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].

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&#39;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...

The authors have nothing to disclose.

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&#160;&#8211;&#160;a murine&#160;element within&#160;this network &#8211;&#160;and perform subsequent analyses on RNA, protein, and cellular level.
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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 cornerstone in the treatment of patients with VA for decades5,6. But despite pharmacological and catheter-based interventions, a relevant number of patients still suffers from recurrent VA7.
Sympathetic input to the heart is mostly mediated via neuronal cell bodies in the stellate ganglia (SG), bilateral star-shaped structures of the sympathetic chain, which relay information via numerous intrathoracic nerves from the brainstem to the heart8,9,10. Nerve sprouting from the SG after injury is associated with VA and sudden cardiac death11,12, emphasizing the SG as a target for autonomic modulation13,14. A reduction of sympathetic input to the heart can be achieved temporarily via percutaneous injection of local anesthetics or permanently by partial removal of the SG via video-assisted thoracoscopy15,16. Cardiac sympathetic denervation presents an option for patients with therapy-refractory VA with promising results14,16,17. We have learned from explanted SG of these patients that neuronal and neurochemical remodeling, neuro-inflammation and glial activation are hallmarks of sympathetic remodeling that might contribute or aggravate autonomic dysfunction18,19. Still, the underlying cellular and molecular processes in these neurons remain obscure to date, for example, the role of neuronal transdifferentiation into a cholinergic phenotype20,21. Experimental studies present novel approaches to treat VA, for example, the reduction of sympathetic nerve activity via optogenetics22, but in-depth characterization of the SG is still lacking in many cardiac pathologies that go in hand with VA. Mouse models mimicking these pathologies allow to study neuronal remodeling that potentially precedes the onset of arrhythmias12,23. These can be completed by further morphological and functional analyses for autonomic characterization of the heart and the nervous system. In the present protocol, we provide a basic repertoire of methods allowing to dissect and characterize the murine SG to improve the understanding of VA.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plate</td>
			<td>TPP</td>
			<td>92097</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>Adhesion Slides SuperFrost plus&#160; 25 x 75 x 1 mm</td>
			<td>R. Langenbrinck</td>
			<td>03-0060</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Albumin bovine Fraction V receptor grade lyophil.</td>
			<td>Serva</td>
			<td>11924.03</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>bisBenzimide H33342 trihydrochloride (Hoechst)</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>B2261</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Chicken anti neurofilament</td>
			<td>EMD Millipore</td>
			<td>AB5539</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>D8418</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey anti chicken IgY Alexa 647&#160;</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>AP194SA6</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey anti goat IgG Alexa 568&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11057</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey anti rabbit IgG Alexa 488&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21206</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Drying block 37-100 mm</td>
			<td>Whatman (Sigma Aldrich)</td>
			<td>WHA10310992&#160;</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma Aldrich</td>
			<td>E4009</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Ethanol 99 % denatured with MEK, IPA and Bitrex (min. 99,8 %)</td>
			<td>Th.Geyer</td>
			<td>2212.5000</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Eukitt mounting medium</td>
			<td>AppliChem</td>
			<td>253681.0008</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Fluoromount-G + DAPI</td>
			<td>Southern Biotech</td>
			<td>0100-20</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Goat anti choline acetyltransferase</td>
			<td>EMD Millipore</td>
			<td>AP144P</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>H2O2 30% (w/w)</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>H1009</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Heparin Sodium 25.000 UI / 5ml</td>
			<td>Rotexmedica</td>
			<td>PZN: 3862340</td>
			<td>Preparation SG</td>
		</tr>
		<tr>
			<td>High-capacity cDNA reverse transctiption kit</td>
			<td>Life technologies</td>
			<td>&#160;4368813</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Isoflurane (Forene)</td>
			<td>Abbott Laboratories</td>
			<td>2594.00.00</td>
			<td>Preparation SG</td>
		</tr>
		<tr>
			<td>Mayer&#39;s hemalum solution</td>
			<td>Merck</td>
			<td>1.09249.0500</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Microscope cover glasses 20x20 mm or smaller</td>
			<td>Marienfeld</td>
			<td>0101040</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>miRNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>NanoDrop 2000c</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000C</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Opal 570 Reagent Pack</td>
			<td>Akoya Bioscience</td>
			<td>FP1488001KT</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16% w/v aq. soln., methanol free&#160;</td>
			<td>Alfa Aesar</td>
			<td>43368</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Pasteur pipettes, LDPE, unsterile, 3 ml, 154 mm</td>
			<td>Th.Geyer</td>
			<td>7691202</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline tablets</td>
			<td>Gibco</td>
			<td>18912-014</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Pinzette Dumont SS Forceps</td>
			<td>FineScienceTools</td>
			<td>11203-25</td>
			<td>Preparation SG</td>
		</tr>
		<tr>
			<td>QIAzol Lysis Reagent</td>
			<td>Qiagen&#160;</td>
			<td>79306</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Rabbit anti tyrosine hydroxylase</td>
			<td>EMD Millipore</td>
			<td>AB152</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Merck</td>
			<td>R0901-100ML</td>
			<td>RNA isolation (optional)</td>
		</tr>
		<tr>
			<td>RNAscope Multiplex Fluorescent Reagent Kit v2</td>
			<td>biotechne (ACD)</td>
			<td>323100</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>RNAscope Probe-Mm-S100b-C2</td>
			<td>biotechne (ACD)</td>
			<td>431738-C2</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>RNAscope Probe-Mm-Tubb3</td>
			<td>biotechne (ACD)</td>
			<td>423391</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>Stainless steel beads 7 mm&#160;</td>
			<td>Qiagen&#160;</td>
			<td>69990</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Sudan black B</td>
			<td>Roth</td>
			<td>0292.2</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay Cdkn1b (Mm00438168_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay Choline acetyltransferase (Mm01221880_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay MKi67 (Mm01278617_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay PTPCR (Mm01293577_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay S100b (Mm00485897_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay Tyrosin Hydroxylase (Mm00447557_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan mastermix</td>
			<td>Applied biosystems</td>
			<td>4370074</td>
			<td>Gene Expression analysis&#160;</td>
		</tr>
		<tr>
			<td>Tissue Lyser II</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Triton X-100 10% solution</td>
			<td>Sigma-Aldrich</td>
			<td>93443-100ml</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416-100ML</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>Wacom bamboo pen</td>
			<td>Wacom</td>
			<td>CTL-460/K</td>
			<td>Cell size measurements</td>
		</tr>
		<tr>
			<td>Whatman prepleated qualitative filter paper, Grade 595 1/2</td>
			<td>Sigma-Aldrich</td>
			<td>WHA10311647</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>W21404</td>
			<td>RNAscope</td>
		</tr>
	</tbody>
</table>

location, dissection, and analysis of the murine stellate ganglion

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;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)&#160;&#8211;&#160;bilateral star-shaped structures of the sympathetic chain &#8211;&#160;are an important component of the sympathetic infrastructure. The SG are a recognized target for&#160;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.

Figure 1&#160;visualizes how to identify and dissect the SG. Figure 1A shows a schematic drawing of the location, while Figure 1B presents the view into&#160;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 ...

Watch this Scientific Journal Video about Location, Dissection, and Analysis of the Murine Stellate Ganglion at JoVE.com

Location, Dissection, and Analysis of the Murine Stellate Ganglion

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&#160;the role of its sympathetic branch is an ongoing matter ...

Every heartbeat is under tight control of the sympathetic nervous system. Most neurons within cardiac neurocontrol interact with the stellate ganglia. Our protocol makes it possible to study the underlying processes.With the video and images at hand, even the unexperienced investigator can find the stellate ganglion in mice and apply the techniques to different disease models. The sympathetic nervous system is a key component of cardiac arrhythmogenesis. Modulation of the stellate ganglia appears promising in the treatment of patients with life-threatening ventricular arrhythmias.Understanding the cellular and molecular processes in neurons and glial cells might pave the way for targeted therapies. The main challenge of this protocol is to locate the stellate ganglia. We recommend performing control staining for tyrosine hydroxylase in the beginning to assess the quality of preparation.After extraction of heart and lungs, place the torso of the mouse under a stereo microscope. Ensure good lighting with an external light source and flush the thorax with PBS. Locate the first rib in the longus colli muscles, then gently expose the connective tissue lateral to the longus colli muscle using the tip of the Dumont forceps.Repeat this process on the opposite side to locate the second stellate ganglion, then turn the forceps around by 180 degrees and using the flat side, grip the stellate ganglion and pull it out with minimal pressure. Place both stellate ganglia in a dish filled with PBS and inspect them with the appropriate magnification. Remove excess vessels, fat tissue, and larger nerves.Fix the stellate ganglia in 4%methanol-free paraformaldehyde for two hours at room temperature. Prepare Sudan Black stock solution for reduction of autofluorescence and improvement of signal to background ratio. Dissolve it for two to three hours on a magnetic stirrer at room temperature.Next, prepare Sudan Black working solution by centrifuging the stock solution for 30 minutes at 13, 000 times G to remove debris. Dilute the stock in 70%ethanol to a final concentration of 0.25%Sudan Black. Prepare dense bleach by mixing methanol, 30%hydrogen peroxide, and dimethyl sulfoxide at a ratio of four to one to one.Add 200 microliters of the bleach per stellate ganglion to improve antibody permeabilization and place the plate on a shaker for one hour at room temperature. Perform a descending series of rehydration using 100, 75, 50, and 25%methanol on a shaker, incubating for 10 minutes in each solution. For permeabilization, incubate the stellate ganglia twice in PBS with 1%Triton for 60 minutes each at room temperature.Remove the permeabilization solution from the stellate ganglia and add Sudan Black working solution, then incubate for two hours on a shaker. Carefully remove the Sudan Black solution by tilting the plate and pipetting from the upright side, then add 200 microliters of PBST. Wash for five minutes on a shaker.Remove PBST by aspirating it with a pipette and repeat the wash twice. After the last wash, add 200 microliters of blocking solution. Incubate at four degrees Celsius overnight on a shaker.On the next day, add primary antibodies to the blocking solution, adapting antibody concentrations from established protocols. Remove blocking solution from the stellate ganglia and add the primary antibodies. Incubate the stellate ganglia for 36 to 48 hours at four degrees Celsius on a shaker.Remove the antibody solution carefully and add 200 microliters of PBST. Place the plate on a shaker for 30 minutes. Remove the PBST and repeat the wash five more times.Prepare the secondary antibody working solution by centrifuging fluorescent Alexa labeled secondary antibodies for one minute at 13, 000 times G.Dilute the antibodies in blocking solution at a ratio of one to 500 and add them to the stellate ganglia. Incubate the plate for 12 to 24 hours at four degrees Celsius on a shaker. Remove the antibody solution carefully and add 200 microliters of PBST.For embedding, add fluorescent mounting medium on a glass slide and place it under a stereo microscope. Using Dumont forceps, pick up the stellate ganglion, remove excess liquid by dipping one end on a filter paper, and place it on the mounting medium. If necessary, correct the position of the stellate ganglion using Dumont forceps, then gently place a glass cover slip.Let the slides dry in the dark overnight at room temperature. Image the stellate ganglia stained for tyrosine hydroxylase at 200 times magnification for cell size measurement, taking four to six random images for every stellate ganglion. Analyze the images using ImageJ software to estimate cell size.An overview of a murine stellate ganglion stained positive for the sympathetic marker tyrosine hydroxylase and choline acetyltransferase in glial cells ensheathing neuronal cell bodies can be visualized by staining for S100B in combination with neuronal marker PGP9.5. An exemplary analysis of whole mount in situ hybridization and immunofluorescent co-staining of a stellate ganglion shows that tyrosine hydroxylase protein and mRNA molecules of beta-3 tubulin are expressed in large neuronal cell bodies, while mRNA of S100B is also detectable in surrounding glial cells. In the merge, it is visible that some neurons are negative for tyrosine hydroxylase, but express beta-3 tubulin, while S100B mRNA can also be detected in surrounding cells, as depicted in the magnification here.Images of tyrosine hydroxylase stained stellate ganglia were used for cell size measurements with ImageJ, and the data was compared for neuronal somata from control stellate ganglia versus diabetic stellate ganglia using the Mann-Whitney test. The expression of genes from different cell types of the stellate ganglia are shown here. Hematoxylin and eosin staining of a formalin fixed stellate ganglion visualizes connective tissue and cells on top.When attempting this protocol, remember to locate the strip and dissect along the longus colli muscle to identify the stellate ganglia, especially if the spine broke during cervical dislocation. Little is known about glial cells in the cardiac nervous system. Our techniques can be adapted to study their role in the stellate ganglia.

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9.1K visualizações.  EVK Düsseldorf, cNEP, cardiac Neuro- and Electrophysiology Research Consortium. Every heartbeat is under tight control of the sympathetic nervous system. Most neurons within cardiac neurocontrol interact with the stellate ganglia. Our protocol makes it possible to study the underlying processes.With the video and images at hand, even the unexperienced investigator can find the stellate ganglion in mice and apply the techniques to different disease models. The sympathetic nervous system is a key component of cardiac arrhythmogenesis. Modulation of the stellate gang...