Name: visualizing impairment of the endothelial and glial barriers of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo
Uploaded: 2019-03-26T13:00:00
Description: Watch this Scientific Journal Video about Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo at JoVE.com

我们感谢莉迪亚·索罗金, 她与我们分享了她最初的现场酶学协议10.

所有研究都是根据这些准则根据瑞士关于保护动物的立法进行的, 并得到了瑞士伯尔尼州兽医办公室的批准 (许可号: be31/和 BE 77/18)。
1. 在特定无病原体 (SPF) 条件下的 c57bl6 小鼠的外壳
<ol><li>在每一个通风笼子里, 以 12/半小时的光暗周期的家庭老鼠。提供食物和水的。为了监督小鼠的微生物质量, 实验组根据 FELASA 建议13, 采用肮脏床上用品的哨兵进行季度健?...

<ol>
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J., 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=Molecular+characterization+of+perivascular+drainage+pathways+in+the+murine+brain.">Molecular characterization of perivascular drainage pathways in the murine brain.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 38, 669-686 (2018).</li><li>Kermode, A. 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=Breakdown+of+the+blood-brain+barrier+precedes+symptoms+and+other+MRI+signs+of+new+lesions+in+multiple+sclerosis.+Pathogenetic+and+clinical+implications.">Breakdown of the blood-brain barrier precedes symptoms and other MRI signs of new lesions in multiple sclerosis. Pathogenetic and clinical implications.</a> Brain. 113 (Pt 5), 1477-1489 (1990).</li><li>Tommasin, S., Gianni, C., De Giglio, L., Pantano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroimaging+techniques+to+assess+inflammation+in+Multiple+Sclerosis.">Neuroimaging techniques to assess inflammation in Multiple Sclerosis.</a> Neuroscience. , (2017).</li><li>Lopes Pinheiro, M. 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I., 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=Visualizing+context-dependent+calcium+signaling+in+encephalitogenic+T+cells+in+vivo+by+two-photon+microscopy.">Visualizing context-dependent calcium signaling in encephalitogenic T cells in vivo by two-photon microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 114, E6381-E6389 (2017).</li><li>Lodygin, D., 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+combination+of+fluorescent+NFAT+and+H2B+sensors+uncovers+dynamics+of+T+cell+activation+in+real+time+during+CNS+autoimmunity.">A combination of fluorescent NFAT and H2B sensors uncovers dynamics of T cell activation in real time during CNS autoimmunity.</a> Nature Medicine. 19, 784-790 (2013).</li><li>Agrawal, S., 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=Dystroglycan+is+selectively+cleaved+at+the+parenchymal+basement+membrane+at+sites+of+leukocyte+extravasation+in+experimental+autoimmune+encephalomyelitis.">Dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation in experimental autoimmune encephalomyelitis.</a> Journal of Experimental Medicine. 203, 1007-1019 (2006).</li><li>Song, J., 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=Focal+MMP-2+and+MMP-9+activity+at+the+blood-brain+barrier+promotes+chemokine-induced+leukocyte+migration.">Focal MMP-2 and MMP-9 activity at the blood-brain barrier promotes chemokine-induced leukocyte migration.</a> Cell Reports. 10, 1040-1054 (2015).</li><li>Tietz, S., 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=Lack+of+junctional+adhesion+molecule+(JAM)-B+ameliorates+experimental+autoimmune+encephalomyelitis.">Lack of junctional adhesion molecule (JAM)-B ameliorates experimental autoimmune encephalomyelitis.</a> Brain, Behavior, and Immunity. 73, 3-20 (2018).</li><li>Mähler Convenor, M., Berard, M., Feinstein, R., Gallagher, A., Illgen-Wilcke, B., Pritchett-Corning, K., Raspa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+recommendations+for+the+health+monitoring+of+mouse,+rat,+hamster,+guinea+pig+and+rabbit+colonies+in+breeding+and+experimental+units.">FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units.</a> Lab Anim. 48 (3), 178-192 (2014).</li><li>Bittner, S., Afzali, A. M., Wiendl, H., Meuth, S. G. <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+(MOG35-55)+induced+experimental+autoimmune+encephalomyelitis+(EAE)+in+C57BL/6+mice.">Myelin oligodendrocyte glycoprotein (MOG35-55) induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice.</a> Journal of Visual Experiments. , (2014).</li><li>Tietz, S. M., 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=Refined+clinical+scoring+in+comparative+EAE+studies+does+not+enhance+the+chance+to+observe+statistically+significant+differences.">Refined clinical scoring in comparative EAE studies does not enhance the chance to observe statistically significant differences.</a> European Journal of Immunology. 46, 2481-2483 (2016).</li><li>Mendel, I., Kerlero de Rosbo, N., Ben-Nun, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+myelin+oligodendrocyte+glycoprotein+peptide+induces+typical+chronic+experimental+autoimmune+encephalomyelitis+in+H-2b+mice:+fine+specificity+and+T+cell+receptor+V+beta+expression+of+encephalitogenic+T+cells.">A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice: fine specificity and T cell receptor V beta expression of encephalitogenic T cells.</a> European Journal of Immunology. 25, 1951-1959 (1995).</li><li>Miller, S. D., Karpus, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+encephalomyelitis+in+the+mouse.">Experimental autoimmune encephalomyelitis in the mouse.</a> Current Protocols in Immunology. 15, (2007).</li><li>Sobel, R. A., Tuohy, V. K., Lu, Z. J., Laursen, R. A., Lees, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+experimental+allergic+encephalomyelitis+in+SJL/J+mice+induced+by+a+synthetic+peptide+of+myelin+proteolipid+protein.">Acute experimental allergic encephalomyelitis in SJL/J mice induced by a synthetic peptide of myelin proteolipid protein.</a> Journal of Neuropathology &amp; Experimental Neurology. 49, 468-479 (1990).</li><li>Westarp, M. E., 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=T+lymphocyte+line-mediated+experimental+allergic+encephalomyelitis--a+pharmacologic+model+for+testing+of+immunosuppressive+agents+for+the+treatment+of+autoimmune+central+nervous+system+disease.">T lymphocyte line-mediated experimental allergic encephalomyelitis--a pharmacologic model for testing of immunosuppressive agents for the treatment of autoimmune central nervous system disease.</a> Journal of Pharmacology and Experimental Therapeutics. 242, 614-620 (1987).</li><li>Klotz, L., 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=B7-H1+shapes+T-cell-mediated+brain+endothelial+cell+dysfunction+and+regional+encephalitogenicity+in+spontaneous+CNS+autoimmunity.">B7-H1 shapes T-cell-mediated brain endothelial cell dysfunction and regional encephalitogenicity in spontaneous CNS autoimmunity.</a> Proceedings of the National Academy of Sciences of the United States of America. 113, E6182-E6191 (2016).</li><li>Tietz, S. M., 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=MK2+and+Fas+receptor+contribute+to+the+severity+of+CNS+demyelination.">MK2 and Fas receptor contribute to the severity of CNS demyelination.</a> PLoS One. 9, e100363 (2014).</li><li>Coisne, C., Mao, W., Engelhardt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+Natalizumab+blocks+adhesion+but+not+initial+contact+of+human+T+cells+to+the+blood-brain+barrier+in+vivo+in+an+animal+model+of+multiple+sclerosis.">Cutting edge: Natalizumab blocks adhesion but not initial contact of human T cells to the blood-brain barrier in vivo in an animal model of multiple sclerosis.</a> Journal of Immunology. 182, 5909-5913 (2009).</li><li>Korner, 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=Critical+points+of+tumor+necrosis+factor+action+in+central+nervous+system+autoimmune+inflammation+defined+by+gene+targeting.">Critical points of tumor necrosis factor action in central nervous system autoimmune inflammation defined by gene targeting.</a> Journal of Experimental Medicine. 186, 1585-1590 (1997).</li><li>Krueger, M., 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=Blood-brain+barrier+breakdown+involves+four+distinct+stages+of+vascular+damage+in+various+models+of+experimental+focal+cerebral+ischemia.">Blood-brain barrier breakdown involves four distinct stages of vascular damage in various models of experimental focal cerebral ischemia.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 35, 292-303 (2015).</li><li>Hawkins, B. T., Egleton, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+imaging+of+blood-brain+barrier+disruption.">Fluorescence imaging of blood-brain barrier disruption.</a> Journal of Neuroscience Methods. 151, 262-267 (2006).</li><li>Graesser, D., 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=Altered+vascular+permeability+and+early+onset+of+experimental+autoimmune+encephalomyelitis+in+PECAM-1-deficient+mice.">Altered vascular permeability and early onset of experimental autoimmune encephalomyelitis in PECAM-1-deficient mice.</a> Journal of Clinical Investigation. 109, 383-392 (2002).</li><li>Engelhardt, B., Sorokin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+and+the+blood-cerebrospinal+fluid+barriers:+function+and+dysfunction.">The blood-brain and the blood-cerebrospinal fluid barriers: function and dysfunction.</a> Seminars in Immunopathology. 31, 497-511 (2009).</li><li>Owens, T., Bechmann, I., Engelhardt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+spaces+and+the+two+steps+to+neuroinflammation.">Perivascular spaces and the two steps to neuroinflammation.</a> Journal of Neuropathology &amp; Experimental Neurology. 67, 1113-1121 (2008).</li><li>Sixt, M., 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=Endothelial+cell+laminin+isoforms,+laminins+8+and+10,+play+decisive+roles+in+T+cell+recruitment+across+the+blood-brain+barrier+in+experimental+autoimmune+encephalomyelitis.">Endothelial cell laminin isoforms, laminins 8 and 10, play decisive roles in T cell recruitment across the blood-brain barrier in experimental autoimmune encephalomyelitis.</a> Journal of Cell Biology. 153, 933-946 (2001).</li><li>Grewal, I. S., 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=CD62L+is+required+on+effector+cells+for+local+interactions+in+the+CNS+to+cause+myelin+damage+in+experimental+allergic+encephalomyelitis.">CD62L is required on effector cells for local interactions in the CNS to cause myelin damage in experimental allergic encephalomyelitis.</a> Immunity. 14, 291-302 (2001).</li></ol>

在这里, 我们提出了一个方案诱导和监测 EAE 在雌性 c57bl6 小鼠。女性优先被选择, 并且有妇女的发生率: 男性在 m s 中 3: 1。为了评估 EAE 的严重程度, 我们使用了3分评分表。EAE 的严重程度通常是根据运动功能障碍的严重程度来评分的。应牺牲 EAE 晚期的小鼠, 即表现出2分以上的小鼠, 以避免动物遭受不必要的痛苦。因此, 建议对老鼠进行近距离的评分, 例如, 如协议中所解释的那样, 每天两次。 有几...

中枢神经系统 (CNS) 协调脊椎动物的所有身体和精神功能, 中枢神经系统的稳态对于神经元的适当沟通至关重要。中枢神经系统的稳态是由神经血管单位 (NVU) 保证的, 它保护中枢神经系统免受血液变化环境的影响。NVU 由中枢神经系统微血管内皮细胞组成, 具有生物化学性质的独特性, 并在具有周细胞、星形胶质细胞、神经元和细胞外基质 (ECM) 成分的连续串扰中建立血脑屏障 (BBB), 建立两个独特的基底膜1。内皮基底膜, 使 BBB 内皮细胞的腔侧含有大量的周周细胞, 并由层压蛋白α4和层压蛋白α5组成, 以及其他 ECM 蛋白 2.相反, 实质基底膜由层蛋白α1和层蛋白α2组成, 并被星形胶质细胞终脚所接受。实质基底膜与星形胶质细胞终脚共同构成胶质细胞限制, 将中枢神经系统神经元网络与充满血管周围或蛛网膜下腔的脑脊液分离3。由于 NVU 的独特结构, 免疫细胞贩运到中枢神经系统是不同于, 进入外周组织, 因为它需要一个两步的过程与免疫细胞, 首先违反内皮 BBB, 然后胶质限制, 以到达中枢神经系统。
多发性硬化症 (MS) 是中枢神经系统常见的神经炎症性疾病, 其中大量循环免疫细胞进入中枢神经系统, 引起神经炎症、脱髓鞘和 BBB 完整性的局灶性丧失4。BBB 完整性的丧失是 MS 的一个早期特征, 磁共振成像 (MRI) 5 显示中枢神经系统中存在的增强铝对比度病变就表明了这一点.白细胞向中枢神经系统的外渗发生在毛细血管后静脉水平;然而, BBB 基底膜上的免疫细胞直径所涉及的精确机制以及随后的胶质屏障仍有待探索。实验性自身免疫性脑炎 (EAE) 是 MS 的动物模型, 对我们目前对 MS 发病机制的了解做出了显著贡献。例如, 使用 EAE 模型, 已经发现白细胞外渗发生在一个多步骤的过程中, 包括由选择和粘液样分子 (p-选择蛋白糖蛋白配体 (PSGL)-1 介导的初始捕获和滚动步骤,其次是整合依赖的牢固的逮捕和在 BBB 内皮细胞上的 T 细胞爬行到允许的边的 diapdesis6。
一旦 T 细胞穿过内皮 BBB 和内皮基底膜, 它们就需要在巨噬细胞或树突状细胞上遇到它们的同源抗原, 这些细胞战略性地定位在平生细胞或血管周围的空间。这种相互作用诱导促进炎症介质的聚焦产生, 从而触发中枢神经系统组织通过胶质细胞7,8,9侵入免疫细胞所需的后续机制。基质-金属蛋白酶 (MMP)-2 和 MMP-9 的焦点活化改变趋化因子活化并诱导星形胶质细胞末端脚部细胞外基质受体的降解, 这是免疫细胞在胶质细胞中迁移到胶质细胞中的先决条件。中枢神经系统实质和诱导的临床症状的 OAE10,11。
结合中枢神经系统浸润免疫细胞的检测与 BBB 泄漏和中枢神经系统组织切片中的明胶酶活性相结合, 为神经炎症背景下内皮屏障和胶质屏障的功能完整性提供了有价值的信息。例如, 我们最近研究了内皮紧密结合分子连接粘附分子 (JAM)-b 在 EAE 环境下向中枢神经系统的导入中的本构损失。与健康的野生型 c57bl6 小鼠相比, 健康的 Jam-b 缺乏的同学没有显示出 BBB 完整性的损伤, 这表明在体内的通量评估使用内源性和外源示踪剂12。在 EAE 方面, 缺乏 Jam-b 的 c57bl6 小鼠表现出改善的疾病症状, 这与炎症细胞捕获在小孔和血管周围空间12有关.为了研究这种现象, 我们在原位酶学, 允许识别明胶酶活性, 以测试缺乏正义酶酶活性的族洗钱蛋白酶活性是否可能导致能够突破胶质限制12。
鉴于缺乏不同的转基因小鼠模型, 例如, 可能导致 BBB 功能变化的不同 BBB 紧密连接分子的可用性, 研究 BBB 完整性的方法非常重要。此外, 新开发的药物可能会对 NVU 的壁垒产生影响。在这里, 我们展示了如何诱导 eae c57bl6 小鼠积极免疫髓鞘少突胶质细胞糖蛋白 (MOG)-肽 aaaa35-55 在完整的 Freund 的佐剂。然后, 我们解释了如何定位免疫细胞浸润的内皮和胶质屏障的 NVU, 以及如何研究在体内内皮和胶质屏障完整性的原位检测外源示踪剂和明胶酶活性, 分别。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >AMCA anti-rabbit antibody</td>
			<td >Jackson ImmunoResearch</td>
			<td >111-156-045</td>
			<td >Store at 4 &#176;C; protect from light</td>
		</tr>
		<tr >
			<td >Anti-CD45 Antibody (30F11)</td>
			<td >Pharmingen</td>
			<td >07-1401</td>
			<td >Store at 4 &#176;C</td>
		</tr>
		<tr >
			<td >Anti-Laminin Antibody</td>
			<td >DAKO</td>
			<td >Z0097</td>
			<td >Store at 4 &#176;C</td>
		</tr>
		<tr >
			<td >Breeding food</td>
			<td >e.g. PROVIMI KLIBA SA</td>
			<td >3336</td>
			<td ></td>
		</tr>
			<tr >
			<td >Individually ventilated cages, Blue line Type II or III</td>
			<td >e.g. Tecniplast	</td>
			<td >1145T, 1285L</td>
			<td ></td>
		</tr>	
		<tr >
			<td >BSA fraction V</td>
			<td >Applichem</td>
			<td >A1391</td>
			<td >Store at 4 &#176;C</td>
		</tr>
		<tr >
			<td >Cold gelatine</td>
			<td >Sigma-Aldrich</td>
			<td >G 9391</td>
			<td ></td>
		</tr>
		<tr >
			<td >Coplin jar + rack</td>
			<td >e.g. Carl Roth GmbH + Co. KG</td>
			<td >H554.1; H552.1</td>
			<td ></td>
		</tr>
		<tr >
			<td >Cy3 anti-rat antibody</td>
			<td >Jackson ImmunoResearch</td>
			<td >111-156-144</td>
			<td >Store at 4 &#176;C; protect from light</td>
		</tr>
		<tr >
			<td >Cover slips 24 x 40 mm # 1</td>
			<td >e.g. Thermo Scientific</td>
			<td >85-0186-00</td>
			<td></td>
		</tr>
		<tr >
			<td >Dextran Alexa Fluor 488 (10,000 MW)</td>
			<td >e.g. Molecular probes</td>
			<td >D22910</td>
			<td >Store at -20 &#176;C; protect from light</td>
		</tr>
		<tr >
			<td >Dextran Texas Red (3000 MW)</td>
			<td >Invitrogen</td>
			<td >D3328</td>
			<td >Store at -20 &#176;C; protect from light</td>
		</tr>
		<tr >
			<td >EnzChek Gelatinase/Collagenase Assay Kit&#160;</td>
			<td >Thermo Fisher Scientific; EnzCheck</td>
			<td >E12055</td>
			<td >Store at -20 &#176;C; protect form light</td>
		</tr>
		<tr >
			<td >Female C57BL/6J mice (8-12 weeks)</td>
			<td >e.g. Janvier Labs</td>
			<td ></td>
			<td >Females, 8-12 weeks</td>
		</tr>
		<tr >
			<td >Freezing box for histology slides</td>
			<td >e.g. Carl Roth GmbH + Co. KG</td>
			<td >2285.1</td>
			<td ></td>
		</tr>
		<tr >
			<td >18G x 1&#189;&#8217;&#8217; (1.2mm x 40mm) injection needle</td>
			<td >e.g. BD, BD Microlance 3</td>
			<td >304622</td>
			<td ></td>
		</tr>
		<tr >
			<td >27G x &#190;&#8217;&#8217; &#8211; Nr. 20 (0.4mm x 19mm) injection needle</td>
			<td >e.g. BD, BD Microlance 3</td>
			<td >302200</td>
			<td ></td>
		</tr>
		<tr >
			<td >30G x &#189;&#8217;&#8217; (0.3 mm x 13 mm) injection needle</td>
			<td >e.g. BD, BD Microlance 3</td>
			<td >304000</td>
			<td ></td>
		</tr>
		<tr >
			<td >Incomplete Freund&#8217;s adjuvant (IFA)</td>
			<td >e.g. Santa Cruz Biotechnology</td>
			<td >sc-24648</td>
			<td >Store at 4&#176;C</td>
		</tr>
		<tr >
			<td >Maintenance food</td>
			<td >e.g. PROVIMI KLIBA SA</td>
			<td >3436</td>
			<td ></td>
		</tr>
		<tr >
			<td >MOGaa35-55 peptide</td>
			<td >e.g. GenScript</td>
			<td ></td>
			<td >Store at -80 &#176;C</td>
		</tr>
		<tr >
			<td >microscope slides (Superfrost Plus )</td>
			<td >Thermo Scientific</td>
			<td >J1800AMNZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mycobacterium tuberculosis H37RA</td>
			<td >e.g. BD</td>
			<td >231141</td>
			<td >Store at 4 &#176;C&#160;</td>
		</tr>
		<tr >
			<td >NaCl 0.9 %</td>
			<td >B. Braun</td>
			<td >3535789</td>
			<td ></td>
		</tr>
		<tr >
			<td >O.C.T. compound (Tissue-Tek )</td>
			<td >Sakura</td>
			<td >4583</td>
			<td ></td>
		</tr>
		<tr >
			<td >Omnican 50 30G x &#189;&#8217;&#8217;</td>
			<td >B. Braun</td>
			<td >9151125S</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde</td>
			<td >Merck</td>
			<td >30525-89-4</td>
			<td ></td>
		</tr>
		<tr >
			<td >Pertussis toxin</td>
			<td >e.g. List biological laboratories, Inc.</td>
			<td >180</td>
			<td >Store at 4 &#176;C</td>
		</tr>
		<tr >
			<td >poly(vinyl alcohole) (Mowiol 4-88)</td>
			<td >Sigma-Aldrich</td>
			<td >81381</td>
			<td ></td>
		</tr>
		<tr >
			<td >Protease Inhibitor EDTA free (Roche)</td>
			<td >Sigma-Aldrich</td>
			<td >4693132001</td>
			<td >Store at 4 &#176;C</td>
		</tr>
		<tr >
			<td >repelling pen e.g. DAKO Pen</td>
			<td >e.g. DAKO</td>
			<td >S2002</td>
			<td ></td>
		</tr>
		<tr >
			<td >sealing film e.g. Parafilm M</td>
			<td >e.g Sigma-Aldrich</td>
			<td >P7793</td>
			<td ></td>
		</tr>
		<tr >
			<td >Silica gel</td>
			<td >e.g. Carl Roth GmbH + Co. KG</td>
			<td >9351.1</td>
			<td ></td>
		</tr>
		<tr >
			<td >Stitch scissor</td>
			<td >F.S.T</td>
			<td >15012-12</td>
			<td ></td>
		</tr>
		<tr >
			<td >syringe 1 ml</td>
			<td >e.g. PRIMO</td>
			<td >62.1002</td>
			<td ></td>
		</tr>
		<tr >
			<td >syringe 10 ml</td>
			<td >e.g. CODAN Medical ApS</td>
			<td >2022-05</td>
			<td ></td>
		</tr>
		<tr >
			<td >vaporizer system Univentor 400</td>
			<td >UNO.BV</td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing impairment of the endothelial and glial barriers of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo

神经血管单元 (NVU) 由微血管内皮细胞组成, 形成血脑屏障 (BBB)、内皮基底膜 (嵌入过周) 和胶质细胞限制细胞, 由实质基底膜和星形胶质细胞组成端部饲料包括中枢神经系统 (CNS) 微血管的烧蚀面。除了维持中枢神经系统的稳态外, NVU 还控制着免疫细胞贩运进入中枢神经系统。在对中枢神经系统进行免疫监测期间, 低活性淋巴细胞的数量可以穿过内皮屏障, 而不会导致 BBB 功能障碍或临床疾病。相反, 在多发性硬化症或其动物模型实验性自身免疫性脑炎 (EAE) 等神经炎症期间, 大量的免疫细胞可以穿过 BBB, 随后胶质细胞最终到达中枢神经系统实质导致临床疾病。
因此, 免疫细胞迁移到中枢神经系统实质是一个两步的过程, 涉及连续迁移整个内皮和胶质屏障的 NVU 采用不同的分子机制。如果在通过内皮屏障后, T 细胞在血管周围抗原表达细胞上遇到认知抗原, 它们的局部激活将启动随后的机制, 导致明胶酶的局灶性激活,将使 T 细胞越过胶质屏障, 进入中枢神经系统实质。因此, 在 EAE 期间, 评估 BBB 通透性和 MMP 活性与中枢神经系统免疫细胞积累的空间相关性, 可以指定 NVU 内皮屏障和胶质屏障完整性的丧失。 
本文介绍了如何通过主动免疫诱导 c57bl6 小鼠的 EAE, 以及如何利用外源荧光示踪剂联合分析 BBB 在体内的通透性。我们进一步展示了如何可视化和定位明胶酶活性在 EAE 大脑中的原位酶结合到免疫荧光污渍的 bbb 基底膜和 CD45 + 入侵免疫细胞。

评估 c57bl6 小鼠 EAE 的临床过程应导致如图 2 a 所示的疾病曲线和图 2A所示的小鼠体重变化。接受 MOGaa35-55 免疫的 c57bl6 小鼠通常在主动免疫后10-12 天左右开始出现疾病症状 (图 1 a)。通常情况下, 免疫小鼠在注射乳剂和第一剂百日咳毒素的第二天显示出短暂的体重下降 (图 1 b)。从 EAE 诱导后的第2天开始, 免疫?...

Watch this Scientific Journal Video about Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo at JoVE.com

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

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

在这里, 我们提出的方案, 以调查损伤的神经血管单位在实验性自身免疫性脑髓炎在体内。我们专门讨论了如何确定血液脑屏障通透性和明胶酶活性与白细胞迁移跨越胶质细胞限制。

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

这里介绍的方法使我们能够研究免疫细胞在实验性自身免疫性脑脊髓炎（多发性硬化症的动物模型）中跨越大脑屏障迁移的分子机制。通过研究血脑屏障渗透性和基质-金属蛋白酶活性，同时在空间相关性与免疫细胞渗透在脑组织部分，我们可以准确地将这些神经炎症事件与EE的临床症状关联。专业处理患病小鼠需要深入训练，以及正确评分EE的临床症状。因此，视觉演示是有益的。从 C57-Black/6 小鼠的麻醉开始，这些小鼠已被安置在文本协议中描述的特定无病原体条件下。一只手固定麻醉小鼠，将30微升MOG肽注射，将Freund的辅助乳液皮下注射到每个后腿侧翼，靠近内结淋巴结。现在，把鼠标放在它的腹部，将20微升的MOG-肽乳液注射到尾部左右的软脂肪组织中。此外，将乳液的一小滴注入小鼠的颈部。将100微升百日咳毒素溶液内向小鼠注射。将鼠标头放在身体中心下方，以避免注射到肠道中。将维持饮食替换为育种饮食，以便在预期的临床疾病之前和期间为小鼠提供能量含量较高的食物。在第一次治疗后48小时重复百日咳注射。每天早上在笼子里看看EAE老鼠的健康状况。每天下午给 EAE 老鼠评分。将 EAE 实验中的每只鼠标都从笼子里拿出来，并检查尾巴是否有鼻塞。用手指向上移动尾巴。健康的老鼠会保持它的尾巴。如果临床 EAE 已经开始，尾部的尾部将降低，通过尾部的逐渐下降可见。最终，鼠标将无法抬起尾巴。将 EAE 实验中的每只鼠标都放在干净的长凳上，以观察和记录行走行为。有关疾病严重程度的评估（即 EAE 评分）的评分标准，请参阅文本。评估和记录实验中包含的每只鼠标的重量。对于制备脱氧体库存溶液，在 500 微升 0.9% 氯化钠溶液中溶解 10 毫克三千克顿 Dextran 德州红，在 250 微升 0.9% 氯化钠溶液中溶解 5 毫克 10 千克多克脱氧货量。对于 dextran 工作溶液，就在注射前，移液器 55 微升 10 千克顿 Dextran Alexa Fluor 488 库存溶液上一块密封膜上。然后，添加55微升的三千元德斯特兰德州红股票解决方案。混合并收集100微升到一次性精细注射器中。接下来，将麻醉小鼠放在表上的横向位置。在鼠标唤醒之前，静脉注射100微升荧光示踪剂到小鼠中。让示踪器循环15分钟，然后执行文本协议中详述的小鼠灌注。用碎干冰填充平坦的脱华容器，加入2-甲基丁烷，直到液体达到干燥冰袋上方约一厘米。盖上松动的盖子，如冰桶盖。用锋利的剪刀从被刺穿的鼠标的头部剪下来后，用一套较小的剪刀去除皮肤和耳朵。小心解剖头骨，从前身巨无霸切向左侧和右侧的前面，允许头骨盖向上折叠在大脑上。然后，小心地从头骨底部抬起完整的大脑，同时用扁平的金属铲切断光学神经。将大脑放在铝箔上。通过放置两个日冕切口，将大脑切成三块。这三块代表前脑、中脑和脑干小脑。使用最佳温度切割矩阵将 Cryomold 填充到第一季度。然后，将大脑切片放入 Cryomold 中，每个大脑片的前部朝下。用矩阵完全覆盖组织。将具有组织水平方向的 Cryomold 放入 2 甲基丁烷浴中。避免将整个组织块浸入浴缸中，确保组织在一分钟内从底部冻结到顶部。将冷冻组织转移到零下80摄氏度进行储存。有关冷冻组织部分的准备，请参阅文本协议。检索从EAE小鼠身上准备的六微米非固定脑组织部分。在烟罩中，解冻塑料冷冻箱内的部分，盖子内含有硅胶，以避免将水留在组织中。使用防水笔绘制线条，在一张幻灯片上分离两个组织部分。在室温下，在13，300倍g下，在5分钟内进行离心反应溶液，使用水浴预热至37摄氏度。然后，使用1X反应缓冲液在37摄氏度下补充组织部分5分钟。五分钟后，倒掉1X反应缓冲液。在组织部分添加反应溶液，在37摄氏度下孵育4小时。然后，用双蒸馏水清洗滑梯五次。现在，在零下20摄氏度的温度下用冰冷的甲醇将部分固定5分钟，然后用PBS在室温下洗涤一次。在 PBS 中加入 1%BSA，并在室温下孵育 20 分钟。然后，通过将幻灯片翻转纸巾，将缓冲区从该部分丢弃。将原发抗体鸡尾酒加入该部分，并在室温下孵育一小时。用PBS洗两次后，加入二次抗体鸡尾酒，在室温下孵育一小时。然后，使用 PBS 清洗部分两次，然后使用嵌入解决方案安装幻灯片。让安装部分在室温下干燥过夜后，使用荧光显微镜分析染色组织部分。EAE 疾病的严重程度增加，直到疾病达到峰值，预计在 EAE 诱导后的第 16 天至 20 天之间。在临床EE的高峰期，小鼠的体重也最低，这增加了与EE在疾病缓解阶段的改善的相关性。这里展示的是一个患有EAE的C57-Black/6小鼠的胆小毛症和相邻大脑帕伦奇玛的代表性图片。在牺牲前15分钟，小鼠静脉注射。追踪器很容易通过微纤维扩散到胆齿丛的气孔中，而血脑屏障不允许循环的示踪剂扩散到大脑的面皮瘤中，因此没有任何荧光信号。这些图像显示了患有EAE的C57-Black/6小鼠小脑中具有代表性的漏血血管。箭头表示示踪迹通过大脑微壁扩散到中枢机屏障中，表明这些血管中的血脑屏障功能受损。在中枢系统，在血管壁外可以看到绿色和红色的荧光信号。正确皮下放置 MOG-CFA 乳液对于安装导致 EAE 的适当免疫反应至关重要。应避免在脑内注射和肌肉内注射。在烟气罩下准备 CFA，以避免吸入衰减的分枝杆菌。此外，避免使用MOG-CFA乳液进行自我注射，因为这可能导致结节瘤，甚至导致基因易感个体的EE。脑渗透免疫细胞可以从患有EAE的小鼠的CNS中分离，通过流动细胞学在神经刺激期间量化渗透免疫细胞子集。评估内皮和胶质脑屏障的完整性可以转化为转基因小鼠模型的分析或其他神经系统疾病的模型。

Research

JoVE Journal

Immunology and Infection

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

鼠标垂体免疫原的制备诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

科研

免疫与感染

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

SJL小鼠诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

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

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

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 ...

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

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

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

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

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 ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

简单而高效的生产和鼠标髓鞘少突胶质糖蛋白的纯化的实验性自身免疫性脑脊髓炎研究

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

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

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 ...

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

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

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