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>0.1M 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>P0180&#8211;199 peptide</td>
			<td>Wuxi Nordisk Biotech Co. Lt. SHG, CHN</td>
			<td></td>
			<td>P0180&#8211;199, sequence S&#8211;S&#8211;K&#8211;R&#8211;G&#8211;R&#8211; Q&#8211;T&#8211;P&#8211;V&#8211;L&#8211;Y&#8211;A&#8211;M&#8211;L&#8211;D&#8211;H&#8211;S&#8211;R&#8211;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&#39;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL syringe with 301/2 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&#39; 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

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

粉条和Capellini处理测试：在大鼠和小鼠的灵巧的前爪功能简单的定量措施

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

科研

神经科学

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)

There are many unanswered questions about cerebral lateralization. In particular, it remains unclear which aspects of language and nonverbal ability are lateralized, whether there are any disadvantages associated with atypical patterns of cerebral lateralization, and whether cerebral lateralization develops with age. In the past, researchers interested in these questions tended to use handedness as a proxy measure for cerebral lateralization, but this is unsatisfactory because handedness is only a weak and indirect indicator of laterality of cognitive functions1. Other methods, such as fMRI, are expensive for large-scale studies, and not always feasible with children2.
Here we will describe the use of functional transcranial Doppler ultrasound (fTCD) as a cost-effective, non-invasive and reliable method for assessing cerebral lateralization. The procedure involves measuring blood flow in the middle cerebral artery via an ultrasound probe placed just in front of the ear. Our work builds on work by Rune Aaslid, who co-introduced TCD in 1982, and Stefan Knecht, Michael Deppe and their colleagues at the University of M&uuml;nster, who pioneered the use of simultaneous measurements of left- and right middle cerebral artery blood flow, and devised a method of correcting for heart beat activity. This made it possible to see a clear increase in left-sided blood flow during language generation, with lateralization agreeing well with that obtained using other methods3.
The middle cerebral artery has a very wide vascular territory (see Figure 1) and the method does not provide useful information about localization within a hemisphere. Our experience suggests it is particularly sensitive to tasks that involve explicit or implicit speech production. The 'gold standard' task is a word generation task (e.g. think of as many words as you can that begin with the letter 'B') 4, but this is not suitable for young children and others with limited literacy skills. Compared with other brain imaging methods, fTCD is relatively unaffected by movement artefacts from speaking, and so we are able to get a reliable result from tasks that involve describing pictures aloud5,6. Accordingly, we have developed a child-friendly task that involves looking at video-clips that tell a story, and then describing what was seen.

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound fTCD

Functional transcranial Doppler sonography (fTCD) is a simple and non-invasive ultrasound technique which can be used to assess the lateralization of cognitive functions, especially language, and is suitable for use with children.

儿童使用功能经颅多普勒超声评估大脑偏侧（fTCD）

There are many unanswered questions about cerebral lateralization.  In particular, it remains unclear which aspects of language and nonverbal ability are ...

Assessing Burrowing, Nest Construction, and Hoarding in Mice

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening of possible treatments for AD currently largely focuses on cognitive testing, which frequently demands expensive equipment and lots of experimenter time. However, human episodic memory (the most severely affected aspect of memory in AD) is different to rodent memory, which seems to be largely non-episodic. Therefore the present ways of screening for new AD treatments for AD in rodents are intrinsically unlikely to succeed. A new approach to preclinical screening would be to characterise the ADL of mice. Fortuitously, several such assays have recently been developed at Oxford, and here the three most sensitive and well-characterised are presented.
Burrowing was first developed in Oxford13. It evolved from a need to develop a mouse hoarding paradigm. Most published rodent hoarding paradigms required a distant food source to be linked to the home cage by a connecting passage. This would involve modifying the home cage as well as making a mouse-proof connecting passage and food source. So it was considered whether it would be possible to put the food source inside the cage. It was found that if a container was placed on the floor it was emptied by the next morning., The food pellets were, however, simply deposited in a heap at the container entrance, rather than placed in a discrete place away from the container, as might be expected if the mice were truly hoarding them. Close inspection showed that the mice were performing digging ("burrowing") movements, not carrying the pellets in their mouths to a selected place as they would if truly hoarding them.6



Food pellets are not an essential substrate for burrowing; mice will empty tubes filled with sand, gravel, even soiled bedding from their own cage. Moreover, they will empty a full tube even if an empty one is placed next to it8.



Several nesting protocols exist in the literature. The present Oxford one simplifies the procedure and has a well-defined scoring system for nest quality5. 



A hoarding paradigm was later developed in which the mice, rather than hoarding back to the real home cage, were adapted to living in the "home base" of a hoarding apparatus. This home base was connected to a tube made of wire mesh, the distal end of which contained the food source. This arrangement proved to yield good hoarding behaviour, as long as the mice were adapted to living in the "home base" during the day and only allowed to enter the hoarding tube at night.

Burrowing, nesting, and hoarding are species-typical activities that mice readily perform in the laboratory. This article describes how they can be easily and cheaply assessed. These protocols are extremely sensitive to mouse strain, brain lesions and diseases. Moreover they constitute &#x201C;environmental enrichment&#x201D; for the mice, and embody the &#x201C;Refinement&#x201D; aspect of the &#x201C;3 Rs&#x201D;18.

评估挖洞，在小鼠的鸟巢建设和囤积

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening ...

Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This is mostly due to the difficulty of microdissection of fetal brains and their sectioning for determination of apoptotic cells caused by prenatal exposure to alcohol. The experiments described here provide visualized techniques from mice breeding to the identification of cell death in fetal brain tissue. This study used C57BL/6 mice as the animal model for studying fetal alcohol exposure and the role of trophic peptide against alcohol-induced apoptosis. The breeding consists of a 2-hr matting window to determine the exact stage of embryonic age. An established fetal alcohol exposure model has been used in this study to determine the effects of prenatal alcohol exposure in fetal brains. This involves free access to alcohol or pair-fed liquid diets as the sole source of nutrients for the pregnant mice.
The techniques involving dissection of fetuses and microdissection of fetal brains are described carefully, since the latter can be challenging. Microdissection requires a stereomicroscope and ultra-fine forceps. Step-by-step procedures for dissecting the fetal brains are provided visually. The fetal brains are dissected from the base of the primordium olfactory bulb to the base of the metencephalon.
For investigating apoptosis, fetal brains are first embedded in gelatin using a peel-away mold to facilitate their sectioning with a vibratome apparatus. Fetal brains embedded and fixed in paraformaldehyde are easily sectioned, and the free floating sections can be mounted in superfrost plus slides for determination of apoptosis or cell death.
TUNEL (TdT-mediated dUTP Nick End Labeling; TdT: terminal deoxynucleotidyl transferase) assay has been used to identify cell death or apoptotic cells. It is noteworthy that apoptosis and cell-mediated cytotoxicity are characterized by DNA fragmentation. Thus, the visualized TUNEL-positive cells are indicative of cell death or apoptotic cells.
The experimental designs here provide information about the use of an established liquid diet for studying the effects of alcohol and the role of neurotrophic peptides during pregnancy in fetal brains. This involves breeding and feeding pregnant mice, microdissecting fetal brains, and determining apoptosis. Together, these visual and textual techniques might be a source for investigating prenatal exposure of harmful agents in fetal brains.

The experimental designs proposed here focus on studying the effects of alcohol exposure in apoptosis and the application of neurotrophic peptide during pregnancy in fetal brain. A detailed description from the breeding to the collection of fetal brains is described. Techniques for determination of apoptosis are also described in detail.

实验方法测试的影响，在怀孕期间对酒精诱导的细胞凋亡在C57BL / 6小鼠神经营养肽，ADNF-9

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

前子宫穿孔和整个半球的外植体早期皮质发育的研究:一个简单的实验方法

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用<em&gt;在子宫内</em&gt;电穿孔的C57BL / 6小鼠

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

正常小鼠β淀粉样肽侧脑室注射敏锐地诱导老年痴呆样认知缺陷

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

MOG35-55 诱发自身免疫性脑脊髓炎 C57BL/6 小鼠矢状面运动步态分析

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

c57bl6 小鼠耳蜗植入术与电唤起脑干反应记录

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...