Sympathetic nerve fibers and ganglia in canine cervical vagus nerves: Localization and quantitation

doi:10.1016/j.hrthm.2012.12.015

Heart Rhythm

Volume 10, Issue 4, April 2013, Pages 585-591

https://doi.org/10.1016/j.hrthm.2012.12.015 Get rights and content

Background

Cervical vagal nerve (CVN) stimulation may improve left ventricular ejection fraction in patients with heart failure.

Objectives

To test the hypothesis that sympathetic structures are present in the CVN and to describe the location and quantitate these sympathetic components of the CVN.

Methods

We performed immunohistochemical studies of the CVN from 11 normal dogs and simultaneously recorded stellate ganglion nerve activity, left thoracic vagal nerve activity, and subcutaneous electrocardiogram in 2 additional dogs.

Results

A total of 28 individual nerve bundles were present in the CVNs of the first 11 dogs, with an average of 1.87±1.06 per dog. All CVNs contain tyrosine hydroxylase-positive (sympathetic) nerves, with a total cross-sectional area of 0.97±0.38 mm². The sympathetic nerves were nonmyelinated, typically located at the periphery of the nerve bundles and occupied 0.03%–2.80% of the CVN cross-sectional area. Cholineacetyltransferase-positive nerve fibers occupied 12.90%–42.86% of the CVN cross-sectional areas. Ten of 11 CVNs showed tyrosine hydroxylase and cholineacetyltransferase colocalization. In 2 dogs with nerve recordings, we documented heart rate acceleration during spontaneous vagal nerve activity in the absence of stellate ganglion nerve activity.

Conclusions

Sympathetic nerve fibers are invariably present in the CVNs of normal dogs and occupy in average up to 2.8% of the cross-sectional area. Because sympathetic nerve fibers are present in the periphery of the CVNs, they may be susceptible to activation by electrical stimulation. Spontaneous activation of the sympathetic component of the vagal nerve may accelerate the heart rate.

Introduction

Cervical vagal nerve (CVN) stimulation has been available for the treatment of resistant partial-onset seizures in Europe since 1994 and in the United States since 1997.1, 2, 3, 4 Animal studies in heart failure models have shown improvement in left ventricular ejection fraction, ventricular arrhythmia suppression, and survival with CVN stimulation.5, 6, 7, 8 The early results of a human heart failure clinical trial showed that CVN stimulation improves left ventricular ejection fraction and New York Heart Association functional class for up to 1-year follow-up.⁹ However, in the same study, CVN stimulation was associated with 26 serious adverse events that occurred in 13 of 32 (40.6%) patients, including deaths and cardiac arrhythmias. In addition to heart failure therapy, the clinicaltrials.gov Web site lists a number of active clinical trials that use CVN for other cardiac and noncardiac diseases. Two of these ongoing trials involve CVN stimulation in patients with heart failure. While there is much enthusiasm for CVN in treating human diseases, only limited information is available regarding the anatomy of the CVN. Foly and DuBois¹⁰ have shown that within felines the CVN is 65%–80% sensory and 20%–35% motor. On average, there are more nerve fibers on the left CVN than on the right CVN. Agostoni et al¹¹ state that myelinated nerves compose about 16% of the CVN. While the CVN is largely considered to be a parasympathetic structure that sends fibers to numerous parts of the body (heart, esophagus, and stomach),¹² previous research has noted a sympathetic component.13, 14, 15, 16, 17, 18 Only one of these studies used specific immunohistochemical markers to identify the sympathetic nerves in the CVN.¹⁸ This single study reported the presence of tyrosine hydroxylase (TH)-positive structures in human vagus nerves, but the study did not provide quantitative information. Studies from our laboratory recently confirmed these findings by documenting the presence of both TH-positive nerve fibers and ganglion cells in the CVN of dogs that underwent rapid pacing to induce atrial fibrillation.¹⁹ However, because rapid atrial pacing is known to cause cardiac sympathetic nerve sprouting and sympathetic hyperinnervation,20, 21 these observations may not be applicable to dogs without rapid atrial pacing. That study did not quantify the amount of TH-positive nerves in the canine CVN. The purpose of the present study was to perform immunohistochemical studies on canine CVN to test the hypothesis that sympathetic nerves are invariably present in all CVN and that the location of these nerves makes them susceptible to activation by CVN stimulation. A secondary aim is to provide quantitative information on the amount of sympathetic nerve fibers in the CVN. We also recorded stellate ganglion nerve activity (SGNA) and thoracic vagal nerve activity (VNA) in ambulatory dogs to document that VNA alone can be associated with heart rate acceleration.

Section snippets

Histological studies of the CVN

We studied CVN from 11 normal healthy dogs weighing 25–30 kg. Both the right and left CVNs were available for analysis from 4 dogs, while from additional 7 dogs, 4 right and 3 left CVNs were available for study. A total of 8 right and 7 left nerves were analyzed. The nerves were fixed with 4% formalin for 45–60 minutes before transfer to 70% ethyl alcohol.²² CVN sections from formalin-fixed, paraffin-embedded, 5-μm-thick cross sections were stained with TH for sympathetic (adrenergic) nerves²³

Histological studies of CVN

As shown in Figure 1A, each CVN may contain more than 1 nerve bundle. There are, on average, 1.87±1.06 distinctly isolated nerve bundles per CVN (range 1–5 bundles). Among a total of 28 individual nerve bundles, 25 contained sympathetic components. Figure 1, Figure 1 show TH-positive nerves located in the upper edge of the small and large bundles, respectively. Figure 1, Figure 1 show ChAT stains. The ChAT-positive nerve structures were widely distributed in the vagus nerves. While the

Sympathetic component of the CVN

Previous studies have shown that there is a sympathetic component of the CVN.13, 14, 15, 16, 17 Therefore, the CVN has also been referred to as the vagosympathetic trunk.²⁹ Electrical stimulation of the vagosympathetic trunk heterogeneously shortens the atrial refractory periods, which was thought to underlie the mechanism of AF.³⁰ More recent studies, however, show that sympathetic nerve activity is also important in the induction of AF and that simultaneous sympathovagal activation is

Conclusions

Sympathetic nerve fibers are invariably present in the CVN of normal dogs and occupy in average up to 2.8% of the cross-sectional area. The sympathetic nerve fibers within the vagal nerve may activate alone and accelerate heart rate. Because sympathetic nerve fibers are present in the periphery of the CVN, these sympathetic nerve fibers may be susceptible to activation by electrical stimulation.

Acknowledgments

We thank Lei Lin, Nicole Courtney, Jessica Warfel, and Janet Hutcheson for their assistance.

References (37)

R.D. Janes et al.
Anatomy of human extrinsic cardiac nerves and ganglia
Am J Cardiol
(1986)
K. Kawagishi et al.
Tyrosine hydroxylase-immunoreactive fibers in the human vagus nerve
J Clin Neurosci
(2008)
M. Ogawa et al.
Left stellate ganglion and vagal nerve activity and cardiac arrhythmias in ambulatory dogs with pacing-induced congestive heart failure
J Am Coll Cardiol
(2007)
O.F. Sharifov et al.
Roles of adrenergic and cholinergic stimulation in spontaneous atrial fibrillation in dogs
J Am Coll Cardiol
(2004)
E. Patterson et al.
Triggered firing in pulmonary veins initiated by in vitro autonomic nerve stimulation
Heart Rhythm
(2005)
E. Patterson et al.
Sodium-calcium exchange initiated by the Ca²⁺ transient: an arrhythmia trigger within pulmonary veins
J Am Coll Cardiol
(2006)
J.P. Beekwilder et al.
Overview of the clinical applications of vagus nerve stimulation
J Clin Neurophysiol
(2010)
A.P. Amar et al.
An institutional experience with cervical vagus nerve trunk stimulation for medically refractory epilepsy: rationale, technique, and outcome
Neurosurgery
(1998)
E. Ben-Menachem et al.
First International Vagus Nerve Stimulation Study Group. Vagus nerve stimulation for treatment of partial seizures, 1: A controlled study of effect on seizures
Epilepsia
(1994)
A. Handforth et al.
Vagus nerve stimulation therapy for partial-onset seizures: a randomized active-control trial
Neurology
(1998)

M. Li et al.

Vagal nerve stimulation markedly improves long-term survival after chronic heart failure in rats

Circulation

(2004)

C. Zheng et al.

Vagal stimulation markedly suppresses arrhythmias in conscious rats with chronic heart failure after myocardial infarction

Conf Proc IEEE Eng Med Biol Soc

(2005)

H.N. Sabbah et al.

Vagus nerve stimulation in experimental heart failure

Heart Fail Rev

(2011)

Y. Zhang et al.

Chronic vagus nerve stimulation improves autonomic control and attenuates systemic inflammation and heart failure progression in a canine high-rate pacing model

Circ Heart Fail

(2009)

G.M. De Ferrari et al.

Chronic vagus nerve stimulation: a new and promising therapeutic approach for chronic heart failure

Eur Heart J

(2011)

J.O. Foly et al.

Quantitative studies of the vagus nerve in the cat, I: The ratio of sensory and motor fibers

J Comp Neurol

(1937)

E. Agostoni et al.

Functional and histological studies of the vagus nerve and its branches to the heart, lungs and abdominal viscera in the cat

J Physiol

(1957)

T.R. Henry

Therapeutic mechanisms of vagus nerve stimulation

Neurology

(2002)

Cited by (39)

Autonomic Modulation of Atrial Fibrillation
2023, JACC: Basic to Translational Science
The autonomic nervous system plays a vital role in cardiac arrhythmias, including atrial fibrillation (AF). Therefore, reducing the sympathetic tone via neuromodulation methods may be helpful in AF control. Myocardial ischemia is associated with increased sympathetic tone and incidence of AF. It is an excellent disease model to understand the neural mechanisms of AF and the effects of neuromodulation. This review summarizes the relationship between autonomic nervous system and AF and reviews methods and mechanisms of neuromodulation. This review proposes that noninvasive or minimally invasive neuromodulation methods will be most useful in the future management of AF.
Organ- and function-specific anatomical organization of vagal fibers supports fascicular vagus nerve stimulation
2023, Brain Stimulation
Vagal fibers travel inside fascicles and form branches to innervate organs and regulate organ functions. Existing vagus nerve stimulation (VNS) therapies activate vagal fibers non-selectively, often resulting in reduced efficacy and side effects from non-targeted organs. The transverse and longitudinal arrangement of fibers inside the vagal trunk with respect to the functions they mediate and organs they innervate is unknown, however it is crucial for selective VNS. Using micro-computed tomography imaging, we tracked fascicular trajectories and found that, in swine, sensory and motor fascicles are spatially separated cephalad, close to the nodose ganglion, and merge caudad, towards the lower cervical and upper thoracic region; larynx-, heart- and lung-specific fascicles are separated caudad and progressively merge cephalad. Using quantified immunohistochemistry at single fiber level, we identified and characterized all vagal fibers and found that fibers of different morphological types are differentially distributed in fascicles: myelinated afferents and efferents occupy separate fascicles, myelinated and unmyelinated efferents also occupy separate fascicles, and small unmyelinated afferents are widely distributed within most fascicles. We developed a multi-contact cuff electrode to accommodate the fascicular structure of the vagal trunk and used it to deliver fascicle-selective cervical VNS in anesthetized and awake swine. Compound action potentials from distinct fiber types, and physiological responses from different organs, including laryngeal muscle, cough, breathing, and heart rate responses are elicited in a radially asymmetric manner, with consistent angular separations that agree with the documented fascicular organization. These results indicate that fibers in the trunk of the vagus nerve are anatomically organized according to functions they mediate and organs they innervate and can be asymmetrically activated by fascicular cervical VNS.
Subcutaneous nerve stimulation reduces sympathetic nerve activity in ambulatory dogs with myocardial infarction
2020, Heart Rhythm
Subcutaneous nerve stimulation (ScNS) remodels the stellate ganglion and reduces stellate ganglion nerve activity (SGNA) in dogs. Acute myocardial infarction (MI) increases SGNA through nerve sprouting.
The purpose of this study was to test the hypothesis that ScNS remodels the stellate ganglion and reduces SGNA in ambulatory dogs with acute MI.
In the experimental group, a radio transmitter was implanted during the first sterile surgery to record nerve activity and an electrocardiogram, followed by a second sterile surgery to create MI. Dogs then underwent ScNS for 2 months. The average SGNA (aSGNA) was compared with that in a historical control group (n = 9), with acute MI monitored for 2 months without ScNS.
In the experimental group, the baseline aSGNA and heart rate were 4.08±0.35 μV and 98±12 beats/min, respectively. They increased within 1 week after MI to 6.91±1.91 μV (P=.007) and 107±10 beats/min (P=.028), respectively. ScNS reduced aSGNA to 3.46±0.44 μV (P<.039) and 2.14±0.50 μV (P<.001) at 4 and 8 weeks, respectively, after MI. In comparison, aSGNA at 4 and 8 weeks in dogs with MI but no ScNS was 8.26±6.31 μV (P=.005) and 10.82±7.86 μV (P=0002), respectively. Immunostaining showed confluent areas of remodeling in bilateral stellate ganglia and a high percentage of tyrosine hydroxylase–negative ganglion cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling was positive in 26.61%±11.54% of ganglion cells in the left stellate ganglion and 15.94%±3.62% of ganglion cells in the right stellate ganglion.
ScNS remodels the stellate ganglion, reduces SGNA, and suppresses cardiac nerve sprouting after acute MI.
Subcutaneous nerve stimulation for rate control in ambulatory dogs with persistent atrial fibrillation
2019, Heart Rhythm
Subcutaneous nerve stimulation (ScNS) damages the stellate ganglion and improves rhythm control of atrial fibrillation (AF) in ambulatory dogs.
The purpose of this study was to test the hypothesis that thoracic ScNS can improve rate control in persistent AF.
We created persistent AF in 13 dogs and randomly assigned them to ScNS (n = 6) and sham control (n = 7) groups. ¹⁸F-2-Fluoro-2-deoxyglucose positron emission tomography/magnetic resonance imaging of the brain stem was performed at baseline and at the end of the study.
The average stellate ganglion nerve activity reduced from 4.00 ± 1.68 μV after the induction of persistent AF to 1.72 ± 0.42 μV (P = .032) after ScNS. In contrast, the average stellate ganglion nerve activity increased from 3.01 ± 1.26 μV during AF to 5.52 ± 2.69 μV after sham stimulation (P = .023). The mean ventricular rate during persistent AF reduced from 149 ± 36 to 84 ± 16 beats/min (P = .011) in the ScNS group, but no changes were observed in the sham control group. The left ventricular ejection fraction remained unchanged in the ScNS group but reduced significantly in the sham control group. Immunostaining showed damaged ganglion cells in bilateral stellate ganglia and increased brain stem glial cell reaction in the ScNS group but not in the control group. The ¹⁸F-2-fluoro-2-deoxyglucose uptake in the pons and medulla was significantly (P = .011) higher in the ScNS group than the sham control group at the end of the study.
Thoracic ScNS causes neural remodeling in the brain stem and stellate ganglia, controls the ventricular rate, and preserves the left ventricular ejection fraction in ambulatory dogs with persistent AF.
Antiarrhythmic effects of vagal nerve stimulation after cardiac sympathetic denervation in the setting of chronic myocardial infarction
2018, Heart Rhythm
Neuraxial modulation with cardiac sympathetic denervation (CSD) can potentially reduce burden of ventricular tachyarrhythmia (VT). However, despite catheter ablation and CSD, VT can recur in patients with cardiomyopathy and the role of vagal nerve stimulation (VNS) in this setting is unclear.
The purpose of this study was to evaluate the electrophysiological effects of VNS after CSD in normal and infarcted hearts.
In 10 normal and 6 infarcted pigs, electrophysiological and hemodynamic parameters were evaluated before and during intermittent VNS pre-CSD (bilateral stellectomy and T2–T4 thoracic ganglia removal) as well as post-CSD. The effect of VNS during isoproterenol was also assessed pre- and post-CSD. Multielectrode ventricular activation recovery interval (ARI) recordings, a surrogate of action potential duration, were obtained. VT inducibility was tested during isoproterenol infusion after CSD with and without VNS.
VNS increased the global ARI by 4% ± 4% pre-CSD and by 5% ± 6% post-CSD, with enhanced effects observed during isoproterenol infusion (10% ± 8% pre-CSD and 12% ± 9% post-CSD) in normal animals. In infarcted animals pre-CSD, VNS increased ARI by 6% ± 7% before and by 13% ± 8% during isoproterenol infusion. Post-CSD, VNS increased ARI by 6% ± 5% before and by 11% ± 7% during isoproterenol infusion. VT was inducible in all infarcted animals post-CSD during isoproterenol infusion; this inducibility was reduced by 67% with VNS (P = .01). In all animals, the hemodynamic effects of VNS remained after CSD.
After CSD, the beneficial electrophysiological effects of VNS remain. Furthermore, VNS can reduce VT inducibility beyond CSD in the setting of circulating catecholamines, suggesting a role for additional parasympathetic modulation in the treatment of ventricular arrhythmias.
Antiarrhythmic effects of stimulating the left dorsal branch of the thoracic nerve in a canine model of paroxysmal atrial tachyarrhythmias
2018, Heart Rhythm
Stellate ganglion nerve activity (SGNA) precedes paroxysmal atrial tachyarrhythmia (PAT) episodes in dogs with intermittent rapid left atrial (LA) pacing. The left dorsal branch of the thoracic nerve (LDTN) contains sympathetic nerves originating from the stellate ganglia.
The purpose of this study was to test the hypothesis that high-frequency electrical stimulation of the LDTN can cause stellate ganglia damage and suppress PATs.
We performed long-term LDTN stimulation in 6 dogs with and 2 dogs without intermittent rapid LA pacing while monitoring SGNA.
LDTN stimulation reduced average SGNA from 4.36 μV (95% confidence interval [CI] 4.10–4.62 μV) at baseline to 3.22 μV (95% CI 3.04–3.40 μV) after 2 weeks (P = .028) and completely suppressed all PAT episodes in all dogs studied. Tyrosine hydroxylase staining showed large damaged regions in both stellate ganglia, with increased percentages of tyrosine hydroxylase–negative cells. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that 23.36% (95% CI 18.74%–27.98%) of ganglion cells in the left stellate ganglia and 11.15% (95% CI 9.34%–12.96%) ganglion cells in the right stellate ganglia were positive, indicating extensive cell death. A reduction of both SGNA and heart rate was also observed in dogs with LDTN stimulation but without rapid LA pacing. Histological studies in the 2 dogs without intermittent rapid LA pacing confirmed the presence of extensive stellate ganglia damage, along with a high percentage of terminal deoxynucleotidyl transferase dUTP nick end labeling–positive cells.
LDTN stimulation damages both left and right stellate ganglia, reduces left SGNA, and is antiarrhythmic in this canine model of PAT.

View all citing articles on Scopus

: This article was processed by a guest editor. This study was supported in part by National Institutes of Health grants P01HL78931 and R01HL71140, a Piansky Endowment (to Dr Fishbein), a Medtronic-Zipes Endowment (to Dr Chen), and the Indiana University Health-Indiana University School of Medicine Strategic Research Initiative.

: The first 2 authors contributed equally to this project.

: Dr Chen is a consultant to Cyberonics. Our laboratory received equipment donations from Cyberonics, Medtronic, St Jude Medical, and Cryocath.

View full text

Sympathetic nerve fibers and ganglia in canine cervical vagus nerves: Localization and quantitation

Background

Objectives

Methods

Results

Conclusions

Introduction

Section snippets

Histological studies of the CVN

Histological studies of CVN

Sympathetic component of the CVN

Conclusions

Acknowledgments

Am J Cardiol

J Clin Neurosci

J Am Coll Cardiol

J Am Coll Cardiol

Heart Rhythm

J Am Coll Cardiol

Overview of the clinical applications of vagus nerve stimulation

J Clin Neurophysiol

An institutional experience with cervical vagus nerve trunk stimulation for medically refractory epilepsy: rationale, technique, and outcome

Neurosurgery

First International Vagus Nerve Stimulation Study Group. Vagus nerve stimulation for treatment of partial seizures, 1: A controlled study of effect on seizures

Epilepsia

Vagus nerve stimulation therapy for partial-onset seizures: a randomized active-control trial

Neurology

Vagal nerve stimulation markedly improves long-term survival after chronic heart failure in rats

Circulation

Vagal stimulation markedly suppresses arrhythmias in conscious rats with chronic heart failure after myocardial infarction

Conf Proc IEEE Eng Med Biol Soc

Vagus nerve stimulation in experimental heart failure

Heart Fail Rev

Chronic vagus nerve stimulation improves autonomic control and attenuates systemic inflammation and heart failure progression in a canine high-rate pacing model

Circ Heart Fail

Chronic vagus nerve stimulation: a new and promising therapeutic approach for chronic heart failure

Eur Heart J

Quantitative studies of the vagus nerve in the cat, I: The ratio of sensory and motor fibers

J Comp Neurol

Functional and histological studies of the vagus nerve and its branches to the heart, lungs and abdominal viscera in the cat

J Physiol

Therapeutic mechanisms of vagus nerve stimulation

Neurology