实验性变态反应性脑脊髓炎大鼠模型的复制

目的：观察不同的遗传背景（远交系和近交系）和饲养环境对Wistar大鼠实验性变态反应性脑脊髓炎（EAE）发病率的影响，摸索EAE模型制作的实验方法与技术。方法： 采用动物行为学、常规HE和LFB染色方法，观察不同的遗传背景和饲养环境下豚鼠全脊髓匀浆诱导大鼠EAE的发病情况与中枢神经系统（CNS）的病理变化。结果： （1）动物行为学的改变：各组大鼠EAE发病率、潜伏期和症状评分无显著差异，但近交系组发病大鼠可出现四肢瘫痪。（2）病理学改变：每组发病大鼠CNS内可见不同程度的炎性细胞浸润；髓鞘染色可见广泛的神经髓鞘变性、脱失；而其中近交系组的5只EAE大鼠髓鞘脱失部位大多局限在炎症改变明显的血管套周围的白质区。结论： 远交系与近交系，或清洁级与普通级的Wistar大鼠的EAE的发病率无明显差异，而远交系与近交系的不同遗传背景可能会影响大鼠EAE的体征和病理表现。     

糖皮质激素受体阻断剂对Wistar大鼠EAE的影响

目的：观察糖皮质激素受体阻断剂（RU486）对EAE大鼠的影响。方法：雌性Wistar大鼠23只分成三组：对照（Contr01）组、模型（EAE）组、RU486组，采用豚鼠脊髓匀浆／完全弗氏佐剂（GPSCH／CFA）免疫Wistar大鼠建立EAE模型，采用行为学变化观察动物的发病情况，常规HE和Kluver＆Barrera染色法观察中枢神经系统（（INS）的病理变化情况。结果：①EAE组7／8大鼠出现典型的EAE行为学改变、CNS炎性细胞浸润（Okuda评分）和髓鞘脱失。②RU486组3／9大鼠出现行为学改变、CNS炎性细胞浸润和髓鞘脱失，均重于模型组（P〈0．01）。结论：RU486未增加大鼠EAE的发病率，但可使EAE大鼠的行为学改变、CNS炎性细胞浸润和髓鞘脱失加重。     

2-BFI对EAE大鼠的神经保护作用及其免疫调节机制

目的探讨2-（2-苯并呋喃基）-2-咪唑啉[2-（-2-benzofuranyl）-2-imidazoline,2-BFI]干预大鼠实验性自身免疫性脑脊髓炎（ EAE）免疫调节机制。方法50只雌性SD大鼠随机分为5组：对照组、EAE组、2-BFI 低剂量组（1．5 mg/kg ）、2-BFI 中剂量组（3 mg/kg ）和2-BFI 高剂量组（6 mg/kg）。采用豚鼠脊髓匀浆（ GPSCH）免疫诱导建立EAE模型。通过观察大鼠行为学变化进行临床症状评分;采用HE染色观察中枢神经系统（ CNS）的炎性浸润;采用免疫组化观察腰髓中激活的星形胶质细胞和小胶质细胞细胞数量;用酶联免疫吸附法（ ELISA ）检测颈髓中IL-1β、IFN-γ、IL-4和IL-10的含量。结果与EAE组比较,大、中、小剂量2-BFI组大鼠EAE发病率下降,临床症状减轻,潜伏期延长,CNS内炎性细胞浸润减少,但仅中剂量3 mg/kg 2-BFI组在EAE临床症状和病理学改变方面差异有统计学意义（P＜0．05）;免疫组化结果显示,与EAE组比较,中剂量2-BFI组的活化的小胶质细胞数量显著减少,活化的星形胶质细胞数量显著增多（P＜0．05）;组织ELISA结果显示,与EAE组比较,2-BFI干预后,脊髓中的IL-1β、IFN-γ下降, IL-4和IL-10水平上升,中剂量2-BFI 组最为显著（ P＜0．05）。结论中剂量2-BFI对EAE大鼠具有神经保护作用,其作用机制可能与免疫调节作用有关。     

咪唑克生对实验性自身免疫性脑脊髓炎大鼠脊髓内胶质细胞变化的影响

目的:探讨咪唑克生(Idazoxan,Ida)对实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis.EAE)大鼠脊髓内神经胶质细胞变化的影响及其意义.方法:采用临床症状评分、病理学检查和免疫组化观察咪唑克生处理条件下豚鼠全脊髓匀浆诱导的Lewis大鼠EAE临床表现、病理改变及脊髓内星形胶质细胞和小胶质细胞变化.结果:咪唑克生虽不减低Lewis大鼠EAE的发病率,但可减轻其临床症状和病理学改变.在免疫后15天,炎性脱髓鞘病灶内和周围星形胶质细胞数量增多,胞体肥大,与此相反,小胶质细胞数量是减少的.结论:Ida对实验性自身免疫性脑脊髓炎大鼠具有保护作用,其作用机制可能为咪唑克生对CNS的免疫机制有一定影响.     

RhoA和Rock-Ⅱ在Lewis大鼠EAE模型中的表达

目的研究RhoA和Rock-Ⅱ在Lewis大鼠实验性自身免疫性脑脊髓炎（EAE）模型中的表达,探索其在EAE模型中的作用。方法把36只Lewis大鼠随机分为两组：A组（空白对照组）,B组（EAE模型组）。以豚鼠全脊髓为抗原混合福氏完全佐剂（CFA）,诱导Lewis大鼠EAE模型,观察实验动物神经功能损害的情况;采用免疫组织化学染色法观察实验动物各组脑、脊髓组织中RhoA、Rock-Ⅱ的表达。结果成功建立Lewis大鼠EAE模型,RhoA和Rock-Ⅱ在EAE模型组的表达较空白对照组明显增加,差异有统计学意义（P〈0.01）。结论 RhoA和Rock-Ⅱ在EAE模型中过量表达,可能在EAE模型的发病过程中起关键作用。     

MOG1-125诱发的实验性自身免疫性脑脊髓炎大鼠模型

目的建立髓鞘少突胶质细胞糖蛋白多肽1-125(MOG1-125)诱发的实验性自身免疫性脑脊髓炎(EAE)大鼠模型。方法采用大鼠MOG1-125和福氏完全佐剂作为抗原。在第1天和第15天分别进行两次皮内注射抗原,免疫SD大鼠建立EAE的动物模型,观察其临床症状,取脊髓组织进行冰冻切片、半薄切片和超薄切片,光镜和电镜观察脊髓组织学改变。结果 SD大鼠在两次免疫后8～10 d发病,其发病率为15%。在快蓝染色和甲苯胺蓝染色后,发现脊髓白质存在广泛的脱髓鞘浅染区。通过透射电镜观察进一步证实脊髓白质存在脱髓鞘现象。结论采用大鼠MOG1-125和完全弗氏佐剂作为抗原对SD大鼠进行两次免疫,能够成功诱发出EAE模型。     

实验性自身免疫性脑脊髓炎脑干听觉通路损害的研究

目的： 探讨实验性自身免疫性脑脊髓炎（EAE）大鼠听觉通路损害的情况，为临床研究提供实验依据。方法： 以豚鼠全脊髓匀浆（GPSCH）为抗原免疫Wistar 大鼠建立 EAE的动物模型，采用诱发电位仪检测实验组与对照组的脑干听觉诱发电位，通过脑干及耳蜗神经核HE染色及Weil髓鞘染色从解剖病理的角度证实脑干及耳蜗神经核损害。通过大鼠行为学的变化及光镜下脑和脊髓的病理改变来确定EAE的成功。结果： EAE大鼠的BAEP改变：左耳：Ⅳ、Ⅴ波潜伏期及Ⅰ-Ⅴ波峰间伏期IPL长于对照组（P<0.05）,右耳：Ⅰ、Ⅱ波潜伏期及Ⅰ-Ⅲ Ⅲ-Ⅴ Ⅰ-Ⅴ波峰间伏期IPL长于对照组（P<0.05）；而Ⅲ、Ⅳ、Ⅴ等波的潜伏期显著长于对照组（P<0.01）。病理图像显示脑干耳蜗神经核HE染色有大量淋巴细胞浸润，呈袖套样改变，髓鞘染色显示弥漫性脱髓鞘病变。结论： Wistar大鼠实验性自身免疫性脑脊髓炎（EAE）听神经通路有损害。     

CD4~+CD25~+调节性T细胞移植对自身免疫性脑脊髓膜炎大鼠的保护作用

目的观察输注CD4~+CD25~+调节性T细胞对实验性自身免疫性脑脊髓膜炎(EAE)模型大鼠后脑和脊髓组织的保护作用及相关机制。方法取SD大鼠30只,随机均分为空白对照组、模型组(实验性自身免疫性脑脊髓炎模型)和实验组(CD4~+CD25~+调节性T细胞输注),对EAE大鼠进行神经功能评分及体质量测量,通过病理学HE染色和免疫组化观察脑和脊髓炎症浸润,实时荧光定量PCR(qPCR)检测鼠脑和脊髓IFN-γ、IL-17、IL-23和Foxp3 m RNA水平表达。结果实验组SD大鼠神经功能评分明显改善,体质量明显增加;与模型组比较,实验组神经症状和病理改变减轻,鼠脑和脊髓IFN-γ、IL-17、IL-23 mRNA表达下降,而Foxp3 mRNA表达增加(P0.05)。结论 CD4~+CD25~+调节性T细胞通过调节免疫系统的脑和脊髓IFN-γ、IL-17、IL-23和Foxp3 mRNA水平的表达对EAE起保护作用。     

EAE诱导过程中脾脏及CNS浸润Treg/Teff的变化

探讨实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)诱导过程中脾脏及中枢神经系统(central nervous system,CNS)CD4+CD25+Foxp3+Treg、效应T细胞(effector T cell,Teff)的变化及作用。用髓鞘少突胶质细胞糖蛋白(myelin oligodendrocyte glycoprotein,MOG35-55)免疫C57BL/6小鼠诱导EAE;用流式细胞仪分析EAE发病初期及高峰期脾脏和CNS浸润Treg、Th1和Th17的变化。MOG35-55肽诱导产生典型的EAE症状,脊髓可见大量的单个核细胞浸润;EAE发病前,脾脏Treg、Th1、Th17数量增加,随着EAE临床症状的出现,脾脏Treg、Teff均减少;EAE发病前既有少量Teff迁移至CNS,主要为Th17;EAE发病高峰期,CNS内Th17比例降低,Th1比例及数量显著增加;EAE发病前CNS可见少量Treg存在,发病后大量Treg迁移至CNS,但不能抑制CNS的炎症反应。以上结果提示,Th17可能参与了CNS免疫损伤的启动,而CNS浸润的Thl与疾病的严重程度密切相关;EAE发病高峰期脾脏Treg伴随着Teff大量迁移至CNS,但不能控制EAE的进展。     

PPARγ激动剂对实验性自身免疫性脑脊髓炎钙激活中性蛋白酶表达的影响

目的： 探讨过氧化物体增殖剂激活的受体γ激动剂对大鼠实验性自身免疫性脑脊髓炎(EAE)的治疗作用, 以及对其脑组织中钙激活的中性蛋白酶（calpain）表达的影响。方法： 建立大鼠EAE动物模型，分别给予PPARγ激动剂罗格列酮和非甾体类抗炎药布洛芬预处理，通过各组间临床表现评分评价药物疗效，RT-PCR法检测calpain mRNA 的表达，Western blotting法检测calpain蛋白表达水平。结果: 罗格列酮和布洛芬治疗组大鼠的临床表现评分显著低于EAE模型组；3组间calpain mRNA表达水平无显著差异（P>0.05），但罗格列酮和布洛芬组calpain蛋白质的表达明显低于EAE模型组(P<0.05)。结论： PPARγ激动剂能够显著减轻EAE大鼠的临床症状，抑制calpain蛋白质的表达，表明PPARγ激动剂对实验性自身免疫性脑脊髓炎大鼠具有脑保护作用。     

Immune regulatory mechanisms influence early pathology in spinal cord injury and in spontaneous autoimmune encephalomyelitis

Injuries to the central nervous system (CNS) trigger an inflammatory reaction with potentially devastating consequences. In this report we compared the characteristics of the inflammatory response on spinal cord injury (SCI) caused by a stab wound between the T7 and T9 vertebrae and spontaneous experimental autoimmune encephalomyelitis (EAE). SCI and EAE were compared in two types of myelin basic protein Ac1-11-specific T-cell receptor transgenic mice: T/R+ mice harbor regulatory T cells, and T/R- mice lack regulatory T cells. Our results show that 8 days after SCI, T/R- mice developed a strong T-cell infiltrate in the spinal cord, with remarkable down-modulation of CD4 expression that was accompanied by a local increase in Mac-3+ and F4/80+ reactivity and diffuse local and distal astrogliosis. In contrast, T/R+ mice exhibited a modest increase in CD4+ cells localized to the site of injury, without CD4 down-modulation; focal astrogliosis was restricted to the site of the lesion, although Mac-3+ and F4/80+ cells were also present. Similarly to T/R- mice that underwent SCI, T cells displaying down-modulated CD4 expression were found in the CNS of older T/R- mice afflicted by spontaneous EAE. Overall, our results suggest that common mechanisms regulate T-cell accumulation in CNS lesions of different causes, such as mechanic lesion or autoimmune-mediated damage.     

Immunohistochemical Study of Netrin-1 in the Spinal Cord with Rat Experimental Autoimmune Encephalomyelitis

To investigate whether netrin-1 is involved in autoimmune injury of the central nervous system, the expression of netrin-1 protein was analyzed in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis revealed significantly increased content of netrin-1 in the spinal cords of rats at the peak stage of EAE, as compared with the levels in normal control animals (p < 0.01). Immunohistochemistry detected the netrin-1 protein in neurons, oligodendrocytes, astrocytes and vascular endothelial cells in the spinal cords of normal controls. In EAE-affected spinal cords, netrin-1 immunoreactivity was detected in infiltrating inflammatory cells at the peak stage as well as in neurons, oligodendrocytes and astrocytes. These results suggest that netrin-1 is transiently increased in rat EAE lesions, where it contributes to the modulation of rat acute EAE.     

Downregulation of microglial keratan sulfate proteoglycans coincident with lymphomonocytic infiltration of the rat central nervous system.

The monoclonal antibody (MAb) 5D4 against a keratan sulfate (KS) epitope of bovine cartilage proteoglycan stains ramified microglia in the rat brain. In this study we show that 5D4-positive microglia is abundant in the normal rat spinal cord and nearly absent during both the active and recovery phase of experimental autoimmune encephalomyelitis (EAE) in myelin-immunized Lewis rats. In contrast, during Wallerian degeneration of the optic nerve the density of KS-immunoreactive microglia remains constant. KS immunoreactivity is absent from both normal and transected sciatic nerves, and spinal nerve roots. On immunoblots of spinal cord extracts MAb 5D4 stains a novel type of KS proteoglycans (KSPGs) with an apparent molecular weight mainly between 140 and 200 kd, which significantly decrease in acute EAE. Our data suggest that high levels of KSPG expression correlate to a downregulated immunophenotype of resident macrophages in the nervous system. The lack of detectable KS in peripheral nerve points to a divergent differentiation of bone marrow-derived resident macrophages in the peripheral and central nervous systems and may partially account for the rapid macrophage response to axonal injury in the peripheral nervous system. Downregulation of microglial KSPG could be a prerequisite for a rapid inflammatory response in the central nervous system.     

Immunohistochemical study of netrin-1 in the spinal cord with rat experimental autoimmune encephalomyelitis

To investigate whether netrin-1 is involved in autoimmune injury of the central nervous system, the expression of netrin-1 protein was analyzed in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis revealed significantly increased content of netrin-1 in the spinal cords of rats at the peak stage of EAE, as compared with the levels in normal control animals (p < 0.01). Immunohistochemistry detected the netrin-1 protein in neurons, oligodendrocytes, astrocytes and vascular endothelial cells in the spinal cords of normal controls. In EAE-affected spinal cords, netrin-1 immunoreactivity was detected in infiltrating inflammatory cells at the peak stage as well as in neurons, oligodendrocytes and astrocytes. These results suggest that netrin-1 is transiently increased in rat EAE lesions, where it contributes to the modulation of rat acute EAE.     

T-cell-mediated disruption of the neuronal microtubule network: correlation with early reversible axonal dysfunction in acute experimental autoimmune encephalomyelitis

During the course of the central nervous system autoimmune disease multiple sclerosis (MS), damage to myelin leads to neurological deficits attributable to demyelination and conduction failure. However, accumulating evidence has indicated that axonal injury is also a predictor of MS clinical disease. Using the animal model of MS, experimental autoimmune encephalomyelitis (EAE), we examined whether axonal dysfunction occurred early in disease and correlated with disease symptoms. We tracked axons during EAE by using transgenic mice that express yellow fluorescent protein (YFP) in neurons. At the onset of disease, we observed a loss of YFP fluorescence in the spinal cord in areas that coincided with immune cell infiltration, before prominent demyelination. These inflammatory lesions also exhibited evidence of axonal injury but not axonal loss. During the recovery phase of EAE, the return of YFP fluorescence occurred in parallel with the resolution of inflammation. Using in vitro cultured neurons expressing YFP, we demonstrated that encephalitogenic T cells alone directed the destabilization of microtubules within neurites, resulting in a change in the pattern of YFP fluorescence. This study provides evidence that encephalitogenic T cells directly cause reversible axonal dysfunction at the onset of neurological deficits during an acute central nervous system inflammatory attack.     

T-bet is essential for the progression of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is mediated by myelin-specific CD4+ T helper 1 (Th1) cells, while recovery from the disease is associated with the presence of Th2 cells. Here we used animals with targeted deletion of the T-bet gene to determine its role in the progression of EAE. T-bet regulates the production of interferon-gamma (IFN-gamma) in CD4+ and natural killer cells, and CD4+ T cells from T-bet-deficient mice were unable to differentiate into a Th1 phenotype. Moreover BALB/c mice deficient in T-bet were resistant to the induction of EAE disease, with minimal inflammatory infiltrates in the central nervous system. These mice were resistant to EAE induction even when PLP(180-199) peptide specific effector T cells from BALB/c wild type were transferred to BALB/c T-bet-deficient mice. This resistance to EAE is may be caused by the production of the anti-inflammatory cytokine interleukin-10 (IL-10) from the spleen cells upon ex vivo stimulation with PLP(180-199) peptide and in vivo presence in the central nervous system. There was no difference in the recall responses in spleen cells from T-bet-deficient and wild type mice; however, less secretion of IFN-gamma was observed from primed splenocytes. The expression of IFN-gamma was less in the central nervous system of T-bet-deficient mice whereas IL-10 was significantly higher in T-bet-deficient as compared to wild type mice. These data indicate that T-bet genes play a critical role in maintaining the encephalitogenic nature of CD4+ T cells in autoimmune responses during EAE disease progression.     

Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions

Recent magnetic resonance (MR) studies of multiple sclerosis lesions indicate that axonal injury is a major correlate of permanent clinical deficit. In the present study we systematically quantified acute axonal injury, defined by immunoreactivity for beta-amyloid-precursor-protein in dystrophic neurites, in the central nervous system of 22 multiple sclerosis patients and 18 rats with myelin-oligodendrocyte glycoprotein (MOG)-induced chronic autoimmune encephalomyelitis (EAE). The highest incidence of acute axonal injury was found during active demyelination, which was associated with axonal damage in periplaque and in the normal appearing white matter of actively demyelinating cases. In addition, low but significant axonal injury was also observed in inactive demyelinated plaques. In contrast, no significant axonal damage was found in remyelinated shadow plaques. The patterns of axonal pathology in chronic active EAE were qualitatively and quantitatively similar to those found in multiple sclerosis. Our studies confirm previous observations of axonal destruction in multiple sclerosis lesions during active demyelination, but also indicate that ongoing axonal damage in inactive lesions may significantly contribute to the clinical progression of the disease. The results further emphasize that MOG-induced EAE may serve as a suitable model for testing axon-protective therapies in inflammatory demyelinating conditions.     

盐酸青藤碱对实验性自身免疫性脑脊髓炎大鼠脑内核转录因子-κB活性的作用

目的研究青藤碱的免疫抑制作用是否与核因子-κB的活性有关。方法以实验性自身免疫性脑脊髓炎大鼠为免疫异常模型，观察青藤碱对其临床表现和病理特点的改变，检测了脑组织中核转录因子-κB的活性及血浆中皮质醇、泌乳素水平的改变。结果青藤碱可降低EAE大鼠临床评分，改善病理变化，抑制脑组织中核转录因子-κB活性，增加血浆中皮质醇水平，降低血浆泌乳素水平。结论青藤碱的免疫抑制作用与抑制脑组织核因子-κB活性有关，其对核因子-κB活性的抑制作用可能与增加皮质醇分泌、抑制泌乳素分泌有关。     

Apoptosis of T lymphocytes in experimental autoimmune encephalomyelitis. Evidence for programmed cell death as a mechanism to control inflammation in the brain.

In experimental autoimmune encephalomyelitis (EAE) myelin-specific T lymphocytes attack the myelinated tissue of the central nervous system (CNS). In the Lewis rat, EAE as a rule has an acute, monophasic course. With spontaneous clinical recovery the inflammatory CNS infiltrates are cleared from the nervous tissue within a few days. This is well in line with the remarkably low incidence of myelin-specific T cells present in EAE infiltrate. Combining immunocytochemical techniques, ultrastructural criteria and in situ nick translation we found up to 49% of T lymphocytes in EAE lesions showing signs of apoptosis at the time of recovery from disease. Our results suggest that apoptosis of T lymphocytes may be one possible mechanism to eliminate T lymphocytes from inflammatory brain lesions.     

Lymphocytes utilise sialylated surface molecules to accumulate in developing lesions of autoimmune encephalomyelitis.

The accumulation of desialylated radiolabelled normal spleen cells and non-neuroantigen specific CD4 T-lymphocytes was measured in the lumbosacral spinal cord of Lewis rats with autoimmune encephalomyelitis (EAE) induced with myelin basic protein in Freund's adjuvant. The labelled cells were preincubated with sialidase and thoroughly washed prior to intravenous injection into rats exhibiting early clinical signs of EAE. Four hours later, the rats were killed and blood and spinal cord samples were radioassayed. Compared with untreated cells, desialylation markedly reduced the accumulation of both normal spleen cells and memory T-lymphocytes in the spinal cord, despite similar levels of cells being present in the blood. In another experiment, the accumulation of desialylated, macrophage-depleted spleen lymphocytes was measured during the onset, recovery and short-term "relapse" phases of acute EAE. Again, compared with controls the accumulation of desialylated lymphocytes was always significantly less, despite similar numbers of cells in the circulation. Lastly, intravenous injections of sialidase produced delayed onset of both clinical and histological signs in rats with passively-transferred EAE. These data confirm and extend previous findings, using a different animal model, that sialyl residues on the lymphocyte surface are important to the accumulation of such cells at inflammatory sites in the central nervous system. The possible relevance of these findings to human demyelinating disease is discussed.