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Neuroscience

来自成人大脑的短期自由浮动切片培养

Published: November 05, 2019
doi:
10.3791/59845
, , , , , , , , ,
1Department of Biochemistry and Immunology, Ribeirão Preto Medical School,University of São Paulo, 2Department of Physiology, Ribeirão Preto Medical School,University of São Paulo, 3Department of Pathology and Forensic Medicine, Ribeirão Preto Medical School,University of São Paulo, 4Department of Anatomy, Institute of Biosciences,São Paulo State University, 5Clinical Hospital at the Ribeirão Preto Medical School,University of São Paulo

Summary

提出了一种从成人大脑中制备自由浮动切片培养物的协议。该协议是使用膜插入物广泛使用的切片培养方法的变体。它简单，经济高效，建议进行短期检测，旨在解开与年龄相关的脑疾病背后的神经退化机制。

Abstract

组织，或切片培养，已被广泛用于模拟中枢神经系统在体外功能的各个方面。尽管在神经科学中切片培养物的潜力，使用成人神经组织来制备这种培养物的研究仍然很少，尤其是那些来自人类受试者的研究。使用成人人体组织来制备切片培养物对增进人类神经病变的理解特别有吸引力，因为它们具有独特的特性，这些特性是典型的成熟人类大脑，缺乏由啮齿动物（通常是新生儿）生产的切片。神经组织。该协议描述了如何使用从活体人类捐赠者那里收集的脑组织提交到切除脑外科，以准备短期的，自由浮动的切片培养物。还介绍了使用这些培养物维护和执行生化和细胞生物学测定的程序。代表性结果表明，典型的人体皮质层压在体外4天（DIV4）后保存在切片中，预期存在主要神经细胞类型。此外，DIV4的切片在受到毒性刺激（H2O2）的挑战时会经历强细胞死亡，这表明该模型有可能成为细胞死亡检测的平台。该方法是使用膜插入物广泛使用的协议的更简单、经济有效的替代方法，主要推荐用于运行短期检测，旨在解开与年龄相关的脑疾病背后的神经退化机制。最后，虽然该协议专门用于使用从患者那里收集的皮质组织，这些患者接受药物治疗，但认为从其他大脑区域/条件收集的组织也应被视为生成类似自由浮动切片区域性的源。

Introduction

在研究中使用人类样本无疑是研究人类大脑病理学的绝佳选择，现代技术为使用患者衍生组织进行稳健和合乎道德的实验开辟了新途径。从成人人脑制备的有机体/切片培养物等方法已越来越多地用于光遗传学1、电生理学2、3、4、5、可塑性等范式中。6，7，8，9，神经毒性/神经保护10，11，12，13，细胞治疗14，药物筛选15，16，17， 遗传学和基因编辑12，18，19，20，等等， 作为一个策略，以更好的了解成年后的神经疾病。

理解人类大脑病变背后的机制取决于需要大量的实验策略。相反，在切片培养的情况下，虽然获取人类样本仍然很困难，但从单个皮质样本中生成多达 50 个切片的可能性部分地规避了通过增加每个收集的组织复制和进行测定的数量21。

几个方案的大脑器官/切片培养已经描述，从经典的oculo拔模22，23到辊管24，25，26，半渗透膜接口27，28，29，30和自由浮动切片31，32。根据实验设计的特殊性，每种技术都有其优点和缺点。从成人大脑短期的，自由浮动的切片培养在某些情况下比Stoppini等人使用的方法27有利，如果考虑到事实，虽然长期细胞在体外生存通常是一个主要的关注，在评估一种文化方法，在许多实验中只需要很短的时间在文化需要12，31，32，33，34，35。在这些条件下，使用自由浮动培养物的优点是更简单、更具成本效益，并且比在培养中保存的切片在 2-3 周内更精确地类似于原始人体组织状况。

尽管切片培养对神经科学有潜力，但利用成人神经组织来制备这种培养物的研究仍然很少，尤其是来自人类的研究。本文介绍了一种协议，使用从活体人类捐赠者那里收集的脑组织提交到切除脑外科，准备自由浮动切片培养物。使用这些培养物维护和执行生化和细胞生物学测定的程序是详细的。该协议已被证明对分析与成年期相关的神经病变机制的生存能力和神经元功能有价值。

Protocol

活成人脑组织是从接受切除神经外科治疗药理性叶癫痫的患者身上获得的（图1A）。所有程序都由里贝里诺普雷托医学院诊所医院（17578/2015）的伦理委员会批准，患者（或其法律责任人员）同意并签署了知情同意条款。组织采集工作由巴西圣保罗大学里贝里奥普雷托医学院的神经外科小组进行。 1. 材料灭菌 注：所有材料…

Representative Results

评估培养切片的质量和健康的一个关键方面是预期神经细胞类型、神经元和胶质细胞的存在和典型形态。在DIV4的切片中观察到人类皮质层压的典型结构，通过神经元免疫标记（图2D）显示。此外，还观察到了微胶质和星体的预期存在（图2B，C）。这些结果表明，组织结构没有受到外科手术/样品处理或体外短期期的显著影响。?…

Discussion

此协议用于生产自由浮动的短期切片培养法是培养成年人类新皮质切片的替代方法。这种切片培养方案可用于研究（但不限于）光遗传学1，44，45，电生理学2，3，4，5，短期可塑性46，47，</s…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到FAPESP（25681-3/2017年授予AS）、CAPES（博士后研究金PNPD/INCT-HSM到A.F.和博士前奖学金到N.D.M.）和FAEPA的支持。G.M.A. 持有 FAPESP （MS 2018/06614-4） 的硕士奖学金。N.G.C. 持有 CNPq 研究奖学金。我们感谢患者及其家属为这项研究捐赠被切除的纸巾。我们要感谢圣保罗大学里贝里奥普雷托医学院临床医院的居民、护士、技术人员和CIREP团队的支持，他们帮助参与了这一进程的各个阶段。

Materials

2-Propanol Merck 1096341000
Acrylamide/Bis-Acrylamide, 30% solution Sigma Aldrich A3449 
Agarose Sigma Aldrich A9539
Ammonium persulfate Sigma A3678-25G
Amphotericin B Gibco 15290-018
Antibody anti-ERK 2 (rabbit) Santa Cruz Biotecnology sc-154 Dilution 1:1,000 in BSA 2.5%
Antibody anti-pERK (mouse) Santa Cruz Biotecnology sc-7383 Dilution 1:1,000 in BSA 2.5%
B27 Gibco 17504-044
BDNF Sigma Aldrich SRP3014
Bovine Serum Albumin Sigma Aldrich A7906
Bradford 1x Dye Reagent BioRad 500-0205
EDTA Sigma T3924 Used in RIPA buffer
Glucose Merck 108337
Glutamax Gibco 35050-061
Hank's Balanced Salts Sigma Aldrich H1387-10X1L
Hepes Sigma Aldrich H4034
Hydrochloric acid Merck 1003171000
Hydrogen Peroxide (H2O2) Vetec 194
Mouse IgG, HRP-linked whole Ab (anti-mouse) GE NA931-1ML
NaCl Merck 1064041000 Used in RIPA buffer
Neurobasal A Gibco 10888-022
Non-fat dry milk (Molico) Nestlé Used for membrane blocking
PBS Buffer pH 7,2 Laborclin 590338
Penicilin/Streptomicin Sigma Aldrich P4333
Potassium Chloride Merck 1049361000
Prime Western Blotting Detection Reagent GE RPN2232
Rabbit IgG, HRP-linked whole Ab (anti-rabbit) GE NA934-1ML
SDS Sigma L5750 Used in RIPA buffer
TEMED GE 17-1312-01
Thiazolyl Blue Tetrazolium Bromide (MTT) Sigma Aldrich M5655
Tris Sigma T-1378 Used in RIPA buffer
Triton x-100 Sigma X100 Used in RIPA buffer
Ultrapure Water Millipore Sterile water, derived from MiliQ water purification system
Equipment and Material
24-well plates Corning CL S3526 Flat Bottom with Lid
Amersham Potran Premium (nitrocellulose membrane)  GE 29047575
Carbogen Mixture White Martins 95% O2, 5% CO2
CO2 incubator New Brunswick Scientific CO-24 Incubation of slices 5% CO2, 36ºC
Microplate Reader Molecular Devices
Microtubes Greiner 001608 1,5mL microtube
Motorized pestle Kimble Chase
Plastic spoon Size of a dessert spoon
Razor Blade Bic Chrome Platinum, used in slicing with vibratome
Scalpel Blade Becton Dickinson (BD) Number 24 Used for slicing of tissue; recommended same size or smaller
Superglue (Loctite Super Bonder) Henkel Composition: Etilcianoacrilato; 2-Propenoic acid; 6,6'-di-terc-butil-2,2'-metilenodi-p-cresol; homopolymer
Vibratome  Leica 14047235612 – VT1000S
Name of Material/ Equipment for Immunohistochemistry
Antibody anti-NeuN (mouse) Millipore  MAB377 Dilution 1:1,000 in Phosphate Buffer
Antibody anti-GFAP (mouse) Merck MAB360 Dilution 1:1,000 in Phosphate Buffer
Antibody anti-Iba1 (rabbit) Abcam EPR16588 – ab178846 Dilution 1:2,000 in Phosphate Buffer
Biotinylated anti-mouse IgG Antibody (H+L) Vector BA-9200
DAB Sigma Aldrich D-9015
Entellan Merck 107960
Ethanol Merck 1.00983.1000
Gelatin Synth 00G1002.02.AE Used for coating slides
Microtome Leica SM2010R Equipped with Freezing Stage (BFS-10MP, Physiotemp), set to -40ºC
Normal Donkey Serum Jackson Immuno Research 017-000-121
Paraformaldehyde Sigma Aldrich 158127
Rabbit IgG, HRP-linked whole Ab (anti-rabbit) GE NA934-1ML
Slides (Star Frost) Knittel Glaser Gelatin coated slides
Sucrose Vetec 60REAVET017050
Vectastain ABC HRP Kit (Peroxidase, Standard) Vector PK-4000, Kit Standard
Xylene Synth 01X1001.01.BJ
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Brain Slice CultureFree-floating Slice CultureBrain SurgeryNeurodegenerationCell BiologyImmunohistochemistryVibratomeAgaroseSlicingTransportCarbogen

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Fernandes, A., Mendes, N. D., Almeida, G. M., Nogueira, G. O., Machado, C. d. M., Horta-Junior, J. d. A. d. C., Assirati Junior, J. A., Garcia-Cairasco, N., Neder, L., Sebollela, A. Short-Term Free-Floating Slice Cultures from the Adult Human Brain. J. Vis. Exp. (153), e59845, doi:10.3791/59845 (2019).
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