Search Results (71)

Background/Objectives: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system characterized by inflammation, demyelination, and axonal loss. Despite available therapies, there is currently no effective cure for MS. Ibudilast (IBD), a phosphodiesterase inhibitor, and 6-shogaol (SH), a bioactive compound from ginger, have independently shown therapeutic potential in MS models. This study aimed to evaluate the therapeutic efficacy of combining SH and IBD in modulating neuroinflammation and improving functional recovery in a cuprizone (CPZ) mouse model of MS. Methods: Male SWR/J mice were exposed to 0.3% CPZ for 5 weeks to induce demyelination, followed by 4 weeks of spontaneous remyelination after CPZ withdrawal. During remyelination, the CPZ group was subdivided into four groups: no therapy, SH (25 mg/kg), IBD (10 mg/kg), and SH + IBD. Behavioral tests were used to assess locomotion, muscle strength, coordination, and memory. Gene expression of proinflammatory and anti-inflammatory cytokines was analyzed in brain tissue. Results: The combined treatment significantly improved locomotor activity, muscle strength, and memory during remyelination phases while suppressing proinflammatory gene expression and enhancing anti-inflammatory pathways in the brain. Conclusions: SH and IBD combination therapy provides enhanced anti-inflammatory and functional benefits compared with monotherapies, supporting its potential as a promising multi-target therapeutic strategy for improving functional recovery and modulating neuroinflammation during the spontaneous remyelination phase following CPZ withdrawal. Full article

Reactive astrocytes are a hallmark of several neurological diseases in multiple sclerosis and experimental demyelination models. Their morphological alterations are commonly assessed by qualitative histopathology, yet quantitative tools are required to better capture astrocytic heterogeneity and to allow correlations with imaging-derived biomarkers. Here, we present a workflow for the quantitative analysis of Glial Fibrillary Acidic Protein (GFAP) network remodeling in astrocytes in the cuprizone model of demyelination. C57BL/6 mice were intoxicated with cuprizone for 3 or 5 weeks to induce progressive demyelination, microglial activation, and reactive astrogliosis. Brain sections were processed for anti-GFAP immunohistochemistry, and individual astrocytes from the stratum oriens of the hippocampus were digitally reconstructed. Diverse parameters of GFAP topology, including soma size, process length, branching order, convex hull area, and ramification index, were extracted using either the commercial Neurolucida^® 360 software or the open-source Simple Neurite Tracer (SNT) plugin in ImageJ. Principal component analysis revealed clear differences between control astrocytes and astrocytes in cuprizone-intoxicated animals, with reactive astrocytes displaying increased numbers of primary processes, enhanced bifurcation, and process complexity. Comparative evaluation of Neurolucida^® 360 and SNT demonstrated that both tools are suitable for astrocyte reconstruction, although Neurolucida^® 360 enabled faster and more detailed tracing. This protocol provides a reproducible pipeline for the quantitative assessment of astrocyte morphology under control and pathological conditions, thereby supporting future efforts to link cellular remodeling to functional outcomes in neuroinflammatory disease models. Full article

Demyelinating disorders such as multiple sclerosis are characterized by oligodendrocyte loss and insufficient remyelination. The cuprizone model provides a well-established experimental system for studying these processes. The apelinergic system, including the apelin receptor (APLNR), has been implicated in neuroprotection and central nervous system homeostasis. However, its role in white matter demyelination and repair remains incompletely understood. This study aimed to characterize the spatial and temporal dynamics of APLNR expression in relation to oligodendrocyte lineage cells in the corpus callosum (CC) during demyelination and remyelination. Demyelination was induced in 8-week-old C57BL/6 mice by 0.2% cuprizone supplementation in their drinking water for 5 weeks, followed by 5 weeks remyelination phase after toxin withdrawal. Histological assessment using Luxol Fast Blue/Cresyl violet staining was performed to evaluate structural changes in the CC. Immunohistochemistry and confocal microscopy were used to analyze APLNR expression, GST-π⁺ cells, and NG2⁺ cells, including their spatial distribution and co-localization. Quantitative analyses and correlation tests were conducted to assess relationships between cellular markers and CC area. Demyelination resulted in significant reduction in CC area and a marked decrease in GST-π⁺ cells, accompanied by a robust increase in NG2⁺ cells, while remyelination led to partial structural and cellular recovery. APLNR expression increased progressively from control to demyelination and further during remyelination, exhibiting pronounced regional heterogeneity with higher levels in lateral CC regions. Confocal analysis demonstrated increasing co-localization of APLNR with NG2⁺ cells, particularly during remyelination. Correlation analyses identified GST-π⁺ cell density as the strongest predictor of CC area, whereas APLNR showed phase-dependent associations, including a positive correlation with GST-π⁺ cells during remyelination and a negative relationship with NG2⁺ cells during demyelination. APLNR expression is dynamically regulated during cuprizone-induced demyelination and remyelination and is closely associated with oligodendrocyte lineage cell responses. Its increased expression and enhanced co-localization with NG2⁺ cells during remyelination suggest a potential role in endogenous repair processes. However, as the findings are based on descriptive analyses, further functional studies are required to determine the mechanistic contribution of APLNR signaling and its potential as a therapeutic target in demyelinating diseases. Full article

Background/Objectives: Demyelination and neuroinflammation are central features of multiple sclerosis (MS), contributing to motor deficits and cognitive decline. Cuprizone (CPZ)-induced demyelination is a well-established model for studying multiple sclerosis-like neurotoxicity. This study investigated the neuroprotective and immunomodulatory effects of self-nanoemulsifying drug delivery systems (SNEDDSs) incorporating curcumin, piperine, and Zanthoxylum rhetsa seed oil. Methods: Male mice were divided into five groups: control, CPZ-only, and CPZ co-treated with three nanoformulations BFZ (blank SNEDDS), CFZ (curcumin-SNEDDS), and PFZ (curcumin–piperine SNEDDS). CPZ was administered for 5 weeks, followed by a 2-week recovery or treatment phase. Key neuroinflammatory markers like CD4, CD8, cholinergic (acetylcholinesterase, AChE), myelin integrity (MBP), BDNF, CREB, TNFα, Il-1β were assessed at weeks 5 and 7 using ELISA. Alterations in antioxidant enzymes, brain histology, and behavioral outcomes were also investigated. Results: At week 5, CPZ significantly increased CD4 and CD8 expression and reduced AChE and MBP levels, indicating neuroinflammation, cholinergic impairment, and demyelination. Nanoformulation treatments (both prophylactic and therapeutic) markedly reduced CD4 and CD8 levels, with PFZ showing the most pronounced effect. AChE activity was significantly restored in all treatment groups, with PFZ and CFZ exceeding baseline levels, suggesting enhanced cholinergic function. MBP levels were highest in PFZ-treated mice, surpassing control values and indicating strong remyelination potential. These improvements persisted and further advanced at week 7, especially in PFZ and CFZ groups. Conclusions: Curcumin-based SNEDDS, particularly PFZ, significantly mitigated CPZ-induced neuroinflammation, promoted remyelination, and restored cholinergic activity in the frontal cortex. These findings highlight the therapeutic potential of bioenhanced curcumin nanoformulations for treating demyelinating and neuroinflammatory disorders. Full article

Multiple sclerosis (MS) is a chronic, immune-mediated disease of the central nervous system (CNS) characterized by oligodendrocyte (OLG) degeneration, myelin loss, and impaired remyelination. Apolipoprotein D (Apo D), a glia-derived lipocalin, has emerged in recent decades as a neuroprotective molecule involved in lipid transport, oxidative stress regulation, and inflammation control during aging and neurodegenerative diseases like MS. However, its role in demyelination/remyelination dynamics remains poorly defined. In this study, we used the cuprizone (CPZ)-induced demyelination model in C57BL/6 mice to analyze Apo D expression patterns in the corpus callosum during de- and remyelination. We also assessed whether the atypical antipsychotic clozapine (CLO), previously shown to upregulate Apo D in vivo, could modulate its expression and influence myelin recovery in this pathological context. Using a combination of magnetic resonance imaging, Luxol fast blue staining, and double immunohistochemistry, we demonstrated that CPZ treatment for 3 or 6 weeks led to significant demyelination, hydrocephalus, and reduced motor cortex thickness, which were partially reversed after treatment cessation. Apo D expression in OLGs was significantly reduced by CPZ exposure, both at the protein level and in terms of immunoreactive cell counts, but was restored following treatment withdrawal. Notably, co-administration of CLO prevented the CPZ-induced reduction in Apo D expression in OLGs, although it did not attenuate myelin loss. In this way, our results reveal a strong correlation between Apo D expression and OLG/myelin integrity in vivo. While CLO did not exert remyelinating effects, it preserved Apo D levels under demyelinating conditions, suggesting a potential indirect neuroprotective mechanism. These findings support the relevance of Apo D in CNS myelin homeostasis and highlight its potential as a molecular target for therapeutic intervention in demyelinating diseases such as MS. Full article

Background/Objectives: Demyelination occurs to a variable extent in various neurological diseases, such as multiple sclerosis. Physical exercise benefits central neural functions that depend on the brain’s electrophysiological and glial activity. It is unclear whether both factors—i.e., demyelination and exercise—interact in the brain. We aimed to investigate if this interaction occurs during brain development. Methods: Developing rats were subjected to a cuprizone-induced demyelination. Part of these rats were treadmill-exercised for five weeks. After this period, some demyelinated animals were allowed to remyelinate by receiving a similar diet, without cuprizone, for six weeks. The exercised groups were compared with the corresponding sedentary groups. All groups were evaluated electrophysiologically (cortical spreading depression features), and their brains were processed for immunohistochemical labeling with four specific glial antibodies (anti-APC, MBP, GFAP, and Iba1). Results: Compared with the corresponding controls, cuprizone demyelination and treadmill exercise accelerated and decelerated CSD propagation. Cuprizone reduced APC, MBP, and GFAP immunolabeling and increased Iba1 immunostaining. Remyelination reverted the cuprizone effects. Exercise counteracted the cuprizone-induced changes in GFAP- and Iba1-containing cells but not in MBP- and APC-containing ones. Conclusions: Our data confirmed the effectiveness of the cuprizone demyelination paradigm. They evidenced the potential neuroprotective effect of regular physical exercise, suggesting that this non-pharmacological intervention could benefit patients with central demyelination-dependent diseases. Full article

Gait disturbances are among the most prominent motor symptoms in multiple sclerosis (MS), yet their functional characterization in preclinical models remains limited. In this study, we used high-speed ventral plane videography (DigiGait™) to analyze locomotor behavior during 5 weeks of cuprizone-induced demyelination in 10 male C57BL/6 mice. Gait analysis revealed significant alterations in stride time (left front paw from 0.303 ± 0.01 s to 0.257 ± 0.007 s; p = 0.003), paw angle (right fore paw from −13.78 ± 0.928° to 5.456 ± 2.146°; p = 0.003), and midline distance (right hind paw from 1.889 ± 0.099 cm to 1.216 ± 0.096 cm; p = 0.013), particularly in the hind limbs. These behavioral impairments correlated with histopathological findings of reduced myelination and elevated microglial activation in motor-relevant brain regions, including the corpus callosum, caudate-putamen, and motor cortex. Notably, specific gait parameters showed strong correlations with the degree of demyelination, supporting their relevance as functional biomarkers. Our data demonstrate that high-resolution gait analysis provides a sensitive, non-invasive tool to monitor functional deficits in demyelinating models and may aid in evaluating therapeutic efficacy in future studies. Full article

The occurrence of demyelination in the central nervous system (CNS) causes neurodegenerative lesions. The occurrence and development of demyelination involve multiple pathological mechanisms, including the generation of reactive oxygen species (ROS) caused by mitochondrial dysfunction in microglia and subsequent neuroinflammation. Scutellarin is a natural flavonoid drug with significant neuroprotective effects, including antioxidant, anti-inflammatory, and anti-apoptotic properties, and is widely used in the treatment of neurological diseases. However, the protective effects and mechanisms of scutellarin on demyelination have not yet been elucidated. This study aims to investigate the neuroprotective effects of scutellarin on demyelination and its underlying molecular mechanisms. Our results showed that treatment with scutellarin significantly alleviated Cuprizone-induced myelin damage, neuronal apoptosis, and neurological deficits in mice. In in vitro experiments, scutellarin significantly reduced Cuprizone–copper-induced pro-inflammatory microglia formation and inhibited the secretion of TNF-α, thereby reducing myelin cell damage. Mechanism studies revealed that scutellarin inhibited the secretion of TNF-α by microglia and alleviated myelin cell damage by reducing the excessive production of mitochondrial reactive oxygen species (Mito-ROS), reactive oxygen species (ROS), and malondialdehyde (MDA) induced by Cuprizone–copper in microglia. Finally, scutellarin improved mitochondrial dysfunction in microglia and significantly alleviated myelin cell damage by inhibiting the expression of p38MAPK. In conclusion, our findings demonstrate that scutellarin exerts significant neuroprotective effects on Cuprizone-induced mice by improving mitochondrial dysfunction in microglia, thereby reducing inflammatory responses. This effect is closely associated with the inhibition of the p38MAPK pathway. Full article

Demyelination in the central nervous system (CNS) disrupts neuronal communication and promotes neurodegeneration. Despite the widespread use of cuprizone-induced demyelination models to study myelin injury and repair, the mechanisms underlying oligodendrocyte apoptosis and regeneration are poorly understood. This study investigated the dynamic cellular and molecular changes that occur during demyelination and remyelination, with a focus on glial cell responses, blood-brain barrier (BBB) integrity, and neuroimmune interactions. C57BL/6J mice exposed to cuprizone exhibited weight loss, sensorimotor deficits, and cognitive decline, which were reversed during remyelination. Histological and immunofluorescence analyses revealed reduced myelin protein levels, including myelin basic protein (MBP) and myelin-associated glycoprotein (MAG), and decreased oligodendrocyte populations during demyelination, with recovery during repair. The BBB permeability increases during demyelination, is associated with the decreased expression of tight junction proteins (ZO-1, Occludin), and normalizes during remyelination. Single-cell RNA sequencing revealed dynamic shifts in glial cell populations and upregulated Psap-Gpr37l1 signaling. Neuroimmune activation and oxidative stress peak during demyelination, characterized by elevated ROS, MDA, and immune cell infiltration, followed by recovery. Transcriptomic profiling revealed key inflammatory pathways (JAK-STAT, NF-κB) and hub genes associated with demyelination and repair. These findings provide insights into myelin repair mechanisms and highlight potential therapeutic targets for treating demyelinating diseases. Full article

Under conditions of amino acid deficiency, mammalian cells activate a nutrient-sensing kinase known as general control nonderepressible 2 (GCN2). The activation of GCN2 results in the phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2α), which can be phosphorylated by three other three integrated stress response (ISR) kinases, reducing overall protein synthesis. GCN2 activation also promotes the translation of specific mRNAs, some of which encode transcription factors that enhance the transcription of genes involved in the synthesis, transport, and metabolism of amino acids to restore cellular homeostasis. The phosphorylation of eIF2α has been shown to protect oligodendrocytes, the cells responsible for producing myelin in the central nervous system during remyelination. Here, we explore the potential role of the kinase GCN2 in the myelination process. We challenged mice deficient in the GCN2-encoding gene with a pharmacological demyelinating stimulus (cuprizone) and evaluated the recovery of myelin as well as ISR activation through the levels of eIF2α phosphorylation. Our findings indicate that GCN2 controls the establishment of myelin by fine-tuning its abundance and morphology in the central nervous system. We also found that GCN2 is essential for remyelination. Surprisingly, we discovered that GCN2 is necessary to maintain eIF2α levels during remyelination. Full article

The cuprizone (CPZ) model of multiple sclerosis (MS) is excellent for studying the molecular differences behind the damage caused by poisoning. Metabolic differences in the kynurenine pathway (KP) of tryptophan (TRP) degradation are observed in both MS and a CPZ mouse model. Our goal was to analyze the kynurenine, serotonin, and indole pathways of TRP degradation on the periphery, in the neurodegenerative processes of inflammation. In our study, mice were fed with 0.2% CPZ toxin for 5 weeks. We examined the metabolites in the three pathways of TRP breakdown in urine, plasma, and relevant visceral organs with bioanalytical measurements. In our analyses, we found a significant increase in plasma TRP, 5-hydroxytryptophan (5-HTP), and indole-3-acetic acid (IAA) levels, while a decrease in the concentrations of 3-hydroxy-L-kynurenine (3-HK), xanthurenic acid (XA), kynurenic acid (KYNA), and quinaldic acid in the plasma of toxin-treated group was found. A marked decrease in the levels of 3-HK, XA, KYNA, quinaldic acid, and indole-3-lactic acid was also observed in the visceral organs by the end of the poisoning. Furthermore, we noticed a decrease in the urinary levels of the TRP, KYNA, and XA metabolites, while an increase in serotonin and 5-hydroxyindoleacetic acid in the CPZ group was noticed. The toxin treatment resulted in elevated tryptamine and indoxyl sulfate levels and reduced IAA concentration. Moreover, the urinary para-cresyl sulfate concentration also increased in the treated group. In the present study, we showed the differences in the three main metabolic pathways of TRP degradation in the CPZ model. We confirmed the relationship and correlation between the content of the kynurenine metabolites in the plasma and the tissues of the visceral organs. We emphasized the suppression of the KP and the activity of the serotonin and indole pathways with a particular regard to the involvement of the microbiome by the indole pathway. Consequently, this is the first study to analyze in detail the distribution of the kynurenine, serotonin, and indole pathways of TRP degradation in the periphery. Full article

GABAergic signaling and GABA_B receptors play crucial roles in regulating the physiology of oligodendrocyte-lineage cells, including their proliferation, differentiation, and myelination. Therefore, they are promising targets for studying how spinal oligodendrocyte precursor cells (OPCs) respond to injuries and neurodegenerative diseases like multiple sclerosis. Taking advantage of the temporally controlled and cell-specific genetic downregulation of GABA_B receptors from OPCs, our investigation addresses their specific influence on OPC behavior in the gray and white matter of the mouse spinal cord. Our results show that, while GABA_B receptors do not significantly alter spinal cord myelination under physiological conditions, they distinctly regulate the OPC differentiation and Ca²⁺ signaling. In addition, we investigate the impact of OPC-GABA_B receptors in two models of toxic demyelination, namely, the cuprizone and the lysolecithin models. The genetic downregulation of OPC-GABA_B receptors protects against demyelination and oligodendrocyte loss. Additionally, we observe the enhanced resilience to cuprizone-induced pathological alterations in OPC Ca²⁺ signaling. Our results provide valuable insights into the potential therapeutic implications of manipulating GABA_B receptors in spinal cord OPCs and deepen our understanding of the interplay between GABAergic signaling and spinal cord OPCs, providing a basis for future research. Full article

Multiple sclerosis (MS) is a chronic neurodegenerative disorder involving demyelination. The cuprizone model is commonly used to study MS by inducing oligodendrocyte stress and demyelination. The subventricular zone (SVZ) plays a key role in neurogenesis, while the neuronal/glial antigen 2 (NG2) is a marker for immature glial cells, involved in oligodendrocyte differentiation. The apelin receptor (APLNR) is linked to neurogenesis and behavior modulation. This study explores the role of APLNR in NG2-positive cells during de- and remyelination phases in the experimental cuprizone mouse model. Thirty male C57BL/6 mice were divided into control (not treated), demyelination (5 weeks cuprizone administration), and remyelination (5 weeks cuprizone administration + 5 weeks recovery) groups. Histological examinations, immunohistochemistry, and immunofluorescence on serial coronal sections were conducted to evaluate corpus callosum (CC) morphology and APLNR and NG2 expression in the SVZ, in addition to behavioral assessments. The histological analysis showed a significant reduction in the CC’s thickness and area after five weeks of cuprizone exposure, followed by recovery five weeks post-exposure. During the demyelination phase, APLNR-expressing cells peaked while NG2-positive cells decreased. In the remyelination phase, APLNR-expressing cells declined, and NG2-positive cells increased. Confocal microscopy confirmed the co-localization of NG2 and APLNR markers. Statistically significant differences were observed across experimental groups. Correlation analyses highlighted associations between APLNR/NG2 cell counts and CC changes. Behavioral tests revealed impaired motor coordination and memory during demyelination, with gradual recovery during remyelination. Significant changes in the CC structure and the number of APLNR and NG2-positive cells were observed during de- and remyelination, suggesting that NG2-positive cells expressing APLNR may play a key role in remyelination. Full article

Demyelination and axonal degeneration are fundamental pathological characteristics of multiple sclerosis (MS), an inflammatory disease of the central nervous system (CNS). Although the molecular mechanisms driving these processes are not fully understood, hepatocyte growth factor (HGF) has emerged as a potential regulator of neuroinflammation and tissue protection in MS. Elevated HGF levels have been reported in MS patients receiving immunomodulatory therapy, indicating its relevance in disease modulation. This study investigated HGF’s neuroprotective effects using transgenic mice that overexpressed HGF. The experimental autoimmune encephalomyelitis (EAE) model, which mimics MS pathology, was employed to assess demyelination and axonal damage in the CNS. HGF transgenic mice showed delayed EAE progression, with reduced CNS inflammation, decreased demyelination, and limited axonal degeneration. Scanning electron microscopy confirmed the preservation of myelin and axonal integrity in these mice. In addition, we explored HGF’s effects using a cuprizone-induced demyelination model, which operates independently of the immune system. HGF transgenic mice exhibited significant protection against demyelination in this model as well. We also investigated the expression of key HGF receptors, particularly c-Met and KAI-1. While c-Met, which is associated with increased inflammation, was upregulated in EAE, its expression was significantly reduced in HGF transgenic mice, correlating with decreased neuroinflammation. Conversely, KAI-1, which has been linked to axonal protection and stability, showed enhanced expression in HGF transgenic mice, suggesting a protective mechanism against axonal degeneration. These findings underscore HGF’s potential in preserving CNS structure and function, suggesting it may be a promising therapeutic target for MS, offering new hope for mitigating disease progression and enhancing neuroprotection. Full article

Several studies have proposed a potential role for oxidative stress in the development of multiple sclerosis (MS). For this reason, it seems tentative to think that treatment with antioxidant substances could be useful in the treatment of this disease. In this narrative review, we provide a summary of the current findings on antioxidant treatments, both in experimental models of MS, especially in experimental autoimmune encephalomyelitis (EAE) and in the cuprizone-induced demyelination model, and clinical trials in patients diagnosed with MS. Practically all the antioxidants tested in experimental models of MS have shown improvement in clinical parameters, in delaying the evolution of the disease, and in improving histological and biochemical parameters, including decreased levels of markers of inflammation and oxidative stress in the central nervous system and other tissues. Only a few clinical trials have been carried out to investigate the potential efficacy of antioxidant substances in patients with MS, most of them in the short term and involving a short series of patients, so the results of these should be considered inconclusive. In this regard, it would be desirable to design long-term, randomized, multicenter clinical trials with a long series of patients, assessing several antioxidants that have demonstrated efficacy in experimental models of MS. Full article