Cuprizone Administration Research Articles

Quetiapine enhances oligodendrocyte regeneration and myelin repair after cuprizone-induced demyelination

Myelin and oligodendrocyte dysfunctions have been consistently found in patients with schizophrenia. The effect of antipsychotics on myelin disturbances is unknown. The present study examined the effects of quetiapine on oligodendrocyte regeneration and myelin repair in a demyelination animal model.C57BL/6 mice were fed with cuprizone (0.2% w/w) for 12weeks to induce chronic demyelination and oligodendrocyte degeneration, after which cuprizone was withdrawn to allow recovery. Quetiapine (10mg/kg/day) or vehicle (water) was administrated orally to mice for 0, 2, 3, or 4weeks after cuprizone withdrawal. Locomotor activity and Y-maze tests were used to evaluate behavioral changes in the mice. Immunohistochemical staining was used to detect morphological and biological changes in the brains. Cuprizone administration for 12weeks resulted in severe demyelination, locomotor hyperactivity, and working memory impairment in mice. Remyelination occurred when cuprizone was withdrawn. Quetiapine treatment during the recovery period significantly improved the spatial working memory and increased myelin restoration. Quetiapine treatment also enhanced the repopulation of mature oligodendrocytes in the demyelinated lesions, which was associated with down-regulation of transcription factor olig2 in the process of cell maturation. The results of this study demonstrated that quetiapine treatment during the recovery period improves spatial working memory and promotes oligodendrocyte development and remyelination. This study supports the role of oligodendrocyte dysfunction in memory deficits in a schizophrenia mouse model and suggests that quetiapine may target oligodendrocytes and improve cognitive function.

The cuprizone model: regional heterogeneity of pathology

The cuprizone model is a model of de- and remyelination secondary to oligodendrocyte death, likely to be mediated by an inhibition of mitochondrial function. The aim of this study was to characterize histopathological changes associated with de/remyelination in grey and white matter at different disease stages in C57Bl/6 mice after per oral administration of cuprizone. Oligodendrocyte loss, astrocytosis and complement activation was detected in areas of demyelination. Demyelination, astrocytosis and complement activation occurred earlier in the cerebral cortex than in the corpus callosum. There was no perivascular lymphocyte infiltration. Microglia- and macrophage activation was observed in the corpus callosum, but not in the cerebral cortex. After cuprizone exposure was stopped, remyelination was extensive in the corpus callosum, but scarce in the cortex. In conclusion, cortical demyelination and oligodendrocyte loss in the cuprizone model may be due to a direct effect on oligodendrocyte mitochondrial function, as it occurs in the absence of microglial activation. The histopathology of de/remyelination in the cuprizone treated mice show regional heterogeneities which suggest differences in the underlying pathophysiology. Cuprizone-induced demyelination is a relevant model for the study of regional heterogeneity of demyelination and lesion pathology in multiple sclerosis.

Myelin copper and the cuprizone model of schizophrenia

In recent years increasing evidence is pointing toward white matter abnormalities in schizophrenia and other psychiatric disorders. The present paper will provide an overview over the role of myelin in cognition and brain function, and its potential involvement in brain disorders. Furthermore, we will examine one particular experimental model for the study of dysmyelination, created by the administration of the toxin cuprizone. Cuprizone, a copper chelator, causes white matter abnormalities in rodents. The administration of cuprizone during specific developmental periods allows for the targeting of specific brain areas for dysmyelination. Thus, cuprizone can be used to study the pathogenesis and pathophysiology of myelin deficiencies in the central nervous system, and its effect on behaviors relevant to psychiatric disorders.

Dynamic expression of Cx47 in mouse brain development and in the cuprizone model of myelin plasticity

The study shows the dynamic expression of connexin47 (Cx47) in oligodendrocytes and myelin of mice, either in myelinogenesis occurring in early development or in an experimental model of new-myelinogenesis of adult mice. Cx47 first appeared in the embryonic mouse brain at E10.5 successively the expression increased, principally in regions populated by developing oligodendrocytes. The expression declined postnatally toward adulthood and immunoreactivity was restricted to a few specific areas, such as the corpus callosum, the striatum, the cerebellum, and the spinal cord. Since the expression of Cx47 in developing oligodendrocytes preceded those of Cx32 and Cx29, a role of Cx47 in myelinogenesis was postulated. This hypothesis was tested in a model of re-myelination, which principally involved the corpus callosum, occurring in adult mice by treatment with cuprizone. Cx47 was upregulated during demyelination and recovered during the remyelination phase. During demyelination, Cx47 was first over-expressed in the corpus callosum and later, when the myelin virtually disappeared in the injured areas, Cx47 was expressed in astrocytes located inside and closely around the demyelinated areas. The remyelination of injured areas occurred after stopping the administration of cuprizone and continued to complete recovery. In this period the expression of Cx47 shifted from astrocytes to newly-formed myelin. Thus, Cx47 exhibits in this model a transient and de novo expression in astrocytes with a topographic segregation in the injured areas, only when oligodendrocytes and the myelin were most severely affected. Taken as a whole the evidence suggests that Cx47 play a key role in myelination.

Proteomic Analysis of Demyelinated and Remyelinating Brain Tissue following Dietary Cuprizone Administration

Cuprizone intoxication is a commonly used model of demyelination that allows the temporal separation of demyelination and remyelination. The underlying biochemical alterations leading to demyelination, using this model, remain unclear and may be multifold. Analysis of proteomic changes within the brains of cuprizone-exposed animals may help elucidate key cellular processes. In the current study, we report the results of the liquid chromatography tandem mass spectrometry analysis of total protein from the brain hemispheres of control and toxin-exposed mice at 6 weeks of exposure and after 3 and 6 weeks of recovery to identify protein changes during the remyelination phase. We found that at 6 weeks of cuprizone exposure, myelin proteins were reduced compared to controls and increased throughout the course of recovery, as expected. In contrast, other protein groups, such as proteins related to mitochondrial function, were increased at 6 weeks of treatment compared to untreated controls and returned toward control levels following withdrawal of toxin. These results suggest that a global proteomic analysis of the brain tissue of cuprizone-treated mice can identify changes related to the demyelination/remyelination process.

Regional differences between grey and white matter in cuprizone induced demyelination

Cuprizone feeding is a commonly used model to study experimental de- and remyelination, with the corpus callosum being the most frequently investigated white matter tract. We have previously shown that demyelination is also extensive in the cerebral cortex in the cuprizone model. In the current study, we have performed a detailed analysis of the dynamics of demyelination in the cortex in comparison to the corpus callosum. Prominent and almost complete demyelination in the corpus callosum was observed after 4.5–5 weeks of 0.2% cuprizone feeding, whereas complete cortical demyelination was only observed after 6 weeks of cuprizone feeding. Interestingly, remyelination in the corpus callosum occurred even before the termination of cuprizone administration. Accumulation of microglia in the corpus callosum started as early as week 3 reaching its maximum at week 4.5 and was still significantly elevated at week 6 of cuprizone treatment. Within the cortex only a few scattered activated microglial cells were found. Furthermore, the intensity of astrogliosis, accumulation of oligodendrocyte progenitor cells and nestin positive cells differed between the two areas investigated. The time course and dynamics of demyelination differ in the corpus callosum and in the cortex, suggesting different underlying pathomechanisms.

Statin Therapy Inhibits Remyelination in the Central Nervous System

Remyelination of lesions in the central nervous system contributes to neural repair following clinical relapses in multiple sclerosis. Remyelination is initiated by recruitment and differentiation of oligodendrocyte progenitor cells (OPCs) into myelinating oligodendrocytes. Simvastatin, a blood-brain barrier-permeable statin in multiple sclerosis clinical trials, has been shown to impact the in vitro processes that have been implicated in remyelination. Animals were fed a cuprizone-supplemented diet for 6 weeks to induce localized demyelination in the corpus callosum; subsequent return to normal diet for 3 weeks stimulated remyelination. Simvastatin was injected intraperitoneally during the period of coincident demyelination and OPC maturation (weeks 4 to 6), throughout the entire period of OPC responses (weeks 4 to 9), or during the remyelination-only phase (weeks 7 to 9). Simvastatin treatment (weeks 4 to 6) caused a decrease in myelin load and both Olig2(strong) and Nkx2.2(strong) OPC numbers. Simvastatin treatment (weeks 4 to 9 and 7 to 9) caused a decrease in myelin load, which was correlated with a reduction in Nkx2.2(strong) OPCs and an increase in Olig2(strong) cells, suggesting that OPCs were maintained in an immature state (Olig2(strong)/Nkx2.2(weak)). NogoA+ oligodendrocyte numbers were decreased during all simvastatin treatment regimens. Our findings suggest that simvastatin inhibits central nervous system remyelination by blocking progenitor differentiation, indicating the need to monitor effects of systemic immunotherapies that can access the central nervous system on brain tissue-repair processes.

Discoidin Domain Receptor 1, a Tyrosine Kinase Receptor, is Upregulated in an Experimental Model of Remyelination and During Oligodendrocyte Differentiation In Vitro

The discoidin domain receptor (DDR1) is highly expressed in oligodendrocytes during the neurodevelopmental myelination process and is genetically associated to schizophrenia. In this study, we aimed to further assess the involvement of DDR1 in both remyelination and oligodendrocyte differentiation. In the mouse model of demyelination-remyelination induced by oral administration of cuprizone, in situ hybridization showed an upregulation of the DDR1 gene in three different white matter areas (corpus callosum, dorsal fornix, and external capsule) during the remyelination period. Moreover, real time reverse transcriptase polymerase chain reaction showed that the increase in DDR1 messenger RNA (mRNA) was strongly correlated with the number of DDR1-positive cells in the corpus callosum (Spearman coefficient = 0.987, P = 0.013). Cells positive for DDR1 mRNA were also positive for oligodendrocyte markers (OLIG2, carnosine, and APC) but not for markers of oligodendrocyte precursors (NG2), myelin markers (CNPase), microglia (CD11b), or reactive glia (GFAP). Differentiation of a human oligodendroglial cell line, HOG16, was associated with an increase in mRNA expression of DDR1 and several myelin proteins (MBP and MOBP) but not other proteins (APC and CNPase). Here, we demonstrate that DDR1 is upregulated in vitro and in vivo when oligodendrocyte myelinating machinery is activated. Further studies are needed to identify the specific molecular pathway.

Expression of an acyl‐CoA synthetase, lipidosin, in astrocytes of the murine brain and its up‐regulation during remyelination following cuprizone‐induced demyelination

Lipidosin is an 80-kDa protein with long-chain acyl-CoA synthetase activity expressed in the brain, adrenal gland, testis, and ovary, which are selectively damaged in X-linked adrenoleukodystrophy (X-ALD). Western blot analysis of the cerebrum and cerebellum revealed a gradual increase in the expression of lipidosin postnatally. Light microscopic immunohistochemistry using a panel of specific monoclonal antibodies showed that the lipidosin-immunopositive cells were ubiquitously distributed in the brain and were denser in the gray matter than in the white matter. Lipidosin immunoreactivity was colocalized with GFAP immunoreactivity but not with ubiquitin C-terminal hydrolase 1 (= PGP9.5) immunoreactivity, a neuronal marker, and lipidosin-producing cells detected by an antisense probe specific for lipidosin mRNA were also GFAP immunopositive. These data together with Western blot analysis of primary cultured astrocytes indicate that lipidosin is expressed in astrocytes. Immunoelectron microscopic analysis revealed that lipidosin immunoreactivity was widely distributed from perivascular endfeet to perisynaptic processes without being limited to peroxisomes. Lipidosin immunoreactivity was greatly increased in astrocytes in the area of remyelination following experimental demyelination induced by the administration of cuprizone to mice. These data suggest that lipidosin was involved in fatty acid metabolism during reconstruction of the myelin sheath.

Platelet-derived growth factor regulates oligodendrocyte progenitor numbers in adult CNS and their response following CNS demyelination

To design therapies for demyelinating diseases such as multiple sclerosis, it will be important to understand the mechanisms that control oligodendrocyte progenitor cell (OPC) numbers in the adult central nervous system (CNS). During development, OPC numbers are limited by the supply of platelet-derived growth factor-A (PDGF-A). Here, we examine the role of PDGF-A in regulating OPC numbers in normal and demyelinated adult CNS using transgenic mice that overexpress PDGF-A in astrocytes under the control of the glial fibrillary acidic protein (GFAP) gene promoter (GFAP-PDGF-A mice). In adult GFAP-PDGF-A mice, there was a marked increase in OPC density, particularly in white matter tracts, indicating that the PDGF-A supply controls OPC numbers in the adult CNS as well as during development. To discover whether increasing PDGF expression increases the number of OPCs following demyelination and whether this enhances the efficiency of remyelination, we induced demyelination in GFAP-PDGF-A transgenic mice by intraspinal injection of lysolecithin or dietary administration of cuprizone. In both demyelinating models, OPC density within lesions was significantly increased compared to wild-type mice. However, morphological analysis of lysolecithin lesions did not reveal any difference in the time course or extent of remyelination between GFAP-PDGF-A and wild-type mice. We conclude that the availability of OPCs is not rate limiting for remyelination of focal demyelinated lesions in the mouse. Nevertheless, our experiments show that it is possible to increase OPC population density in demyelinated areas by artificially increasing the supply of PDGF.

Zinc supplementation does not prevent cuprizone toxicity in the brain of mice

The metal ion chelator cuprizone (bis(cyclohexanone)oxalyldihydrazone) is an effective agent for inducing experimental demyelination in rodents. The current work was undertaken to assess whether this demyelination was a result of the ability of cuprizone to sequester metal ions thereby leading to a reduction of tissue concentrations of essential trace metals. After cuprizone administration, demyelination in mouse brain was followed using biochemical techniques and it was shown that mice fed cuprizone for 49 days had reduced blood concentrations of copper, zinc and iron but that the brain contents of these cations were not decreased. Also, that loss of carbonic anhydrase activity (CAII)(a zinc metalloenzyme) in cuprizone fed mice could not be prevented by the feeding of a zinc supplemented diet. Indeed, zinc supplementation itself had an deletrious effect on kidney CA11 activity. The findings are discussed in relation to possible roles for CA in the CNS and kidney.

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.

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.

Phosphatidylethanolamine methyltransferase activity in developing, demyelinating, and diabetic mouse brain.

The catalysis by phosphatidylethanolamine methyltransferase (PEMT) of phosphatidylcholine (PC) synthesis by the successive methylation of phosphatidylethanolamine in the presence of S-adenosylmethionine (AdoMet) as methyl donor, was detected in actively myelinating mouse brains. PEMT activity in the microsome fraction of fetal mouse brain at 17 days of gestation was 253 mu u/mg protein and that of adult brain after 7 days of remyelination following 6 weeks cuprizone administration was 148 mu u/mg. These figures are much higher than found in normal adult brains (1.7 mu u/mg). An increase in PEMT activity was observed in the brains of genetically transmitted diabetic mice, C57BL/KsJ-db/db, and streptozotocin-induced diabetic mice; 16.3 and 9.2 mu u/mg, respectively. The methyl group of mecobalamin was transferred to homocysteine producing AdoMet and was further metabolized into choline and acetylcholine in brain slices. These results suggest that in the diabetic state, an increase in PC synthesis is probably required in order to replace damaged myelin or to supply choline or acetylcholine essential to for nerve functions. Mecobalamin might serve as the source of the methyl group utilized for PC synthesis.

Evidence for a "dying-back" gliopathy in demyelinating disease.

Recurrent demyelination was produced in mice by Cuprizone administration. During the second course of Cuprizone, the animals showed greater resistance to the toxin and demyelination occurred slowly and was complete only after prolonged periods. The earliest changes in oligodendrocytes occurred in the most distal processes, the inner cytoplasmic tongues, which showed degenerative changes 3 to 4 weeks before degeneration of the oligodendrocyte cell bodies or demyelination occurred. The results show for the first time that in demyelinating disease, a "dying-back" process similar to that described in axonal disease can affect the oligodendrocyte.

The demonstration of recurrent demyelination and remyelination of axons in the central nervous system.

A model for studying recurrent demyelination and remyelination in the central nervous system was developed by means of repeated administration of Cuprizone to mice. In contrast to the demyelination seen during the first course of Cuprizone, the recurrent demyelination was markedly protracted, displayed features of a "dying-back" gliopathy, and resulted in a greatly reduced inflammatory and glial reaction. The repeat remyelination also occurred at a slower tempo, varied markedly in completeness, and was associated with a diminished regeneration of oligodendrocytes. These results demonstrated that axons of the central nervous system in this model can be recurrently remyelinated if oligodendrocytes are available, that regeneration of oligodendrocytes is dependent upon the tissue reaction to demyelination, and that remyelinated axons are not more susceptible to demyelination than normal ones.

The perineuronal satellite oligodendrocyte. A role in remyelination.

Demyelination was induced in the superior cerebellar peduncles of weanling mice by the administration of Cuprizone. Remyelination occurred when the animals were replaced on a normal diet. Perineuronal satellite oligodendrocytes in the periventricular gray were clearly seen to be remyelinating axons. This study demonstrates for the first time the role of these cells in remyelination, and raises the possibility that they may be involved in normal myelination of the central nervous system.

ENZYMIC ACTIVITIES AND SODIUM, POTASSIUM AND COPPER CONCENTRATIONS IN MOUSE BRAIN AND LIVER AFTER CUPRIZONE TREATMENT IN VIVO

Abstract— The oral administration of cuprizone to mice induces status spongiosus in the CNS and giant mitochondria in the liver. The activities of monoamine oxidase and cytochrome oxidase in brain and liver are inhibited and succinate dehydrogenase activity is increased. There is an increase in sodium and decrease in potassium concentrations; brain dry weight and copper content are decreased. Animals treated with copper‐chelated cuprizone do not show any changes.