RESUMEN
Demyelination is a common pathological feature in a wide range of diseases, characterized by the loss of myelin sheath and myelin-supporting oligodendrocytes. These losses lead to impaired axonal function, increased vulnerability of axons to damage, and result in significant brain atrophy and neuro-axonal degeneration. Multiple pathomolecular processes contribute to neuroinflammation, oligodendrocyte cell death, and progressive neuronal dysfunction. In this study, we use the cuprizone mouse model of demyelination to investigate long-term non-invasive gamma entrainment using sensory stimulation as a potential therapeutic intervention for promoting myelination and reducing neuroinflammation in male mice. Here, we show that multisensory gamma stimulation mitigates demyelination, promotes oligodendrogenesis, preserves functional integrity and synaptic plasticity, attenuates oligodendrocyte ferroptosis-induced cell death, and reduces brain inflammation. Thus, the protective effects of multisensory gamma stimulation on myelin and anti-neuroinflammatory properties support its potential as a therapeutic approach for demyelinating disorders.
Asunto(s)
Cuprizona , Enfermedades Desmielinizantes , Modelos Animales de Enfermedad , Vaina de Mielina , Oligodendroglía , Animales , Cuprizona/toxicidad , Masculino , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/terapia , Enfermedades Desmielinizantes/patología , Ratones , Oligodendroglía/metabolismo , Oligodendroglía/patología , Vaina de Mielina/metabolismo , Ratones Endogámicos C57BL , Ferroptosis , Plasticidad Neuronal , Encéfalo/patología , Encéfalo/metabolismo , Enfermedades Neuroinflamatorias/inducido químicamente , Enfermedades Neuroinflamatorias/patologíaRESUMEN
In people with multiple sclerosis (MS), newborn and surviving oligodendrocytes (OLs) can contribute to remyelination, however, current therapies are unable to enhance or sustain endogenous repair. Low intensity repetitive transcranial magnetic stimulation (LI-rTMS), delivered as an intermittent theta burst stimulation (iTBS), increases the survival and maturation of newborn OLs in the healthy adult mouse cortex, but it is unclear whether LI-rTMS can promote remyelination. To examine this possibility, we fluorescently labelled oligodendrocyte progenitor cells (OPCs; Pdgfrα-CreER transgenic mice) or mature OLs (Plp-CreER transgenic mice) in the adult mouse brain and traced the fate of each cell population over time. Daily sessions of iTBS (600 pulses; 120 mT), delivered during cuprizone (CPZ) feeding, did not alter new or pre-existing OL survival but increased the number of myelin internodes elaborated by new OLs in the primary motor cortex (M1). This resulted in each new M1 OL producing ~ 471 µm more myelin. When LI-rTMS was delivered after CPZ withdrawal (during remyelination), it significantly increased the length of the internodes elaborated by new M1 and callosal OLs, increased the number of surviving OLs that supported internodes in the corpus callosum (CC), and increased the proportion of axons that were myelinated. The ability of LI-rTMS to modify cortical neuronal activity and the behaviour of new and surviving OLs, suggests that it may be a suitable adjunct intervention to enhance remyelination in people with MS.
Asunto(s)
Cuprizona , Enfermedades Desmielinizantes , Vaina de Mielina , Oligodendroglía , Remielinización , Estimulación Magnética Transcraneal , Animales , Estimulación Magnética Transcraneal/métodos , Oligodendroglía/metabolismo , Enfermedades Desmielinizantes/terapia , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Ratones , Vaina de Mielina/metabolismo , Modelos Animales de Enfermedad , Ratones Transgénicos , Corteza Motora/patología , Corteza Motora/metabolismo , Supervivencia Celular , Ratones Endogámicos C57BL , Esclerosis Múltiple/terapia , Esclerosis Múltiple/patologíaAsunto(s)
Antibacterianos , Humanos , Antibacterianos/efectos adversos , Fluoroquinolonas/efectos adversos , Polineuropatías/inducido químicamente , Masculino , Enfermedades Desmielinizantes/inducido químicamente , Femenino , Persona de Mediana Edad , Anciano , Conducción Nerviosa/efectos de los fármacosRESUMEN
AIMS: The mechanism underlying the reversible unconsciousness induced by general anesthetics (GA) remains unclear. Recent studies revealed the critical roles of myelin and oligodendrocytes (OLs) in higher functions of the brain. However, it is unknown whether myelin actively participates in the regulation of GA. The aim of this study is to investigate the roles and possible mechanisms of myelin in the regulation of consciousness alterations induced by isoflurane anesthesia. METHODS: First, demyelination models for the entire brain and specific neural nuclei were established to investigate the potential role of myelination in the regulation of GA, as well as its possible regional specificity. c-Fos staining was then performed on the demyelinated nuclei to verify the impact of myelin loss on neuronal activity. Finally, the activity of neurons during isoflurane anesthesia in demyelinated mice was recorded by optical fiber photometric calcium signal. The related behavioral indicators and EEG were recorded and analyzed. RESULTS: A prolonged emergence time was observed from isoflurane anesthesia in demyelinated mice, which suggested the involvement of myelin in regulating GA. The demyelination in distinct nuclei by LPC further clarified the region-specific roles of isoflurane anesthesia regulation by myelin. The effect of demyelination on isoflurane anesthesia in the certain nucleus was consistent with that in neurons towards isoflurane anesthesia. Finally, we found that the mechanism of myelin in the modulation of isoflurane anesthesia is possibly through the regulation of neuronal activity. CONCLUSIONS: In brief, myelin in the distinct neural nucleus plays an essential role in regulating the process of isoflurane anesthesia. The possible mechanism of myelin in the regulation of isoflurane anesthesia is neuronal activity modification by myelin integrity during GA. Our findings enhanced the comprehension of myelin function, and offered a fresh perspective for investigating the neural mechanisms of GA.
Asunto(s)
Anestésicos por Inhalación , Isoflurano , Ratones Endogámicos C57BL , Vaina de Mielina , Neuronas , Isoflurano/farmacología , Animales , Anestésicos por Inhalación/farmacología , Ratones , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/metabolismo , Masculino , Neuronas/efectos de los fármacos , Enfermedades Desmielinizantes/inducido químicamente , Electroencefalografía , Encéfalo/efectos de los fármacosRESUMEN
Multiple sclerosis is a chronic inflammatory demyelinating disorder of the CNS characterized by continuous myelin damage accompanied by deterioration in functions. Clobetasol propionate (CP) is the most potent topical corticosteroid with serious side effects related to systemic absorption. Previous studies introduced CP for remyelination without considering systemic toxicity. This work aimed at fabrication and optimization of double coated nano-oleosomes loaded with CP to achieve brain targeting through intranasal administration. The optimized formulation was coated with lactoferrin and chitosan for the first time. The obtained double-coated oleosomes had particle size (220.07 ± 0.77 nm), zeta potential (+30.23 ± 0.41 mV) along with antioxidant capacity 9.8 µM ascorbic acid equivalents. Double coating was well visualized by TEM and significantly decreased drug release. Three different doses of CP were assessed in-vivo using cuprizone-induced demyelination in C57Bl/6 mice. Neurobehavioral tests revealed improvement in motor and cognitive functions of mice in a dose-dependent manner. Histopathological examination of the brain showed about 2.3 folds increase in corpus callosum thickness in 0.3 mg/kg CP dose. Moreover, the measured biomarkers highlighted the significant antioxidant and anti-inflammatory capacity of the formulation. In conclusion, the elaborated biopolymer-integrating nanocarrier succeeded in remyelination with 6.6 folds reduction in CP dose compared to previous studies.
Asunto(s)
Quitosano , Clobetasol , Cuprizona , Enfermedades Desmielinizantes , Modelos Animales de Enfermedad , Lactoferrina , Esclerosis Múltiple , Remielinización , Animales , Lactoferrina/química , Lactoferrina/farmacología , Quitosano/química , Ratones , Clobetasol/farmacología , Clobetasol/química , Remielinización/efectos de los fármacos , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/tratamiento farmacológico , Enfermedades Desmielinizantes/patología , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inducido químicamente , Liposomas/química , Ratones Endogámicos C57BL , Masculino , Tamaño de la Partícula , Encéfalo/efectos de los fármacos , Encéfalo/patología , Encéfalo/metabolismo , Antioxidantes/farmacología , Antioxidantes/química , Liberación de FármacosRESUMEN
The research aims to study the therapeutic impact of HEK293-XPack-Olig2 cell-derived exosomes on remyelination of the corpus callosum in a cuprizone-induced demyelinating disease model. A lentiviral vector expressing Olig2 was constructed using XPack technology. The highly abundant Olig2 exosomes (ExoOs) were isolated by centrifugation for subsequent experiments. Western blot, nanoparticle tracking analysis (NTA), and electron microscopy showed no significant difference in particle size and morphology between Exos and ExoOs, and a high level of Olig2 expression could be detected in ExoOs, indicating that exosome modification by XPack technology was successful. The Black Gold/Fluromyelin staining analysis showed that the ExoOs group significantly reduced the demyelination area in the corpus callosum compared to the PBS and Exos groups. Additionally, the PDGFRα/APC staining of the demyelinating region revealed an increase in APC+ oligodendrocytes and a decrease in PDGFRα+ oligodendrocyte progenitor cells (OPCs) in the ExoOs group. Furthermore, there was evident myelin regeneration in the demyelinated areas after ExoOs treatment, with better g-ratio and a higher number of intact myelin compared to the other treatment groups. The level of Sox10 expression in the brain tissue of the ExoOs group were higher compared to those of the PBS and Exos groups. The demyelination process can be significantly slowed down by the XPack-modified exosomes, the differentiation of OPCs promoted, and myelin regeneration accelerated under pathological conditions. This process is presumed to be achieved by changing the expression level of intracellular differentiation-related genes after exosomes transport Olig2 enriched into oligodendrocyte progenitors.
Asunto(s)
Cuprizona , Enfermedades Desmielinizantes , Exosomas , Factor de Transcripción 2 de los Oligodendrocitos , Exosomas/metabolismo , Cuprizona/toxicidad , Animales , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Humanos , Células HEK293 , Vaina de Mielina/metabolismo , Vaina de Mielina/patología , Remielinización/fisiología , Ratones , Células Precursoras de Oligodendrocitos/metabolismo , Ratones Endogámicos C57BL , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Masculino , Oligodendroglía/metabolismo , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: Multiple sclerosis (MS) is an irreversible progressive CNS pathology characterized by the loss of myelin (i.e. demyelination). The lack of myelin is followed by a progressive neurodegeneration triggering symptoms as diverse as fatigue, motor, locomotor and sensory impairments and/or bladder, cardiac and respiratory dysfunction. Even though there are more than fourteen approved treatments for reducing MS progression, there are still no cure for the disease. Thus, MS research is a very active field and therefore we count with different experimental animal models for studying mechanisms of demyelination and myelin repair, however, we still lack a preclinical MS model assembling demyelination mechanisms with relevant clinical-like signs. RESULTS: Here, by inducing the simultaneous demyelination of both callosal and cerebellar white matter fibers by the double-site injection of lysolecithin (LPC), we were able to reproduce CNS demyelination, astrocyte recruitment and increases levels of proinflammatory cytokines levels along with motor, locomotor and urinary impairment, as well as cardiac and respiratory dysfunction, in the same animal model. Single site LPC-injections either in corpus callosum or cerebellum only, fails in to reproduce such a complete range of MS-like signs. CONCLUSION: We here report that the double-site LPC injections treatment evoke a complex MS-like mice model. We hope that this experimental approach will help to deepen our knowledge about the mechanisms of demyelinated diseases such as MS.
Asunto(s)
Cerebelo , Cuerpo Calloso , Enfermedades Desmielinizantes , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Esclerosis Múltiple , Animales , Esclerosis Múltiple/patología , Cuerpo Calloso/patología , Cerebelo/patología , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/inducido químicamente , Ratones , Masculino , Lisofosfatidilcolinas , Citocinas/metabolismo , Vaina de Mielina/patologíaRESUMEN
The kappa opioid receptor has been identified as a promising therapeutic target for promoting remyelination. In the current study, we evaluated the ability of nalfurafine to promote oligodendrocyte progenitor cell (OPC) differentiation and myelination in vitro, and its efficacy in an extended, cuprizone-induced demyelination model. Primary mouse (C57BL/6J) OPC-containing cultures were treated with nalfurafine (0.6-200 nM), clemastine (0.01-100 µM), T3 (30 ng/mL), or vehicle for 5 days. Using immunocytochemistry and confocal microscopy, we found that nalfurafine treatment increased OPC differentiation, oligodendrocyte (OL) morphological complexity, and myelination of nanofibers in vitro. Adult male mice (C57BL/6J) were given a diet containing 0.2% cuprizone and administered rapamycin (10 mg/kg) once daily for 12 weeks followed by 6 weeks of treatment with nalfurafine (0.01 or 0.1 mg/kg), clemastine (10 mg/kg), or vehicle. We quantified the number of OLs using immunofluorescence, gross myelination using black gold staining, and myelin thickness using electron microscopy. Cuprizone + rapamycin treatment produced extensive demyelination and was accompanied by a loss of mature OLs, which was partially reversed by therapeutic administration of nalfurafine. We also assessed these mice for functional behavioral changes in open-field, horizontal bar, and mouse motor skill sequence tests (complex wheel running). Cuprizone + rapamycin treatment resulted in hyperlocomotion, poorer horizontal bar scores, and less distance traveled on the running wheels. Partial recovery was observed on both the horizontal bar and complex running wheel tests over time, which was facilitated by nalfurafine treatment. Taken together, these data highlight the potential of nalfurafine as a remyelination-promoting therapeutic.
Asunto(s)
Cuprizona , Enfermedades Desmielinizantes , Ratones Endogámicos C57BL , Morfinanos , Vaina de Mielina , Sirolimus , Compuestos de Espiro , Animales , Morfinanos/farmacología , Masculino , Compuestos de Espiro/farmacología , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/tratamiento farmacológico , Ratones , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/patología , Vaina de Mielina/metabolismo , Sirolimus/farmacología , Cuprizona/toxicidad , Células Cultivadas , Modelos Animales de Enfermedad , Células Precursoras de Oligodendrocitos/efectos de los fármacos , Células Precursoras de Oligodendrocitos/metabolismo , Diferenciación Celular/efectos de los fármacosRESUMEN
Demyelination is generated in several nervous system illnesses. Developing strategies for effective clinical treatments requires the discovery of promyelinating drugs. Increased GABAergic signaling through γ-aminobutyric acid type A receptor (GABAAR) activation in oligodendrocytes has been proposed as a promyelinating condition. GABAAR expressed in oligodendroglia is strongly potentiated by n-butyl-ß-carboline-3-carboxylate (ß-CCB) compared to that in neurons. Here, mice were subjected to 0.3% cuprizone (CPZ) added in the food to induce central nervous system demyelination, a well-known model for multiple sclerosis. Then ß-CCB (1 mg/Kg) was systemically administered to analyze the remyelination status in white and gray matter areas. Myelin content was evaluated using Black-Gold II (BGII) staining, immunofluorescence (IF), and magnetic resonance imaging (MRI). Evidence indicates that ß-CCB treatment of CPZ-demyelinated animals promoted remyelination in several white matter structures, such as the fimbria, corpus callosum, internal capsule, and cerebellar peduncles. Moreover, using IF, it was observed that CPZ intake induced an increase in NG2+ and a decrease in CC1+ cell populations, alterations that were importantly retrieved by ß-CCB treatment. Thus, the promyelinating character of ß-CCB was confirmed in a generalized demyelination model, strengthening the idea that it has clinical potential as a therapeutic drug.
Asunto(s)
Carbolinas , Cuprizona , Enfermedades Desmielinizantes , Modelos Animales de Enfermedad , Remielinización , Animales , Cuprizona/toxicidad , Remielinización/efectos de los fármacos , Ratones , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/metabolismo , Carbolinas/farmacología , Carbolinas/administración & dosificación , Vaina de Mielina/metabolismo , Vaina de Mielina/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/inducido químicamente , Esclerosis Múltiple/patología , Sustancia Blanca/efectos de los fármacos , Sustancia Blanca/metabolismo , Sustancia Blanca/patología , Imagen por Resonancia MagnéticaRESUMEN
BACKGROUND: Lipid droplet (LD)-laden microglia is a key pathological hallmark of multiple sclerosis. The recent discovery of this novel microglial subtype, lipid-droplet-accumulating microglia (LDAM), is notable for increased inflammatory factor secretion and diminished phagocytic capability. Lipophagy, the autophagy-mediated selective degradation of LDs, plays a critical role in this context. This study investigated the involvement of microRNAs (miRNAs) in lipophagy during demyelinating diseases, assessed their capacity to modulate LDAM subtypes, and elucidated the potential underlying mechanisms involved. METHODS: C57BL/6 mice were used for in vivo experiments. Two weeks post demyelination induction at cervical level 4 (C4), histological assessments and confocal imaging were performed to examine LD accumulation in microglia within the lesion site. Autophagic changes were observed using transmission electron microscopy. miRNA and mRNA multi-omics analyses identified differentially expressed miRNAs and mRNAs under demyelinating conditions and the related autophagy target genes. The role of miR-223 in lipophagy under these conditions was specifically explored. In vitro studies, including miR-223 upregulation in BV2 cells via lentiviral infection, validated the bioinformatics findings. Immunofluorescence staining was used to measure LD accumulation, autophagy levels, target gene expression, and inflammatory mediator levels to elucidate the mechanisms of action of miR-223 in LDAM. RESULTS: Oil Red O staining and confocal imaging revealed substantial LD accumulation in the demyelinated spinal cord. Transmission electron microscopy revealed increased numbers of autophagic vacuoles at the injury site. Multi-omics analysis revealed miR-223 as a crucial regulatory gene in lipophagy during demyelination. It was identified that cathepsin B (CTSB) targets miR-223 in autophagy to integrate miRNA, mRNA, and autophagy gene databases. In vitro, miR-223 upregulation suppressed CTSB expression in BV2 cells, augmented autophagy, alleviated LD accumulation, and decreased the expression of the inflammatory mediator IL-1ß. CONCLUSION: These findings indicate that miR-223 plays a pivotal role in lipophagy under demyelinating conditions. By inhibiting CTSB, miR-223 promotes selective LD degradation, thereby reducing the lipid burden and inflammatory phenotype in LDAM. This study broadens the understanding of the molecular mechanisms of lipophagy and proposes lipophagy induction as a potential therapeutic approach to mitigate inflammatory responses in demyelinating diseases.
Asunto(s)
Autofagia , Catepsina B , Enfermedades Desmielinizantes , Gotas Lipídicas , Lisofosfatidilcolinas , Ratones Endogámicos C57BL , MicroARNs , Microglía , Animales , MicroARNs/genética , MicroARNs/metabolismo , Microglía/metabolismo , Microglía/patología , Ratones , Gotas Lipídicas/metabolismo , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/patología , Catepsina B/metabolismo , Catepsina B/genética , Lisofosfatidilcolinas/metabolismo , Modelos Animales de Enfermedad , Masculino , Regulación de la Expresión Génica , Línea CelularRESUMEN
Dietary administration of a copper chelator, cuprizone (CPZ), has long been reported to induce intense and reproducible demyelination of several brain structures such as the corpus callosum. Despite the widespread use of CPZ as an animal model for demyelinating diseases such as multiple sclerosis (MS), the mechanism by which it induces demyelination and then allows robust remyelination is still unclear. An intensive mapping of the cell dynamics of oligodendrocyte (OL) lineage during the de- and remyelination course would be particularly important for a deeper understanding of this model. Here, using a panel of OL lineage cell markers as in situ hybridization (ISH) probes, including Pdgfra, Plp, Mbp, Mog, Enpp6, combined with immunofluorescence staining of CC1, SOX10, we provide a detailed dynamic profile of OL lineage cells during the entire course of the model from 1, 2, 3.5 days, 1, 2, 3, 4,5 weeks of CPZ treatment, as well as after 1, 2, 3, 4 weeks of recovery from CPZ treatment. The result showed an unexpected early death of mature OLs and response of OL progenitor cells (OPCs) in vivo upon CPZ challenge, and a prolonged upregulation of myelin-forming OLs compared to the intact control even 4 weeks after CPZ withdrawal. These data may serve as a basic reference system for future studies of the effects of any intervention on de- and remyelination using the CPZ model, and imply the need to optimize the timing windows for the introduction of pro-remyelination therapies in demyelinating diseases such as MS.
Asunto(s)
Linaje de la Célula , Cuprizona , Enfermedades Desmielinizantes , Oligodendroglía , Cuprizona/toxicidad , Animales , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/patología , Oligodendroglía/metabolismo , Modelos Animales de Enfermedad , Hibridación in Situ/métodos , Ratones Endogámicos C57BL , Ratones , Remielinización/efectos de los fármacos , Remielinización/fisiología , Masculino , Quelantes/toxicidad , Quelantes/farmacología , Vaina de Mielina/patología , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/metabolismoRESUMEN
Cognitive impairment (CI) and memory deficit are prevalent manifestations of multiple sclerosis (MS). This study explores the therapeutic potential of arbutin on memory deficits using a rat hippocampal demyelination model induced by lysophosphatidylcholine (LPC). Demyelination was induced by bilateral injection of 1% LPC into the CA1 area of the hippocampus, and the treated group received daily arbutin injections (50â¯mg/kg, i.p) for two weeks. Arbutin significantly improved memory impairment 14 days post-demyelination as assessed by Morris water maze test. Histological and immunohistochemical analyses demonstrated that arbutin reduced demyelination suppressed pro-inflammatory markers (IL-1ß, TNF-α) and increased anti-inflammatory cytokine IL-10. Arbutin also diminished astrocyte activation, decreased iNOS, enhanced anti-oxidative factors (Nrf2, HO-1), and exhibited neuroprotective effects by elevating myelin markers (MBP) and brain derived neurotrophic factor (BDNF). These findings propose arbutin as a potential therapeutic candidate for multiple sclerosis-associated memory deficits, warranting further clinical exploration.
Asunto(s)
Antiinflamatorios , Arbutina , Enfermedades Desmielinizantes , Modelos Animales de Enfermedad , Lisofosfatidilcolinas , Trastornos de la Memoria , Fármacos Neuroprotectores , Animales , Lisofosfatidilcolinas/farmacología , Ratas , Trastornos de la Memoria/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/administración & dosificación , Masculino , Arbutina/farmacología , Arbutina/administración & dosificación , Enfermedades Desmielinizantes/tratamiento farmacológico , Enfermedades Desmielinizantes/inducido químicamente , Antiinflamatorios/farmacología , Antiinflamatorios/administración & dosificación , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) with inflammation and immune dysfunction. OBJECTIVES: We compared the remyelination and immunomodulation properties of mesenchymal stem cells (MSCs) with their conditioned medium (CM) in the cuprizone model. METHODS: Twenty-four C57BL/ 6 mice were divided into four groups. After cuprizone demyelination, MSCs and their CM were injected into the right lateral ventricle of mice. The expression level of IL-1ß, TNF-α, and BDNF genes was evaluated using the qRT-PCR. APC antibody was used to assess the oligodendrocyte population using the immunofluorescent method. The remyelination and axonal repair were studied by specific staining of the LFB and electron microscopy techniques. RESULTS: Transplantation of MSCs and CM increased the expression of the BDNF gene and decreased the expression of IL-1ß and TNF-α genes compared to the cuprizone group, and these effects in the cell group were more than CM. Furthermore, cell transplantation resulted in a significant improvement in myelination and axonal repair, which was measured by luxol fast blue and transmission electron microscope images. The cell group had a higher number of oligodendrocytes than other groups. CONCLUSIONS: According to the findings, injecting MSCs intraventricularly versus cell-conditioned medium can be a more effective approach to improving chronic demyelination in degenerative diseases like MS.
Asunto(s)
Cuprizona , Enfermedades Desmielinizantes , Modelos Animales de Enfermedad , Inflamación , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Ratones Endogámicos C57BL , Animales , Trasplante de Células Madre Mesenquimatosas/métodos , Ratones , Células Madre Mesenquimatosas/metabolismo , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Medios de Cultivo Condicionados/farmacología , Inflamación/patología , Inflamación/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Oligodendroglía/metabolismo , Remielinización , Esclerosis Múltiple/patología , Esclerosis Múltiple/terapia , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/inducido químicamente , Factor de Necrosis Tumoral alfa/metabolismo , Masculino , Vaina de Mielina/metabolismoRESUMEN
Addressing inflammation, demyelination, and associated neurodegeneration in inflammatory demyelinating diseases like multiple sclerosis (MS) remains challenging. ACT-1004-1239, a first-in-class and potent ACKR3 antagonist, currently undergoing clinical development, showed promise in preclinical MS models, reducing neuroinflammation and demyelination. However, its effectiveness in treating established disease and impact on remyelination after the occurrence of demyelinated lesions remain unexplored. This study assessed the therapeutic effect of ACT-1004-1239 in two demyelinating disease models. In the proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) model, ACT-1004-1239 administered upon the detection of the first signs of paralysis, resulted in a dose-dependent reduction in EAE disease severity, concomitant with diminished immune cell infiltrates in the CNS and reduced demyelination. Notably, efficacy correlated with elevated plasma concentrations of CXCL11 and CXCL12, two pharmacodynamic biomarkers of ACKR3 antagonism. Combining ACT-1004-1239 with siponimod, an approved immunomodulatory treatment for MS, synergistically reduced EAE severity. In the cuprizone-induced demyelination model, ACT-1004-1239 administered after 5 weeks of cuprizone exposure, significantly accelerated remyelination, already quantifiable one week after cuprizone withdrawal. Additionally, ACT-1004-1239 penetrated the CNS, elevating brain CXCL12 concentrations. These results demonstrate that ACKR3 antagonism significantly reduces the severity of experimental demyelinating diseases, even when treatment is initiated therapeutically, after the occurrence of lesions. It confirms the dual mode of action of ACT-1004-1239, exhibiting both immunomodulatory effects by reducing neuroinflammation and promyelinating effects by accelerating myelin repair. The results further strengthen the rationale for evaluating ACT-1004-1239 in clinical trials for patients with demyelinating diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Ratones Endogámicos C57BL , Remielinización , Animales , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Remielinización/efectos de los fármacos , Ratones , Femenino , Enfermedades Desmielinizantes/tratamiento farmacológico , Enfermedades Desmielinizantes/inducido químicamente , Cuprizona , Azetidinas/farmacología , Azetidinas/uso terapéutico , Agentes Inmunomoduladores/farmacología , Agentes Inmunomoduladores/uso terapéutico , Compuestos de Bencilo/uso terapéutico , Compuestos de Bencilo/farmacología , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/metabolismoRESUMEN
Dysregulation of oligodendrocyte progenitor cell (OPC) recruitment and oligodendrocyte differentiation contribute to failure of remyelination in human demyelinating diseases such as multiple sclerosis (MS). Deletion of muscarinic receptor enhances OPC differentiation and remyelination. However, the role of ligand-dependent signaling versus constitutive receptor activation is unknown. We hypothesized that dysregulated acetylcholine (ACh) release upon demyelination contributes to ligand-mediated activation hindering myelin repair. Following chronic cuprizone (CPZ)-induced demyelination (male and female mice), we observed a 2.5-fold increase in ACh concentration. This increase in ACh concentration could be attributed to increased ACh synthesis or decreased acetylcholinesterase-/butyrylcholinesterase (BChE)-mediated degradation. Using choline acetyltransferase (ChAT) reporter mice, we identified increased ChAT-GFP expression following both lysolecithin and CPZ demyelination. ChAT-GFP expression was upregulated in a subset of injured and uninjured axons following intraspinal lysolecithin-induced demyelination. In CPZ-demyelinated corpus callosum, ChAT-GFP was observed in Gfap+ astrocytes and axons indicating the potential for neuronal and astrocytic ACh release. BChE expression was significantly decreased in the corpus callosum following CPZ demyelination. This decrease was due to the loss of myelinating oligodendrocytes which were the primary source of BChE. To determine the role of ligand-mediated muscarinic signaling following lysolecithin injection, we administered neostigmine, a cholinesterase inhibitor, to artificially raise ACh. We identified a dose-dependent decrease in mature oligodendrocyte density with no effect on OPC recruitment. Together, these results support a functional role of ligand-mediated activation of muscarinic receptors following demyelination and suggest that dysregulation of ACh homeostasis directly contributes to failure of remyelination in MS.
Asunto(s)
Enfermedades Desmielinizantes , Oligodendroglía , Transducción de Señal , Animales , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Ratones , Oligodendroglía/metabolismo , Oligodendroglía/efectos de los fármacos , Femenino , Masculino , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ratones Endogámicos C57BL , Acetilcolina/metabolismo , Cuprizona/toxicidad , Lisofosfatidilcolinas/toxicidad , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Colina O-Acetiltransferasa/metabolismo , Remielinización/fisiología , Remielinización/efectos de los fármacos , Vaina de Mielina/metabolismo , Vaina de Mielina/efectos de los fármacos , Ratones TransgénicosRESUMEN
INTRODUCTION: Demyelination is a key factor in axonal degeneration and neural loss, leading to disability in multiple sclerosis (MS) patients. Transforming growth factor beta activated kinase 1 (TAK1) is a critical molecule involved in immune and inflammatory signaling pathways. Knockout of microglia TAK1 can inhibit autoimmune inflammation of the brain and spinal cord and improve the outcome of MS. However, it is unclear whether inhibiting TAK1 can alleviate demyelination. METHODS: Eight-week-old male c57bl/6j mice were randomly divided into five groups: (a) the control group, (b) the group treated with cuprizone (CPZ) only, (c) the group treated with 5Z-7-Oxozaenol (OZ) only, and (d) the group treated with both cuprizone and 15 µg/30 µg OZ. Demyelination in the mice of this study was induced by administration of CPZ (ig) at a daily dose of 400 mg/kg for consecutive 5 weeks. OZ was intraperitoneally administered at mentioned doses twice a week, starting from week 3 after beginning cuprizone treatment. Histology, rotarod test, grasping test, pole test, Western blot, RT-PCR, and ELISA were used to evaluate corpus callosum demyelination, behavioral impairment, oligodendrocyte differentiation, TAK1 signaling pathway expression, microglia, and related cytokines. RESULTS: Our results demonstrated that OZ protected against myelin loss and behavior impairment caused by CPZ. Additionally, OZ rescued the loss of oligodendrocytes in CPZ-induced mice. OZ inhibited the activation of JNK, p65, and p38 pathways, transformed M1 polarized microglia into M2 phenotype, and increased brain-derived neurotrophic factor (BDNF) expression to attenuate demyelination in CPZ-treated mice. Furthermore, OZ reduced the expression of proinflammatory cytokines and increases anti-inflammatory cytokines in CPZ-treated mice. CONCLUSION: These findings suggest that inhibiting TAK1 may be an effective approach for treating demyelinating diseases.
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Cuprizona , Enfermedades Desmielinizantes , Lactonas , Ratones Endogámicos C57BL , Microglía , Resorcinoles , Zearalenona/administración & dosificación , Animales , Cuprizona/farmacología , Microglía/efectos de los fármacos , Microglía/metabolismo , Enfermedades Desmielinizantes/tratamiento farmacológico , Enfermedades Desmielinizantes/inducido químicamente , Ratones , Masculino , Quinasas Quinasa Quinasa PAM/metabolismo , Zearalenona/farmacología , Zearalenona/análogos & derivados , Polaridad Celular/efectos de los fármacos , Cuerpo Calloso/efectos de los fármacos , Cuerpo Calloso/patología , Cuerpo Calloso/metabolismo , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: Current treatments for Multiple Sclerosis (MS) poorly address chronic innate neuroinflammation nor do they offer effective remyelination. The vagus nerve has a strong regulatory role in inflammation and Vagus Nerve Stimulation (VNS) has potential to affect both neuroinflammation and remyelination in MS. OBJECTIVE: This study investigated the effects of VNS on demyelination and innate neuroinflammation in a validated MS rodent model. METHODS: Lysolecithin (LPC) was injected in the corpus callosum (CC) of 46 Lewis rats, inducing a demyelinated lesion. 33/46 rats received continuously-cycled VNS (cVNS) or one-minute per day VNS (1minVNS) or sham VNS from 2 days before LPC-injection until perfusion at 3 days post-injection (dpi) (corresponding with a demyelinated lesion with peak inflammation). 13/46 rats received cVNS or sham from 2 days before LPC-injection until perfusion at 11 dpi (corresponding with a partial remyelinated lesion). Immunohistochemistry and proteomics analyses were performed to investigate the extend of demyelination and inflammation. RESULTS: Immunohistochemistry showed that cVNS significantly reduced microglial and astrocytic activation in the lesion and lesion border, and significantly reduced the Olig2+ cell count at 3 dpi. Furthermore, cVNS significantly improved remyelination with 57.4 % versus sham at 11 dpi. Proteomic gene set enrichment analyses showed increased activation of (glutamatergic) synapse pathways in cVNS versus sham, most pronounced at 3 dpi. CONCLUSION: cVNS improved remyelination of an LPC-induced lesion. Possible mechanisms might include modulation of microglia and astrocyte activity, increased (glutamatergic) synapses and enhanced oligodendrocyte clearance after initial injury.
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Enfermedades Desmielinizantes , Lisofosfatidilcolinas , Ratas Endogámicas Lew , Remielinización , Estimulación del Nervio Vago , Animales , Ratas , Remielinización/fisiología , Remielinización/efectos de los fármacos , Lisofosfatidilcolinas/toxicidad , Enfermedades Desmielinizantes/terapia , Enfermedades Desmielinizantes/inducido químicamente , Estimulación del Nervio Vago/métodos , Masculino , Enfermedades Neuroinflamatorias/terapia , Enfermedades Neuroinflamatorias/inducido químicamente , Enfermedades Neuroinflamatorias/etiología , Modelos Animales de Enfermedad , Esclerosis Múltiple/terapia , Esclerosis Múltiple/inducido químicamente , Cuerpo CallosoRESUMEN
Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease of the central nervous system that injures the myelin sheath, provoking progressive axonal degeneration and functional impairments. No efficient therapy is available at present to combat such insults, and hence, novel safe and effective alternatives for MS therapy are extremely required. Rutin (RUT) is a flavonoid that exhibits antioxidant, anti-inflammatory, and neuroprotective effects in several brain injuries. The present study evaluated the potential beneficial effects of two doses of RUT in a model of pattern-III lesion of MS, in comparison to the conventional standard drug; dimethyl fumarate (DMF). Demyelination was induced in in male adult C57BL/6 mice by dietary 0.2% (w/w) cuprizone (CPZ) feeding for 6 consecutive weeks. Treated groups received either oral RUT (50 or 100 mg/kg) or DMF (15 mg/kg), along with CPZ feeding, for 6 consecutive weeks. Mice were then tested for behavioral changes, followed by biochemical analyses and histological examinations of the corpus callosum (CC). Results revealed that CPZ caused motor dysfunction, demyelination, and glial activation in demyelinated lesions, as well as significant oxidative stress, and proinflammatory cytokine elevation. Six weeks of RUT treatment significantly improved locomotor activity and motor coordination. Moreover, RUT considerably improved remyelination in the CC of CPZ + RUT-treated mice, as revealed by luxol fast blue staining and transmission electron microscopy. Rutin also significantly attenuated CPZ-induced oxidative stress and inflammation in the CC of tested animals. The effect of RUT100 was obviously more marked than either that of DMF, regarding most of the tested parameters, or even its smaller tested dose. In silico docking revealed that RUT binds tightly within NF-κB at the binding site of the protein-DNA complex, with a good negative score of -6.79 kcal/mol. Also, RUT-Kelch-like ECH-associated protein 1 (Keap1) model clarifies the possible inhibition of Keap1-Nrf2 protein-protein interaction. Findings of the current study provide evidence for the protective effect of RUT in CPZ-induced demyelination and behavioral dysfunction in mice, possibly by modulating NF-κB and Nrf2 signaling pathways. The present study may be one of the first to indicate a pro-remyelinating effect for RUT, which might represent a potential additive benefit in treating MS.
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Enfermedades Desmielinizantes , Esclerosis Múltiple , Enfermedades Neurodegenerativas , Fármacos Neuroprotectores , Masculino , Animales , Ratones , Esclerosis Múltiple/inducido químicamente , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/metabolismo , Cuprizona/efectos adversos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/tratamiento farmacológico , Enfermedades Desmielinizantes/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , FN-kappa B/metabolismo , Rutina/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
Demyelination is the loss of myelin in CNS, resulting in damaged myelin sheath. Oxidative stress and neuroinflammation play a key role in inducing demyelinating diseases like MS; hence, controlling oxidative stress and neuroinflammation is important. Cuprizone (CPZ), a copper chelator, generates oxidative stress and neuroinflammation, thereby inducing demyelination. Therefore, the CPZ-induced demyelinating mouse model (CPZ model) is widely used in research. The present study was intended to unravel a mechanism of inhibition of demyelination by arsenic in a CPZ model, which is otherwise known for its toxicity. We investigated an alternative mechanism of inhibition of demyelination by arsenic through the reversal of SOD1 activity employing in silico analysis, analytical chemistry techniques, and in vitro and in vivo experiments. In vivo experiments showed protection of body weight, survivability, and myelination of the corpus callosum in CPZ and arsenic-co-exposed animals, where neuroinflammation was apparently not involved. In vitro experiments revealed that arsenic-mediated reversal of impaired SOD1 activity leads to reduced cellular ROS levels and better viability of primary oligodendrocytes. Reversal of SOD1 activity was also observed in the corpus callosum tissue isolated from experimental animals. In silico and analytical chemistry studies revealed that similar to copper, arsenic can potentially bind to CPZ and thereby make the copper freely available for SOD1 activity. Suitable neurobehavior tests further validated the protective effect of arsenic. Taken together, the present study revealed that arsenic protects oligodendrocytes and demyelination of corpus callosum by reversing CPZ-induced impaired SOD1 activity.
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Arsénico , Cuerpo Calloso , Cuprizona , Enfermedades Desmielinizantes , Modelos Animales de Enfermedad , Microglía , Animales , Cuprizona/toxicidad , Cuerpo Calloso/patología , Cuerpo Calloso/efectos de los fármacos , Cuerpo Calloso/metabolismo , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Microglía/efectos de los fármacos , Microglía/patología , Microglía/metabolismo , Arsénico/toxicidad , Ratones Endogámicos C57BL , Ratones , Enfermedades Neuroinflamatorias/patología , Enfermedades Neuroinflamatorias/inducido químicamente , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Masculino , Superóxido Dismutasa-1/metabolismo , Oligodendroglía/efectos de los fármacos , Oligodendroglía/patología , Oligodendroglía/metabolismo , Vaina de Mielina/metabolismo , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In the adult brain, activity-dependent myelin plasticity is required for proper learning and memory consolidation. Myelin loss, alteration, or even subtle structural modifications can therefore compromise the network activity, leading to functional impairment. In multiple sclerosis, spontaneous myelin repair process is possible, but it is heterogeneous among patients, sometimes leading to functional recovery, often more visible at the motor level than at the cognitive level. In cuprizone-treated mouse model, massive brain demyelination is followed by spontaneous and robust remyelination. However, reformed myelin, although functional, may not exhibit the same morphological characteristics as developmental myelin, which can have an impact on the activity of neural networks. In this context, we used the cuprizone-treated mouse model to analyze the structural, functional, and cognitive long-term effects of transient demyelination. Our results show that an episode of demyelination induces despite remyelination long-term cognitive impairment, such as deficits in spatial working memory, social memory, cognitive flexibility, and hyperactivity. These deficits were associated with a reduction in myelin content in the medial prefrontal cortex (mPFC) and hippocampus (HPC), as well as structural myelin modifications, suggesting that the remyelination process may be imperfect in these structures. In vivo electrophysiological recordings showed that the demyelination episode altered the synchronization of HPC-mPFC activity, which is crucial for memory processes. Altogether, our data indicate that the myelin repair process following transient demyelination does not allow the complete recovery of the initial myelin properties in cortical structures. These subtle modifications alter network features, leading to prolonged cognitive deficits in mice.