Low-level transcutaneous electrical stimulation of the auricular branch of the vagus nerve:A noninvasive approach to treat
the initial pha of atrialfibrillation
Lilei Yu,MD,*Benjamin J.Scherlag,PhD,†Shaolong Li,MD,†Youqi Fan,MD,†John Dyer,PhD,†Shailesh Male,MD,†Vandana Varma,MD,†Yong Sha,MD,†Stavros Stavrakis,MD,PhD,†
Sunny S.Po,MD,PhD†
From the*Department of Cardiology,Renmin Hospital of Wuhan University,Wuhan,China and y Heart Rhythm Institute and Department of Medicine,University of Oklahoma Health Sciences Center,Oklahoma City,Oklahoma.
BACKGROUND We studied the effects of transcutaneous electrical stimulation at the tragus,the anterior protuberance of the outer ear,for inhibiting atrialfibrillation(AF).
OBJECTIVE To develop a noninvasive transcutaneous approach to deliver low-level vagal nerve stimulation to the tragus in order to treat cardiac arrhythmias such as AF.
METHODS In16pentobarbital anesthetized dogs,multielectrode catheters were attached to pulmonary veins and atria.Three tungsten-coated microelectrodes were inrted into the anterior right ganglionated plexi to record neural activity.Tragus stimula-tion(20Hz)in the right ear was accomplished by attaching2 alligator clips onto the tragus.The voltage slowing the sinus rate or atrioventricular conduction was ud as the threshold for tting the low-level tragus stimulation(LL-TS)at80%below the thresh-old.At baline,programmed stimulation determined the effective refractory period(ERP)and the window of vulnerability(WOV),a measure of AF inducibility.For hours1–3,rapid atrial pacing(RAP) was applied alone,followed by concomitant RAPþLL-TS for hours 4–6(N¼6).The same parameters were measured during sinus rhythm when RAP stopped after each hour.In4other animals, bivagal tranction was performed before LL-TS.
RESULTS During hours1–3of RAP,there was a progressive and significant decrea in ERP,increa in WOV,and increa in neural activity vs baline(all P o.05).With RAPþLL-TS during hours 4–6,there was a linear return of ERP,WOV,and neural activity toward baline levels(all P o.05,compared to the third-hour values).In4dogs,bivagal tranction prevented the reversal of ERP and WOV despite3hours of RAPþLL-TS. CONCLUSIONS LL-TS can rever RAP-induced atrial remodeling and inhibit AF inducibility,suggesting a potential noninvasive treatment of AF.
KEYWORDS Atrialfibrillation;Autonomic nervous system; Transcutaneous stimulation
ABBREVIATIONS ABVN¼auricular branch of the vagus nerve; AF¼atrialfibrillation;ANS¼autonomic nervous system; ARGP¼anterior right ganglionated plexi;AV¼atrioventricular;CANS¼cardiac autonomic nervous system; ERP¼effective refractory period;LL-TS¼low-level tragus stimulation;LL-VNS¼low-level vagal nerve stimulation;NTS¼nucleus tractus solitarius;PV¼pulmonary vein;RAP¼rapid atrial pacing;TENS¼transcutaneous electrical nerve stimulation;WOV¼window of vulnera-bility
(Heart Rhythm2013;10:428–435)I2013Heart Rhythm Society. All rights rerved.
Introduction
In previous studies from our laboratory,we found that low-level vagus nerve stimulation(LL-VNS),at voltages sub-stantially below that which slowed the sinus rate or atrioventricular(AV)conduction,significantly increas the effective refractory period(ERP)in the atria and in the pulmonary vein(PV)myocardium.1–5Furthermore,atrial fibrillation(AF)inducibility at the sites was significantly suppresd and AF duration was also shortened substan-tially.In tho experiments,LL-VNS was applied to both vagal trunks discted in the neck and the vagal pregangli
o-nics at the posterior wall of the superior vena cava.3Direct neural recordings also indicate that the antiarrhythmic effects of LL-VNS is mediated by suppressing the activity of the intrinsic cardiac autonomic nervous system(CANS).3 Several previous reports have documented the effects of transcutaneous electrical stimulation to reduce the amount of anesthetic ud during operative procedures,6suppress psis in a murine model of endotoxemia7or elicit evoked potentials
This work was supported in part by a grant from the Helen and Wil
Webster Arrhythmia Rearch Fund(to Dr Scherlag)and the Heart Rhythmspectating
Institute of the University of Oklahoma(to Dr Po).Address reprint
requests and correspondence:Dr Sunny S.Po,Heart Rhythm Institute,
University of Oklahoma Health Sciences Center,1200Everett Dr(6E103),
Oklahoma City,Oklahoma73104.E-mail address:sunny-po@ouhsc.edu.
1547-5271/$-e front matter B2013Heart Rhythm Society.All rights rerved.dx.doi/10.1016/j.hrthm.2012.11.019
惊喜的英文翻译in the vagal nucleus in the brain in volunteer subjects.8,9 In some of the reports,stimulation of the auricular branch of the vagus nerve(ABVN)located at the tragus,the anterior protuberance of the outer ear,was capable of affecting neural pathways at a distance.6,8–10The purpo of the prent study was to develop a noninvasive transcutaneous approach to deliver LL-VNS to the tragus in order to treat cardiac arrhythmias such as AF.We cho right tragus stimulation becau the ABVN is easily accessible8,9and LL-VNS of the right vagus nerve had the same antiarrhythmic effects as bilateral vagal stimulation.3,4
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Methods
All animal studies were reviewed and approved by the Institutional Animal Care and U Committee of the Uni-versity of Oklahoma Health Sciences Center.Ten adult mongrel dogs,weighing22–26kg,were anesthetized with sodium pentobarbital(30mg/kg),and general anesthesia was maintained by hourly intravenous injection of50–100mg. Dogs were intubated and attached to positive pressure ventilation with a mixture of room air and100%oxygen. The right and left femoral veins were discted and8-F sheaths inrted into each vesl to deliver drugs and saline as well as catheter inrtion.An electrode catheter was inrted into the left femoral artery and pasd into the aortic root to record the His bundle potential.A nsor-controlled heating pad was ud under the do
g to regulate body temperature at37.01CϮ0.51C.
Initially,a left thoracotomy was performed at the fourth intercostal space and the left atrium and left superior and inferior PVs were expod by incising and reflecting t he pericardium as previously described.1–5Multielectrode catheters were attached to the PVs and left atrial appendage. The pericardium and thoracotomy were then sutured clod. The dog was then turned to the right side and a similar thoracotomy and pericardiotomy allowed exposure of the right atrium and right superior and inferior PVs.Again, multielectrode catheters were attached at the sites. Tragus stimulation
The stimulation of the tragus in the right ear(Figure1)was accomplished by attaching2alligator clips side by side on the right tragus or by using a light spring loaded plastic clip with electrodes on opposite sides of the inner and outer portions of the tragus.Incremental voltages were applied to the tragus(20Hz,1-ms square wave)until slowing of the
sinus rate or AV conduction was achieved.The voltage necessary to achieve a slowing of the sinus rate or AV conduction(measured by the AH interval)was ud as the threshold for tting the low-level tragus stimulation(LL-TS)in each experiment.In6experiments,LL-TS was t at 80%below the voltage required to slow the sinus rate or AV conduction.In4other experiments,LL-TS t at80%belo
w the threshold was delivered to the right tragus after tranc-tion of both vagi at the level just below the junction of the innominate vein and superior vena cava.In all experiments,the stimulation threshold was checked at the end of each hour of rapid atrial pacing(RAP)to ensure that LL-TS was t appropriately.
RAP simulating AF
The left atrial appendage was paced for6hours at1200 beats/min(2Âthreshold)to induce acute atrial remodeling. After each hour of RAP,pacing was temporarily stopped for 5–10minutes.After AF terminated and sinus
唯爱的英文rhythm Snout
Neck
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Without Tragus Stimulation
With Tragus Stimulation
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Figure1Reprentative examples of(A)the location of the right tragus highlighted by the red arrow;(B)suprathreshold tragus stimulation that shortened the sinus rate from440to390ms,suggesting the activation of the vagus nerve;(C)a nerve bundle in the tragus stained with HE(hematoxylin and eosin;left panel)and acetylcholine estera(AChE;middle and right panels).The dark brown color in the middle panel and thefluorescent spots in the right panel reprent sites showing immunoreactivity to AChE.
Yu et al Transcutaneous Vagal Stimulation429
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resumed,we determined the ERP and AF inducibility.Using programmed stimulation S1–S1¼330ms and decremental S1–S2at 10Âdiastolic threshold,ERP at atrial and PV sites were determined.The S1–S2intervals were decread from 150ms initially by decrements of 10ms and then 1ms when approaching ERP.5The difference between the longest and the shortest S1–S2interval (in ms)at which AF was induced was defined as the window of vulnerability (WOV),which rved as a quantitative measurement of AF inducibility.The cumulative WOV (P WOV)was the sum of WOVs at all sites in each dog.1–3ERP dispersion was calculated off-line as the coefficient of variation (standa
rd deviation/mean)of the ERP at all recording sites.2,5RAP was performed in the first 3hours without the application of LL-TS,whereas during the last 3hours,both RAP and LL-TS were applied simultaneously.
Neural recording
Three tungsten-coated microelectrodes were inrted into the fat pad located at the caudal end of the sinus node containing the anterior right ganglionated plexi (ARGP).The 3micro-electrodes were positioned such that they would contact different areas of the ARGP but would not be displaced by either cardiac or respiratory movement.The 3microelec-trodes were connected by a common lead to a preamplifier (Princeton Applied Rearch,model 113,Princeton,NJ).Bandpass filters were t between 300Hz and 10kHz,with amplification ranging from 100Âto 500Â.The sampling rate was 1kHz.Further amplification (50–200Â)was obtained by u of a hardwired amplifier (Spike 2,CED,Ltd,Cambridge,England,UK).A 1-minute recording of the ARGP neural activity during sinus rhythm was acquired immediately before LL-TS and after each hour of LL-TS for comparison.The neural activity was characterized by the recorded amplitude and frequency.Neural activity was defined as deflections with a signal-to-noi ratio greater than 3:1and the amplitude and frequency were manually determined as previously described.5
Immunohistochemical staining of the tragus
In 6animals,the right tragus was excid and 5-m m ctions were cut from paraffin blocks of the tragus.The ctions were air dried and fixed in acetone for 10minutes and then washed in Tris-buffered saline.Hydrogen peroxida block (Dako,Carpenteria,CA)was placed on the ctions for 10minutes,and the slides were washed in Tris-buffered saline.Protein block was placed on the ctions for 30minutes.Primary antibodies were then incubated overnight at 41C.Antibodies for acetylcholine estera (Chemicon,Leverkun,Germany)were ud to stain cholinergic nerves.Quantification of the nerve density in the tragus area was assisted by a commercially available software (ImagePro,Media Cybernetics,Inc,Rockville,MD).The nerve density bad on the immunoreactivity of each slide was determined by the average of 3fields with the highest nerve density.The
nerve density was expresd as the total area of positive staining per square millimeter (m m 2/mm 2).
Statistical analysis2013年考研政治真题
Data are expresd as mean ϮSD.Repeated measures analysis of variance (ANOVA)was ud to ex
amine the effect of each intervention on the respective parameters over time.Post hoc analysis,with the Tukey method to adjust for multiple comparisons,was performed to compare the follow-ing parameters measured hourly to the values in the baline state or the values at the end of the third hour of RAP before the initiation of LL-TS:(a)PV and atrial ERPs (Figures 2and 3),(b)ERP dispersion and S WOV (Figures 4and 5),and (c)the frequency and amplitude of the neural activity (Figure 6).P values o .05were considered statistical significant.
Results
The average stimulation threshold,which induced any slowing of the sinus rate or AV conduction,was 9.8Ϯ2.6V (N ¼10;Figure 1B ).In 6dogs,immunohistochemical studies showed the prence of nerve bundles in the tragus area,which was positive for acetylcholine estera (Figure 1C ).The density of acetylcholine estera (þ)neural elements in the right tragus was 45,658.3Ϯ7338.2m m 2/mm 2(N ¼6).Figure 2shows the consistent pattern of a significant decrea in the ERP during the first 3hours of RAP at all PV and atrial recording sites.With the addition of LL-TS t at 80%below the threshold for the next 3hours along with RAP,all sites showed a reversal of the ERP decrea and return toward baline values.In contrast,LL-TS after tranction of both vagus nerves failed to rever the ERP changes (Figure 3).LL-TS did not affect the AF duration or cycle length induced by RAP.After R
AP was stopped every hour,AF continued for 28Ϯ13,27Ϯ6,and 31Ϯ7conds after 1,3,and 6hours of RAP,respectively (n ¼6;P 4.05).In the prence of LL-TS,the AF duration after RAP was 30Ϯ4,31Ϯ6,and 35Ϯ2conds after 1,3,and 6hours of RAP,respectively (n ¼6;P 4.05).The AF cycle after RAP was stopped was 101Ϯ11,101Ϯ9,and 99Ϯ15ms after 1,3,and 6hours of RAP,respectively (n ¼6;P 4.05).In the prence of LL-TS,the AF cycle length after RAP was 109Ϯ6,103Ϯ10,and 106Ϯ9ms after 1,3,and 6hours of RAP,respectively (n ¼6;P 4.05).LL-TS had a similar effect on the dispersion of refractori-ness,which incread progressively during the first 3hours of RAP (Figure 4A ).The values at this time were signifi-cantly greater than at baline.With the continued applica-tion of RAP þLL-TS for the next 3hours,there was a reversal of the ERP dispersion toward baline levels.In contrast,LL-TS after tranction of both vagus nerves failed to rever the changes in ERP dispersion (Figure 4B ).Using the same programmed stimulation protocol to deter-mine ERP curves,the width of P WOV was determined as a function of the same time periods of 3hours of RAP and 3hours of combined RAP þLL-TS.Figure 5A shows the progressive
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and statistically significant increa in P
WOV during the first 3hours and the reversal toward control values during the next 3hours with the delivery of LL-TS t at 80%below the threshold.Again,LL-TS after tranction of both vagus nerves failed to rever the changes in P
WOV (Figure 5B ).
A typical example of the continuous monitoring of the neural activity recorded from the ARGP for 6hours of RAP is shown in Figure 6A .Figures 6
B and 6
C show a progressive increa in the frequency and amplitude of neural firing compared to baline,which was reverd by LL-TS t at 80%below the threshold introduced during hours 4–6(n ¼6).
Discussion
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Major findings
In this report,transcutaneous electrical stimulation of the ABVN at the right tragus was capable of suppressing AF and
reversing acute atrial remodeling (eg,shortening of ERP and increa in ERP dispersion)induced by RAP.The salutary effects are likely the result of inhibition of the activity of the intrinsic CANS.Elimination of the effects by tranction of both vagus nerves indicates that the vagal efferent fibers are part of the final pathway responsible for the inhibition of the intrinsic CANS.
In the prent study,we could not record the vagus nerve activity during LL-TS becau of the noi introduced by LL-TS.The antiarrhythmic effects of LL-TS that we attributed to the stimulation of the ABVN thus indirectly supported by the prence of acetylcholine estera-positive nerve bundles in the tragus.However,the neural connection between the tragus and the atrium was demonstrated by the changes in the neural activity of the ARGP and the associated electrophysiological properties.Three hours of RAP resulted in a progressive and significant increa in AF inducibility as measured by P WOV and a
ctyconcomitant
Figure 2Mean ERP values at PV and atrial sites during 6hours of RAP.In the last 3hours,LL-TS t at 80%below the thresh-old was applied with RAP (N ¼6).At all sites,mean ERP decread significantly after 3hours of RAP (*P o .05,**P o .01,com-pared to baline).After 3hours of RAP þLL-TS,mean ERP at all sites showed a significant reversal toward baline values (#P o .05,##
P o .01,compared with the end of the third hour of RAP).3H LL-TS ¼3hours of low-level tragus stimulation;6H RAP ¼6hours of rapid atrial pacing;ERP ¼effective refractory period;PV ¼pulmonary vein;RA and LA ¼right and left atrium,respectively;RAP ¼rapid atrial pacing;RSPV,LSPV,RIPV,and LIPV ¼right superior,left super-ior,right inferior,and left inferior pulmonary vein,respectively;TH ¼threshold.
Yu et al Transcutaneous Vagal Stimulation 431
significant decrea in the ERP at all tested PV and atrial sites.The initial progressive increa in neural firing was directly associated with the changes.LL-TS during the next 3hours resulted in a progressive return of ERP and WOV toward baline values and an associated decrea in neural activity recorded from the ARGP.Notably,the antiarrhythmic effects of LL-TS were eliminated by trans
比尔克林顿ec-tion of both vagus nerves at the level of the junction of the innominate vein and the superior vena cava (Figures 3–5),underlying the critical role of the efferent vagal fibers in AF suppression.
Tragus stimulation has been shown to activate the nuclei in the brain.Polak et al 9showed that vagus somatonsory evoked potentials can be elicited by transcutaneous tragus stimulation at intensities that did not produce perception of pain.Fallgatter et al 8demonstrated that the stimulation of the tragus area innervated by ABVN-elicited nsory evoked potentials that can be recorded from the scalp overlying the brainstem in human volunteers.The evoked potentials presumably originated from the vagal nuclei in the nucleus tractus solitarius (NTS).In mammals,the ABVN cours through the mastoid canaliculus and then between the internal jugular vein and the bony wall of the jugular foramen through which it reaches the brain stem.11Although the anatomy of this nerve had been studied in detail,the physiology of it remains poorly understood.Nomura and Mizuno 12applied horradish peroxida to trace the cranial cour of the auricular branch of the vagus nerve and found that the afferent fibers of this nerve terminate mainly in the NTS as well as other brain stem nuclei such as the trigeminal nucleus.It is well known that a large number of autonomic nerve fibers,including the fibers from the heart and lungs,project to the NTS.10,13,14The reflex loop formed by the ABVN,NTS,and autonomic nerves of the lungs has been propod to cau a unique f
orm of cough.This reflex (Arnold’s ear-cough reflex)is induced by the stimulation of the posterior or anterior aspect of the inferior wall of the external ear canal.The role of the NTS in this reflex may provide insight into antiarrhythmic effects we obrved in our study.It is known that the NTS receives afferent
vagal
Figure 3Mean ERP values at PV and atrial recording sites during 6hours of RAP.In the last 3hours of RAP,LL-TS was t at 80%below the threshold after tranction of both vagus nerves (N ¼4).At all sites,mean ERP decread significantly at 3hours compared to baline (*P o .05,**P o .01,***P o .001).LL-TS after vagal tranction failed to rever ERP shortening (P 4.05,compared with the end of third hour of RAP).All abbreviations as in Figure 2.
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