J Neural Transm DOI 10.1007/s00702-014-1299-7
NEUROLOGY AND PRECLINICAL NEUROLOGICAL STUDIES - ORIGINAL ARTICLE
The effect of transcutaneous vagus nerve stimulation on cortical excitability Fioravante Capone • Giovanni Assenza • Giovanni Di Pino Gabriella Musumeci • Federico Ranieri • Lucia Florio • Carmen Barbato • Vincenzo Di Lazzaro
•
Received: 27 May 2014 / Accepted: 16 August 2014 Ó Springer-Verlag Wien 2014
Abstract There is great interest about the therapeutic potentialities of transcutaneous vagus nerve stimulation (tVNS) applied to neuropsychiatric disorders. However, the mechanisms of action of tVNS and its impact on cortical excitability are unclear. To this regard, transcranial magnetic stimulation (TMS) can be useful because it is able of evaluating non-invasively excitatory and inhibitory circuitry of the human cortex. Aim of the present study is to investigate the effects of tVNS on cerebral cortex excitability in healthy volunteers by means of TMS. Ten healthy subjects participated in this randomized placebo-controlled double-blind study. Real tVNS was administered at left external acoustic meatus, while sham stimulation was performed at left ear lobe, both of them for 60 min. We evaluated motor thresholds, motor evoked potential amplitude, recruitment curves, and short-interval intracortical inhibition (SICI) in right and left motor cortex. Such parameters were evaluated before and 60 min after the exposure to tVNS, for both the real and the sham stimulation. Cardiovascular parameters were monitored during the stimulation. A generalized linear model for repeated measures was implemented to assess the effect of time and stimulation type on cardiovascular and neurophysiological variables. SICI, a double-pulse TMS paradigm informative of
F. Capone (&) G. Assenza G. Di Pino G. Musumeci F. Ranieri L. Florio C. Barbato V. Di Lazzaro Institute of Neurology, Campus Bio-Medico University, Via Alvaro del Portillo 200, 00128 Rome, Italy e-mail: [email protected] F. Capone G. Di Pino G. Musumeci F. Ranieri L. Florio V. Di Lazzaro Fondazione Alberto Sordi, Research Institute for Ageing, Rome, Italy
GABA-A activity, was significantly increased in right motor cortex after real tVNS. Other neurophysiological parameters, as well as cardiovascular variables, remained unchanged. Our findings confirm that tVNS is a safe and effective way to stimulate vagus nerve and provide innovative data about the possible mechanisms of action that supports the potential therapeutic application of this technique. Keywords Transcutaneous vagus nerve stimulation Cortical excitability Transcranial magnetic stimulation Intracortical inhibition
Introduction Vagus nerve stimulation (VNS) is approved as adjunctive treatment for refractory epilepsy and depression (BenMenachem 2002; Grimm and Bajbouj 2010) and its application is currently under investigation for a wide range of neuropsychiatric diseases (Groves and Brown 2005). However, the diffusion of this technique has been hampered by its invasiveness: indeed, VNS requires the surgical implantation of a stimulator connected to an electrode located along the cervical branch of the vagus nerve. Recently, to reduce the discomfort of traditional VNS and its possible side-effects, an alternative strategy to stimulate non-invasively the vagus nerve has been proposed (Ventureyra 2000). This technique consists of transcutaneous stimulation (tVNS) of the external auditory channel at the inner side of the tragus. Such stimulation activates the auricular branch of the vagus nerve and via this pathway, the nuclei of the nerve located in the brainstem. In particular, the nucleus of the tractus solitarius is a crucial structure because it is the target of the main
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component of afferent vagal inputs and because it projects widespread toward a variety of key brain areas, including thalamus, amygdala, hippocampus, and neocortex (Kraus et al. 2007). The effectiveness of tVNS has been demonstrated in animal models (He et al. 2013) and it has been also confirmed in humans by functional magnetic resonance imaging (fMRI) studies showing that the patterns of brain activation induced by tVNS are quite similar to those observed during invasive VNS (Kraus et al. 2007, 2013). These observations raised the attention toward the therapeutic applications of tVNS, to such an extent that this technique has been evaluated for the treatment of several neuropsychiatric disorders such as epilepsy (Rong et al. 2014; He et al. 2013; Stefan et al. 2012), depression (Rong et al. 2012; Hein et al. 2013), and tinnitus (Lehtima¨ki et al. 2013). Nevertheless, the mechanisms of action of tVNS remain unclear especially because, so far, little is known about the functional connections between the vagus brain stem nuclei and the cortical structures (Fallgatter et al. 2003). Indeed, the study of tVNS influences on the cortical functional dynamic is worthwhile. To this regard, the excitatory and inhibitory circuits of the human cortex can be evaluated non-invasively by means of transcranial magnetic stimulation (TMS). TMS investigation is well-suited to test the mechanisms of action of VNS, as previously showed by Di Lazzaro and coworkers for traditional VNS. They reported an enhanced activity of intracortical inhibitory circuits, as assessed by a paired-pulse TMS protocol, in five patients affected by epilepsy (Di Lazzaro et al. 2004). Along this line, aim of the present study is to evaluate the effects of tVNS on cerebral cortex excitability in healthy volunteers, by means of TMS.
Subjects Ten (3 males and 7 females) healthy volunteers [mean age 30.2 ± 3.6 (SD) years] participated in the experiments. All of them gave their informed consent. All the subjects were naive to tVNS. The study was performed accordingly to the Declaration of Helsinki and was approved by the Local Ethics Committee. tVNS procedure The bipolar stimulation of the auricular branch of the vagus nerve was performed through an electric stimulator (Twister—EBM) and two Ag–AgCl electrodes (5 mm in diameter), placed in the left external acoustic meatus at the inner side of the tragus. The distance between the cathode and anode was 5 mm. For sham stimulation, electrodes were attached to the left ear lobe, an anatomical area that is known to be outside the innervation of the auricular branch of the vagus nerve (Fallgatter et al. 2003). To reduce cardiac side-effects, electrodes were placed on the left ear because vagal fibers to the heart are supposed to originate from the right side (Nemeroff et al. 2006). tVNS was delivered as trains lasting 30 s and composed by 600 pulses (intra-train pulse frequency = 20 Hz; pulse duration = 0.3 ms) repeated every 5 min for 60 min. These parameters were chosen accordingly to previous studies in animals (Sun et al. 2012) and humans (Binnie 2000; Sackeim et al. 2001). The intensity of stimulation was individually adjusted to a level ranging above the detection threshold and below the pain threshold. In this range, when possible, we chose an intensity of 8 mA as suggested by Polak et al. (2009) who demonstrated that such intensity is able to activate the vagus brainstem nuclei without perception of pain.
Methods Safety evaluation Experimental design This is a randomized placebo-controlled double-blind study. Real tVNS was performed at left external acoustic meatus, while sham stimulation was performed at left ear lobe. All subjects underwent both real and sham tVNS for 60 min. The order by which the conditions were administered was pseudo-randomized across participants. Interval between sessions was kept above 48 h. Participants were blind to conditions. The electrophysiological parameters under evaluation were tested before (baseline), and 60 min after the beginning of the exposure to tVNS or to sham stimulation. Vital parameters, such as heart rate (HR) and blood pressure (BP), were monitored throughout the whole duration of the stimulation.
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Even if tVNS was performed on left side, a potential harmful effect on cardiovascular function cannot be ruled out a priori. Therefore, during the stimulation, subjects were monitored by multimodal monitor that measures HR and BP. Such parameters were recorded every 15 min. Moreover, to test the tolerability of tVNS, subjects were questioned, every 5 min, about the presence of unpleasant sensations or other discomforts. Cortical excitability evaluation Magnetic stimulation was performed with a high-power Magstim 200 (Magstim Co., Whitland, Dyfed, UK). A figure-of-eight coil, with external loop diameter of 9 cm,
The effect of transcutaneous vagus nerve
was held over the motor cortex at the optimum scalp position to elicit motor evoked potentials (MEPs) in the contralateral first dorsal interosseous muscle (FDI). The induced current flowed in a posteroanterior direction. MEPs were recorded via two 9-mm diameter Ag–AgCl surface electrodes with the active electrode set over the motor point of the contralateral FDI and the reference set on the metacarpophalangeal joint of the index finger. EMG signal was amplified and filtered (bandwidth 3 Hz–3 kHz) by D360 amplifiers (Digitimer, Welwyn Garden City, Herts, UK). Data were collected on a computer with a sampling rate of 10 kHz per channel and stored for later off-line analysis using a CED 1401 A/D converter (Cambridge Electronic Design, Cambridge, UK).We evaluated motor thresholds (MTs), MEP amplitude, recruitment curves, and short interval intracortical inhibition (SICI) in right and left motor cortex. Such parameters were evaluated before (baseline) and 60 min after the tVNS. Resting motor threshold (RMT) was defined as the minimum stimulus intensity that produced a liminal MEP (about 50 lV in at least 50 % of ten trials) at rest. Active motor threshold (AMT) was defined as the minimum stimulus intensity that produced a liminal MEP (about 200 lV in at least 50 % of ten trials) during isometric contraction of the tested muscle. A constant level of voluntary contraction was maintained with reference to an oscilloscope display of the EMG signal in front of the subject. Auditory feedback of the EMG activity was also provided. RMT and AMT are given in percentage of maximum stimulator output (%MSO). We evaluated the recruitment curve of EMG responses using magnetic stimuli of increasing intensities. Recruitment curves were recorded with target muscles at rest and magnetic stimuli were applied at RMT, 10, 20, 30, 40, and 50 % of maximum stimulator output above RMT and 10 % of maximum stimulator output below RMT. Five stimuli were delivered at each intensity. MEP amplitude was evaluated using a stimulus intensity of 120 % RMT with the muscle at rest. Ten sweeps of the data were collected, and the mean peak-to-peak amplitude of the MEPs was calculated. SICI was studied using the technique of Kujirai et al. (1993). Two magnetic stimuli were given through the same stimulating coil over the motor cortex and the effect of the first (conditioning) stimulus on the second (test) stimulus was investigated. The conditioning stimulus was set at an intensity of 5 % of MSO below AMT. The intensity of the test stimulus was adjusted to elicit an unconditioned test MEP in the relaxed contralateral FDI of approximately 1 mV in peak-to-peak amplitude. Inter-stimulus intervals (ISIs) of 2 and 3 ms (for SICI) were investigated. Five stimuli were delivered at each ISI in pseudorandomized order. Subjects were provided with audio-visual feedback
of the EMG at high gain (50 lV/D) to assist them in maintaining complete muscular relaxation. The SICI data were averaged across the two inhibitory ISIs to obtain one grand mean single value for SICI. After tVNS, the intensity of the test stimulus was adjusted, whenever necessary, to ensure that the test MEP matched the amplitude to the baseline test MEP, as measured before stimulation. Statistical analysis All data are expressed as mean ± standard deviation if not differently reported. Instantaneous HR, systolic BP, diastolic BP, and mean BP were normally distributed (Kolgmorow-Smirnov test p [ 0.5). A generalized Linear Model (GLM) for repeated measures (5 times: basal value, after 15, 30, 45, 55 min) was implemented to assess the time effect (pre-stimulation vs post-stimulation) and the stimulation type effect (tVNS vs sham) on instantaneous HR, systolic BP, diastolic BP, and mean BP. A restricted GLM was designed to compare pre-stimulation values of basal condition with those of the last measure (60 min after). A GLM was designed to verify the combined effect of time, stimulation type and hemisphere on neurophysiological variables (RMT, AMT, MEP amplitude, SICI, slope of recruitment curve).
Results Effect of tVNS on cardiovascular variables Instantaneous HR, systolic BP, diastolic BP, and mean BP were not modified by tVNS, as revealed by the lack of
Table 1 The effect of tVNS on cardiovascular variables Source
Measure
F
Sig.
Stimulation type
HR
0.004
0.954
BPs
0.034
0.858
BPd
0.575
0.470
BPm HR
0.252 0.406
0.629 0.803
Stimulation
Stimulation 9 stimulation type
BPs
0.726
0.581
BPd
1.944
0.127
BPm
1.804
0.152
HR
1.337
0.278
BPs
2.292
0.081
BPd
0.774
0.550
BPm
1.218
0.323
HR heart rate, BPs systolic blood pressure, BPd diastolic blood pressure, BPm mean blood pressure
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F. Capone et al. Table 2 The effect of tVNS on cortical excitability Source
Measure
Time
RMT
1.714
0.223
AMT
0.078
0.787
MEP
0.201
0.664
SICI
0.161
0.698
I–O slope
0.708
0.422
RMT
0.056
0.818
AMT
0.885
0.371
MEP
0.012
0.916
SICI
3.629
0.089
Side
I–O slope Stimulation type
Fig. 1 The effect of tVNS on cardiovascular variables. HR heart rate (bpm), BPs systolic blood pressure (mmHg), BPd diastolic blood pressure (mmHg), BPm mean blood pressure (mmHg) Time 9 side
interaction between stimulation type and time effects (p [ 0.05, see Table 1). The restricted GLM including only the first and the last measurements did not show any effect of tVNS too (Fig. 1).
Time 9 stimulation type
Effect of tVNS on cortical excitability The analysis of MTs did not reveal any interaction either between time and stimulation type or among time, stimulation type and hemisphere. The univariate analysis in this GLM showed only a significant stimulation type interaction for both the rMT and aMT, because pre-stimulation and post-stimulation session of sham tVNS show higher values of those of real tVNS (Table 2). Time, stimulation type and hemisphere simple and combined effects did not affect MEP amplitude. SICI was affected by a triple interaction of time, stimulation type and hemisphere, showing an increased cortical inhibitory modulation of the right motor cortex after real tVNS (Fig. 2). Recordings in a representative subject are shown in Fig. 3. Neither simple nor double effects between these three factors were found for SICI. GLM analysis for repeated measures did not reveal any simple or compound effect of time, stimulation type and hemisphere on the slope of the recruitment curve (Table 2).
Discussion This is the first study that evaluated the effects of tVNS on cortical excitability in healthy subjects. We found a selective and pronounced increase of intracortical
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Side 9 stimulation type
Time 9 side 9 stimulation type
F
Sig.
0.584
0.464
RMT AMT
17.028 12.716
0.003* 0.006*
MEP
0.326
0.582
SICI
0.066
0.803
I–O slope
0.211
0.657
RMT
2.087
0.182
AMT
1.601
0.238
MEP
0.258
0.624
SICI
0.092
0.769
I–O slope
1.769
0.216
RMT
0.393
0.546
AMT
0.090
0.771
MEP
0.674
0.433
SICI
2.164
0.175
I–O slope
1.952
0.196
RMT
0.325
0.582
AMT MEP
0.095 1.340
0.764 0.277
SICI
0.125
0.732
I–O slope
0.954
0.354
RMT
0.042
0.842
AMT
3.418
0.098
MEP
0.023
0.884
SICI
5.306
0.047*
I–O slope
0.091
0.769
Repeated measure ANOVA generalized linear model, significance was set at p \ 0.05 (*) RMT resting motor threshold, AMT active motor threshold, MEP motor evoked potential, SICI short interval intra-cortical inhibition, I– O slope input–output curve slope
inhibition produced by paired-pulse stimulation (SICI), while other parameters (RMT, AMT, MEP amplitude, and recruitment curves) were not modified. Sham stimulation had no effect. Moreover, the evaluation of cardiovascular parameters confirms that tVNS is a safe and well-tolerated procedure in humans. Indeed, instantaneous HR, systolic BP, diastolic BP, and mean BP were not significantly modified by tVNS.
The effect of transcutaneous vagus nerve
Fig. 2 The effect of tVNS on cortical excitability. %MSO percentage of maximal stimulator output, RMT resting motor threshold, AMT active motor threshold, MEP motor evoked potential, SICI short
interval intra-cortical inhibition. Asterisk repeated measure ANOVA revealed a triple interaction of time 9 side 9 stimulation type
Taken together, these findings confirm that tVNS is a reliable method to stimulate vagus nerve and support the hypothesis that the mechanisms behind tVNS action are mostly the same of traditional VNS. Indeed, the effect on SICI showed in the present paper is quite similar to the effect of an implanted vagus nerve stimulator in epileptic patients, formerly described by Di Lazzaro and colleagues (Fallgatter et al. 2003). The existence of a tight analogy between the effect on brain activity of traditional and transcutaneous VNS, perfectly fits with previous neuroimaging findings: Kraus et al. (2007) demonstrated that the changes in fMRI brain activation induced by tVNS strongly resemble those observed with invasive VNS.
On top of that, the present study provides original data about the possible mechanism of action of tVNS by using the innovative approach of paired-pulse TMS. SICI is a cortical phenomenon (Di Lazzaro et al. 1998) strongly related to the activity of GABA-A inhibitory circuits of motor cortex (Di Lazzaro et al. 2000). Therefore, our data suggest that one action of tVNS could be the increase in GABAergic cortical activity. This hypothesis is consistent with the results of previous studies. First of all, BenMenachem et al. (1995) found increased GABA levels in cerebrospinal fluid during VNS. Moreover, Kraus et al. (2007) exploited fMRI to investigate the influences of tVNS on motor cortex activity. Interestingly, they found a
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F. Capone et al. Fig. 3 The effect of tVNS on SICI in a representative subject. Traces on the left show the average (of 5 trials each) of EMG responses evoked in the FDI by cortical stimulation alone and cortical stimulation conditioned by a cortical stimulus subthreshold for motor responses (5 % of maximum magnetic stimulator output below active motor threshold) given 2–3 ms earlier. Traces on the right show recordings after tVNS. After tVNS, SICI is increased
significant BOLD signal increase in the precentral gyrus of both sides induced by left tVNS. Conversely, the effect on cortical excitability we report is limited to the contralateral motor cortex. A possible reason that may account for this difference could be related with the different physiological mechanisms of which the two methods are more informative, as suggested by a recent paper on transcranial direct current stimulation (Antal et al. 2011). In particular, SICI could reflect specific changes in GABA-A inhibitory circuits while the BOLD signal could represent a general measure of synaptic activity. In spite of that, the alternative hypothesis that sees such differences related to different employed paradigms of tVNS cannot be definitely ruled out. It may be argued that the effect of tVNS on cortical excitability showed in the present paper could be indirectly mediated by the subcortical activation of brainstem structures. In particular, the nucleus of the tractus solitarius projects to the locus coeruleus and to the raphe nuclei. The interaction with these pontine and medullary nuclei, which provide widespread noradrenergic and serotonergic innervation to the neocortex, seems potentially relevant to explain VNS mechanisms (Groves and Brown 2005). Indeed, since SICI is also influenced by both noradrenergic (Korchounov et al. 2003) and serotonergic (Langguth et al. 2009) neurotransmission, the effects of tVNS on cortical excitability, described in the present paper, could be mediated by these neurotransmitter ascending systems. Finally, our study could have important implications for clinical applications of tVNS. Indeed, given that the inhibitory effect we report is limited to the contralateral motor cortex and tVNS is commonly used only on the left
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vagus nerve, it could be speculated that the present findings reduce the target for the clinical applications of this technique to epileptic patients with the focus located only in the right hemisphere. Against this conclusion, it should be considered that the tool we exploited to test tVNS effects has undoubtedly a limited vision, insofar the enhancement of GABAergic activity in cortex, as expressed by the increase in SICI, is only one possible mechanism of tVNS effect. For these effects, indeed, different sites (thalamus, brainstem structures) and mechanisms (reduced repetitive firing, neuromodulation of synaptic vesicular release) have been proposed so far (Bergey 2013). To better clarify this point, we auspicate additional, specifically-designed future studies. In conclusion, this study confirms that tVNS is a safe, reliable and effective way to stimulate vagus nerve and provides innovative data about the possible mechanisms of action of this procedure. Future research is warranted to explore the therapeutic implications of these findings on a wide range of neuropsychiatric disorders.
References Antal A, Polania R, Schmidt-Samoa C, Dechent P, Paulus W (2011) Transcranial direct current stimulation over the primary motor cortex during fMRI. Neuroimage 55:590–596 Ben-Menachem E (2002) Vagus-nerve stimulation for the treatment of epilepsy. Lancet Neurol 1:477–482 Ben-Menachem E, Hamberger A, Hedner T, Hammond EJ, Uthman BM, Slater J et al (1995) Effects of vagus nerve stimulation on amino acids and other metabolites in the CSF of patients with partial seizures. Epilepsy Res 20:221–227
The effect of transcutaneous vagus nerve Bergey GK (2013) Neurostimulation in the treatment of epilepsy. Exp Neurol 244:87–95 Binnie CD (2000) Vagus nerve stimulation for epilepsy: a review. Seizure 9:161–169 Di Lazzaro V, Restuccia D, Oliviero A, Profice P, Ferrara L, Insola A et al (1998) Magnetic transcranial stimulation at intensities below active motor threshold activates intracortical inhibitory circuits. Exp Brain Res 2:265–268 Di Lazzaro V, Oliviero A, Meglio M, Cioni B, Tamburrini G, Tonali P et al (2000) Direct demonstration of the effect of lorazepamon the excitability of the human motor cortex. Clin Neurophysiol 111:794–799 Di Lazzaro V, Oliviero A, Pilato F, Saturno E, Dileone M, Meglio M et al (2004) Effects of vagus nerve stimulation on cortical excitability in epileptic patients. Neurology 12:2310–2312 Fallgatter AJ, Neuhauser B, Herrmann MJ, Ehlis AC, Wagener A, Scheuerpflug P et al (2003) Far field potentials from the brain stem after transcutaneous vagus nerve stimulation. J Neural Transm 12:1437–1443 Grimm S, Bajbouj M (2010) Efficacy of vagus nerve stimulation in the treatment of depression. Expert Rev Neurother 10:87–92 Groves DA, Brown VJ (2005) Vagal nerve stimulation: a review of its applications and potential mechanisms that mediate its clinical effects. Neurosci Biobehav Rev 3:493–500 He W, Jing X, Wang X, Rong P, Li L, Shi H et al (2013) Transcutaneous auricular vagus nerve stimulation as a complementary therapy for pediatric epilepsy: a pilot trial. Epilepsy Behav 3:343–346 Hein E, Nowak M, Kiess O, Biermann T, Bayerlein K, Kornhuber J et al (2013) Auricular transcutaneous electrical nerve stimulation in depressed patients: a randomized controlled pilot study. J Neural Transm 5:821–827 Korchounov A, Ilic TV, Ziemann U (2003) The alpha2-adrenergic agonist guanfacine reduces excitability of human motor cortex through disfacilitation and increase of inhibition. Clin Neurophysiol 114:1834–1840 Kraus T, Ho¨sl K, Kiess O, Schanze A, Kornhuber J, Forster C (2007) BOLD fMRI deactivation of limbic and temporal brain structures and mood enhancing effect by transcutaneous vagus nerve stimulation. J Neural Transm 11:1485–1493 Kraus T, Kiess O, Ho¨sl K, Terekhin P, Kornhuber J, Forster C (2013) CNS BOLD fMRI effects of sham-controlled transcutaneous
electrical nerve stimulation in the left outer auditory canal: a pilot study. Brain Stimul 5:798–804 Kujirai T, Caramia MD, Rothwell JC, Day BL, Thompson PD, Ferbert A et al (1993) Corticocortical inhibition in human motor cortex. J Physiol 471:501–519 Langguth B, Sand P, Marek R, Landgrebe M, Frank E, Hajak G et al (2009) Allelic variation in the serotonin transporter promoter modulates cortical excitability. Biol Psychiatry 3:283–286 Lehtima¨ki J, Hyva¨rinen P, Ylikoski M, Bergholm M, Ma¨kela¨ JP, Aarnisalo A et al (2013) Transcutaneous vagus nerve stimulation in tinnitus: a pilot study. Acta Otolaryngol 4:378–382 Nemeroff CB, Mayberg HS, Krahl SE, McNamara J, Frazer A, Henry TR et al (2006) VNS therapy in treatment-resistant depression: clinical evidence and putative neurobiological mechanisms. Neuropsychopharmacology 7:1345–1355 Polak T, Markulin F, Ehlis AC, Langer JB, Ringel TM, Fallgatter AJ (2009) Far field potentials from brain stem after transcutaneous vagus nerve stimulation: optimization of stimulation and recording parameters. J Neural Transm 10:1237–1242 Rong PJ, Fang JL, Wang LP, Meng H, Liu J, Ma YG et al (2012) Transcutaneous vagus nerve stimulation for the treatment of depression: a study protocol for a double blinded randomized clinical trial. BMC Complement Altern Med 12:255 Rong P, Liu A, Zhang J, Wang Y, Yang A, Li L et al (2014) An alternative therapy for drug-resistant epilepsy: transcutaneous auricular vagus nerve stimulation. Chin Med J (Engl) 2:300–304 Sackeim HA, Rush AJ, George MS, Marangell LB, Husain MM, Nahas Z, Johnson CR, Seidman S, Giller C, Haines S, Simpson RK Jr, Goodman RR (2001) Vagus nerve stimulation (VNS) for treatment-resistant depression: efficacy, side effects, and predictors of outcome. Neuropsychopharmacology 5:713–728 Stefan H, Kreiselmeyer G, Kerling F, Kurzbuch K, Rauch C, Heers M et al (2012) Transcutaneous vagus nerve stimulation (t-VNS) in pharmacoresistant epilepsies: a proof of concept trial. Epilepsia 7:115–118 Sun Z, Baker W, Hiraki T, Greenberg JH (2012) The effect of right vagus nerve stimulation on focal cerebral ischemia: an experimental study in the rat. Brain Stimul 1:1–10 Ventureyra EC (2000) Transcutaneous vagus nerve stimulation for partial onset seizure therapy. A new concept. Childs Nerv Syst 16:101–102
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NEUROLOGY AND PRECLINICAL NEUROLOGICAL STUDIES - ORIGINAL ARTICLE
The effect of transcutaneous vagus nerve stimulation on cortical excitability Fioravante Capone • Giovanni Assenza • Giovanni Di Pino Gabriella Musumeci • Federico Ranieri • Lucia Florio • Carmen Barbato • Vincenzo Di Lazzaro
•
Received: 27 May 2014 / Accepted: 16 August 2014 Ó Springer-Verlag Wien 2014
Abstract There is great interest about the therapeutic potentialities of transcutaneous vagus nerve stimulation (tVNS) applied to neuropsychiatric disorders. However, the mechanisms of action of tVNS and its impact on cortical excitability are unclear. To this regard, transcranial magnetic stimulation (TMS) can be useful because it is able of evaluating non-invasively excitatory and inhibitory circuitry of the human cortex. Aim of the present study is to investigate the effects of tVNS on cerebral cortex excitability in healthy volunteers by means of TMS. Ten healthy subjects participated in this randomized placebo-controlled double-blind study. Real tVNS was administered at left external acoustic meatus, while sham stimulation was performed at left ear lobe, both of them for 60 min. We evaluated motor thresholds, motor evoked potential amplitude, recruitment curves, and short-interval intracortical inhibition (SICI) in right and left motor cortex. Such parameters were evaluated before and 60 min after the exposure to tVNS, for both the real and the sham stimulation. Cardiovascular parameters were monitored during the stimulation. A generalized linear model for repeated measures was implemented to assess the effect of time and stimulation type on cardiovascular and neurophysiological variables. SICI, a double-pulse TMS paradigm informative of
F. Capone (&) G. Assenza G. Di Pino G. Musumeci F. Ranieri L. Florio C. Barbato V. Di Lazzaro Institute of Neurology, Campus Bio-Medico University, Via Alvaro del Portillo 200, 00128 Rome, Italy e-mail: [email protected] F. Capone G. Di Pino G. Musumeci F. Ranieri L. Florio V. Di Lazzaro Fondazione Alberto Sordi, Research Institute for Ageing, Rome, Italy
GABA-A activity, was significantly increased in right motor cortex after real tVNS. Other neurophysiological parameters, as well as cardiovascular variables, remained unchanged. Our findings confirm that tVNS is a safe and effective way to stimulate vagus nerve and provide innovative data about the possible mechanisms of action that supports the potential therapeutic application of this technique. Keywords Transcutaneous vagus nerve stimulation Cortical excitability Transcranial magnetic stimulation Intracortical inhibition
Introduction Vagus nerve stimulation (VNS) is approved as adjunctive treatment for refractory epilepsy and depression (BenMenachem 2002; Grimm and Bajbouj 2010) and its application is currently under investigation for a wide range of neuropsychiatric diseases (Groves and Brown 2005). However, the diffusion of this technique has been hampered by its invasiveness: indeed, VNS requires the surgical implantation of a stimulator connected to an electrode located along the cervical branch of the vagus nerve. Recently, to reduce the discomfort of traditional VNS and its possible side-effects, an alternative strategy to stimulate non-invasively the vagus nerve has been proposed (Ventureyra 2000). This technique consists of transcutaneous stimulation (tVNS) of the external auditory channel at the inner side of the tragus. Such stimulation activates the auricular branch of the vagus nerve and via this pathway, the nuclei of the nerve located in the brainstem. In particular, the nucleus of the tractus solitarius is a crucial structure because it is the target of the main
123
F. Capone et al.
component of afferent vagal inputs and because it projects widespread toward a variety of key brain areas, including thalamus, amygdala, hippocampus, and neocortex (Kraus et al. 2007). The effectiveness of tVNS has been demonstrated in animal models (He et al. 2013) and it has been also confirmed in humans by functional magnetic resonance imaging (fMRI) studies showing that the patterns of brain activation induced by tVNS are quite similar to those observed during invasive VNS (Kraus et al. 2007, 2013). These observations raised the attention toward the therapeutic applications of tVNS, to such an extent that this technique has been evaluated for the treatment of several neuropsychiatric disorders such as epilepsy (Rong et al. 2014; He et al. 2013; Stefan et al. 2012), depression (Rong et al. 2012; Hein et al. 2013), and tinnitus (Lehtima¨ki et al. 2013). Nevertheless, the mechanisms of action of tVNS remain unclear especially because, so far, little is known about the functional connections between the vagus brain stem nuclei and the cortical structures (Fallgatter et al. 2003). Indeed, the study of tVNS influences on the cortical functional dynamic is worthwhile. To this regard, the excitatory and inhibitory circuits of the human cortex can be evaluated non-invasively by means of transcranial magnetic stimulation (TMS). TMS investigation is well-suited to test the mechanisms of action of VNS, as previously showed by Di Lazzaro and coworkers for traditional VNS. They reported an enhanced activity of intracortical inhibitory circuits, as assessed by a paired-pulse TMS protocol, in five patients affected by epilepsy (Di Lazzaro et al. 2004). Along this line, aim of the present study is to evaluate the effects of tVNS on cerebral cortex excitability in healthy volunteers, by means of TMS.
Subjects Ten (3 males and 7 females) healthy volunteers [mean age 30.2 ± 3.6 (SD) years] participated in the experiments. All of them gave their informed consent. All the subjects were naive to tVNS. The study was performed accordingly to the Declaration of Helsinki and was approved by the Local Ethics Committee. tVNS procedure The bipolar stimulation of the auricular branch of the vagus nerve was performed through an electric stimulator (Twister—EBM) and two Ag–AgCl electrodes (5 mm in diameter), placed in the left external acoustic meatus at the inner side of the tragus. The distance between the cathode and anode was 5 mm. For sham stimulation, electrodes were attached to the left ear lobe, an anatomical area that is known to be outside the innervation of the auricular branch of the vagus nerve (Fallgatter et al. 2003). To reduce cardiac side-effects, electrodes were placed on the left ear because vagal fibers to the heart are supposed to originate from the right side (Nemeroff et al. 2006). tVNS was delivered as trains lasting 30 s and composed by 600 pulses (intra-train pulse frequency = 20 Hz; pulse duration = 0.3 ms) repeated every 5 min for 60 min. These parameters were chosen accordingly to previous studies in animals (Sun et al. 2012) and humans (Binnie 2000; Sackeim et al. 2001). The intensity of stimulation was individually adjusted to a level ranging above the detection threshold and below the pain threshold. In this range, when possible, we chose an intensity of 8 mA as suggested by Polak et al. (2009) who demonstrated that such intensity is able to activate the vagus brainstem nuclei without perception of pain.
Methods Safety evaluation Experimental design This is a randomized placebo-controlled double-blind study. Real tVNS was performed at left external acoustic meatus, while sham stimulation was performed at left ear lobe. All subjects underwent both real and sham tVNS for 60 min. The order by which the conditions were administered was pseudo-randomized across participants. Interval between sessions was kept above 48 h. Participants were blind to conditions. The electrophysiological parameters under evaluation were tested before (baseline), and 60 min after the beginning of the exposure to tVNS or to sham stimulation. Vital parameters, such as heart rate (HR) and blood pressure (BP), were monitored throughout the whole duration of the stimulation.
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Even if tVNS was performed on left side, a potential harmful effect on cardiovascular function cannot be ruled out a priori. Therefore, during the stimulation, subjects were monitored by multimodal monitor that measures HR and BP. Such parameters were recorded every 15 min. Moreover, to test the tolerability of tVNS, subjects were questioned, every 5 min, about the presence of unpleasant sensations or other discomforts. Cortical excitability evaluation Magnetic stimulation was performed with a high-power Magstim 200 (Magstim Co., Whitland, Dyfed, UK). A figure-of-eight coil, with external loop diameter of 9 cm,
The effect of transcutaneous vagus nerve
was held over the motor cortex at the optimum scalp position to elicit motor evoked potentials (MEPs) in the contralateral first dorsal interosseous muscle (FDI). The induced current flowed in a posteroanterior direction. MEPs were recorded via two 9-mm diameter Ag–AgCl surface electrodes with the active electrode set over the motor point of the contralateral FDI and the reference set on the metacarpophalangeal joint of the index finger. EMG signal was amplified and filtered (bandwidth 3 Hz–3 kHz) by D360 amplifiers (Digitimer, Welwyn Garden City, Herts, UK). Data were collected on a computer with a sampling rate of 10 kHz per channel and stored for later off-line analysis using a CED 1401 A/D converter (Cambridge Electronic Design, Cambridge, UK).We evaluated motor thresholds (MTs), MEP amplitude, recruitment curves, and short interval intracortical inhibition (SICI) in right and left motor cortex. Such parameters were evaluated before (baseline) and 60 min after the tVNS. Resting motor threshold (RMT) was defined as the minimum stimulus intensity that produced a liminal MEP (about 50 lV in at least 50 % of ten trials) at rest. Active motor threshold (AMT) was defined as the minimum stimulus intensity that produced a liminal MEP (about 200 lV in at least 50 % of ten trials) during isometric contraction of the tested muscle. A constant level of voluntary contraction was maintained with reference to an oscilloscope display of the EMG signal in front of the subject. Auditory feedback of the EMG activity was also provided. RMT and AMT are given in percentage of maximum stimulator output (%MSO). We evaluated the recruitment curve of EMG responses using magnetic stimuli of increasing intensities. Recruitment curves were recorded with target muscles at rest and magnetic stimuli were applied at RMT, 10, 20, 30, 40, and 50 % of maximum stimulator output above RMT and 10 % of maximum stimulator output below RMT. Five stimuli were delivered at each intensity. MEP amplitude was evaluated using a stimulus intensity of 120 % RMT with the muscle at rest. Ten sweeps of the data were collected, and the mean peak-to-peak amplitude of the MEPs was calculated. SICI was studied using the technique of Kujirai et al. (1993). Two magnetic stimuli were given through the same stimulating coil over the motor cortex and the effect of the first (conditioning) stimulus on the second (test) stimulus was investigated. The conditioning stimulus was set at an intensity of 5 % of MSO below AMT. The intensity of the test stimulus was adjusted to elicit an unconditioned test MEP in the relaxed contralateral FDI of approximately 1 mV in peak-to-peak amplitude. Inter-stimulus intervals (ISIs) of 2 and 3 ms (for SICI) were investigated. Five stimuli were delivered at each ISI in pseudorandomized order. Subjects were provided with audio-visual feedback
of the EMG at high gain (50 lV/D) to assist them in maintaining complete muscular relaxation. The SICI data were averaged across the two inhibitory ISIs to obtain one grand mean single value for SICI. After tVNS, the intensity of the test stimulus was adjusted, whenever necessary, to ensure that the test MEP matched the amplitude to the baseline test MEP, as measured before stimulation. Statistical analysis All data are expressed as mean ± standard deviation if not differently reported. Instantaneous HR, systolic BP, diastolic BP, and mean BP were normally distributed (Kolgmorow-Smirnov test p [ 0.5). A generalized Linear Model (GLM) for repeated measures (5 times: basal value, after 15, 30, 45, 55 min) was implemented to assess the time effect (pre-stimulation vs post-stimulation) and the stimulation type effect (tVNS vs sham) on instantaneous HR, systolic BP, diastolic BP, and mean BP. A restricted GLM was designed to compare pre-stimulation values of basal condition with those of the last measure (60 min after). A GLM was designed to verify the combined effect of time, stimulation type and hemisphere on neurophysiological variables (RMT, AMT, MEP amplitude, SICI, slope of recruitment curve).
Results Effect of tVNS on cardiovascular variables Instantaneous HR, systolic BP, diastolic BP, and mean BP were not modified by tVNS, as revealed by the lack of
Table 1 The effect of tVNS on cardiovascular variables Source
Measure
F
Sig.
Stimulation type
HR
0.004
0.954
BPs
0.034
0.858
BPd
0.575
0.470
BPm HR
0.252 0.406
0.629 0.803
Stimulation
Stimulation 9 stimulation type
BPs
0.726
0.581
BPd
1.944
0.127
BPm
1.804
0.152
HR
1.337
0.278
BPs
2.292
0.081
BPd
0.774
0.550
BPm
1.218
0.323
HR heart rate, BPs systolic blood pressure, BPd diastolic blood pressure, BPm mean blood pressure
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F. Capone et al. Table 2 The effect of tVNS on cortical excitability Source
Measure
Time
RMT
1.714
0.223
AMT
0.078
0.787
MEP
0.201
0.664
SICI
0.161
0.698
I–O slope
0.708
0.422
RMT
0.056
0.818
AMT
0.885
0.371
MEP
0.012
0.916
SICI
3.629
0.089
Side
I–O slope Stimulation type
Fig. 1 The effect of tVNS on cardiovascular variables. HR heart rate (bpm), BPs systolic blood pressure (mmHg), BPd diastolic blood pressure (mmHg), BPm mean blood pressure (mmHg) Time 9 side
interaction between stimulation type and time effects (p [ 0.05, see Table 1). The restricted GLM including only the first and the last measurements did not show any effect of tVNS too (Fig. 1).
Time 9 stimulation type
Effect of tVNS on cortical excitability The analysis of MTs did not reveal any interaction either between time and stimulation type or among time, stimulation type and hemisphere. The univariate analysis in this GLM showed only a significant stimulation type interaction for both the rMT and aMT, because pre-stimulation and post-stimulation session of sham tVNS show higher values of those of real tVNS (Table 2). Time, stimulation type and hemisphere simple and combined effects did not affect MEP amplitude. SICI was affected by a triple interaction of time, stimulation type and hemisphere, showing an increased cortical inhibitory modulation of the right motor cortex after real tVNS (Fig. 2). Recordings in a representative subject are shown in Fig. 3. Neither simple nor double effects between these three factors were found for SICI. GLM analysis for repeated measures did not reveal any simple or compound effect of time, stimulation type and hemisphere on the slope of the recruitment curve (Table 2).
Discussion This is the first study that evaluated the effects of tVNS on cortical excitability in healthy subjects. We found a selective and pronounced increase of intracortical
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Side 9 stimulation type
Time 9 side 9 stimulation type
F
Sig.
0.584
0.464
RMT AMT
17.028 12.716
0.003* 0.006*
MEP
0.326
0.582
SICI
0.066
0.803
I–O slope
0.211
0.657
RMT
2.087
0.182
AMT
1.601
0.238
MEP
0.258
0.624
SICI
0.092
0.769
I–O slope
1.769
0.216
RMT
0.393
0.546
AMT
0.090
0.771
MEP
0.674
0.433
SICI
2.164
0.175
I–O slope
1.952
0.196
RMT
0.325
0.582
AMT MEP
0.095 1.340
0.764 0.277
SICI
0.125
0.732
I–O slope
0.954
0.354
RMT
0.042
0.842
AMT
3.418
0.098
MEP
0.023
0.884
SICI
5.306
0.047*
I–O slope
0.091
0.769
Repeated measure ANOVA generalized linear model, significance was set at p \ 0.05 (*) RMT resting motor threshold, AMT active motor threshold, MEP motor evoked potential, SICI short interval intra-cortical inhibition, I– O slope input–output curve slope
inhibition produced by paired-pulse stimulation (SICI), while other parameters (RMT, AMT, MEP amplitude, and recruitment curves) were not modified. Sham stimulation had no effect. Moreover, the evaluation of cardiovascular parameters confirms that tVNS is a safe and well-tolerated procedure in humans. Indeed, instantaneous HR, systolic BP, diastolic BP, and mean BP were not significantly modified by tVNS.
The effect of transcutaneous vagus nerve
Fig. 2 The effect of tVNS on cortical excitability. %MSO percentage of maximal stimulator output, RMT resting motor threshold, AMT active motor threshold, MEP motor evoked potential, SICI short
interval intra-cortical inhibition. Asterisk repeated measure ANOVA revealed a triple interaction of time 9 side 9 stimulation type
Taken together, these findings confirm that tVNS is a reliable method to stimulate vagus nerve and support the hypothesis that the mechanisms behind tVNS action are mostly the same of traditional VNS. Indeed, the effect on SICI showed in the present paper is quite similar to the effect of an implanted vagus nerve stimulator in epileptic patients, formerly described by Di Lazzaro and colleagues (Fallgatter et al. 2003). The existence of a tight analogy between the effect on brain activity of traditional and transcutaneous VNS, perfectly fits with previous neuroimaging findings: Kraus et al. (2007) demonstrated that the changes in fMRI brain activation induced by tVNS strongly resemble those observed with invasive VNS.
On top of that, the present study provides original data about the possible mechanism of action of tVNS by using the innovative approach of paired-pulse TMS. SICI is a cortical phenomenon (Di Lazzaro et al. 1998) strongly related to the activity of GABA-A inhibitory circuits of motor cortex (Di Lazzaro et al. 2000). Therefore, our data suggest that one action of tVNS could be the increase in GABAergic cortical activity. This hypothesis is consistent with the results of previous studies. First of all, BenMenachem et al. (1995) found increased GABA levels in cerebrospinal fluid during VNS. Moreover, Kraus et al. (2007) exploited fMRI to investigate the influences of tVNS on motor cortex activity. Interestingly, they found a
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F. Capone et al. Fig. 3 The effect of tVNS on SICI in a representative subject. Traces on the left show the average (of 5 trials each) of EMG responses evoked in the FDI by cortical stimulation alone and cortical stimulation conditioned by a cortical stimulus subthreshold for motor responses (5 % of maximum magnetic stimulator output below active motor threshold) given 2–3 ms earlier. Traces on the right show recordings after tVNS. After tVNS, SICI is increased
significant BOLD signal increase in the precentral gyrus of both sides induced by left tVNS. Conversely, the effect on cortical excitability we report is limited to the contralateral motor cortex. A possible reason that may account for this difference could be related with the different physiological mechanisms of which the two methods are more informative, as suggested by a recent paper on transcranial direct current stimulation (Antal et al. 2011). In particular, SICI could reflect specific changes in GABA-A inhibitory circuits while the BOLD signal could represent a general measure of synaptic activity. In spite of that, the alternative hypothesis that sees such differences related to different employed paradigms of tVNS cannot be definitely ruled out. It may be argued that the effect of tVNS on cortical excitability showed in the present paper could be indirectly mediated by the subcortical activation of brainstem structures. In particular, the nucleus of the tractus solitarius projects to the locus coeruleus and to the raphe nuclei. The interaction with these pontine and medullary nuclei, which provide widespread noradrenergic and serotonergic innervation to the neocortex, seems potentially relevant to explain VNS mechanisms (Groves and Brown 2005). Indeed, since SICI is also influenced by both noradrenergic (Korchounov et al. 2003) and serotonergic (Langguth et al. 2009) neurotransmission, the effects of tVNS on cortical excitability, described in the present paper, could be mediated by these neurotransmitter ascending systems. Finally, our study could have important implications for clinical applications of tVNS. Indeed, given that the inhibitory effect we report is limited to the contralateral motor cortex and tVNS is commonly used only on the left
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vagus nerve, it could be speculated that the present findings reduce the target for the clinical applications of this technique to epileptic patients with the focus located only in the right hemisphere. Against this conclusion, it should be considered that the tool we exploited to test tVNS effects has undoubtedly a limited vision, insofar the enhancement of GABAergic activity in cortex, as expressed by the increase in SICI, is only one possible mechanism of tVNS effect. For these effects, indeed, different sites (thalamus, brainstem structures) and mechanisms (reduced repetitive firing, neuromodulation of synaptic vesicular release) have been proposed so far (Bergey 2013). To better clarify this point, we auspicate additional, specifically-designed future studies. In conclusion, this study confirms that tVNS is a safe, reliable and effective way to stimulate vagus nerve and provides innovative data about the possible mechanisms of action of this procedure. Future research is warranted to explore the therapeutic implications of these findings on a wide range of neuropsychiatric disorders.
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