从微解剖小鼠脉络丛中分离和表征免疫细胞
Summary
本研究使用流式细胞术和两种不同的门控策略对分离的灌注小鼠脑脉络丛;该协议确定了填充该大脑结构的主要免疫细胞亚群。
Abstract
大脑不再被视为孤立运作的器官;越来越多的证据表明，外周免疫系统的变化可以间接塑造大脑功能。在大脑和体循环之间的界面，构成血脑脊液屏障的脉络丛（CP）被强调为脑外交流的关键部位。脑脊髓质酸产生脑脊液、神经营养因子和信号分子，这些分子可以塑造大脑稳态。CP也是一个活跃的免疫学利基。与在生理条件下主要由小胶质细胞填充的脑实质相反，CP免疫细胞的异质性概括了在其他外周器官中发现的多样性。脑瘫免疫细胞的多样性和活性随衰老、应激和疾病而变化，调节脑瘫上皮的活性，从而间接塑造大脑功能。该协议的目标是分离小鼠CP并确定填充它们的主要免疫亚群的约90%。该方法是表征CP免疫细胞并了解它们在协调外围到大脑通信中的功能的工具。拟议的方案可能有助于破译CP免疫细胞如何在健康和各种疾病条件下间接调节大脑功能。
Introduction
自从保罗·埃尔利希（Paul Erhlich）在19世纪末 发现血脑屏障以来，大脑一直被认为与其他器官和血液几乎分离。然而，在过去的十年中，出现了大脑功能由各种生物因素塑造的概念，例如肠道微生物群和全身免疫细胞和信号1，2，3，4。与此同时，其他脑边界，如脑膜和脉络丛（CP）已被确定为活动免疫 – 脑串扰的界面，而不是惰性屏障组织5，6，7，8。
脑瘫构成血脑脊液屏障，是分隔大脑和外围的边界之一。它们位于大脑的四个心室中，即第三个、第四个和两个侧心室，并且与参与神经发生的区域（例如海马体的脑室下区和粒下区）相邻3。在结构上，CP由由单层上皮细胞包围的开窗状毛细血管网络组成，这些细胞通过紧密和粘附连接相互连接9，10。脑瘫上皮的主要生理作用涉及脑脊液的产生，脑脊液从废物代谢物和蛋白质聚集体中冲洗大脑，以及产生和控制各种信号分子的血脑通道，包括激素和神经营养因子11，12，13。来自CP的分泌分子通过调节神经发生和小胶质细胞功能来塑造大脑的活动14，15，16，17，18，19，这使得CP对大脑稳态至关重要。CP还从事各种免疫活动;虽然在非病理条件下脑实质中的主要免疫细胞类型是小胶质细胞，但CP免疫细胞群的多样性与外周器官一样广泛3，7，这表明免疫调节和信号传导的各种通道在CP中起作用。
内皮细胞和上皮细胞之间的空间，CP基质，主要由边界相关巨噬细胞（BAM）填充，其表达促炎细胞因子和与抗原呈递相关的分子，以响应炎症信号3。巨噬细胞的另一种亚型，Kolmer’s epiplexus细胞，存在于CP上皮的顶端表面20。CP基质也是树突状细胞，B细胞，肥大细胞，嗜碱性粒细胞，嗜中性粒细胞，先天淋巴细胞和T细胞的利基，这些细胞主要是能够识别中枢神经系统抗原的效应记忆T细胞7，21，22，23，24。此外，CP处免疫细胞群的组成和活性会随着全身或脑部的扰动而变化，例如在衰老过程中10，14，15，21，25，微生物群扰动7，stress26和疾病27，28。值得注意的是，这些变化被认为间接塑造了大脑功能，即CP CD4 + T细胞向Th2炎症的转变发生在大脑衰老中，并触发CP的免疫信号传导，可能塑造衰老相关的认知能力下降14，15，21，25，29.因此，阐明CP免疫细胞的特性对于更好地了解它们对CP上皮生理和分泌的调节功能至关重要，从而破译它们在健康和疾病条件下对大脑功能的间接影响。
脑瘫是仅包含少数免疫细胞的小结构。它们的隔离需要在灌注的初步步骤后进行显微解剖;否则，血液中的免疫细胞将构成主要污染物。该协议旨在使用流式细胞术表征CP的髓细胞和T细胞亚群。该方法鉴定了在非炎性条件下构成小鼠CP的约90%的免疫细胞群，根据最近发表的使用其他方法解剖免疫CP异质性的工作7，10，28。该方案可用于表征CP免疫细胞区室中疾病的变化以及 体内其他实验范式。
Protocol
所有程序均符合欧盟委员会关于处理实验动物的指南，指令86/609/EEC。它们由第59号伦理委员会批准，由CETEA / CEEA第089号批准，编号为dap210067和APAFIS #32382-2021070917055505 v1。 1. 材料的准备 将所有抗体（材料表）储存在4°C，避免光照。 DAPI储备溶液（1mg / mL）：将粉末重悬于PBS-/- （材料表），等分试样，并储存在-20°C。…
Representative Results
这里介绍的流式细胞术分析以高度可重复的方式成功地揭示了髓细胞和T细胞的主要亚群（分别为图1和图2），以及它们每只小鼠的相对总数（图3）。 对骨髓细胞的流式细胞术分析表明，CP由CD11b + CX3CR1 + F4 / 80high BAM填充，占CP处CD45 +免疫细胞的近80%。这些BAM根据其MHC-II…
Discussion
旨在了解对大脑稳态和疾病的免疫学贡献的研究主要集中在脑实质内的细胞上，而忽略了脑边界，如CP，但这些边界仍然是大脑功能的关键贡献者2，3。由于CP的小尺寸，驻留免疫细胞的数量少以及访问这种组织的复杂，CP免疫细胞群的分析具有挑战性。对全脑免疫细胞（CD45 +）进行的流式细胞术不允许表征驻留在CP中的罕见免疫群体。为了高精?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢巴斯德动物研究所和CB-UTechS设施成员的帮助。这项工作得到了巴斯德研究所的财政支持。
Materials
| anti-mouse CD16/CD32 | BD Biosciences | 553142 | Flow cytometry antibody |
| Albumin, bovine | MP Biomedicals | 160069 | Blocking reagent |
| APC anti-mouse CX3CR1 | BioLegend | 149008 | Flow cytometry antibody |
| APC anti-mouse TCRb | BioLegend | 109212 | Flow cytometry antibody |
| APC-Cy7 anti-mouse CD4 | BioLegend | 100414 | Flow cytometry antibody |
| APC-Cy7 anti-mouse IA-IE | BioLegend | 107628 | Flow cytometry antibody |
| BD FACSymphony A5 Cell Analyzer | BD Biosciences | Flow cytometry analyzer | |
| BV711 anti-mouse Ly6C | BioLegend | 128037 | Flow cytometry antibody |
| Collagenase IV | Gibco | 17104-019 | Enzyme to dissociate CP tissue |
| DAPI | Thermo Scientific | 62248 | Live/dead marker |
| EDTA | Ion chelator | ||
| fine scissors | FST | 14058-11 | Dissection tool |
| FITC anti-mouse CD45 | BioLegend | 103108 | Flow cytometry antibody |
| Flow controller infusion inset | CareFusion | RG-3-C | Blood perfusion inset |
| FlowJo software | BD Biosciences | Analysis software | |
| forceps | FST | 11018-12 | Dissection tool |
| Heparin | Sigma-Aldrich | H3149-10KU | Anticoagulant |
| Imalgene | Boehringer Ingelheim | Ketamine, anesthesic | |
| OneComp eBeads | Invitrogen | 01-1111-42 | Control beads to realize compensation |
| PBS-/- | Gibco | 14190-094 | Buffer |
| PBS+/+ | Gibco | 14040-091 | Buffer |
| PE anti-mouse CD8a | BioLegend | 100708 | Flow cytometry antibody |
| PE anti-mouse F4/80 | BioLegend | 123110 | Flow cytometry antibody |
| PE-Dazzle 594 anti-mouse CD11b | BioLegend | 101256 | Flow cytometry antibody |
| Rompun | Bayer | Xylazine, anesthesic | |
| thin forceps | Dumoxel Biology | 11242-40 | Dissection tool |
| Vetergesic | Ceva | Buprenorphin, analgesic |
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Dominguez-Belloso, A., Schmutz, S., Novault, S., Travier, L., Deczkowska, A. Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses. J. Vis. Exp. (180), e63487, doi:10.3791/63487 (2022).