Minocycline reduces remyelination by suppressing ciliary neurotrophic factor expression after cuprizone‐induced demyelination

Remyelination is disrupted in demyelinating diseases such as multiple sclerosis, but the underlying pathogenetic mechanisms are unclear. In this study, we employed the murine cuprizone model of demyelination, in which remyelination occurs after removal of the toxin from the diet, to examine the cellular and molecular changes during demyelination and remyelination. Microglia accumulated in the corpus callosum during weeks 2–4 of the cuprizone diet, and these cells remained activated 2 weeks after the change to the normal diet. To examine the role of microglia in remyelination, mice were treated with minocycline to inactivate these cells after cuprizone‐induced demyelination. Minocycline treatment reduced the number of CC1‐positive oligodendrocytes, as well as levels of myelin basic protein (MBP) and CNPase in the remyelination phase. The expression of CNTF mRNA in the corpus callosum increased after 4 weeks on the cuprizone diet and remained high 2 weeks after the change to the normal diet. Minocycline suppressed CNTF expression during the remyelination phase on the normal diet. Primary culture experiments showed that CNTF was produced by microglia in addition to astrocytes. In vitro, CNTF directly affected the differentiation of oligodendrocytic cells. These findings suggest that minocycline reduces remyelination by suppressing CNTF expression by microglia after cuprizone‐induced demyelination.     

Investigation of neuro-inflammatory parameters in a cuprizone induced mouse model of multiple sclerosis

Cuprizone, copper chelator, treatment of mouse is a toxic model of multiple sclerosis (MS) in which oligodendrocyte death, demyelination and remyelination can be observed. Understanding T and B cell subset as well as their cytokines involved in MS pathogenesis still requires further scrutiny to better understand immune component of MS. The study presented here, aimed to evaluate relevant cytokines, lymphocytes, and gene expressions profiles during demyelination and remyelination in the cuprizone mouse model of MS. Eighty male C57BL/6J mice fed with 0.2% cuprizone for eight weeks. Cuprizone has been removed from the diet in the following eight weeks. Cuprizone treated and control mice sacrificed biweekly, and corpus callosum of the brain was investigated by staining. Lymphocyte cells of mice analyzed by flow cytometry with CD3e, CD11b, CD19, CD80, CD86, CD4, CD25 and FOXP3 antibodies. IFN-gamma, IL-1alpha, IL-2, IL-5, IL-6, IL-10, IL-17, TNF-alpha cytokines were analyzed in plasma samples. Neuregulin 1 (Nrg1), ciliary neurotrophic factor (Cntf) and C-X-C chemokine receptor type 4 (Cxcr4) gene expressions in corpus callosum sections of the mice brain were quantified. Histochemistry analysis showed that demyelination began at the fourth week of cuprizone administration and total demyelination occurred at the twelfth week in chronic model. Remyelination occurred at the fourth week of following withdrawal of cuprizone from diet. The level of mature and activated T cells, regulatory T cells, T helper cells and mature B cells increased during demyelination and decreased when cuprizone removed from diet. Further, both type 1 and type 2 cytokines together with the proinflammatory cytokines increased. The level of oligodendrocyte maturation and survival genes showed differential gene expression in parallel to that of demyelination and remyelination. In conclusion, for the first-time, involvement of both cellular immune response and antibody response as well as oligodendrocyte maturation and survival factors having role in demyelination and remyelination of cuprizone mouse model of MS have been shown.     

Astrogliosis during acute and chronic cuprizone demyelination and implications for remyelination

In multiple sclerosis, microglia/macrophage activation and astrocyte reactivity are important components of the lesion environment that can impact remyelination. The current study characterizes these glial populations relative to expression of candidate regulatory molecules in cuprizone demyelinated corpus callosum. Importantly, periods of recovery after acute or chronic cuprizone demyelination are examined to compare conditions of efficient versus limited remyelination, respectively. Microglial activation attenuates after early demyelination. In contrast, astrocyte reactivity persists throughout demyelination and a 6-week recovery period following either acute or chronic demyelination. This astrocyte reaction is characterized by (a) early proliferation, (b) increased expression of GFAP (glial fibrillary acidic protein), Vim (vimentin), Fn1 (fibronectin) and CSPGs (chondroitin sulphate proteoglycans) and (c) elaboration of a dense network of processes. Glial processes elongated in the axonal plane persist throughout lesion areas during both the robust remyelination that follows acute demyelination and the partial remyelination that follows chronic demyelination. However, prolonged astrocyte reactivity with chronic cuprizone treatment does not progress to barrier formation, i.e. dense compaction of astrocyte processes to wall off the lesion area. Multiple candidate growth factors and inflammatory signals in the lesion environment show strong correlations with GFAP across the acute cuprizone demyelination and recovery time course, yet there is more divergence across the progression of chronic cuprizone demyelination and recovery. However, differential glial scar formation does not appear to be responsible for differential remyelination during recovery in the cuprizone model. The astrocyte phenotype and lesion characteristics in this demyelination model inform studies to identify triggers of non-remyelinating sclerosis in chronic multiple sclerosis lesions.     

Spatio-Temporal Patterns of Demyelination and Remyelination in the Cuprizone Mouse Model

Cuprizone administration in mice provides a reproducible model of demyelination and spontaneous remyelination, and has been useful in understanding important aspects of human disease, including multiple sclerosis. In this study, we apply high spatial resolution quantitative MRI techniques to establish the spatio-temporal patterns of acute demyelination in C57BL/6 mice after 6 weeks of cuprizone administration, and subsequent remyelination after 6 weeks of post-cuprizone recovery. MRI measurements were complemented with Black Gold II stain for myelin and immunohistochemical stains for associated tissue changes. Gene expression was evaluated using the Allen Gene Expression Atlas. Twenty-five C57BL/6 male mice were split into control and cuprizone groups; MRI data were obtained at baseline, after 6 weeks of cuprizone, and 6 weeks post-cuprizone. High-resolution (100μm isotropic) whole-brain coverage magnetization transfer ratio (MTR) parametric maps demonstrated concurrent caudal-to-rostral and medial-to-lateral gradients of MTR decrease within corpus callosum (CC) that correlated well with demyelination assessed histologically. Our results show that demyelination was not limited to the midsagittal line of the corpus callosum, and also that opposing gradients of demyelination occur in the lateral and medial CC. T₂-weighted MRI gray/white matter contrast was strong at baseline, weak after 6 weeks of cuprizone treatment, and returned to a limited extent after recovery. MTR decreases during demyelination were observed throughout the brain, most clearly in callosal white matter. Myelin damage and repair appear to be influenced by proximity to oligodendrocyte progenitor cell populations and exhibit an inverse correlation with myelin basic protein gene expression. These findings suggest that susceptibility to injury and ability to repair vary across the brain, and whole-brain analysis is necessary to accurately characterize this model. Whole-brain parametric mapping across time is essential for gaining a real understanding of disease processes in-vivo. MTR increases in healthy mice throughout adolescence and adulthood were observed, illustrating the need for appropriate age-matched controls. Elucidating the unique and site-specific demyelination in the cuprizone model may offer new insights into in mechanisms of both damage and repair in human demyelinating diseases.     

Cuprizone demyelination of the corpus callosum in mice correlates with altered social interaction and impaired bilateral sensorimotor coordination

For studies of remyelination in demyelinating diseases, the cuprizone model of CC (corpus callosum) demyelination has experimental advantages that include overall size, proximity to neural stem cells of the subventricular zone, and correlation with a lesion predilection site in multiple sclerosis. In addition, cuprizone treatment can be ended to allow more direct analysis of remyelination than with viral or autoimmune models. However, CC demyelination lacks a useful functional correlate in rodents for longitudinal analysis throughout the course of demyelination and remyelination. In the present study, we tested two distinct behavioural measurements in mice fed 0.2% cuprizone. Running on a ‘complex'' wheel with varied rung intervals requires integration between cerebral hemispheres for rapid bilateral sensorimotor coordination. Maximum running velocity on the ‘complex'' wheel decreased during acute (6 week) and chronic (12 week) cuprizone demyelination. Running velocity on the complex wheel distinguished treated (for 6 weeks) from non-treated mice, even after a 6-week recovery period for spontaneous remyelination. A second behavioural assessment was a resident–intruder test of social interaction. The frequency of interactive behaviours increased among resident mice after acute or chronic demyelination. Differences in both sensorimotor coordination and social interaction correlated with demonstrated CC demyelination. The wheel assay is applicable for longitudinal studies. The resident–intruder assay provides a complementary assessment of a distinct modality at a specific time point. These behavioural measurements are sufficiently robust for small cohorts as a non-invasive assessment of demyelination to facilitate analysis of subsequent remyelination. These measurements may also identify CC involvement in other mouse models of central nervous system injuries and disorders.     

Spatial and temporal profiles of growth factor expression during CNS demyelination reveal the dynamics of repair priming

Demyelination is the cause of disability in various neurological disorders. It is therefore crucial to understand the molecular regulation of oligodendrocytes, the myelin forming cells in the CNS. Growth factors are known to be essential for the development and maintenance of oligodendrocytes and are involved in the regulation of glial responses in various pathological conditions. We employed the well established murine cuprizone model of toxic demyelination to analyze the expression of 13 growth factors in the CNS during de- and remyelination. The temporal mRNA expression profile during demyelination and the subsequent remyelination were analyzed separately in the corpus callosum and cerebral cortex using laser microdissection and real-time PCR techniques. During demyelination a similar pattern of growth factor mRNA expression was observed in both areas with a strong up-regulation of NRG1 and GDNF and a slight increase of CNTF in the first week of cuprizone treatment. HGF, FGF-2, LIF, IGF-I, and TGF-ß1 were up-regulated mainly during peak demyelination. In contrast, during remyelination there were regional differences in growth factor mRNA expression levels. GDNF, CNTF, HGF, FGF-2, and BDNF were elevated in the corpus callosum but not in the cortex, suggesting tissue differences in the molecular regulation of remyelination in the white and grey matter. To clarify the cellular source we isolated microglia from the cuprizone lesions. GDNF, IGF-1, and FGF mRNA were detected in the microglial fraction with a temporal pattern corresponding to that from whole tissue PCR. In addition, immunohistochemical analysis revealed IGF-1 protein expression also in the reactive astrocytes. CNTF was located in astrocytes. This study identified seven different temporal expression patterns for growth factors in white and grey matter and demonstrated the importance of early tissue priming and exact orchestration of different steps during callosal and cortical de- and remyelination.     

Alterations in metabolism and gene expression in brain regions during cuprizone-induced demyelination and remyelination

Exposure of mice to the copper chelator, cuprizone, results in CNS demyelination. There is remyelination after removal of the metabolic insult. We present brain regional studies identifying corpus callosum as particularly severely affected; 65% of cerebroside is lost after 6 weeks of exposure. We examined recovery of cerebroside and ability to synthesize cerebroside and cholesterol following removal of the toxicant. The temporal pattern for concentration of myelin basic protein resembled that of cerebroside. We applied Affymetrix GeneChip technology to corpus callosum to identify temporal changes in levels of mRNAs during demyelination and remyelination. Genes coding for myelin structural components were greatly down-regulated during demyelination and up-regulated during remyelination. Genes related to microglia/macrophages appeared in a time-course (peaking at 6 weeks) correlating with phagocytosis of myelin and repair of lesions. mRNAs coding for many cytokines had peak expression at 4 weeks, compatible with intercellular signaling roles. Of interest were other genes with temporal patterns correlating with one of the three above patterns, but of function not obviously related to demyelination/remyelination. The ability to correlate gene expression with known pathophysiological events should help in elucidating further function of such genes as related to demyelination/remyelination.     

Characterization of Glial Populations in the Aging and Remyelinating Mouse Corpus Callosum

Cells in the white matter of the adult brain have a characteristic distribution pattern in which several cells are contiguously connected to each other, making a linear array (LA) resembling pearls-on-a-string parallel to the axon axis. We have been interested in how this pattern of cell distribution changes during aging and remyelination after demyelination. In the present study, with a multiplex staining method, semi-quantitative analysis of the localization of oligodendrocyte lineage cells (oligodendrocyte progenitors, premyelinating oligodendrocytes, and mature oligodendrocytes), astrocytes, and microglia in 8-week-old (young adult) and 32-week-old (aged) corpus callosum showed that young adult cells still include immature oligodendrocytes and that LAs contain a higher proportion of microglia than isolated cells. In aged mice, premyelinating oligodendrocytes were decreased, but microglia continued to be present in the LAs. These results suggest that the presence of microglia is important for the characteristic cell localization pattern of LAs. In a cuprizone-induced demyelination model, we observed re-formation of LAs after completion of cuprizone treatment, concurrent with remyelination. These re-formed LAs again contained more microglia than the isolated cells. This finding supports the hypothesis that microglia contribute to the formation and maintenance of LAs. In addition, regardless of the distribution of cells (LAs or isolated cells), astrocytes were found to be more abundant than in the normal corpus callosum at 24 weeks after cuprizone treatment when remyelination is completed. This suggests that astrocytes are involved in maintaining the functions of remyelinated white matter.

     

A New Model of Cuprizone-Mediated Demyelination/Remyelination

In the central nervous system, demyelinating diseases, such as multiple sclerosis, result in devastating long-term neurologic damage, in part because of the lack of effective remyelination in the adult human brain. One model used to understand the mechanisms regulating remyelination is cuprizone-induced demyelination, which allows investigation of remyelination mechanisms in adult animals following toxin-induced demyelination. Unfortunately, the degree of demyelination in the cuprizone model can vary, which complicates understanding the process of remyelination. Previous work in our laboratory demonstrated that the Akt/mTOR pathway regulates active myelination. When given to young postnatal mice, the mTOR inhibitor, rapamycin, inhibits active myelination. In the current study, the cuprizone model was modified by the addition of rapamycin during cuprizone exposure. When administered together, cuprizone and rapamycin produced more complete demyelination and provided a longer time frame over which to investigate remyelination than treatment with cuprizone alone. The consistency in demyelination will allow a better understanding of the mechanisms initiating remyelination. Furthermore, the slower rate of remyelination provides a longer window of time in which to investigate the diverse contributing factors that regulate remyelination. This new model of cuprizone-induced demyelination could potentially aid in identification of new therapeutic targets to enhance remyelination in demyelinating diseases.     

Deep Gray Matter Demyelination Detected by Magnetization Transfer Ratio in the Cuprizone Model

In multiple sclerosis (MS), the correlation between lesion load on conventional magnetic resonance imaging (MRI) and clinical disability is weak. This clinico-radiological paradox might partly be due to the low sensitivity of conventional MRI to detect gray matter demyelination. Magnetization transfer ratio (MTR) has previously been shown to detect white matter demyelination in mice. In this study, we investigated whether MTR can detect gray matter demyelination in cuprizone exposed mice. A total of 54 female C57BL/6 mice were split into one control group () and eight cuprizone exposed groups (). The mice were exposed to (w/w) cuprizone for up to six weeks. MTR images were obtained at a 7 Tesla Bruker MR-scanner before cuprizone exposure, weekly for six weeks during cuprizone exposure, and once two weeks after termination of cuprizone exposure. Immunohistochemistry staining for myelin (anti-Proteolopid Protein) and oligodendrocytes (anti-Neurite Outgrowth Inhibitor Protein A) was obtained after each weekly scanning. Rates of MTR change and correlations between MTR values and histological findings were calculated in five brain regions. In the corpus callosum and the deep gray matter a significant rate of MTR value decrease was found, per week () and per week () respectively. The MTR values correlated to myelin loss as evaluated by immunohistochemistry (Corpus callosum: . Deep gray matter: ), but did not correlate to oligodendrocyte density. Significant results were not found in the cerebellum, the olfactory bulb or the cerebral cortex. This study shows that MTR can be used to detect demyelination in the deep gray matter, which is of particular interest for imaging of patients with MS, as deep gray matter demyelination is common in MS, and is not easily detected on conventional clinical MRI.     

Beneficial Effects of Minocycline on Cuprizone Induced Cortical Demyelination

In this study, we investigated the potential of minocycline to influence cuprizone induced demyelination in the grey and white matter. To induce demyelination C57BL/6 mice were fed with cuprizone for up to 6 weeks and were analysed at different timepoints (week 0, 4, 5, 6). Mice treated with minocycline had less demyelination of the cortex and corpus callosum compared with sham treated animals. In the cortex decreased numbers of activated and proliferating microglia were found after 6 weeks of cuprizone feeding, while there were no significant effects for microglial infiltration of the corpus callosum. In addition to the beneficial effects on demyelination, minocycline prevented from motor coordination disturbance as shown in the beam walking test. For astrogliosis and the numbers of OPC and oligodendrocytes no treatment effects were found. In summary, minocycline treatment diminished the course of demyelination in the grey and white matter and prevented disturbances in motor coordination.     

Oligodendrocyte Progenitor Cells Directly Utilize Lactate for Promoting Cell Cycling and Differentiation

Oligodendrocyte progenitor cells (OPCs) undergo marked morphological changes to become mature oligodendrocytes, but the metabolic resources for this process have not been fully elucidated. Although lactate, a metabolic derivative of glycogen, has been reported to be consumed in oligodendrocytes as a metabolite, and to ameliorate hypomyelination induced by low glucose conditions, it is not clear about the direct contribution of lactate to cell cycling and differentiation of OPCs, and the source of lactate for remyelination. Therefore, we evaluated the effect of 1,4‐dideoxy‐1,4‐imino‐d‐arabinitol (DAB), an inhibitor of the glycogen catabolic enzyme glycogen phosphorylase, in a mouse cuprizone model. Cuprizone induced demyelination in the corpus callosum and remyelination occurred after cuprizone treatment ceased. This remyelination was inhibited by the administration of DAB. To further examine whether lactate affects proliferation or differentiation of OPCs, we cultured mouse primary OPC‐rich cells and analyzed the effect of lactate. Lactate rescued the slowed cell cycling induced by 0.4 mM glucose, as assessed by the BrdU‐positive cell ratio. Lactate also promoted OPC differentiation detected by monitoring the mature oligodendrocyte marker myelin basic protein, in the presence of both 36.6 mM and 0.4 mM glucose. Furthermore, these lactate‐mediated effects were suppressed by the reported monocarboxylate transporter inhibitor, α‐cyano‐4‐hydroxy‐cinnamate. These results suggest that lactate directly promotes the cell cycling rate and differentiation of OPCs, and that glycogen, one of the sources of lactate, contributes to remyelination in vivo. J. Cell. Physiol. 232: 986–995, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

环己酮二腙诱导大鼠脑白质脱髓鞘及细胞凋亡的实验研究

目的:探讨双环己酮草酰二腙(cuprizone)诱导大鼠脑白质脱髓鞘及其病因,证实双环己酮草酰二腙引起脑白质脱髓鞘与细胞凋亡有关。方法:用环己酮草酰二腙制备大鼠脑白质脱髓鞘模型(酮腙组),与安定(安定组)、苯巴比妥(苯巴比妥组)、生理盐水对照组(对照组)比较,应用电镜技术及caspase3免疫组化染色,观察各组第14天、28天、42天时脑组织结构变化及细胞凋亡的信号传导通路。结果:电镜显示,安定组、苯巴比妥组、酮腙14天组和对照组白质结构完整致密,无脱髓鞘现象;酮腙28天组可见髓鞘排列较紊乱,部分结构松解变性,但无典型脱髓鞘改变;酮腙42天组胼胝体压部可见髓鞘肿胀,多部位髓鞘被涡轮状空泡所裂解。caspase3染色:酮腙28天、42天组可见皮层下白质、胼胝体、脑干及小脑白质caspase3阳性染色,与安定组、苯巴比妥组、对照组和酮腙14天组比较差异具有统计学意义(P<0.05);酮腙42天组caspase3阳性染色明显多于28天组,差异具有统计学意义(P<0.05)。结论:环己酮草酰二腙可诱导大鼠脑白质脱髓鞘、空泡样变;病变白质区存在大量caspase3阳性染色,且早于脱髓鞘。提示:caspase蛋白酶级联反应参与了环己酮草酰二腙诱导脑白质脱髓鞘的过程,进一步说明细胞凋亡可能是脑白质脱髓鞘原因之一。     

Oligoprogenitor Cells Derived from Spermatogonia Stem Cells Improve Remyelination in Demyelination Model

Embryonic stem (ES) like cells-derived testis represents a possible alternative to replace of neurons and glia. Here, we differentiated spermatogonia cells to oligoprogenitor (OP) like cells and transplanted them to demyelination model and assess their recovery potential in a demyelinated corpus callosum model in rats. ES like cells were differentiated to OP like cells using appropriate inducers and were transplanted in situ to demyelinated corpus callosum. Cell integration as well as demyelination extension and myelination intensity changes were evaluated using histologic studies and immunocytochemistry after 2 and 4 weeks post transplantation. Investigation of Nestin, NF68, Olig2, and NG2 by immunocytochemical technique indicated the differentiation of ES like cells to neuroprogenitor and oligodendrocyte like cells in each induction stage. Histologic findings showed a significant decrease in demyelination extension and a significant increase in remyelination intensity in cell transplanted groups. Also on the base of PLP expression, differentiation of transplanted cells was confirmed to myelinogenic cells using immunocytochemistry technique. We conclude that ES like cells derived from spermatogonia cells can be differentiated to OP like cells that can form myelin after transplantation into the demyelination model in rat, this represents recovery potential of spermatogonia cells which opens new window for cell therapeutic approaches using spermatogonial stem cells.     

Toll-Like Receptor 2-Mediated Glial Cell Activation in a Mouse Model of Cuprizone-Induced Demyelination

Multiple sclerosis (MS) is a chronic degenerative disease of the central nervous system that is characterized by myelin abnormalities, oligodendrocyte pathology, and concomitant glia activation. The factors triggering gliosis and demyelination are currently not well characterized. New findings suggest an important role of the innate immune response in the initiation and progression of active demyelinating lesions. Especially during progressive disease, aberrant glia activation rather than the invasion of peripheral immune cells is accountable for progressive neuronal injury. The innate immune response can be induced by pathogen-associated or danger-associated molecular patterns, which are identified by pattern recognition receptors (PRRs), including the Toll-like receptors (TLRs). In this study, we used the cuprizone model in mice to investigate the expression of TLR2 during the course of cuprizone-induced demyelination. In addition, we used TLR2-deficient mice to analyze the functional role of TLR2 activation during cuprizone-induced demyelination and reactive gliosis. We show a significantly increased expression of TLR2 in the corpus callosum and hippocampus of cuprizone-intoxicated mice. The absence of receptor signaling in TLR2-deficient mice resulted in less severe reactive astrogliosis in the corpus callosum and cortex. In addition, microglia activation was ameliorated in the corpus callosum of TLR2-deficient mice, but augmented in the cortex compared to wild-type littermates. Extent of demyelination and loss of mature oligodendrocytes was comparable in both genotypes. These results suggest that the TLR2 orchestrates glia activation during gray and white matter demyelination in the presence of an intact blood-brain barrier. Future studies now have to address the underlying mechanisms of the region-specific TLR2-mediated glia activation.     

Thyroid hormone alleviates demyelination induced by cuprizone through its role in remyelination during the remission period

Multiple sclerosis (MS) is a disease induced by demyelination in the central nervous system, and the remission period of MS is crucial for remyelination. In addition, abnormal levels of thyroid hormone (TH) have been identified in MS. However, in the clinic, insufficient attention has been paid to the role of TH in the remission period. Indeed, TH not only functions in the development of the brain but also affects myelination. Therefore, it is necessary to observe the effect of TH on remyelination during this period. A model of demyelination induced by cuprizone (CPZ) was used to observe the function of TH in remyelination during the remission period of MS. Through weighing and behavioral tests, we found that TH improved the physical symptoms of mice impaired by CPZ. Supplementation of TH led to the repair of myelin as detected by immunohistochemistry and western blot. In addition, a sufficient TH supply resulted in an increase in myelinated axons without affecting myelin thickness and g ratio in the corpus callosum, as detected by electron microscopy. Double immunostaining with myelin basic protein and neurofilament 200 (NF200) showed that the CPZ-induced impairment of axons was alleviated by TH. Conversely, insufficient TH induced by 6-propyl-2-thiouracil resulted in the enlargement of mitochondria. Furthermore, we found that an adequate supply of TH promoted the proliferation and differentiation of oligodendrocyte lineage cells by immunofluorescence, which was beneficial to remyelination. Further, we found that TH reduced the number of astrocytes without affecting microglia. Conclusively, it was shown that TH alleviated demyelination induced by CPZ by promoting the development of oligodendrocyte lineage cells and remyelination. The critical time for remyelination is the remission period of MS. TH plays a significant role in alleviating demyelination during the remission period in the clinical treatment of MS.     

Expression of carbonic anhydrase II mRNA and protein in oligodendrocytes during toxic demyelination in the young adult mouse

The aim of this study was to identify events that might take place in oligodendrocytes early in the process of demyelination, i.e., before the occurrence of massive loss of myelin. It was considered important to focus on demyelination and remyelination in young adults, in whose brains there would be relatively few juvenile glial precursor cells. CAII mRNA and protein were used to monitor changes in oligodendrocytes during cuprizone intoxication in the mice. After four or eight weeks of cuprizone feeding CAII message became less plentiful in oligodendrocyte processes. Two days after removal of cuprizone CAII message had appeared in those cell processes. Four or eight weeks after beginning cuprizone feeding CAII protein had decreased∼25% in forebrain homogenates. The loss of CAII protein was reversible after four weeks on cuprizone, but not after eight weeks. After four weeks of cuprizone feeding the numbers of CAII mRNA-prositive oligodendrocytes had decreased by ∼50%m and after eight weeks, by ∼80%. By 12 weeks, however, the number of oligodendrocytes expressing CAII mRNA had spontaneously returned to normal levels. Before eight weeks of cuprizone feeding, loss of myelinated tracts in the corpus striatum was reversible. Demyelination appreared to become irreversible after nine weeks of intoxication, although expression of CAII mRNA remained reversible. The results suggest that in the brain of the young adult, oligodendrocytes expressing message for CAII can be generated spontaneously shortly before demyelination becomes irreversible, and can survive and continue to express CAII mRNA but not CAII protein. Special issue dedicated to Dr. Marion E. Smith.     

Strain differences in cuprizone induced demyelination

Background

Multiple sclerosis (MS) is a severe neurological disorder, characterized by demyelination of the central nervous system (CNS), and with a prevalence of greater than 2 million people worldwide. In terms of research in MS pathology, the cuprizone toxicity model is widely used. Here we investigated the contribution of genetic differences in response to cuprizone-induced demyelination in two genetically different mouse strains: CD1 and C57BL/6.

Results

We demonstrate that exposure to a diet containing 0.2% cuprizone resulted in less severe demyelination in the midline of the corpus callosum over the fornix in CD1 mice than C57BL/6 mice. With continuous cuprizone feeding, demyelination in CD1 mice was not prominent until after 7 weeks, in contrast to C57BL/6 mice, which showed prominent demyelination after 4 weeks of exposure. Concomitantly, immunohistochemical analysis demonstrated more oligodendrocytes, as well as fewer oligodendrocyte progenitor cells, microglia and astrocytes in cuprizone treated CD1 mice. We also analyzed 4-weeks-cuprizone treated corpus callosum tissue samples and found that cuprizone treated CD1 mice showed a smaller reduction of myelin-associated glycoprotein (MAG) and a smaller increase of Iba1 and NG2.

Conclusions

These observations suggest that CD1 mice are less vulnerable to cuprizone-induced demyelination than C57BL/6 mice and thus genetic background factors appear to influence the susceptibility to cuprizone-induced demyelination.

     

GAS6 enhances repair following cuprizone-induced demyelination

Growth arrest-specific protein 6 (gas6) activities are mediated through the Tyro3, Axl, and Mer family of receptor tyrosine kinases. Gas6 is expressed and secreted by a wide variety of cell types, including cells of the central nervous system (CNS). In this study, we tested the hypothesis that administration of recombinant human Gas6 (rhGas6) protein into the CNS improves recovery following cuprizone withdrawal. After a 4-week cuprizone diet, cuprizone was removed and PBS or rhGas6 (400 ng/ml, 4 μg/ml and 40 μg/ml) was delivered by osmotic mini-pump into the corpus callosum of C57Bl6 mice for 14 days. Nine of 11 (82%) PBS-treated mice had abundant lipid-associated debris in the corpus callosum by Oil-Red-O staining while only 4 of 19 (21%) mice treated with rhGas6 had low Oil-Red-O positive droplets. In rhGas6-treated mice, SMI32-positive axonal spheroids and APP-positive deposits were reduced in number relative to PBS-treated mice. Compared to PBS, rhGas6 enhanced remyelination as revealed by MBP immunostaining and electron microscopy. The rhGas6-treated mice had more oligodendrocytes expressing Olig1 in the cytoplasm, indicative of oligodendrocyte progenitor cell maturation. Relative to PBS-treated mice, rhGas6-treated mice had fewer activated microglia in the corpus callosum by Iba1 immunostaining. The data show that rhGas6 treatment resulted in more efficient repair following cuprizone-induced injury.