Name: a simple approach to induce experimental autoimmune neuritis in c57bl/6 mice for functional and neuropathological assessments
Uploaded: 2017-11-09T13:00:00.000+00:00
Duration: 7 min 30 s
Description: Watch this Scientific Journal Video about A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments at JoVE.com

DGG 是一个澳洲彼得多尔蒂和多发性硬化研究澳大利亚 (微软亚洲研究院) 早期职业研究员。JLF 得到微软亚洲研究院博士后奖学金的支持。这项工作得到澳大利亚国家卫生和医学研究理事会 (澳洲) 项目赠款 #APP1058647 JX。

此处描述的所有程序均由弗洛里神经科学和精神健康研究所 (墨尔本脑中心) 动物伦理委员会批准, 并遵循澳大利亚的《使用动物科学守则》目的. 
 1. 可诱导性 注意: 在6-8 周的雄性 C57BL/6 小鼠中可以成功的诱导。归纳协议总共需要9天。0天指的是第一次免疫的那天。对于这个协议, 注射进行了麻醉和 #160;(见下面的步骤 1.1.2). 
<ol> <li> 在天-1: <ol> <li> 准备百日咳毒素 (参见 材料表 ) 解决方案的1.6 和 #181; 使用无菌0.1 米小鼠-等渗磷酸盐缓冲盐水 (MT PBS)。
		<li> 麻醉与异氟醚: <ol> <li> 在麻醉室中放置鼠标 (C57BL/6、雄性、6-8 周), 并将氧气流量调整到1升/分. </li>
			<li> 打开异氟醚蒸发器, 调整它为麻醉 2.5%, 并监测呼吸和等待2分钟, 或直到初级反射 (角膜和后肢) 不再响应和 #160; </li> </ol>
		</li> <li> 从麻醉腔中取出鼠标, 并管理250和 #181; 上述百日咳毒素溶液通过腹腔 (ip) 注射使用0.5 毫升注射器与 30 1/2 G 针. </li>
		<li> 准备注射接种免疫: <ol> <li> 准备 解决方案 使用2毫克/毫升的 P0 180-199 多肽解决方案 (带和 #62; 98% 纯度, 在0.9% 盐水中, 序列 s-s--r-g--r-Q-T-对-钒--l--s--M--d--s-秒--s. </li>
			<li> 准备 解决方案 B 使用20毫克/mL 解决方案 结核分枝杆菌 (见 材料表 ) 在弗氏和 #39; 完全佐剂 (包括: 15% mannide monoolate + 85% 的石蜡油和0.5 毫克/毫升干燥杀死和干的 M 分枝杆菌酸 ). </li>
			<li> 将最终接种用于注射, 将解决方案 a 和 B 的相等体积部分组合在一个磁珠搅拌器中, 并在室温下以最高速度混合1分钟. 
			注: 解决方案 a 和 B 可以保存为库存, 并存储在4和 #176; C 为最多一个月, 但组合的最终接种必须在鼠标注射当天新鲜准备. </li>
		在0天的 </ol> </li> </ol> </li> <li> 中, 管理50和 #181; 接种 (步骤1.1.4 中概述) 的 L, 通过皮下注射使用23克针的皮下注射进入小鼠. 
	注: 注射可以放在肩胛骨之间, 也可放在后肢和尾部之间. </li>
	<li> 在1天, 使百日咳毒素溶液的1.2 和 #181; g/毫升 (在 MT PBS) 和管理250和 #181; L 通过 ip 注射使用0.5 毫升注射器与 30 1/2 g 针的一只老鼠. </li>
	<li> 在3天, 重复上面的步骤 1.3. 
	注: 库存解决方案 a 和 B 可以组成, 并存储在4和 #730; C 为最多一个月。然而, 最终注射接种, 生产的结合股票解决方案 a 和 B, 必须在当天进行注射. </li>
	<li> 在8天, 管理50和 #181; 接种 (步骤1.1.4 中概述) 的 L, 通过皮下注射 (参见上面的步骤 1.2), 在与步骤 1.2 (每种动物) 相同的注射地点, 在同一注入的小鼠上. </li> </ol>
 2. 临床评分 <ol> <li> 每天从0开始执行临床评分. </li>
	<li> 根据已发布的条件为每个鼠标提供0、1、2、3或4的分数 4 。请参见 表 1 以了解临床评分. 
	注意: 为了保持一致性, 应该努力在同一时间每天同时对小鼠进行评分. </li>
</ol> 3。电机功能评估 注意: 对相同队列的动物进行临床评分, 同时评估其运动性能。电机功能评估装置必须连接到装有步态功能成像系统的计算机 (参见 材料表 )。此外, 还建议所有被评估的小鼠都应该习惯于在诱导前进行的跑步任务。为此, 练习运行 (每只鼠标2测试运行) 在疾病诱导前三天执行 (天-3). 
<ol> <li> 打开马达功能评估设备并在指示灯按钮上切换. </li>
	<li> 将鼠标牢牢地放在它的脚上, 然后将其放到一个装满红色墨水的容器中, 并保持其尾部的墨水. 
	注意: 此步骤对于 C57BL/6 应变是必需的, 但如果使用白大褂颜色的鼠标应变, 则可以跳过. </li>
	<li> 将鼠标放到步行车厢中, 并将跑步机速度设置为15厘米/秒. </li>
	<li> 打开跑步机并单击 #34; 记录和 #34; 按钮以使用步态功能成像系统捕获鼠标的运行动作 (请参阅 材料表 ). </li>
	<li> 使用计时器并测量每个运行三十六年代的时间。三十六年代以后, 停止记录和停止跑步机. 
	注意: 无法成功完成三十六年代时间间隔的运行任务的鼠标被认为失败. </li>
	<li> 将视频文件保存在指定的文件夹中. </li>
	<li> 对每个要评估的鼠标重复上述步骤。每3天测试一次相同的运行任务的小鼠. </li>
	<li> 使用软件为步态功能参数分析编辑过的视频文件 (请参阅 材料表 ). 
	注: 有关如何分析不同的马达参数的详细信息, 请参阅制造商和 #39 的说明, 然后再进行分析。通过免疫组化或电子显微镜获得轴突和髓鞘损伤的组织学证据, 动物组织可以根据研究的具体目的, 在任何阶段后运动功能评估中进行. </li>
</ol>

<ol>
	<li>Newswanger, D. L., Warren, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guillain-Barre+syndrome.">Guillain-Barre syndrome.</a> Am Fam Physician. 69 (10), 2405-2410 (2004).</li><li>Ruiz, E., Ramalle-Gomara, E., Quinones, C., Martinez-Ochoa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+Guillain-Barre+syndrome+mortality+in+Spain+from+1999+to+2013.">Trends in Guillain-Barre syndrome mortality in Spain from 1999 to 2013.</a> Int J Neurosci. 126 (11), 985-988 (2016).</li><li>Walling, A. D., Dickson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guillain-Barre+syndrome.">Guillain-Barre syndrome.</a> Am Fam Physician. 87 (3), 191-197 (2013).</li><li>Gonsalvez, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+and+Neuropathological+Testing+Paradigm+Reveals+New+Disability-Based+Parameters+and+Histological+Features+for+P0180-199-Induced+Experimental+Autoimmune+Neuritis+in+C57BL/6+Mice.">A Functional and Neuropathological Testing Paradigm Reveals New Disability-Based Parameters and Histological Features for P0180-199-Induced Experimental Autoimmune Neuritis in C57BL/6 Mice.</a> J Neuropathol Exp Neurol. 76 (2), 89-100 (2017).</li><li>Berg, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guillain-Barre+syndrome:+pathogenesis,+diagnosis,+treatment+and+prognosis.">Guillain-Barre syndrome: pathogenesis, diagnosis, treatment and prognosis.</a> Nat Rev Neurol. 10 (8), 469-482 (2014).</li><li>Koller, H., Schroeter, M., Kieseier, B. C., Hartung, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+inflammatory+demyelinating+polyneuropathy--update+on+pathogenesis,+diagnostic+criteria+and+therapy.">Chronic inflammatory demyelinating polyneuropathy--update on pathogenesis, diagnostic criteria and therapy.</a> Curr Opin Neurol. 18 (3), 273-278 (2005).</li><li>Griffin, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+the+motor-sensory+axonal+Guillain-Barre+syndrome.">Pathology of the motor-sensory axonal Guillain-Barre syndrome.</a> Ann Neurol. 39 (1), 17-28 (1996).</li><li>Taylor, W. A., Hughes, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+allergic+neuritis+induced+in+SJL+mice+by+bovine+P2.">Experimental allergic neuritis induced in SJL mice by bovine P2.</a> J Neuroimmunol. 8 (2-3), 153-157 (1985).</li><li>Zou, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P0+protein+peptide+180-199+together+with+pertussis+toxin+induces+experimental+autoimmune+neuritis+in+resistant+C57BL/6+mice.">P0 protein peptide 180-199 together with pertussis toxin induces experimental autoimmune neuritis in resistant C57BL/6 mice.</a> J Neurosci Res. 62 (5), 717-721 (2000).</li><li>Miletic, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P0(106-125)+is+a+neuritogenic+epitope+of+the+peripheral+myelin+protein+P0+and+induces+autoimmune+neuritis+in+C57BL/6+mice.">P0(106-125) is a neuritogenic epitope of the peripheral myelin protein P0 and induces autoimmune neuritis in C57BL/6 mice.</a> J Neuropathol Exp Neurol. 64 (1), 66-73 (2005).</li><li>Hafer-Macko, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+motor+axonal+neuropathy:+an+antibody-mediated+attack+on+axolemma.">Acute motor axonal neuropathy: an antibody-mediated attack on axolemma.</a> Ann Neurol. 40 (4), 635-644 (1996).</li><li>Griffin, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+nodal+changes+in+the+acute+motor+axonal+neuropathy+pattern+of+the+Guillain-Barre+syndrome.">Early nodal changes in the acute motor axonal neuropathy pattern of the Guillain-Barre syndrome.</a> J Neurocytol. 25 (1), 33-51 (1996).</li></ol>

作者对这项工作没有利益冲突。

本报告概述了一个简单的方法, 诱导使用 P0180-199多肽在 C57BL/6 小鼠, 使量化的关键病理和功能缺陷的小鼠的免疫诱导。不同的是, 这里所描述的归纳协议是使用麻醉, 而执行免疫注射。使用异氟醚麻醉, 大大提高了能力, 以确保总体积的接种被注入皮下在理想的位置, 最小的错误和压力的动物。
小鼠将开始显示疾病的临床症状从 6 dpi4使用这种方法的诱?...

外周神经病可以是遗传的起源或后天, 与后天神经病有代谢, 缺血性, 炎症, 或毒性沉淀。这些疾病也可以被归类为轴突或鞘的来源。最常见的后天性脱髓鞘外周神经病是急性炎症性脱髓鞘神经病 (AIDP, 也称为格林-巴利综合征, GBS) 和慢性炎症性脱髓鞘神经病 (CIDP)1,2,3,4;两者都是 pathogenetically 的特点是对髓鞘的自身免疫反应, 导致鞘周围神经。在这些疾病中, 活化的 T 细胞穿过血神经屏障, 在三七皂苷内产生免疫反应。激活的巨噬细胞, 然后导致鞘要么直接通过吞噬的攻击或间接通过分泌炎症介质, 导致临床残疾, 如麻痹和感觉障碍5。虽然鞘轴突保留 remyelinated 跟随鞘的能力, 髓通常是延迟或不完整的, 导致裸轴突的易感性到不可逆转的损害, 这是永久临床的主要原因残疾.目前, 最有效的治疗方法是免疫调节, 但尽管他们的疗效, 在许多情况下, 恢复往往是缓慢的和〜25% 患者将体验到剩余的功能赤字, 大大降低他们的生活质量6, 7。
是一种广泛使用的脱髓鞘周围神经病的动物模型, 它提供了有价值的洞察机制和评估新的治疗药物的方法4。该模型可诱导不同种类的家兔、大鼠、小鼠和豚鼠, 并由神经源性抗原免疫诱发。然而, 最终成功的细胞诱导依赖于适当的免疫反应的疾病发生。鉴于免疫功能的种类 (和间/应变) 的变化, 多种抗原和佐剂的组合已被开发成功地诱导了。在小鼠遗传工具方面, C57BL/6 是最广泛使用的;然而, 传统的 P2 蛋白肽 57-81 (P257-81), 导致在易感 SJL 小鼠菌株的疾病8是不能非法发病导致功能性赤字的 C57BL/6 菌株。幸运的是, 使用 P0106-125或 P0180-199多肽的敏化范例, 连同注射百日咳毒素的佐剂一起提供, 可以克服这一障碍, 使复杂的遗传工具能够用于小鼠模型。
本文介绍了一种简单的 C57BL/6 小鼠的诱导方法。此外, 还提供了一个综合详细的方法来评估与疾病相关的功能和病理的缺陷。P0180-199多肽9首选于 P057-81备选10。与 P057-81备选10相比, P0180-199模型已被描述为产生较不严重的临床症状, 因此可能会承受引入潜在有害的遗传摄动, 从外科手术中恢复 (如渗透泵植入), 并经得起跑步机的步态功能测试4。然而, 在 P0 的57-81诱导变异中研究这种疾病时, 可以很容易地使用在这里描述的跑步机步态功能测试和组织学协议。

<table><tbody><tr><td>C57BL/6, male, 6-8 weeks old</td><td>Australian Bioresources Cenre, WA, Australia</td><td></td><td></td></tr><tr><td>Pertussis toxin</td><td>List Biological Laboratories, Inc., CA, USA</td><td>#181</td><td></td></tr><tr><td>0.1 M mouse-isotonic phosphated buffered salined (MT-PBS)</td><td></td><td></td><td>Laboratories will have their own protocol.</td></tr><tr><td>Isoflurane</td><td>Pharmachem, QLD, Australia</td><td></td><td>Laboratories will have their own protocol for administration.</td></tr><tr><td>P0&lt;sub&gt;180&amp;ndash;199 &lt;/sub&gt;peptide</td><td>Wuxi Nordisk Biotech Co. Lt. SHG, CHN</td><td></td><td>P0&lt;sub&gt;180&amp;ndash;199&lt;/sub&gt;, sequence S&amp;ndash;S&amp;ndash;K&amp;ndash;R&amp;ndash;G&amp;ndash;R&amp;ndash; Q&amp;ndash;T&amp;ndash;P&amp;ndash;V&amp;ndash;L&amp;ndash;Y&amp;ndash;A&amp;ndash;M&amp;ndash;L&amp;ndash;D&amp;ndash;H&amp;ndash;S&amp;ndash;R&amp;ndash;S</td></tr><tr><td>Heat killed Mycobacterium tuberculosis (strain H37RA)</td><td>Difco, MI, USA</td><td>#231141</td><td></td></tr><tr><td>Freund&#039;s complete adjuvant (FCA)</td><td>Difco, MI, USA</td><td>#263910</td><td></td></tr><tr><td>16% Paraformaldehyde (PFA)</td><td>Electron Microscopy Services</td><td>#15710</td><td>Dilute to 4% PFA day of tissue collection.</td></tr><tr><td>25% glutaraldheyde</td><td>ProSciTech Pty Ltd, QLD, Australia</td><td>#11-30-8</td><td>Dilute to 2.5% glutaraldehyde day of fixation.</td></tr><tr><td>Sodium azide</td><td>Chem-Supply Pty Ltd, SA, Australia</td><td>SL189</td><td>Create 10% (w/v) stock in 0.1M MT-PBS. Use at 0.03% (v/v).</td></tr><tr><td>Sucrose</td><td>Chem-Supply Pty Ltd, SA, Australia</td><td>SA030</td><td>Use at 30% (w/v).</td></tr><tr><td>Optimum cutting temperature (OCT) medium</td><td>Sakura Finetek, CA, USA</td><td>#4583</td><td></td></tr><tr><td>Normal donkey serum</td><td>Merck Millipore, MA, USA</td><td>#S30-100</td><td>Use as antibody diluent at 10% (v/v) or other concentration determined by own laboratory.</td></tr><tr><td>Triton-X 100</td><td>Sigma Aldrich, MI, USA</td><td>#90o2-31-1</td><td>Use in antibody diluent at 0.3% (v/v) or other concentration determined by own laboratory.</td></tr><tr><td>rabbit anti-amyloid precursor protein (APP)</td><td>Invitrogen (Life Technologies), CA, USA</td><td>S12700</td><td>Used at 1:400 or titrate in own lab.</td></tr><tr><td>rabbit anti-contactin-associated protein-1 (Caspr)</td><td>Gift from Prof Elior Peles, Wiezmann Institute of Science, Israel</td><td></td><td>Used at 1:500 or titrate in own lab.</td></tr><tr><td>Appropriate Alexa Fluor conjugated secondary antibodies</td><td>Molecular Probes (Life Technologies), OR, USA</td><td>Various</td><td>Use at 1:200 or titrate in own lab. Choice of species the antibody was raised in and Alexa Fluor chosen is at the discretion of each laboratory.</td></tr><tr><td>Aqueous mounting solution</td><td>Dako (Agilent), CA, USA</td><td>#S3023</td><td>Each laboratory will have their own preference.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.5 mL syringe with 30&lt;sup&gt;1/2&lt;/sup&gt; g needles</td><td>BD</td><td>#326105</td><td></td></tr><tr><td>23 g needles</td><td>BD</td><td>#305143</td><td></td></tr><tr><td>Red ink pad</td><td></td><td></td><td>Any red ink pad or red food dye could be used to mark the animals&#039; feet.</td></tr><tr><td>DigiGate apparatus (includes treadmill)</td><td>eMouse Specifics Inc. Framingham, MA</td><td></td><td></td></tr><tr><td>DigiGate Imaging System</td><td>eMouse Specifics Inc. Framingham, MA</td><td></td><td></td></tr><tr><td>Stopwatch</td><td></td><td></td><td>Any timer may be used.</td></tr><tr><td>DigiGait 8 Software</td><td>eMouse Specifics Inc. Framingham, MA</td><td></td><td></td></tr><tr><td>Dissecting microscope</td><td>Zeiss</td><td></td><td>Any appropriate dissecting microscope may be used.</td></tr><tr><td>Charged slides</td><td>Superfrost Plus, Lomb Scientific Pty Ltd</td><td>SF41296SP</td><td></td></tr><tr><td>Cyrostat</td><td>Leica</td><td></td><td>Any suitable cyrostat may be used.</td></tr><tr><td>Perfusion equipment and dissecting instruments</td><td></td><td></td><td>Labs will have their own perfusion protoctols.</td></tr><tr><td>Opaque humified chamber</td><td></td><td></td><td>Labs may produce their own using an opaque plastic container.</td></tr><tr><td>PAP pene</td><td>GeneTex (USA)</td><td></td><td>Wax pencil, or surface tension may also be used to create a well around the tissue section.</td></tr><tr><td>Confocal microscope</td><td>Zeis LSM780</td><td></td><td>Any confocal microscope with appropriate laser lines may be used.</td></tr><tr><td>FIJI/Image J</td><td>National Institues of Health</td><td></td><td>Available from www.fiji.sc</td></tr></tbody></table>

a simple approach to induce experimental autoimmune neuritis in c57bl/6 mice for functional and neuropathological assessments

实验性自身免疫神经炎 (欢迎) 是自身免疫性外周脱髓鞘疾病的实验模型。通过免疫小鼠的神经源性多肽诱导炎症, 使其对周围神经系统的成分有直接的攻击作用。最近的进展使得在相对耐药的 C57BL/6 小鼠线使用髓鞘蛋白零 (P0)106-125或 P0 的180-199多肽在佐剂和注射百日咳毒素的结合。在 C57BL/6 菌株中诱导药物的能力允许使用在这个老鼠背景上存在的众多的基因工具, 从而允许对疾病的发病机制和对新疗法的机械作用的审讯的复杂研究与转基因方法相结合。在本研究中, 我们展示了一个简单的方法, 成功地诱导使用 P0180-199肽的 C57BL/6 小鼠。我们还概述了在这个模型中出现的功能缺陷评估的一个协议, 同时还有一系列的病理特征。因此, 该模型是研究人外周脱髓鞘神经病的一个强有力的实验模型, 并确定了旨在促进髓鞘修复和预防自身免疫性神经损伤的潜在疗法的疗效。炎.

P0180-199多肽诱导的 C57BL/6 小鼠, 导致一个单相疾病的临床评分开始从6天后第一次免疫 (dpi), 和最大评分严重性观察从 25 dpi, 然后从 40 dpi 的一些临床评分改善持续时间 (图 1)4,9。在步态功能方面, 早在 6 dpi 时, 小鼠就开始在一个简单的跑步任务上失败, 35 dpi 小鼠几乎没有能力完成一个简单的跑步机运?...

Watch this Scientific Journal Video about A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments at JoVE.com

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

本报告概述了一个简单的方法, 成功诱导实验性自身免疫神经炎 (一) 使用髓磷脂蛋白零 (P0)180-199肽结合弗氏的完整佐剂和百日咳毒素。我们提出了一个复杂的范例, 能够准确地评估在这一过程中出现的功能性赤字和神经病的程度。

C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

a-simple-approach-to-induce-experimental-autoimmune-neuritis-c57bl6

Research

JoVE Journal

Neuroscience

9.2K 次观看.  The University of Melbourne. 本报告概述了一个简单的方法, 成功诱导实验性自身免疫神经炎 (一) 使用髓磷脂蛋白零 (P0)180-199肽结合弗氏的完整佐剂和百日咳毒素。我们提出了一个复杂的范例, 能够准确地评估在这一过程中出现的功能性赤字和神经病的程度。

视频: A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

神经干/祖细胞在小鼠EAE的慢性全身性注射

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

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 ...

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

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

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) ...

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing motor function deficits are basic readouts of the immune-mediated inflammation of the spinal cord. However, scores and body weight do not allow for an in vivo assessment of brain inflammation and optic neuritis. The latter is an early and frequent manifestation in about 2/3 of MS patients. Here, we show methods for bioluminescence and near-infrared live imaging to assess EAE evoked optic neuritis, brain inflammation, and blood-brain barrier (BBB) disruption in living mice using an in vivo imaging system. A bioluminescent substrate activated by oxidases primarily showed optic neuritis. The signal was specific and allowed the visualization of medication effects and disease time courses, which paralleled the clinical scores. Pegylated fluorescent nanoparticles that remained within the vasculature for extended periods of time were used to assess the BBB integrity. Near-infrared imaging revealed a BBB leak at the peak of the disease. The signal was the strongest around the eyes. A near-infrared substrate for matrix metalloproteinases was used to assess EAE-evoked inflammation. Auto-fluorescence interfered with the signal, requiring spectral unmixing for quantification. Overall, bioluminescence imaging was a reliable method to assess EAE-associated optic neuritis and medication effects and was superior to the near-infrared techniques in terms of signal specificity, robustness, ease of quantification, and cost.

We show a technique for in vivo live bioluminescence and near-infrared imaging of optic neuritis and encephalitis in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis in SJL/J mice.

多发性硬化症的小鼠模型EAE视神经炎及脑部炎症的生物发光和近红外成像

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing ...

医学

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 ...

A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely used as a model to identify new drug targets for better patient treatment. MS is characterized by several different disease courses: relapsing-remitting MS (RRMS), primary progressive MS (PPMS), secondary progressive MS (SPMS), and a rare progressive-relapsing form of MS (PRMS). Although animal models do not accurately mimic all of these contrasting human disease phenotypes, there are EAE models that reflect some of the different clinical manifestations of MS. For example, myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6J mice mimics human PPMS, while myelin proteolipid protein (PLP)-induced EAE in SJL/J mice resembles RRMS. Other autoantigens, such as myelin basic protein (MBP), and a number of different mouse strains are also used to study EAE. To induce disease in these autoantigen-immunization EAE models, a water-in-oil emulsion is prepared and injected subcutaneously. The majority of EAE models also require an injection of pertussis toxin for the disease to develop. For consistent and reproducible EAE induction, a detailed protocol to prepare the reagents to produce antigen/adjuvant emulsions is necessary. The method described here takes advantage of a standardized method to generate water-in-oil emulsions. It is simple and fast and uses a shaking homogenizer instead of syringes to prepare quality-controlled emulsions.

To induce experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, mice are immunized with a water-in-oil emulsion containing an autoantigen and complete Freund's adjuvant. While several protocols exist for the preparation of these emulsions, a rapid, simple, and standardized homogenization protocol for emulsion preparation is presented here.

一种快速、简单、标准化的均质化制备抗原/辅助乳剂以诱导实验性自身免疫性脑脊髓炎的方法

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely ...

小鼠实验性自身免疫性脑脊髓炎的诱导和评价

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

作者聚焦：创建与雄性和雌性小鼠相关的多功能实验性自身免疫性脑脊髓炎模型 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

两性多发性硬化症建模：MOG35-55 诱导的实验性自身免疫性脑脊髓炎

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different ...

神经科学

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and axonal damage. Currently, there are no measures to cure MS completely, but multiple disease-modifying therapies (DMT) are available to control and mitigate disease progression. There are significant similarities between the CNS pathological features of experimental autoimmune encephalomyelitis (EAE) and MS patients. EAE has been widely used as a representative model to determine MS drugs' efficacy and explore the development of new therapies for MS disease. Active induction of EAE in mice has a stable and reproducible effect and is particularly suitable for studying the effects of drugs or genes on autoimmune neuroinflammation. The method of immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) and the daily assessment of disease symptoms using a clinical scoring system is mainly shared. Given the complex etiology of MS with diverse clinical manifestations, the existing clinical scoring system can't satisfy the assessment of disease treatment. To avoid the shortcomings of a single intervention, new indicators to assess EAE based on clinical manifestations of anxiety-like moods and osteoporosis in MS patients are created to provide a more comprehensive assessment of MS treatment.

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

实验性自身免疫性脑脊髓炎的诱导及多样评价指标

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...

C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法

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此视频中的章节

神经干/祖细胞在小鼠EAE的慢性全身性注射

髓鞘少突胶质细胞糖蛋白（MOG

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

多发性硬化症的小鼠模型EAE视神经炎及脑部炎症的生物发光和近红外成像

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

一种快速、简单、标准化的均质化制备抗原/辅助乳剂以诱导实验性自身免疫性脑脊髓炎的方法

两性多发性硬化症建模：MOG_35-55 诱导的实验性自身免疫性脑脊髓炎

两性多发性硬化症建模：MOG_35-55 诱导的实验性自身免疫性脑脊髓炎

两性多发性硬化症建模：MOG_35-55 诱导的实验性自身免疫性脑脊髓炎

实验性自身免疫性脑脊髓炎的诱导及多样评价指标