Experimental Autoimmune Encephalomyelitis Group Research Articles

Therapeutic Efficacy of Mesenchymal Stem Cells and Mesenchymal Stem Cells-derived Neural Progenitors in Experimental Autoimmune Encephalomyelitis.

Background and ObjectivesThe goal of treatment for MS is to reduce the inflammation and induce the regeneration of degenerated axons. Considering the anti-inflammatory and regenerative capacity of mesenchymal stem cell (MSCs), in this study the therapeutic efficacy of allogeneic MSCs and MSCs-derived neural progenitor cells (MSCs-NPs) was investigated in cellular therapy of chronic experimental autoimmune encephalomyelitis (EAE).Methods and ResultsMSCs, MSCs-NPs and MSCs+MSCs-NP were administered intravenously to EAE mice on days 22, 29, and 36 post immunization. The levels of cytokines and PGE2 in sera or supernatant of in vitro cultured splenocytes derived from treated mice were measured by ELISA. The results of this study showed that in comparison to MSCs monotherapy, MSCs-NPs administration had a more profound capability of inhibiting the proliferation of pathogenic MOG35–55-specific T cells, decreasing IFN-γ production and increasing anti-inflammatory IL-10 cytokine production. These findings could be explained by higher ability of in vitro cultured MSCs-NPs in production of PGE2 compared to MSCs. In line with these findings, while the administration of MSCs and MSCs-NPs significantly decreased the clinical scores of EAE in comparison with the untreated EAE group, MSCs-NPs were significantly more efficient in reducing clinical score compared to MSCs. Of interest, combined therapy with MSCs and MSCs-NPs did not provide any benefit over monotherapy with MSCs-NPs.ConclusionsIn comparison to MSCs, allogenic MSCs-NPs are more potent in the attenuation of EAE.

Exploring experimental autoimmune optic neuritis using multimodal imaging

BackgroundNeuro-axonal injury is a key contributor to non-reversible long-term disability in multiple sclerosis (MS). However, the underlying mechanisms are not yet fully understood. Visual impairment is common among MS patients, in which episodes of optic neuritis (ON) are often followed by structural retinal damage and sustained functional impairment. Alterations in the optic nerve and retina have also been described in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. Thus, investigating structural anterior visual pathway damage may constitute a unique model for assessing mechanisms and temporal sequence of neurodegeneration in MS. We used a multimodal imaging approach utilizing optical coherence tomography (OCT) and diffusion tensor imaging (DTI) to explore the mechanisms and temporal dynamics of visual pathway damage in the animal model of MS. Methods7 EAE-MOG35-55 and 5 healthy female C57BL/6J mice were used in this study. Ganglion cell complex (GCC) thickness was derived from an OCT volume scan centred over the optic nerve head, while the structure of the optic nerve and tracts was assessed from DTI and co-registered T2-weighted sequences performed on a 7T MRI scanner. Data was acquired at baseline, disease onset, peak of disease and recovery. Linear mixed effect models were used to account for intra-subject, inter-eye dependencies, group and time point. Correlation analyses assessed the relationship between GCC thickness and DTI parameters. Immunofluorescence staining of retina and optic nerve sections was used to assess distribution of marker proteins for microglia and neurodegeneration (nerve filaments). ResultsIn EAE mice, a significant increase in GCC thickness was observed at disease onset (p < 0.001) followed by a decrease at recovery (p < 0.001) compared to controls. The EAE group had significant GCC thinning at recovery compared to all other time points (p < 0.001 for each). Signal increase on T2-weighted images around the optic nerves indicative of inflammation was seen in most of the EAE mice but in none of the controls. A significant decrease in axial diffusivity (AD) and increase in radial diffusivity (RD) values in EAE optic nerves (AD: p = 0.02, RD: p = 0.01) and tract (AD: p = 0.02, RD: p = 0.006) was observed compared to controls. GCC at recovery was positively correlated with AD (optic nerve: rho = 0.74, p = 0.04, optic tract: rho = 0.74, p = 0.04) and negatively correlated with RD (optic nerve: rho = −0.80, p = 0.02, optic tract: rho = −0.75, p = 0.04). Immunofluorescence analysis indicated the presence of activated microglia in the retina and optic nerves in addition to astrocytosis and axonal degeneration in the optic nerve of EAE mice. ConclusionOCT detected GCC changes in EAE may resemble what is observed in MS-related acute ON: an initial phase of swelling (indicative of inflammatory edema) followed by a decrease in thickness over time (representative of neuro-axonal degeneration). In line with OCT findings, DTI of the visual pathway identifies EAE induced pathology (decreased AD, and increased RD). Immunofluorescence analysis provides support for inflammatory pathology and axonal degeneration. OCT together with DTI can detect retinal and optic nerve damage and elucidate to the temporal sequence of neurodegeneration in this rodent model of MS in vivo.

The Modulatory Effects of Vitamin D on the Expression of IL-12 and TGF-β in the Spinal Cord and Serum of Mice with Experimental Autoimmune Encephalomyelitis

The immunoregulatory effects of transforming growth factor (TGF)-βand interleukin-12 (IL-12) and immunomodulatory actions of vitamin D (VD) were reported in several studies. This study aims to evaluate VD effects on IL-12 and TGF-β expression in experimental autoimmune encephalomyelitis (EAE). EAE was induced in three groups of C57BL/6 mice by immunization with MOG and administered intra-peritoneally 200 ngVD, PBS or olive oil (OO) from day +3 to +30. One group was also considered as healthy control group. At day 31, cytokines expression in the spinal cord and their serum levels were determined using real time-PCR and ELISA, respectively. IL-12 gene expression and its serum levels in PBS-injected- or OO-administrated EAE groups were significantly higher than healthy group. IL-12 gene expression in EAE group treated with VD was significantly decreased compared to PBS-injected- or OO-administrated EAE groups (P<0.001 and P<0.02, respectively). Serum IL-12 levels were significantly reduced in VD-treated EAE mice compared to PBS-injected- or OO-administrated EAE groups (P<0.004 and P<0.05, respectively). No significant difference was observed between PBS-injected-EAE, OO-administrated EAE and healthy groups regarding the expression of TGF-β. In EAE mice treated with VD, TGF-β expression was significantly higher than healthy group and PBS-injected EAE mice (P<0.01 and P<0.04, respectively). VD-treated EAE group had also higher serum TGF-β levels than PBS-injected-, OO-administrated and healthy groups (P<0.05). VD modulates the expression of IL-12 and TGF-β in spinal cord and serum of EAE mice.

S-allyl cysteine improves clinical and neuropathological features of experimental autoimmune encephalomyelitis in C57BL/6 mice

Multiple sclerosis (MS) is a deleterious autoimmune and demyelinating disorder of the central nervous system with debilitating sensory and motor complications. There is still no definite cure for it and the main focus for its treatment mostly pivots around subsiding its severity and recurrence. Experimental autoimmune encephalomyelitis (EAE) is an established animal model of MS. S-allyl cysteine (SAC) is the active and main constituent of aged garlic extract with anti-inflammatory and neuroprotective property. This study was conducted to evaluate its possible protective effect in EAE model of MS. SAC was administered p.o. at a dose of 50 mg/kg/day to female C57BL/6 mice immunized with myelin oligodendrocytic glycoprotein (MOG35-55). Results showed that SAC is capable to alleviate clinical signs and severity of the disease and improved lumbar spinal cord tissue level of tumor necrosis factor α (TNFa), interleukin 17 (IL-17), activity-dependent neuroprotector homeobox (ADNP), microtubule-associated proteins 1A/1 B light chain 3A (MAP1LC3A), and matrix metalloproteinase 9 (MMP-9). In addition, SAC attenuated inflammatory cell infiltration, axonal demyelination, and axonal loss in lumbar spinal cord in EAE group, as demonstrated by H & E, Luxol fast blue (LFB), and Bielschowsky silver staining, respectively. Taken together, SAC could mitigate severity of MOG35-55-induced EAE as a valid model of MS via amelioration of pathogenic molecular mechanisms responsible for neuroinflammation and axonal damage.

Effects of Tripterygium glycoside treatment on experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease mediated by CD4+ T cells. It is characterized by mononuclear cell infiltration around the small blood vessels in the central nervous system (CNS). Previous investigations have found that apoptosis is associated with the occurrence and development of autoimmune disease, and that mononuclear cell apoptosis and clearance from the CNS is one of the repair mechanisms of EAE. Tripterygiumwilfordii glycoside (TWP) is an organic matter isolated from Tripterygiumwilfordii, which has anti‑inflammatory and immunosuppressive effects. In the present study, male Lewis rats were randomly divided into a normal control, EAE and TWP groups. Rats in EAE and TWP groups received injections of emulsified EAE antigen (myelin protein) at two points on the footpad while control group received PBS. The TWP group was then treated with TWP daily for 21days. Symptoms and nerve function scores were observed and evaluated. Specimens of blood, brain and spinal cord were collected for further pathological examination, Tunel assay, ELISA and immunohistochemistry were performed to examine the effect of TWP on the onset of EAE, and changes in CNS inflammatory infiltration, cell apoptosis, and the expression of nuclear factor (NF)‑κB P65 and interleukin (IL)‑2. The results showed that the TWP treatment group exhibited decreased EAE and delayed onset, compared with the control. The clinical symptoms were significantly reduced and alleviation of inflammatory cell infiltration was observed. Compared with the EAE group, a higher inflammatory cell apoptotic rate, and reduced serum levels of IL‑2 and NF‑κB p65‑positive cells were observed in the TWP treatment group. Therefore, TWP effectively inhibited EAE via the inhibition of CNS inflammatory cell infiltration, enhancement of inflammatory cell apoptosis, and downregulation of the expression of NF‑κB and IL‑2.

Ginger Extract Modulates the Expression of Chemokines CCL20 and CCL22 and Their Receptors (CCR6 and CCR4) in the Central Nervous System of Mice with Experimental Autoimmune Encephalomyelitis.

Background Chemokines facilitate the leukocytes infiltration into the central nervous system (CNS) which is an essential step in the pathogenesis of multiple sclerosis. Ginger has also a broad anti-inflammatory properties. The aim was to evaluate the effects of ginger extract on the expression of CCL20 and CCL22 and their receptors (CCR6 and CCR4, respectively) in experimental autoimmune encephalomyelitis (EAE). Material and Methods Female C57BL/6 mice used for EAE induction by immunization with myelin oligodendroglial glycoprotein. Then, the EAE mice were treated with PBS or ginger extract, from day +3 to +30. At day 31, mice were scarified and the expression of CCL20 and CCL22 and their receptors in the spinal cord measured using real time-PCR. Results The expression of CCL20, CCL22 and CCR4 in the spinal cord of PBS-administrated EAE mice was significantly higher than healthy group (P<0.04, P<0.05 and P<0.02, respectively). In 200- and 300 mg/kg ginger extract-treated EAE mice, the expression of CCL20, CCL22 and CCR4 were significantly reduced as compared with PBS-administrated EAE group (P<0.04, P<0.01 and P<0.002 for 200 mg/kg ginger extract and P<0.01, P<0.005 and P<0.004 for 300 mg/kg ginger extract, respectively). The CCR6 expression in EAE mice treated with 200- or 300 mg/kg ginger extracts was lower than PBS-administrated EAE mice (P<0.01 and P=0.07, respectively). Conclusion Treatment of EAE mice with ginger extract down-regulate the expression of CCL20 and CCL22 and their receptors in EAE mice. The possible therapeutic potential of ginger for treatment of MS can be considered in future investigations.

Neutralization of Interleukin-9 Decreasing Mast Cells Infiltration in Experimental Autoimmune Encephalomyelitis.

Background:Th9 cells are a newly discovered CD4+ T helper cell subtype, characterized by high interleukin (IL)-9 secretion. Growing evidences suggest that Th9 cells are involved in the pathogenic mechanism of multiple sclerosis (MS). Mast cells are multifunctional innate immune cells, which are perhaps best known for their role as dominant effector cells in allergies and asthma. Several lines of evidence point to an important role for mast cells in MS and its animal models. Simultaneously, there is dynamic “cross-talk” between Th9 and mast cells. The aim of the present study was to examine the IL-9-mast cell axis in experimental autoimmune encephalomyelitis (EAE) and determine its interaction after neutralizing anti-IL-9 antibody treatment.Methods:Female C57BL/6 mice were randomly divided into three groups (n = 5 in each group): mice with myelin oligodendrocyte glycoprotein (MOG)-induced EAE (EAE group), EAE mice treated with anti-IL-9 antibody (anti-IL-9 Abs group), and EAE mice treated with IgG isotype control (IgG group). EAE clinical score was evaluated. Mast cells from central nervous system (CNS) were detected by flow cytometry. The production of chemokine recruiting mast cells in the CNS was explored by reverse transcription-polymerase chain reaction (RT-PCR). In mice with MOG-induced EAE, the expression of IL-9 receptor (IL-9R) complexes in CNS and spleen mast cells was also explored by RT-PCR, and then was repeating validated by immunocytochemistry. In vitro, spleen cells from EAE mice were cultured with anti-IL-9 antibody, and quantity of mast cells was counted by flow cytometry after co-culture.Results:Compared with IgG group, IL-9 blockade delayed clinical disease onset and ameliorated EAE severity (t = −2.217, P = 0.031), accompany with mast cells infiltration decreases (day 5: t = −8.005, P < 0.001; day 15: t = −11.857, P < 0.001; day 20: t = −5.243, P = 0.001) in anti-IL-9 Abs group. The messenger RNA expressions of C-C motif chemokine ligand 5 (t = −5.932, P = 0.003) and vascular cell adhesion molecule-1 (t = −4.029, P = 0.004) were significantly decreased after IL-9 neutralization in anti-IL-9 Abs group, compared with IgG group. In MOG-induced EAE, the IL-9R complexes were expressed in CNS and spleen mast cells. In vitro, splenocytes cultured with anti-IL-9 antibody showed significantly lower levels of mast cells in a dose-dependent manner, compared with splenocytes cultured with anti-mouse IgG (5 μg/ml: t = −0.894, P = 0.397; 10 μg/ml: t = −3.348, P = 0.019; 20 μg/ml: t = −7.639, P < 0.001).Conclusions:This study revealed that IL-9 neutralization reduced mast cell infiltration in CNS and ameliorated EAE, which might be relate to the interaction between IL-9 and mast cells.

Epigenetic and gene expression alterations of FOXP3 in the T cells of EAE mouse model of multiple sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease with demyelination and neurodegeneration of the central nervous system. It has been shown that the regulatory T (Treg) cells are responsible for maintaining tolerance to self-antigens and can suppress the autoimmune process in several animal models such as experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Recent basic studies have demonstrated that forkhead box P (FOXP3) and BTB domain and CNC homolog 2 (BACH2) are the master transcription factors of these cells playing a pivotal role in the polarization of naïve T cells into Treg cells. In the current study, the expression of FOXP3 and BACH2 genes and FOXP3 promoter methylation were evaluated in T cells of the EAE-induced mice. The results of this study showed a prominent and significant hypermethylation of the FOXP3 gene promoter in the EAE-induced mice compared to the sham and control groups. The expression of FOXP3 and BACH2 genes was significantly decreased in the EAE group in comparison with the sham and control groups. This study suggests that the epigenetic modification of FOXP3 gene is involved in the pathogenesis of EAE and this could be important in therapy in an appropriate and logical statement.

The Anti-Inflammatory Effects of Oral-Formulated Tacrolimus in Mice with Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a T-lymphocyte-mediated autoimmune disease that is characterized by inflammation in the central nervous system (CNS). Although many disease-modifying therapies (DMTs) are presumed effective in patients with MS, studies on the efficacy and safety of DMTs for preventing MS relapse are limited. Therefore, we tested the immunosuppressive anti-inflammatory effects of oral-formulated tacrolimus (FK506) on MS in a mouse model of experimental autoimmune encephalomyelitis (EAE). The mice were randomly divided into 3 experimental groups: an untreated EAE group, a low-dose tacrolimus-treated EAE group, and a high-dose tacrolimus-treated EAE group. After autoimmunization of the EAE mice with myelin oligodendrocyte glycoprotein, symptom severity scores, immunohistochemistry of the myelination of the spinal cord, and western blotting were used to evaluate the EAE mice. After the autoimmunization, the symptom scores of each EAE group significantly differed at times. The group treated with the larger tacrolimus dose had the lowest symptom scores. The tacrolimus-treated EAE groups exhibited less demyelination and inflammation and weak immunoreactivity for all of the immunization biomarkers. Our results revealed that oral-formulated tacrolimus inhibited the autoimmunization in MS pathogenesis by inactivating inflammatory cells.

Ginger extract modulates the expression of IL-12 and TGF-β in the central nervous system and serum of mice with experimental autoimmune encephalomyelitis.

The main function of IL-12 is differentiation of naive T cells intoTh1 cells and TGF-β is a powerful immunoregulatory cytokine. The immunomodulatory and anti-inflammatory properties of ginger have also been reported in some studies. The aim of this study was to evaluate the effects of ginger extract on the expression of IL-12 and TGF-β in a model of experimental autoimmune encephalomyelitis (EAE). EAE was induced in C57BL/6 mice by immunization with myelin oligodendroglial glycoprotein emulsified in complete Freund's adjuvant. The mice were administered intra-peritoneally with ginger extracts or PBS, from day +3 to +30. On day 31, mice were scarified and the expression of IL-12 and TGF-β mRNA in the spinal cord were determined by using real time-PCR. The serum levels of cytokines were measured by ELISA. In PBS-treated EAE mice, the expression of IL-12 P35 and IL-12 P40 mRNA in the CNS and the mean serum levels of IL-12 were significantly higher than those of healthy group (p<0.001). In ginger-treated EAE mice, the expression of IL-12 mRNA and its serum levels were significantly lower as compared to PBS-treated EAE mice. No significant difference was observed between PBS-treated EAE mice and healthy group regarding the expression of TGF-β mRNA. In ginger (300 mg/kg)-treated EAE group, the expression of TGF-β mRNA and its serum levels were significantly higher in comparison to PBS-treated EAE mice (p<0.01 and p<0.05, respectively). These results indicated that ginger extract modulates the expression of IL-12 and TGF-β in CNS and serum of EAE mice.

Piperlongumine attenuates experimental autoimmune encephalomyelitis through inhibition of NF-kappaB activity

Multiple sclerosis (MS) is a chronic, autoimmune and neurodegenerative disease in which demyelination sporadically and repeatedly occurs in the central nervous system (CNS). The activity of nuclear factor kappa B (NF-κB), a family of transcription factors, was increased in the cerebrospinal fluid (CSF) and/or the serum and brain and/or spinal cord of MS patients than in a healthy donors. In our study, we investigated whether piperlongumine (PL), which is known to have inhibitory effect on activity of NF-κB, can alleviate an experimental autoimmune encephalomyelitis (EAE). The mice were immunized with myelin oligodendrocyte glycoprotein 35–55 (MOG35–55), and then we injected PL (1.5mg/kg/day or 3.0mg/kg/day) into the mice intraperitoneally on every second day from days 2 to 28. For in vitro study, we treated PL (0.5, 1 and 2.5μM) to RAW 264.7 and Jurkat cells with each stimulator. We observed that the paralytic severity and neuropathology of EAE in PL-treated group were decreased compared with the EAE group. PL showed a suppressed effect on demyelination, immune cells infiltration, astrocytes/microglials activation, level of inflammatory cytokines and proteins as well as NF-κB activity. Production of inflammatory cytokines and proteins as well as translocation of NF-κB into nucleus by treatment stimulators in RAW 264.7 and Jurkat cells were reduced by PL. Moreover, treatment of NF-κB inhibitor further inhibited production of inflammatory cytokines and proteins. These results suggest that PL can mitigate MOG-induced EAE symptoms and activation of macrophages and T cells by inhibiting NF-κB signaling. Therefore, PL could be useful for the treatment for MS.

A Novel Combination of Docosahexaenoic Acid, All-Trans Retinoic Acid, and 1, 25-Dihydroxyvitamin D3 Reduces T-Bet Gene Expression, Serum Interferon Gamma, and Clinical Scores but Promotes PPARγ Gene Expression in Experimental Autoimmune Encephalomyelitis.

Vitamins are immunologically interesting due to their significant immunomodulatory activities. Experimental autoimmune encephalomyelitis (EAE) is one of the most commonly used experimental models for studying autoimmune disorder in multiple sclerosis (MS). The aim of this study was to evaluate the protective and ameliorative effects of novel combination of all-trans retinoic acid (ATRA), 1,25-dihydroxyvitamin D3 (D3), and docosahexaenoic acid (DHA) on EAE-specific determinants and target gene expressions. Mice were randomly categorized into three groups before EAE induction [non-treated EAE (Group E), treated EAE (Group T), and healthy mice (Group H)]. Encephalomyelitis was induced in female C57BL/6 mice by subcutaneous immunization using commercial kits. Preceding day of EAE induction, combination of ATRA, D3, and DHA was administered with a single IP injection every 48h and continued until day 26. Findings of present study showed that administration of vitamins A, D, and DHA significantly decreased average clinical scores, cumulative EAE score, and EAE incidence in Group T, compared to Group E (p values <0.001). Interferon γ secretion in serum and T-bet mRNA expression in splenocytes were significantly reduced (p=0.004, p=0.029, respectively) while PPARγ mRNA expression was significantly increased in Group T compared to Group E (p=0.021). These findings highlighted that ATRA, D3, and DHA combination modulated PPARγ and T-bet gene expression and resulted in decrease in Th1 response and lymphocyte invasion into the central nervous system (CNS) and resultant inflammation. In conclusion, the results of this study suggested the potential use of this intervention in treatment and/or prevention of EAE/MS and probably other Th1 cell-mediated autoimmune diseases.

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

BackgroundMultiple 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.MethodsSixty-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.ResultsLoss 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.ConclusionFive 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.

Human chorionic gonadotropin (hCG) sub-chronic administration mediated MMP-9 activities and cytokine association deteriorate experimental autoimmune encephalomyelitis (EAE) condition in mice model

We investigated the role of sub-chronic administration of human chorionic gonadotropin (hCG) in an experimental autoimmune encephalomyelitis (EAE) mice model as a multiple sclerosis (MS) model. MS is a disease of malfunctioned immune system where infiltrating immune cells are stimulated by matrix metalloproteinase (MMP) activation and cause blood–brain barrier disruption followed by the destruction of myelin sheath by concurrent activation of cytokines. hCG is responsible for the expression and activation of MMPs and regulation of cytokines in different cells. Mice were implanted with the osmotic pump containing hCG and luteinizing hormone (LH) which maintain their release for 4 weeks with constant velocity. The activities of MMP-9 and NADPH oxidase and the levels of cytokines were measured in the plasma and the CNS tissues. The hCG treated EAE mice showed a decrease in body weights, increased mortality, accelerated onset stage and amplified EAE severity. MMP-9 activity was increased significantly in the EAE+hCG group compared to EAE group in both CNS and plasma. We also found increased activity of NADPH oxidase in plasma at onset stage and in brain tissues at day 38. IL-6 and IL-4 levels in brain tissue were found significantly higher in EAE+hCG group than in EAE group. EAE+LH group mice showed early onset patterns like EAE+hCG group and MMP-9 and NADPH oxidase levels were higher than EAE group, but cytokines levels were not significantly different from EAE group. We suggest that hCG treatment should be considered carefully as multiple or constant hCG treatment could exacerbate aggravation of MS induction and clinical symptoms.

You-Gui pills promote nerve regeneration by regulating netrin1, DCC and Rho family GTPases RhoA, Racl, Cdc42 in C57BL/6 mice with experimental autoimmune encephalomyelitis

Ethnopharmacological relevanceYou-Gui pills (YGPs) are an effective traditional Chinese formula being used clinically for the treatment of multiple sclerosis (MS). Previous studies demonstrated that YGPs exerted the potent neuroprotective effects in murine models of experimental autoimmune encephalomyelitis (EAE), which is an equivalent animal model for multiple sclerosis (MS). However, the mechanism of YGPs functions remained unclear. Aim of this studyThe aim of this study was to evaluate the therapeutic effect of YGPs in MOG35–55-induced EAE mice and to further elucidate the underlying molecular mechanism. Methods: Female C57BL/6 mice were divided into six groups, including the non-treated EAE model, prednisone acetate- and 1.2, 2.4 or 4.8g/kg YGPs-treated EAE groups, and a normal control group. The EAE model was established by injecting the mice subcutaneously with MOG35–55 antigen. The body weights were measured and the neurological functions were scored in each group. The pathology and morphology of the brain and spinal cord was examined. The expression of MAP-2 was detected by immunofluorescent staining. The levels of netrin1, DCC, RhoA, Rac1, and Cdc42 were assayed by immunohistochemistry, qRT-PCR and Western blot on day 40 post-immunization (PI). ResultsYGPs treatments significantly reduced neurological function scores in EAE mice, where the inflammatory infiltration was reduced and the axon and myelin damage in both brain and spinal cord was alleviated. In the brain and spinal cord tissues, YGPs increased the expression of neuronal factors MAP-2, netrin1 and DCC. The expression of Rac1 and Cdc42 were increased, while RhoA was reduced following YGPs treatments. ConclusionOur results demonstrated that YGPs exhibited a neuroprotective effect on promoting nerve regeneration at the brain and spinal cord in EAE mice induced by MOG35–55. Netrin1, DCC and the Rho family GTPases of RhoA, Racl, Cdc42 were involved in mediating the effects of YGPs on nerve regeneration.

Ginger Extract Reduces the Expression of IL-17 and IL-23 in the Sera and Central Nervous System of EAE Mice.

IL-17/IL-23 axis plays an important role in the pathogenesis of several autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). The immunomodulatory properties of ginger are reported in previous studies. To evaluate the effects of ginger extract on the expression of IL-17 and IL-23 in a model of EAE. EAE was induced in C57BL/6 mice by immunization with myelin oligodendroglial glycoprotein and then treated with PBS or ginger extracts, from day +3 to +30. At day 31, mice were scarificed and the expression of IL-17 and IL-23 mRNA in spinal cord were determined by using real time-PCR. The serum levels of cytokines were measured by ELISA. The mRNA expression of IL-17, IL-23 P19 and IL-23 P40 in CNS and serum levels of IL-17 and IL-23 were significantly higher in PBS-treated EAE mice than non-EAE group (p<0.003, p<0.001, p<0.001, p<0.05 and p<0.01, respectively). In 200 mg/kg ginger-treated EAE mice the mRNA expression of IL-17, P19 and P40 in CNS and serum IL-23 levels were significantly decreased as compared to PBS-treated EAE mice (p<0.05, p<0.001, p<0.001 and p<0.05, respectively). Moreover, 300 mg/kg ginger-treated EAE group had significantly lower expression of IL-17, P19 and P40 in CNS and lower serum IL-17 and IL-23 levels than PBS-treated EAE group (p<0.02, p<0.001, p<0.001, p<0.03 and p<0.004, respectively). Ginger extract reduces the expression of IL-17 and IL-23 in EAE mice. The therapeutic potential of ginger for treatment of MS could be considered in further studies.

The role of CNS TLR2 activation in mediating innate versus adaptive neuroinflammation

Toll-like receptor 2 (TLR2) is expressed on immune cells in the periphery and the CNS and mediates both innate and adaptive immune responses. Recent studies have implicated TLR2 in systemic pathogenesis of adaptive immunity in experimental autoimmune encephalomyelitis (EAE). In addition, TLR2 is expressed on oligodendrocyte progenitor cells and its activation inhibits their differentiation and myelination. We investigated the roles of CNS TLR2 activation in mediating neuro-inflammatory responses in intact versus EAE animals. We examined the effects of intra-cerebro-ventricular (ICV) injection of Zymosan, a TLR2 agonist, on naive versus EAE animals. The neuro-inflammatory response was characterized by immune-fluorescent staining for IBA-1+ microglia/macrophages and CD3+ T cells, and by semi-quantitative real time PCR for TLR2 and immune cytokines. The nature of the immune cells isolated from EAE brain tissue was assessed by their proliferative response to the PLP peptide autoantigen. Survival and clinical scores were monitored; demyelination and axonal loss were quantified by Gold-Black and Bielschowsky stains. Our findings showed that Zymosan injection in naïve mice induced a massive neuro-inflammatory response without any clinical manifestations. In EAE mice, ICV Zymosan induced a severe acute toxic response with 80% mortality. Surviving animals returned to pre-injection clinical score, and their course of disease was not altered as compared to control EAE group. Demyelination and axonal loss were not affected by ICV Zymosan injection. Quantification of immune response in the brain by real time PCR, immunofluorescent stains and proliferative response to PLP peptide suggested that TLR2 activation induces innate but not adaptive immune response. We conclude that EAE mice are hypersensitive to CNS TLR2 activation with a severe toxic response. This might represent the susceptibility of multiple sclerosis patients to even trivial infections. As CNS TLR2 activation does not alter the clinical and pathological course of EAE, it implies that CNS TLR2 activation affects the innate but not adaptive brain immune responses.

Promotion of experimental autoimmune encephalomyelitis upon neutrophil granulocytes’ stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP) peptide

It has been acknowledged that neutrophil granulocytes, the common mediators of immune responses against extracellular bacteria, can also intercede autoimmune reactions such as experimental autoimmune encephalomyelitis (EAE). Formyl-methionyl-leucyl-phenylalanine (fMLP) is a microbial peptide that can be well-tolerated when intravenously administered and can directly lead to activation and accumulation of neutrophils into the blood circulation. Here, this antigenic peptide was injected to the mice at the induction of EAE, and the immunological and pathological outcomes were assessed. As a peripheral immune organ, spleen of the animals that received fMLP contained considerably high percentages of Gr-1hiLy7/4hi mature neutrophils. In the sera samples, only a slight difference was determined in Th1/Th2/Th17-related cytokine levels. Expression of CXCR1 or CXCR2 chemokine receptors was not significantly modulated in EAE with or without fMLP which might indicate a direct role for this antigenic peptide on neutrophil accumulation. Even though fMLP administration did not propone the clinical (symptomatic) onset of the disease, the animals showed severe body conditions with higher EAE scores. Accordingly, the expression of TNF-α and CXCL1 inflammatory mediators in the brain was increased in fMLP–EAE group. In conclusion, with its potent capacity to mobilize neutrophils and to stimulate innate immune cells, fMLP peptide can be used to aggravate immune reactions in EAE. This observation may indicate that the strength of innate immune responses particularly at the induction phase of EAE might influence the clinical course of the disease.

Combination therapy with lenalidomide and nanoceria ameliorates CNS autoimmunity

ObjectiveMultiple sclerosis (MS) is a debilitating neurological disorder involving an autoimmune reaction to oligodendrocytes and degeneration of the axons they ensheath in the CNS. Because the damage to oligodendrocytes and axons involves local inflammation and associated oxidative stress, we tested the therapeutic efficacy of combined treatment with a potent anti-inflammatory thalidomide analog (lenalidomide) and novel synthetic anti-oxidant cerium oxide nanoparticles (nanoceria) in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. MethodsC57BL/6 mice were randomly assigned to a control (no EAE) group, or one of the four myelin oligodendrocyte glycoprotein-induced EAE groups: vehicle, lenalidomide, nanoceria, or lenalidomide plus nanoceria. During a 23day period, clinical EAE symptoms were evaluated daily, and MRI brain scans were performed at 11–13days and 20–22days. Histological and biochemical analyses of brain tissue samples were performed to quantify myelin loss and local inflammation. ResultsLenalidomide treatment alone delayed symptom onset, while nanoceria treatment had no effect on symptom onset or severity, but did promote recovery; lenalidomide and nanoceria each significantly attenuated white matter pathology and associated inflammation. Combined treatment with lenalidomide and nanoceria resulted in a near elimination of EAE symptoms, and reduced white matter pathology and inflammatory cell responses to a much greater extent than either treatment alone. InterpretationBy suppressing inflammation and oxidative stress, combined treatment with lenalidomide and nanoceria can reduce demyelination and associated neurological symptoms in EAE mice. Our preclinical data suggest a potential application of this combination therapy in MS.

The Use of Agmatine Provides the New Insight in an Experimental Model of Multiple Sclerosis.

The aim of the study is to investigate the hypothesis that agmatine (AGM) enhances blood brain barrier (BBB) compounds properties in experimental autoimmune encephalomyelitis (EAE), which is an established animal model for studying multiple sclerosis (MS). Wild-type (WT) and knockout (KO) CBA/H iNOS-/- mice, 3 months old (15 ± 5 g) were used for EAE induction by myelin basic protein (MBP) dissolved in complete Freund's adjuvant (CFA). The animals were divided into control, CFA, EAE, EAE + AGM and AGM groups. After the development of full clinical remission, the animals were sacrificed and the immunohistochemical and biochemical examinations were performed in brain homogenates. We had noticed the increased expressions of occludin in WT and KO mice with EAE + AGM, compared to EAE groups in which these expressions were significantly decreased compared to the controls. The significant elevations of matrix metalloproteinases (MMPs)-MMP-3 and MMP-9 in WT and KO EAE animals were decreased during AGM treatment in both groups. AGM application post EAE in WT and KO mice caused decreased level of Iba-1 stain, compared to EAE groups. The obtained results suggest beneficial AGM effects in EAE on BBB components, which might be useful for novel therapeutic strategies in MS.