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Neurosciences

脑室微注射脂多糖到斑马鱼幼虫中以评估神经炎症和神经毒性

Published: August 23, 2022
doi:
10.3791/64313
,
1State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences,University of Macau, Avenida da Universidade, 2Department of Pharmaceutical Sciences, Faculty of Health Sciences,University of Macau, Avenida da Universidade

Summary

该协议展示了在斑马鱼幼虫模型中将脂多糖显微注射到脑室区域，以研究由此产生的神经炎症反应和神经毒性。

Abstract

神经炎症是各种神经系统疾病的关键因素，包括神经退行性疾病。因此，研究和开发替代 体内 神经炎症模型以了解神经炎症在神经变性中的作用具有极大的意义。本研究开发并验证了通过心室显微注射脂多糖（LPS）介导的神经炎症幼虫斑马鱼模型，以诱导免疫反应和神经毒性。采用转基因斑马鱼品系elavl3：mCherry、ETvmat2：GFP和mpo：EGFP通过荧光实时成像结合荧光强度分析实时定量脑神经元活力。使用视频跟踪记录器自动记录斑马鱼幼虫的运动行为。研究一氧化氮（NO）含量和白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）和人肿瘤坏死因子α（TNF-α）等炎性细胞因子的mRNA表达水平，以评估斑马鱼幼虫头部LPS诱导的免疫应答。脑室注射LPS后24 h，斑马鱼幼虫出现神经元丧失和运动缺陷。此外，LPS诱导的神经炎症增加了受精后6天斑马鱼幼虫头部NO释放和IL-6、IL-1β和TNF-α的mRNA表达，导致斑马鱼脑中中性粒细胞的募集。在这项研究中，注射浓度为2.5-5mg / mL的斑马鱼，浓度为5dpf，是该药理学神经炎症测定的最佳条件。该协议提供了一种新的，快速有效的LPS脑室显微注射方法，以诱导斑马鱼幼虫中LPS介导的神经炎症和神经毒性，这对于研究神经炎症很有用，也可以用作高通量 体内 药物筛选测定。

Introduction

神经炎症已被描述为参与中枢神经系统（CNS）几种神经退行性疾病发病机制的关键抗神经源性因素1。病理损伤后，神经炎症可能导致各种不良后果，包括抑制神经发生和诱导神经元细胞死亡2，3。在炎症诱导反应的基础过程中，多种炎症细胞因子（如TNF-α，IL-1β和IL-6）分泌到细胞外空间，并作为神经元死亡和抑制神经发生的关键成分4，5，6。

将炎症介质（如IL-1β、L-精氨酸和内毒素）显微注射到脑中可引起神经元细胞减少和神经炎症7，8，9。脂多糖（LPS，图1）是存在于革兰氏阴性细菌细胞壁中的致病性内毒素，可诱导神经炎症，加剧神经变性，并减少动物的神经发生10。LPS直接注射到小鼠大脑的CNS中，增加了一氧化氮，促炎细胞因子和其他调节剂的水平11。此外，将LPS立体定向注射到局部脑环境中可诱导神经毒性分子的过量产生，导致神经元功能受损并随后发展为神经退行性疾病10，12，13，14，15。在神经科学领域，对生物体中细胞和生物过程的实时和时程显微镜观察对于理解发病机制和药理作用至关重要16。然而，神经炎症和神经毒性小鼠模型的实时成像从根本上受到显微镜有限的光学穿透深度的限制，这排除了功能成像和对发育过程的实时观察17，18，19。因此，开发替代神经炎症模型对于促进通过活体成像研究病理发展以及神经炎症和神经变性的潜在机制具有重要意义。

斑马鱼（Danio rerio）由于其进化上保守的先天免疫系统，光学透明度，大胚胎离合器尺寸，遗传可追踪性和体内成像的适用性，已成为研究神经炎症和神经变性的有前途的模型19，20，21，22，23.以前的方案要么在没有机制评估的情况下直接将LPS注射到斑马鱼幼虫的卵黄和后脑室中，要么简单地将LPS添加到鱼水（培养基）中以诱导致命的全身免疫反应24，25，26，27。在此，我们开发了一种将LPS显微注射到脑室的方案，以在受精后5天（dpf）斑马鱼幼虫中触发先天免疫反应或神经毒性。这种反应通过神经元细胞丢失，运动行为缺陷，亚硝酸盐氧化物释放增加，炎症基因表达的激活以及注射后24小时斑马鱼大脑中嗜中性粒细胞的募集来证明。

Protocol

AB野生型斑马鱼和转基因斑马鱼系elavl3：mCherry，ETvmat2：GFP和mpo：EGFP来自中国医学研究所（ICMS）。澳门大学动物研究伦理委员会授予动物实验的伦理批准（UMARE-030-2017），该协议遵循机构动物护理指南。 1. 斑马鱼胚胎和幼虫饲养 通过自然配对交配产生斑马鱼胚胎（每次交配 200-300 个胚胎），如之前报道的那样28. 通过将 34.8 克 NaCl?…

Representative Results

这里描述的工作流程提供了一种新的、快速的、有效的方法，用于诱导斑马鱼幼虫中LPS介导的神经炎症和神经毒性。在该描述的方案中，使用显微注射器将5 dpf斑马鱼用LPS（图1）注射到脑室中（图2A-C）。使用1%埃文斯蓝色染色剂验证成功注射到脑室部位（图2D）。使用注射器将斑马鱼头与其眼睛和身体分?…

Discussion

越来越多的流行病学和实验数据表明，慢性细菌和病毒感染是神经退行性疾病的可能危险因素36.感染触发炎症过程和宿主免疫反应的激活37。即使反应作为一种防御机制，过度激活的炎症也不利于神经发生，炎症环境不允许新生神经元存活38。结果，它会损害宿主神经元的功能和活力。研究表明，炎症在神经变性的病理生理学中起重要作用<sup c…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项研究获澳门特别行政区科学技术发展基金（参考编号）资助。FDCT0058/2019/A1和0016/2019/AKP），澳门大学研究委员会（MYRG2020-00183-ICMS和CPG2022-00023-ICMS）和中国国家自然科学基金（第81803398号）。

Materials

Agarose Sigma-Aldrich A6361
Agarose, low gelling temperature Sigma-Aldrich A9414
Drummond Nanoject III Programmable Nanoliter Injector Drummond Scientific 3-000-207
Fluorescence stereo microscopes Leica M205 FA
GraphPad Prism software GraphPad Software Ver. 7.04
Lipopolysaccharides from Escherichia coli O111:B4 Sigma-Aldrich L3024
Manual micromanipulator World Precision Instruments M3301
Mineral oil Sigma-Aldrich M5904
Mx3005P qPCR system Agilent Technologies Mx3005P
Nanovue plus spectrophotometer Biochrom 80-2140-46
Nitrite concentration assay kit Beyotime Biotechnology S0021M
Phosphate-buffered saline Sigma-Aldrich P4417
Programmable Horizontal Pipette Puller World Precision Instruments PMP-102
PTU (N-Phenylthiourea) Sigma-Aldrich P7629
Random primers Takara 3802
SuperScript II Reverse Transcriptase Invitrogen 18064014
SYBR Premix Ex Taq II kit Accurate Biology AG11701
The 3rd Gen Tgrinder Tiangen OSE-Y30
Thin wall glass capillaries (4”) with filament, OD 1.5 mm World Precision Instruments TW150F-4
Tricaine (3-amino benzoic acid ethyl ester) Sigma-Aldrich A-5040
TRNzol Universal reagent Tiangen DP424
Zebrafish tracking box ViewPoint Behavior Technology
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Tags

Keywords: Brain VentricleMicroinjectionLipopolysaccharideNeuroinflammationNeurotoxicityZebrafishIn Vivo ModelAnti-neuroinflammatory DrugsGlass CapillaryMicromanipulatorAgaroseAnesthesia
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He, Y., Lee, S. M. Y. Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity. J. Vis. Exp. (186), e64313, doi:10.3791/64313 (2022).
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