Name: induction of experimental autoimmune encephalomyelitis in mice and evaluation of the disease-dependent distribution of immune cells in various tissues
Uploaded: 2016-05-08T14:00:00.000+00:00
Duration: 8 min 47 s
Description: 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

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?...

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

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

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JoVE Journal

Immunology and Infection

23.5K 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

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 ...

科研

免疫与感染

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 ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

确定免疫系统抑制中枢神经系统对保护药物干预自身免疫性脱髓鞘

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

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 ...

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 ...

研究自身免疫性脱髓鞘中的体液反应

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

作者聚焦:在多发性硬化症研究中研究 B 细胞反应的新型检测方法

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

实验性自身免疫性脑脊髓炎后小鼠自身反应性抗体的定量

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...

从自身免疫小鼠中同时分离所有 CNS 驻留细胞类型

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

作者聚焦:通过同时分离所有主要的 CNS 驻留细胞类型来研究神经炎症通路 

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

从成年自身免疫性脑脊髓炎小鼠中同时分离主要中枢神经系统驻留细胞类型

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...

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

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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

髓鞘少突胶质细胞糖蛋白（MOG

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实验性自身免疫性脑脊髓炎后小鼠自身反应性抗体的定量

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