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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| AMCA anti-rabbit antibody | Jackson ImmunoResearch | 111-156-045 | Store at 4 °C; protect from light |
| Anti-CD45 Antibody (30F11) | Pharmingen | 07-1401 | Store at 4 °C |
| Anti-Laminin Antibody | DAKO | Z0097 | Store at 4 °C |
| Breeding food | e.g. PROVIMI KLIBA SA | 3336 | |
| Individually ventilated cages, Blue line Type II or III | e.g. Tecniplast | 1145T, 1285L | |
| BSA fraction V | Applichem | A1391 | Store at 4 °C |
| Cold gelatine | Sigma-Aldrich | G 9391 | |
| Coplin jar + rack | e.g. Carl Roth GmbH + Co. KG | H554.1; H552.1 | |
| Cy3 anti-rat antibody | Jackson ImmunoResearch | 111-156-144 | Store at 4 °C; protect from light |
| Cover slips 24 x 40 mm # 1 | e.g. Thermo Scientific | 85-0186-00 | |
| Dextran Alexa Fluor 488 (10,000 MW) | e.g. Molecular probes | D22910 | Store at -20 °C; protect from light |
| Dextran Texas Red (3000 MW) | Invitrogen | D3328 | Store at -20 °C; protect from light |
| EnzChek Gelatinase/Collagenase Assay Kit | Thermo Fisher Scientific; EnzCheck | E12055 | Store at -20 °C; protect form light |
| Female C57BL/6J mice (8-12 weeks) | e.g. Janvier Labs | Females, 8-12 weeks | |
| Freezing box for histology slides | e.g. Carl Roth GmbH + Co. KG | 2285.1 | |
| 18G x 1½’’ (1.2mm x 40mm) injection needle | e.g. BD, BD Microlance 3 | 304622 | |
| 27G x ¾’’ – Nr. 20 (0.4mm x 19mm) injection needle | e.g. BD, BD Microlance 3 | 302200 | |
| 30G x ½’’ (0.3 mm x 13 mm) injection needle | e.g. BD, BD Microlance 3 | 304000 | |
| Incomplete Freund’s adjuvant (IFA) | e.g. Santa Cruz Biotechnology | sc-24648 | Store at 4°C |
| Maintenance food | e.g. PROVIMI KLIBA SA | 3436 | |
| MOGaa35-55 peptide | e.g. GenScript | Store at -80 °C | |
| microscope slides (Superfrost Plus ) | Thermo Scientific | J1800AMNZ | |
| Mycobacterium tuberculosis H37RA | e.g. BD | 231141 | Store at 4 °C |
| NaCl 0.9 % | B. Braun | 3535789 | |
| O.C.T. compound (Tissue-Tek ) | Sakura | 4583 | |
| Omnican 50 30G x ½’’ | B. Braun | 9151125S | |
| Paraformaldehyde | Merck | 30525-89-4 | |
| Pertussis toxin | e.g. List biological laboratories, Inc. | 180 | Store at 4 °C |
| poly(vinyl alcohole) (Mowiol 4-88) | Sigma-Aldrich | 81381 | |
| Protease Inhibitor EDTA free (Roche) | Sigma-Aldrich | 4693132001 | Store at 4 °C |
| repelling pen e.g. DAKO Pen | e.g. DAKO | S2002 | |
| sealing film e.g. Parafilm M | e.g Sigma-Aldrich | P7793 | |
| Silica gel | e.g. Carl Roth GmbH + Co. KG | 9351.1 | |
| Stitch scissor | F.S.T | 15012-12 | |
| syringe 1 ml | e.g. PRIMO | 62.1002 | |
| syringe 10 ml | e.g. CODAN Medical ApS | 2022-05 | |
| vaporizer system Univentor 400 | UNO.BV |
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