Therapeutic effects of dasatinib in mouse model of multiple sclerosis

Abstract

Background: Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis (MS). EAE is mainly mediated by adaptive and innate immune responses that lead to an inflammatory demyelination and axonal damage. Dasatinib (Sprycel) is a selective protein tyrosine kinase inhibitor with immunomodulatory properties that abrogates multiple signal transduction pathways in immune cells. In the present research, our aim was to test the therapeutic efficacy of dasatinib in experimental model of MS.

Methods: We performed EAE induction in female C57BL/6 mice by myelin oligodendrocyte glycoprotein_35–55 (MOG_35–55) in Complete Freund’s Adjuvant (CFA) emulsion, and used dasatinib for the treatment of EAE. During the course of study, clinical evaluation was assessed, and on day 21 post-immunization blood samples were taken from the heart of mice for tumor necrosis factor-alpha (TNF-α), nitric oxide (NO) and antioxidants capacity evaluation. The mice were sacrificed and brains and cerebellums of mice were removed for histological analysis. Also for in vitro analysis, we used C6 astrocytoma cell line to evaluate the inhibitory effects of dasatinib in cell proliferation and matrix metalloproteinase-2 (MMP-2) activity.

Results: Our findings demonstrated that dasatinib had beneficial effects on EAE by lower incidence, attenuation in the severity and a delay in the onset of disease. The serum level of NO and TNF-α in dasatinib treated mice was significantly lower than control mice. In vitro, dasatinib inhibited cell proliferation and MMP-2 activity.

Conclusion: Dasatinib with its potential therapeutic effects and immunomodulatory properties may be recommended, after additional necessary tests and trials, for the treatment of MS.     

Low-level laser therapy ameliorates disease progression in a mouse model of multiple sclerosis

Multiple sclerosis (MS) is an autoimmune demyelinating inflammatory disease characterized by recurrent episodes of T cell-mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability. The existing therapies for MS are only partially effective and are associated with undesirable side effects. Low-level laser therapy (LLLT) has been clinically used to treat inflammation, and to induce tissue healing and repair processes. However, there are no reports about the effects and mechanisms of LLLT in experimental autoimmune encephalomyelitis (EAE), an established model of MS. Here, we report the effects and underlying mechanisms of action of LLLT (AlGaInP, 660?nm and GaAs, 904?nm) irradiated on the spinal cord during EAE development. EAE was induced in female C57BL/6 mice by immunization with MOG_35–55 peptide emulsified in complete Freund’s adjuvant. Our results showed that LLLT consistently reduced the clinical score of EAE and delayed the disease onset, and also prevented weight loss induced by immunization. Furthermore, these beneficial effects of LLLT seem to be associated with the down-regulation of NO levels in the CNS, although the treatment with LLLT failed to inhibit lipid peroxidation and restore antioxidant defense during EAE. Finally, histological analysis showed that LLLT blocked neuroinflammation through a reduction of inflammatory cells in the CNS, especially lymphocytes, as well as preventing demyelination in the spinal cord after EAE induction. Together, our results suggest the use of LLLT as a therapeutic application during autoimmune neuroinflammatory responses, such as MS.     

地塞米松对实验性自身免疫性脑脊髓炎小鼠髓鞘保护作用的研究

目的:分析地塞米松对实验性自身免疫性脑脊髓炎(EAE)小鼠炎症反应、髓鞘脱失及髓鞘再生的影响,探讨地塞米松治疗多发性硬化的新作用。方法:应用MOG35-55免疫C57BL/6小鼠建立EAE模型。小鼠随机分为正常对照组、EAE组及地塞米松组,观察各组临床症状;采用HE染色、LFB染色、透射电镜扫描及免疫组化染色方法,检测免疫后第13、20、30 d各组小鼠脊髓组织炎症反应、髓鞘脱失及髓鞘再生情况。结果:地塞米松显著降低EAE小鼠发病率、延缓起病时间、减轻疾病严重程度。各个时间点地塞米松组脊髓组织炎性细胞浸润、髓鞘脱失及轴索变性程度较EAE组明显减轻。免疫后第20、30 d,EAE组Olig2阳性细胞数较正常对照组明显增加;免疫后各时间点,地塞米松组Olig2阳性细胞数较正常对照组均明显增加,第13、20 d较EAE组明显增加。结论:地塞米松可增加脊髓组织Olig2表达、促进髓鞘再生,这可能为地塞米松治疗EAE及多发性硬化的效应途径。     

Methylprednisolone induces reversible clinical and pathological remission and loss of lymphocyte reactivity to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of human multiple sclerosis (MS). EAE, induced by immunisation with myelin-associated autoantigens, is characterised by an inflammatory infiltrate in the central nervous system (CNS) associated with axonal degeneration, demyelination and damage. We have recently shown in an experimental mouse model of autoimmune gastritis that methylprednisolone treatment induces a reversible remission of gastritis with regeneration of the gastric mucosa. Here, we examined the effect of oral methylprednisolone on the mouse EAE model of human MS induced by immunisation with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)). We examined the clinical scores, CNS pathology and lymphocyte reactivity to MOG(35-55) following treatment and withdrawal of the steroid. Methylprednisolone remitted the clinical signs of EAE and the inflammatory infiltrate in the CNS, accompanied by loss of lymphocyte reactivity to MOG(35-55) peptide. Methylprednisolone withdrawal initiated relapse of the clinical features, a return of the CNS inflammatory infiltrate and lymphocyte reactivity to MOG(35-55) peptide. This is the first study to show that methylprednisolone induced a reversible remission in the clinical and pathological features of EAE in mice accompanied by loss of lymphocyte reactivity to the encephalitogen. This model will be useful for studies directed at a better understanding of mechanisms associated with steroid-induced disease remission, relapse and remyelination and also as an essential adjunct to an overall curative strategy.     

Methylprednisolone induces reversible clinical and pathological remission and loss of lymphocyte reactivity to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of human multiple sclerosis (MS). EAE, induced by immunisation with myelin-associated autoantigens, is characterised by an inflammatory infiltrate in the central nervous system (CNS) associated with axonal degeneration, demyelination and damage. We have recently shown in an experimental mouse model of autoimmune gastritis that methylprednisolone treatment induces a reversible remission of gastritis with regeneration of the gastric mucosa. Here, we examined the effect of oral methylprednisolone on the mouse EAE model of human MS induced by immunisation with myelin oligodendrocyte glycoprotein peptide (MOG_35–55). We examined the clinical scores, CNS pathology and lymphocyte reactivity to MOG_35–55 following treatment and withdrawal of the steroid. Methylprednisolone remitted the clinical signs of EAE and the inflammatory infiltrate in the CNS, accompanied by loss of lymphocyte reactivity to MOG_35–55 peptide. Methylprednisolone withdrawal initiated relapse of the clinical features, a return of the CNS inflammatory infiltrate and lymphocyte reactivity to MOG_35–55 peptide. This is the first study to show that methylprednisolone induced a reversible remission in the clinical and pathological features of EAE in mice accompanied by loss of lymphocyte reactivity to the encephalitogen. This model will be useful for studies directed at a better understanding of mechanisms associated with steroid-induced disease remission, relapse and remyelination and also as an essential adjunct to an overall curative strategy.     

St. John’s wort and its component hyperforin alleviate experimental autoimmune encephalomyelitis through expansion of regulatory T-cells

Multiple sclerosis (MS) is a central nervous system disorder mainly characterized by inflammation, demyelination and axonal injury. Anti-inflammatory agents can be used to ameliorate the disease process. Hypericum perforatum L or St. John’s wort is widely used as an anti-depressant and anti-inflammatory remedy in traditional and?herbal medicine. Based on St. John’s wort properties, the therapeutic potentials of an H. perforatum extract (HPE) and a single component,?hyperforin were evaluated for effectiveness against MOG_35–55-induced experimental autoimmune encephalomyelitis (EAE), an animal model for?human multiple sclerosis. Female C57BL/6 mice were immunized with specific antigen MOG_35–55 and then administered different doses of?hyperforin or HPE post-immunization. Clinical symptoms/other relevant parameters were assessed daily. Histological analysis of the spinal cord was performed. T-cell proliferative activity was also evaluated using a BrdU assay. The effect of?hyperforin on regulatory T-cells (T_reg cells) was assessed using flow cytometry. The results indicate?hyperforin and HPE reduced the incidence and severity of EAE, an outcome that closely correlated with an inhibition of pathological features (leukocyte infiltration and demyelination) and antigen-specific T-cell proliferation. The study also showed that?hyperforin caused increased T_reg cell levels in the spleen. These results indicated that?hyperforin and HPE could attenuate EAE autoimmune responses by inhibiting immune cell infiltration and expansion of T_reg cell and could eventually be considered as a potential candidate for use in the treatment of MS.     

Therapeutic approach by Aloe vera in experimental model of multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) that leads to an inflammatory demyelination, axonal damage, and progressive neurologic disability that affects ～2.5 million people worldwide. The aim of the present research was to test the therapeutic effect of Aloe vera in experimental model of MS. All experiments were conducted on C57BL/6 male mice aged 6–8 weeks. To induce the experimental autoimmune encephalomyelitis (EAE), 250 µg of the myelin oligodendrocyte glycoprotein 35–55 peptide emulsified in complete freund’s adjuvant was injected subcutaneously on day 0 over two flank areas. In addition, 200?ng of pertussis toxin in 100 µL phosphate buffered saline was injected intraperitoneally on days 0 and 2. The therapeutic protocol was carried out intragastrically using 120?mg/kg/day Aloe vera from 7 days before to 21 days after EAE induction. The mice were killed 21 days after EAE induction. The brains of mice were removed for histological analysis and their isolated splenocytes were cultured. The results indicated that treatment with Aloe vera caused a significant reduction in severity of the disease in experimental model of MS. Histological analysis showed 3?±?2 plaques in Aloe vera-treated mice compared with 5?±?1 plaques in control group. The density of mononuclear infiltration in the CNS of Aloe vera-treated mice (500?±?200) was significantly less in comparison to 700?±?185 cells in control group. Moreover, the serum level of nitric oxide in treatment group was significantly less than control animals. The level of interferon-γ in cell culture supernatant of treated mice splenocytes was lower than control group, whereas decrease in serum level of interleukin-10 in treatment group was not significant in comparison with control mice. These data indicate that Aloe vera therapy can attenuate the disease progression in experimental model of MS.     

银杏提取物对实验性自身免疫性脑脊髓炎小鼠的影响

目的:探讨银杏提取物(GBE)对实验性自身免疫性脑脊髓炎(EAE)小鼠炎症脱髓鞘病变的影响。方法:应用髓鞘少突胶质细胞糖蛋白33-55(MOG33-55)配以完全弗氏佐剂(CFA)免疫小鼠,诱发EAE模型。将小鼠分为CFA对照组、EAE模型组和GBE治疗组(每日腹腔注射GBE70mg/kg)。通过神经功能评分、行为学实验以及免疫荧光染色,观察GBE对EAE小鼠的影响。结果:GBE组小鼠各时间段神经功能评分均低于EAE组(P0.05),行为学检测显示发病高峰期falling latency时间较EAE组延长10s;GBE组较EAE组视神经髓鞘碱性蛋白(MBP)表达水平增高,可见MBP阳性髓鞘结构包绕轴突;海马伞矢状切片免疫荧光染色证实GBE组CD11b阳性小胶质细胞较EAE组明显减少,但是GFAP阳性星形胶质细胞数量与EAE组无明显差别。结论:GBE可能通过抑制小胶质细胞激活从而延缓EAE小鼠脱髓鞘进程,提示GBE对多发性硬化具有一定的治疗作用。     

补阳还五汤对实验性自身免疫性脑脊髓炎单核巨噬细胞的免疫调控作用

目的:探讨补阳还五汤(BYHWD)治疗实验性自身免疫性脑脊髓炎(EAE)的有效性及对单核巨噬细胞免疫调控的作用及机制。方法:雌性C57BL/6小鼠用小鼠髓鞘少突胶质细胞糖蛋白35-55肽段(MOG_(35-55))免疫制作慢性EAE模型,随机分为生理盐水处理组和BYHWD组。在免疫后第3天开始分别予以生理盐水和BYHWD灌胃,500μL/d,持续观察临床症状和体质量变化。免疫后17 d各组统一处死部分动物,HE染色观察炎性细胞浸润情况,髓鞘染色观察脊髓髓鞘脱失比例,流式细胞术检测脾细胞M1型和M2型巨噬细胞表型;免疫荧光组织化学染色和Western blotting检测脊髓巨噬细胞iNOS、TNF-α、arginase及IL-10的表达。结果:BYHWD推迟EAE起病,减轻EAE症状,抑制中枢神经系统脊髓的炎性浸润和髓鞘脱失,促进脊髓及脾组织中M1型巨噬细胞转化为M2型。结论:BYHWD干预可缓解EAE行为学和病理学的改变,其作用机制可能与其诱导巨噬细胞极性转化相关。     

Caspase-mediated oligodendrocyte cell death in the pathogenesis of autoimmune demyelination

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory diseases of the central nervous system (CNS) characterized by localized areas of demyelination. MS is believed to be an autoimmune disorder mediated by activated immune cells such as T- and B-lymphocytes and macrophages/microglia. Lymphocytes are primed in the peripheral tissues by antigens, and clonally expanded cells infiltrate the CNS. They produce large amounts of inflammatory and cytokines that lead to demyelination and axonal degeneration. Although several studies have shown that oligodendrocytes (OLGs), the myelin-forming glial cells in the CNS, are sensitive to cell death stimuli, such as cytotoxic cytokines, anti-myelin antibodies, nitric oxide, and oxidative stress, in vitro, the mechanisms underlying injury to the OLGs in MS/EAE remain unclear. Transgenic mice that express the anti-apoptotic protein specifically in OLGs and caspase-11-deficient mice are significantly resistant to EAE induction. Histopathological analyses show that the number of caspase-activated OLGs and dead OLGs are reduced in the CNS of these mice. The numbers of infiltrating immune cells and the amounts of cytokines are also markedly reduced in EAE lesions. Therefore, caspase-mediated OLG death leads to the exacerbation of demyelination and the deterioration of neurological manifestations by inducing local inflammatory events.     

Bax-ablation attenuates experimental autoimmune encephalomyelitis in mice

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by demyelination and axonal damage. Although the exact pathophysiology is unknown, apoptosis plays a crucial role. Here, we studied the role of the pro-apoptotic gene Bax in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), the animal model for MS. We demonstrate that the clinical signs were markedly reduced in the EAE Bax-deficient mice as compared to wild type (2.3 +/- 0.5 vs. 1.02 +/- 0.32, respectively, P < 0.05). Bax-deficient mice demonstrated less inflammatory infiltration and axonal damage, although they showed similar T-cell immune potency. In conclusion, ablation of the bax gene attenuates the severity of MOG-induced EAE and emphasizes the importance of apoptosis in the pathogenesis of EAE and MS.     

Correlation of nitric oxide levels in the cerebellum and spinal cord of experimental autoimmune encephalomyelitis rats with clinical symptoms

Experimental autoimmune encephalomyelitis (EAE) is a well-established cell-mediated autoimmune inflammatory disease of the CNS, which has been used as a model of the human demyelinating disease. EAE is characterized by infiltration of the CNS by lymphocytes and mononuclear cells, microglial and astrocytic hypertrophy, and demyelination which cumulatively contribute to clinical expression of the disease. EAE was induced in female Sprague-Dawley rats, 3 months old (300 g ± 20 g), by immunization with myelin basic protein (MBP) in combination with Complete Freund's adjuvant (CFA). The animals were divided into 7 groups: control, EAE, CFA, EAE + aminoguanidine (AG), AG, EAE + N-acetyl-L-cysteine (NAC) and NAC. The animals were sacrificed 15 days after EAE induction, and the level of nitric oxide (NO(·)) production was determined by measuring nitrite and nitrate concentrations in 10% homogenate of cerebellum and spinal cord. Obtained results showed that the level of NO(·) was significantly increased in all examined tissues of the EAE rats compared to the control and CFA groups. Also, AG and NAC treatment decreased the level of NO(·) in all tissues compared to the EAE group. The level of NO(·) is increased significantly in the spinal cord compared to the cerebellum. The clinical course of the EAE was significantly decreased in the EAE groups treated with AG and NAC during the development of the disease compared to EAE group and its correlates with the NO(·) level in cerebellum and spinal cord. The findings of our work suggest that NO(·) and its derivatives play an important role in multiple sclerosis (MS). It may be the best target for new therapies in human demyelinating disease and recommend the new therapeutic approaches based on a decreased level of NO(·) during the course of MS.     

Recombinant thrombomodulin ameliorates experimental autoimmune encephalomyelitis by suppressing high mobility group box 1 and inflammatory cytokines

Recombinant thrombomodulin (rTM) has pleiotrophic properties, including anti‐coagulation and anti‐inflammation; however, its effectiveness as a treatment for multiple sclerosis (MS) has not been evaluated fully. High mobility group box 1 (HMGB1) and proinflammatory cytokines, working as inflammatory mediators, are reportedly involved in the inflammatory pathogenesis of MS. The aim of this study was to determine whether rTM can be a potential therapeutic agent for experimental autoimmune encephalomyelitis (EAE). EAE mice received rTM treatment (1 mg or 0·1 mg/kg/day) from days 11 to 15 after immunization. The clinical variables, plasma levels of inflammatory cytokines and HMGB1 and pathological findings in EAE were evaluated. rTM administration ameliorated the clinical and pathological severity of EAE. An immunohistochemical study of the spinal cord showed weaker cytoplasmic HMGB1 staining in the rTM‐treated EAE mice than in the untreated EAE mice. Plasma levels of inflammatory cytokines and HMGB1 were suppressed by rTM treatment. In conclusion, rTM down‐regulated inflammatory mediators in the peripheral circulation and prevented HMGB1 release from nuclei in the central nervous system, suppressing EAE‐related inflammation. rTM could have a novel therapeutic potential for patients with MS.     

骨髓间充质干细胞异体移植对小鼠实验性自身免疫性脑脊髓炎的治疗作用

目的探讨大鼠骨髓间充质干细胞(BMSCs)异体移植对小鼠实验性自身免疫性脑脊髓炎(EAE)的治疗作用。方法全骨髓贴壁培养法获得大鼠BMSCs,流式细胞术检测细胞免疫表型,并诱导成骨方向分化。取雌性C57BL/6小鼠,随机分为3组:正常对照组、PBS组和大鼠BMSCs组,用髓鞘少突胶质细胞糖蛋白(MOG)35~55联合完全弗氏佐剂诱导建立EAE模型。小鼠免疫后38d和48d腹腔注射PBS或大鼠BMSCs进行治疗,神经功能评分观察各组小鼠神经功能变化。二次治疗12d后取各组小鼠脊髓、脾脏和外周血。HE染色及Luxol fast blue染色观察脊髓炎性细胞浸润及髓鞘脱失情况;脾脏制成单细胞悬液,细胞CFSE标记后10mg/L刀豆球蛋白(Con A)和MOG_(35~55)刺激培养3d,观察脾细胞增殖情况。ELISA检测大鼠BMSCs移植后外周血细胞因子干扰素γ(IFN-γ)、白细胞介素17(IL-17)含量。结果流式细胞术显示,大鼠BMSCs第3代(P3)细胞表达抗原CD29、CD90、CD106,不表达CD45。体外诱导其能向成骨分化。小鼠发病后,大鼠BMSCs组小鼠神经功能缺损症状较PBS组减轻,评分降低。HE染色和Luxol fast blue染色结果显示,大鼠BMSCs组脊髓炎细胞浸润和脱髓鞘比同时间点PBS组减轻(P0.05)。Con A和MOG_(35~55)刺激培养后,PBS组和大鼠BMSCs组脾细胞增殖增加,而大鼠BMSCs组又较PBS组降低。与PBS组相比,大鼠BMSCs组血浆细胞因子IFN-γ、IL-17含量降低(P0.05)。结论全骨髓贴壁培养法能有效分离纯化BMSCs,大鼠BMSCs异体移植对小鼠EAE模型有治疗作用。     

Targeting deoxycytidine kinase improves symptoms in mouse models of multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease driven by lymphocyte activation against myelin autoantigens in the central nervous system leading to demyelination and neurodegeneration. The deoxyribonucleoside salvage pathway with the rate-limiting enzyme deoxycytidine kinase (dCK) captures extracellular deoxyribonucleosides for use in intracellular deoxyribonucleotide metabolism. Previous studies have shown that deoxyribonucleoside salvage activity is enriched in lymphocytes and required for early lymphocyte development. However, specific roles for the deoxyribonucleoside salvage pathway and dCK in autoimmune diseases such as MS are unknown. Here we demonstrate that dCK activity is necessary for the development of clinical symptoms in the MOG_35–55 and MOG_1–125 experimental autoimmune encephalomyelitis (EAE) mouse models of MS. During EAE disease, deoxyribonucleoside salvage activity is elevated in the spleen and lymph nodes. Targeting dCK with the small molecule dCK inhibitor TRE-515 limits disease severity when treatments are started at disease induction or when symptoms first appear. EAE mice treated with TRE-515 have significantly fewer infiltrating leukocytes in the spinal cord, and TRE-515 blocks activation-induced B and T cell proliferation and MOG_35–55-specific T cell expansion without affecting innate immune cells or naïve T and B cell populations. Our results demonstrate that targeting dCK limits symptoms in EAE mice and suggest that dCK activity is required for MOG_35–55-specific lymphocyte activation-induced proliferation.     

Infection with Mycobacterium bovis BCG diverts traffic of myelin oligodendroglial glycoprotein autoantigen-specific T cells away from the central nervous system and ameliorates experimental autoimmune encephalomyelitis

Infectious agents have been proposed to influence susceptibility to autoimmune diseases such as multiple sclerosis. We induced a Th1-mediated central nervous system (CNS) autoimmune disease, experimental autoimmune encephalomyelitis (EAE) in mice with an ongoing infection with Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) to study this possibility. C57BL/6 mice infected with live BCG for 6 weeks were immunized with myelin oligodendroglial glycoprotein peptide (MOG(35-55)) to induce EAE. The clinical severity of EAE was reduced in BCG-infected mice in a BCG dose-dependent manner. Inflammatory-cell infiltration and demyelination of the spinal cord were significantly lessened in BCG-infected animals compared with uninfected EAE controls. ELISPOT and gamma interferon intracellular cytokine analysis of the frequency of antigen-specific CD4(+) T cells in the CNS and in BCG-induced granulomas and adoptive transfer of MOG(35-55)-specific green fluorescent protein-expressing cells into BCG-infected animals indicated that nervous tissue-specific (MOG(35-55)) CD4(+) T cells accumulate in the BCG-induced granuloma sites. These data suggest a novel mechanism for infection-mediated modulation of autoimmunity. We demonstrate that redirected trafficking of activated CNS antigen-specific CD4(+) T cells to local inflammatory sites induced by BCG infection modulates the initiation and progression of a Th1-mediated CNS autoimmune disease.     

Distinct pathological patterns in relapsing-remitting and chronic models of experimental autoimmune enchephalomyelitis and the neuroprotective effect of glatiramer acetate

The respective roles of inflammatory and neurodegenerative processes in the pathology of multiple sclerosis (MS) and in its animal model experimental autoimmune encephalomyelitis (EAE) are controversial. Novel treatment strategies aim to operate within the CNS to induce neuroprotection and repair processes in addition to their anti-inflammatory properties. In this study we analyzed and compared the in situ pathological manifestations of EAE utilizing two different models, namely the relapsing-remitting PLP-induced and the chronic MOG-induced diseases. To characterize pathological changes, both transmission electron microscopy (TEM) and immunohistochemistry were employed. The effect of the approved MS drug glatiramer acetate (GA, Copaxone) on myelin damage/repair and on motor neuron loss/preservation was studied in both EAE models. Ultrastructural spinal cord analysis revealed multiple white matter damage foci, with different patterns in the two EAE models. Thus, the relapsing-remitting model was characterized mainly by widespread myelin damage and by remyelinating fibers, whereas in the chronic model axonal degeneration was more prevalent. Loss of lower motor neurons was manifested only in mice with chronic MOG-induced disease. In the GA-treated mice, smaller lesions, increased axonal density and higher prevalence of normal appearing axons were observed, as well as decreased demyelination and degeneration. Furthermore, quantitative analysis of the relative remyelination versus demyelination, provides for the first time evidence of significant augmentation of remyelination after GA treatment. The loss of motor neurons in GA-treated mice was also reduced in comparison to that of EAE untreated mice. These effects were obtained even when GA treatment was applied in a therapeutic schedule, namely after the appearance of clinical symptoms. Hence, the remyelination and neuronal preservation induced by GA are in support of the neuroprotective consequences of this treatment.     

Optical neuritis induced by different concentrations of myelin oligodendrocyte glycoprotein presents different profiles of the inflammatory process

Abstract

Optical neuritis (ON) is characterized by inflammation of the optic nerve, and is one of the first clinical signs of multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is the animal model used to study MS and ON. The present study evaluated the induction, development and progression of ON using an EAE model induced by 100?μg or 300?μg of MOG_35–55. An EAE model was induced in C57BL/6 mice by tail base injection of 100?μg or 300?μg of MOG_35–55 in complete Freund’s adjuvant, supplemented with Mycobacterium tuberculosis. On the day of injection and 48?h later, animals received intraperitoneally 300?ng of pertussis toxin. On days 7, 10, 14, 21 and 58 the optic nerve was dissected for histological analysis, production of CCL5 and immunohistochemical detection of CD4 and CD8. The histological changes observed in the optic nerves consisted of inflammatory cell infiltrates showing varying degrees of ON in the two groups. The onset of ON in the 300?μg of MOG_35–55 group was coincident with higher production of CCL5, on day 10 after induction. However, the 100?μg MOG_35–55 group showed more intense inflammatory infiltrate on day 14 after induction, with higher amounts of CD4 and CD8, reaching an excessive demyelination process on days 21 and 58 after induction. The results suggest that two different concentrations of MOG_35–55 lead to different forms of evolution of optic neuritis.     

Leukocyte recruitment during onset of experimental allergic encephalomyelitis is CCR1 dependent

We have shown that macrophages and microglia present within demyelinating plaques of patients with multiple sclerosis (MS) are immunoreactive for the chemokine receptor CCR1 and its ligand, macrophage inflammatory protein-1alpha. To test the importance of CCR1 to the pathogenesis of MS, we studied the progression of experimental allergic encephalomyelitis (EAE) in CCR1(+/+) vs. CCR1(-/-) mice. After immunization with myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide, nearly all CCR1(+/+) mice developed EAE (95% incidence, severity 2.5+/-0.1), whereas CCR1(-/-) mice had less severe disease (55% incidence, p<0.001; severity 1. 2+/-0.2, p<0.001). CCR1(+/+) mice showed elevated brain mRNA for the chemokines immune protein (IP)-10, RANTES and monocyte chemoattractant protein-1 prior to disease onset, whereas only IP-10 mRNA was elevated in CCR1(-/-) mice. Both groups of mice had comparable in vitro lymphocyte proliferation and cytokine production upon stimulation with MOG peptide, and similar cutaneous hypersensitivity responses to 2,4-dinitrofluorobenzene, suggesting that CCR1(-/-) mice were not systemically immunosuppressed. These data demonstrate that deletion of a chemokine receptor is at least partially protective in EAE, and suggest that targeting of CCR1 may be of therapeutic significance clinically.     

Glatiramer acetate (Copaxone) regulates nitric oxide and related cytokine secretion in experimental autoimmune encephalomyelitis

Nitric oxide (NO) is an important mediator involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). We examined the effect of glatiramer acetate (GA), an agent with suppressing effect on EAE and of therapeutic value for the treatment of MS, on the secretion of NO, as well as of the NO regulating cytokines. We observed that induction of EAE leads to 4-fold elevation in NO secretion and that treatment of the EAE mice by GA indeed leads to a significant reduction in the NO secretion by the splenocytes in response to the encephalitogen. A parallel decrease was observed in the secretion of the NO inducing cytokine IL-1beta. On the other hand, the secretion level of NO modulating cytokines IL-10 and IL-13 was significantly augmented. The correlation between these findings and the therapeutic effect of GA is discussed.