Experimental Allergic Encephalomyelitis Model Research Articles

The role of vitamin D through SphK1/S1P in the regulation of MS progression

Sphingosine-1-phosphate (S1P) is biologically active lipid, leading to neuroinflammation and macrophage invasion in central nervous system, plays an important role in the development of multiple sclerosis (MS) model in experimental allergic encephalomyelitis (EAE) rats. Vitamin D is observed to be a key factor in regulating cell S1P levels. We detected vitamin D can alleviate the symptoms of EAE rats, but the exact mechanism is unclear. In PC12 cells, vitamin D can reverse S1P-induced cell death, but the signaling pathway unclear. This study was aimed to investigate S1P regulation mechanism or signaling pathway mediated by vitamin D in EAE and PC12 model. In our experiments, S1P and Sphingosine kinase type 1 (SphK1) mRNA and protein expression in EAE rats group, control group, vitamin D feeding group were detected by HPLC, ELISA, RT-PCR and western blot. PC12 cell death was detected by Propidium (PI) staining. VDR plasmid overexpression and RNA interference, immunofluorescence, real-time cell analysis, protein immunoblotting was used to detect SphK1 transcriptional regulation, cell-substrate attachment quality, the signaling pathway of cell apoptosis and inflammation related gene expression (Bax/Bcl-2, Casepase-3, Il-6, TGF-β, TNF-α). Our study showed vitamin D can reverse the elevation of S1P level in EAE rats, reduce the severity and shorten the course of EAE. 1,25-(OH) 2D3 coupled with vitamin D receptor (VDR) inhibited SphK1 transcription. 1,25-(OH)2D3 significantly reduced PC12 cell death rate induced by S1P, in addition improved the cell substrate attachment quality. 1,25-(OH) 2D3 can block S1P-induced p-ERK activation and PI3K /Akt signaling pathway reduced Il-6, TGF-β, TNF-α cytokine release and Bax/Bcl-2, Casepase-3 apoptosis protein expression. On the other hand, immunofluorescence staining showed 1,25-(OH) 2D3 can increase the expression of neuronal perinuclear protein MAP2 in PC12 cells probably protect nerve cells further. In summary, the ameliorative effect of vitamin D was derived from its ability to reduce S1P levels, provides an idea for vitamin D as a combination therapy for disease.

Perivascular PDGFRB+ cells accompany lesion formation and clinical evolution differentially in two different EAE models.

Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that may lead to progressive disability. Here, we explored the behavioral pattern and the role of vasculature especially PDGFRB+ pericytes/ perivascular cells, in MS pathogenesis. We have evaluated vascular changes in two different experimental allergic encephalomyelitis (EAE) mice models (MOG and PLP-induced). PDGFRB+ cells demonstrated distinct and different behavioral patterns. In both models, fibrosis formation was detected via collagen, fibronectin, and extracellular matrix accumulation. The PLP-induced animal model revealed that fibrosis predominantly occurs in perivascular locations and that PDGFRB+ cells are accumulated around vessels. Also, the expression of fibrotic genes and genes coding extracellular matrix (ECM) proteins are upregulated. Moreover, the perivascular thick wall structures in affected vessels of this model presented primarily increased PDGFRB+ cells but not NG2+ cells in the transgenic NG2-DsRed transgenic animal model. On the other hand, in MOG induced model, PDGFRB+ perivascular cells were accumulated at the lesion sites. PDGFRB+ cells colocalized with ECM proteins (collagen, fibronectin, and lysyl oxidase L3). Nevertheless, both MOG and PLP-immunized mice showed increasing EAE severity, and disability parallel with enhanced perivascular cell accumulation as the disease progressed from earlier (day 15) to later (day 40). As a result, we have concluded that PDGFRB+ perivascular cells may be participating in lesion progression and as well as demonstrating different responses in different EAE models.

Вивчення корекції соматичного болю за умов експериментальної патології розсіяного склерозу

The purpose of the study was to experimentally substantiate the ways of pharmacological correction of somatic pain syndrome in conditions of the experimental equivalent of multiple sclerosis through a comparative system analysis and the use of complex methodological approaches. Materials and methods. To study multiple sclerosis, we used an experimental model with autoimmune mechanisms of inflammatory demyelination – a model of experimental allergic encephalomyelitis. To assess the antinociceptive activity of painkillers, we used the method of electrical stimulation of the rats’ tail root. The activity of the enzyme prostaglandin H-synthetase was also determined. Results and discussion. A comparative analysis of the analgesic activity indicators of combinations of methylprednisolone with analgesics under the condition of the formed experimental allergic encephalomyelitis showed that their antinociceptive potential (taking into account the basic therapy with methylprednisolone) decreased in the series meloxicam > lornoxicam ≈ ketorolac ≈ paracetamol > celecofenacoxib ≈ sodium diclofupene ≈ diclofupene ≈ diclofupene. Accordingly, the maximum effect on the threshold of nociception under these experimental conditions was exerted by meloxicam and lornoxicam. The combined administration of methylprednisolone with diclofenac sodium, celecoxib and meloxicam reduced the activity of prostaglandin N-synthetase in the brain structures by 49.8% (p <0.05), 50.4% (p <0.05) and 51% (p <0.05), respectively, compared with the indicators of the control group. The same drugs markedly reduced the activity of prostaglandin N-synthetase in the spinal cord by 23.9% (p <0.05) (Methylprednisolone + diclofenac), by 34% (p <0.05) (Methylprednisolone + celecoxib) and by 47.4% (p <0.05) (Methylprednisolone + meloxicam) compared with the control group. Our analysis of the analgesic activity of antidepressants and anticonvulsants as means of correcting nociceptive pain in experimental allergic encephalomyelitis found that their antinociceptive potential was inferior to the severity of the analgesic effect of nonsteroidal anti-inflammatory drugs. Conclusion. Among the studied non-steroidal anti-inflammatory drugs, antidepressants and anticonvulsants, the maximum therapeutic efficacy as a means of correcting nociceptive pain in experimental allergic encephalomyelitis against the background of basic methylprednisolone therapy was shown by meloxicam, which gives grounds to recommend it as the analgesic of choice for eliminating somatic pain syndromes

Фармакологическая коррекция винкамином системы антиоксидантной защиты в мозге в модели рассеянного склероза

Objective. In the course of the research, the effect of vincamine (nootropic drug) on neurological status, as well as the activity of antioxidant enzymes and the level of their coding genes’ expression in the somatosensory cortex of rats within the model of experimental allergic encephalomyelitis (EAE) were being studied. Relevance: the topicality of studying the mechanisms of multiple sclerosis in the early stages of its development is dictated by the need to search for markers of the disease and its therapy before the onset of its clinical manifestation. Materials and methods. The animals’ neurological status was studied using muscular strength, balance, tenacity and traction tests. Rates of survival and the rats’ body weight were also being evaluated. The study of activity antioxidant enzymes: superoxide dismutase (SOD), glutathione peroxidase (GPO), and glutathione reductase (GR), as well as the expression of SOD1, GPX4, GPX6, and GSR genes was conducted on the 14th and 30th day of immunization. Results. The percentage of vincamine-injected animals’ survival was 100% versus 87% among rats that were not injected with the nootropic drug during immunization (р<0,05) . Besides, after the injection of vincamine, a less significant decrease in body weight (р<0,01) and a less pronounced neurological deficit (р<0,05) in comparison with immunized non-injected animals were reported. The vincamine injection contributed to an increase in all studied antioxidant enzymes’ activity and the level of their genes’ expression in the somatosensory cortex. Conclusion. Against the background of vincamine injection, a minimization of neurological deficit is being observed, probably due to a decrease in oxidative stress in the rat brain during the clinical stage of experimental allergic encephalomyellitis.

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines.

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund's adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund's adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

Morpho-functional parameters of neurons in the sensorimotor cortex and neuroapoptosis under conditions of an induced experimental allergic encephalomyelitis in rats and a course of intranasal gel administration containing n-phenylacetyl-l-prolylglycine (N

Purpose. The aim of the research is to evaluate a neuroprotective effect of the nasal dosage form containing Noopept (n-phenylacetyl-l-prolylglycine ethyl ester) by the degree of influence on morphofunctional parameters of neurons in the sensorimotor cortex of animals with experimental allergic encephalomyelitis.Methods. The experiments were conducted on 260 outbred albino rats weighing 190-220 g which were obtained from the nursery of the State Institution «Institute ofPharmacology and Toxicology of the Academy of Medical Sciences of Ukraine». The quarantine (acclimatization period) for all animals lasted 14 days. An animal model of experimental allergic encephalomyelitis (EAE) which has clinical manifestations and pathogenic mechanisms similar to multiple sclerosis was used for the study. There were five groups of animals in the experiment: 1) the intact group (10 rats); 2) the control group – non-treated subjects with EAE which received saline solution (20 rats); 3) the animals with EAE which received basic treatment – 3,4 mg / kg of Methylprednisolone (MP) intraperitoneally, administered slowly in saline solution with a volume of not more than 1/10 of the blood volume of a rat (20 rats); 4) the animals with EAE which received MP and a nasal gel containing n-phenylacetyl-l-prolylglycine at a dose of 10 mg / kg (20 rats); 5) the animals with EAE which received MP and Citicolinum (Ceraxon, «Ferrer Internacional S.A.», Spain) series D003U1, 500 mg / kg, intragastrically (20 rats). Morphometric studies were carried out with the help of Axioskop microscope (Ziess,Germany), a magnification of x40. The image of neurons in the area of the CA-1 zone of the hippocampus, obtained from the microscope using a highly sensitive video camera COHU-4922 (COCHU Inc.,USA).Results. It has been shown that the combination therapy with Methylprednisolone and an intranasal gel containing n-phenylacetyl-l-prolylglycine intensified neuroprotectivity in the animals with EAE. In particular, a neuronal density in rats with the experimental pathology, which received a combination of Methylprednisolone and a gel containing n-phenylacetyl-l-prolylglycine, increased by 14,7 (р <0,05) and their area equaled in values with animals from the intact group; at the same time, the amount of RNA increased by 9,2%. In addition, in conditions of this experiment n-phenylacetyl-l-prolylglycine decreased density of apoptotic and destructive cells by 32,4% (р <0,05) in relation to the control and the MP groups, and the amount of apoptotic neurons – by 44,6% (р <0,05). Judging by effect on the neuronal density and the density of apoptotic and destructively altered neurons in the sensorimotor cortex of outbred albino rats with EAE, a combination of Methylprednisolone and a gel containing n-phenylacetyl-l-prolylglycine reliably exceeds the combined therapy of Methylprednisolone and Citicolinum and the MP monotherapy. A higher, according to some indicators, neuroprotective, nootropic, anxiolytic activity of a gel containing n-phenylacetyl-l-prolylglycine is due to its intranasal route of administration. Conclusions: For the first time, we have obtained reliable data on the neuroprotective effect of the new dosage form containing n-phenylacetyl-l-prolylglycine (Noopept) – an intranasal gel – under conditions of an induced experimental multiple sclerosis. It has been shown that a course of intranasal administration of a gel containing n-phenylacetyl-l-prolylglycine (Noopept) at a dose of 10 mg / kg in combination with Methylprednisolone leads to a decrease in the death of neurons in the sensorimotor cortex of rats with EAE and an increase in the amount of RNA in them. It has been shown that a course of intranasal administration of a gel containing n-phenylacetyl-l-prolylglycine (Noopept) at a dose of 10 mg / kg in combination with Methylprednisolone leads to neuroapoptosis inhibition – a decrease in the density of apoptotic and destructively altered neurons in the sensorimotor cortex of outbred albino rats with EAE. It has been found that judging by effect on the neuronal density and the density of apoptotic and destructively altered neurons in the sensorimotor cortex of the outbred albino rats with EAE, a combination of Methylprednisolone and a gel containing n-phenylacetyl-l-prolylglycine reliably exceeds the combined therapy of Methylprednisolone and Citicolinum and the MP monotherapy.

Sarm1 knockout protects against early but not late axonal degeneration in experimental allergic encephalomyelitis.

Programmed axonal degeneration, also known as Wallerian degeneration, occurs in immune-mediated central nervous system (CNS) inflammatory disorders such as multiple sclerosis and the animal model experimental allergic encephalomyelitis (EAE). Sterile alpha and TIR domain containing protein 1 (SARM1) functions to promote programmed axonal degeneration. To test the hypothesis that loss of SARM1 will reduce axonal degeneration in immune-mediated CNS inflammatory disorders, the course and pathology of EAE was compared in Sarm1 knockout mice and wild type littermates. The clinical course of EAE was similar in Sarm1 knockout and wild type. Analysis of EAE in mice expressing neuronal yellow fluorescent protein (YFP) showed significantly less axonal degeneration in Sarm1 knockout mice compared to wild type littermates at 14 days post-induction of EAE. At 21 days post-induction, however, difference in axonal degeneration was not significant. At 42 days post-induction, Sarm1 knockout mice were indistinguishable from wild type with respect to markers of axonal injury, and were similar with respect to axonal density in the lumbar cords. There was no significant change in peripheral immune activation or CNS inflammatory cell infiltration associated with EAE in Sarm1 knockout mice. In conclusion, Sarm1 deletion delayed axonal degeneration early in the course of CNS inflammation, but did not confer long-term protection from axonal degeneration in an animal model of immune-mediated CNS inflammation.

Intravenous C16 and angiopoietin-1 improve the efficacy of placenta-derived mesenchymal stem cell therapy for EAE

The placenta has emerged as an attractive source of mesenchymal stem cells (MSCs) because of the absence of ethical issues, non-invasive access, and abundant yield. However, inflammatory cell invasion into grafts negatively impacts the survival and efficacy of transplanted cells. Previous studies have shown that synthetic C16 peptide can competitively block the transmigration of leukocytes into the central nerve system, while angiopoietin-1 (Ang-1) can inhibit inflammation-induced blood vessel leakage and inflammatory cell infiltration in rats with experimental allergic encephalomyelitis (EAE). In this study, we investigated the effects of intravenous administration of C16 and Ang-1 on the efficacy of placenta-derived MSC (PMSC) transplantation in a rat model of EAE. We found that, compared with PMSCs alone, treatment with PMSCs along with intravenously administered C16 and Ang-1 was more effective at ameliorating demyelination/neuronal loss and neurological dysfunction, reducing inflammatory cell infiltration, perivascular edema, and reactive astrogliosis (p < 0.05). Mechanistic studies revealed that intravenous C16 and Ang-1 increased PMSC engraftment in the central nervous system and promoted expression of the neurotropic proteins brain-derived neurotrophic factor, growth-associated protein 43, and p75 neurotrophin receptor as well as the neuronal-glial lineage markers neurofilament protein 200 and myelin basic protein in the engrafted PMSCs.

Evaluate Toxocara Canis Excretory-Secretory Antigens in Experimental Allergic Encephalomyelitis (EAE)

BACKGROUND: Toxocara canis is the most prevalent intestinal roundworm of canid species. OBJECTIVES: This study aims to evaluate the effects of Toxocara canis excretory-secretory antigens (TcES Ag) on modulating the immune system in Experimental Allergic Encephalomyelitis (EAE) model. METHODS: Adult worms of T.canis were collected from dogs to obtain excretory-secretory antigens. Female C57BL/6mice divided to four groups (5 mice in each) including: group 1 (MOG +TcES Ag), 2 (MOG), 3 (normal) and 4 (TcES Ag). EAE was induced in groups 1 and 2 using myelin oligodendrocyte glycoprotein peptide. Before EAE induction, TcES Ag was injected in group 1. Twenty nine after EAE induction, mice spleens were removed. Mononuclear cells were cultured and used for RNA extraction.Real Time PCR was performed to evaluate RNA expression levels of T-bet (Th1 lineage-specific transcription factor), GATA-3 (Th2 transcription factor), and FOXP3 (Treg transcription factor). RESULTS: Our results indicated the clinical signs (disability score) of mice in group 1 significantly were decreased as compared to the control group. Gene expression of T-bet in the TcES Ag treatment groupmarkedly were diminished compared to MOG and normal group. The expression of GATA-3 gene in group 1 was lower than that in group 2. CONCLUSIONS: It seems that the TcES Ag may reduce disability score in multiple sclerosis EAE model, and other recombinant antigens should be examined.

Establish and optimize the model of experimental allergic encephalomyelitis in mice

Objective To explore the appropriate dosage of drugs inducing experimental allergic encephalomyelitis (EAE) in mice, and evaluate the modified model mice. Methods Different doses of myelin oligodendrocyte glycoprotein (MOG35-55: 200 μg, 100 μg, 50 μg, 25 μg), together with different doses of inactivation of mycobacterium tuberculosis (H37RA: 800 μg, 250 μg, 100 μg) and pertussis toxin(500 ng, 200 ng), were used to induce the EAE model. After immunized, the clinical disease severity of EAE mice was measured by the standard EAE grading clinical score daily. The open field test was used to detect the locomotion of mice. The Western blot and immunofluorescence were used to detect the level of myelin basic proteins (MBP) in different brain regions of mice. Results Compared with the EAE mice induced with high-dose drugs, the mice with low-dose drugs (25 μg MOG35-55, 100 μg H37RA, 200 ng pertussis toxin) had low neurological scores. And they displayed normal locomotion compared with the control mice (day 16: group EAE(8.885±0.772)cm/s vs control group (8.933±0.567)cm/s, P>0.05; day 31: group EAE (11.130±0.630)cm/s vs control group (10.670±0.959)cm/s, P>0.05; day 55: group EAE (7.686±0.428)cm/s vs control group (8.313±0.918)cm/s, P>0.05). Moreover, there was a significant decrease of MBP in the parahippocampal cortex (PHC) and fimbria-fornix of EAE mice induced with low-dose of drugs (PHC: group EAE (0.369±0.096) vs control group (1.000±0.163), P<0.05; fimbria-fornix: group EAE (0.494±0.071) vs control group (1.000±0.143), P<0.05). Conclusion The EAE mice induced with low-dose drugs(25 μg MOG35-55, 100 μg H37RA, 200 ng pertussis toxin) have low neurological scores, normal locomotion, and myelin impairment in the central neuronal system. And it can be used in the cognitive behavioral research of demyelination disease, such as multiple sclerosis. Key words: Experimental allergic encephalomyelitis (EAE); Neurological score; Locomotion; Myelin impairment

Capparis ovata treatment suppresses inflammatory cytokine expression and ameliorates experimental allergic encephalomyelitis model of multiple sclerosis in C57BL/6 mice

Since ancient times, Capparis species have been widely used in traditional medicine to treat various diseases. Our recent investigations have suggested Capparis ovata's potential anti-neuroinflammatory application for the treatment of multiple sclerosis (MS). The present study was designed to precisely determine the underlying mechanism of its anti-neuroinflammatory effect in a mouse model of MS. C. ovata water extract (COWE) was prepared using the plant's fruit, buds, and flower parts (Turkish Patent Institute, PT 2012/04,093). We immunized female C57BL/6J mice with MOG35–55/CFA. COWE was administered at a daily dose of 500mg/kg by oral gavage either from the day of immunization (T1) or at disease onset (T2) for 21days. Gene expression analysis was performed using a Mouse Multiple Sclerosis RT² Profiler PCR Array, and further determinations and validations of the identified genes were performed using qPCR. Whole-genome transcriptome profiling was analyzed using Agilent SurePrint G3 Mouse GE 8X60K microarrays. Immunohistochemical staining was applied to brain sections of the control and treated mice to examine the degree of degeneration. COWE was further fractionated and analyzed phytochemically using the Zivak Tandem Gold Triple Quadrupole LC/MS–MS system. COWE remarkably suppressed the development of EAE in T1, and the disease activity was completely inhibited. In the T2 group, the maximal score was significantly reduced compared with that of the parallel EAE group. The COWE suppression of EAE was associated with a significantly decreased expression of genes that are important in inflammatory signaling, such as TNFα, IL6, NF-κB, CCL5, CXCL9, and CXCK10. On the other hand, the expression of genes involved in myelination/remyelination was significantly increased. Immunohistochemical analysis further supported these effects, showing that the number of infiltrating immune cells was decreased in the brains of COWE-treated animals. In addition, differential expression profiling of the transcriptome revealed that COWE treatment caused the down regulation of a group of genes involved in the immune response, inflammatory response, antigen processing and presentation, B-cell-mediated immunity and innate immune response. Collectively, these results suggest anti-neuroinflammatory mechanisms by which COWE treatment delayed and suppressed the development of EAE and ameliorated the disease in mice with persistent clinical signs.

Peripheral and central neuronal ATF3 precedes CD4 + T-cell infiltration in EAE

Experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis produced by immunization with myelin oligodendrocyte glycoprotein (MOG) and adjuvants, results from profound T-cell mediated CNS demyelination. EAE is characterized by progressive, ascending motor dysfunction and symptoms of ongoing pain and hypersensitivity, in some cases preceding or concomitant with the motor deficits. In this regard, the EAE model mimics major features of multiple sclerosis, where a central neuropathic pain state is common. Although the latter condition is presumed to arise from a CNS loss of inhibitory controls secondary to the demyelination, dysfunction of sensory neurons may also contribute. Based on our previous studies that demonstrated the utility of monitoring expression of activating transcription factor 3 (ATF3), a sensitive marker of injured sensory neurons, here we followed both ATF3 and CD4+ T cells invasion of sensory ganglia (as well as the CNS) at different stages of the EAE model. We found that ATF3 is induced in peripheral sensory ganglia and brainstem well before the appearance of motor deficits. Unexpectedly, the ATF3 induction always preceded T cell infiltration, typically in adjacent, but non-overlapping regions. Surprisingly, control administration of the pertussis toxin and/or Complete Freund's adjuvants, without MOG, induced ATF3 in sensory neurons. In contrast, T cell infiltration only occurred with MOG. Taken together, our results suggest that the clinical manifestations in the EAE result not only from central demyelination but also from neuronal stress and subsequent pathophysiology of sensory neurons.

Analysis of the direct injury effector of oligodendroglia cells or myelin sheath in an experimental allergic encephalomyelitis model induced by the MOG35-55 peptide

The aim of the present study was to investigate the possible role of cytotoxic T lymphocytes (CTL) and mononuclear macrophages in the pathogenic processes of experimental animals. To construct a chronic experimental allergic encephalomyelitis (EAE) model, an artificially synthesized myelin oligodendrocyte glycoprotein (MOG)35‑55 peptide was used to induce C57BL/6 mice. Subsequently, the experimental animals were investigated at the level of their nervous function, and histopathological, immunohistochemical and fluorescence immunohistochemical experiments were performed at different time points following immunization. The expression of immune molecules and cytokines associated with the activation of the mononuclear macrophages and CTL during the different stages was assessed by western blotting and reverse transcription‑quantitative polymerase chain reaction. As a result, the MOG35‑55 peptide was identified as being successful at inducing C57BL/6 mice for the development of the EAE model. A modest level of mononuclear macrophage and lymphocyte infiltration was observed in the central nervous system (CNS), although no infiltration of neutrophils was observed. A sporadic flaky deletion of the myelin sheath was also identified. The activation and proliferation of mononuclear macrophages, including microglia cells, was clearly demonstrated. Furthermore, the expression levels of major histocompatibility complex class I and II molecules and interleukin‑12 in the brain, which is associated with the activation and proliferation of mononuclear macrophages, increased over the duration of the experiment compared with less pronounced changes in the expression levels of interferon (IFN)‑γ, Fas and perforin in the CNS, which are associated with the function of CTL. The secretion of IFN‑γ in the spleen increased during the morbidity peak, however, any noticeable activation and proliferation of CD8+ T cells was absent. These results demonstrated that the induced immune response mediated by mononuclear macrophages made a more important contribution compared with CTL towards the pathological process of myelin sheath injury. Mononuclear macrophages are therefore, identified as being one of the most significant effector cell types to directly injure the myelin sheath in the CNS.

Effects of human Wharton’s jelly-derived mesenchymal stem cells and Interleukin-10 on behavioural responses of rats with experimental allergic encephalomyelitis

On the rat model of experimental allergic encephalomyelitis (EAE), as analogue of multiple sclerosis of human, we studied the effect of Wharton’s jelly-derived mesenchymal stem cells and interleukin-10 on the functional parameters of the CNS.Materials and methods. EAE was induced with spinal cord homogenate of rats with complete Freund’s adjuvant. MSCs were isolated by the explants technique from Wharton’s jelly of the human umbilical cord and cultured up to two passages. Recombinant IL-10 was administered intravenously on the day 10 after induction of EAE and subocipitally on the day 17 at a dose of 0.2 mg per animal. Cell transplantation performed subocipitally on the day 17 at a dose of 106 cells per animal. Behavioral reactions studied in the “open field” test three times: on the day 12, 15 and 24 after the induction of EAE.Results. Induced EAE results in significant changes in adaptive behavior of rats on the 12th day as suppression of orienting-exploratory and activation of emotional activity. The application of MSCs and IL-10 has normalized the rates of cognitive activities (orienting-exploratory activity) and emotional sphere (the level of fear, anxiety).Conclusion. Combined treatment of induced EAE in rats with interleukin-10 and human Wharton’s jelly-derived mesenchymal stem cells is effective at correction of behavioral responses of animals.

Hypomethylating Agent 5‐Aza‐2′‐deoxycytidine (DAC) Ameliorates Multiple Sclerosis in Mouse Models

Increasing evidence supports the role of epigenetics in the development of autoimmune disorders and the possibility of using epigenetic modifying drugs in the context of MS has not yet been investigated. We have explored the effect of the hypomethylating agent 5-aza-2'-deoxycytidine (DAC) in two murine models of experimental allergic encephalomyelitis (EAE). DAC treatment was associated with a significant amelioration of the clinical and histological hallmarks of EAE in both models. These effects were observed both in prophylactic and therapeutic regimens. The milder course of the disease was associated with a reduction in the number of spinal cord infiltrating lymphocytes and amelioration of the histopathological signs associated with EAE. In addition, increased transcript levels of anti-inflammatory cytokines and decreased mRNA expression of pro-inflammatory mediators were also observed. Finally, DAC treatment increased the percentage of circulating regulatory T cells by inducing Foxp3 expression via demethylation of a CpG island in Foxp3.

All‐trans‐retinoic acid ameliorates experimental allergic encephalomyelitis by affecting dendritic cell and monocyte development

Experimental allergic encephalomyelitis (EAE) can be induced in animal models by injecting the MOG35-55 peptide subcutaneously. Dendritic cells (DCs) that are located at the immunization site phagocytose the MOG35-55 peptide. These DCs mature and migrate into the nearest draining lymph nodes (dLNs), then present antigen, resulting in the activation of naive T cells. T helper type 1 (Th1) and Th17 cells are the primary cells involved in EAE progression. All-trans-retinoic acid (AT-RA) has been shown to have beneficial effects on EAE progression; however, whether AT-RA influences DC maturation or mediates other functions is unclear. In the present study, we showed that AT-RA led to the down-regulation of MHC class II, CD80 (B7-1) and CD86 (B7-2) expressed on the surface of DCs that were isolated from dLNs or spleen 3 days post-immunization in an EAE model. Changes to DC function influenced Th1/Th17 subset polarization. Furthermore, the number of CD44(+) monocytes (which might trigger EAE progression) was also significantly decreased in dLNs, spleen, subarachnoid space and the spinal cord parenchyma after AT-RA treatment. These findings are the first to demonstrate that AT-RA impairs the antigen-presenting capacity of DCs, leading to down-regulation of pathogenic Th1 and Th17 inflammatory cell responses and reducing EAE severity.

Dose-Dependent Anti-Inflammatory and Neuroprotective Effects of anανβ3 Integrin-Binding Peptide

Previous studies have shown that prevention of leukocyte infiltration by targeting integrins involved in transendothelial migration may suppress the clinical and pathological features of neuroinflammatory disease. This study was designed to investigate the effects of C16, an α ν β3 integrin-binding peptide, in an acute experimental allergic encephalomyelitis (EAE) rat model. Multiple histological and immunohistochemical staining, electron microscopy observation, ELISA assay, Western blot, and magnetic resonance imaging (MRI) were employed to assess the degree of inflammation, axonal loss, neuronal apoptosis, white matter demyelination, and extent of gliosis in the brain and spinal cord of differently treated EAE models. The results showed that C16 treatment could inhibit extensive leukocyte and macrophage accumulation and infiltration and reduce cytokine tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) expression levels. A significantly lower clinical score at the peak time of disease was also demonstrated in the C16 treated group. Moreover, astrogliosis, demyelination, neuronal death, and axonal loss were all alleviated in C16 treated EAE animals, which may be attributed to the improvement of microenvironment. The data suggests that C16 peptide may act as a protective agent by attenuating inflammatory progression and thus affecting the expression of some proinflammatory cytokines during neuroinflammatory disease.

Antineuroinflammatory and neurotrophic effects of CNTF and C16 peptide in an acute experimental autoimmune encephalomyelitis rat model

Experimentalallergic encephalomyelitis (EAE) is an animal model for inflammatory demyelinating autoimmune disease, i.e., multiple sclerosis (MS). In the present study, we investigated the antineuroinflammatory/neuroprotective effects of C16, an ανβ3 integrin-binding peptide, and recombinant rat ciliary neurotrophic factor (CNTF), a cytokine that was originally identified as a survival factor for neurons, in an acute rodent EAE model. In this model, C16 peptide was injected intravenously every day for 2 weeks, and CNTF was delivered into the cerebral ventricles with Alzet miniosmotic pumps. Disease severity was assessed weekly using a scale ranging from 0 to 5. Multiple histological and molecular biological assays were employed to assess inflammation, axonal loss, neuronal apoptosis, white matter demyelination, and gliosis in the brain and spinal cord of different groups. Our results showed that the EAE induced rats revealed a significant increase in inflammatory cells infiltration, while C16 treatment could inhibit the infiltration of leukocytes and macrophages down to 2/3–1/3 of vehicle treated EAE control (P < 0.05). The delayed onset of disease, reduced clinical score (P < 0.01) in peak stage and more rapid recovery also were achieved in C16 treated group. Besides impairing inflammation, CNTF treatment also exerted direct neuroprotective effects, decreasing demyelination and axon loss score (P < 0.05 versus vehicle treated EAE control), and reducing the neuronal death from 40 to 50% to 10 to 20% (P < 0.05). Both treatments suppressed the expression of cytokine tumor necrosis factor-α and interferon-γ when compared with the vehicle control (P < 0.05). Combined treatment with C16 and CNTF produced more obvious functional recovery and neuroprotective effects than individually treatment (P < 0.05). These results suggested that combination treatment with C16 and CNTF, which target different neuroprotection pathways, may be an effective therapeutic alternative to traditional therapy.

Arterial, venous and other vascular risk factors in multiple sclerosis

Purpose of review: This article reviews vascular risk factors with specific emphasis on lipid abnormalities reported to be associated with multiple sclerosis (MS).Recent findings: The current paradigm of MS, supported only partially by MS lesion histopathology and its animal model (experimental allergic encephalomyelitis) considers MS to be a predominantly autoimmune disease. Until recently, most of the known risk factors for MS were interpreted in the context of the autoimmune theory, which still fails to explain why genetically close populations exposed to similar pathogens and/or environmental risk factors have different incidences of MS. Therapies which partially modulate the inflammatory arm of MS pathogenesis, fail to achieve similar benefits in later disease stages, when less inflammatory lesions and more neurodegeneration are present. Several studies have reported an increased cardiovascular morbidity in MS patients and that vascular comorbidity at any time during the disease course also increased the risk of progressive disability. A condition named chronic cerebrospinal venous insufficiency provided a different perspective, on the possible association of MS with the abnormalities of the venous system. Our recent findings revealed increased prevalence of chronic cerebrospinal venous insufficiency associated with MS disease progression as well as with other neurologic disorders. On the other hand, recently emerging evidence indicates that there is an association between lipoproteins and cholesterol metabolism and MS disease progression. Expanded disability status scale worsening was associated with higher baseline low-density lipoprotein and total cholesterol, and higher serum high-density lipoprotein levels were associated with lower contrast-enhancing T1-weigthed lesion volume. It is thought that apolipoprotein A-1 and paraoxonase anti-oxidant enzyme are associated with high-density lipoprotein and contribute to its anti-oxidant and anti-inflammatory properties. A significant inter-dependence was also recently demonstrated between vitamin D, one of the best known environmental risk factors for MS and MS disease progression and the serum lipid profile. Future work in this direction is required in order to better elucidate the role of lipid metabolism and vascular pathology in pathogenesis of MS.