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.
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.
