Relationship between the clinical scoring and demyelination in central nervous system with total antioxidant capacity of plasma during experimental autoimmune encephalomyelitis development in mice

Experimental autoimmune encephalomyelitis (EAE) was induced in a mouse model (C57/BL6) to investigate the antioxidant status of animals at various clinical stages of the disease. For this purpose, blood, brain and spinal cord samples from EAE mice were collected and examined at different scores following post-immunization with myelin oligodendrocyte glycoprotein (MOG). The clinical sign of mobility of animals on different days was associated with gradual increase in lipid peroxidation products (malondialdehyde, i.e. MDA) in brain and spinal cord. Changes in lipid peroxidation during EAE progression was inversely related to superoxide dismutase (SOD) activity in erythrocyte preparation. However, suppression of catalase in erythrocytes, tissue glutathione (GSH) and plasma total antioxidant capacity (FRAP assay) were the early events in EAE, occurred during scores 1 and 2. Biochemical alterations were corroborated with histopathological observations showing demyelination and inflammatory foci in central nervous system (CNS) of animals suffering from partial hind limb paralysis (score 3). These data suggest that generation of MDA in CNS is a continuous process during EAE induction and suppression of antioxidant factors are early events of the disease, but crucial in increasing the vulnerability of CNS to demyelinating lesions.     

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。     

细胞因子在实验性自身免疫性脑脊髓炎耐受中的作用

细胞因子(CK)在实验性自身免疫性脑脊髓炎(EAE)的免疫机制中起着重要作用。Th细胞的不同转化决定EAE的发生、发展或抑制。由多种CK构成的免疫调节网络操纵着Th细胞的免疫应答。通过作用Th细胞使之向抑制EAE方向转化,从而寻找对EAE耐受的途径,是目前EAE研究的一个重要方面。以下就与EAE耐受相关的CK研究进行综述,探讨EAE免疫病理机制。     

T细胞定向迁移诱导实验性自身免疫性脑脊髓炎小鼠胸腺萎缩

目的 初步探讨实验性自身免疫性脑脊髓炎(EAE)小鼠胸腺萎缩的机制.方法 髓鞘少突胶质细胞糖蛋白(MOG)免疫C57BL/6小鼠诱导EAE,卵清白蛋白(OVA)免疫的小鼠作为对照;不同时间点计数胸腺、脾脏、淋巴结细胞总数,检测脾脏中胸腺来源细胞及中枢神经系统(CNS)浸润细胞.结果 MOG肽成功诱导EAE动物模型,小鼠出现典型的肢体运动功能障碍,脊髓可见大量炎性细胞浸润;MOG和OVA免疫均诱导胸腺细胞增加,第5天达到高峰,随后逐渐下降;EAE发病后胸腺细胞迅速减少,发病高峰期几乎完全消失,胸腺严重萎缩;MOG和OVA免疫后脾脏和淋巴结细胞总数持续升高,新近胸腺来源的T细胞增加尤其明显;EAE发病后脾脏T细胞总数减少,CNS浸润淋巴细胞总数增加.结论 大量T细胞在胸腺发育成熟并释放到外周,进而定向迁移至CNS诱导EAE是胸腺萎缩的主要原因.     

Effect of geranylgeranylacetone on optic neuritis in experimental autoimmune encephalomyelitis

Optic neuritis is an acute inflammatory demyelinating syndrome of the central nervous system (CNS) that often occurs in multiple sclerosis (MS). Since it can cause irreversible visual loss, especially in the optic-spinal form of MS or neuromyelitis optica (NMO), the present study was conducted to assess the effects of geranylgeranylacetone (GGA) on optic neuritis in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Myelin oligodendrocyte glycoprotein-induced EAE mice received oral administration of GGA at 500 mg/kg or vehicle once daily for 22 days. The effects of GGA on the severity of optic neuritis were examined by morphological analysis on day 22. Visual functions were measured by the multifocal electroretinograms (mfERG). In addition, the effects of GGA on severity of myelitis were monitored both on clinical signs and morphological aspects. The visual function, as assessed by the second-kernel of mfERG, was significantly improved in GGA-treated mice compared with vehicle-treated mice. GGA treatment decreased the number of degenerating axons in the optic nerve and prevented cell loss in the retinal ganglion cell layer. However, the severity of demyelination in the spinal cord remained unaffected with the treatment of GGA. These results suggest that oral GGA administration has beneficial effect on the treatment for optic neuritis in the EAE mouse model of MS.     

Exacerbation of experimental autoimmune encephalomyelitis in rodents infected with murine gammaherpesvirus-68

Viral infections have long been suspected to play a role in the pathogenesis of multiple sclerosis. In the present study, two different rodent models of experimental autoimmune encephalomyelitis (EAE) were used to demonstrate the ability of murine gammaherpesvirus-68 (gammaHV-68) to exacerbate development of neurological symptoms. SJL mice received UV-inactivated gammaHV-68 or intranasalgammaHV-68, followed by immunization against proteolipid-protein peptide 139-151. Infected mice became moribund within 10 days post-immunization, whereas mice exposed to UV-inactivated gammaHV-68 recovered. In the second model, Lewis rats were exposed to UV-inactivated gammaHV-68 or to gammaHV-68, followed by passive transfer of encephalitogenic T lymphocytes specific for myelin basic protein. Consistently, infected rats had higher clinical scores, and this result was observed during acute or latent gammaHV-68 infection. It is unlikely that this gammaHV-68-induced exacerbation was due to significant viral replication within the central nervous system since nested PCR, viral plaque assays, and infectious-centers assays demonstrated no detectable virus in spinal cords or brains of infected rodents undergoing EAE. Taken together, these studies demonstrate increased clinical symptoms of EAE in rodents infected by a gammaherpesvirus that has a limited ability to invade the central nervous system.     

红景天苷对实验性自身免疫性脑脊髓炎小鼠的干预作用与机制

目的:观察不同浓度红景天苷(salidroside)对实验性自身免疫脑脊髓炎(experimental autoimmuneencephalomyelitis,EAE)小鼠模型的作用,并探讨初步的作用机制.方法:建立实验性自身免疫性脊髓炎小鼠模型,免疫当天起给予高中低剂量的红景天苷灌胃,1次/d,共给药35 d.给药完成后,进行神经功能评分,TUNEL染色检测脊髓髓鞘组织的细胞凋亡情况,ATP水平检测试剂盒检测脊髓组织ATP含量水平,免疫印迹法检测线粒体内细胞色素c氧化酶(mito cyto-c)、胞浆中细胞色素c氧化酶(cyto cyto-c)、活化的含半胱氨酸的天冬氨酸蛋白水解酶-3(cleaved cas-3)与活化的含半胱氨酸的天冬氨酸蛋白水解酶-9(cleaved cas-9)蛋白表达水平.结果:红景天苷降低EAE小鼠的神经功能评分,后延发病时间;随红景天苷浓度升高,脊髓髓鞘组织的细胞凋亡数逐渐降低,脊髓组织ATP水平显著升高;EAE小鼠脊髓组织细胞胞浆中的cyto-c蛋白表达显著升高,同时cleavedcas-3与cleaved cas-9蛋白表达显著升高,红景天苷干预后,胞浆中cyto-c蛋白表达显著下降,同时cleaved cas-3与cleaved cas-9蛋白表达下降.结论:红景天苷对缓解实验性自身免疫性脊髓炎症状具有良好的效果,可能通过抑制内源性线粒体通路而降低脊髓组织细胞的凋亡而发挥作用.     

实验性自身免疫性脑脊髓炎小鼠发病高峰期外周及中枢淋巴细胞亚群的变化

目的:观察髓鞘少突胶质细胞糖蛋白MOG35-55诱导的实验性自身免疫性脑脊髓炎小鼠发病高峰期中枢及外周淋巴细胞亚群的变化,探讨EAE发病高峰期细胞与体液免疫学的变化。方法:用MOG35-55免疫诱导雌性C57BL/6小鼠制作EAE模型,记录小鼠行为学变化,HE染色观察CNS炎症组织病理变化,使用流式细胞仪检测小鼠中枢及外周脾脏淋巴细胞中CD3+CD4+、CD3+CD8+、CD4+CD25+、B220+细胞亚群变化情况。结果:EAE组小鼠中枢神经系统有CD3+CD4+、CD3+CD8+、CD4+CD25+、B220+淋巴细胞的浸润,CFA阴性对照组中枢神经系统未检测到淋巴细胞浸润。EAE组小鼠外周脾细胞中CD3+CD4+、CD3+CD8+细胞较CFA阴性对照组减少(P0.05),B220+细胞较CFA阴性对照组明显升高(P0.01),CD4+CD25+细胞较CFA阴性对照组升高但无统计学差异。结论:小鼠在EAE发病高峰期,外周脾细胞中CD3+CD4+、CD3+CD8+阳性细胞明显减少,B220+明显升高,CD4+CD25+也开始有升高趋势,表明EAE发病高峰期细胞免疫及体液免疫共同调控了EAE的病理过程,T淋巴细胞与B淋巴细胞都起了很重要的主导作用。     

间充质干细胞改善自身免疫性脑脊髓炎的作用研究

目的 探讨间充质干细胞(MSC)治疗自身免疫性脑脊髓炎(EAE)的机制.方法 用MOG35-55肽和弗氏完全佐剂乳化剂诱导建立C57BL/6小鼠的EAE模型;分离纯化培养骨髓来源MSC细胞;临床评分和脊髓组织切片评估小鼠的发病情况;酶联免疫吸附试验(ELISA)检测EAE组,MSC治疗组和对照组小鼠外周血细胞因子TNF-α,IFN-γ,IL-4和TGF-β的含量;流式细胞术分析3组小鼠脾脏细胞中CD4+ Foxp3+调节性T细胞(Treg)的比例变化.结果 分离纯化C57BL/6小鼠MSC成功;MSC治疗组小鼠的临床评分明显降低,且脊髓组织切片显示T细胞浸润显著减少;外周血中细胞因子IL-4,TGF-β显著高于对照组(t=7.719、17.17,P均＜0.01)和EAE组(t=54.45、48.36,P均＜0.01),IFN-γ,TNF-α低于对照组(t=104.90、1.998,P均＜0.01)和EAE组(t=270.1、13.58,P均＜0.01);脾脏细胞中CD4+ Foxp3+ Treg细胞的比例明显高于对照组(t=15.91,P＜0.01)和EAE组(t=33.39,P＜0.01).结论 MSC能够有效改善EAE小鼠的症状;其对EAE小鼠的治疗作用是通过上调抗炎细胞因子(IL-4,TGF-β)和Treg细胞并下调促炎因子(IFN-γ,TNF-α)的水平,来发挥免疫调节作用.     

Deletion of both ICAM-1 and C3 enhances severity of experimental autoimmune encephalomyelitis compared to C3-deficient mice

Multiple sclerosis (MS) is an autoimmune disease characterized by central nervous system (CNS) inflammation and leukocyte infiltration, demyelination of neurons, and blood-brain barrier breakdown. The development of experimental autoimmune encephalomyelitis (EAE), the animal model for MS is dependent on a number of components of the immune system including complement and adhesion molecules. Previous studies in our lab have examined the role of C3, the central complement component, and intercellular adhesion molecule-1 (ICAM-1) a key cell adhesion molecule involved in leukocyte trafficking to sites of inflammation including the CNS. In these studies we demonstrated that myelin oligodendrocyte glycoprotein (MOG)-induced EAE is markedly attenuated in both ICAM-1(-/-) and C3(-/-) mice. Given the pivotal role that these proteins play in EAE, we hypothesized that EAE in ICAM-1(-/-) and C3(-/-) double mutant mice would likely fail to develop. Unexpectedly, EAE in ICAM-1(-/-)xC3(-/-) mice was only modestly attenuated compared to wild type mice and significantly worse than C3(-/-) mice. Leukocyte infiltration was commensurate with disease severity between the three groups of mice. Spinal cord T cells from ICAM-1(-/-)xC3(-/-) mice produced the highest levels of IFN-gamma and TNF-alpha, despite reduced disease severity compared to wild type mice. The mechanisms behind the elevated EAE severity in ICAM-1(-/-)xC3(-/-) mice may relate to altered homing of leukocytes or processing of self-antigens in the double mutant background.     

Fasudil联合骨髓源神经干细胞对EAE小鼠的神经保护作用

目的:探讨Rho激酶抑制剂法舒地尔(fasudil)联合骨髓源神经干细胞(bone marrow-derived neural stem cells,BM-NSCs)对实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠的神经保护作用。方法:32只雌性C57BL/6小鼠(8~10周龄),用髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)免疫,制备EAE,随机分为对照(dd H_2O)组、fasudil组、BM-NSCs组和fasudil+BM-NSCs组,并分别给予相应处理。免疫后检测小鼠临床症状和相关神经营养因子的表达,Image-Pro Plus 6.0软件进行阳性细胞计数,Graph Pad Prism 5软件统计分析。结果:与dd H_2O组比较,fasudil+BM-NSCs处理组明显延迟小鼠的平均起病时间,降低最高临床评分,并缓解EAE小鼠的临床症状;fasudil组、BM-NSCs组和fasudil+BM-NSCs组中脑源性神经营养因子、胶质细胞源性神经营养因子、神经生长因子、神经营养因子3和睫状神经营养因子阳性细胞数均有不同程度的增加,其中,fasudil+BM-NSCs组上述神经营养因子的表达明显多于dd H_2O组、fasudil组和BM-NSCs组(P0.01)。结论:Fasudil联合BM-NSCs通过协同和叠加效应促进神经营养因子表达,改善中枢神经系统微环境,发挥神经保护作用,从而缓解EAE的临床症状。     

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。     

Anti-TWEAK monoclonal antibodies reduce immune cell infiltration in the central nervous system and severity of experimental autoimmune encephalomyelitis

TWEAK is a member of the TNF family, constitutively expressed in the central nervous system (CNS), with pro-inflammatory, proliferative or apoptotic effects depending upon cell types. Its receptor, Fn14, is expressed in CNS by endothelial cells, reactive astrocytes and neurons. We showed that TWEAK and Fn14 mRNA expression increased in spinal cord during experimental autoimmune encephalomyelitis (EAE). We investigated the role of TWEAK during EAE using neutralizing anti-TWEAK antibody in myelin oligodendrocyte glycoprotein (MOG) induced EAE in C57BL/6 mice. We observed a reduction of disease severity and leukocyte infiltration when mice were treated after the priming phase.     

The protective effect of total glucosides of white paeony capsules on experimental autoimmune encephalomyelitis

AimsTo learn about the effect and mechanism of total glucosides of white peony capsule (TGP), on experimental autoimmune encephalomyelitis (EAE), an acknowledged animal model of multiple sclerosis (MS).MethodsThe rat model of EAE was induced by subcutaneous injection with guinea pig spinal cord homogenate. The severity of the disease model was assessed by clinical score, hematoxylin and eosin (H&E) and luxol fast blue (LFB). Immunohistochemical assay was used to observe the types of inflammatory cells and adhesive molecule expression. Enzyme-linked immunosorbent assay (ELISA) was applied to detect content of the stem cell growth factor / mast cell growth factor (scf/MGF), interleukin-6 (IL-6) and IL-2. Immunofluorescence assay was applied to observe the expression of connexin43 (Cx43), glial fibrillary acidic protein (GFAP), connexin47 (Cx47) and the monoclonal antibody anti-adenomatous polyposis coli (APC) clone CC1.ResultsCompare with the animals in EAE model group, TGP treated rats (particularly those treated with high doses) showed a significant decrease in morbidity, clinical scores, CNS infiltration of inflammatory cells (including mononuclear macrophages, CD4⁺ and CD8⁺ T cells) and demyelination. The key adhesion molecule ICAM-1, cytokines IL-2、IL-6 and scf/MGF were significantly decreased with TGP treatment. Oppositely, PD-1, connexin47 in oligodendrocytes and connexin43 in astrocytes were elevated with TGP treatment.ConclusionTo sum up, TGP exhibited a significantly prevention and treatment effect on EAE rat model, and this improvement was achieved through a combination way composed of glial and inflammatory cells, junction proteins, various factors including adhesion factors, interleukins and scf/MGF.     

Suppression of experimental autoimmune encephalomyelitis in Lewis rats by antibodies against CD2

The immunotherapeutic potential of three anti-rat CD2 monoclonal antibodies (mAb) (OX34, OX54, OX55) and the combination of OX54 with OX55 was tested in Lewis rat experimental autoimmune encephalomyelitis (EAE). In actively induced EAE, a single injection of OX34 2 days before immunization with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) completely prevented or greatly attenuated EAE in all animals. Injection of OX54 acted moderately suppressive while OX55 or OX54/55 did not affect disease severity. Abrogation of EAE by OX34 was not restricted to its application before immunization. Therapeutic administration of all three mAb and the Ab combination from onset of first clinical signs efficiently blocked progression of disease and prevented all animals from developing hind limb paresis. In adoptive transfer EAE induced with in vitro activated cells of an encephalitogenic T helper line, clinical and histological signs were completely prevented by injection of OX34 on the day of cell transfer and 4 days later, underlining the strong impact of anti-CD2 mAb on the effector phase of disease. Immunocytofluorometric analysis of peripheral blood lymphocytes after a single Ab injection demonstrated that all mAb induced a variable degree of transient reduction in T cell numbers and modulation of CD2 antigens. In contrast to the other mAb, OX34 persisted on lymphocytes for at least 11 days, which may explain its unique suppressive effect on EAE after a single injection before immunization. The assumption that prophylactic administration of OX34 also inhibits MBP-induced EAE, due to persistence into the effector phase, was substantiated by the finding that none of the mAb prevented generation of an antigen-specific cellular response in MBP/CFA-immunized animals. Since none of the Ab induced T cell unresponsiveness or inhibited T cell activation by antigen- or Ab-mediated stimulation of the T cell receptor, we suggest that their marked action on the effector phase of EAE may rely on inhibition of T cell infiltration into the central nervous system. The demonstrated efficacy of these anti-CD2 mAb in EAE suggests a potential therapeutic role that may be equal to that of anti-CD4 or anti-T cell receptor Ab.     

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。     

外源性TGF-β1对实验性自身免疫性脑脊髓炎模型小鼠TLR4表达的影响

目的:观察外源性TGF-β1对实验性自身免疫性脑脊髓炎模型小鼠TLR4和MD-2表达的影响,探讨其可能的作用机制。方法:以MOG35-55免疫C57BL/6J小鼠建立EAE模型,多时间点皮下注射TGF-β1作为干预,RT-PCR检测实验小鼠脑中TLR4的mRNA表达;ELISA法检测外周血清中MD-2的水平。结果:与EAE组相比,TGF-β1组在发病初期TLR4和MD-2表达差异无统计学意义(P>0.05,P>0.05);高峰期TLR4表达增高(P<0.05),MD-2表达无差异(P>0.05);慢性期TLR4和MD-2表达均降低(P<0.05,P<0.05)。结论:外源性TGF-β1可能通过上调TLR4的表达而影响EAE小鼠的病情。     

The influence and impact of ageing and immunosenescence (ISC) on adaptive immunity during multiple sclerosis (MS) and the animal counterpart experimental autoimmune encephalomyelitis (EAE)

The human ageing process encompasses mechanisms that effect a decline in homeostasis with increased susceptibility to disease and the development of chronic life-threatening illness. Increasing age affects the immune system which undergoes a progressive loss of efficiency, termed immunosenescence (ISC), to impact on quantitative and functional aspects of innate and adaptive immunity. The human demyelinating disease multiple sclerosis (MS) and the corresponding animal model experimental autoimmune encephalomyelitis (EAE) are strongly governed by immunological events that primarily involve the adaptive arm of the immune response. MS and EAE are frequently characterised by a chronic pathology and a protracted disease course which thereby creates the potential for exposure to the inherent, on-going effects and consequences of ISC. Collective evidence is presented to confirm the occurrence of established and unendorsed biological markers of ISC during the development of both diseases. Moreover, results are discussed from studies during the course of MS and EAE that reveal a premature upregulation of ISC-related biomarkers which indicates untimely alterations to the adaptive immune system. The effects of ISC and a prematurely aged immune system on autoimmune-associated neurodegenerative conditions such as MS and EAE are largely unknown but current evaluation of data justifies and encourages further investigation.