compatible with present data -- and which can be tested more rigorously with the cuprizone model. The reproducibility of the model indicates that it may allow for testing of manipulations (e.g. available knockouts or transgenics on the common genetic background, or pharmacological treatments) which may accelerate or repress the process of demyelination and or remyelination.

Animals↗

PubMed2004

K+ channel blockade impairs remyelination in the cuprizone model.

The adult CNS has the capacity to remyelinate following metabolic, toxic and autoimmune demyelinating insults. In cuprizone-induced demyelination, spontaneous remyelination occurs after the cessation of cuprizone diet. We used the cuprizone model to investigate the role of glial K(+) channels in oligodendroglial (OLG) regeneration and remyelination in vivo. We found that treatment with 4-aminopyridine (4-AP), a broad-spectrum K(+) channel antagonist, results in: (1) decreased number of oligodendroglial progenitors (OP) and OLGs; (2) diminished astrogliosis; and (3) decreased remyelination in the corpus callosum based on the immunoreactivity to myelin basic protein (MBP), Rip monoclonal antibody, and by electron microscopy. Our findings support the concept that glial K(+) channels play an important role during OLG regeneration and remyelination, a crucial factor to be considered during the development of therapeutic strategies to facilitate recovery in demyelinating diseases and spinal cord injury.

4-Aminopyridine↗

PubMed1993

Atypical localization of the oligodendrocytic isoform (PI) of glutathione-S-transferase in astrocytes during cuprizone intoxication.

Immunocytochemical staining for the Pi and Mu isoforms of glutathione-S-transferase was used to investigate changes in the glial cells in the mouse forebrain. During early development in mouse forebrains the localizations of carbonic anhydrase, Pi and Mu were similar to the respective cellular localizations that had been observed in neonatal rat brain. That is, Pi was found in oligodendrocyte precursors, Mu in astrocytes, and carbonic anhydrase in both oligodendrocyte precursors and astrocytes. In forebrains of 6-week-old mice the neurotoxicant, cuprizone, induced oligodendrocyte degeneration, gliosis, and partial demyelination. Degeneration, gliosis, and partial demyelination. Degeneration of oligodendrocytes, and astrocytosis, began during the initial week of cuprizone feeding, and by the end of the eighth week some demyelination was observed. After mice were fed cuprizone for 4 to 7 weeks, Pi appeared in some of the reactive astrocytes, and Pi-positive astrocytes were present for at least 7 additional weeks. Normally, Pi appeared only in oligodendrocytes. Very few Pi-positive oligodendrocytes remained after the second week. During the eighth week healthy-looking carbonic anhydrase-positive oligodendrocytes reappeared and began to accumulate, and a few small patches of Pi-positive oligodendrocytes were also observed. In summary, some novel findings about glial cells were the observation of an enzyme (Pi) that is lost earlier from oligodendrocytes than is carbonic anhydrase, the apparently unique shift in Pi expression from oligodendrocytes to astrocytes and the greater temporal dissociation between loss of oligodendrocytes and demyelination in the older mice.

Animals↗

PubMed2000

Interferon-gamma protects against cuprizone-induced demyelination.

Evidence suggests that interferon-gamma (IFN-gamma), a proinflammatory cytokine secreted by activated T lymphocytes, contributes a deleterious effect to immune-mediated demyelinating disorders such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Nevertheless, mouse strains that are normally resistant to EAE induction become susceptible when the gene encoding either IFN-gamma or its receptor is mutated, demonstrating that the role that this cytokine plays in demyelinating disorders is complex. We have examined the effect of IFN-gamma in a chemically induced model of CNS demyelination. Mice that receive through their diet the copper chelator cuprizone display extensive demyelination of the corpus callosum. Remarkably, transgenic mice that ectopically express low levels of IFN-gamma in the CNS did not display evidence of demyelination when treated with cuprizone, nor did they shows signs of oligodendroglial death, astrogliosis, or microgliosis, which are typically seen in treated animals. Myelin protein gene expression was, however, dramatically reduced in both the treated control and the transgenic animals, indicating that demyelination is not an obligatory consequence of a large diminution of myelin protein synthesis. Interestingly, the CNS of the IFN-gamma-expressing mice contained elevated levels of insulin-like growth factor I, which has been demonstrated to have a protective effect against the demyelinating action of cuprizone.

Animals↗

PubMed1983

Biochemical changes in Cuprizone-induced spongiform encephalopathy. I. Changes in the activities of 2',3'-cyclic nucleotide 3'-phosphohydrolase, oligodendroglial ceramide galactosyl transferase, and the hydrolysis of the alkenyl group of alkenyl, acyl-glycerophospholipids by plasmalogenase in different regions of the brain.

Cuprizone (biscyclohexanone oxaldihydrazone) which is known to produce a status spongiosus and demyelination in the CNS was administered in the diet of weanling male mice at a concentration of 0.4% by weight for a period of six weeks before returning animals to a normal diet. Changes in body weight but not brain weight were reversible. Based on the decline in CNP'ase activity and the concentration of galactocerebroside, the loss of myelin was around 70% in those sections of the cerebrum with a high content of white matter while the cerebellum was less affected. The activity of oligodendroglial HFA-ceramide galactosyl transferase was also reduced. These biochemical parameters of myelination were increased after withdrawal of Cuprizone. Remyelination in the cerebrum but not the cerebellum was incomplete. The activity of plasmalogenase hydrolysing the alkenyl group of alkenyl, acyl-phospholipids increased 2-fold in those sections in which myelin loss was most severe. The increase preceded the greatest loss of myelin components (3 to 6 weeks on Cuprizone). The origin of the increased phospholipase activity in demyelinating tissue is discussed. Following myelination, there was a deficit in plasmalogenase activity particularly in the frontal cortex of the cerebrum, where the plasmalogen concentration was higher than in controls.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗

PubMed2005

Copper and zinc dismetabolism in the mouse brain upon chronic cuprizone treatment.

Recent reports describe successful treatment using copper chelation therapy in neurodegenerative animal models. However, the success claimed for chelation therapy in neurodegenerative diseases is still rather controversial. To acquire new information on copper metabolism/homeostasis, we utilized cuprizone, a very sensitive and selective copper-chelating agent with well-known neurotoxic properties, as a relevant chemical model in mice. Upon cuprizone treatment, mice developed a pronounced astrocytosis, with brain oedema and spongiosis characterised by vacuolisations of the neuropil predominantly in the white matter. In addition, cuprizone treatment severely altered copper and zinc homeostasis in the central nervous system (CNS) as well as in all other tissues examined, with increasing metal ion concentrations particularly in the CNS. Concomitant with this increase in the Cu and Zn concentration in the brain, metallothionein-I and -II were also highly immunoreactive in astrocyte, consistent with the astrocytosis and demyelination observed in our and other laboratories.

Animals↗

PubMed1987

Blood-brain barrier permeability during Cuprizone-induced demyelination. Implications for the pathogenesis of immune-mediated demyelinating diseases.

Blood vessels in the superior cerebellar peduncles were studied in Cuprizone-fed mice for leakage of proteins into the parenchyma. The status of the blood-brain barrier was determined at various stages of demyelination both by tracer methods using horseradish peroxidase and immunochemically using antisera to extravasated serum proteins and was compared to three positive and negative control conditions. The results showed no evidence of significant protein leakage into the subendothelial basement membrane or extravascular space in Cuprizone mice, during the development of demyelination. The apparent lack of damage to the blood-brain barrier found in Cuprizone animals as compared to the blood-brain barrier alterations previously reported for immune-mediated demyelinating diseases, confirms the theory that these alterations are not a non-specific association of any demyelinating process, but are of primary pathogenetic importance in immune-mediated demyelination.

Animals↗