This work was supported by the Else Kr&#246;ner-Fresenius Foundation (EKFS) Research Training Group Translational Research Innovation - Pharma (TRIP) and by the "Landesoffensive zur Entwicklung wissenschaftlich-&#246;konomischer Exzellenz (LOEWE), Schwerpunkt: Anwendungsorientierte Arzneimittelforschung" of the State of Hesse.

道德守则:我们的实验过程由Regierungspräsidium达姆施塔特（德国）的伦理委员会批准并确认国家和欧洲法规。所有作出了努力，以尽量减少动物的痛苦，减少使用动物的数量。 1. EAE模型<ol><li> EAE模型的感应<ol><li>使用10至13周龄的雌性129S4 / SvJae×C57BL / 6小鼠为EAE的诱导。 </li><li>给小鼠皮下注射，到上部和下背部，所述致脑炎MOG 35 - 55（髓鞘少突胶质糖蛋白）的肽（200微克），在200微升完全Freund`s佐剂（CFA）含有400微克结核杆菌乳化。 注意:作为一个负，非疾病诱导假控制，含有结核杆菌 ，在相同的体积和通过相同的途径不完全弗氏佐剂，都可以使用。 </li><li>疗法eafter，并再次24小时后，给小鼠百日咳毒素的腹膜内注射（总共0.2微克，在200μl磷酸盐缓冲盐水，PBS稀释）。使用未经处理的小鼠作为对照组，以能够比较患病健康的动物。 注意:也可以以诱导EAE 200 - 300微克MOG 35 - 55肽，300 -在CFA 500 ​​微克结核杆菌和0.2 - 0.3微克百日咳毒素中的0.1体积- 0.2毫升，每注射。 </li><li>每天开始一周​​注射后，检查小鼠的临床症状（见步骤1.2.1） 注意:发病的一天中不同的实验而变化，但在我们的实验室条件下，这是大约11天，因此，在这里14天被定义为后3天发病。在本研究中所有小鼠开发临床症状。 </li></ol></li><li>小鼠的评分<ol><li>通过分类临床评分临床症状如下:0）没有招牌，0.5）的尾巴末端瘫痪，1）尾部完全瘫痪，1.5）跛行的尾巴和后腿轻度乏力，2）跛行的尾巴和后腿严重虚弱，2.5）柔弱的尾部和瘫痪一只后腿，3）柔弱的尾部和两条后腿瘫痪，3.5）两个后肢和一个前肢无力瘫痪（小鼠达到这个分数进行安乐死，与当地的道德准则保持）。 </li></ol></li></ol> 2.流式细胞仪分析单细胞的制备注:抗体混合物中含有1微升CD45-Vioblue，2微升的CD8 eFluor650，0.5微升的CD11b-eFluor605，0.5微升F4 / 80-PE-Cy7的，1微升CD3-PE-CF594，CD4-V500，0.5微升的CD11c的-AlexaFluor700，1μl的CD19-APC-H7和1μlLy6G-APC-Cy7的。 注意:如果你把血液，淋巴结，脾和脊髓那么程序如下:小鼠深深isofl的组合麻醉urane和氯胺酮（100mg / kg的体重）（以每分钟卡波金2％）。接下来打开胸腔，与心脏内取出粘血，用冷PBS灌注心脏内的老鼠。然后取出淋巴结其次是脾，最后脊髓。如果不需要心内灌注小鼠然后通过颈部脱位安乐死深度麻醉下的小鼠。 <ol><li>脾细胞的分离<ol><li>麻醉小鼠用异氟烷（在每分钟卡波金2％）和氯胺酮（100mg / kg的体重）的组合。 </li><li>用80％异丙醇湿切口区，以避免与毛发的任何污染和纵向打开胸腔，无需穿刺深层组织，用剪刀。 </li><li>用冷PBS pH 7.0的23心内灌注的小鼠。 注:脾脏位于左肋下的左上腹部象限。如果只脾进行研究，这个器官可能被灌注前，为了去除retain的所有细胞类型的兴趣。 </li><li>取出脾切除大约1/8和称重。储存在冰上在PBS样品。 </li><li>通过一个70微米的目筛（置于50ml管）挤压脾组织，使用2毫升注射器的柱塞。 </li><li>用5毫升PBS洗网格。离心1800 XG 3分钟。重悬500微升裂解液沉淀。 注意:在此阶段，可能有必要使如果，以后，一个替代FACS分析方法用于不采用珠粒（见第3节）的差分细胞计数。 </li><li>孵育在室温（RT）下10分钟，并加入500μlPBS中。在室温下（RT）650 XG离心6分钟。 </li><li>重复用500μl的PBS洗涤步骤。弃上清，重悬细胞沉淀在100微升0.2％牛血清白蛋白（BSA）/ PBS中，加入2微升Fc受体-1（FCRI）封闭缓冲液，并在室温下孵育在黑暗中15分钟。 </li><li>加入13微升的tibody混合物，孵育在室温15分钟在黑暗中，在650 XG在RT加入500μlPBS中并离心6分钟。弃去上清液。 注意:生产商的染色程序建议的单个洗涤，但背景染色的额外减少可以由一个或两个以上的倍任选重复洗涤步骤来实现。 </li><li>悬浮于500μlPBS中的细胞沉淀（对于蛋白质分离或可能运行缓冲液，以潜在地减少细胞聚集），并将其转移到流式细胞管的流动。保持细胞在冰上。 </li></ol></li><li>淋巴结细胞的分离<ol><li>麻醉小鼠用异氟烷（在每分钟卡波金2％）和氯胺酮（100mg / kg的体重）的组合。 </li><li>用80％异丙醇湿切口区域和纵向打开胸部用剪刀。用冷PBS pH 7.0的23心内灌注的小鼠。 </li><li>为了分离腹股沟淋巴结，小心取出皮肤喜的区域p和仔细挑选淋巴结出来的脂肪组织与镊子。权衡腹股沟淋巴结和样品在PBS中储存在冰上。 注意:我们在研究，因为O`Connor 等人用腹股沟淋巴结肿大。发现活化的单核细胞，巨噬细胞，中性粒细胞和T细胞的高数量均出现在EAE诱导24后腹股沟淋巴结。 </li><li>挤压通过一个70微米的筛网筛使用2毫升注射器的柱塞的淋巴结（置于50ml管）。 </li><li>用5毫升PBS洗网格。离心2400 XG 8分钟。重悬在100μl0.2％BSA / PBS将细胞沉淀，加入2微升FCRI封闭缓冲液，并在室温下孵育在黑暗中15分钟。 </li><li>添加13微升抗体混合物，在黑暗中孵育在室温15分钟后，在室温650 XG加入500μlPBS中并离心6分钟。 </li><li>弃上清，重悬在300微升PBS中细胞沉淀（或一个合适的运行缓冲液），并将其传送到一个流式细胞管。 </li></ol></li><li>血细胞分离<ol><li>加入50微升20毫米的HEPES至50微升的血液（用EDTA稳定），并加入500μl裂解液。在室温下孵育10分钟，加入500μlPBS中。离心在在RT 650 xg离心6分钟。弃上清，重复洗涤步骤。 </li><li>重悬在100μl0.2％BSA / PBS将细胞沉淀，加入2微升FCRI封闭缓冲液，并在室温下孵育在黑暗中15分钟。 </li><li>添加13微升抗体混合物，在黑暗中孵育在室温15分钟后，在室温650 XG加入500μlPBS中并离心6分钟。弃上清，重悬细胞沉淀在300微升PBS中，并转移到流式细胞管的流动。 </li></ol></li><li>脊髓细胞的分离<ol><li>麻醉小鼠用异氟烷（在每分钟卡波金2％）和氯胺酮（100mg / kg的体重）的组合。 </li><li>用80％异丙醇湿切口区域和纵向打开胸腔用剪刀。用冷PBS pH 7.0的23心内灌注的小鼠。 </li><li>湿切口区域的背面并重新进行纵行剪开用手术刀和去除皮肤。 </li><li>切出脊柱的腰椎部分（其支配后肢），并将其与PBS填充注射器冲洗提取脊髓。切出大约腰脊髓的1/3，权衡这和样品在PBS中储存在冰上。 </li><li>离心机在250×g离心在4℃下2分钟。弃上清，加入500微升细胞脱离溶液和HBSS（1:1）。加入3毫克/毫升胶原酶A和1单位/ ml的DNA酶I，decollat​​e通过反复上下吹打细胞和以400rpm在37℃振荡孵育30分钟。 注意:如果优选的，所述细胞悬浮液可以污染髓鞘和细胞碎片通过密度梯度离心可提高流式细胞测量流量的灵敏度，从而减少背景自发荧光的的被清除ð需要被收购的事件数（参见3.5节）。 </li><li>离心在250 xg离心在RT 2分钟。弃上清，重悬在1ml 10％胎牛血清（FCS）的丸粒/ Dulbecco氏最小基本培养基（DMEM）中，并通过重复的向上和向下的移液再次decollat​​e细胞。 </li><li>离心在250 xg离心在RT 2分钟。重复洗涤步骤。 </li><li>重悬在1ml的PBS将细胞沉淀，并通过使用2毫升注射器的柱塞70微米网状筛（置于50ml管）挤压脊髓。 </li><li>用4毫升的PBS洗涤网格。离心在1800 xg离心在RT 3分钟。弃去上清液，重悬在100微升0.2％BSA细胞沉淀/ PBS，加入2微升FCRI封闭剂，并在室温下孵育在黑暗中15分钟。 </li><li>添加13微升抗体混合物，在黑暗中孵育在室温15分钟后，在室温650 XG加入500μlPBS中并离心6分钟。 </li><li>弃去上清液，重悬细胞PELLE吨500μl的PBS（或一个合适的运行缓冲液），并将其转移到流式细胞管的流动。 </li></ol></li></ol> 3.流式细胞分析<ol><li>滴定所有抗体事先确定由Olesch 等 25描述最佳浓度。 </li><li>使用抗体捕获补偿珠单色补偿，根据manufacturer`s说明创建多色彩补偿矩阵。 </li><li>控制日常使用根据manufacturer`s指示特定珠的仪器校准。 </li><li>流式细胞仪直接测量流量前，加入30微升流式细胞仪绝对计数标准的细胞（隔离见2.1，2.2，2.3，2.4），以确定绝对细胞计数。代替使用计数珠的细胞进行计数。 </li><li>占用样品（从脾脏，淋巴结和血细胞:100000事件，脊髓500000事件）到血细胞计数器的流次分析使用特定的流式细胞仪软件。 <ol><li>打开流式细胞仪软件，并通过按按钮"添加样本"添加FCS 3.0文件。 </li><li>双击打开添加的文件。调整为x轴和y轴的通道。选择感兴趣的细胞群创建栅极（ 见图4）。 </li></ol></li></ol> 4.定量PCR分析<ol><li> mRNA的分离和转录成cDNA <ol><li>通过酚氯仿从腰脊髓（1/3），脾（1/8）和腹股沟淋巴结中提取基因，并用乙醇沉淀26。 <ol><li>为此，均质化在1ml硫氰酸胍 - 苯酚混合物中的组织，孵育10分钟。加入200μl的氯仿和10分钟再次孵育。 </li><li>离心步骤之后（18,000 xg离心4℃15分钟），洗上层水相是用500μl异丙醇和离心机（18,000 xg离心在4 8分钟C）。 </li><li>用500μl乙醇和离心步骤（18,000 xg离心在4℃下5分钟）后洗涤沉淀，干燥在真空浓缩的粒料（在30℃下5分钟）。此后，重悬在30微升水中沉淀。 </li></ol></li><li>从血液中提取的mRNA，离心机将500μlEDTA稳定的血液​​（300 xg离心10分钟，4℃）。 <ol><li>取血沉棕黄层，包含白血球，并稀释在1ml裂解溶液（150mM的氯化铵 ，100mM的碳酸氢钠 ，0.1毫钠EDTA pH 7.4）中。 </li><li> 10分钟温育在RT后，离心细胞，在650×g离心6分钟，之后用PBS洗两次。 </li><li>提取根据制造商的说明使用试剂盒提取RNA白血细胞（白细胞）的mRNA的表达。 </li></ol></li><li>执行使用用于cDNA合成的试剂盒，包括根据制造商的说明随机六聚体合成cDNA。使用200毫微克的mRNA的CDN综合。 </li></ol></li><li>定量PCR <ol><li>量化的特定mRNA的量，则使用1微升的cDNA，1μM的正向/反向引物和荧光DNA根据制造商的说明27结合染料。请参阅表1的引物组的序列。测量的IL-1β的CT值，IL-6，IL-23，IFNγ，TNFα和PPIA（肽基脯氨酰异构酶）使用定量PCR系统。 </li><li>相对确定的表达使用比较CT（循环阈值）方法27。 <ol><li>对于这一点，正常化的靶基因PPIA的表达水平的CT值通过从目标基因中减去PPIA的平均CT值，如在以前的研究（巴塞尔姆斯等人 28，Schiffmann 等人 29，Schiffmann描述等人 30）。随后，计算出所谓的ΔΔCT值，使用的，标准化ΔCT值从EAE小鼠的靶基因未处理的小鼠的靶基因的表达水平的，再由在从EAE小鼠的ΔCT值的未处理小鼠中减去平均ΔCT值。 </li><li>获得靶基因的相对mRNA表达，使用以下公式计算:2的比例- ΔΔCT。 </li></ol></li></ol></li><li>测试每个引物的特异性。确定使用2％琼脂糖凝胶31中的扩增片段的大小。片段大小示于表 1。 </li></ol>

MJP is a consultant to Xellia and to Leo Pharmaceuticals.

这里所描述的EAE模型得到最多关注，MS的模式，并在测试的治疗策略为MS 32是经常使用。鼠标疾病具有MS的许多临床和组织学特征，是由自身免疫诱导神经元抗原引起的。致敏髓鞘抗原与血脑屏障功能障碍，因此，免疫细胞浸润到中枢神经系统有关。我们的研究结果显示，免疫细胞在急性期淋巴结瞬时增加。脾T细胞和B细胞减少的疾病的临床前阶段。在循环血液，T细胞，B细胞，树突细胞和单核细胞在临床前阶段瞬时增加，而粒细胞增加症依赖性。最后，在脊髓，增加免疫细胞是可检测的（ 图 2）。这些数据表明，T细胞和B细胞从脾，其作用迁移贮存器为这些细胞，在那里它们被激活和增殖的淋巴结。随后，他们通过循环进入脊髓迁移。嗜中性粒细胞，单核细胞和树突状细胞，在另一方面，从骨髓迁移通过循环进入CNS。 我们的数据表明，在脊髓，IFNγ，TNFα和IL-1β中的一种疾病相关的方式进行调节，而IL-6是在疾病的发作瞬时上调。这些数据支持这样的假说，即EAE病理是由Th1和Th17细胞驱动。 Th1细胞释放IFNγ，这反过来又激活巨噬细胞释放的TNFα和IL-1β33。此外，IL-6的已知驱动Th1和Th17细胞的分化，从而促进EAE 34的发展。 的临床症状发作和疾病的严重程度的日子是在EAE小鼠的变量。根据我们的经验，有几种řeasons此。在临床前阶段暴露于应力小鼠显示疾病的降低严重程度和在疾病发作的延迟。小鼠的壳体也影响EAE的严重性，也发病的一天。最近，已表明小鼠的共生菌群影响EAE 35和不同外壳的情况的发展可能影响微生物，由此，在EAE病理。此外，这是非常重要的总是使用新鲜制备的百日咳毒素。百日咳毒素损伤血脑屏障这对于EAE的发展的先决条件。在有效期后，不要使用乳化MOG 35-55肽。小鼠的年龄也影响EAE的病理。因此，存在应，以产生一个可重复的EAE模型中满足一些规则。使用10岁和13周，小鼠之间来自同一供应商，如果有可能的小鼠。如果将小鼠从外部供应商订购，允许小鼠适应新环境两周在动物房子。轻轻EAE诱导前处理的老鼠，让他们习惯了处理。避免EAE诱导后出现不必要的处理。提供食物和水，他们可以很容易，只要他们表现出临床症状到达老鼠。如果小鼠对与药物治疗，最好是它混入食物或饮用水，以避免混淆应力的效果。如果管饲法或喷射为用于药物施用必不可少的，它使用相同的路线的车辆是很重要的。 除了这里所描述的原发进行性EAE模型，复发 - 缓解EAE模型也存在。不同的病程是由不同的小鼠品系不同抗原的给药引起。因此，MOG 35的应用- 55在小鼠品系，如C57BL / 6，Sv129，B10，NOD和Biozzi小鼠，导致原发进行性疾病形式。有趣的是，发病的当天的不同之处的各种小鼠品系。而C57BL / 6，SV129和B10小鼠发展第一临床症状，从约11天到12，Biozzi小鼠显示第一次临床症状后21天36。在先前的研究中，它表明，129S4 / SvJae×C57BL / 6杂种首先从约11第28天表现出的临床症状。蛋白脂质蛋白的注射（PLP）131-151在SJL小鼠中导致的诱导复发-缓和病程37。最好的EAE模型中使用将取决于研究的重点。因此，如果复发相是感兴趣，复发 - 缓解模式应该被采用，而如果疾病发作是主要的焦点，应使用渐进模型。如果一个特定的蛋白质的作用是在EAE模型进行调查，使用敲除小鼠，它记住，不是所有的菌株可用于诱导EAE和可能的敲除菌株的回交到一个很重要敏感菌株是必要的。 三种最普遍的TMS的动物模型ypes是:（1）自身抗原诱导的EAE; （2）病毒诱导的自发的慢性脱髓鞘疾病，和（3）毒素诱发的脱髓鞘38。 EAE模型的特征在于由一个自身抗原肽的应用诱导炎症和自身免疫应答的如髓鞘少突胶质蛋白或蛋白脂质蛋白38。自发的EAE的发展中的转基因小鼠过表达T细胞受体对髓鞘少突胶质蛋白39的肽。为了诱导病毒诱导的慢性炎症，中枢神经系统的神经营养性感染可以被诱导，例如，与Theiler`s鼠脑脊髓炎病毒。感染了病毒的细胞被两个T细胞和体液应答攻击，从而导致慢性脱髓鞘40,41。毒素诱发的脱髓鞘可以被诱导，例如与铜螯合剂cuprizone 42。这种模式专门导致demyeli由于国家攻击cuprizone少突胶质细胞的和潜在客户，从而为星形胶质细胞和小胶质细胞的活化，而炎症过程只起到次要作用。毒素的清除后，产生了新的少突胶质细胞，并形成一个新的髓鞘。使用这三种模式的，以互补方式，允许在MS的发病机理的不同方面的药物的效果的评价，​​因为这些模型对于炎症，和免疫和脱髓鞘过程不同。在EAE和病毒诱导的模型，炎症和自身免疫过程主要驱动的疾病，而在毒素诱发的模型，脱髓鞘和髓鞘再生过程是主要的。对用于MS的治疗潜在药物的临床前试验，在至少三种模式，毒素诱导的模型中，复发 - 缓解EAE和原发进行性脑脊髓炎或病毒诱发的模型应该被用来验证该药物的有效性。 EAE模型描述了她E可以用来获得进一步洞察MS样疾病的发展过程中的作用机制，确定该疾病的关键细胞驱动程序，如Th1时，Th17细胞和B细胞43,44。参与MS的发展并促进和解决的病变的处理的炎症和免疫过程的更好的理解，更可能的是，新的治疗策略，可开发。 然而，这里描述的EAE模型只是在与MS共同的一些特征，并具有从人类疾病的几个明显的差异。最突出的区别在于，MS患者中疾病的介绍，这是不是在EAE模型再现广泛地变化。这可能是由于在MS患者和EAE小鼠和这样的事实，小鼠近交系不同病变的图案。在EAE小鼠，同质病灶中的脊髓最为突出，而在MS患者，主要异构病变在大脑45,46找到。此外，MS和EAE分享Th1细胞和Th17细胞的为他们的发展的重要贡献，但与MS，CD8 + T细胞在EAE病理33次要作用。总之，EAE模型主要集中于自身免疫，炎性过程和神经变性的联合作用而脱髓鞘过程是选择性评估较难。因此，其他动物模型如cuprizone模型可以用于脱髓鞘的不同研究处理47。 EAE模型是不完善的，但尽管如此，MS 33的一个有用的模型。

多发性硬化症（MS）及其相应的动物模型，实验性自身免疫性脑脊髓炎（EAE），显示出在中枢神经系统（CNS）的自身免疫性神经炎症的变化。早期主动MS和EAE的病变被浸润免疫细胞的存在表征。 MS的病因至今不明，但市场普遍认为涉及髓鞘由自身反应性T细胞介导的​​破坏。这些自身反应性T细胞分泌促炎性细胞因子和趋化因子，以吸引其他免疫细胞诸如B细胞，单核细胞和嗜中性粒细胞从循环。单核细胞分化成巨噬细胞。由自身反应性T细胞分泌干扰素γ（IFNγ）的极化将巨噬细胞促炎巨噬细胞。促进少突胶质细胞的细胞凋亡的促炎性巨噬细胞释放细胞因子和活性氧。少突胶质细胞的死亡导致脱髓鞘。此外，B细胞分化为plasma细胞和针对髓鞘释放的自身抗体，最终导致髓鞘退化。髓鞘的丧失导致轴突和神经元的退化和由此在CNS代表MS 1的主要特征的病变部位的形成。在外围，T细胞和B细胞中的淋巴结被激活，它们在脾增殖并通过循环迁移到中枢神经系统。单核细胞和嗜中性粒细胞增殖的骨髓和也通过循环迁移到中枢神经系统。 从骨髓，脾和淋巴结进入血液或从血流进入CNS白细胞外渗是一个多步骤的过程，依赖于几个因素，包括白细胞和内皮趋化因子和趋化因子受体介导的分子间的相互作用。由各种细胞类型产生的趋化因子可免疫reactio期间被诱导通过像肿瘤坏死因子α（TNFα），IFNγ和白细胞介素-6（IL-6），随后募集免疫细胞炎症2,3的部位的细胞因子Ñ。免疫细胞呈现在其表面上的趋化因子受体的子集，这取决于细胞类型和迁移途径的炎症部位。因此，CXCR2，CCR1和CXCR1是在骨髓和血液4成熟嗜中性粒细胞中表达，和它的配体，CXCL2，CCL5或CXCL6结合，分别激活嗜中性粒细胞，并促进其粘附到内皮和随后的细胞的迁移进入组织5-9。 CCL2和CCL20吸引单核细胞和Th1 / Th17细胞10，其分别表示CCR2 11和CCR6 12。 CCR1和CCR5，通过不同的细胞类型，包括T细胞，单核细胞和巨噬细胞13表示，结合CCL3，CCL5和CCL7和MS 14期间上调。 CXCR3表达于T细胞结合CCL9，CCL10和CCL11 15。 以MS治疗的一个主要策略是免疫细胞的耗竭或预防免疫细胞浸润进入CNS。因此，具体的趋化因子受体的阻断已经在EAE影响。拮抗或CCR1 16，CCR2 17的基因缺失，CCR7 18或CXCR2 19降低EAE病理学，而拮抗或CCR1 20的基因缺失，CCR5 20或CXCR3 21没有降低的病理学。因此，具体的趋化因子受体的白细胞上表达为后者的浸润进入CNS关键和决定EAE的过程。 免疫细胞的耗竭为MS患者的有效治疗策略，因为渗透免疫细胞释放细胞因子，如TNFα，IL-6和IL-1β，这反过来，促进炎症过程或神经元22的降解。此外，自动反应性Th1细胞释放IFNγ，这反过来又刺激巨噬细胞释放的TNFα，IL-1β和IL-23。 这个手稿描述的EAE，免疫细胞分布以及在EAE小鼠的各种组织中的细胞因子水平（mRNA）的测定的诱导。细胞在不同时间点的疾病过程中被分离，以提供所述炎性过程并最终导致在CNS损伤形成的时间依赖性的概述。

<table border="0" cellpadding="0" cellspacing="0" width="913">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">ABI Prism 7500 Sequence Detection System&#160;</td>
			<td style="width:305px;">Applied Biosystems, Austin, USA</td>
			<td style="width:119px;"></td>
			<td style="width:195px;">quantitative PCR system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Accutase</td>
			<td>Sigma Aldrich Munich, Germany</td>
			<td>A6964</td>
			<td>cell detachment solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD3-PE-CF594</td>
			<td>BD, Heidelberg, Germany</td>
			<td>562286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD4-V500</td>
			<td>BD, Heidelberg, Germany</td>
			<td>560782</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD8-eFluor650</td>
			<td>eBioscience, Frankfurt, Germany</td>
			<td>95-0081-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD11b-eFluor605</td>
			<td>eBioscience, Frankfurt, Germany</td>
			<td>93-0112-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD11c-AlexaFluor700</td>
			<td>BD, Heidelberg, Germany</td>
			<td>560583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD19-APC-H7&#160;</td>
			<td>BD, Heidelberg, Germany</td>
			<td>560143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD45-Vioblue&#160;</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-092-910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CompBeads</td>
			<td>BD, Heidelberg, Germany</td>
			<td>552843</td>
			<td>compensation beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase A</td>
			<td>Sigma Aldrich Munich, Germany</td>
			<td>C0130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytometric absolute count standard&#160;</td>
			<td>Polyscience, Eppelheim, Germany</td>
			<td>BLI-580-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytometer Setup and Tracking beads&#160;</td>
			<td>BD, Heidelberg, Germany</td>
			<td>642412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>Sigma Aldrich Munich, Germany</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">EAE Kit</td>
			<td>Hooke Laboratories, Lawrence, USA</td>
			<td>EK2110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">F4/80-PE-Cy7&#160;</td>
			<td>BioLegend, Fell, Germany</td>
			<td>123114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">First Strand cDNA-Synthesis kit&#160;</td>
			<td>Thermo Scientific, Schwerte, Germany</td>
			<td>K1612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fc receptor-1 blocking buffer&#160;</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow cytometric absolute count standard</td>
			<td>Polyscience, Eppelheim, Germany</td>
			<td>580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo software v10&#160;</td>
			<td>Treestar, Ashland, USA</td>
			<td></td>
			<td>flow cytometry software</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">LSRII/Fortessa&#160;</td>
			<td>BD, Heidelberg, Germany</td>
			<td></td>
			<td>flow cytometer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ly6G-APC-Cy7&#160;</td>
			<td>BD, Heidelberg, Germany</td>
			<td>560600</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Lysing solution&#160;</td>
			<td>BD, Heidelberg, Germany</td>
			<td>349202</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Maxima SYBR Green&#160;</td>
			<td>Thermo Scientific, Schwerte, Germany</td>
			<td>K0221</td>
			<td>fluorescent DNA binding dye&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy Mini Kit&#160;</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>74104</td>
			<td>RNA extraction kit</td>
		</tr>
	</tbody>
</table>

induction of experimental autoimmune encephalomyelitis in mice and evaluation of the disease-dependent distribution of immune cells in various tissues

多发性硬化症是推定为炎症性自身免疫疾病，它在中枢神经系统（CNS），导致认知和运动功能障碍的特征是病变的形成。实验性自身免疫性脑脊髓炎（EAE）是MS的有用的动物模型，是因为它的特征还在于通过损伤形成在CNS，运动功能障碍，并且也由自身免疫和炎性反应驱动。一项所述的EAE模型被诱导与来自髓鞘少突胶质蛋白（MOG）35-55小鼠衍生的肽。在EAE小鼠制定一个渐进的病程。这当然分为三个阶段:临床前阶段（第0天 - 9），疾病发作（10天 - 11）和急性期（12天 - 14）。 MS和EAE是由渗透到中枢神经系统自身反应性T细胞诱导。这些T细胞分泌趋化因子和细胞因子而导致进一步的免疫细胞的募集。因此，在脊髓D中的免疫细胞的分布uring这三种疾病的阶段进行了研究。以突出的T细胞，B细胞和单核细胞的活化/增殖/累积开始时的疾病的时间点，在淋巴结，脾和血液中的免疫细胞的分布也被评估。此外，一些细胞因子（IL-1β，IL-6，IL-23，TNFα，IFNγ）的三种疾病相的水平进行测定，以深入了解该疾病的炎性过程。最后，该数据提供了免疫细胞的EAE病理过程中的功能信息的概述。

图1给出了本文中介绍的不同方法的示意图1）小鼠接受MOG 35-55抗原注射后10.7±0.3天28开发初期临床症状。 EAE小鼠的代表性疾病过程示于图1。2）各种组织（脾，淋巴结，脊髓）和血液从在不同时间点的控制和EAE小鼠中的临床前阶段（第2天，4天萃取6天），该疾病的发病过程中（10天）和在疾病（12天，14天）; 3）细胞因子，从而调节EAE发展的mRNA表达谱的急性期，在各个确定的所述疾病过程中的组织在不同时间点提取，使用定量PCR。4）单细胞从提取的各种组织中分离在疾病过程和免疫细胞分布在不同的时间点使用流式细胞仪测定。 在脾和淋巴结细胞，T细胞和B细胞中观察到的最显着的细胞类型。在对比淋巴结，脾脏，此外，大量的单核细胞和嗜中性粒细胞的存在。所有免疫细胞显示在急性期在淋巴结暂时升高，而只有巨噬细胞，B细胞，T细胞和嗜中性粒细胞在脾增加。在血液中，所有的免疫细胞在临床前阶段瞬时增加。在腰脊髓，在所有免疫细胞的疾病依赖性增加观察到，除了单核细胞，这大概分化的巨噬细胞的入口进入脊髓（ 图2）之后。 CD4 +和CD8 + T细胞显示了在不同的病程中脾脏，淋巴结和血液类似的变化。上午翁细胞浸润脊髓，在CD4 + T细胞的增加是最明显（ 图3）。在图4中，对于不同的细胞群的确定门控策略被示出。详细地说，在这个手稿中描述的细胞群被确定为如下:单核细胞（CD45hi，CD3-，Ly6G-，CD19-，CD11c-，细胞CD11b +），巨噬细胞（CD45hi，CD3-，Ly6G-，CD11c-，CD11b的+，F4 / 80hi），嗜中性粒细胞（CD45hi，CD3-，细胞CD11b + Ly6G +），树突细胞（CD45hi，CD3-，Ly6G-，CD19-，的CD11c +），B细胞（CD45hi，CD3-，CD19 +），T细胞（CD45hi，CD3 +） ，CD4 + T细胞（CD45hi，CD3 +，CD4 +）和CD8 + T细胞（CD45hi，CD3 +，CD8 +）。细胞的数量相关的组织的量（重量脾脏，淋巴结，脊髓）或血容量，从而反映绝对细胞数。然而，有可能作为总细胞的百分比衡量来表达的细胞类型。 在LYMpH值的节点，IL-23 mRNA的表达在临床前阶段瞬时增加，而IL-6的mRNA的表达的疾病依赖性增加。在脾，IL-6和IL-23的mRNA表达在临床前阶段瞬时增加，而在该疾病的发作，IL-1β的mRNA表达被提高。在血液，TNFαmRNA的表达的一种疾病相关的方式增加。在腰脊髓，TNFα，IL-1β和在急性期的IFNγ增加，而IL-6在发病期（ 图5）上调。 <img alt="图1" src="/files/ftp_upload/53933/53933fig1.jpg" /> 图1:工作流程进行诱导和EAE的免疫学评估方案。 1）EAE的小鼠导致的临床症状的发展引起的。临床症状群分类通过临床评分编，如下:0）没有迹象，0.5）尾的远端麻痹，1）的尾部的完全瘫痪，1.5）柔弱的尾部和后肢轻度虚弱，2）柔弱的尾部和后腿严重的弱点，2.5）柔弱的尾部和一个后腿，3）柔弱的尾部和两条后腿瘫痪麻痹。在所示的图中使用的小鼠的数量为7的数字已从巴塞尔姆斯等人 28修改。2）将小鼠的疾病过程中，脾，腹股沟淋巴结，血液和腰脊髓中在不同时间点处死提取和单细胞从这些组织中分离出来。3）在各种组织免疫细胞分布，流式细胞仪检测; 4）在不同组织中的各种细胞因子，通过定量PCR检测mRNA的表达。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53933/53933fig1large.jpg" target="_blank">请点击此处查看大图此F的</a>igure。 <img alt="图2" src="/files/ftp_upload/53933/53933fig2.jpg" /> 图2:免疫细胞分布，通过FACS（荧光激活细胞分选）分析，脾脏，淋巴结，从不同疾病阶段EAE小鼠血液和脊髓样品测定在实验中所示，每组小鼠的数目为2 - 10中性粒细胞的分布/血中性粒细胞和单核细胞/脊髓巨噬细胞改编并从巴塞尔姆斯等[28]修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53933/53933fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53933/53933fig3.jpg" /> 图3:CD4 + T细胞和CD8 <su从EAE小鼠样品 P&gt; + T细胞的分布不同疾病阶段，流式细胞仪分析确定。 A）淋巴结，B）脾，C）血液和D）脊髓。在所示的实验中，每组小鼠的数目为2 - 10。结果以平均值±标准差（SEM）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53933/53933fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53933/53933fig4.jpg" /> 图4: 门控策略T细胞，B细胞，中性粒细胞，单核细胞，树突状细胞和巨噬细胞从12日A）SSC / CD45的所有细胞中的情节EAE小鼠脊髓的流式细胞仪分析 。门:从CD45 +细胞CD45 +细胞B）FSC-H /的情节FSC-W秒。门:单细胞C）SSC-A的剧情/ FSC-A从单个细胞。门:活细胞。 D）FSC-H / CD3从活细胞的情节。门:CD3 +和CD3 -细胞E）CD4 / CD8从CD3 +细胞中的情节。门:CD4 + CD8 - （CD4 + T细胞 ）和CD4 - CD8 +（CD8 + T细胞 ）F）CD19和Ly6G / CD11b的从CD3一个图景-细胞。门:CD19 +细胞CD11b -细胞（B细胞 ），Ly6G +的CD11b +细胞（ 中性粒细胞 ）和CD19 - Ly6G -细胞G）的CD11c / CD11b的细胞从CD19作图- Ly6G -细胞:门的CD11c +细胞（ 树突状细胞 ）和CD11c的-细胞高）SSC / F4 / 80 fr的情节。OM的CD11c -细胞。门:F4 / 80 -细胞（ 单核 ）和F4 / 80 +细胞（ 巨噬细胞 ）。 9一位代表的情节所示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53933/53933fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53933/53933fig5.jpg" /> 图 5:细胞因子分布（IL-1β，IL-6，IL-23，TNFα，IFNγ） 的不同疾病阶段从EAE小鼠样品 A）淋巴结，B）的脾，C）的血液和D）脊髓。 mRNA的表达水平标准化为肽基丙基异构酶（PPIA），并使用相同年龄的未处理小鼠的mRNA水平进行了计算。测量一式三份进行。每组小鼠的数目是2 - 3。结果表示为平均值±站ndard误差（SEM）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53933/53933fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 基因 </td><td colspan="3"> 前锋 </td><td colspan="3"> 相反 </td><td> fragement大小（BP） </td></tr><tr><td> IL-1β的</td><td colspan="3"> CTGGTGTGTGACGTTCCCATTA </td><td colspan="3"> CCGACAGCACGAGGCTTT </td><td> 76 </td></tr><tr><td> IL-6的</td><td colspan="3"> TAGTCCTTCCTACCCCAATTTCC </td><td colspan="3"> TTGGTCCTTAGCCACTCCTTC </td><td> 76 </td></tr><tr><td> IL-23的</td><td colspan="3"> GAGCAGCAACTCTGACTGAGCC </td><td colspan="3"> GAACAGCACAAGTCCTAATGGGTTA </td><td> 127 </td></tr><tr><td> IFNγ </td><td colspan="3"> CACGGCACAGTCATTGAAAGC </td><td colspan="3"> CACCATCCTTTTGCCAGTTCC </td><td> 118 </td></tr><tr><td> TNFα </td><td colspan="3"> TGACAAGCCTGTAGCCCACG </td><td colspan="3"> GCCTTGTCCCTTGAAGAGAACC </td><td> 178 </td></tr><tr><td> PPIA </td><td colspan="3"> GCTGGACCAAACACAAACGG </td><td colspan="3"> GCCATTCCTGGACCCAAAAC </td><td> 144 </td></tr></tbody></table> 表1:引物对。

Watch this Scientific Journal Video about Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues at JoVE

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

induction-experimental-autoimmune-encephalomyelitis-mice-evaluation

Nanyang Technological University

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Immunology and Infection

23.4K 次观看.  Goethe University Hospital Frankfurt. This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

视频: Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

鼠标垂体免疫原的制备诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

科研

免疫与感染

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

SJL小鼠诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

在实验性自身免疫性脑脊髓炎（EAE）抗性品系小鼠继转移和抗原挑战克服反应迟钝

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

髓鞘少突胶质细胞糖蛋白（MOG<sub&gt; 35-55</sub&gt;）诱导实验性自身免疫性脑脊髓炎（EAE）的C57BL / 6小鼠

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

诱导BALB/c小鼠药物诱导的自身免疫性肝炎研究其致病机制

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

葡萄糖-6-磷酸异构酶（G6PI）诱导RA小鼠中iNKT细胞的采用免疫治疗

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...