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Abstract
Introduction
Protocol
Results
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신경과학

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

Published: August 27, 2017
doi:
10.3791/55359
, , ,
1Department of Anatomy and Cell Biology,Martin Luther University Halle-Wittenberg, 2Department of Anatomy,University of Otago

Summary

器官海马切片培养 (OHSC) 代表一个体外模型, 它能很好地模拟体内的情况。在这里, 我们描述一个 vibratome-based 改进的切片协议, 以获得高质量的切片, 用于评估新物质的神经保护潜能或肿瘤细胞的生物学行为。

Abstract

在器官海马切片培养 (OHSC) 中, 神经元和胶质细胞的形态和功能特征得到了很好的保存。该模型适用于处理不同的研究问题, 涉及神经保护、神经元电生理实验、神经元网络或肿瘤侵袭等研究。multisynaptic 回路中的海马结构和神经元活动在 OHSC 中很好保存, 尽管切片程序本身最初会损伤并导致胶质瘢痕的形成。瘢痕的形成可能改变了小分子的力学性质和扩散行为,等。切片允许在没有动物手术的情况下监测脑损伤后的时间依赖性过程, 并研究各种脑源性细胞类型的相互作用, 即在生理和病理上条件.这个模型的一个矛盾的方面是缺乏血流和免疫血细胞。在神经元损伤过程中, 从血液中迁移免疫细胞起着重要作用。当这些细胞在切片中丢失时, 就可以观察到文化中的内在过程, 而不受外界干扰。此外, 在 OHSC 中, 中外部环境的组成是精确控制的。这个方法的一个进一步好处是被牺牲的动物的数量比标准准备更低。几个 OHSC 可以获得从一个动物做同时研究与多个治疗在一个动物可能。由于这些原因, OHSC 很适合分析新的保护疗法在组织损伤或肿瘤侵袭过程中的作用。

这里介绍的协议描述了一种 OHSC 的制备方法, 它允许产生高重复性的、保存完好的切片, 可用于各种实验研究, 如神经保护或肿瘤侵袭研究。

Introduction

OHSC 是一个良好的体外模型, 研究神经元、星形胶质细胞和小鼠的生理和病理性质,1。对细胞外环境进行控制, 对各种刺激后的细胞学和形态学变化进行监测是很容易的。在准备2,3之后, 海马神经元的组织及其连接保存完好。在一些优势, OHSC 允许监测脑损伤和肿瘤侵袭没有动物手术。六至八 OHSC 可从单个啮齿动物的大脑中获得。因此, OHSC 有助于显著减少动物数量, 并允许在同一动物体内检测多种药物浓度、基因操纵或不同的损伤模型。在 slice-based 测定中, 可以精确控制实验条件。此外, 时间依赖性发展的病理条件, 如次生损害, 可以很容易地监测的延时成像。

在给定的协议中, 最初由 Stoppini et al.建立4, 描述了准备步骤, 并突出显示了选择适当切片的重要形态学标志。我们建议在后天准备7-9 大鼠或产后日4-5 只小鼠。在这些时期, OHSC 表现出对机械创伤的强大抵抗力和神经元回路重组的高电位。相比之下, 从胚胎或成年大鼠的准备工作迅速改变其结构, 并失去其器官形态学在种植期间, 因此更不适合研究长期的过程中的基础研究5,6,7,8,9,10,11. OHSC 生存率的另一个关键点是切片本身的厚度, 作为扩散, 因而养分供应是有限的12,13,14。

Protocol

动物实验是按照欧洲共同体理事会 2010/63/欧共体欧洲议会指令批准的《关于在神经科学研究中使用动物的政策和政策》进行的。欧洲联盟理事会关于保护用于科学目的的动物的问题. 1. 准备仪器和培养基 准备 OHSC 使用以下工具集: 两个小剪刀, 两个弯曲的镊子, 一个镊子与罚款提示, 三刀片 (两个大小 11, 一个大小 15),三手术刀夹, 圆形滤纸 (直径:35 毫米), 琼脂, 一刀?…

Representative Results

神经保护研究:为了确定神经元损伤, 计数的 PI 阳性核和 IB4阳性小胶质细胞在每三个光学部分的颗粒胞层 (协鑫) 的齿状回 (DG) 的数量。对于肿瘤侵袭实验, 采用最大强度 z 投影法计算肿瘤细胞覆盖面积, 作为入侵的测量手段, 并对不同的入侵模式进行可视化 (图 6)。 在未经治疗的控制 O…

Discussion

本议定书描述了 OHSC 的准备工作。这个模型允许测试的内在能力和脑组织的反应后, 应用的生理和病理刺激。除了对电生理参数的分析外, OHSC 可以损伤, 并可确定损伤对所有细胞类型的影响。治疗与不同的物质和详细描述毁损过程或愈合在没有巨噬细胞和淋巴细胞是可能的。

成功的准备和培养的最关键的步骤是: 1) 在无菌条件下工作, 2) 正确准备介质, 考虑温度和 pH 值, 和 3) ?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者想感谢克里斯汀· Auste 的支持, 她的视频录音和 Chalid Ghadban 为他的出色技术援助。Urszula Grabiec 是支持 FKZ 29/18。

Materials

6-Well Falcon 35-3046
Agar Fluka 5040
Autoclav Systec DX-45
CFDA  Thermo Fisher V12883
Confocal laser scanning microscope (CLSM) LSM700 Carl Zeiss
Eagle´s Minimal Essential Medium  Invitrogen 32360-034
Fluorescein labeled Griffonia (Bandeiraea) Simplicifolia Lectin I Vector Labs FL-1101
Fluorescein labeled GSL I – isolectin B4 Vector Labs FL-1201  
Glucose Merk 1083371000
Glutamin Invitrogen 25030-024
Hank´s Balanced Salt Solution (with Ca2+ and Mg2+) Invitrogen 24020-133
Hank´s Balanced Salt Solution (without Ca2+  and Mg2+) Invitrogen 14170-138
Insulin Sigma Aldrich I5500
L-ascorbic acid Sigma Aldrich A5960
L-Glutamin Invitrogen 25030-024
LN229 Cell-Lines-Service 300363
Medical cyanoacrylate glue (Histoacryl glue) B.Braun 1050052
Millicell Culture Inserts Millipore PICMORG50
NMDA N-methyl-D-aspartic acid Sigma Aldrich M3262
Normal Horse Seum Invitrogen 26050-088
Penicillin Streptomycin Invitrogen 15140-122
Petri dishes (all sizes) Greiner 627160/664160/628160
PFA Roth 0335.1 toxic
Propidium iodid (PI) Sigma Aldrich 81845-25MG toxic
U138 ATCC HTB-14
Vibratome Leica Leica VT 1200
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Tags

Organotypic Hippocampal Slice CulturesNeuroprotectionTumor Cell InvasivenessVibratomeAgar BlocksPreparation MediumCytoarchitectureCell Culture InsertCulture MediumInflammatory ReactionsNeuroprotection Studies
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Grabiec, U., Hohmann, T., Hammer, N., Dehghani, F. Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells. J. Vis. Exp. (126), e55359, doi:10.3791/55359 (2017).
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