一般捐赠基金，国家 - 这项工作是由NINDS P30-NS069324，国家多发性硬化症SocietyRG 4587-A-1，Civitan国际研究基金会，麦克L. Jezdimir横贯性脊髓炎基金会，阿拉巴马州卫生服务基金会资助的大学科学基金会1355183，和T32 AI007051从国家过敏和传染病研究所，美国国立卫生研究院。

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作者什么都没有透露。

MS患者继续经历疾病复发而采取任何减弱T细胞活化和/或渗透进入CNS药物，warranting的直接保护中枢神经系统的治疗方案的开发。 EAE已经典被用于MS的症状进行建模和研究在体内的免疫系统和中枢神经系统之间相互作用的性质时可以是一个功能强大的工具。使用对EAE的治疗方面的考虑， 例如 ，定时之前或疾病开始后，在与检查在CNS的免疫细胞浸润和增殖和活化在周边相结合，有可能描绘免疫系统和两个上的治疗效果中枢神经系统。 而EAE在C57BL / 6小鼠被更广泛地利用，EAE在SJL小鼠可能是更具有代表性的大多数的MS的情况下，因为这些小鼠具有在实质一个复发 - 缓解型和免疫细胞的浸润脑10。 SJL小鼠缓解期有明显的复苏，以及，使病情已经呈现后，可以开始治疗，但在减少炎症倍。考虑到SJL小鼠并不总是复发和汇中同步，从而导致潜在的大量变异时的结果汇集是很重要的。因此，一些研究人员可能会选择从一个动物显示临床评分代表性的结果，而在疾病进展为服用F​​ACS分析和组织学小鼠个体化分。 当操作被到EAE小鼠制成可以协助的治疗如何影响免疫系统或中枢神经系统的确定考虑。有用于治疗开始时许多选项，每个都有其自己的内涵免疫细胞是否已进入CNS和它们如何与中枢神经系统进行交互。发病前处理意味着免疫细胞尚未进入或造成中枢神经系统的损害。出现症状后治疗意味着免疫细胞已进入中枢神经系统，并已造成了一定的损害。使用SJL小鼠中，治疗还可以复发，其中免疫细胞正在积极浸润和引起炎症，或缓解，其中免疫细胞可以是在CNS中不太普遍用更少的炎症过程中开始。考虑，其中免疫细胞在治疗期间是在病理过程时可以进行关于治疗如何影响中枢神经系统和免疫系统的初始假设。 有许多，其中处理可能会影响免疫细胞和中枢神经系统，每减少EAE症状的严重程度的最终结果的方法。因此，有必要使用流式细胞术分析和免疫组织化学来看看如何免疫细胞中的外周和中枢神经系统受到影响，免疫细胞是否已进入中枢神经系统，以及中枢神经系统如何响应治疗。而脊髓的流式细胞术分析可确定有多少细胞哈已经输入的中枢神经系统在给定的时间，不能确定，这种效果是由于减少的免疫细胞的运输除非免疫细胞的增殖是在脾不受影响。因此，有必要分析外周和中枢神经组织，并确定什么样的结果意味着机械地当两个组织进行了比较。另外，也可以对免疫细胞的活性曲线以通过处理而改变，例如具有在致病辅助性T细胞重分布的开关来调节T细胞重分布。看为不同的细胞类型的标记和处理的和未处理的动物之间进行比较百分比表达因此也是一个重要的考虑因素。在MS研究中的一个新兴的概念表明，B细胞发挥在自身免疫性脱髓鞘了重要作用。这是基于研究表明，B细胞所必需的T细胞20的激活。这个概念是通过处理如利妥昔单抗，针对CD20前的抗体的成功支持压B细胞21,22的表面上。这表现在单克隆抗体的ocrelizumab的在临床试验的成功，药物靶向CD20的不同表位可以提高B细胞靶向疗法23的功效。 这里提出的技术的一个限制是，它有可能为免疫细胞进入CNS，但无法在实质的旅行。免疫组织化学可用于检测免疫细胞的血管周围袖套和评价处理和未处理的动物之间的实质行进距离。另一个潜在的限制涉及对EAE发病中的微生物的效果。共生的肠道菌群可严重影响疾病的发病机制24;因此，小鼠放置在不同的殖民地，甚至在不同的笼子可以在疾病严重程度的巨大差异。因此，它总是优选尽可能使用在同一笼饲养同窝对照实验涉及EAE。最后需要说明的是，如果它是实验需要消除的在外围免疫细胞增殖的改变的影响，有可能这样做使用被动转移感应，而不是在此协议中描述的活性诱导。 用于神经保护进一步的确认可以使用共培养系统11来测试细胞死亡或通过使用条件性敲除小鼠的其允许蛋白的缺失选择性上的细胞类型特异性的机制来实现。此外，为了扩大是神经保护药剂的探索，轴突横断和神经元死亡的标志物应该包括在内。重要性的另一领域是髓鞘再生。受伤的轴突无法remyelinate进一步支持了神经保护疗法应该是髓鞘再生疗法的重要组成部分。此外，无髓鞘的轴突更容易受到伤害比myelina特德轴突。这表明，当一个轴突成为促进髓鞘及时将防止性轴索损伤脱髓鞘治疗干预。探索这些途径中，可以使用其他的体内模型用于脱髓鞘和髓鞘再生（ 即 cuprizone和溶血卵磷脂）。本文的方法描述侧重于量化髓鞘损失评估保护作用。为髓鞘再生的评价祖细胞的数目以及它们的增殖和成熟的也将是非常重要的研究的能力。有了这些替代车型的提，还必须考虑脑炎的不同型号被病毒介导的。有两个产生髓磷脂损失充分表征RNA病毒的模型:一个是Theiler鼠脑脊髓炎，无包膜小核糖核酸病毒，而另一个是鼠肝炎病毒，该冠状病毒家族25,26中的一员。 EAE是ST的有价值的工具的操纵或治疗如何影响免疫系统和中枢神经系统体内 udies。这里描述可以有助于确定治疗方法影响疾病过程中，无论是在外围，在血脑屏障，或在CNS的协议。为MS无电流疗法治愈疾病和患者经常遇到下降一段时间。同样，涉及免疫细胞浸润到中枢神经系统髓鞘的退化，包括急性播散性脑脊髓炎，横断性脊髓炎和视神经脊髓炎，保护中枢神经系统，因为它直接受到攻击由浸润的免疫细胞缺乏治疗其他疾病。考虑到治疗的时间和使用脾脏的流式细胞术分析和脊髓结合CNS的免疫组织化学以评估炎症和损害将使机械测定关于治疗，以制成。

多发性硬化（MS）主要在大脑中疾病早期的白质的区域的特征在于炎症性病变。长期进展后，灰质萎缩通过MRI成像检测并标记该疾病的神经变性阶段。反应性神经胶质增生，脱髓鞘，并在白质轴索损害归因​​于中枢神经系统浸润免疫细胞。目前使用中的MS逆转或直接防止神经变性CNS中处理无 - 代替，它们通过减弱T细胞活化和/或渗透进入CNS减少炎症。由于没有特效药MS和利用现有治疗的患者继续经历疾病进展的防止脱髓鞘和神经元丢失的药物发现是非常重要的。然而，对免疫细胞的作用以及对中枢神经系统区分可能是困难的实验，作为结果- 即 ，降低至CNS损伤-看起来在SAME无论通过其发生的机制。因此，保护​​中枢神经系统的评估必须以中枢神经系统浸润的免疫细胞和免疫细胞的增殖在外围，以确定如何剂药物影响疾病的机制来评估合作。 实验性自身免疫性脑脊髓炎（EAE）是自身免疫性炎症性疾病，这是用于药物目前用于治疗的MS 1-4的发现直接负责的一个公认的动物模型。小鼠通常用于EAE，与C57BL / 6小鼠是基于遗传变异体的可用性的流行株。患有EAE诱导C57BL / 6小鼠表现出与围绕第10天诱导后发病的慢性疾病的进展。脊髓实质和小脑的浸润是这些动物的组织病理学的特性，与在皮层薄壁5缺席浸润。此外，皮质损伤和脱髓鞘在B雨是疾病6-9的标志，这是在C57BL / 6小鼠相对缺席。因此，它可能是优选的，当可能使用SJL小鼠，其具有复发-缓解疾病和出现类似于在MS 10中的脑和脊髓都发现病变。 治疗不能被归类为神经保护，如果免疫细胞不会到达中枢神经系统。因此，此协议利用大脑，脊髓，并从EAE小鼠的脾的流式细胞术分析，以确定治疗对免疫细胞浸润到CNS和外周免疫细胞增殖的作用，如先前证实11。中枢神经系统组织的免疫组化分析，以确定程度和神经保护性质也被描述。结合这些方法允许对免疫细胞是否被激活，并在外围增殖，免疫细胞是否进入中枢神经系统的确定，以及中枢神经系统是否公关炎症或损害otected。如果神经保护作用被怀疑尽管对免疫系统的影响，实验者可以改变处理后的免疫细胞浸润的开始时间到发生在CNS。 这里，我们提出使用主动EAE，MS的T细胞介导的​​动物模型两种不同型号的协议，并且该疾病期间流式细胞仪分析在不同时间点免疫结合，以确定在不同方面的实验性疗法的功效MS的发病机制。这种方法将帮助研究人员在免疫细胞增殖和浸润对中枢神经系统的保护作用之间进行区分，从而更容易来缩小药物对疾病发病机理如何行动。

<table><tbody><tr><td>22 x 22 x 20 mm embedding mold</td><td>Fisher Scientific</td><td>NC9719245</td><td></td></tr><tr><td>22 x 30 x 20 mm embedding mold</td><td>Fisher Scientific</td><td>NC9531194</td><td></td></tr><tr><td>2-Mercaptoethanol (55 mM)</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>21985-023</td><td></td></tr><tr><td>2-Methylbutane</td><td>Fisher Scientific</td><td>O3551-4</td><td></td></tr><tr><td>30 x 22 x 20 mm embedding mold</td><td>Fisher Scientific</td><td>18-30</td><td></td></tr><tr><td>ACK Lysing Buffer</td><td>Quality Biological</td><td>118-156-101</td><td></td></tr><tr><td>anti-CD4 PE-Cy7</td><td>BD Biosciences</td><td>552775</td><td>0.2 mg/ml stock concentration</td></tr><tr><td>anti-Foxp3-FITC</td><td>eBioscience</td><td>11-5773-82</td><td>0.5 mg/ml stock concentration</td></tr><tr><td>anti-GFAP (Cocktail)</td><td>Biolegend</td><td>835301</td><td>1 - 3 mg/ml stock concentration</td></tr><tr><td>anti-Iba-1 Polyclonal Antibody (50 &amp;#181;g)</td><td>Wako</td><td>019-19741</td><td>0.5 mg/ml stock concentration</td></tr><tr><td>anti-IFN-&amp;gamma; APC</td><td>eBioscience</td><td>17-7311-82</td><td>0.2 mg/ml stock concentration</td></tr><tr><td>anti-IL-17A PerCP-Cy5.5</td><td>eBioscience</td><td>45-7177-82</td><td>0.2 mg/ml stock concentration</td></tr><tr><td>anti-Ki-67 PE</td><td>eBioscience</td><td>12-5698-82</td><td>0.2 mg/ml stock concentration</td></tr><tr><td>anti-MBP (D-18)</td><td>Santa Cruz Biotechnology</td><td>sc-13912</td><td>0.2 mg/ml stock concentration</td></tr><tr><td>anti-TCR&amp;beta; FITC</td><td>eBioscience</td><td>11-5961-85</td><td>0.5 mg/ml stock concentration</td></tr><tr><td>anti-TCR&amp;beta; PE</td><td>eBioscience</td><td>12-5961-83</td><td>0.2 mg/ml stock concentration</td></tr><tr><td>Biotinylated Goat Anti-Rabbit IgG</td><td>Vector Labs</td><td>BA-1000</td><td>1.5 mg/ml stock concentration</td></tr><tr><td>Biotinylated Horse Anti-Mouse IgG</td><td>Vector Labs</td><td>BA-2000&amp;nbsp;</td><td>1.5 mg/ml stock concentration</td></tr><tr><td>Citric Acid, Anhydrous, 99.5%</td><td>Fisher Scientific</td><td>AC42356-5000</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA), tetrasodium salt dihydrate, 99%</td><td>Fisher Scientific</td><td>AC446085000</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>HyClone</td><td>SH30071.03</td><td></td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides, case of 10</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Golgi Plug</td><td>BD Biosciences</td><td>555029</td><td>protein transport inhibitor</td></tr><tr><td>Immedge Hydrophobic Barrier Pen</td><td>Fisher Scientific</td><td>NC9545623</td><td></td></tr><tr><td>Ionomycin</td><td>EMD Millipore</td><td>407952-5mg</td><td></td></tr><tr><td>L-Glutamine, 100x</td><td>Corning</td><td>25-005-Cl</td><td></td></tr><tr><td>MEM Nonessential Amino Acids</td><td>Corning</td><td>25-025-Cl</td><td></td></tr><tr><td>Near IR Live/Dead Staining Kit</td><td>Life Technologies</td><td>L10119</td><td>viability dye</td></tr><tr><td>Normal goat serum</td><td>Vector Labs</td><td>S-1000</td><td></td></tr><tr><td>Normal horse serum</td><td>Vector Labs</td><td>S-2000</td><td></td></tr><tr><td>Paraformaldehyde, 96%</td><td>Fisher Scientific</td><td>AC416785000</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution, 100x</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-01</td><td>density gradient</td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-500</td><td>resinous mounting medium</td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA)</td><td>Sigma</td><td>P1585-1mg</td><td></td></tr><tr><td>Purified anti-Myelin Basic Protein Antibody</td><td>BioLegend</td><td>808401</td><td></td></tr><tr><td>RPMI 1640</td><td>Corning</td><td>10-040-CM</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Corning</td><td>25-000-Cl</td><td></td></tr><tr><td>Tissue-Tek CRYO-OCT Compound</td><td>Fisher Scientific</td><td>14-373-65</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td>nonionic detergent</td></tr><tr><td>Vectastain Elite ABC Kit (Standard)</td><td>Fisher Scientific</td><td>NC9206402</td><td>avidin-biotin-peroxidase&amp;nbsp;complex (ABC) in immunoperoxidase&amp;nbsp;</td></tr><tr><td>Vector Laboratories Peroxidase Substrate Kit (DAB)</td><td>Fisher Scientific</td><td>NC9276270</td><td>DAB solution</td></tr></tbody></table>

determining immune system suppression versus cns protection for pharmacological interventions in autoimmune demyelination

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

在这里，我们使用的EAE两个模型来理解，如果药剂通过提供CNS保护要么减弱CNS浸润T细胞或预防炎症性免疫细胞浸润袭港期间髓鞘和轴索损伤。以确定是否治疗剂阻止免疫细胞浸润到脊髓，用于慢性EAE的C57BL / 6小鼠模型，其中免疫细胞浸润和疾病病理学主要位于在脊髓（ 图1A）。以确定是否治疗药物的免疫细胞的侵入中枢神经系统，复发-缓解EAE的SJL动物模型被使用，这表明疾病病理中的脑和脊髓（ 图1B）二者中提供的CNS的保护。 临床评估 相关的临床评估是根据下面的栏目典型（ 图1制成C）或非典型的（ 图1D）的EAE。对于典型的临床疾病，0分是无异常行为。当由尾基部拾起，尾部可以快速旋转（很像直升机转子）和后腿将分开。的1的临床评分是部分柔弱的尾部，其可通过由尾根部抬起鼠标来确定。正常的直升机般旋转可减弱或消失，而尾部的一部分可能是完全瘫软。一个有用的方法来确定尾部麻痹程度是运行一个人的手指尾的长度，作为unparalyzed尾巴通常会围绕手指卷曲而部分瘫痪的尾巴将无法这样做。 2临床分代表完全瘫痪的尾巴。尾部没有移动采摘在尾巴的基部鼠标向上时，会发生在所有。 3临床分数代表局部后肢麻痹。这个分数的确定需要鼠标可以自由地在移动FLAŤ面。如果一个后肢拖随着鼠标向前移动，或者如果一个或两个后肢似乎是部分瘫痪，可给予3分。 4临床分表示完全后肢瘫痪。有了这个分数，鼠标将无法移动它的后肢，并会拖累本身向前使用它的前肢。 5临床评分代表一个垂死的老鼠，或困难一只老鼠从笼子里，或呼吸运动本身。如果鼠标不能沿笼底或者如果呼吸吃力拖累本身，鼠标应该安乐死。 6临床分数代表在笼子里发现死老鼠。 6分是不寻常的，比其他EAE死亡原因应进行调查。 非典型临床疾病可能会或可能不会瘫痪陪同。如果鼠标非典型疾病加上典型症状呈现，可能有必要对包括两个单独的评分系统。 0分无异常行为，一s的典型的评分系统。 1临床分代表轻微的头部转动或倾斜而鼠标走。这可通过允许鼠标向前走，并观察一个恒定左或右方向性其运动来确定。 2临床分代表更明显的头部转动和扶正能力差。作为具有1非典型得分，鼠标具有方向性的移动，并且可以具有与平衡轻微困难。 3临床分数代表没有能力走在一条直线上。鼠标将难以平衡，并可以使用笼子的一边，帮助权利本身，因为它散步。 4临床分代表鼠标铺设在其一侧，无法行走，由于平衡的问题。鼠标可能能够拖动本身沿笼的底部，但可以具有​​方向性其运动。 5临床分数代表，除非支持连续轧制。到达这个分数鼠标应安乐死。一所诊所人得分6代表在笼子里发现死老鼠。 6分是不寻常的，比其他EAE死亡原因应进行调查。 可能有必要以允许分数， 如 "在中间"的，添加0.5〜分数如果鼠标的病情变化略，或者如果两个分数之间进行选择是困难的。例如，开始一个鼠标更缓慢移动比其正常的对应，但显示不麻痹，或当由所述尾拾取可给予一个分数的0.5该搂抱其后腿其前代替张开腿出来的小鼠。鼠标只能拖本身沿着笼子的底部，并且只能抽搐其后肢定期或在触摸时可能给予评分3.5。 评估免疫细胞浸润减少 EAE在C57BL / 6小鼠模型（ 图1A，第0天），抗原presentati诱导后对T细胞在脾和增殖发生在天1 - 5随后的免疫细胞浸润到CNS中围绕第7天，大约存在与临床评分的初始免疫细胞浸润小鼠后3至5天。为了评估如果治疗剂是阻断免疫细胞浸润到脊髓，药物或媒介物被引入在脾抗原呈递和增殖后7一天，但之前的免疫细胞开始渗透到脊髓。如果免疫细胞浸润已衰减，所述临床疾病过程中应反映在疾病的从天上升阶段为10〜15（ 图2）改善的临床得分。 在免疫细胞浸润的减少也将导致削弱神经炎症。活性星形细胞增生和小神经胶质细胞被认为是神经发炎的主要特点。染色用GFAP和小胶质细胞与伊巴-1-星形细胞然后可用于评估昌在平均面积率染色ES量化神经炎症（ 图3）。 以确定是否免疫细胞浸润减少，脊髓被除去并在疾病的峰（ 图1A，约18天）流式细胞分析处理。这确保了免疫细胞的数量最多的已进入到脊髓。 T细胞进入CNS的入口被认为是引发炎症事件和Th1和Th17细胞中EAE的动物模型，以及MS患者中找到。两者合计，流式细胞仪分析应该包括这两种类型的病原性T细胞的评估。此外，调节性T细胞是抑制疾病充分表征抑制性T细胞。因此，调节性T细胞的从总的CD4 +群体的百分比必须相比效应T细胞群体的百分比进行评估。这将或透露是否出现在T细胞浸润总体上减少如存在e是在中枢神经系统的T细胞表型的倾斜。代表点图（ 图4A）展示的 CD4 +的总数量的减少与载体处理的小鼠（在右上象限数字）脊髓相比浸润T细胞在脊髓从药物处理的小鼠。分别IFN-γ+ IL-17 +，和Foxp3 +和应减少（ 图4A）:评价Th1时，Th17细胞和Treg细胞以下签名蛋白进行评价。统计分析应该对CD4 + IFN-γ+ IL-17 +，和Foxp3 +细胞数目进行展示一个显著减少（ 图4B）。为了排除T细胞亚群的偏斜，IFN-γ+ IL-17 + IFN-γ+ IL-17的比例的统计评估- ，IL-17 + IFN-γ - ，并且执行的Foxp3 +细胞（ 网络连接古尔4C）。 以消除在中枢神经系统浸润的T细胞的减少是在外围抑制增殖，活化和分化的结果的可能性，需要进行评估的活跃增殖的T细胞除了T细胞亚型的比例数。如果活化和分化不受影响（ 图 5A）中的 CD4 + IFN-γ+的百分比没有变化，IL-17 +，或FOXP3 +应当找到。此外，如果扩散不受影响（ 图 5B）中的Ki67 + CD4 +细胞没有变化应被发现。药物治疗被引入第7天或更高，以避免改变初始抗原呈递和T细胞活化中的周边。然而，在遗传模型蛋白质通常组成胚胎发育过程中删除或使脾ASSE EAE诱导前诱导的高度重视ssment。 评估CNS保护 为了证明如果一个特定的治疗剂调节免疫后细胞浸润中枢神经系统疾病的病理，药物干预应在临床疾病得分的第一个高峰期间进行管理。因为这些小鼠表现出复发 - 缓解型EAE的SJL模型是用于这些实验是有利的。如果药物治疗预防髓鞘轴突变性，在临床评分的改善将观察（ 图6）。髓鞘的病理评估必须确证髓鞘损伤与改善临床分数一致的减少。定量评价髓鞘完整性，髓鞘碱性蛋白的DAB染色（MBP）进行，接着对这种染色的光密度的统计分析（ 图7）。为了进一步证实该神经炎症是持续或therape降低UTIC干预，反应性神经胶质增生，可以通过测量平均分数面积为上述（ 图3）所描述的反应性胶质进行评估。为了证实该治疗干预是直接保护CNS无免疫调节作用，免疫细胞浸润到中枢神经系统和增殖脾脏衰减必须打折扣。 （5图4和 ），以解决这个问题，对脑和免疫细胞浸润和外周T细胞增殖和活化的评估脊髓评估方法应该如上所述来进行。两者合计，阻断在中枢神经系统中的细胞损伤，没有在中枢神经系统浸润T细胞或T细胞中的周边的增殖减少的证据的治疗剂是中枢神经系统的保护处理。 <img alt="图1" src="/files/ftp_upload/54348/54348fig1.jpg" /> 图1. 代表性的地区环境部门一道从EAE在C57BL / 6小鼠SJL临床评分的LTS（ 一 ）临床得分（平均值±SEM）C57BL / 6小鼠（n = 10）与MOG 35-55诱导产生EAE的慢性疾病。 （ 二 ）临床得分（平均值±SEM）与PLP 139-151诱导产生EAE与复发缓解型疾病SJL小鼠（n = 3）。 （C）的用于追踪在EAE小鼠典型疾病进展的临床得分栏目。 （D）用于追踪EAE小鼠非典型疾病进展的临床评分栏目。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54348/54348fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/54348/54348fig2.jpg" /> 图2. 药理治疗免疫前细胞浸润与EAE C57BL / 6小鼠。 </str翁&gt;临床分数（平均值±SEM）与PBS从第7天接种后处理C57BL / 6小鼠（n = 20）或SAS（N = 19）与MOG 35-55。数据是来自三个池独立的实验。使用非参数双尾曼 - 惠特尼U检验确定统计学差异，* P &lt;0.05。与许可（11）重新打印。 <img alt="图1" src="/files/ftp_upload/54348/54348fig3.jpg" /> 图3. 免疫荧光染色和反应性胶质的控制，EAE的脊髓的定量和处理C57BL / 6小鼠。（ 一 ）控制的脊髓荧光标记为GFAP（星形胶质细胞）和IBA-1（小胶质细胞）（未免疫）小鼠（左小图）和EAE小鼠用PBS（中图）或SAS（右图）处理。比例尺= 100微米。使用面积分数技术测量百分之immunop测定染色的量化对于GFAP（B）和IBA-1（C）的ositive区域。平均值±SEM，n = 3的对照组，n = 3 SAS处理，或n = 4 PBS处理的小鼠，每只小鼠6部分。使用单向ANOVA确定统计学差异，* P &lt;0.05，** P &lt;0.01，*** P &lt;0.001。重新打印与（11）权限。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54348/54348fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/54348/54348fig4.jpg" /> 图4. FACS EAE C57BL / 6小鼠脊髓的研究表明减少T细胞浸润在处理的小鼠中，C57BL / 6小鼠SAS或PBS处理，开始7天EAE的postinduction。在第15天（A）代表散点图获得脊髓表明Th1细胞（IFN-γ+ / IL-17 - ）和Th17（IFN-γ- / IL-17 +）细胞中的CD4 +栅极（上图）和T调节细胞（FOXP3 +）（下图）。点图显示在右上象限的百分比。 （B）中的CD4 +细胞的绝对数量，以及IFN-γ+ IL-17A +和Foxp3的+细胞进行统计学分析。 （C）的SAS-和PBS处理的EAE小鼠之间的T细胞群的百分比的变化也受到审查。平均值±SEM，每组10对PBS处理，和n = 9用于从两个独立的实验治疗的SAS。双尾t检验用于所有条形图。 ** P &lt;0.01。重新打印与（11）权限。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54348/54348fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/54348/54348fig5.jpg" /> 图5. FAC演示EAE C57BL / 6小鼠脾脏S分析等效性T细胞的表达谱和增殖处理和未处理小鼠。脾脏从PBS-和SAS治疗的小鼠进行了分析EAE的15天postinduction。 （A）的CD4 + T细胞的百分比，Th1型（IFN-γ+ / IL-17 - ），Th17细胞（IFN-γ - / IL-17 +）在从PBS-脾脏，和调节性T细胞（的Foxp3 +）组（n = 10），并从两个独立实验的SAS-组（n = 9）的小鼠。 （B，左图）中Ki-67 +细胞在从幼稚脾脏的CD4 +群体的百分比（n = 4时），以及从PBS-（N = 5）和SAS处理的小鼠（n = 5）诱导与EAE。单向ANOVA检验证明中Ki-67 +幼稚从脾脏细胞或者PBS-或SAS处理的EAE脾相比的比例之间的统计学意义。 PBS-和SAS处理的EAE脾间无显着性。 （B，右图）代表点图;数字表明增殖比例。散点图显示百分比。柱状图代表双尾t检验，*** P &lt;0.001。重新打印与（11）权限。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54348/54348fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/54348/54348fig6.jpg" /> 图6. 药物治疗后，免疫细胞浸润SJL小鼠EAE临床得分（平均值±SEM）用PBS处理SJL小鼠（N = 8）或SAS（N = 8）从24日接种后（虚线）与PLP 139-151。数据是平均临床得分±SEM。使用非参数双尾曼 - 惠特尼U检验确定统计学差异，*** P &lt;0.001。上面一行表示用于统计值分析。与许可（11）重新打印。 <img alt="图1" src="/files/ftp_upload/54348/54348fig7.jpg" /> 图7. MBP染色的定量使用光密度。（A）在胸段脊髓MBP的代表从染色相比于同窝出生的控制C57BL / 6小鼠EAE诱导不明基因敲除小鼠。方括号表示减少MBP染色表明脱髓鞘的代表区域。从一个未指定的遗传敲除的C57BL / 6小鼠胸脊髓（B）中的MBP染色。 （C）用的EAE诱导的未指定的遗传基因敲除小鼠（KO; N = 6只小鼠，2 - 4腰椎和每只动物胸椎部分）显示在脊髓比野生型（WT MBP染色的较高的光密度（OD）; N = 3只，2 - 4腰椎和每个动物的胸段）与EAE诱发小鼠。统计学分析USI纳克双尾t检验，* P &lt;0.05。误差棒表示SEM。比例尺为100μm。

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确定免疫系统抑制与中枢神经系统保护对自身免疫性脱髓鞘药物干预的影响免疫学和感染|视频

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

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

确定免疫系统抑制中枢神经系统对保护药物干预自身免疫性脱髓鞘

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

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12.1K 次观看.  University of Alabama at Birmingham. This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

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

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 ...

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免疫与感染

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 ...

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) ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

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

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

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

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

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

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

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

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

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

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

研究自身免疫性脱髓鞘中的体液反应

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

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

作者聚焦:在多发性硬化症研究中研究 B 细胞反应的新型检测方法

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

实验性自身免疫性脑脊髓炎后小鼠自身反应性抗体的定量

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

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 ...

确定免疫系统抑制中枢神经系统对保护药物干预自身免疫性脱髓鞘

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

神经干/祖细胞在小鼠EAE的慢性全身性注射

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

使用的淋巴细胞外渗分析

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

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

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

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

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