Longitudinal in vivo evaluation of retinal ganglion cell complex layer and dendrites in mice with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), induced by the immunization of myelin oligodendrocyte glycoprotein (MOG), is related to human MOG antibody-associated disease (MOGAD). Neuroinflammation and demyelination of the optic nerve can lead to retinal ganglion cell (RGC) death and axonal damage in MOGAD. Here, we aimed to evaluate the structural changes in RGCs longitudinally by in vivo imaging in mice with RGCs expressing yellow fluorescent protein along the course of EAE. Successful induction of EAE was confirmed by the neurological function scores and histology analyses. The changes in the thickness of ganglion cell complex (GCC) layer and RGC survival and dendrites were monitored longitudinally along the course of EAE. Before the onset of EAE, there were no significant changes in the number and morphology of RGCs and the thickness of the GCC layer as compared to the mice without EAE induction. After the onset of EAE, the thickness of the GCC layer and the RGC number and dendritic network all gradually decreased along the course of EAE. Notably, dendritic shrinkage could be detected earlier than the thinning of the GCC layer. In summary, this study delineated the longitudinal profile of RGC structural changes in EAE mice, providing an assessment platform for monitoring outcomes of RGC treatments.

Cannabis and Cannabinoids in Multiple Sclerosis: From Experimental Models to Clinical Practice-A Review.

BACKGROUND: As far as 80% of people diagnosed with multiple sclerosis (MS) experience disabling symptoms in the course of the disease, such as spasticity and neuropathic pain. As first-line symptomatic therapy is associated with important adverse reactions, cannabinoids have become increasingly popular among patients with MS. This review intends to provide an overview of the evidence of the role of cannabinoids in treating symptoms related to MS and to encourage further research on this matter. AREAS OF UNCERTAINTY: To date, the evidence supporting the role of cannabis and its derivatives in alleviating the MS-related symptoms comes only from studies on experimental models of demyelination. To the best of our knowledge, relatively few clinical trials inquired about the therapeutic effects of cannabinoids on patients with MS, with variable results. DATA SOURCES: We conducted a literature search through PubMed and Google Scholar from the beginning until 2022. We included articles in English describing the latest findings regarding the endocannabinoid system, the pharmacology of cannabinoids, and their therapeutic purpose in MS. RESULTS: Evidence from preclinical studies showed that cannabinoids can limit the demyelination process, promote remyelination, and have anti-inflammatory properties by reducing immune cell infiltration of the central nervous system in mice with experimental autoimmune encephalomyelitis. Moreover, it has been established that experimental autoimmune encephalomyelitis mice treated with cannabinoids experienced a significant reduction of symptoms and slowing of the disease progression. Given the complexity of human immune and nervous systems, cannabinoids did not have the anticipated effects on human subjects. However, data obtained from clinical trials showed some beneficial results of cannabinoids as a single or as add-on therapy in reducing the spasticity and pain related to MS. CONCLUSION: Considering their various mechanisms of action and good tolerability, cannabinoids remain an interesting therapy for spasticity and chronic pain related to MS.

Constipation induced gut microbiota dysbiosis exacerbates experimental autoimmune encephalomyelitis in C57BL/6 mice.

BACKGROUND: Constipation is a common gastrointestinal dysfunction which has a potential impact on people's immune state and their quality of life. Here we investigated the effects of constipation on experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). METHODS: Constipation was induced by loperamide in female C57BL/6 mice. The alternations of gut microbiota, permeability of intestinal barrier and blood-brain barrier, and histopathology of colon were assessed after constipation induction. EAE was induced in the constipation mice. Fecal microbiota transplantation (FMT) was performed from constipation mice into microbiota-depleted mice. Clinical scores, histopathology of inflammation and demyelination, Treg/Th17 and Treg17/Teff17 imbalance both in the peripheral lymphatic organs and central nervous system, cytokines include TGF-ß, GM-CSF, IL-10, IL-17A, IL-17F, IL-21, IL-22, and IL-23 in serum were assessed in different groups. RESULTS: Compared with the vehicle group, the constipation mice showed gut microbiota dysbiosis, colon inflammation and injury, and increased permeability of intestinal barrier and blood-brain barrier. We found that the clinical and pathological scores of the constipation EAE mice were severer than that of the EAE mice. Compared with the EAE mice, the constipation EAE mice showed reduced percentage of Treg and Treg17 cells, increased percentage of Th17 and Teff17 cells, and decreased ratio of Treg/Th17 and Treg17/Teff17 in the spleen, inguinal lymph nodes, brain, and spinal cord. Moreover, the serum levels of TGF-ß, IL-10, and IL-21 were decreased while the GM-CSF, IL-17A, IL-17F, IL-22, and IL-23 were increased in the constipation EAE mice. In addition, these pathological processes could be transferred via their gut microbiota. CONCLUSIONS: Our results verified that constipation induced gut microbiota dysbiosis exacerbated EAE via aggravating Treg/Th17 and Treg17/Teff17 imbalance and cytokines disturbance in C57BL/6 mice.

Regulatory T Cells and Their Derived Cytokine, Interleukin-35, Reduce Pain in Experimental Autoimmune Encephalomyelitis.

Sensory problems such as neuropathic pain are common and debilitating symptoms in multiple sclerosis (MS), an autoimmune inflammatory disorder of the CNS. Regulatory T (Treg) cells are critical for maintaining immune homeostasis, but their role in MS-associated pain remains unknown. Here, we demonstrate that Treg cell ablation is sufficient to trigger experimental autoimmune encephalomyelitis (EAE) and facial allodynia in immunized female mice. In EAE-induced female mice, adoptive transfer of Treg cells and spinal delivery of the Treg cell cytokine interleukin-35 (IL-35) significantly reduced facial stimulus-evoked pain and spontaneous pain independent of disease severity and increased myelination of the facial nociceptive pathway. The effects of intrathecal IL-35 therapy were Treg-cell dependent and associated with upregulated IL-10 expression in CNS-infiltrating lymphocytes and reduced monocyte infiltration in the trigeminal afferent pathway. We present evidence for a beneficial role of Treg cells and IL-35 in attenuating pain associated with EAE independently of motor symptoms by decreasing neuroinflammation and increasing myelination.SIGNIFICANCE STATEMENT Pain is a highly prevalent symptom affecting the majority of multiple sclerosis (MS) patients and dramatically affects overall health-related quality of life; however, this is a research area that has been largely ignored. Here, we identify for the first time a role for regulatory T (Treg) cells and interleukin-35 (IL-35) in suppressing facial allodynia and facial grimacing in animals with experimental autoimmune encephalomyelitis (EAE). We demonstrate that spinal delivery of Treg cells and IL-35 reduces pain associated with EAE by decreasing neuroinflammation and increasing myelination independently of motor symptoms. These findings increase our understanding of the mechanisms underlying pain in EAE and suggest potential treatment strategies for pain relief in MS.

Quantifying immunoregulation by autoantigen-specific T-regulatory type 1 cells in mice with simultaneous hepatic and extra-hepatic autoimmune disorders.

Nanoparticles (NPs) displaying autoimmune disease-relevant peptide-major histocompatibility complex class II molecules (pMHCII-NPs) trigger cognate T-regulatory type 1 (Tr1)-cell formation and expansion, capable of reversing organ-specific autoimmune responses. These pMHCII-NPs that display epitopes from mitochondrial protein can blunt the progression of both autoimmune hepatitis (AIH) and experimental autoimmune encephalomyelitis (EAE) in mice carrying either disease. However, with co-morbid mice having both diseases, these pMHCII-NPs selectively treat AIH. In contrast, pMHCII-NPs displaying central nervous system (CNS)-specific epitopes can efficiently treat CNS autoimmunity, both in the absence and presence of AIH, without having any effects on the progression of the latter. Here, we develop a compartmentalized population model of T-cells in co-morbid mice to identify the mechanisms by which Tr1 cells mediate organ-specific immunoregulation. We perform time-series simulations and bifurcation analyses to study how varying physiological parameters, including local cognate antigenic load and rates of Tr1-cell recruitment and retention, affect T-cell allocation and Tr1-mediated immunoregulation. Various regimes of behaviour, including 'competitive autoimmunity' where pMHCII-NP-treatment fails against both diseases, are identified and compared with experimental observations. Our results reveal that a transient delay in Tr1-cell recruitment to the CNS, resulting from inflammation-dependent Tr1-cell allocation, accounts for the liver-centric effects of AIH-specific pMHCII-NPs in co-morbid mice as compared with mice exclusively having EAE. They also suggest that cognate autoantigen expression and local Tr1-cell retention are key determinants of effective regulatory-cell function. These results thus provide new insights into the rules that govern Tr1-cell recruitment and their autoregulatory function.

Influenza infection triggers disease in a genetic model of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system. Most MS patients experience periods of symptom exacerbation (relapses) followed by periods of partial recovery (remission). Interestingly, upper-respiratory viral infections increase the risk for relapse. Here, we used an autoimmune-prone T-cell receptor transgenic mouse (2D2) and a mouse-adapted human influenza virus to test the hypothesis that upper-respiratory viral infection can cause glial activation, promote immune cell trafficking to the CNS, and trigger disease. Specifically, we inoculated 2D2 mice with influenza A virus (Puerto Rico/8/34; PR8) and then monitored them for symptoms of inflammatory demyelination. Clinical and histological experimental autoimmune encephalomyelitis was observed in ∼29% of infected 2D2 mice. To further understand how peripheral infection could contribute to disease onset, we inoculated wild-type C57BL/6 mice and measured transcriptomic alterations occurring in the cerebellum and spinal cord and monitored immune cell surveillance of the CNS by flow cytometry. Infection caused temporal alterations in the transcriptome of both the cerebellum and spinal cord that was consistent with glial activation and increased T-cell, monocyte, and neutrophil trafficking to the brain at day 8 post infection. Finally, Cxcl5 expression was up-regulated in the brains of influenza-infected mice and was elevated in cerebrospinal fluid of MS patients during relapse compared with specimens acquired during remission. Collectively, these data identify a mechanism by which peripheral infection may exacerbate MS as well as other neurological diseases.

Acid sensing ion channel 2: A new potential player in the pathophysiology of multiple sclerosis.

Acid-sensing ion channels (ASICs) are proton-gated channels involved in multiple biological functions such as: pain modulation, mechanosensation, neurotransmission, and neurodegeneration. Earlier, we described the genetic association, within the Nuoro population, between Multiple Sclerosis (MS) and rs28936, located in ASIC2 3'UTR. Here we investigated the potential involvement of ASIC2 in MS inflammatory process. We induced experimental autoimmune encephalomyelitis (EAE) in wild-type (WT), knockout Asic1-/- and Asic2-/- mice and observed a significant reduction of clinical score in Asic1-/- mice and a significant reduction in the clinical score in Asic2-/- mice in a limited time window (i.e., at days 20-23 after immunization). Immunohistochemistry confirmed the reduction in adaptive immune cell infiltrates in the spinal cord of EAE Asic1-/- mice. Analysis of mechanical allodynia, showed a significant higher pain threshold in Asic2-/- mice under physiological conditions, before immunization, as compared to WT mice and Asic1-/- . A significant reduction in pain threshold was observed in all three strains of mice after immunization. More importantly, analysis of human autoptic brain tissue in MS and control samples showed an increase of ASIC2 mRNA in MS samples. Subsequently, in vitro luciferase reporter gene assays, showed that ASIC2 expression is under possible miRNA regulation, in a rs28936 allele-specific manner. Taken together, these findings suggest a potential role of ASIC2 in the pathophysiology of MS.

Early alpha-lipoic acid therapy protects from degeneration of the inner retinal layers and vision loss in an experimental autoimmune encephalomyelitis-optic neuritis model.

BACKGROUND: In multiple sclerosis (MS), neurodegeneration is the main reason for chronic disability. Alpha-lipoic acid (LA) is a naturally occurring antioxidant which has recently been demonstrated to reduce the rate of brain atrophy in progressive MS. However, it remains uncertain if it is also beneficial in the early, more inflammatory-driven phases. As clinical studies are costly and time consuming, optic neuritis (ON) is often used for investigating neuroprotective or regenerative therapeutics. We aimed to investigate the prospect for success of a clinical ON trial using an experimental autoimmune encephalomyelitis-optic neuritis (EAE-ON) model with visual system readouts adaptable to a clinical ON trial. METHODS: Using an in vitro cell culture model for endogenous oxidative stress, we compared the neuroprotective capacity of racemic LA with the R/S-enantiomers and its reduced form. In vivo, we analyzed retinal neurodegeneration using optical coherence tomography (OCT) and the visual function by optokinetic response (OKR) in MOG35-55-induced EAE-ON in C57BL/6J mice. Ganglion cell counts, inflammation, and demyelination were assessed by immunohistological staining of retinae and optic nerves. RESULTS: All forms of LA provided equal neuroprotective capacities in vitro. In EAE-ON, prophylactic LA therapy attenuated the clinical EAE score and prevented the thinning of the inner retinal layer while therapeutic treatment was not protective on visual outcomes. CONCLUSIONS: A prophylactic LA treatment is necessary to protect from visual loss and retinal thinning in EAE-ON, suggesting that a clinical ON trial starting therapy after the onset of symptoms may not be successful.

Anti-CD52 antibody treatment depletes B cell aggregates in the central nervous system in a mouse model of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) for which several new treatment options were recently introduced. Among them is the monoclonal anti-CD52 antibody alemtuzumab that depletes mainly B cells and T cells in the immune periphery. Considering the ongoing controversy about the involvement of B cells and in particular the formation of B cell aggregates in the brains of progressive MS patients, an in-depth understanding of the effects of anti-CD52 antibody treatment on the B cell compartment in the CNS itself is desirable. METHODS: We used myelin basic protein (MBP)-proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 (B6) mice as B cell-dependent model of MS. Mice were treated intraperitoneally either at the peak of EAE or at 60 days after onset with 200 µg murine anti-CD52 vs. IgG2a isotype control antibody for five consecutive days. Disease was subsequently monitored for 10 days. The antigen-specific B cell/antibody response was measured by ELISPOT and ELISA. Effects on CNS infiltration and B cell aggregation were determined by immunohistochemistry. Neurodegeneration was evaluated by Luxol Fast Blue, SMI-32, and Olig2/APC staining as well as by electron microscopy and phosphorylated heavy neurofilament serum ELISA. RESULTS: Treatment with anti-CD52 antibody attenuated EAE only when administered at the peak of disease. While there was no effect on the production of MP4-specific IgG, the treatment almost completely depleted CNS infiltrates and B cell aggregates even when given as late as 60 days after onset. On the ultrastructural level, we observed significantly less axonal damage in the spinal cord and cerebellum in chronic EAE after anti-CD52 treatment. CONCLUSION: Anti-CD52 treatment abrogated B cell infiltration and disrupted existing B cell aggregates in the CNS.

Spasticity Treatment Ameliorates the Efficacy of Melatonin Therapy in Experimental Autoimmune Encephalomyelitis (EAE) Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease in the central nervous system (CNS). Melatonin is an effective treatment in MS patients and experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Melatonin secretion peaks at 2 AM, concomitant with the time at which the muscles are resting and the body is exerting its antioxidant activity. The current study was designed to investigate combination treatment of baclofen, a muscle relaxant drug, and melatonin in EAE mice. Results showed that melatonin (Mel) alone or in combination with baclofen (Bac + Mel) reduced clinical scores and demyelination by significantly increasing myelin oligodendrocyte glycoprotein (MOG) levels, a marker for mature oligodendrocytes, compared to EAE mice. Moreover, Mel or Bac + Mel therapy caused a significant increase in IL-4 serum levels, an anti-inflammatory cytokine, whereas IFN-γ serum levels, a pro-inflammatory cytokine, were significantly reduced. On the other hand, Mel or Bac + Mel caused a significant reduction in malondialdehyde (MDA) levels, a marker of oxidative stress, in comparison to EAE mice. In contrast, the activity of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) was significantly increased in Mel and Bac + Mel groups. In summary, combination therapy improved clinical scores and tend to enhance the efficiency of melatonin treatment by further promoting remyelination, decreasing inflammation, and stimulating the activity of antioxidant enzymes, which suggests that prior spasticity treatment increases the efficacy of melatonin therapy in EAE mouse model of MS. Further experimental and clinical studies are needed to ensure the beneficial role of this combination strategy.

Combination of cuprizone and experimental autoimmune encephalomyelitis to study inflammatory brain lesion formation and progression.

Brain-intrinsic degenerative cascades are a proposed factor driving inflammatory lesion formation in multiple sclerosis (MS) patients. We recently described a model combining noninflammatory cytodegeneration (via cuprizone) with the classic active experimental autoimmune encephalomyelitis (Cup/EAE model), which exhibits inflammatory forebrain lesions. Here, we describe the histopathological characteristics and progression of these Cup/EAE lesions. We show that inflammatory lesions develop at various topographical sites in the forebrain, including white matter tracts and cortical and subcortical grey matter areas. The lesions are characterized by focal demyelination, discontinuation of the perivascular glia limitans, focal axonal damage, and neutrophil granulocyte extravasation. Transgenic mice with enhanced green fluorescent protein-expressing microglia and red fluorescent protein-expressing monocytes reveal that both myeloid cell populations contribute to forebrain inflammatory infiltrates. EAE-triggered inflammatory cerebellar lesions were augmented in mice pre-intoxicated with cuprizone. Gene expression studies suggest roles of the chemokines Cxcl10, Ccl2, and Ccl3 in inflammatory lesion formation. Finally, follow-up experiments in Cup/EAE mice with chronic disease revealed that forebrain, but not spinal cord, lesions undergo spontaneous reorganization and repair. This study underpins the significance of brain-intrinsic degenerative cascades for immune cell recruitment and, in consequence, MS lesion formation.

A role for cathepsin Z in neuroinflammation provides mechanistic support for an epigenetic risk factor in multiple sclerosis.

BACKGROUND: Hypomethylation of the cathepsin Z locus has been proposed as an epigenetic risk factor for multiple sclerosis (MS). Cathepsin Z is a unique lysosomal cysteine cathepsin expressed primarily by antigen presenting cells. While cathepsin Z expression has been associated with neuroinflammatory disorders, a role for cathepsin Z in mediating neuroinflammation has not been previously established. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced in both wildtype mice and mice deficient in cathepsin Z. The effects of cathepsin Z-deficiency on the processing and presentation of the autoantigen myelin oligodendrocyte glycoprotein, and on the production of IL-1ß and IL-18 were determined in vitro from cells derived from wildtype and cathepsin Z-deficient mice. The effects of cathepsin Z-deficiency on CD4+ T cell activation, migration, and infiltration to the CNS were determined in vivo. Statistical analyses of parametric data were performed by one-way ANOVA followed by Tukey post-hoc tests, or by an unpaired Student's t test. EAE clinical scoring was analyzed using the Mann-Whitney U test. RESULTS: We showed that mice deficient in cathepsin Z have reduced neuroinflammation and dramatically lowered circulating levels of IL-1ß during EAE. Deficiency in cathepsin Z did not impact either the processing or the presentation of MOG, or MOG- specific CD4+ T cell activation and trafficking. Consistently, we found that cathepsin Z-deficiency reduced the efficiency of antigen presenting cells to secrete IL-1ß, which in turn reduced the ability of mice to generate Th17 responses-critical steps in the pathogenesis of EAE and MS. CONCLUSION: Together, these data support a novel role for cathepsin Z in the propagation of IL-1ß-driven neuroinflammation.

Influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type I T cells infiltration into central nervous system.

Multiple sclerosis starts with increased migration of auto-reactive lymphocytes across the blood-brain barrier, resulting in persistent neurodegeneration. Clinical and epidemiological studies indicated upper respiratory viral infections are associated with clinical exacerbation of multiple sclerosis. However, so far there is no any direct evidence to support it. Using the experimental autoimmune encephalomyelitis mice as the model for multiple sclerosis, we demonstrated that mice experienced with influenza virus infection were unable to recover from experimental autoimmune encephalomyelitis with a long-term exacerbation. The exacerbated disease was due to more type I T cells, such as CD45highCD4+CD44high, CD45highCD4+CCR5+, CD45high IFNγ+CD4+, MOG35-55-specific IFNγ+CD4+ and influenza virus-specific IFNγ+CD4+ T cells, infiltrating central nervous system in mice with prior influenza virus infection. Influenza virus infection created a notable inflammatory environment in lung and mediastinal lymph node after influenza virus inoculation, suggesting the lung may constitute an inflammatory niche in which auto-aggressive T cells gain the capacity to enter CNS. Indeed, the early stage of EAE disease was accompanied by increased CCR5+CD4+, CXCR3+CD4+ T cell and MOG35-55 specific CD4+ T cells localized in the lung in influenza virus-infected mice. CCL5/CCR5 might mediate the infiltration of type I T cells into CNS during the disease development after influenza infection. Administration of CCR5 antagonist could significantly attenuate the exacerbated disease. Our study provided the evidence that the prior influenza virus infection may promote the type I T cells infiltration into the CNS, and subsequently cause a long-term exacerbation of experimental autoimmune encephalomyelitis.

Genetic prion disease: no role for the immune system in disease pathogenesis?

Prion diseases, which can manifest by transmissible, sporadic or genetic etiologies, share several common features, such as a fatal neurodegenerative outcome and the aberrant accumulation of proteinase K (PK)-resistant PrP forms in the CNS. In infectious prion diseases, such as scrapie in mice, prions first replicate in immune organs, then invade the CNS via ascending peripheral tracts, finally causing death. Accelerated neuroinvasion and death occurs when activated prion-infected immune cells infiltrate into the CNS, as is the case for scrapie-infected mice induced for experimental autoimmune encephalomyelitis (EAE), a CNS inflammatory insult. To establish whether the immune system plays such a central role also in genetic prion diseases, we induced EAE in TgMHu2ME199K mice, a line mimicking for late onset genetic Creutzfeldt Jacob disease (gCJD), a human prion disease. We show here that EAE induction of TgMHu2ME199K mice neither accelerated nor aggravated prion disease manifestation. Concomitantly, we present evidence that PK-resistant PrP forms were absent from CNS immune infiltrates, and most surprisingly also from lymph nodes and spleens of TgMHu2ME199K mice at all ages and stages of disease. These results imply that the mechanism of genetic prion disease differs widely from that of the infectious presentation, and that the conversion of mutant PrPs into PK resistant forms occurs mostly/only in the CNS. If the absence of pathogenic PrP forms form immune organs is also true for gCJD patients, it may suggest their blood is devoid of prion infectivity.

VPAC1 receptor (Vipr1)-deficient mice exhibit ameliorated experimental autoimmune encephalomyelitis, with specific deficits in the effector stage.

BACKGROUND: Vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating polypeptide (PACAP) are two highly homologous neuropeptides. In vitro and ex vivo experiments repeatedly demonstrate that these peptides exert pronounced immunomodulatory (primarily anti-inflammatory) actions which are mediated by common VPAC1 and VPAC2 G protein-coupled receptors. In agreement, we have shown that mice deficient in PACAP ligand or VPAC2 receptors exhibit exacerbated experimental autoimmune encephalomyelitis (EAE). However, we observed that VIP-deficient mice are unexpectedly resistant to EAE, suggesting a requirement for this peptide at some stage of disease development. Here, we investigated the involvement of VPAC1 in the development of EAE using a VPAC1-deficient mouse model. METHODS: EAE was induced in wild-type (WT) and VPAC1 knockout (KO) mice using myelin oligodendrocyte glycoprotein 35-55 (MOG35-55), and clinical scores were assessed continuously over 30 days. Immune responses in the spinal cords were determined by histology, real-time PCR and immunofluorescence, and in the draining lymph nodes by antigen-recall assays. The contribution of VPAC1 expression in the immune system to the development of EAE was evaluated by means of adoptive transfer and bone marrow chimera experiments. In other experiments, VPAC1 receptor analogs were given to WT mice. RESULTS: MOG35-55-induced EAE was ameliorated in VPAC1 KO mice compared to WT mice. The EAE-resistant phenotype of VPAC1 KO mice correlated with reduced central nervous system (CNS) histopathology and cytokine expression in the spinal cord. The immunization phase of EAE appeared to be unimpaired because lymph node cells from EAE-induced VPAC1 KO mice stimulated in vitro with MOG exhibited robust proliferative and Th1/Th17 responses. Moreover, lymph node and spleen cells from KO mice were fully capable of inducing EAE upon transfer to WT recipients. In contrast, WT cells from MOG-immunized mice did not transfer the disease when administered to VPAC1 KO recipients, implicating a defect in the effector phase of the disease. Bone marrow chimera studies suggested that the resistance of VPAC1-deficient mice was only minimally dependent on the expression of this receptor in the immunogenic/hematopoietic compartment. Consistent with this, impaired spinal cord inductions of several chemokine mRNAs were observed in VPAC1 KO mice. Finally, treatment of WT mice with the VPAC1 receptor antagonist PG97-269 before, but not after, EAE induction mimicked the clinical phenotype of VPAC1 KO mice. CONCLUSIONS: VPAC1 gene loss impairs the development of EAE in part by preventing an upregulation of CNS chemokines and invasion of inflammatory cells into the CNS. Use of VPAC1 antagonists in WT mice prior to EAE induction also support a critical role for VPAC1 signaling for the development of EAE.

Oxidative damage and chemokine production dominate days before immune cell infiltration and EAE disease debut.

BACKGROUND: Multiple sclerosis is widely accepted as an inflammatory disease. However, studies indicate that degenerative processes in the CNS occur prior to inflammation. In the widely used animal model experimental autoimmune encephalomyelitis (EAE), we investigated the significance of degenerative processes from mitochondrial membrane potentials, reactive oxidative species, cell death markers, chemokines, and inflammatory cell types in brain, spinal cord, and optic nerve tissue during the effector phase of the disease, before clinical disease was evident. METHODS: Sixty-two rats were placed in eight groups, n = 6 to 10. Four groups were immunized with spinal cord homogenate emulsified in complete Freund's adjuvant (one served as EAE group), three groups were immunized with complete Freund's adjuvant only, and a control group was injected with phosphate buffered saline only. Groups were sacrificed 3, 5, 7, or 12-13 days after the intervention and analyzed for early signs of CNS degeneration. RESULTS: Loss of mitochondrial membrane potential and oxidative changes was observed days before clinical disease debut at day 9.75 ± 0.89. The early mitochondrial changes were not associated with cytochrome C release, cleavage of caspases 9 (38/40 kDa) and 3 (17/19 kDa), and cleavage of PARP (89 kDa) or spectrin (120/150 kDa), and apoptosis was not initiated. Axonal degeneration was only present at disease onset. Increases in a range of cytokines and chemokines were observed systemically as a consequence of immunization with complete Freund's adjuvant, whereas the encephalitogenic emulsion induced an upregulation of the chemokines Ccl2, Ccl20, and Cxcl1, specifically in brain tissue, 7 days after immunization. CONCLUSION: Five to seven days after immunization, subtle decreases in the mitochondrial membrane potential and an increased reactive oxygen species burden in brain tissue were observed. No cell death was detected at these time-points, but a specific expression pattern of chemokines indicates activity in the CNS, several days before clinical disease debut.

Nogo receptor complex expression dynamics in the inflammatory foci of central nervous system experimental autoimmune demyelination.

BACKGROUND: Nogo-A and its putative receptor NgR are considered to be among the inhibitors of axonal regeneration in the CNS. However, few studies so far have addressed the issue of local NgR complex multilateral localization within inflammation in an MS mouse model of autoimmune demyelination. METHODS: Chronic experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. Analyses were performed on acute (days 18-22) and chronic (day 50) time points and compared to controls. The temporal and spatial expression of the Nogo receptor complex (NgR and coreceptors) was studied at the spinal cord using epifluorescent and confocal microscopy or real-time PCR. Data are expressed as cells/mm2, as mean % ± SEM, or as arbitrary units of integrated density. RESULTS: Animals developed a moderate to severe EAE without mortality, followed by a progressive, chronic clinical course. NgR complex spatial expression varied during the main time points of EAE. NgR with coreceptors LINGO-1 and TROY was increased in the spinal cord in the acute phase whereas LINGO-1 and p75 signal seemed to be dominant in the chronic phase, respectively. NgR was detected on gray matter NeuN+ neurons of the spinal cord, within the white matter inflammatory foci (14.2 ± 4.3 % NgR+ inflammatory cells), and found to be colocalized with GAP-43+ axonal growth cones while no ß-TubIII+, SMI-32+, or APP+ axons were found as NgR+. Among the NgR+ inflammatory cells, 75.6 ± 9.0 % were microglial/macrophages (lectin+), 49.6 ± 14.2 % expressed CD68 (phagocytic ED1+ cells), and no cells were Mac-3+. Of these macrophages/monocytes, only Arginase-1+/NgR+ but not iNOS+/NgR+ were present in lesions both in acute and chronic phases. CONCLUSIONS: Our data describe in detail the expression of the Nogo receptor complex within the autoimmune inflammatory foci and suggest a possible immune action for NgR apart from the established inhibitory one on axonal growth. Its expression by inflammatory macrophages/monocytes could signify a possible role of these cells on axonal guidance and clearance of the lesioned area during inflammatory demyelination.

Altered excitatory-inhibitory balance within somatosensory cortex is associated with enhanced plasticity and pain sensitivity in a mouse model of multiple sclerosis.

BACKGROUND: Chronic neuropathic pain is a common symptom of multiple sclerosis (MS). MOG35-55-induced experimental autoimmune encephalomyelitis (EAE) has been used as an animal model to investigate the mechanisms of pain in MS. Previous studies have implicated sensitization of spinal nociceptive networks in the pathogenesis of pain in EAE. However, the involvement of supraspinal sites of nociceptive integration, such as the primary somatosensory cortex (S1), has not been defined. We therefore examined functional, structural, and immunological alterations in S1 during the early stages of EAE, when pain behaviors first appear. We also assessed the effects of the antidepressant phenelzine (PLZ) on S1 alterations and nociceptive (mechanical) sensitivity in early EAE. PLZ has been shown to restore central nervous system (CNS) tissue concentrations of GABA and the monoamines (5-HT, NA) in EAE. We hypothesized that PLZ treatment would also normalize nociceptive sensitivity in EAE by restoring the balance of excitation and inhibition (E-I) in the CNS. METHODS: We used in vivo flavoprotein autofluorescence imaging (FAI) to assess neural ensemble responses in S1 to vibrotactile stimulation of the limbs in early EAE. We also used immunohistochemistry (IHC), and Golgi-Cox staining, to examine synaptic changes and neuroinflammation in S1. Mechanical sensitivity was assessed at the clinical onset of EAE with Von Frey hairs. RESULTS: Mice with early EAE exhibited significantly intensified and expanded FAI responses in S1 compared to controls. IHC revealed increased vesicular glutamate transporter (VGLUT1) expression and disrupted parvalbumin+ (PV+) interneuron connectivity in S1 of EAE mice. Furthermore, peri-neuronal nets (PNNs) were significantly reduced in S1. Morphological analysis of excitatory neurons in S1 revealed increased dendritic spine densities. Iba-1+ cortical microglia were significantly elevated early in the disease. Chronic PLZ treatment was found to normalize mechanical thresholds in EAE. PLZ also normalized S1 FAI responses, neuronal morphologies, and cortical microglia numbers and attenuated VGLUT1 reactivity-but did not significantly attenuate the loss of PNNs. CONCLUSIONS: These findings implicate a pro-excitatory shift in the E-I balance of the somatosensory CNS, arising early in the pathogenesis EAE and leading to large-scale functional and structural plasticity in S1. They also suggest a novel antinociceptive effect of PLZ treatment.

Interaction between interleukin-1ß and type-1 cannabinoid receptor is involved in anxiety-like behavior in experimental autoimmune encephalomyelitis.

BACKGROUND: Mood disorders, including anxiety and depression, are frequently diagnosed in multiple sclerosis (MS) patients, even independently of the disabling symptoms associated with the disease. Anatomical, biochemical, and pharmacological evidence indicates that type-1 cannabinoid receptor (CB1R) is implicated in the control of emotional behavior and is modulated during inflammatory neurodegenerative diseases such as MS and experimental autoimmune encephalomyelitis (EAE). METHODS: We investigated whether CB1R could exert a role in anxiety-like behavior in mice with EAE. We performed behavioral, pharmacological, and electrophysiological experiments to explore the link between central inflammation, mood, and CB1R function in EAE. RESULTS: We observed that EAE-induced anxiety was associated with the downregulation of CB1R-mediated control of striatal GABA synaptic transmission and was exacerbated in mice lacking CB1R (CB1R-KO mice). Central blockade of interleukin-1ß (IL-1ß) reversed the anxiety-like phenotype of EAE mice, an effect associated with the concomitant rescue of dopamine (DA)-regulated spontaneous behavior, and DA-CB1R neurotransmission, leading to the rescue of striatal CB1R sensitivity. CONCLUSIONS: Overall, results of the present investigation indicate that synaptic dysfunction linked to CB1R is involved in EAE-related anxiety and motivation-based behavior and contribute to clarify the complex neurobiological mechanisms underlying mood disorders associated to MS.

Siponimod (BAF312) prevents synaptic neurodegeneration in experimental multiple sclerosis.

BACKGROUND: Data from multiple sclerosis (MS) and the MS rodent model, experimental autoimmune encephalomyelitis (EAE), highlighted an inflammation-dependent synaptopathy at the basis of the neurodegenerative damage causing irreversible disability in these disorders. This synaptopathy is characterized by an imbalance between glutamatergic and GABAergic transmission and has been proposed to be a potential therapeutic target. Siponimod (BAF312), a selective sphingosine 1-phosphate1,5 receptor modulator, is currently under investigation in a clinical trial in secondary progressive MS patients. We investigated whether siponimod, in addition to its peripheral immune modulation, may exert direct neuroprotective effects in the central nervous system (CNS) of mice with chronic progressive EAE. METHODS: Minipumps allowing continuous intracerebroventricular (icv) infusion of siponimod for 4 weeks were implanted into C57BL/6 mice subjected to MOG35-55-induced EAE. Electrophysiology, immunohistochemistry, western blot, qPCR experiments, and peripheral lymphocyte counts were performed. In addition, the effect of siponimod on activated microglia was assessed in vitro to confirm the direct effect of the drug on CNS-resident immune cells. RESULTS: Siponimod administration (0.45 µg/day) induced a significant beneficial effect on EAE clinical scores with minimal effect on peripheral lymphocyte counts. Siponimod rescued defective GABAergic transmission in the striatum of EAE, without correcting the EAE-induced alterations of glutamatergic transmission. We observed a significant attenuation of astrogliosis and microgliosis together with reduced lymphocyte infiltration in the striatum of EAE mice treated with siponimod. Interestingly, siponimod reduced the release of IL-6 and RANTES from activated microglial cells in vitro, which might explain the reduced lymphocyte infiltration. Furthermore, the loss of parvalbumin-positive (PV+) GABAergic interneurons typical of EAE brains was rescued by siponimod treatment, providing a plausible explanation of the selective effects of this drug on inhibitory synaptic transmission. CONCLUSIONS: Altogether, our results show that siponimod has neuroprotective effects in the CNS of EAE mice, which are likely independent of its peripheral immune effect, suggesting that this drug could be effective in limiting neurodegenerative pathological processes in MS.