作者感谢杰登史密斯严格审查和编辑校对的稿子。这项工作已获得支持，从国家多发性硬化症协会（NMSS，部分补助RG-4001-A1），意大利多发性硬化症协会（AISM，授予2010/R/31），意大利卫生部（GR08-7），翅膀生命，Banca银行阿格里科拉POPOLARE二拉古萨（BAPR），欧洲研究委员会（ERC）下的ERC-2010-STG赠款协定没有260511-SEM_SEM和欧洲共同体（欧共体）第七框架计划（FP7/2007-2013）根据赠款协议N *度; 280772 - 卓智。

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作者什么都没有透露。

成体干细胞的疗法正在成为最有希望的策略，用于治疗慢性炎性CNS疾病如MS2 11中的一个。同时维持其治疗作用的机制仍有待完全阐明，全国人大移植在神经退行性疾病的不同的实验模型的显著影响，已经引起了几分挑衅的信念，干细胞可能很快被应用到人类的研究。然而，设想我们需要面对的一些关键问题，并解决一些悬而未决的问题，如理想的干细胞来源的移植（自体与异体，?...

强有力的证据已经出现从体内研究，证明在中枢神经系统疾病1-8的动物模型中的体细胞的神经干/祖细胞（的NPC）的移植的治疗效果。不过，也有一些与干细胞输送到主机的问题需要仔细考虑之前，这些实验结果可以转化为临床应用。向的（非造血）恢复性干细胞疗法对于多灶性，慢性炎症性脑疾病的发展的一个特别重大的障碍是怪物注射的理想的路径的识别。目标疾病的病理生理学有相当的了解（焦或多灶性;主要炎症或退行性小学），以及与交付相关的技术可行性和风险问题谨慎的分析是在确定干细胞传递最优协议。 而焦点（ <em&gt;如。进入神经 ​​系统实质）造血干细胞移植是一种合理的方法为特征的破坏空间狭窄的区域中枢神经系统疾病的治疗（ 如帕金森氏症和亨廷顿氏病，脑和脊髓外伤和中风），同样的方法可以证明是几乎不可行的，如MS，其中多灶性，慢性的，空间上传播中枢神经系统损伤会随时间积累的条件。在后一种情况下，针对局灶性细胞注射到病变的个体，也阻碍了移植的NPC的能力有限迁移的中枢神经系统实质内长的距离，从而促使中枢神经系统的替代，更合适的方法识别与微创全国人大针对移植。 从小鼠的NPC针对颅内肿瘤（ 例如 ，神经胶质瘤）观测了很大的希望出现的CNS9外血管内注射时。根据这一开创性在干细胞pathotrophism 10的体内证据，大量的数据被积累有关实验性自身免疫性脑脊髓炎（EAE）的实验动物的可行性和NPC的全身移植的治疗功效，作为模型的炎性中枢神经系统损伤， 通过静脉内（iv）或侧脑室（ICV）全国人大注射1,2,5,6,8 ......我们首先证明，这是依赖于移植的NPC的能力为目标，进入中枢神经系统发炎，并随后参与多间在体内 11项具体的微环境中的通信程序。为了特异性靶向中枢神经系统，的NPC被直接输送到脑脊髓液（CSF）中通过侧脑室注射循环，或到通过静脉注射的血液。一旦进入无论是血液或脑脊液，移植的NPC积极互动血脑屏障（BBB）或血脑脊液（BCSFB）壁垒和进入CNS实质。这人大移植和血脑屏障（或BCSFB）之间的相互作用是通过特定的NPC表面细胞粘附分子（CAMS）的监管，并通过高水平的CAM反配体对活化的内皮/室管膜细胞12-14的表达提供便利。这些凸轮的例子包括受体，透明质酸，CD44和胞间粘附分子（ICAM）-1配体很迟抗原（VLA）-4 5,15,16（即，在白细胞，负责与活化室管膜的相互作用细胞和内皮细胞），并以一个低得多的程度淋巴细胞功能相关抗原（LFA）-1和P-选择素糖蛋白配体（PSGL）-1。的NPC也表达多种趋化因子受体，包括CCR1，CCR2，CCR5，CXCR3和CXCR4（但不表达CCR3和CCR7），它在功能上是积极的，无论是在体外和体内 5,16。因此，systemica莱注入的NPC用这些凸轮，以及G-蛋白偶联受体（GPCR的），积累在发炎的中枢神经系统的电平。相反，注入的NPC进入全身健康小鼠不通过血管或脑脊髓液空间的路由2进入中枢神经系统。中枢神经系统炎症，或内皮/室管膜细胞活化之后的全身细胞因子或lypopolisaccharide（LPS）注射作为化学诱导的脑炎的模型，因此，有必要对全身注射的NPC积累到大脑和脊髓2。因此，中枢神经系统的成功定位与全身治疗鼻咽癌是依赖于机会（胡）的一种疾病特定的窗口中，大脑和脊髓的环境，有利于积累和NPC的跨内皮迁移的鉴定。这种情况一般出现在急性和亚急性炎症17的范围内。一旦已经进入中枢神经系统，移植未分化的NPC已经显示出改善的小鼠的临床病理特征以及更大的，非人灵长类动物与EAE。这已被描述为依赖从最小的细胞替代2和内血管周围的CNS 2,5,6,18免疫调节和神经保护性旁分泌因子与非CNS发炎的区域19,20（ 例如淋巴结）显着分泌响应于炎性细胞信号引起了浸润的免疫细胞5。 在这里，我们描述成慢性EAE的小鼠模型的全身注射体细胞的NPC的关键方法问题。更具体地说，我们定义我们已经建立了以（i）本协议派生，扩展和移植体的NPC准备从成年C57BL / 6小鼠的脑室下区（SVZ）; （ⅱ）引起的慢性EAE在这种小鼠及（iii）进行治疗有效的全身性（ⅳ或ICV）怪物移植我NTO EAE小鼠。

<table><tbody><tr><td>&lt;strong&gt;Cell culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EBSS</td><td>Sigma</td><td>E2888</td><td></td></tr><tr><td>L-Cystein</td><td>Sigma Aldrich</td><td>C7352</td><td></td></tr><tr><td>Papain</td><td>Worthington</td><td>30H11965</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>D/0700/50</td><td></td></tr><tr><td>Mouse NeuroCult basal medium</td><td>Stem Cell Technologies</td><td>05700</td><td></td></tr><tr><td>NeuroCult proliferation supplements</td><td>Stem Cell Technologies</td><td>05701</td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3393</td><td></td></tr><tr><td>Basic fibroblast growth factor</td><td>Peprotech</td><td>100-18B-1000</td><td></td></tr><tr><td>Epidermal growth factor</td><td>Peprotech</td><td>AF-100-15-1000</td><td></td></tr><tr><td>Pen/Strep</td><td>Invitrogen</td><td>1514012</td><td></td></tr><tr><td>Matrigel (coating solution)</td><td>BD Biosciences</td><td>354230</td><td></td></tr><tr><td>NeuroCult&amp;reg; Differentiation Kit (Mouse)</td><td>Stem Cell Technologies</td><td>05704</td><td></td></tr><tr><td>Accumax</td><td>eBioscience</td><td>00-4666-56</td><td></td></tr><tr><td>Dulbecco&#039;s PBS (DPBS) (10x) without Ca and Mg</td><td>PAA Laboratories</td><td>H15-011</td><td></td></tr><tr><td>Myco trace</td><td>PAA Laboratories</td><td>Q052-020</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>&lt;strong&gt;Immunofluorescence&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Normal goat serum</td><td>PAA Laboratories</td><td>B11-035</td><td></td></tr><tr><td>Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether</td><td>Sigma Aldrich</td><td>T8787</td><td></td></tr><tr><td>Mouse anti Nestin</td><td>Abcam</td><td>ab11306</td><td></td></tr><tr><td>Rabbit anti GFAP</td><td>DAKO</td><td>203344</td><td></td></tr><tr><td>Mouse anti Histone H3 (phospho S10)&amp;nbsp;</td><td>Abcam</td><td>ab14955</td><td></td></tr><tr><td>Rabbit anti MAP-2</td><td>Abcam</td><td>ab32454</td><td></td></tr><tr><td>Rat anti MBP</td><td>AbD SEROTEC</td><td>MCA409S</td><td></td></tr><tr><td>Anti-O4 Antibody, clone 81 | MAB345</td><td>Millipore</td><td>MAB345</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>Mounting solution</td><td>DAKO</td><td>S3023</td><td></td></tr><tr><td>&lt;strong&gt;EAE&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Freund&#039;s Adjuvant Incomplete</td><td>Sigma Aldrich</td><td>F5506</td><td></td></tr><tr><td>&lt;em&gt;Mycobacterium tuberculosis&lt;/em&gt;</td><td>DIFCO</td><td>H37Ra</td><td></td></tr><tr><td>MOG(35&amp;ndash;55)&amp;nbsp;</td><td>Espikem</td><td></td><td></td></tr><tr><td>Pertussis toxin</td><td>List Biological Laboratories</td><td>181</td><td></td></tr><tr><td>&lt;strong&gt;Tissue processing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Iris scissor straight</td><td>Fine Sciences Tools</td><td>14060-09</td><td></td></tr><tr><td>Blunt/bended forceps</td><td>Fine Sciences Tools</td><td>11080-02</td><td></td></tr><tr><td>Brain slicer</td><td>Zivic Instruments</td><td>BSMAS005-1</td><td></td></tr><tr><td>Surgical blades</td><td>Swann-Morton</td><td>324</td><td></td></tr><tr><td>P200, P1000 pipettes</td><td></td><td></td><td></td></tr><tr><td>Ketamine (Vetalar)</td><td>Boehringer Ingelheim</td><td>01LC0030</td><td></td></tr><tr><td>Xylazine (Rompun)</td><td>Bayer</td><td>32371</td><td></td></tr><tr><td>Stereotaxic frame</td><td>KOPF</td><td>Model 900</td><td></td></tr><tr><td>Hamilton syringe</td><td>Hamilton</td><td>7762-04</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>158127</td><td></td></tr><tr><td>VECTASTAIN Elite ABC Kit</td><td>Vector Laboratories</td><td>PK-6100</td><td></td></tr></tbody></table>

systemic injection of neural stem/progenitor cells in mice with chronic eae

神经干/祖细胞（的NPC）是一种很有前途的干细胞来源，移植方法，旨在大脑修复或修复再生神经内科。该指令已产生的大量证据显示，大脑修复局灶性或全身性鼻咽癌移植后实现了神经系统疾病的几种临床前模型。 这些实验数据已经确定了细胞传递路线为恢复性干细胞疗法的脑部疾病，需要紧急评估的主要障碍之一。脑实质内干细胞移植术代表逻辑态度来对待这些特点是孤立的和可访问的脑病变，如脊髓损伤和帕金森氏病的病症。不幸的是，这个原则是很差适用特点是多灶性，炎症和传播（无论是在时间和空间）性质，包括多发性硬化症（MS）的条件。因此，脑是由系统·瞄准主位NPC交付已经成为一种低侵入性治疗有效协议，提供细胞到大脑，影响中枢神经系统（CNS）的实验性慢性炎症性损害啮齿类动物和灵长类动物的脊髓。 小区交付这种替代方法依赖于人大pathotropism，特别是他们与生俱来的能力（一） 通过感知功能细胞黏附分子和炎症细胞因子和趋化因子受体的环境; （二）交叉后静脉注射（IV）的泄漏解剖障碍或脑室内（ICV）注射液; （ⅲ）积聚在炎性脑和脊髓损伤血管周围的多个位点（S）的水平;及（iv）发挥卓越组织营养及免疫调节作用到不同的主机的靶细胞在体内 。 在这里，我们描述了我们已经开发了IV的方法。和&lt;em&gt;的同基因的NPC对小鼠实验性自身免疫性脑脊髓炎（EAE）侧脑室递送，作为慢性中枢神经系统炎性脱髓鞘模型，并设想全身干细胞递送作为有价值的技术发炎的大脑中神经再生的选择性靶向。

全国人大推导和表征 SVZ解剖是由机械和酶解（ 图1A）方法为在池进行6-8周龄C57BL / 6小鼠（n = 5-7只小鼠/池）。经过培养的CGM几天，自由浮动的神经球开始形成（ 图1A和1B）。主球被收集和机械传代，每4-5格。传代后，活细胞和死细胞的数目是确定的和累积的细胞数作图以生成生长曲线（ 图1C）。这使传播速率的指示，从而提供了一个间接的参...

Watch this Scientific Journal Video about Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE at JoVE.com

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

神经干/祖细胞（的NPC）的移植持有再生神经内科伟大的应许。全身交付的NPC已经变成有效的，低侵入性的，而且治疗非常有效的协议，提供干细胞在大脑和受中枢神经系统的实验性慢性炎症性损害啮齿类动物和灵长类动物的脊髓。

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 ...

systemic-injection-neural-stemprogenitor-cells-mice-with-chronic

Research

JoVE Journal

Immunology and Infection

17.6K 次观看.  University of Cambridge, UK. 神经干/祖细胞（的NPC）的移植持有再生神经内科伟大的应许。全身交付的NPC已经变成有效的，低侵入性的，而且治疗非常有效的协议，提供干细胞在大脑和受中枢神经系统的实验性慢性炎症性损害啮齿类动物和灵长类动物的脊髓。 

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

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

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

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

科研

免疫与感染

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

髓鞘少突胶质细胞糖蛋白（MOG<sub&gt; 35-55</sub&gt;）诱导实验性自身免疫性脑脊髓炎（EAE）的C57BL / 6小鼠

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

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) ...

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

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

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

确定免疫系统抑制中枢神经系统对保护药物干预自身免疫性脱髓鞘

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

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

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

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

多发性硬化症的小鼠模型EAE视神经炎及脑部炎症的生物发光和近红外成像

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

医学

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

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

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

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

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

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 ...

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 ...

神经干/祖细胞在小鼠EAE的慢性全身性注射

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

髓鞘少突胶质细胞糖蛋白（MOG

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

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

多发性硬化症的小鼠模型EAE视神经炎及脑部炎症的生物发光和近红外成像

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

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

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