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Summary
Abstract
Introduction
Protocol
Résultats
Discussion
Divulgations
Acknowledgements
Materials
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Médecine

穆林·斯特拉特·甘利翁的位置、解剖和分析

Published: December 22, 2020
doi:
10.3791/62026
, , , , ,
1Division of Cardiology,EVK Düsseldorf, cNEP, cardiac Neuro- and Electrophysiology Research Consortium, 2DZHK (German Centre for Cardiovascular Research), 3Institute of Neural and Sensory Physiology, Medical Faculty,Heinrich Heine University Düsseldorf, 4Clinic for Cardiology, University Heart & Vascular Centre,University Hospital Hamburg-Eppendorf, 5Department of Cardiology,Leiden University Medical Center

Summary

心脏自主神经系统的病理生理变化，特别是其交感分支的病理生理变化，有助于心室心律失常的发病和维持。在本协议中，我们展示了如何描述粘膜结节，以增进对基础分子和细胞过程的理解。

Abstract

自主神经系统是心脏电生理学的一大驱动力。特别是其同情分支的作用是心室心律失常（VA）病理生理学中正在进行的研究。交感链的双子星形结构（SG）   中的神经元   是交感基础设施的重要组成部分。SG 是治疗难治 VA 患者通过心脏交感衰减而公认的治疗目标。虽然在SG的神经元重塑和胶质激活已经描述了VA患者，潜在的潜在的细胞和分子过程，潜在的心律失常开始之前，只是不够了解，应该阐明，以改善自主调节。小鼠模型允许我们研究交感神经元改造，但对缺乏经验的研究者来说，识别穆林SG是具有挑战性的。因此，许多常见的心脏病缺乏对穆林SG的深入细胞和分子生物学研究。在这里，我们描述了解剖和研究成年小鼠SG的基本剧目，用于RNA水平分析（基因表达分析的RNA分离，原位杂交）、蛋白质水平（免疫荧光全安装染色）和细胞水平（基本形态、细胞大小测量）。我们提出潜在的解决方案，以克服制备技术中的挑战，以及如何通过淬火自流来改善染色。这允许通过既定标记对神经元和胶质细胞进行可视化，以确定细胞组成和重塑过程。这里介绍的方法允许SG的特征获得关于容易发生VA的小鼠自主功能障碍的进一步信息，并可以辅之以额外的技术来研究心脏自主神经系统的神经元和胶质成分。

Introduction

心脏自主神经系统是一种受严格控制的交感、寄生和感官成分的平衡，使心脏能够适应环境变化，并具有适当的生理反应1，2。这种平衡的干扰，例如，同情活动的增加，已被确定为发病和维持心室心律失常（VA）3，4的关键驱动因素。因此，自主调制，通过药理学减少与β阻滞剂的交感活性，一直是治疗VA患者几十年的基石5，6。但是，尽管药理学和导管为基础的干预，相关数量的患者仍然患有复发性VA7。

对心脏的交感输入主要通过交感链的双星形结构——神经元细胞体进行中介，这种结构通过无数的胸内神经从脑干传递信息到心脏8、9、10。受伤后从SG发芽的神经与VA和心脏骤死11，12有关，强调SG作为自主调制的目标13，14。通过局部麻醉剂的皮下注射或通过视频辅助胸腔镜检查15、16部分切除SG，可以暂时减少对心脏的交感输入。心脏交感衰竭为治疗难治VA的患者提供了一个选择，结果有希望的结果14，16，17。我们从这些患者的外植SG中了解到，神经元和神经化学改造、神经炎症和胶质活化是同情性重塑的标志，可能有助于或加重自主功能障碍18，19。尽管如此，这些神经元中潜在的细胞和分子过程至今仍然模糊不清，例如，神经元分化成胆碱表型20，21的作用。实验研究提出了治疗VA的新方法，例如，通过光遗传学22减少交感神经活动，但SG的深度表征仍然缺乏许多心脏病理与VA齐头并进。模仿这些病理学的小鼠模型可以研究神经元重塑，这可能先于心律失常12，23的发生。这些可以通过进一步的形态和功能分析来完成，以实现心脏和神经系统的自主特征。在本协议中，我们提供了一系列基本的方法，允许解剖和描述 Murine SG，以增进对 VA 的理解。

Protocol

所有涉及动物的程序都得到汉堡州动物护理和使用委员会（ORG870，959）和北莱茵-威斯特法伦州自然、环境和消费者保护局（LANUV，07/11）的批准，并符合国家卫生研究院的实验室动物护理和使用指南（2011年）。研究使用男性和女性（10-24周）C57BL/6小鼠（库存号000664，杰克逊实验室）和小鼠同源性（db/db）或异质性（db/het：控制）为糖尿病自发突变（Leprdb：Bks.Cg-Dock7m+/+ 勒普db/J，股?…

Representative Results

图 1可视化如何识别和解剖 SG.图 1A显示了位置的示意图图，而图 1B在切除心肺包后将视图呈现到胸腔中。左和右长部的科利肌肉从 SG 和肋骨笼是方向的重要地标。解剖是沿着肌肉和第一根肋骨之间的虚线进行的。SG 和交感链变得可见为白色结构（图 1C）。图 1D显示了左长腹肌和第一根…

Discussion

对VA发病前交感神经系统神经元和胶质细胞的细胞和分子过程的理解是高度关注的，因为心脏骤停仍然是全世界最常见的死因。因此，在当前的手稿中，我们提供了一系列基本的方法来识别该网络中的穆林 SG – 喃喃元素 ， 并随后对 RNA、蛋白质和细胞水平进行分析。

Murine SG的一个挑战是它的大小和有限的细胞数量39。因此，不同的动物模型?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

作者要感谢哈特维格·维博尔德的出色技术援助，以及汉堡-埃彭多夫大学医学中心的UKE显微镜成像设施（Umif）提供显微镜和支持。这项研究由DZHK（德国心血管研究中心）[FKZ 81Z4710141]资助。

Materials

96-well plate TPP 92097 RNAscope
Adhesion Slides SuperFrost plus  25 x 75 x 1 mm R. Langenbrinck 03-0060 Microscopy
Albumin bovine Fraction V receptor grade lyophil. Serva 11924.03 Whole mount staining
bisBenzimide H33342 trihydrochloride (Hoechst) Sigma-Aldrich, St. Louis, MO, USA B2261 Whole mount staining
Chicken anti neurofilament EMD Millipore AB5539 Whole mount staining
Dimethyl sulfoxide (DMSO) Merck, KGA, Darmstadt, Germany D8418 Whole mount staining
Donkey anti chicken IgY Alexa 647  Merck, KGA, Darmstadt, Germany AP194SA6 Whole mount staining
Donkey anti goat IgG Alexa 568  Thermo Fisher Scientific A11057 Whole mount staining
Donkey anti rabbit IgG Alexa 488  Thermo Fisher Scientific A21206 Whole mount staining
Drying block 37-100 mm Whatman (Sigma Aldrich) WHA10310992  Whole mount staining
Eosin Y Sigma Aldrich E4009 Whole mount staining
Ethanol 99 % denatured with MEK, IPA and Bitrex (min. 99,8 %) Th.Geyer 2212.5000 Whole mount staining
Eukitt mounting medium AppliChem 253681.0008 Whole mount staining
Fluoromount-G Southern Biotech 0100-01 Whole mount staining
Fluoromount-G + DAPI Southern Biotech 0100-20 Whole mount staining
Goat anti choline acetyltransferase EMD Millipore AP144P Whole mount staining
H2O2 30% (w/w) Merck, KGA, Darmstadt, Germany H1009 Whole mount staining
Heparin Sodium 25.000 UI / 5ml Rotexmedica PZN: 3862340 Preparation SG
High-capacity cDNA reverse transctiption kit Life technologies  4368813 RNA isolation
Isoflurane (Forene) Abbott Laboratories 2594.00.00 Preparation SG
Mayer's hemalum solution Merck 1.09249.0500 Whole mount staining
Methanol Sigma-Aldrich 34860 Whole mount staining
Microscope cover glasses 20×20 mm or smaller Marienfeld 0101040 Whole mount staining
miRNeasy Mini Kit Qiagen 217004 RNA isolation
NanoDrop 2000c Thermo Fisher Scientific ND-2000C RNA isolation
Opal 570 Reagent Pack Akoya Bioscience FP1488001KT RNAscope
Paraformaldehyde, 16% w/v aq. soln., methanol free  Alfa Aesar 43368 Whole mount staining
Pasteur pipettes, LDPE, unsterile, 3 ml, 154 mm Th.Geyer 7691202 Whole mount staining
Phosphate-buffered saline tablets Gibco 18912-014 Whole mount staining
Pinzette Dumont SS Forceps FineScienceTools 11203-25 Preparation SG
QIAzol Lysis Reagent Qiagen  79306 RNA isolation
Rabbit anti tyrosine hydroxylase EMD Millipore AB152 Whole mount staining
RNAlater Merck R0901-100ML RNA isolation (optional)
RNAscope Multiplex Fluorescent Reagent Kit v2 biotechne (ACD) 323100 RNAscope
RNAscope Probe-Mm-S100b-C2 biotechne (ACD) 431738-C2 RNAscope
RNAscope Probe-Mm-Tubb3 biotechne (ACD) 423391 RNAscope
Stainless steel beads 7 mm  Qiagen  69990 RNA isolation
Sudan black B Roth 0292.2 Whole mount staining
TaqMan Gene Expression Assay Cdkn1b (Mm00438168_m1) Thermo Fisher Scientific 4331182 Gene expression analysis
TaqMan Gene Expression Assay Choline acetyltransferase (Mm01221880_m1) Thermo Fisher Scientific 4331182 Gene expression analysis
TaqMan Gene Expression Assay MKi67 (Mm01278617_m1) Thermo Fisher Scientific 4331182 Gene expression analysis
TaqMan Gene Expression Assay PTPCR (Mm01293577_m1) Thermo Fisher Scientific 4331182 Gene expression analysis
TaqMan Gene Expression Assay S100b (Mm00485897_m1) Thermo Fisher Scientific 4331182 Gene expression analysis
TaqMan Gene Expression Assay Tyrosin Hydroxylase (Mm00447557_m1) Thermo Fisher Scientific 4331182 Gene expression analysis
TaqMan mastermix Applied biosystems 4370074 Gene Expression analysis 
Tissue Lyser II Qiagen 85300 RNA isolation
Triton X-100 10% solution Sigma-Aldrich 93443-100ml Whole mount staining
Tween-20 Sigma-Aldrich P9416-100ML RNAscope
Wacom bamboo pen Wacom CTL-460/K Cell size measurements
Whatman prepleated qualitative filter paper, Grade 595 1/2 Sigma-Aldrich WHA10311647 Whole mount staining
Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate Thermo Fisher Scientific W21404 RNAscope
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Tags

Keywords: Stellate GanglionSympathetic Nervous SystemCardiac NeurocontrolCardiac ArrhythmogenesisTyrosine HydroxylaseLongus Colli MuscleParaformaldehydeSudan BlackDense BleachAntibody Permeabilization
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Scherschel, K., Bräuninger, H., Glufke, K., Jungen, C., Klöcker, N., Meyer, C. Location, Dissection, and Analysis of the Murine Stellate Ganglion. J. Vis. Exp. (166), e62026, doi:10.3791/62026 (2020).
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