Temporal and spatial evolution of various functional neurons during demyelination induced by cuprizone.

Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS). Here we report the temporal and spatial evolution of various functional neurons during demyelination in a cuprizone (CPZ)-induced mouse model. CPZ did not significantly induce the damage of axons and neurons after 2 wk of feeding. However, after 4-6 wk of CPZ feeding, axons and neurons were markedly reduced in the cortex, posterior thalamic nuclear group, and hippocampus. Simultaneously, the expression of TPH+ tryptophan neurons and VGLUT1+ glutamate neurons was obviously decreased, and the expression of TH+ dopaminergic neurons was slightly decreased in the tail part of the substantia nigra striatum, whereas the number of ChAT+ cholinergic neurons was not significantly different in the brain. In the second week of feeding, CPZ caused a higher level of glutamate secretion and upregulated the expression of EAAT2 on astrocytes, which should contribute to rapid and sufficient glutamate uptake and removal. This finding reveals that astrocyte-driven glutamate reuptake protected the CNS from excitotoxicity by rapid reuptake of glutamate in 4-6 wk of CPZ feeding. At this stage, although NG2+ oligodendroglia progenitor cells (OPCs) were enhanced in the demyelination foci, the myelin sheath was still absent. In conclusion, we comprehensively observed the temporal and spatial evolution of various functional neurons. Our results will assist with understanding how demyelination affects neurons during CPZ-induced demyelination and provide novel information for neuroprotection in myelin regeneration and demyelinating diseases.NEW & NOTEWORTHY Our results further indicate temporal and spatial evolution of various functional neurons during the demyelination in a cuprizone (CPZ)-induced mouse model, which mainly occur 4-6 wk after CPZ feeding. At the same time, the axonal compartment is damaged and, consequently, neuronal death occurs, while glutamate neurons are lost obviously. The astrocyte-mediated glutamate reuptake could protect the neurons from the excitatory effects of glutamate.

Antagonizing astrocytic platelet activating factor receptor-neuroinflammation for total flavone of epimedium in response to cuprizone demyelination.

Demyelinating diseases of the central nervous system are characterized by recurrent demyelination and progressive neurodegeneration, but there are no clinical drugs targeting myelin regeneration or improving functional disability in the treatment of multiple sclerosis. Total flavone of Epimedium (TFE) is the main active components of Epimedium, which exhibits the beneficial biological activities in the treatment of diseases, but there is no report in the treatment of demyelinating disorder. The purpose of this study was to explore the therapeutic potential and possible mechanism of TFE in the treatment of demyelination. The results showed that TFE efficiently improved the behavioural performance and histological demyelination in cuprizone (CPZ)-induced demyelinating model. In terms of action, TFE increased astrocytes enrichment in corpus callosum, striatum and cortex, and promoted astrocytes to express neurotrophic factors. Furthermore, the expression of platelet-activating factor receptor (PAFR) in astrocytes was induced by CPZ feeding and LPS stimulation, accompanied by the increase of inflammatory cytokines TNF-α,IL-6 and IL-1ß. TFE declined the expression of PAFR, and inhibited inflammatory response. At the same time, TFE also antagonized PAFR activation and inflammatory response triggered by PAF, which further confirmed that TFE, as a new PAFR antagonist, inhibited the astrocyte-derived inflammatory response by antagonizing PAFR-neuroinflammation axis, thus contributing to myelin protection and regeneration.

Trifluoperazine reduces cuprizone-induced demyelination via targeting Nrf2 and IKB in mice.

Multiple sclerosis (MS) is one of the most common neurodegenerative diseases. In this disease, the immune system attacks oligodendrocyte cells and the myelin sheath of myelinated neurons in the central nervous system, causing their destruction. These conditions lead to impaired conduction of nerve impulses and are manifested by symptoms such as weakness, fatigue, visual and motor disorders. This study aimed to evaluate the ability of trifluoperazine (TF) to improve cuprizone-induced behavioral and histopathological changes in the prefrontal cortex of C57BL/6 male mice. Demyelination was induced by adding 0.2% cuprizone (CPZ) to the standard animal diet for 6 weeks. Three doses of TF (0.5, 1 and 2 mg/kg/day; i.p.) were given once daily for the last 2 weeks of treatment. Treatment with CPZ induced a weight loss during 6 weeks of treatment compared to the control group, which was reversed by the administration of TF. Behavioral tests (pole test and rotarod performance test) showed a decrease in motor coordination and balance in the group treated with CPZ (P < 0.01). Treatment with TF during the last two weeks was able to improve these motor deficiencies. Histopathological examination also evidenced an increase in demyelination in the CPZ group, which was improved by TF administration. In addition, CPZ intake significantly decreased the cerebral cortex levels of p-Nrf2 (P < 0.001) and increased the levels of p-IKB (P < 0.001) and, these changes were normalized in the TF groups. TF administration also reversed the increased levels of nitrite and the reduced activity of the antioxidant enzyme superoxide dismutase associated with CPZ exposure. TF can to reduce the harmful effects of CPZ by reducing the demyelination and modulating the Nrf2 and NF-kB signaling pathways.

Cuprizone and EAE mouse frontal cortex proteomics revealed proteins altered in multiple sclerosis.

Two pathophysiological different experimental models for multiple sclerosis were analyzed in parallel using quantitative proteomics in attempts to discover protein alterations applicable as diagnostic-, prognostic-, or treatment targets in human disease. The cuprizone model reflects de- and remyelination in multiple sclerosis, and the experimental autoimmune encephalomyelitis (EAE, MOG1-125) immune-mediated events. The frontal cortex, peripheral to severely inflicted areas in the CNS, was dissected and analyzed. The frontal cortex had previously not been characterized by proteomics at different disease stages, and novel protein alterations involved in protecting healthy tissue and assisting repair of inflicted areas might be discovered. Using TMT-labelling and mass spectrometry, 1871 of the proteins quantified overlapped between the two experimental models, and the fold change compared to controls was verified using label-free proteomics. Few similarities in frontal cortex between the two disease models were observed when regulated proteins and signaling pathways were compared. Legumain and C1Q complement proteins were among the most upregulated proteins in cuprizone and hemopexin in the EAE model. Immunohistochemistry showed that legumain expression in post-mortem multiple sclerosis brain tissue (n = 19) was significantly higher in the center and at the edge of white matter active and chronic active lesions. Legumain was associated with increased lesion activity and might be valuable as a drug target using specific inhibitors as already suggested for Parkinson's and Alzheimer's disease. Cerebrospinal fluid levels of legumain, C1q and hemopexin were not significantly different between multiple sclerosis patients, other neurological diseases, or healthy controls.

Amide Proton Transfer-weighted 7-T MRI Contrast of Myelination after Cuprizone Administration.

Background Investigations of amide proton signal changes in the white matter of demyelinating diseases may provide important biophysical information for diagnostic and prognostic assessments. Purpose To evaluate amide proton signals in cuprizone-induced rats using amide proton transfer-weighted (APTw) MRI, which provides in vivo image contrast by changing amide proton concentrations during demyelination (DEM) and subsequent remyelination (REM). Materials and Methods In this animal study, APTw 7-T MRI was performed in 21 male Wistar rats divided into cuprizone-induced (n = 14) and control (n = 7) groups from February to August 2020. The cuprizone-induced group was further subdivided into DEM (n = 7) and REM (n = 7) groups. Seven weeks after cuprizone feeding, rats in the DEM group were killed prior to transmission electron microscopy and myelin staining, while rats in the REM group were changed to a normal chow diet and fed for 5 weeks. In each group, the APTw signals were calculated using a conventional magnetization transfer ratio at 3.5 ppm based on regions of interest in the corpus callosum. Statistical differences in APTw signals among the groups were analyzed with one-way analysis of variance followed by Tukey post hoc tests. Results The mean APTw signals in the control and DEM groups were -4.42% ± 0.60 (standard deviation) (95% CI: -4.98, -3.86) and -2.57% ± 0.48 (95% CI: -3.01, -2.12), respectively, indicating higher in vivo APTw signals in the DEM lesion (P < .001). After REM, mean APTw signal in the REM group was -3.83% ± 0.67 (95% CI: -4.45, -3.22), similar to that in the control group (P = .18) and lower than that in the DEM group (P < .001). Conclusion Significant amide proton transfer-weighted (APTw) metric changes coupled with the histologic characteristics of the demyelination and remyelination processes indicate the potential usefulness of APTw 7-T MRI to monitor earlier myelination processes. © RSNA, 2021 Online supplemental material is available for this article. See also the editorial by van Zijl in this issue.

Neuronal hibernation following hippocampal demyelination.

Cognitive dysfunction occurs in greater than 50% of individuals with multiple sclerosis (MS). Hippocampal demyelination is a prominent feature of postmortem MS brains and hippocampal atrophy correlates with cognitive decline in MS patients. Cellular and molecular mechanisms responsible for neuronal dysfunction in demyelinated hippocampi are not fully understood. Here we investigate a mouse model of hippocampal demyelination where twelve weeks of treatment with the oligodendrocyte toxin, cuprizone, demyelinates over 90% of the hippocampus and causes decreased memory/learning. Long-term potentiation (LTP) of hippocampal CA1 pyramidal neurons is considered to be a major cellular readout of learning and memory in the mammalian brain. In acute slices, we establish that hippocampal demyelination abolishes LTP and excitatory post-synaptic potentials of CA1 neurons, while pre-synaptic function of Schaeffer collateral fibers is preserved. Demyelination also reduced Ca2+-mediated firing of hippocampal neurons in vivo. Using three-dimensional electron microscopy, we investigated the number, shape (mushroom, stubby, thin), and post-synaptic densities (PSDs) of dendritic spines that facilitate LTP. Hippocampal demyelination did not alter the number of dendritic spines. Surprisingly, dendritic spines appeared to be more mature in demyelinated hippocampi, with a significant increase in mushroom-shaped spines, more perforated PSDs, and more astrocyte participation in the tripartite synapse. RNA sequencing experiments identified 400 altered transcripts in demyelinated hippocampi. Gene transcripts that regulate myelination, synaptic signaling, astrocyte function, and innate immunity were altered in demyelinated hippocampi. Hippocampal remyelination rescued synaptic transmission, LTP, and the majority of gene transcript changes. We establish that CA1 neurons projecting demyelinated axons silence their dendritic spines and hibernate in a state that may protect the demyelinated axon and facilitates functional recovery following remyelination.

Temporal Changes in In Vivo Glutamate Signal during Demyelination and Remyelination in the Corpus Callosum: A Glutamate-Weighted Chemical Exchange Saturation Transfer Imaging Study.

BACKGROUND: Glutamate-weighted chemical exchange saturation transfer (GluCEST) is a useful imaging tool that can be used to detect changes in glutamate levels in vivo and could also be helpful in the diagnosis of brain myelin changes. We investigated glutamate level changes in the cerebral white matter of a rat model of cuprizone-administered demyelination and remyelination using GluCEST. METHOD: We used a 7 T pre-clinical magnetic resonance imaging (MRI) system. The rats were divided into the normal control (CTRL), cuprizone-administered demyelination (CPZDM), and remyelination (CPZRM) groups. GluCEST data were analyzed using the conventional magnetization transfer ratio asymmetry in the corpus callosum. Immunohistochemistry and transmission electron microscopy analyses were also performed to investigate the myelinated axon changes in each group. RESULTS: The quantified GluCEST signals differed significantly between the CPZDM and CTRL groups (-7.25 ± 1.42% vs. -2.84 ± 1.30%; p = 0.001). The increased GluCEST signals in the CPZDM group decreased after remyelination (-6.52 ± 1.95% in CPZRM) to levels that did not differ significantly from those in the CTRL group (p = 0.734). CONCLUSION: The apparent temporal signal changes in GluCEST imaging during demyelination and remyelination demonstrated the potential usefulness of GluCEST imaging as a tool to monitor the myelination process.

Melatonin improves memory defects in a mouse model of multiple sclerosis by up-regulating cAMP-response element-binding protein and synapse-associated proteins in the prefrontal cortex.

Multiple sclerosis is a progressive autoimmune disorder of the myelin sheath and is the most common inflammatory disease of young adults. Up to 65% of multiple sclerosis patients have cognitive impairments such as memory loss and difficulty in understanding and maintaining attention and concentration. Many pharmacological interventions have been used to reverse motor impairments in multiple sclerosis patients; however, none of these drugs improve cognitive function. Melatonin can diffuse through the blood-brain barrier and has well-known antioxidant and anti-inflammatory properties with almost no side effects; it is, therefore, a promising neuroprotective supplement for many neurological diseases, such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, ischemic stroke, and fibromyalgia. However, only some researches have assessed the effect of melatonin on cognitive dysfunction in multiple sclerosis. Here, we evaluated the effects of melatonin supplementation on memory defects induced by cuprizone in a mouse model of multiple sclerosis. Cuprizone (400 mg/kg) and melatonin (80 mg/kg) were administered to SWR/J mice daily for 5 weeks. Open field, tail-flick, and novel object recognition behavioral tests were performed. Also, expression of cAMP-response element-binding protein, synaptophysin, and postsynaptic density protein 95 were measured in the prefrontal cortex. Melatonin significantly improved the memory defects induced by cuprizone toxicity by up-regulating cAMP-response element-binding protein and by increasing expression of the synapse-associated synaptophysin and postsynaptic density protein 95 genes in the prefrontal cortex. These results indicate that melatonin may provide protective effects against memory impairments associated with multiple sclerosis.

Increased blood-brain barrier hyperpermeability coincides with mast cell activation early under cuprizone administration.

The cuprizone induced animal model of demyelination is characterized by demyelination in many regions of the brain with high levels of demyelination in the corpus callosum as well as changes in neuronal function by 4-6 weeks of exposure. The model is used as a tool to study demyelination and subsequent degeneration as well as therapeutic interventions on these effects. Historically, the cuprizone model has been shown to contain no alterations to blood-brain barrier integrity, a key feature in many diseases that affect the central nervous system. Cuprizone is generally administered for 4-6 weeks to obtain maximal demyelination and degeneration. However, emerging evidence has shown that the effects of cuprizone on the brain may occur earlier than measurable gross demyelination. This study sought to investigate changes to blood-brain barrier permeability early in cuprizone administration. Results showed an increase in blood-brain barrier permeability and changes in tight junction protein expression as early as 3 days after beginning cuprizone treatment. These changes preceded glial morphological activation and demyelination known to occur during cuprizone administration. Increases in mast cell presence and activity were measured alongside the increased permeability implicating mast cells as a potential source for the blood-brain barrier disruption. These results provide further evidence of blood-brain barrier alterations in the cuprizone model and a target of therapeutic intervention in the prevention of cuprizone-induced pathology. Understanding how mast cells become activated under cuprizone and if they contribute to blood-brain barrier alterations may give further insight into how and when the blood-brain barrier is affected in CNS diseases. In summary, cuprizone administration causes an increase in blood-brain barrier permeability and this permeability coincides with mast cell activation.

Laquinimod ameliorates secondary brain inflammation.

Accumulating evidence suggests that a degenerative processes within the brain can trigger the formation of new, focal inflammatory lesions in Multiple Sclerosis (MS). Here, we used a novel pre-clinical MS animal model to test whether the amelioration of degenerative brain events reduces the secondary recruitment of peripheral immune cells and, in consequence, inflammatory lesion development. Neural degeneration was induced by a 3 weeks cuprizone intoxication period. To mitigate the cuprizone-induced pathology, animals were treated with Laquinimod (25 mg/kg) during the cuprizone-intoxication period. At the beginning of week 6, encephalitogenic T cell development in peripheral lymphoid organs was induced by the immunization with myelin oligodendrocyte glycoprotein 35-55 peptide (i.e., Cup/EAE). Demyelination, axonal injury and reactive gliosis were determined by immunohistochemistry. Positron emission tomography (PET) imaging was performed to analyze glia activation in vivo. Vehicle-treated cuprizone mice displayed extensive callosal demyelination, glia activation and enhanced TSPO-ligand binding. This cuprizone-induced pathology was profoundly ameliorated in mice treated with Laquinimod. In vehicle-treated Cup/EAE mice, the cuprizone-induced pathology triggered massive peripheral immune cell recruitment into the forebrain, evidenced by multifocal perivascular inflammation, glia activation and neuro-axonal injury. While anti myelin oligodendrocyte glycoprotein 35-55 peptide immune responses were comparable in vehicle- and Laquinimod-treated Cup/EAE mice, the cuprizone-triggered immune cell recruitment was ameliorated by the Laquinimod treatment. This study clearly illustrates that amelioration of a primary brain-intrinsic degenerative process secondary halts peripheral immune cell recruitment and, in consequence, inflammatory lesion development. These findings have important consequences for the interpretation of the results of clinical studies.

Melatonin ameliorates cuprizone-induced reduction of hippocampal neurogenesis, brain-derived neurotrophic factor, and phosphorylation of cyclic AMP response element-binding protein in the mouse dentate gyrus.

INTRODUCTION: The aim of this study was to investigate the effects of cuprizone on adult hippocampal neurogenesis in naïve mice. Additionally, we also studied how melatonin affects the neuronal degeneration induced by cuprizone. METHODS: Eight-week-old male C57BL/6J mice were randomly divided into three groups: (a) the control group, (b) the group treated with cuprizone only, and (c) the group treated with both cuprizone and melatonin. Cuprizone was administered with food at 0.2% ad libitum for 6 weeks. Melatonin was also administered with tap water at 6 g/L ad libitum for 6 weeks; the animals were then euthanized for immunohistochemistry with Ki67, doublecortin (DCX), glucose transporter 3 (GLUT3), and phosphorylation of cyclic adenosine monophosphate (AMP) response element binding (pCREB); double immunofluorescence of neuronal nuclei (NeuN) and myelin basic protein (MBP); and Western blot analysis of brain-derived neurotrophic factor (BDNF) expression to reveal the effects of cuprizone and melatonin on cell damage and hippocampal neurogenesis. RESULTS: Administration of cuprizone significantly decreased the number of differentiating (DCX-positive) neuroblasts and proliferating (Ki67-positive) cells in the dentate gyrus. Moreover, cuprizone administration decreased glucose utilization (GLUT3-positive cells) and cell transcription (pCREB-positive cells and BDNF protein expression) in the dentate gyrus. Administration of melatonin ameliorated the cuprizone-induced reduction of differentiating neuroblasts and proliferating cells, glucose utilization, and cell transcription. CONCLUSION: The results of the study suggest that cuprizone treatment disrupts hippocampal neurogenesis in the dentate gyrus by reducing BDNF levels and decreasing the phosphorylation of CREB. These effects were ameliorated by melatonin treatment.

Continuous cuprizone intoxication allows active experimental autoimmune encephalomyelitis induction in C57BL/6 mice.

Oligodendrocyte degeneration is a hallmark of multiple sclerosis pathology, and protecting oligodendrocytes and myelin is likely to be of clinical relevance. Traditionally, oligodendrocyte and myelin degeneration are viewed as a direct consequence of an inflammatory attack, but metabolic defects might be equally important. Appropriate animal models to study the interplay of inflammation and metabolic injury are, therefore, needed. Here, we describe that in spite of its immunosuppressive effects, a continuous intoxication with cuprizone allows the induction of active experimental autoimmune encephalomyelitis (EAE) by myelin oligodendrocyte glycoprotein (MOG35-55) immunization. Although the clinical severity of EAE is ameliorated in cuprizone-intoxicated mice, the recruitment of granulocytes, and especially, CD3+ lymphocytes into the forebrain is triggered by the cuprizone insult. Such combined lesions are further characterized by oligodendrocyte apoptosis and microglia activation, closely mimicking type III multiple sclerosis lesions. In summary, we provide a protocol that allows to study the direct interplay of immune-mediated and metabolic oligodendrocyte injury and its consequences for the cerebral white and grey matters.

Five Decades of Cuprizone, an Updated Model to Replicate Demyelinating Diseases.

INTRODUCTION: Demyelinating diseases of the central nervous system (CNS) comprise a group of neurological disorders characterized by progressive (and eventually irreversible) loss of oligodendrocytes and myelin sheaths in the white matter tracts. Some of myelin disorders include: Multiple sclerosis, Guillain-Barré syndrome, peripheral nerve polyneuropathy and others. To date, the etiology of these disorders is not well known and no effective treatments are currently available against them. Therefore, further research is needed to gain a better understand and treat these patients. To accomplish this goal, it is necessary to have appropriate animal models that closely resemble the pathophysiology and clinical signs of these diseases. Herein, we describe the model of toxic demyelination induced by cuprizone (CPZ), a copper chelator that reduces the cytochrome and monoamine oxidase activity into the brain, produces mitochondrial stress and triggers the local immune response. These biochemical and cellular responses ultimately result in selective loss of oligodendrocytes and microglia accumulation, which conveys to extensive areas of demyelination and gliosis in corpus callosum, superior cerebellar peduncles and cerebral cortex. Remarkably, some aspects of the histological pattern induced by CPZ are similar to those found in multiple sclerosis. CPZ exposure provokes behavioral changes, impairs motor skills and affects mood as that observed in several demyelinating diseases. Upon CPZ removal, the pathological and histological changes gradually revert. Therefore, some authors have postulated that the CPZ model allows to partially mimic the disease relapses observed in some demyelinating diseases. CONCLUSION: for five decades, the model of CPZ-induced demyelination is a good experimental approach to study demyelinating diseases that has maintained its validity, and is a suitable pharmacological model for reproducing some key features of demyelinating diseases, including multiple sclerosis.

Defining Changes in the Spatial Distribution and Composition of Brain Lipids in the Shiverer and Cuprizone Mouse Models of Myelin Disease.

Myelin is composed primarily of lipids and diseases affecting myelin are associated with alterations in its lipid composition. However, correlation of the spatial (in situ) distribution of lipids with the disease-associated compositional and morphological changes is not well defined. Herein we applied high resolution matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), immunohistochemistry (IHC), and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) to evaluate brain lipid alterations in the dysmyelinating shiverer (Shi) mouse and cuprizone (Cz) mouse model of reversible demyelination. MALDI-IMS revealed a decrease in the spatial distribution of sulfatide (SHexCer) species, SHexCer (d42:2), and a phosphatidylcholine (PC) species, PC (36:1), in white matter regions like corpus callosum (CC) both in the Shi mouse and Cz mouse model. Changes in these lipid species were restored albeit not entirely upon spontaneous remyelination after demyelination in the Cz mouse model. Lipid distribution changes correlated with the local morphological changes as confirmed by IHC. LC-ESI-MS analyses of CC extracts confirmed the MALDI-IMS derived reductions in SHexCer and PC species. These findings highlight the role of SHexCer and PC in preserving the normal myelin architecture and our experimental approaches provide a morphological basis to define lipid abnormalities relevant to myelin diseases.

Psychological stress effects on myelin degradation in the cuprizone-induced model of demyelination.

Multiple sclerosis (MS) is known as the most common demyelinating disease worldwide in which previous studies have shown that stress is a risk factor for the disease's onset and progression. Nevertheless, further studies are needed to investigate the consequences of stress in MS pathology. In this study, after 5 days of exposure to psychological and physical stress as a repetitive distress modality, rats were treated with cuprizone. The demyelination degree was compared in animal groups using Luxol fast blue staining, immunohistochemical staining for myelin basic protein and transmission electron microscopy. Outcomes revealed that animals exposed to stress prior to cuprizone ingestion, elicit more intense demyelination. Continuous psychological distress has more severe effects on myelin sheath destruction in the preclinical stage.

Changes in neurosteroidogenesis during demyelination and remyelination in cuprizone-treated mice.

Changes of neurosteroids may be involved in the pathophysiology of multiple sclerosis (MS). The present study investigated whether changes of neurosteroidogenesis also occurred in the grey and white matter regions of the brain in mice subjected to cuprizone-induced demyelination. Accordingly, we compared the expression of neurosteroidogenic proteins, including steroidogenic acute regulatory protein (StAR), voltage-dependent anion channel (VDAC) and 18 kDa translocator protein (TSPO), as well as neurosteroidogenic enzymes, including the side chain cleavage enzyme (P450scc), 3ß-hydroxysteroid dehydrogenase/isomerase and 5α-reductase (5α-R), during the demyelination and remyelination periods. Using immunohistochemistry and a quantitative polymerase chain reaction, we demonstrated a decreased expression of StAR, P450scc and 5α-R with respect to an increase astrocytic and microglial reaction and elevated levels of tumor necrosis factor (TNF)α during the cuprizone demyelination period in the hippocampus, cortex and corpus callosum. These parameters, as well as the glial reaction, were normalised after 2 weeks of spontaneous remyelination in regions containing grey matter. Conversely, persistent elevated levels of TNFα and low levels of StAR and P450scc were observed during remyelination in corpus callosum white matter. We conclude that neurosteroidogenesis/myelination status and glial reactivity are inversely related in the hippocampus and neocortex. Establishing a cause and effect relationship for the measured variables remains a future challenge for understanding the pathophysiology of MS.

Low-level laser therapy modulates demyelination in mice.

There are no effective therapies for remyelination. Low-level laser therapy (LLLT) has been found advantageous in neurogenesis promotion, cell death prevention, and modulation of inflammation in central and peripheral nervous system models. The purpose of this study was to analyse LLLT effects on cuprizone-induced demyelination. Mice were randomly distributed into three groups: Control Laser (CTL), Cuprizone (CPZ), and Cuprizone Laser (CPZL). Mice from CPZ and CPZL groups were exposed to a 0.2% cuprizone oral diet for four complete weeks. Six sessions of transcranial laser irradiation were applied on three consecutive days, during the third and fourth weeks, with parameters of 36 J/cm2, 50 mW, 0.028 cm2 spot area, continuous wave, 1 J, 20 s, 1.78 W/cm2 in a single point equidistant between the eyes and ears of CTL and CPZL mice. Motor coordination was assessed by the rotarod test. Twenty-four hours after the last laser session, all animals were euthanized, and brains were extracted. Serum was obtained for lactate dehydrogenase toxicity testing. Histomorphological analyses consisted of Luxol Fast Blue staining and immunohistochemistry. The results showed that laser-treated animals presented motor performance improvement, attenuation of demyelination, increased number of oligodendrocyte precursor cells, modulated microglial and astrocytes activation, and a milder toxicity by cuprizone. Although further studies are required, it is suggested that LLLT represents a feasible therapy for demyelinating diseases.

Changes in the axo-glial junctions of the optic nerves of cuprizone-treated mice.

Demyelination induced by cuprizone in mice has served a useful model system for the study of demyelinating diseases, such as multiple sclerosis. Severity of demyelination by cuprizone, however, varies across different regions of the central nervous system; the corpus callosum is sensitive, while the optic nerves are resistant. Here, we investigated the effects of cuprizone on optic nerves, focusing on the axo-glial junctions. Immunostaining for sodium channels, contactin-associated protein, neurofascins, and potassium channels revealed that there were no massive changes in the density and morphology of the axo-glial junctions in cuprizone-treated optic nerves. However, when we counted the number of incomplete junctional complexes, we observed increased numbers of isolated paranodes. These isolated paranodes were immunopositive for both axonal and glial membrane proteins, indicating that they were the contact sites between axons and glia. These were not associated with sodium channels or potassium channels, suggesting the absence of physiological functions. When teased axons from cuprizone-treated optic nerves were immunostained, the isolated paranodes were found at the internode region of the myelin. From these observations, we conclude that cuprizone induces new contacts between axons and myelins at the internode region.

Cuprizone Administration Alters the Iron Metabolism in the Mouse Model of Multiple Sclerosis.

Cuprizone (CZ) is a widely used copper chelating agent to develop non-autoimmune animal model of multiple sclerosis, characterized by demyelination of the corpus callosum (CC) and other brain regions. The exact mechanisms of CZ action are still arguable, but it seems that the only affected cells are the mature oligodendrocytes, possibly via metabolic disturbances caused by copper deficiency. During the pathogenesis of multiple sclerosis, high amount of deposited iron can be found throughout the demyelinated areas of the brain in the form of extracellular iron deposits and intracellularly accumulated iron in microglia. In the present study, we used the accepted experimental model of 0.2% CZ-containing diet with standard iron concentration to induce demyelination in the brain of C57BL/6 mice. Our aim was to examine the changes of iron homeostasis in the CC and as a part of the systemic iron regulation, in the liver. Our data showed that CZ treatment changed the iron metabolism of both tissues; however, it had more impact on the liver. Besides the alterations in the expressions of iron storage and import proteins, we detected reduced serum iron concentration and iron stores in the liver, together with elevated hepcidin levels and feasible disturbances in the Fe-S cluster biosynthesis. Our results revealed that the CZ-containing diet influences the systemic iron metabolism in mice, particularly the iron homeostasis of the liver. This inadequate systemic iron regulation may affect the iron homeostasis of the brain, eventually indicating a relationship among CZ treatment, iron metabolism, and neurodegeneration.

Targeting demyelination via α-secretases promoting sAPPα release to enhance remyelination in central nervous system.

Remyelination is an endogenous regenerative process of myelin repair in the central nervous system (CNS) with limited efficacy in demyelinating disorders. As strategies enhancing endogenous remyelination become a therapeutic challenge, we have focused our study on α-secretase-induced sAPPα release, a soluble endogenous protein with neuroprotective and neurotrophic properties. However, the role of sAPPα in remyelination is not known. Therefore, we investigated the remyelination potential of α-secretase-induced sAPPα release following CNS demyelination in mice. Acute demyelination was induced by feeding mice with cuprizone (CPZ) for 5weeks. To test the protective effect and the remyelination potential of etazolate, an α-secretase activator, we designed two treatment protocols. Etazolate was administrated either during the last two weeks or at the end of the CPZ intoxication. In both protocols, etazolate restored the number of myelinated axons in corpus callosum with a corresponding increase in the amount of MBP, one of the major myelin proteins in the brain. We also performed ex vivo studies to decipher etazolate's mechanism of action in a lysolecithin-induced demyelination model using organotypic culture of cerebellar slices. Etazolate treatment was able to i) enhance the release of sAPPα in the culture media of demyelinated slices, ii) protect myelinated axons from demyelination, iii) increase the number of mature oligodendrocytes, iv) promote the reappearance of the paired Caspr+ adjacent to the nodes of Ranvier and v) increase the percentage of myelinated axons with short internodes, an indicator of remyelination. Etazolate failed to promote all the aforementioned effects in the presence of GI254023X, an α-secretase inhibitor. Moreover, the protective effects of etazolate in demyelinated slices were mimicked by sAPPα treatment in a dose-dependent manner. In conclusion, etazolate-induced sAPPα release protects myelinated axons from demyelination while also promoting remyelination. This work, thus, highlights the therapeutic potential of strategies that enhance sAPPα release in demyelinating disorders.