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Introduction
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Results
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Immunology and Infection

分离中枢神经系统组织和相关脑膜，用于免疫细胞的下游分析

Published: May 19, 2020
doi:
10.3791/61166
, , , , ,
1Department of Neurology, Geisel School of Medicine,Dartmouth-Hitchcock Medical Center, 2Program in Experimental and Molecular Medicine,Dartmouth College

Summary

本文提出了两种优化的方案，用于检查中枢神经系统内的常驻和外周来源的免疫细胞，包括大脑、脊髓和脑膜。这些协议中的每一个都有助于确定在稳态和炎症条件下占据这些隔室的细胞的功能和组成。

Abstract

中枢神经系统（CNS）由大脑和脊髓组成，被脑膜包裹，膜层是外围和CNS之间的屏障。中枢神经系统是一个免疫学特化的部位，在稳态条件下，免疫特权在中枢神经系统实质中最为明显。相比之下，脑膜含有多种常驻细胞，包括先天性和适应性免疫细胞。在由中枢神经系统损伤、自身免疫、感染甚至神经变性引发的炎症性疾病中，外周来源的免疫细胞可能会进入脑实质并居住在脑膜内。这些细胞被认为在中枢神经系统疾病发病过程中执行有益和有害作用。尽管有这些知识，但在分析中枢神经系统隔室时，脑膜经常被忽视，因为传统的中枢神经系统组织提取方法省略了脑膜层。该协议提出了两种不同的方法，用于快速分离鼠中枢神经系统组织（即脑，脊髓和脑膜），适用于通过单细胞技术，免疫组织化学和原位杂交方法进行下游分析。所描述的方法提供了对CNS组织的全面分析，非常适合评估在稳态条件下和疾病发病期间占据CNS区室的细胞的表型，功能和定位。

Introduction

中枢神经系统（CNS）是一个免疫学专业化的部位。中枢神经系统实质（不包括脑脊液间隙、脑膜和脉管系统）通常被视为免疫特权部位 1,2,3,4,5，在稳态条件下相对缺乏免疫细胞 2,6,7。相比之下，由硬脑膜、蛛网膜和软脑膜层组成的脑膜是中枢神经系统隔室的重要组成部分，在疾病发病机制期间积极参与稳态免疫监视和炎症过程 3,6,7,8。在稳态条件下，脑膜支持许多免疫哨兵细胞，包括先天淋巴细胞（ILC），巨噬细胞，树突状细胞（DC），肥大细胞，T细胞，以及在较小程度上的B细胞9,10,11。

脑膜是高度血管化的结构，含有淋巴管，在中枢神经系统与其外围之间提供淋巴连接8,12,13,14。在由中枢神经系统损伤、感染、自身免疫甚至神经变性引起的炎症性疾病中，外周来源的免疫细胞会浸润脑实质并改变脑膜内的免疫景观。细胞浸润后，脑膜可能代表外周来源免疫细胞的功能生态位，促进免疫细胞聚集、局部免疫细胞活化和中枢神经系统区室的长期存活。在影响中枢神经系统的多种疾病中观察到明显的脑膜炎症，包括多发性硬化症 （MS）15,16,17,18,19、中风 20,21、无菌损伤 22,23（即脊髓损伤和创伤性脑损伤）、偏头痛 24 和微生物感染 25,26，27,28,29.因此，脑膜区室中常驻细胞和外周衍生免疫细胞的表征对于了解这些细胞在稳态条件和疾病发病机制中的作用至关重要。

从颅骨和椎体中提取大脑、脊髓和脑膜在技术上具有挑战性且耗时。目前还没有可用于快速提取大脑的技术，所有三个脑膜层都完好无损。虽然椎板切除术产生出色的脊髓组织形态并保留了脑膜层，但它既非常耗时又复杂30,31。相反，更传统的提取方法，例如从颅骨中取出大脑和脊髓的液压挤出有助于快速提取CNS组织，但蛛网膜和硬脑膜都因这些技术而丢失30,31。在脑和脊髓组织的常规分离过程中，硬脑膜和蛛网膜层的遗漏导致对中枢神经系统隔室内细胞的分析不完整。因此，确定专注于快速提取具有完整脑膜的CNS组织的新技术对于CNS室的最佳分析至关重要。

本手稿介绍了两种从小鼠中快速提取大脑、脊髓和脑膜的方法，有助于对中枢神经系统实质和脑膜中的常驻细胞和外周衍生免疫细胞进行下游分析。这些优化的方案侧重于 1） 分离单细胞悬液以进行下游分析和 2） 准备组织以进行组织学处理。从大脑、脊髓组织以及硬脑膜和蛛网膜脑膜32 获得单细胞悬浮液可以同时分析存在于实质和脑膜隔室中的细胞。单细胞悬浮液可用于不同的应用，包括用于体外刺激的细胞培养测定33、酶联免疫斑点（ELISpot）28、34、35、流式细胞术36、33和单细胞37或批量转录组学。此外，分别具有完整颅骨或椎体的整个大脑和脊髓脱钙的优化方案允许周围骨骼的温和脱钙，使脑膜完整并保留组织形态。该方法允许在实质和脑膜间隙内使用免疫组织化学（IHC）或原位杂交（ISH）技术选择性鉴定蛋白质或RNA。中枢神经系统内驻留细胞和外周来源免疫细胞的表型、活化状态和定位的表征可能为了解中枢神经系统区室中的单个细胞类型如何促进稳态和疾病发病机制提供必不可少的信息。

Protocol

所有动物工作都采用达特茅斯盖泽尔医学院机构动物护理和使用委员会（IACUC）审查和批准的协议。 1. 处理脑和脊髓样本进行脱钙 分离脑和脊髓样本 通过吸入CO2 对小鼠实施安乐死。确保 CO2 流速每分钟置换保持架容积的 10%–30%。 使用镊子提起剑突，并用剪刀在肋骨下方横向切割腹壁，向上拉以避免切割下面的血管或器官?…

Representative Results

该代表性实验旨在量化B和T细胞，并描述B和T细胞在稳态条件下脑膜和实质CNS区室以及小鼠进行性MS模型（即TMEV-IDD）中的定位。如前所述，TMEV-IDD通过5 x 106 斑块形成单位（PFU）的TMEV BeAn的颅内感染在5周龄雌性SJL小鼠中诱导29。 本研究评估了感染后第120天慢性TMEV-IDD期间脑膜，大脑和脊髓中的B和T细胞。使用年龄匹配的假处理小鼠作为对照。该研究由?…

Discussion

在稳态和疾病期间评估CNS区室细胞组成的方法对于了解CNS的生理和病理状态至关重要。然而，尽管是中枢神经系统的重要屏障并容纳多种免疫细胞，但脑膜经常被省略在分析中，因为许多传统的大脑和脊髓组织提取方法不允许收集这些膜。这种遗漏是我们了解脑膜细胞组成和功能及其在稳态和炎症条件下的作用的关键限制。最近的研究表明，驻留在脑膜隔室中的常驻和外周来源的免疫细胞在维持?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢达特茅斯比较医学和研究中心（CCMR）的工作人员对用于这些研究的小鼠的专业护理。伯恩斯坦研究基金资助了这项研究。

Materials

Aluminum foil any N/A
Bovine Serum Albumin ThermoFisher Scientific 37002D
Centrifuge Beckman Coulter Allegra X-12R centrifuge
Collagenase I Worthington LS004196
Conical tube, 15 mL VWR 525-1069
Conical tube, 50 mL VWR 89039-658
Cover glass Hauser Scientific 5000
Cryomold VWR 18000-128
Curved forceps Fine Science Tools 11003-14
Disposable polystyrene tube, 14 mL Fisher Scientific 14-959-1B
Disposable Scalpel Fisher Scientific NC0595256
DNAse I Worthington LS002139
Dry ice Airgas N/A
Durmont #7Forceps Fine Science Tools 11271-30
EDTA disodium salt dihydrate Amresco 0105-500g
Ethanol, 100% any N/A
Fetal Bovine Serum (FBS) Hyclone SH30910.03
Filter top tube, 5 mL VWR 352235
Fixable viability stain 780 Becton Dickinson 565388
Flow cytometer Beckman Coulter Gallios
Glucose Fisher Chemical D16-500
Goat anti-mouse IgG (488 conjugate) Jackson immunoresearch 115-546-146
Goat anti-mouse IgG (594 conjugate) Jackson immunoresearch 115-586-146
Goat anti-rabbit 488 Jackson immunoresearch 111-545-144
Goat anti-rat 594 Jackson immunoresearch 112-585-167
Goat anti-rat 650 Jackson immunoresearch 112-605-167
Hank's Balnced Salt Solution (HBSS) Corning 21-020-CV
Hemacytometer Andwin Scientific 02-671-51B
Hemostat Fine Science Tools 13004-14
HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) ThermoFisher Scientific 15630080
KCl Fisher chemical BP366-500
KH2PO4 (anhydrous) Sigma Aldrich P5655-100G
Liquid Nitrogen Airgas N/A
Mouse FC block (CD16/32) Becton Dickinson 553141
Na2HP04 (anhydrous) Fisher Chemical S374-500
NaCl Fisher chemical S671-500
Needle, 25 gauge Becton Dickinson 305122
Normal mouse serum ThermoFisher Scientific 31881
Nylon mesh strainer VWR 352350
OCT Sakura 4583
Paraformaldehyde, 20% Electron Microscopy Sciences 15713-S Diluted to 4% using 1 x PBS
Pasteur pipette, 9 inch, unplugged Fisher Scientific 13-678-20C
PBS (1x) Corning 21-040-CV
PE Rat Anti-Mouse CD4 Becton Dickinson 553730
PE-CF594 Rat Anti-Mouse CD19 Becton Dickinson 562329
Percoll density gradient media GE healthcare 17-0891-01
PerCP-Cy5.5 Rat Anti-Mouse CD45 Becton Dickinson 550994
Petri dish, 100 mm VWR 353003
pH meter Fisher Scientific 13-636-AB150
Pipet-Aid Drummond Scientific Corporation 4-000-101
Pipette 200 µl Gilson FA10005M
Pipette tips, 1 mL USA Scientific 1111-2831
Pipette tips, 200 µl USA Scientific 1111-1816
Pipette, 1 mL Gilson FA10006M
Prolong Diamond mountant with DAPI ThermoFisher Scientific P36962
Purified Rat Anti-Mouse CD16/CD32 Becton Dickinson 553141
Rabbit anti-mouse CD3 (SP7 clone) Abcam ab16669
Rabbit anti-mouse laminin Abcam ab11575
Rat anti-mouse ERT-R7 Abcam ab51824
RPMI 1640 Corning 10-040-CV
Serological pipet, 1 mL VWR 357521
Serological pipet, 10 mL VWR 357551
Serological pipet, 5 mL VWR 357543
Sodium hydroxide Fisher Scientific S318-100
Sucrose Fisher chemical S5-500
Surgical scissors Fine Science Tools 14001-16
Surgical scissors, extra fine Roboz RS-5882
Syringe, 10 mL Becton Dickinson 302995
Syringe, 5 mL Becton Dickinson 309646
Trypan blue Gibco 15250-061
Vacuum filter system Millipore 20207749
Vacuum flask Thomas Scientific 5340-2L
Vacuum in-line filter Pall Corporation 4402
Vacuum line Cole Palmer EW-06414-20
Water bath ThermoFisher Scientific Versa bath
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Tags

MeningesImmune CellsSingle Cell AnalysisHistological MethodsMicrogliaAstrocytesPericytesEndothelial CellsStromal CellsNeuronsBrainSpinal CordDecalcificationSkull CapVertebrae Column

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DiSano, K. D., Linzey, M. R., Welsh, N. C., Meier, J. S., Pachner, A. R., Gilli, F. Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells. J. Vis. Exp. (159), e61166, doi:10.3791/61166 (2020).
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