实验性自身免疫性脑脊髓炎小鼠神经损害与脑组织中丝裂原活化蛋白激酶表达的关系

目的观察实验性自身免疫性脑脊髓炎(EAE)模型小鼠脑组织中丝裂原活化蛋白激酶(MAPKs)表达变化及其与神经损害的关系。方法 C57BL/6小鼠随机分为:EAE组(n=12),采用髓鞘少突胶质细胞糖蛋白35-55多肽(MOG35-55)制备成抗原乳剂免疫小鼠;对照组(n=10),用生理盐水处理小鼠。每日观察两组小鼠的行为学变化,并进行神经功能障碍评分。于高峰期处死小鼠,冰冻处理脑与脊髓,行苏木精-伊红染色观察脊髓组织的炎症细胞浸润,LFB染色观察脊髓组织的髓鞘脱失,蛋白印迹法检测小鼠脑组织中MAPKs表达。分析EAE小鼠神经功能障碍改变与中枢神经组织MAPKs表达量的相关性。结果 EAE组与对照组比较:日均神经行为学评分增加(P0.01);脊髓炎症细胞浸润增多(P0.001),髓鞘脱失增多(P0.001)。P-ERK(42)、P-ERK(44)、P-JNK(54)表达量均增多(P0.01、P0.05、P0.05)。神经功能障碍与P-ERK(42)、P-ERK(44)、P-JNK(54)表达呈正相关。结论 EAE高峰期神经损伤程度与中枢神经组织中的P-ERK(42)、P-ERK(44)、P-JNK(54)表达增加相平行,提示MOG35-55诱导的EAE中枢神经损伤可能与MAPKs所激活的信号通路有关。     

实验性自身免疫性脑脊髓炎小鼠脊髓中细胞色素C和凋亡诱导因子表达的变化

目的观察实验性自身免疫性脑脊髓炎(EAE)小鼠脊髓中细胞色素C(CytC)和凋亡诱导因子(AIF)表达的变化。方法 C57BL/6小鼠12只随机分为EAE组和正常对照组。使用髓鞘少突胶质细胞糖蛋白和完全弗氏佐剂混合抗原乳剂免疫C57BL/6小鼠制作EAE模型。每天2次记录各组小鼠体重和神经功能评分的变化。采用苏木精-伊红染色、髓鞘LFB染色、免疫组化染色,检测发病高峰期(免疫后第19天)小鼠脊髓的病理变化、髓鞘脱失程度、CytC和AIF的变化。结果与正常对照组比较,EAE组神经功能评分增加(P<0.01)、炎症细胞浸润增加(P<0.01)、脱髓鞘程度严重(P<0.01)、CytC和AIF表达均增加(P<0.01),而且与EAE的病情变化指标(最高症状评分、病理评分)均呈正相关关系。结论 EAE小鼠发病高峰期脊髓中CytC和AIF表达增加,提示EAE小鼠的病情变化与中枢神经系统的线粒体损伤有关。     

实验性自身免疫性脑脊髓炎小鼠脑内主要组织相容性复合体Ⅱ类抗原和分化群3ε的变化

目的观察实验性自身免疫性脑脊髓炎(EAE)小鼠脑内主要组织相容性复合体Ⅱ类抗原(MHC-Ⅱ)和分化群3ε(CD3ε)的变化。方法 25只C57BL/6小鼠随机分为EAE组(n=13)和正常对照组(n=12)。应用髓鞘少突胶质细胞糖蛋白35-55抗原诱导小鼠EAE模型。观察记录小鼠行为学变化;采用常规及髓鞘染色方法观察脊髓损伤和炎症细胞浸润程度;荧光定量PCR检测脑MHC-Ⅱ和CD3εmRNA的表达。结果 EAE组小鼠发病后EAE症状评分逐渐增加;脊髓炎症细胞浸润明显;髓鞘脱失较多;脑组织MHC-Ⅱ和CD3εmRNA表达显著高于正常对照组(均P<0.01),并与EAE症状评分呈正相关。结论 EAE小鼠脑内MHC-Ⅱ及CD3εmRNA表达水平增高与其病情严重程度一致。     

实验性自身免疫性脑脊髓炎小鼠脑组织和脊髓中脑型肌酸激酶、钙泵和钙中性蛋白酶的变化

目的：观察实验性自身免疫性脑脊髓炎（EAE）小鼠模型脑组织和脊髓中脑型肌酸激酶（CK-BB）、钙泵（CaATPase）和钙中性蛋白酶（calpain）的变化。方法：C57BL/6小鼠随机分为：EAE组（n=9）,用髓鞘少突胶质细胞糖蛋白35-55多肽（MOG35-55）及完全弗氏佐剂混合抗原乳剂免疫小鼠;对照组（n=5）,注射生理盐水。用MOG35-55诱导C57BL/6小鼠,建立（EAE）动物模型（即多发性硬化模型）。观察记录EAE小鼠行为学变化;采用苏木精-伊红染色、LFB髓鞘染色,酶标仪和分光光度计法检测发病高峰期（免疫后第19天）的中枢神经组织病理学变化、CK-BB,CaATPase和calpain酶活性的改变。结果：EAE组与对照组相比：①日均神经功能评分和日累积评分增加（P〈0.01）;②苏木精-伊红染色：中枢炎症细胞浸润明显（P〈0.05）;③LFB染色：髓鞘脱失较多;④CK-BB和CaATPase活性降低,calpain活性增加（P〈0.05）;⑤EAE组小鼠（只）日均累积神经功能评分与CK-BB和CaATPase活性呈负相关（P〈0.05）;⑥EAE组小鼠（只）日均累积神经功能评分与calpain酶活性呈正相关（P〈0.05）。结论：EAE高峰期中枢CK-BB和CaATPase活性降低、calpain活性增高是EAE发生发展的后果,提示EAE时中枢能量代谢和CaATPase、calpain的病理性损害。     

2-(2-苯并呋喃基)-2-咪唑啉对实验性自身免疫性脑脊髓炎小鼠小胶质细胞活化及氧化应激的影响

目的观察2-(2-苯并呋喃基)-2-咪唑啉(2-BFI)对实验性自身免疫性脑脊髓炎(EAE)小鼠CNS小胶质细胞活化及氧化应激的影响。方法将27只C57BL/6小鼠随机分为正常对照组、EAE组和2-BFI干预组,每组9只。采用MOG_(35-55)免疫法制作经典EAE模型。2-BFI干预组小鼠于EAE造模后当日起腹腔注射2-BFI 20 mg/kg,2次/d,连续14 d。正常对照组和EAE组以等量生理盐水代替。每日观察并记录动物的行为学变化、进行神经功能缺损评分。免疫后第20 d处死各组小鼠,采用HE染色和LFB染色观察组织学变化;免疫组化法测定诱导型小鼠CNS中一氧化氮合成酶(iNOS)蛋白和离子钙接头分子(Iba-1)的表达,比色法检测丙二醛(MDA)的含量;并对活化的小胶质细胞数量和iNOS蛋白表达水平进行线性相关分析。结果正常对照组小鼠未见神经系统受损的表现。与EAE组相比,2-BFI干预组日均神经功能缺损评分明显降低,CNS脊髓炎性细胞浸润明显减少,髓鞘脱失严重程度明显减轻,活化的小胶质细胞数量减少,iNOS蛋白表达明显减少,MDA含量降低(P0.05~0.01)。相关分析结果显示,iNOS表达水平与活化的小胶质细胞数量呈正相关(r=0.596,P0.01)。结论 2-BFI能减轻EAE小鼠临床症状和组织学改变,2-BFI对EAE小鼠的神经保护作用于小胶质细胞激活和减轻氧化应激相关。     

2-BFI、阿米洛利和咪唑克生对实验性自身免疫性脑脊髓炎小鼠行为学和病理学变化的药效作用比较

目的比较3种咪唑啉Ⅱ类受体(I2R)高选择性配体2-(2-苯并呋喃基)-2-咪唑啉(2-BFI)、阿米洛利和咪唑克生对实验性自身免疫性脑脊髓炎(EAE)模型小鼠行为学和病理学变化的干预效果。方法在相同实验条件下,髓鞘少突胶质细胞糖蛋白35-55多肽诱导EAE小鼠模型后,分为空白对照组、EAE-生理盐水组、EAE-2-BFI组(2-BFI干预)、EAE-阿米洛利组(阿米洛利干预)、EAE-咪唑克生组(咪唑克生干预),每组8只。均采用经实验证实的有效剂量,用动物神经功能评分评价各组间小鼠行为学表现。苏木精-伊红染色和LFB髓鞘染色,观察中枢组织炎性细胞浸润和髓鞘脱失程度。结果 3个不同配体干预组小鼠的神经行为学和炎症病理学改变与EAE-生理盐水组比较均明显减轻,差异有显著统计学意义(P0.01);而3个不同干预组之间的行为学和病理学变化比较无显著差异,2-BFI保护作用具微弱优势。结论 2-BFI、阿米洛利和咪唑克生均能明显减缓小鼠EAE发生与发展,均具有较好抑制中枢炎症反应的药理作用,3者间的药效作用强度未见明显差异。     

实验性自身免疫性脑脊髓炎动物模型早期轴索损伤

目的 研究实验性自身免疫性脑脊髓炎(EAE)动物模型发病早期的轴索损伤.方法 采用髓鞘蛋白脂蛋白(PLP139-151)多肽作为抗原诱发EAE小鼠模型.小鼠经免疫后每天进行神经功能评分及称体质量,于发病后剥离脑及脊髓组织进行病理学研究.结果 7只(23.3%)小鼠于免疫后15～22 d内发病,平均免疫后发病时间为(19.00±2.58)d,平均神经功能评分为(2.14±0.69)分.免疫前小鼠平均体质量[(21.85±0.94)g]与发病后体质量[(23.24±1.55)g]比较差异无统计学意义.HE染色可见发病小鼠软脊膜下脊髓组织炎性细胞浸润明显,以小血管周围为主的血管袖套形成.病变累及脊髓多见,且较大脑病变重.LFB染色可见炎细胞浸润处髓鞘有不同程度脱失,同时Bodian银染可见轴索肿胀、横断.免疫组织化学染色可见髓鞘碱性蛋白着色的相同区域淀粉样前体蛋白浓染,而星形胶质细胞GFAP染色增生不明显.结论 PLP多肽诱发SJL/J小鼠的EAE模型临床症状较轻,病变主要累及脊髓,而在大脑未发现与MS类似的典型病灶.在EAE发病早期,髓鞘还未明显脱失时即可见轴索损伤,且早期轴索损伤与临床症状并不平行.     

氯喹对EAE模型小鼠神经元损伤的影响

目的观察氯喹对实验性自身免疫性脑脊髓炎(EAE)小鼠脊髓神经元存活的影响,并探讨相关机制。方法采用髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)诱导C57BL/6小鼠建立EAE模型,造模成功后给予氯喹干预。应用Knoz法进行临床评分,脊髓腰膨大节段石蜡包埋、冠状切片,行苏木精-伊红、LFB和Nissl染色,分别观察各组小鼠脊髓炎症反应、髓鞘脱失和神经元存活情况。脊髓腰膨大冷冻切片,行免疫荧光双标染色。同时提取脊髓腰膨大组织蛋白检测凋亡相关蛋白表达。结果氯喹缓解EAE小鼠临床症状,减轻脊髓炎性细胞浸润、髓鞘脱失、以及神经元死亡和损伤。与对照组相比,氯喹治疗组脊髓Bax水平降低,Bcl-2水平升高,Bax/Bcl-2比值降低。Neu N与Bcl-2荧光共定位明显增强。结论氯喹可能通过调节中枢神经系统前凋亡蛋白水平、上调神经元Bcl-2表达,从而减轻EAE小鼠神经元损伤以及神经功能缺失症状,发挥神经保护作用。     

2-(2-苯并呋喃基)-2-咪唑啉对实验性自身免疫性脑脊髓炎小鼠脊髓胶质纤维酸性蛋白表达的影响

目的探讨咪唑啉Ⅱ类受体(I2R)高选择性配体2-(2-苯并呋喃基)-2-咪唑啉(2-BFI)对实验性自身免疫性脑脊髓炎(EAE)小鼠脊髓胶质纤维酸性蛋白(GFAP)表达的影响。方法 C57BL/6小鼠30只,随机分成EAE组、2-BFI组和对照组,每组10只。EAE组及2-BFI组给予皮下注射抗原液诱导为EAE模型;2-BFI组同日起腹腔注射2-BFI共14 d。各组每日进行2次神经功能缺损评分;第19 d进行脊髓病理学检查,免疫组化法观察脊髓炎症细胞浸润、髓鞘脱失程度及GFAP表达。结果 2-BFI组神经功能缺损评分(4.17±3.65)明显低于EAE组(10.41±3.02)(P<0.01);脊髓病理评分(1.10±0.59)较EAE组(2.38±0.86)显著减少(P<0.01);GFAP阳性细胞数[(18.83±2.31)个/HP]明显多于EAE组[(12.25±2.66)个/HP](P<0.05)。结论 2-BFI能减缓小鼠EAE疾病发展,可能与促进中枢神经系统中GFAP的表达有关。     

芬戈莫德对实验性自身免疫性脑脊髓炎小鼠脑组织基质金属蛋白酶-9表达影响的研究

目的探讨芬戈莫德对实验性自身免疫性脑脊髓炎(EAE)小鼠脑组织基质金属蛋白酶-9(MMP-9)mRNA及蛋白表达的影响。方法 48只雌性C57BL/6小鼠随机分为完全弗氏佐剂(CFA)组、EAE组、芬戈莫德组。运用髓鞘少突胶质细胞糖蛋白35-55构建EAE小鼠模型,小鼠神经功能缺损评分达到1分或免疫第14天,芬戈莫德组腹腔注射芬戈莫德10 mg·kg-1,相应的CFA组、EAE组给予生理盐水。观察小鼠行为学变化,中枢炎症和脱髓鞘情况,脑组织中的MMP-9 mRNA及蛋白表达。结果芬戈莫德组小鼠临床症状减轻,体重下降减少,潜伏期延长,发病率降低,炎性病灶数减少,脱髓鞘程度减轻。芬戈莫德组MMP-9 m RNA和蛋白表达水平较EAE组降低。结论芬戈莫德能抑制EAE小鼠脑组织中MMP-9 mRNA及蛋白表达水平。     

Claudin-1 induced sealing of blood–brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood–brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.     

咖啡因慢性干预对小鼠实验性自身免疫性脑脊髓炎影响的观察

目的：探讨不同剂量咖啡因慢性干预对小鼠实验性自身免疫性脑脊髓炎（EAE）的影响及其分子免疫学机制。方法：C57BL／6小鼠随机分为CFA阴性对照组,EAE阳性对照组,咖啡因饮水干预组（1mg·k-1,10mg·kg-1,30mg·kg-1）。使用髓鞘少突胶质细胞糖蛋白35-55（MOG35-55）抗原诱导小鼠EAE模型,咖啡因干预至EAE造模后第20天与对照组小鼠同期处死为止。观察小鼠行为学变化、中枢炎症细胞浸润和损害程度、检测中枢细胞因子IL-17、IFN—γ、TGF—β的mRNA表达含量。结果：咖啡因干预组,特别是30mg·kg-1组的病情严重程度减轻,中枢炎症细胞浸润程度下降,促炎因子表达降低,抑炎因子表达上升。结论：慢性咖啡因干预,可通过降低促炎因子的表达,升高抑炎因子的表达,减轻小鼠EAE的炎症损伤。     

阿米洛利对实验性自身免疫性脑脊髓炎小鼠中枢神经系统中细胞因子表达的影响

目的：观察阿米洛利对实验性自身免疫性脑脊髓炎（EAE）小鼠脑组织中白介素17（IL-17）、白介素10（IL-10）和干扰素-γ（IFN-γ）的影响。方法：C57BL/6小鼠27只随机分成3组：EAE-生理盐水组、EAE-阿米洛利组和空白对照组。用髓鞘少突胶质细胞糖蛋白和完全弗氏佐剂混合抗原乳剂诱导小鼠EAE动物模型。EAE-阿米洛利组免疫当日开始给予阿米洛利10 mg.kg-1干预,连续14 d;EAE-生理盐水组给予相同剂量的生理盐水;空白对照组小鼠不做任何处理。每天评估EAE小鼠神经功能变化;采用苏木精-伊红染色观察病理学改变;ELISA法检测发病高峰期（免疫后第19天）脑组织中促炎因子IL-17和IFN-γ、抑炎因子IL-10改变。结果：与EAE-生理盐水组相比,EAE-阿米洛利组①日均神经功能评分降低（P〈0.05）,最大临床症状评分明显降低（P〈0.01）;而两组间发病率无明显差异。②苏木精-伊红染色：中枢神经系统炎症细胞浸润减少（P〈0.01）。③IL-17和IFN-γ表达降低（P〈0.01）,IL-10表达增加（P〈0.05）。结论：阿米洛利能够减缓小鼠EAE病情,其机制可能与下调中枢神经系统IL-17和IFN-γ的表达,上调IL-10的表达有关。     

白芍总苷对EAE大鼠中枢神经系统炎症浸润细胞凋亡及Bcl-2、Bax表达的影响

目的 探讨白芍总苷（TGP）对实验性自身免疫性脑脊髓炎（EAE）大鼠中枢神经系统（CNS）炎症浸润细胞凋亡及Bcl-2、Bax表达的影响.方法 建立大鼠EAE模型,将大鼠随机分为正常对照组、模型组、TGP组、泼尼松组,TGP组免疫后第1天起每天经口灌服白芍总苷悬浊液0.2 g/kg,泼尼松组给予泼尼松,正常对照组、模型组给予同体积生理盐水溶液,第17天处死,病理切片观察CNS炎症细胞浸润情况,TUNEL法检测浸润细胞凋亡情况,免疫组化检测浸润细胞Bcl-2、Bax蛋白的表达.结果 泼尼松组、TGP组与模型组相比,中枢神经系统炎症浸润细胞的数目减少,凋亡率增高.TGP组与模型组相比,Bcl-2表达下降,Bax的表达上调,Bcl-2/Bax的比值下降.泼尼松组与模型组相比,Bcl-2/Bax的比值下降.结论 TGP能减轻EAE的病情,其机制可能是下调Bcl-2的表达,上调Bax蛋白的表达,降低Bcl-2/Bax比值,促进EAE大鼠CNS炎症浸润细胞的凋亡,减少CNS炎症细胞的浸润.     

Kallikrein 6 secreted by oligodendrocytes regulates the progression of experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), and experimental autoimmune encephalomyelitis (EAE) is a well‐established animal model of the disease. Here, we examined the pathophysiological role of Kallikrein 6 (Klk6), a serine protease produced by oligodendrocytes (OLs), in EAE using Klk6 knockout (Klk6?/?) mice. Compared with Klk6+/+ (wild‐type) mice, Klk6?/? mice showed milder EAE symptoms, including delayed onset and milder paralysis. Loss of Klk6 suppressed matrix metalloprotease‐9 expression and diminished the infiltration of peripheral inflammatory cells into the CNS by decreasing blood–brain barrier (BBB) permeability and reducing expression levels of inflammatory cytokines, chemokines and their receptors. Scanning electron microscopic analysis revealed demyelination characterized by myelin detachment from the axons in the early phase of EAE progression (days 3–7) in Klk6+/+ mice but not in Klk6?/? mice. Interestingly, anti‐MOG (myelin oligodendrocyte glycoprotein) autoantibody was also detected in the cerebrospinal fluid (CSF) and spinal cord on day 3 after MOG immunization. Furthermore, treatment of primary cultured OLs with anti‐MOG autoantibody induced oligodendroglial morphological changes and increases in myelin basic protein and Klk6 expression. We also developed a novel enzyme‐linked immunoabsorbent assay method for detecting activated KLK6 in human CSF. In human autopsy brain samples, expression of active KLK6 was detected in OLs using an antibody that specifically recognizes the protein's activated form. Taken together, our findings demonstrate that Klk6 secreted by OLs plays a critical role in the pathogenesis of EAE/MS and that it might serve as a potential therapeutic target for MS.     

Blood-brain barrier disruption and lesion localisation in experimental autoimmune encephalomyelitis with predominant cerebellar and brainstem involvement

The role of the blood–brain barrier (BBB) in determining lesion distribution was assessed in an atypical model of experimental autoimmune encephalomyelitis (EAE) induced in C3H/HeJ mice by immunisation with peptide 190–209 of myelin proteolipid protein, which can result in two distinct types of EAE, each with distinct lesion distribution. Areas of the BBB showing constitutively greater permeability in naïve mice did not correlate with the lesion distribution in EAE. BBB disruption occurred only in sites of inflammatory cell infiltration. Irrespective of the clinical type, the BBB was disrupted in the cerebellum and brainstem. Pertussis toxin had no effect on lesion distribution. Thus, lesion distribution is not influenced solely by BBB permeability.     

Loss of astrocyte connexins 43 and 30 does not significantly alter susceptibility or severity of acute experimental autoimmune encephalomyelitis in mice

We showed previously that mice deficient in astrocyte gap junctions Cx43 and Cx30 exhibit white matter vacuolation and hypomyelination. In this study we tested the hypothesis that loss of astrocytic gap junction proteins leads to exacerbation of the primary demyelinating diseases, using experimental autoimmune encephalomyelitis (EAE) as a model system. To test for this, Cx43 floxed mice were crossed with GFAP:Cre, Cx30 null mice to generate mice lacking astrocytic expression of both Cx43 and Cx30 (dKO). EAE was induced using myelin oligodendrocyte glycoprotein (MOG(35-55)) peptide, and mice were monitored for acute expression of disease. No statistically significant difference in clinical or pathological expression of EAE was observed. Lesion load and susceptibility of different areas of the CNS to inflammation were similar in all genotypes. Moreover, no differences were noted in blood-brain barrier (BBB) permeability, tissue wet weight, axonal pathology, gliosis or demyelination during acute disease. These data show that loss of the astrocytic connexins, Cx43 and Cx30, and the white matter pathology observed in these mice does not statistically affect clinical or pathological expression of EAE and show that astrocyte gap junctions do not regulate autoimmune inflammation and associated BBB disruption in acute EAE.     

Localization of claudin-3 in tight junctions of the blood-brain barrier is selectively lost during experimental autoimmune encephalomyelitis and human glioblastoma multiforme

In the central nervous system (CNS) complex endothelial tight junctions (TJs) form a restrictive paracellular diffusion barrier, the blood-brain barrier (BBB). During inflammation, BBB properties are frequently lost, resulting in brain edema. To investigate whether BBB leakiness correlates with molecular changes at BBB TJs, we performed immunofluorescence stainings for TJ molecules in a mouse model of experimental autoimmune encephalomyelitis (EAE) and in human tissue with glioblastoma multiforme (GBM). In TJs of healthy CNS vessels in both mouse and man we detected occludin, ZO-1, claudin-5 and claudin-3. In EAE brain and spinal cord sections we observed the selective loss of claudin-3 immunostaining from TJs of venules surrounded by inflammatory cuffs, whereas the localization of the other TJ proteins remained unchanged. In addition, selective loss of claudin-3 immunostaining was also observed in altered cerebral microvessels of human GBM. Our data demonstrate the selective loss of claudin-3 from BBB TJs under pathological conditions such as EAE or GBM when the integrity of the BBB is compromised, and therefore suggest that claudin-3 is a central component determining the integrity of BBB TJs in vivo.