The sympathetic and parasympathetic nervous systems are the main branches of the autonomic nervous system (ANS), and they dynamically control the visceral functions to maintain body homeostasis [1
]. A hallmark of ANS is its great ability to react to environmental challenges/stressors (i.e., postural change) in order to properly respond to the metabolic demands of the organism [1
Due to gravitational effects on body fluid distribution, several mechanisms are involved in maintaining cardiac output during orthostatic stress [3
]. Peripheral vasoconstriction and increased heart rate (HR) are the major cardiovascular responses to postural change. These modifications are part of the reflex response elicited by negative feedback mechanisms (e.g., arterial baroreflex and cardiopulmonary reflex) [3
]. Thus, with the fall of venous return, parasympathetic modulation decreases while sympathetic vasomotor activity increases progressively with the angle of body inclination [3
Autonomic dysfunction plays a key role in the onset and progression of many diseases, such as hypertension and heart failure [5
] and it has been shown that it is an independent prognostic factor for adverse cardiovascular outcome [7
]. In addition, autonomic responses to orthostatic stress have clinical implications in cardiovascular disease [9
]. In the study of Folino et al. [10
], heart failure patients with blunted autonomic responses to orthostatic stress showed poor prognosis [10
]. Thus, the ability of the ANS to react to evocative stimuli (e.g., orthostatic maneuver) can indicate a better prognosis in patients with heart failure.
A huge amount of literature has documented the beneficial effects of pharmacological and non-pharmacological treatments aimed at reducing the hyperadrenergic state in cardiovascular diseases, such as beta-blocker therapy and exercise training, respectively [11
]. However, very few options are available to increase vagal modulation, mostly non-pharmacological treatments (such as yoga and respiratory-based techniques) [15
]. In this sense, the possibility to directly and non-invasively modulate the vagus nerve using transcutaneous vagus nerve stimulation (tVNS) seems to be a promising therapy for cardiovascular and non-cardiovascular disorders [17
It has been documented that the auricular branch of the vagus nerve of humans projects to the nucleus tract solitarius (NTS), which is the first central relay of vagal afferents, and to other vagal projections in the brainstem and forebrain [19
]. By means of neuroimaging studies using functional magnetic resonance, Kraus et al. observed that BOLD signal in autonomic neuroregulatory pathways, as limbic structures and the brainstem, decreases during electrical stimulation of the left anterior auditory canal [20
To our knowledge, few studies have assessed the effects of tVNS on the autonomic nervous system in healthy individuals [21
]. In the study by Clancy et al., tVNS acutely reduced efferent sympathetic nerve traffic and sympathovagal balance [22
]. In addition, Antonino et al. also observed a reduction of cardiac sympathovagal balance, likely due to improved arterial baroreflex control of HR [21
]. However, the response of autonomic branches to an orthostatic challenge during tVNS in healthy subjects remains unknown. Thus, we tested the hypothesis that tVNS reduces HR and alters the responsivity of the autonomic nervous system to orthostatic stress in healthy subjects.
The physical and hemodynamic characteristics of the subjects are shown in Table 1
. Data of cardiac and peripheral autonomic control evaluated by linear and nonlinear tools in the resting condition during the baseline recording (Rest_tVNS off) or during tVNS application (Rest_tVNS on) are shown in Table 2
. As to cardiac autonomic control, although tVNS significantly decreased heart rate (HR) (63 (60–66) vs. 66 (61–68) bpm, p
< 0.01), no significant difference was observed in the relative contribution of the low-frequency and high-frequency components at spectral decomposition. By contrast, nonlinear analysis of the R–R interval showed a significant decrease in the frequency of the no variation pattern (0V%), a marker of cardiac sympathetic modulation (17 (5–20) vs. 18 (8–27) %, p
< 0.01). Similarly, when analyzing the effects of tVNS on peripheral autonomic control, the marker of peripheral sympathetic modulation (0V % SAP) was significantly reduced (17 (13–30) vs. 36 (14–47) %, p
< 0.01) (Table 2
The effects of tVNS on arterial baroreflex control of HR are reported in Table 3
. The respiratory rate was significantly coupled with only the HF component of the R–R interval (K2HF
> 85%), as indicated by a high degree of coherence between these variability signals in both conditions (Table 3
). tVNS did not induce any significant change in the cardiorespiratory coupling and baroreflex control.
The autonomic variables evaluated by means of linear and nonlinear approaches were similar in the resting condition when compared between experimental sessions (rest_control vs. rest_tVNS off, supplementary Tables S1–S3
As to the effects of the orthostatic maneuver, we observed a similar relative response (Ä%) during the control and tVNS sessions (Table 4
). In Figure 1
, we can observe that orthostatic stress increases cardiac sympathetic modulation (Figure 1
, Panel A) and decreases cardiac parasympathetic modulation (Figure 1
, Panel C,D), regardless of the stimulation. The response of the peripheral autonomic control to orthostatic stress evaluated by means of a nonlinear approach is shown in Figure 2
. Interestingly, the responsivity of the peripheral sympathetic modulation to orthostatic stress during tVNS was significantly higher compared to the response on the control day (p
= 0.03) (Figure 2
The main findings from the present study are that acute tVNS (1) reduces HR when compared to baseline, (2) decreases cardiac and peripheral sympathetic modulation in the rest condition, and, (3) increases the responsivity of the sympathetic vasomotor modulation to orthostatic change in young healthy subjects.
The vagal system plays a very important role in the regulation and homeostasis of several pathways. A few years ago, Tracey [35
] described the so-called “inflammatory reflex,” i.e., a neural circuit that is elicited by cytokines and activates the vagus nerve to suppress the release of pro-inflammatory cytokines. A large amount of literature has shown that vagal control plays a very important regulatory role for different biological systems, from inflammation to immunity and from the endocrine to cardiovascular systems. In addition, as shown by the elegant study by Weber et al. [36
], a low vagal tone was associated with altered post-stress recovery of different systems (cardiovascular, endocrine, and immune system), thus suggesting that vagal activity is a key homeostatic agent of body systems and vagal withdrawal is a risk factor for stress-related disorders [36
In this study, we focused on the cardiovascular effects of a new and non-invasive technique able to stimulate and modulate vagus nerves in an innovative way.
The reduction in resting HR reveals a direct effect of tVNS with important clinical implication. In this study, we observed that tVNS promoted a decrease greater than 4% in the resting HR. This result may represent the result of direct parasympathetic vagal activity elicited by the auricular stimulation. However, the mechanism by which tVNS—that is, neuromodulation and not a simple stimulation of neural fibers—reduces HR still needs to be elucidated.
Increased HR is a marker of dysautonomia and a reduction in HR is one of the therapeutic targets in several cardiovascular diseases. However, a recent study [8
] demonstrated that according to the parameters of tVNS, the HR effects are time dependent. In particular, by exploring a wide range of pulse widths and pulse frequencies during only one minute of stimulation, Badran et al. [37
] showed that the optimal parameter for stimulating heart rate is 500 µs, 25 Hz. Since we used a pulse width of 200 µs and a pulse frequency of 25 Hz during 10 minutes of stimulation, we observed that these parameters were sufficient to promote a reduction in heart rate in healthy subjects. However, we cannot exclude that different stimulation parameters may also differently affect the cardiac and peripheral autonomic control. Thus, tVNS is a promising adjuvant therapy for a lot of drug-refractory disorders (e.g., refractory hypertension); however, further studies are required to validate this hypothesis.
In the present study, we found no difference in the spectral parameters of the cardiovascular variabilities under resting conditions. However, the symbolic analysis was able to identify a reduction in cardiac sympathetic modulation promoted by tVNS with no changes in cardiac parasympathetic modulation. Another important new finding in our study is that tVNS reduces the sympathetic vasomotor modulation in healthy individuals in the resting condition. These effects of tVNS on cardiac and peripheral sympathetic modulation may explain at least in part the reduction in heart rate and variability in systolic arterial pressure. As all spectral indexes are useful only under conditions characterized by reciprocal changes in sympathetic and parasympathetic modulations, this tool may not be able to detect subtle changes in one of the autonomic branches induced by the tVNS acutely [31
]. In line with this conception, Guzzetti et al. proposed a nonlinear approach of HRV analysis (i.e., symbolic analysis) to quantify the prevalence of sympathetic or parasympathetic cardiac modulation in conditions in which the use of a linear HRV method is limited or even disputable [31
As far as we know, this is the first study that evaluates the cardiac autonomic control during tVNS by means of a nonlinear approach in young healthy subjects. Symbolic analysis has the potential to detect nonreciprocal changes in sympathetic and parasympathetic modulation or reciprocal changes with different magnitudes [31
]. A hallmark of the autonomic nervous system is its ability to induce variations on the target organ (i.e., heart, vessels). In the absence of autonomic dysfunction, the tonic and phasic activities of cardiovascular variabilities are coupled and synchronized in healthy individuals [2
We also investigated the effects of tVNS on arterial baroreflex control in both modulation ranges. In contrast to the findings of Antonino et al. [21
], we did not observe an increase in the gain of arterial baroreflex control of HR during tVNS in healthy subjects under the rest condition. Thus, we believe that the mechanism involved in the reduction of cardiac and peripheral sympathetic modulation promoted by tVNS was a direct effect on NTS. This hypothesis is supported by recent findings in the neuroimaging area. A recent study demonstrated that stimulation at the cymba conchae
produced a significantly stronger activation in the NTS when compared with other locations in the ear [38
]. A classic concept in autonomic neuroscience is that NTS is the first synaptic station of the afferent projections in the central nervous system, and it plays a key role in the modulation of the autonomic efferent activity directed to the cardiovascular system [39
]. In order to produce a proper autonomic response, information from several relay stations must be processed at the NTS level, where all the projections of many and complex neural networks are processed and then organized by means of hierarchical levels into different reflex responses [2
]. We observed significant changes in the sympathetic and not vagal modulation during tVNS in healthy subjects. Due to the absence of autonomic dysfunction, our data reveal the effect of tVNS on directly modulating the cardiac and peripheral sympathetic branch. Based on these findings, we could speculate that the stimulation of the auricular branch of the vagus nerve may activate and modulate not only efferent but also afferent fibers in physiological conditions. Thus, we hypothesized that tVNS acts as a neuro-modulator of ANS, directly at the brainstem level, possibly through a direct action on NTS. Other studies are warranted to confirm this hypothesis.
Besides the resting condition, the autonomic nervous system dynamically adjusts the functions of the cardiovascular system to ensure the adequate levels of cardiac output to meet the perfusion and metabolic requirements of the peripheral organ systems [1
]. In this sense, several authors have emphasized that the autonomic nervous system should also be evaluated under physiological stress with the aim of examining the complexities of neural regulation without artificially isolating the influence of the autonomic branches [31
This dynamic interaction during orthostatic change has been widely used in the investigation of possible autonomic dysfunctions [40
]. Thus, we believe that evaluation of the ANS response to physiological stress during tVNS can be recommended, but further ad hoc studies are needed especially in patients using drugs that act on ANS. As tVNS reduces the adrenergic efferent drive at rest, an increased response of the sympathetic vasomotor modulation to orthostatic challenge is an expected physiological response. It reveals the integrity and ability of the system to adapt and properly react to physiological stress. These findings have important clinical relevance.
This study has strengths and limitations. The major limitation of the present study is that these findings cannot be extrapolated to other populations such as older and/or diseased subjects. Due to the small sample size, another limitation of this study is that the non-Gaussian distributed variables make the use of statistical parametric tests unfeasible. However, it has several strengths. Firstly, we assessed the acute effects of tVNS in resting conditions and in response to an orthostatic challenge and this allowed us to evaluate the dynamics of cardiac and peripheral autonomic control before and after physiological stress under acute effects of tVNS. Finally, we used different tools to provide complementary information of ANS (i.e., spectral and symbolic analysis).