Acamprosate modulates experimental autoimmune encephalomyelitis

Objective  

This pilot study aimed to determine the efficacy of acamprosate (N-acetyl homotaurine) in reducing the pathological features of experimental autoimmune encephalomyelitis (EAE) which is an animal model for multiple sclerosis (MS).     

Anti-inflammatory effects of levetiracetam in experimental autoimmune encephalomyelitis

Levetiracetam (LEV) is an established anticonvulsant with numerous mechanisms of action. Apart from its anti-epileptic effects, recent experimental studies suggest anti-inflammatory properties via modulation of interleukin (IL)-1β and transforming-growth-factor (TGF)-β1. However, its anti-inflammatory properties have not yet been examined in an autoimmune inflammatory disease of the central nervous system (CNS). We investigated LEV anti-inflammatory properties in experimental autoimmune encephalomyelitis, an established mouse model of multiple sclerosis. FACS analyses, ELISA, histology and rt-PCR experiments were done to explore potential anti-inflammatory effects. In line with prior studies, we demonstrate that LEV modulates both the relative gene expression and secretion of IL-1β and TGF-1β. However, these changes were not sufficient to alter the disease course or histological parameters. Additionally, LEV showed no effects on the absolute number of different immune cell subsets. In summary, LEV showed only minor anti-inflammatory effects not sufficient to ameliorate disease course in an autoimmune inflammatory disease of CNS.     

Alendronate alleviates the symptoms of experimental autoimmune encephalomyelitis

Nitrogen-containing bisphosphonates, such as alendronate, have been widely used to treat osteoporosis because they may target multiple signals in the mevalonate cascade. The present study evaluated the therapeutic effects of alendronate on experimental autoimmune encephalomyelitis (EAE), which is a prototypical autoimmune disease model. EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG)_35-55 peptide. The mice were checked daily for clinical symptoms, such as paralysis, and the levels of inflammatory cytokines were analyzed using ELISA, western blot analyses, and immunohistochemistry. The daily oral administration of alendronate to EAE-induced mice significantly reduced the severity of paralysis and lowered T cell proliferation. Additionally, histopathological examinations confirmed that alendronate mitigated inflammation in the spinal cord after EAE induction, suppressed the infiltration of CD68-positive inflammatory cells, and reduced the production of various pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, as well as inducible nitric oxide synthase (iNOS). Furthermore, the alendronate-treated group exhibited a decrease in the number of iNOS-positive inflammatory cells compared to the vehicle-treated group. Taken together, the present results suggest that alendronate alleviated neuro-inflammation in the spinal cords of EAE-induced mice, which is an animal model of multiple sclerosis, possibly by inhibiting the downstream effects of the mevalonate cascade.     

氯马斯汀促进实验性自身免疫性脑脊髓炎小鼠的再髓鞘化

摘要：目的 探讨氯马斯汀对实验性自身免疫性脑脊髓炎（EAE）小鼠髓鞘碱性蛋白（MBP）表达的影响。方法 将50只C57BL/6雌性小鼠适应性喂养后按随机数字表法分成5组,即正常对照组、EAE模型组和氯马斯汀高、中、低剂量组［40、20、10 mg/（kg·d）］,每组10只。EAE模型组及氯马斯汀各剂量组采用抗原髓鞘少突胶质细胞糖蛋白35-55（MOG35-55）诱导EAE模型。氯马斯汀各剂量组每天腹腔注射氯马斯汀预防性给药,正常对照组和EAE模型组腹腔注射生理盐水,连续21 d。建模后每天评判小鼠临床表现,进行神经功能障碍评分。21 d后统一处死小鼠,观察各 组小鼠脊髓组织病理切片的Luxol Fast Blue染色情况并进行脱髓鞘评分。观察各组小鼠脑组织匀浆中MBP及其mRNA表达的变化。结果正常对照组未发病。EAE模型组和氯马斯汀各剂量组从7~8 d开始不同程度发病,在12~16 d逐渐达到发病高峰,且随着干预剂量增大,小鼠发病高峰评分降低（P＜0.01）。正常对照组小鼠脊髓组织未发生脱髓鞘改变;EAE模型组小鼠脊髓组织发生明显脱髓鞘改变;氯马斯汀各剂量组小鼠脱髓鞘程度明显改善,且改善程度呈剂量依赖性（P＜0.01）。与正常对照组比较,EAE模型组与氯马斯汀各剂量组MBP蛋白及mRNA表达明显减少（P＜0.05）;与EAE模型组对比,氯马斯汀各剂量组MBP及其mRNA表达显著升高（P＜0.05）,呈剂量依赖性。结论氯马斯汀对MOG35-55诱导小鼠EAE模型具有防治作用,呈现剂量依赖性,其机制可能与促进MBP表达、改善再髓鞘化有关。     

Reducing suffering in experimental autoimmune encephalomyelitis (EAE)

This report is based on discussions and submissions from an expert working group consisting of veterinarians, animal care staff and scientists with expert knowledge relevant to the field. It aims to facilitate the implementation of the Three Rs (replacement, reduction and refinement) in the use of animal models or procedures involving experimental autoimmune encephalomyelitis (EAE), an experimental model used in multiple sclerosis research. The emphasis is on refinement since this has the greatest potential for immediate implementation. Specific welfare issues are identified and discussed, and practical measures are proposed to reduce animal use and suffering. Some general issues for refinement are summarised to help achieve this, with more detail provided on a range of specific measures to reduce suffering.     

Wistar大鼠实验性变态反应性脑脊髓炎模型的制备

目的　探讨Wistar大鼠诱导实验性变态反应脑脊髓炎 (EAE)动物模型 ,检测EAE中起关键趋化作用的巨噬细胞趋化因子 - 1(MCP- 1)的表达。方法　采用免疫诱导方法制备EAE模型并苏木素 伊红 (HE)染色 ,同时用原位杂交法检测EAE大鼠MCP -1的表达。结果　免疫后 12～ 17d ,发病鼠出现EAE临床症状 ,HE染色 ,光镜下可见血管周围炎性浸润 ,MCP- 1的表达明显增加。结论　Wistar大鼠成功的诱导实验性变态反应脑脊髓炎模型是可信、可用的。     

免疫球蛋白预防给药对实验性自身免疫性脑脊髓炎的保护作用研究

目的研究免疫球蛋白预防给药对实验性自身免疫性脑脊髓炎（EAE）大鼠的保护作用，并初步探讨其保护EAE的机制，为临床治疗多发性硬化提供理论依据。方法建立Lewis大鼠主动免疫EAE实验动物模型，分别于致敏当日给予免疫球蛋白（1g·kg^-1·d^-1×2d）尾静脉静注（预防给药组），并设立相应对照组。动态观察各组大鼠的临床评分，在发病高峰期取大鼠外周血，采用流式细胞检测技术检测T细胞亚群（CD4^＋T细胞和CD8^＋T细胞）数量。结果预防给予免疫球蛋白显著改善EAE临床症状，神经功能评分较对照组显著降低（P〈0．05），发病时间及发病高峰延迟，病程缩短。预防组CD4^＋T细胞较对照组明显降低（P〈0．05），CD88^＋T细胞及CD4^＋／CD8^＋比值2组比较无明显差异。结论预防给予免疫球蛋白可以明显改善EAE大鼠神经缺损症状并缩短病程。免疫球蛋白对EAE的保护作用可能与下调CD4^＋T细胞数量、抑制CD4^＋T细胞增殖、调节其功能有关。     

The effect of ribavirin on reactive astrogliosis in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of CNS inflammatory and demyelinating disease multiple sclerosis. Microglia and astrocytes represent two related cell types involved in the brain pathology in EAE. Accumulations of hypertrophic reactive astrocytes, intensely stained with glial fibrillary acidic protein (GFAP), which also expressed vimentin, are prominent features of EAE lesions. Recent studies from our laboratory reported that ribavirin attenuated the disease process in EAE by reducing clinical and histological manifestations. EAE was induced in genetically susceptible Dark Agouti rats with syngeneic spinal cord homogenate in complete Freund's adjuvant. Real time PCR and immunohistochemistry were used for determination of GFAP and vimentin gene and tissue expression. We have observed the increased gene and tissue expression of GFAP and vimentin in EAE rats. Ribavirin treatment significantly decreased the number of reactive astrocytes at the peak of disease. At the end of the disease, we have observed reactive GFAP(+) and vimentin(+) astrocytes in both immunized and ribavirin-treated groups, accompanied by increased level of GFAP mRNA. The present study indicates that ribavirin may have the ability to attenuate astrocyte proliferation and glial scaring at the peak of the disease and modulate the astroglial response to EAE during the time-course of the disease.     

Effects of human recombinant-interferon β in experimental autoimmune encephalomyelitis in guinea pigs

Context: Although clinical data for beneficial effects of Betaferon, human recombinant-interferon (r-IFN) β-1b, are accumulating, what is less evident is how and why it works.

Objective: The present study was carried out to examine whether Betaferon suppresses progression of experimental autoimmune encephalomyelitis (EAE).

Materials and methods: The EAE model was employed in guinea pigs in vivo, and mononuclear cell proliferation and 2′,5′-oligoadenylate synthetase activity were assessed in vitro.

Results: Betaferon was more reactive in two assays of guinea pigs, mitogen-induced proliferation of peripheral blood mononuclear cells and 2′,5′-oligoadenylate synthetase activity of blood, than in rats and rabbits. Guinea pigs were immunized actively by antigen, porcine myelin basic protein. The neurological deficits were assessed by clinical signs scored daily. Guinea pig Betaferon, replaced with guinea pig albumin (GPA), at 1.2 and 12.0 MIU/kg/day or vehicle was administered subcutaneously daily for 20 days in the immunized guinea pigs. GPA-Betaferon suppressed the manifestation of ataxia or more progression of chronic neurological deficits significantly at 1.2 MIU/kg (p <0.05). Two out of 10 animals manifested no clinical signs in the GPA-Betaferon-treated group with the higher dose, while all animals were worsened with typical clinical signs of EAE in the vehicle group where mononuclear cell infiltrates around blood vessels were seen in the spinal cord of vehicle-treated animals.

Discussion and conclusion: Human r-IFN β-1b attenuates progression of neurological deficits in the EAE model of guinea pigs with evidence for higher susceptibility of animal cells/tissues to the human cytokine, in contrast with rodents and rabbits.     

Ribavirin ameliorates experimental autoimmune encephalomyelitis in rats and modulates cytokine production

To determine the mechanism underlying ribavirin induced amelioration of experimental autoimmune encephalomyelitis (EAE), cytokine profiles were evaluated in draining lymph node (DLN) cell culture supernatants and spinal cord obtained from EAE and/or ribavirin-treated EAE Dark Agouti rats. Administration of ribavirin to EAE rats markedly affected the production of pro-inflammatory cytokines IFN-gamma, IL-1beta and TNF-alpha in DLN and spinal cord, thus shifting the balance towards the anti-inflammatory cytokines IL-10 and TGF-beta. These findings suggest that ribavirin attenuates EAE by limiting cytokine-mediated immunoinflammatory events leading to CNS destruction. The conducted experiments provide rationale for ribavirin to be considered as a candidate drug in the development of new therapeutic strategies for the treatment of autoimmune diseases in humans, such as multiple sclerosis.     

Oral flavonoids delay recovery from experimental autoimmune encephalomyelitis in SJL mice

Flavonoids are food components that appear to have potential beneficial health effects. There is a range of in vitro studies supporting the anti-oxidant and anti-inflammatory properties of flavonoids. Previously, we demonstrated that in vitro flavonoids, including luteolin and apigenin, inhibit proliferation and IFN-gamma production by murine and human autoimmune T cells. In the present study, we examined the effects of oral flavonoids as well as of curcumin on autoimmune T cell reactivity in mice and on the course of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. Continuous oral administration of flavonoids significantly affected antigen-specific proliferation and IFN-gamma production by lymph node-derived T cells following immunization with an EAE-inducing peptide. Both luteolin and apigenin suppress proliferative responses as they did in vitro, whereas IFN-gamma production on the other hand was enhanced. Other flavonoids exerted differential effects on proliferation and IFN-gamma production. The effects of flavonoids and curcumin on EAE were assessed using either passive transfer of autoimmune T cells or active disease induction. In passive EAE, flavonoids led to delayed recovery of clinical symptoms rather than to any reduction in disease. In active EAE, the effects were less pronounced but also, in this case, the flavonoid hesperitin delayed recovery. Oral curcumin had overall mild but beneficial effects. Our results indicate that oral flavonoids fail to beneficially influence the course of EAE in mice but, instead, suppress recovery from acute inflammatory damage.     

Melatonin enhances interleukin-10 expression and suppresses chemotaxis to inhibit inflammation in situ and reduce the severity of experimental autoimmune encephalomyelitis

Melatonin is the major product secreted by the pineal gland at night and displays multifunctional properties, including immunomodulatory functions. In this study, we investigated the therapeutic effect of melatonin in experimental autoimmune encephalomyelitis (EAE). We demonstrated that melatonin exhibits a therapeutic role by ameliorating the clinical severity and restricting the infiltration of inflammatory Th17 cells into the CNS of mice with myelin oligodendrocyte glycoprotein (MOG)-induced EAE. Furthermore, melatonin enhances splenic interleukin (IL)-10 expression in regulatory T cells by inducing IL-27 expression in the splenic DC; it also suppresses the expression of IFN-γ, IL-17, IL-6, and CCL20 in the CNS and inhibits antigen-specific T cell proliferation. However, there were no significant differences in the percentage of splenic regulatory T cells. These data provide the first evidence that the therapeutic administration of melatonin is effective in mice with EAE and modulates adaptive immunity centrally and peripherally. Thus, we suggest that melatonin could play an adjunct therapeutic role in treating human CNS autoimmune diseases such as multiple sclerosis. Melatonin merits further studies in animals and humans.     

C-Phycocyanin ameliorates experimental autoimmune encephalomyelitis and induces regulatory T cells

For decades Experimental Autoimmune Encephalitis (EAE) has remained as an unsurpassed multiple sclerosis (MS) animal model. C-Phycocyanin (C-Pc) has been reported to exhibit pharmacological properties that may be expected to symptomatically improve EAE and MS. However, in this paper we reveal a basic underlying mechanism that may provide a new approach to the rationale of the overall beneficial effect of this natural antioxidant. We demonstrate that C-Pc is able to trigger mechanisms preventing or downgrading EAE expression and induces a regulatory T cell (Treg) response, in peripheral blood mononuclear cells (PBMC) from MS patients. These results agree with reports suggesting that Treg limit acute MS attacks and that C-Pc may act as a neuroprotector and thereby reverts the organic and functional damage in neurodegenerative disorders of the central nervous system (CNS). Moreover, evidence is provided on the antioxidant activity of C-Pc within the CNS, intended to improve the myelin and axonal damage of EAE induced Lewis rats. Our results indicate that specific Treg activation may represent a central and essential mechanism in supporting the therapeutic potential of C-Pc for MS and may lead to new and more effective therapies; this property would then complement and enhance other proven active principles such as interferons (IFN), giving rise to combined therapies.     

氨基葡萄糖对实验性自身免疫性脑脊髓炎大鼠基质金属蛋白酶的影响

目的:探讨不同剂量氨基葡萄糖(GS)对实验性自身免疫性脑脊髓炎(EAE)大鼠临床指标和基质金属蛋白酶-9(MMP-9)的影响.方法:用完全抗原和减毒百日咳杆菌原液免疫大鼠后随机分为三组,①EAE组(n=40):腹腔注射4.5 ml/kg·d-1磷酸盐缓冲溶液;②GS1组(n=40):腹腔注射90 mg/kg·d-1溶液;③Gs2组(n=40):腹腔注射180mg/kg·d-1GS溶液.各组分别于免疫后(pi)6、8、10、12、14、16和18 d取大鼠脑和脊髓制成石蜡切片,行HE染色和MMP-9免疫组化染色,进行组织学观察.结果:pi 14 d疾病发展达高峰,EAE组、GS11和GS2组的发病率分别为77.8%、44.4%和11.1%(n=18),对三组发病率差异行X2检验有统计学意义(P<0.01),两两相互比较有显著统计学差异(P<0.05).症状评分EAE组(2.33±1.57)与GS1组(1.00±1.24)和GS2组(0.60±0.89)行秩和检验比较差异,结果均有显著统计学意义(P<0.05),GS1组与GS2组症状评分比较差异无显著性(P>0.05).同pi 8 d相比,EAE组、GS1组、GS2组体重分别平均减少(24.50±8.42)、(14.00±7.85)和(10.50±5.44)g.免疫组化染色MMP-9在脊膜细胞、炎细胞和内皮细胞内均呈阳性表达.pi 10 d EAE组MMP-9表达积分光密度达最大值,而Gs治疗组pi 12d MMP-9积分光密度表达达高峰.pi 6-16 d GS治疗组MMP-9积分光密度与EAE组差异行方差分析,有显著统计学差异(P<0.01).pi 8-14 d GS2组与GS1组相比变化趋势也有统计学差异(P<0.05).结论:Gs可抑制MMP-9的生成,对EAE发病具有一定程度的保护作用.     

Oral fingolimod rescues the functional deficits of synapses in experimental autoimmune encephalomyelitis

BACKGROUND AND PURPOSE Alterations of glutamate-mediated synaptic transmission occur early during neuroinflammatory insults, and lead to degenerative neuronal damage in multiple sclerosis (MS) and also in experimental autoimmune encephalomyelitis (EAE), which is a murine model of MS. Fingolimod is an effective orally active agent for the treatment of MS, affecting lymphocyte invasion of the brain. However, it is still unclear if fingolimod can be neuroprotective in this disorder. EXPERIMENTAL APPROACH Using neurophysiological recordings and morphological evaluation of dendritic integrity, we evaluated the effects of oral fingolimod on the clinical score of EAE mice in order to determine whether the compound was associated with preservation of synaptic transmission. KEY RESULTS Oral fingolimod prevented and reversed the pre- and postsynaptic alterations of glutamate transmission in EAE mice. These effects were associated with a clear amelioration of the clinical deterioration seen in EAE mice, and with a significant inhibition of neuronal dendritic pathology. Fingolimod did not alter the spontaneous excitatory postsynaptic currents in control animals, suggesting that only the pathological processes behind the inflammation-induced defects in glutamate transmission were modulated by this compound. CONCLUSIONS AND IMPLICATIONS The beneficial effects of fingolimod on the clinical, synaptic and dendritic abnormalities of murine EAE might correlate with the neuroprotective actions of this agent, as observed in MS patients. LINKED ARTICLE This article is commented on by Gillingwater, pp. 858-860 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01612.x.     

黄芩素对实验性自身免疫性脑脊髓炎小鼠的 影响及机制研究

目的 探讨黄芩素（BAI）对实验性自身免疫性脑脊髓炎（EAE）小鼠的防治作用及其对中枢神经系统小胶 质细胞（MG）极化和炎性因子的影响。方法 将50只雌性C57BL/6小鼠按照随机数字表法分为空白对照组,EAE模 型组及BAI低、中、高剂量组,每组10只。空白对照组不做处理,其余各组建立EAE模型。BAI低、中、高剂量组每日 分别给予BAI 75、150、300 mg/kg灌胃,空白对照组及EAE模型组给予等体积生理盐水灌胃,连续14 d。观察小鼠的 神经功能缺损和脊髓组织炎性脱髓鞘情况。采用免疫荧光双重染色法检测小鼠脊髓组织离子钙接头蛋白1（Iba-1） 阳性MG中M1型MG标志物诱导型一氧化氮合酶（iNOS）、M2型MG标志物精氨酸酶1（Arg1）的表达情况及分歧指数 （RI）变化,实时荧光定量PCR法检测小鼠脊髓组织中iNOS、Arg1及炎性因子肿瘤坏死因子-α（TNF-α）、白细胞介 素-10（IL-10）mRNA的表达情况。结果 空白对照组小鼠未见发病,其余各组均不同程度发病。与EAE模型组比 较,BAI各剂量组小鼠发病潜伏期及达高峰期时间延长,神经功能障碍评分降低（均P＜0.05）,脊髓组织脱髓鞘程度 减轻（均P＜0.05）,Iba-1阳性MG中iNOS表达降低,Arg1表达升高,RI值减小（均P＜0.05）,MG的形态倾向于M2型 极化,脊髓组织中iNOS、TNF-α mRNA表达降低,Arg1、IL-10 mRNA表达升高（均P＜0.05）,且BAI剂量越大,变化越 明显。结论 BAI对EAE小鼠发病具有防治作用,且呈剂量依赖性,其机制可能与纠正M1/M2型MG失衡及调节炎 性因子TNF-α、IL-10表达有关。     

The ambivalent role of apoptosis in experimental autoimmune encephalomyelitis and multiple sclerosis

Apoptosis is synonymous to programmed cell death, which occurs in response to a plethora of stimuli and employs a series of highly conserved mediators and pathways. Its ambivalent role in immunology is illustrated by the fact that this process not only serves homeostatic functions but also exerts harmful effects including tissue damage. This is particularly true for neuroinflammatory diseases such as multiple sclerosis (MS), the most frequent neurological disease to afflict adolescents in the western world. Considerable insight into the role of apoptosis in MS has been obtained by using its animal model experimental autoimmune encephalomyelitis (EAE). Experiments using the EAE model have revealed that cell death affects both infiltrating lymphocytes and CNS resident cells, and that it contributes to axonal injury as well as the resolution of inflammation. Furthermore, it was discovered that the molecules involved in inducing and regulating this process are the Fas-FasL system, pro- and anti-apoptotic Bcl-2 family members, 'initiator' and 'effector' caspases, glucocorticoid hormones and various modulatory proteins. The variety of apoptotic mechanisms in combination with their often opposing effects on the disease course highlights the need for a detailed understanding of apoptosis in this context. In the future, this may pave the way to novel approaches aiming at interfering with the apoptotic process to prevent tissue damage or at intentionally inducing cell death in order to ameliorate the disease by deleting autoreactive lymphocytes.