这项研究得到了美国国立卫生研究院 （R03NS131908） 和国防部通过多发性硬化症研究计划的支持，奖励编号为 W81XWH-22-1-0517。意见、解释、结论和建议是作者的观点、解释、结论和建议，并不一定得到国防部的认可。这项研究也得到了东卡罗来纳大学启动基金的支持。

所有涉及小鼠的程序均按照东卡罗来纳大学机构动物护理和使用委员会 （IACUC） 批准的实验指南进行。本研究使用8-10周龄的野生型C57BL/6J雌性小鼠。这些动物是从商业来源获得的（见材料表）。EAE 是在先前发表的报告 12,13,14 之后诱导的。
1. 血清采集
<ol><li>在通过MOG35-55免疫诱导EAE后，在所需的时间点（遵循机构批准的方案）通过CO2窒息或异氟烷过量对实验小鼠实施安乐死。 注:小鼠总数和血清收集的时间点可以根据具体实验而变化。</li>
	<li>在通过脚趾或尾巴捏确认没有生命体征后，将小鼠置于解剖托盘的背部卧位，并用别针或胶带将四肢固定到位。将鼠标身体对准 LED 光源（参见 材料表）以照亮动物的胸部。</li>
	<li>用70%乙醇喷洒小鼠皮毛，并使用解剖剪刀从骨盆到剑突沿腹部切开3-4厘米的中线切口，注意避免任何器官和主要血管（图1A-C）。为了方便该过程，请使用镊子抓住剑突上方的皮肤。</li>
	<li>横向切开横膈膜，然后使用弹簧剪刀在两个侧缘前切肋骨，在到达中线的胸骨之前停下来（图1D）。折叠在小鼠头上创建的肋骨瓣，露出心脏（图1E）。 注意:应避免切割胸骨，因为这会损害靠近骨骼的主要胸动脉并减少可以收集的血液量。</li>
	<li>将连接到1mL注射器的25G针插入左心室，并通过轻轻拉回柱塞来收集血液（图1F）。为了便于穿刺针刺穿，轻轻地将心脏支撑在一副镊子上。 注意:如果血液没有立即进入注射器，则应缓慢旋转针头，并应测试不同的角度。但是，在第一次尝试时应努力正确放置针头，以避免在心脏上产生不必要的孔，使血液渗出。</li>
	<li>将血液转移到无菌 1.5 mL 管中，并在室温下凝结 30 分钟。通过在4°C下以2000×g离心20分钟除去凝块。 收集代表血清分数的上清液，并将一次性等分试样储存到-80°C的低温管中，以备将来检测。</li>
</ol>2. ELISA检测
<ol><li>将冻干的 MOG35-55 肽（参见 材料表）重悬于水中，以获得 10 mg/mL 储备液。在开始实验之前，在包被缓冲液中将储备液稀释至 10 μg/mL（参见 材料表），并将 100 μL/孔的最终溶液移液到 96 孔板中。用粘性薄膜密封板以避免蒸发，并将板在4°C下孵育过夜。 注意:每个样品至少应计算 2 个孔，空白对照也应包括 2 个额外的孔。</li>
	<li>同时，用溶解在包被缓冲液中的100μL/孔牛血清白蛋白（BSA）以10μg/ mL浓度包被相同数量的孔包被。这些额外的孔将用作背景控制。 注意:建议在对照孔中涂覆 BSA，因为涂层和封闭缓冲液具有不同的成分。请注意，其他产脑肽（如PLP139-151或MBP84-104）可用作BSA的无关抗原。</li>
	<li>第二天，用 200 μL/孔的磷酸盐缓冲盐水洗涤板 3 次，盐水补充有 0.05% 吐温 20 （PBS-T）。随后，在PBS（不含吐温20）中加入100μL/孔由3%BSA制成的封闭溶液，并将密封的板在37°C下在杂交炉中孵育1小时。</li>
	<li>用 200 μL/孔的 PBS-T 洗涤板 3 次。在封闭溶液中以 1:100 稀释每个血清样品，并将 100 μL/孔添加到 MOG35-55 和 BSA 包被孔中。将相同体积的封闭溶液添加到指定为空白的孔中。将密封的板在室温下孵育2小时，并不断摇动（250rpm）。</li>
	<li>用 200 μL/孔的 PBS-T 洗涤板 3 次。在由 0.2% BSA 制成的 PBS-T 溶液中稀释 HRP 偶联的二抗（1:2000，参见 材料表），并向所有孔中加入 100 μL/孔。在室温下将密封的板孵育1小时，并不断摇动（250rpm）。</li>
	<li>用 200 μL/孔的 PBS-T 洗涤板 3 次。向所有孔中加入 100 μL/孔的 3,3'，5,5' 四甲基联苯胺 （TMB） 底物（参见 材料表），并在黑暗中孵育 1-5 分钟，监测蓝色的发展。<ol><li>通过加入 100 μL/孔的终止溶液（参见 材料表）终止反应，并使用设置为 450 nm 波长的读板器测量每个孔中的光密度 （OD）。 注意:TMB 底物应在室温下平衡 30-60 分钟，然后再将其添加到孔中。</li>
	</ol></li>
</ol>3. 数据分析
<ol><li>使用从板中读取的 OD 值填充 Excel 电子表格。平均每个样品从重复孔（MOG35-55 和 BSA 涂层）获得的 OD 值。</li>
	<li>对于每个样品，从 MOG35-55 涂层孔的平均值中减去 BSA 涂层孔的平均值。所得的背景校正值将与不同血清样本中抗 MOG35-55 抗体的浓度成正比。 注意:空白孔应产生 0 左右的校正值。</li>
	<li>应用非参数统计检验（如 Mann-Whitney U 检验）来比较两个实验条件之间的平均 OD 值。每个条件至少包括 3 个独立样本。</li>
</ol>

研究自身免疫性脱髓鞘中的体液反应

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+autoantibodies+associated+with+myelin+damage+in+multiple+sclerosis.">Identification of autoantibodies associated with myelin damage in multiple sclerosis.</a> Nat Med. 5 (2), 170-175 (1999).</li><li>Ma, Q., 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=Specific+hypomethylation+programs+underpin+b+cell+activation+in+early+multiple+sclerosis.">Specific hypomethylation programs underpin b cell activation in early multiple sclerosis.</a> Proc Natl Acad Sci U S A. 118 (51), e2111920118 (2021).</li><li>Hauser, S. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+successfully+apply+animal+studies+in+experimental+allergic+encephalomyelitis+to+research+on+multiple+sclerosis.">How to successfully apply animal studies in experimental allergic encephalomyelitis to research on multiple sclerosis.</a> Ann Neurol. 60 (1), 12-21 (2006).</li><li>Khare, 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=Myelin+oligodendrocyte+glycoprotein-specific+antibodies+from+multiple+sclerosis+patients+exacerbate+disease+in+a+humanized+mouse+model.">Myelin oligodendrocyte glycoprotein-specific antibodies from multiple sclerosis patients exacerbate disease in a humanized mouse model.</a> J Autoimmun. 86, 104-115 (2018).</li><li>Marta, C. B., Oliver, A. R., Sweet, R. A., Pfeiffer, S. E., Ruddle, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+myelin+oligodendrocyte+glycoprotein+antibodies+recognize+glycosylated+epitopes+and+perturb+oligodendrocyte+physiology.">Pathogenic myelin oligodendrocyte glycoprotein antibodies recognize glycosylated epitopes and perturb oligodendrocyte physiology.</a> Proc Natl Acad Sci U S A. 102 (39), 13992-13997 (2005).</li><li>Flach, A. 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=Autoantibody-boosted+t-cell+reactivation+in+the+target+organ+triggers+manifestation+of+autoimmune+cns+disease.">Autoantibody-boosted t-cell reactivation in the target organ triggers manifestation of autoimmune cns disease.</a> Proc Natl Acad Sci U S A. 113 (12), 3323-3328 (2016).</li><li>Didonna, A., 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=Ataxin-1+regulates+b+cell+function+and+the+severity+of+autoimmune+experimental+encephalomyelitis.">Ataxin-1 regulates b cell function and the severity of autoimmune experimental encephalomyelitis.</a> Proc Natl Acad Sci U S A. 117 (38), 23742-23750 (2020).</li><li>Didonna, A., 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=Sex-specific+tau+methylation+patterns+and+synaptic+transcriptional+alterations+are+associated+with+neural+vulnerability+during+chronic+neuroinflammation.">Sex-specific tau methylation patterns and synaptic transcriptional alterations are associated with neural vulnerability during chronic neuroinflammation.</a> J Autoimmun. 101, 56-69 (2019).</li><li>Ma, Q., Matsunaga, A., Ho, B., Oksenberg, J. 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提交人声明没有竞争利益。

在这里，报道了一种简单有效的基于 ELISA 的方案，以准确量化 MS 病理学相关动物模型中的体液反应。该方法最近被用于描述 ataxin-1 蛋白在控制 MOG35-55/C57BL6J EAE 范式12 中自身抗体水平方面的新作用。在这方面，应该考虑许多因素，以便使用这种方法获得一致且具有生物学意义的结果。
首先，收集高质量的血清样本并避免溶血至关重要。在严重溶血的标本中?...

多发性硬化症 （MS） 是一种中枢神经系统 （CNS） 的慢性自身免疫性疾病，其特征是免疫细胞局灶性浸润到脑实质、包裹轴突的髓鞘破裂、神经胶质细胞活化和神经元丢失1。除了致病性 T 细胞的公认作用外，多条证据还强调了 B 细胞参与介导针对中枢神经系统的自身免疫反应。B 细胞在 MS 脑中发生克隆扩增，并且在脱髓鞘病变中检测到针对髓鞘成分的抗体 2,3。最近有记录表明，外周 B 细胞在疾病发作时的选择性激活，表明该免疫细胞区室在疾病起始中也具有假定的作用4。B 细胞耗竭疗法（如抗 CD20 单克隆抗体）的成功进一步证实了异常 B 细胞功能与自身免疫性脱髓鞘之间的机制联系 5,6。从分子的角度来看，B 细胞可以通过自身抗原呈递、促炎细胞因子分泌和自身反应性抗体产生来导致疾病。
已经开发了多种动物模型来概括复杂 MS 表型的特定特征。其中，实验性自身免疫性脑脊髓炎（EAE）是 体内 应用最广泛的范式，依赖于实验动物使用源自髓鞘蛋白的短肽进行免疫，如髓鞘少突胶质细胞糖蛋白（MOG）和髓鞘碱性蛋白（MBP）7。EAE 免疫动物发展出脱髓鞘病理学，在许多方面类似于 MS，包括对脑炎肽8 的强大体液反应。出于这个原因，EAE 研究在解剖 B 细胞和自身抗体在疾病背景下的功能方面发挥了重要作用。例如，研究表明，从 MS 患者中分离出的 MOG 特异性抗体可以加重 EAE 模型9 的临床病程。 值得注意的是，人 MOG 中 42 位的脯氨酸残基被证明对于确定自身抗体致病性至关重要10。最近，已发现 MOG 特异性自身抗体不仅通过介导髓鞘丢失来促进疾病，而且还通过促进中枢神经系统11 内自身反应性 T 细胞的再激活。 
考虑到抗体反应在中枢神经系统自身免疫中的重要性，本文提出了一种基于 ELISA 的方案，以有效测量用 MOG35-55 肽免疫的 C57BL/6J 小鼠 EAE 中自身反应性抗体的血清水平。在方案的第一部分，将描述 通过 心内穿刺收集血清的方法。随后，将详细说明设置 ELISA 测定和获取数据的程序。最后，将讨论数据分析和解释。

<table><tbody><tr><td>1 mL syringes</td><td>BD Biosciences</td><td>309628</td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>Fisher</td><td>05-408-129</td><td></td></tr><tr><td>25 G needles</td><td>BD Biosciences</td><td>305122</td><td></td></tr><tr><td>3,3&#039;,5,5&#039;-tetramethylbenzidine (TMB) substrate</td><td>Thermo Fisher</td><td>N301</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Adhesive seals</td><td>Thermo Fisher</td><td>AB0558</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A7906</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>C57BL/6J female mice</td><td>The Jackson Laboratory</td><td>000664</td><td>Animals between 8-10 weeks of age should be used for EAE experiments</td></tr><tr><td>Cryogenic tubes</td><td>Fisher</td><td>10-500-25</td><td></td></tr><tr><td>Dissection tray</td><td>Fisher</td><td>S111022</td><td></td></tr><tr><td>Dissector scissors</td><td>Fine Science Tools</td><td>14082-09</td><td></td></tr><tr><td>ELISA coating buffer</td><td>BioLegend</td><td>421701</td><td>Store at 4&amp;deg;C</td></tr><tr><td>Excel software</td><td>Microsoft</td><td></td><td>Analysis spreadsheet</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11152-10</td><td></td></tr><tr><td>Goat Anti-Mouse IgG, Human ads-HRP</td><td>SouthernBiotech</td><td>1030-05</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>LED light source</td><td>Fisher</td><td>AMPSILED21</td><td></td></tr><tr><td>Microplate reader</td><td>Fisher</td><td>14-377-575&amp;nbsp;</td><td></td></tr><tr><td>Molecular biology grade water</td><td>Corning</td><td>46-000-Cl</td><td></td></tr><tr><td>Mouse MOG35-55 peptide</td><td>EZBiolab</td><td>cp7203</td><td>Store at -80 &amp;deg;C</td></tr><tr><td>Multichannel pipette</td><td>Axygen</td><td>AP-12-200-P</td><td></td></tr><tr><td>Noyes spring scissors</td><td>Fine Science Tools</td><td>15011-12</td><td></td></tr><tr><td>Nunc MaxiSorp 96-well plates</td><td>BioLegend</td><td>423501</td><td></td></tr><tr><td>Orbital shaker</td><td>Fisher</td><td>88-861-023</td><td></td></tr><tr><td>Oven</td><td>VWR</td><td>445-0024</td><td></td></tr><tr><td>Phosphate buffer saline (PBS)</td><td>Thermo Fisher</td><td>14190144</td><td></td></tr><tr><td>Refrigerated tabletop centrifuge</td><td>Thermo Fisher</td><td>75002441</td><td></td></tr><tr><td>Stop solution</td><td>Thermo Fisher</td><td>N600</td><td></td></tr><tr><td>Tween 20</td><td>Bio-Rad</td><td>1706531</td><td></td></tr></tbody></table>

quantification of autoreactive antibodies in mice upon experimental autoimmune encephalomyelitis

实验性自身免疫性脑脊髓炎 （EAE） 是一种疾病模型，在组织病理学和分子水平上概括了自身免疫性疾病多发性硬化症 （MS）。EAE 是通过 皮下注射 短髓鞘肽和特定佐剂对实验动物进行免疫接种来诱导的，以增强免疫反应。与人类对应物一样，EAE小鼠会出现脱髓鞘病变，免疫细胞浸润到中枢神经系统（CNS），神经胶质细胞激活和神经元损伤。一致的证据也支持 B 细胞功能障碍在 MS 和 EAE 病因中的机制作用。B细胞可以作为抗原呈递细胞，也是促炎细胞因子和自身抗体的主要来源。在 EAE 中，产生针对用于诱发疾病的髓鞘肽的抗体。这种自身抗体已被证明可以介导髓鞘丢失或致病性 T 细胞再激活到中枢神经系统中。本文介绍了一种基于 ELISA 的有效方案，用于量化用髓鞘少突胶质细胞糖蛋白 35-55 （MOG35-55） 肽免疫的 C57BL/6J 小鼠血清中的自身抗体。所提出的方法是一种强有力的工具，可用于研究自身免疫性脱髓鞘背景下异常体液反应的特异性和程度。

为了证明本 ELISA 测定的稳健性，在免疫 （dpi） 后 20 天，在 C57BL/6J 雌性小鼠队列中分离的血清样品中测试了该方法，在经过验证的 EAE 诱导方案 12,13,14 之后，在完全弗氏佐剂 （CFA） 中。动物在第 0 天和第 2 天还接受了 400 ng 百日咳毒素。来自模拟免疫动物的血清样本，含有所有物质，但没有肽，作为阴性对照。所有 EAE 小鼠?...

Watch this Scientific Journal Video about Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis at JoVE.com

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

实验性自身免疫性脑脊髓炎 （EAE） 是一种多发性硬化症 （MS） 动物模型，与人类疾病具有强大的体液自身免疫反应。在这里，我们报告了一种简单而灵活的ELISA方案，用于量化EAE免疫小鼠血清中的自身抗体。

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 ...

因此，我们实验室的重点是了解多发性硬化症的分子基础，多发性硬化症是一种中枢神经系统的免疫疾病，其特征是脱髓鞘、神经炎症和轴突损伤。因此，该协议代表了商业试剂盒的经济且灵活的替代方案。我们可以很容易地进行调整，以测试针对不同EEA肽的B细胞抗体反应。我的实验室致力于进一步了解慢性神经系统疾病（如多发性硬化症）中神经炎症和神经退行性变之间的联系。

quantification-autoreactive-antibodies-mice-upon-experimental

Research

JoVE Journal

Immunology and Infection

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

717 次观看.  East Carolina University. 因此，我们实验室的重点是了解多发性硬化症的分子基础，多发性硬化症是一种中枢神经系统的免疫疾病，其特征是脱髓鞘、神经炎症和轴突损伤。因此，该协议代表了商业试剂盒的经济且灵活的替代方案。我们可以很容易地进行调整，以测试针对不同EEA肽的B细胞抗体反应。我的实验室致力于进一步了解慢性神经系统疾病（如多发性硬化症）中神经炎症和神经退行性变...

视频: 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

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

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) ...

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 ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

小鼠体内合成蛋白的定量测量

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

神经科学

脊髓组织学染色用于评估 EAE 疾病严重程度

Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by active immunization with protein components of the myelin sheath and Complete Freund&#39;s adjuvant (CFA) or by the transfer of myelin-specific T effector cells from rodents primed with myelin protein/CFA into na&#239;ve rodents. The severity of EAE is typically scored on a 5-point clinical scale that measures the degree of ascending paralysis, but this scale is not optimal for assessing the extent of recovery from EAE. For example, clinical scores remain high in some EAE models (e.g., myelin oligodendrocyte glycoprotein [MOG] peptide-induced model of EAE) despite the resolution of inflammation. Thus, it is important to complement clinical scoring with histological scoring of EAE, which also provides a means to study the underlying mechanisms of cellular injury in the central nervous system (CNS).
Here, a simple protocol is presented to prepare and stain spinal cord and brain sections from mice and to score inflammation, demyelination, and axonal injury in the spinal cord. The method for scoring leukocyte infiltration in the spinal cord can also be applied to score brain inflammation in EAE. A protocol for measuring soluble neurofilament light (sNF-L) in the serum of mice using a Small Molecule Assay (SIMOA) assay is also described, which provides&#160;feedback on the extent of overall CNS injury in live mice.

作者聚焦：揭示多发性硬化症中肥胖与自身免疫性炎症之间的通路 

Experimental autoimmune encephalomyelitis (EAE) serves as an animal model of multiple sclerosis. This article describes an approach for scoring spinal cord inflammation, demyelination, and axonal injury in EAE. Additionally, a method to quantify soluble neurofilament light levels in the mice serum is presented, facilitating the assessment of axonal injury in live mice.

对实验性自身免疫性脑脊髓炎中的中枢神经系统炎症、脱髓鞘和轴突损伤进行评分

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by ...

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.

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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此视频中的章节

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

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

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

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

小鼠体内合成蛋白的定量测量

对实验性自身免疫性脑脊髓炎中的中枢神经系统炎症、脱髓鞘和轴突损伤进行评分

对实验性自身免疫性脑脊髓炎中的中枢神经系统炎症、脱髓鞘和轴突损伤进行评分

对实验性自身免疫性脑脊髓炎中的中枢神经系统炎症、脱髓鞘和轴突损伤进行评分

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后小鼠自身反应性抗体的定量

副本

摘要

探索更多视频

此视频中的章节

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

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

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

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

小鼠体内合成蛋白的定量测量

对实验性自身免疫性脑脊髓炎中的中枢神经系统炎症、脱髓鞘和轴突损伤进行评分

对实验性自身免疫性脑脊髓炎中的中枢神经系统炎症、脱髓鞘和轴突损伤进行评分

对实验性自身免疫性脑脊髓炎中的中枢神经系统炎症、脱髓鞘和轴突损伤进行评分

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

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

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