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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

Location, Dissection, and Analysis of the Murine Stellate Ganglion

Published: December 22nd, 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

Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes.

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially the role of its sympathetic branch is an ongoing matter of investigation in the pathophysiology of ventricular arrhythmias (VA). Neurons in the stellate ganglia (SG) – bilateral star-shaped structures of the sympathetic chain  are an important component of the sympathetic infrastructure. The SG are a recognized target for treatment via cardiac sympathetic denervation in patients with therapy-refractory VA. While neuronal remodeling and glial activation in the SG have been described in patients with VA, the underlying cellular and molecular processes that potentially precede the onset of arrhythmia are only insufficiently understood and should be elucidated to improve autonomic modulation. Mouse models allow us to study sympathetic neuronal remodeling, but identification of the murine SG is challenging for the inexperienced investigator. Thus, in-depth cellular and molecular biological studies of the murine SG are lacking for many common cardiac diseases. Here, we describe a basic repertoire for dissecting and studying the SG in adult mice for analyses at RNA level (RNA isolation for gene expression analyses, in situ hybridization), protein level (immunofluorescent whole mount staining), and cellular level (basic morphology, cell size measurement). We present potential solutions to overcome challenges in the preparation technique, and how to improve staining via quenching of autofluorescence. This allows for the visualization of neurons as well as glial cells via established markers in order to determine cell composition and remodeling processes. The methods presented here allow characterizing the SG to gain further information on autonomic dysfunction in mice prone to VA and can be complemented by additional techniques investigating neuronal and glial components of the autonomic nervous system in the heart.

The cardiac autonomic nervous system is a tightly regulated equilibrium of sympathetic, parasympathetic, and sensory components that allows the heart to adapt to environmental changes with the appropriate physiological response1,2. Disturbances in this equilibrium, for example, an increase of sympathetic activity, have been established as a key driver for the onset as well as maintenance of ventricular arrhythmias (VA)3,4. Therefore, autonomic modulation, achieved via pharmacological reduction of sympathetic activity with beta-blockers, has been a corn....

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All procedures involving animals were approved by the Animal Care and Use Committee of the State of Hamburg (ORG870, 959) and the North Rhine-Westphalian State Agency for Nature, Environment and Consumer Protection (LANUV, 07/11) and conform to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals (2011). Studies were performed using male and female (aged 10-24 weeks) C57BL/6 mice (stock number 000664, Jackson Laboratories) and mice homozygous (db/db) or heterozygous (db/het; control) f.......

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Figure 1 visualizes how to identify and dissect the SG. Figure 1A shows a schematic drawing of the location, while Figure 1B presents the view into the thorax after removal of the heart-lung-package. The left and right longus colli muscles medial from the SG and the rib cage are important landmarks for orientation. Dissection is performed along the dotted lines between muscles and the first rib. The SG and the sympathetic .......

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The understanding of cellular and molecular processes in neurons and glial cells of the sympathetic nervous system that precede the onset of VA is of high interest, as sudden cardiac arrest remains the most common cause of death worldwide5. Therefore, in the current manuscript, we provide a basic repertoire of methods to identify the murine SG – a murine element within this network – and perform subsequent analyses on RNA, protein, and cellular level.

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The authors would like to thank Hartwig Wieboldt for his excellent technical assistance, and the UKE Microscopy Imaging Facility (Umif) of the University Medical Center Hamburg-Eppendorf for providing microscopes and support. This research was funded by the DZHK (German Centre for Cardiovascular Research) [FKZ 81Z4710141].

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Name Company Catalog Number Comments
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 20x20 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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