Initially one hundred subjects were screened and 52 were excluded considering different causes (smoking, hypothyroidism, use of drugs such as beta-blockers, immunosuppressants, or insulin). A total of 48 subjects underwent new laboratory evaluation, and after the results, 14 did not meet the criteria for diagnosis of MetS. Four patients decided to drop out the study before randomization. Therefore, 30 patients were included, and all then finished the protocol (Fig. 2).
At the beginning of the protocol, clinical, anthropometric, and laboratory data, did not differ between the two groups (p > 0.05) (Table 1).
Table 1
Baseline clinical and fasting laboratory profile of patients in control and the TAVNS groups at baseline.
| Control (n = 10) | TAVNS (n = 20) | p |
Gender (M/F) | 3/7 | 7/13 | |
Age | 46 ± 10 | 43 ± 9 | 0.473 |
Weight (Kg) | 104 ± 22 | 105 ± 18 | 0.960 |
AC (cm) | 122 ± 13 | 119 ± 12 | 0.544 |
BMI (Kg/m2) | 37 ± 6 | 37 ± 5 | 0.854 |
SBP (mmHg) | 135 ± 21 | 137 ± 21 | 0.844 |
DBP (mmHg) | 80 ± 10 | 81 ± 10 | 0.719 |
HR (bpm) | 74 ± 10 | 72 ± 7 | 0.659 |
Glucose (mg/dL) | 105 ± 17 | 100 ± 14 | 0.875 |
Total cholesterol (mg/dL) | 201 ± 49 | 197 ± 42 | 0.832 |
HDL-c (mg/dL) | 48 ± 12 | 46 ± 10 | 0.611 |
LDL-c (mg/dL) | 125 ± 43 | 123 ± 39 | 0.946 |
Triglycerides (mg/dL) | 130 ± 42 | 141 ± 38 | 0.936 |
AST/GOT (U/L) | 24 ± 8 | 23 ± 9 | 0.791 |
ALT/GPT (U/L) | 28 ± 14 | 25 ± 7 | 0.520 |
TSH (uIU/mL) | 2.2 ± 1.0 | 2.3 ± 1.6 | 0.822 |
T4 free (uIU/mL | 1.1 ± 0.2 | 1.1 ± 0.2 | 0.697 |
Insulin (mUI/mL) | 15 ± 7 | 17 ± 7 | 0.438 |
CRP (mg/dL) | 0.6 ± 0.4 | 0.6 ± 0.3 | 0.916 |
HOMA-IR | 3.7 ± 2,0 | 4.2 ± 1.7 | 0.754 |
AC = abdominal circumference; BMI = body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure; HR = heart rate; HDL = high density lipoproteins; LDL = low density lipoproteins; AST/GOT = aspartate aminotransferase; ALT/GPT = alanine aminotransferase; TSH: thyroid-stimulating hormone; T4: thyroxine; CRP = high sensitivity C reactive protein; HOMA-IR: homeostatic model assessment index. Values = mean+/-SD |
A single session of TAVNS reduces HR and improves sympathovagal balance
One of the objectives of this study was to evaluate the effects of a single TAVNS in patients with MetS, considered an acute effect. Thus, office SBP, DBP and HR measurements and beat-to beat blood pressure curves were registered at baseline and after a TAVNS 30-minute session (Table 2). No significant differences in both office and beat-to-beat recording were detected in SBP and DBP measures. However, the office and beat-to-beat analyses of HR showed a small but significant decrease after TAVNS (p = 0.022 and p = 0.011, respectively).
TAVNS also caused a significant increase in HF (nu) component suggesting an increase in vagus nerve activity (vagal modulation of the heart), and a decrease in the LF (nu) component suggesting a lower sympathetic activity. Consequently, the ratio LF/HF decreased pointing to an acute alteration (improvement) in the autonomic balance.
Table 2
Effect of a single TAVNS on hemodynamic parameters and heart rate variability components in MetS patients.
| TAVNS (n = 20) | |
| Baseline | After 30 min of TAVNS | p |
Office parameters | | | |
SBP (mmHg) | 137 ± 21 | 136 ± 22 | 0.859 |
DBP (mmHg) | 81 ± 10 | 80 ± 10 | 0.825 |
HR (bpm) | 72 ± 7 | 69 ± 8 | 0.022* |
Beat-to-beat registry | | | |
SBP (mmHg) | 138 ± 20 | 135 ± 23 | 0.267 |
DBP (mmHg) | 78 ± 8 | 78 ± 9 | 0.755 |
HR (bpm) | 70 ± 8 | 67 ± 8 | 0.011* |
HRV | | | |
VARR | 2749 ± 2793 | 3643 ± 4791 | 0.192 |
RMSSD (ms) | 41 ± 28 | 48 ± 42 | 0.196 |
LF abs (ms2) | 772 ± 839 | 1015 ± 1756 | 0.347 |
HF abs (ms2) | 896 ± 1362 | 1372 ± 2228 | 0.149 |
LF (nu) | 50 ± 13 | 45 ± 15 | 0.040* |
HF (nu) | 50 ± 13 | 55 ± 15 | 0.035* |
LF/HF ratio | 1.4 ± 0.9 | 1.1 ± 0.7 | 0.010* |
SBP = systolic blood pressure; DBP = diastolic blood pressure; HR = heart rate; VARR = total variance of RR interval; RMSSD = square root of the mean of the square of successive differences between adjacent RR intervals; LF abs = low frequency spectral power; HF abs high frequency spectral power; LF (nu) = low frequency in normalized units; HF (nu) = high frequency in normalized units. *p < 0.05 |
Alterations in autonomic neural regulations, such as increased sympathetic activity and/or reduced parasympathetic (vagus) nerve activity as indicated by HRV are not only a powerful and independent predictor of poor prognosis in patients with cardiovascular disease, but also a risk factor for mortality in healthy populations (32) and MetS patients (4, 5, 12, 13). Accordingly, using TAVNS to shift HRV towards increased parasympathetic/vagal activity may be beneficial in these disorders. The electrode was placed at the cymba conchae, an area of the outer ear with highest density afferent fibers within the auricular branch of the afferent vagus nerve (34) which project to the brainstem nucleus tractus solitarius (NTS), thereby providing a physiological substrate for TAVNS efficacy (35). The decrease in HR observed following TAVNS in the present study might be related to an increase in NTS neuronal activity, which subsequently activates signaling of efferent vagus neurons innervating the heart. In the frequency domain analysis of HRV, involving HF, LF and LF/HR ratio, significant differences were observed in the treatment group, which together confirm that a single session of TAVNS affected cardiac activity through changes in both sympathetic and parasympathetic components.
Robust pathophysiological evidence demonstrates that patients with MetS have an increase in cardiac sympathetic modulation with a greater sympathetic vasomotor drive (4, 5, 12, 13). Prior studies, which have investigated effects of vagus nerve stimulation on HRV, have generated various results depending on the study design, such as included population, the region of stimulation and its duration, and methods of HRV analysis. Some studies using TAVNS for shorter periods (5 or 10 min) in healthy subjects have also showed significant improvement in HRV parameters of parasympathetic activity (36, 37). However, regarding the LH component, it is suggested that prolonged TAVNS stimulation (30–60 min) is required to decrease its power. (38). Moreover, other studies using a similar protocol in young health men showed an increase in LF/HF ratio spontaneous baroreflex sensitivity (39, 40). In the present study, we demonstrated an improvement in several autonomic parameters with acute TAVNS in middle-aged MetS patients. Therefore, TAVNS may be an important intervention to rebalance of the autonomic modulation in MetS patients.
Repeated TANVS decreases blood pressure and heart rate and accentuates changes in HR and HRV without changing metabolic parameters
Comparing clinical data of both groups obtained at baseline (V1) and at 8 weeks of follow-up (V8) revealed a significant decrease in systolic and diastolic blood pressures and heart rate in patients that received TAVNS. No changes were detected in the control group (Table 3). Regarding laboratory evaluation, there were no differences detected comparing baseline and follow-up evaluation in both groups (data not shown).
Table 3
Comparison of clinical parameters of the control and TAVNS groups at baseline and after 8-weeks of follow-up.
| Control (n = 10) | TAVNS (n = 20) | |
| Baseline | Follow-up | Baseline | Follow-up | p |
Weight (Kg) | 104 ± 22 | 103 ± 22 | 105 ± 18 | 105 ± 18 | 0.172 |
AC (cm) | 122 ± 13 | 119 ± 15 | 119 ± 12 | 118 ± 11 | 0.332 |
BIM (Kg/m2) | 37 ± 6 | 36 ± 6 | 37 ± 5 | 37 ± 5 | 0.126 |
SBP(mmHg) | 135 ± 21 | 133 ± 21 | 137 ± 21 | 121 ± 11# | 0.028* |
DBP (mmHg) | 80 ± 10 | 83 ± 10 | 81 ± 10 | 77 ± 8# | 0.007* |
HR (bpm) | 74 ± 10 | 76 ± 10 | 72 ± 7 | 68 ± 8# | 0.034* |
HRV | | | | | |
VARR | 2137 ± 1988 | 2060 ± 2065 | 2749 ± 2793 | 3621 ± 4092 | 0.279 |
RMSSD (ms) | 34 ± 21 | 33 ± 24 | 41 ± 28 | 51 ± 41 | 0.181 |
LF abs (ms2) | 666 ± 662 | 576 ± 666 | 772 ± 839 | 710 ± 846 | 0.889 |
HF abs (ms2) | 595 ± 594 | 560 ± 677 | 896 ± 1362 | 1530 ± 2485 | 0.154 |
LF% | 34 ± 6 | 36 ± 9 | 32 ± 7 | 26 ± 8# | 0.037* |
HF% | 29 ± 14 | 28 ± 11 | 34 ± 14 | 43 ± 19# | 0.046* |
SBPV | | | | | |
Variance | 51 ± 34 | 32 ± 24 | 36 ± 28 | 33 ± 16 | 0.204 |
AC = abdominal circumference; BMI = body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure; HR = heart rate; HRV = heart rate variability; VARR = total variance of RR interval; RMSSD = square root of the mean of the square of successive differences between adjacent RR intervals; LF abs = low frequency spectral power; HF abs high frequency spectral power; SBPV = systolic Blood Pressure variability; Variance = total SBPV power; #p < 0.05; *p < 0.05
Additionally, there were significant changes in components of the HRV in the frequency domain and in the systolic blood pressure variability, comparing baseline to follow-up in patients treated with TAVNS. After 8 sessions of TAVNS there was a significant increase in HF % of HRV, indicating an increase in vagal modulation. Moreover, there was a significant decrease in LF%, pointing to a reduction in sympathetic modulation (Table 3). Likewise, the LF/HF ratio, a marker of the sympathovagal balance, was significantly decreased in the treatment group (1.4 ± 0.9 vs 0.8 ± 0.6) but not in the control group (1.5 ± 1.0 vs 1.6 ± 0.6) (Fig. 3A). Additionally, the LF% component of the systolic blood pressure variability component, which suggests sympathetic modulation of the vasculature, decreased in patients that received TAVNS for 8 weeks (36 ± 13 vs 28 ± 13%) with no change in the control group. (27 ± 11 vs 30 ± 9%) (Fig. 3B).
To our knowledge, the present study is the first to investigate the effects of TAVNS once a week of for 8 weeks in MetS patients. The results indicate a beneficial TAVNS effect on cardiovascular autonomic function associated with a further decrease in blood pressure and heart rate. A previous study found that 15-min TAVNS administered every day during two weeks in healthy adults (> 55 years) was associated with increased vagally mediated HRV indexes and improved baroreflex sensitivity at rest. Participants with higher LF/HF at baseline showed a greater reduction in LF/HF at the end of the protocol (41).
In the current study, we observed a lower LF % component of SBPV, a variable that provides information about the contribution of sympathetic activity to vasomotor tone. Specifically, the reduction in LF% SBPV component detected in MetS patients points to reduced systemic sympathetic activity in patients receiving TAVNS. In agreement with this finding, muscle sympathetic nerve activity (MSNA) record is considered a direct measurement of sympathetic activity to the vessels and a previous study demonstrated a decrease MSNA recording in response to a 15 min-stimulation of the tragus of healthy subjects, which reinforce our finding (17). Additionally, studies have indicated that acute TAVNS neuromodulation ameliorates left ventricular strain (38) and 6 months of VNS reduced atrial fibrillation events (42). The antiarrhythmic activity may be a consequence of the TAVNS-mediated restoration of the tonic vagal inhibition over the hyperactivity of the intrinsic cardiac nervous system, ultimately resulting in an electrical and autonomic remodeling phenomenon (32, 38). In view of these observations, long-term TAVNS may be a therapeutic intervention to suppress systemic sympathetic activity and its potential adverse consequences on blood pressure.
Chronic TANVS modulates circulating hematopoietic cells
An example of FACS analysis is presented in Supplementary Fig. 1. Five patients treated with TAVNS did not collect blood samples for this analysis at the end of follow-up, due to logistical problems or difficulties in sample collection. Thus, the study included 15 patients of TSNVS group and 10 controls (Supplementary Table 1). Technical issues during the FACS analysis caused missing data in few specific samples of cells.
Compared to baseline, patients treated with TAVNS exhibited a significant increase in percentage of classical monocytes (CD14++) and a significant decrease in of non-classical monocytes (CD16++) (Fig. 4A,B), which suggests a transition to a systemic anti-inflammatory monocyte profile. There was also a significant increase in the percentages of both circulating (CD31++) and progenitor endothelial cells (CD309++) (Fig. 4C,D) in the treated patients, suggesting that TAVNS modulates this linage to a reparative vascular profile. No significant alterations were observed in the control group (Fig. 4A,B,C,D).
The autonomic nervous system and the vagus nerve are critically implicated in the regulation of cytokine production, inflammatory processes and immune cell migration (4, 14, 16, 19, 20, 23, 26, 43, 44). In this study, there was a significant increase (three times) in the percentage of classic monocytes (CD14++) and a significant decrease (50%) in the percentage of non-classical monocytes (CD16++) after 8 sessions of TAVNS, while no changes were observed in the control group. These subpopulations have different immunological functions (45). The non-classical monocytes (CD16++ - NCM) produce large amounts of interleukin (IL)-1β under baseline conditions or in response to stimulation with lipopolysaccharide (LPS), which led to the notion that NCMs exerts inflammatory functions most importantly in circulation (46). The production of IL-1 β is considered a marker of inflammatory activity in non-classical human monocytes. NCMs in obesity are in a pro-inflammatory state with an increase in intranuclear NF-kB binding, a decrease in IkB-beta (also designated as IKK2) and an increase in the transcription of pro-inflammatory genes regulated by NF-kappa B (47, 48). On the other hand, classical monocytes (CD14++) participate in endothelial adhesion and cell migration, being considered to have an anti-inflammatory profile (47). The results of this study show that activation of a neural signaling with TAVNS can downregulate inflammatory cell phenotypes in blood, supporting the concept of TAVNS as a tool quench proinflammatory mediators while triggering anti-inflammatory signaling.
Of note, MetS subjects treated with 8 weeks of TANVS had an increase in the mean percentage of endothelial progenitor cells (EPCs) compared with baseline (100%), while no significant changes were detected in the control group, EPCs circulate in the blood and appear to reside preferentially at sites of vascular or tissue damage, contributing significantly to re-reendothelialization and physiological neoangiogenesis (49). There is a direct relationship between the EPC and peripheral endothelial function, indicating that the presence of EPCs may be a mechanism used to recover the dysfunctional endothelium (50). The findings suggest that the improvement in the sympathovagal balance altered the hemopoietic release of cells, favoring vascular repair.
There are some limitations of the present study. Regarding the protocol design, we did not use a simulated procedure in the control group, which would give more strength in the comparison between groups. Moreover, there is evidence that stimulation frequency and intensity can affect the extent of VNS. We applied TAVNS once a week. Although we detected significant improvement in frequency domain components of HRV and a decrease in hemodynamic parameters with this treatment, a daily stimulation could cause a stronger cardiovascular autonomic modulation. Nevertheless, it should be noted that TAVNS effects could last longer, even days, after the intervention, and perhaps could have a cumulative effect. Future studies should consider including a sham control group and increasing the frequency of stimulation of TAVNS. More importantly is the small number of patients included in the FACS analysis. Not all TAVNS-treated patients had blood samples for this analysis at the end of follow-up, due to logistical problems or difficulties in the sample collection. In addition, technical issues caused missing data on some specific cell samples. The small sample size increases the risk of type II error. However, the paired analysis showed a very homogeneous response in cell behavior in the treated group, which allowed finding significant differences between groups. Future studies including a larger number of patients are needed to confirm these results