The Heart Knows Best: Baseline Cardiac Parasympathetic Activity as Guide to Transcutaneous Auricular Vagus Nerve Stimulation in Depression

The Heart Knows Best: Baseline Cardiac Parasympathetic Activity as Guide to Transcutaneous Auricular Vagus Nerve Stimulation in Depression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Heart Knows Best: Baseline Cardiac Parasympathetic Activity as Guide to Transcutaneous Auricular Vagus Nerve Stimulation in Depression Sharmili Thanarajah, Carmen Schiweck, Mareike Aichholzer, Emily Brandt, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5366818/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Major Depressive Disorder can be conceptualized as a chronic stress condition associated with autonomic dysregulation, including blunted heart rate reactivity, changes in cortisol levels and peripheral inflammation, pointing to sympathetic activation and parasympathetic withdrawal. Transcutaneous vagus nerve Stimulation (taVNS) offers a non-invasive method to stimulate the vagus nerve – the key component of the parasympathetic system – to restore autonomic balance. Here, we examined whether changes in emotional, cardiac, and inflammatory reactivity are differentially influenced by taVNS in participants with MDD and controls. Additionally, we performed a post-hoc analysis with participants stratified by baseline cardiac parasympathetic activity (CPA) – as a proxy for vagus nerve activity - to evaluate the utility of biological stratification over diagnostic criteria. To assess the effect of chronic stress we conducted a single-blinded, cross-over, randomized controlled trial with 110 participants (51 controls and 59 MDD patients). For the analysis stratified by CPA, we grouped participants into low (n = 54) vs. high (n = 55) CPA regardless of diagnosis. All participants were subjected to an acute stress paradigm, both with taVNS and sham stimulation on two separate days, in a counter-balanced order. There was no difference in any of the outcomes regarding the effect of taVNS in participants with MDD and controls. Analyses split by CPA, however, showed that for those with low CPA, taVNS restored the blunted cardiac stress response and numerically decreased TNF-α levels. Unexpectedly, in people with high CPA, the opposite pattern was observed: heart rate and TNF-α were significantly increased, and vagally mediated heart rate variability was significantly decreased under taVNS compared to sham stimulation. Analyses using CPA as continuous predictors yielded similar results. Our findings suggest that CPA-based stratification may a useful stratification marker for (ta)VNS treatment. We encourage researchers with HRV data to re-evaluate their findings through CPA stratification. Biological sciences/Neuroscience Health sciences/Biomarkers/Prognostic markers Health sciences/Diseases/Psychiatric disorders/Depression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Major depressive disorder (MDD) is a highly prevalent and debilitating condition. Still, 30% of MDD patients do not respond to treatment, highlighting the need for novel treatment options. In the search for new treatment modalities, the conceptualization of MDD as a condition of chronic stress is of interest 1 – 3 . Specifically, people with MDD report excessive subjective levels of chronic stress and show corresponding physiological alterations, including autonomic dysregulation. This is evident in reduced heart rate variability and impaired responsivity to acute stress on both emotional and physiological measures (e.g., resistance, higher heart rate, blunted autonomic reactivity) 4 , 5 . Hence, specifically targeting biological pathways affected by chronic stress may offer new avenues for MDD treatment. The vagus nerve (VN) - the principal parasympathetic component of the autonomous nervous system - is a key player in stress response and recovery. Connecting the brain stem and the inner organs, the VN facilitates the bidirectional communication necessary for rapid adaptations after acute stress exposure. VN efferents influence cardiac reactivity and inflammatory responses via multiple pathways including the cholinergic anti-inflammatory reflex 6 – 8 . Conversely, interoception and the emotional (and attentional) responses are integrated through strong projections that exist between the VN, brain stem nuclei (nucleus tractus solitarii and dorsal-motor nucleus), the hypothalamus, mesolimbic regions, and higher cortical areas including the insular and ventromedial prefrontal cortices 9 – 14 . Hence, impaired vagus function, indicated by low cardiac parasympathetic activity (CPA) 15 significantly affects the stress response and homeostasis. Given the VN’s key role in stress response and recovery, it is noteworthy that (invasive) vagus nerve stimulation is an approved treatment for treatment-resistant cases of MDD. Indeed, VN stimulation has shown promising results in reducing depressive symptoms, but the underlying mechanisms for this effect are not understood. With the introduction of non-invasive techniques, evidence on the physiological mechanisms is accumulating. For instance, non-invasive stimulation of sensory vagal afferents via transcutaneous auricular VNS (taVNS) has been shown to 1) elicit vagal brain stem nuclei and the downstream projections in the brain 16 – 18 , 2) influence behaviour 19 – 21 , and 3) exert anti-inflammatory effects under very specific conditions 22 . While generally positive effects of taVNS on depressive symptoms have been reported 23 , studies (on the working mechanism) in MDD remain scarce. Considering that (1) MDD is a chronic stress condition with impaired CPA and reduced autonomic responses to acute stress and (2) taVNS can selectively activate vagal afferents and modulate the central autonomic network, we hypothesized that taVNS would increase acute stress reactivity, restore blunted HRV, and reduce inflammatory response to acute stress in patients with MDD and healthy controls. After analyzing the groups by diagnosis, we performed a stratification of participants by CPA – a biological marker of vagal impairment - and hypothesized that particularly participants with low CPA will benefit from taVNS compared to those with high CPA. Methods Ethical Approval and Pre-registration and Trial design The single-blind randomized controlled trial “MODULATE” was approved by the Ethical Committee of the Goethe University Frankfurt (Approval number 2021-48) and pre-registered at the German Clinical Trials Register (Identifier: DRKS00024823) and can be retrieved in the WHO Clinical Trial Registration Platform. All participants underwent a screening visit, and two test-days, where sham stimulation or transcutaneous auricular vagus nerve stimulation (taVNS) were administered in a counter-balanced order (see Figure 1. ). On the screening day, participants were assessed for eligibility using the Mini-Neuropsychiatric International Interview (MINI 7.0.2 for DSM-5) and the Montgomery Asberg Depression Rating Scale (MADRS), completed various questionnaires (see below for details) and registered onto the electronic patient-reported outcome system (P1vital® ePRO system). If eligible, participants were invited to two identical test days, which took place in the morning between 07.00 am and 12.00 am and lasted approximately 2 hours. Participants were required to fast overnight (drinking water was permitted). On arrival of the test day, participants provided a blood sample, filled out questionnaires, and were then asked to complete three tasks on the computer: two reward learning tasks (not reported here) and a modified version of the Montreal Imaging Stress Task (MIST). During the MIST, an ECG was recorded. After the stress task, another blood sample was taken, approximately 45 minutes after the stress task onset. In addition, before, shortly after, and 20 minutes after completion of the stress task, cortisol samples were obtained using Salivette ® (Sarstedt). Participants Participants were included at the Department of Psychiatry, Psychotherapy and Psychosomatics at the University Hospital Frankfurt between 24/05/2021 and 27/07/2023. Diagnoses of MDD and potential comorbid diagnoses were assessed with the MINI Diagnostic Interview according to DSM-5 criteria and assessed by trained raters. All participants in the healthy control group had to have an inconspicuous MINI result. All participants were between 18-65 years old. They were excluded if they satisfied the criteria for a bipolar disorder, schizophrenic psychosis, substance abuse disorder or dementia, neurological, autoimmune, rheumatological, neoplastic, dermatologic, or metabolic disorders and for healthy controls only, if they had a previous or current diagnosis of depression. Furthermore, participants with substance-abuse-induced depression were excluded. Both participants and controls were included if they had a BMI below 35, and were excluded if they were pregnant or lactating, or had an acute infection as evidenced by CRP levels above 10 mg/L on testday 1 or 2. In case of any respiratory infection, all procedures were postponed until the participant was symptom-free for at least 48 hours. Intervention Transcutaneous Vagus Nerve and Sham Stimulation Vagus nerve stimulation was applied at the cymba conchae of the right ear , while sham stimulation was applied with identical parameters at the earlobe (see Figure 1). All participants received both interventions (e.g., taVNS either at day 1 or 2) in a randomized order.The NEMOS ® device for research use (tVNS Technologies, Erlangen) was used, with an Impulse duration of 250µs, an impulse frequency of 25Hz, and a stimulation cycle duration of 32 seconds on and 28 seconds off. The stimulation intensity was minimally 100µA and maximally 5000µA and was ramped up during the 32-second stimulation cycle. Stimulation intensity was individually determined for each participant and each test day, by increasing the intensity until the participant reported an uncomfortable sensation, and then brought down in 100µA steps until the participant reported a tingling sensation that was not painful. Individual thresholds were recorded. The stimulation started after the blood sample was taken and the baseline assessments (e.g., BDI, demographic questionnaires) had taken place, approximately 10 minutes before the first reward learning task was initiated. [Figure 1 here] ECG recording and processing An electrocardiogram (ECG) was recorded using a 10-channel isolated bioamplifier (NeXus-10 MKII, Mind Media BV, Netherlands). Three disposable electrodes (Kendall™, H66LG electrodes) in a modified lead II configuration were placed on the chest to record the ECG at a sampling frequency of 1,024Hz. BioTrace+ Software (Mind Media BV, Netherlands) was used to check the signal and export the data. ECG signal processing was performed with Kubios Pro Scientific Analysis software 24 , which has an inbuilt R peak detection and inbuilt artifact and noise correction. A visual inspection of raw data was performed per segment of interest (the 5-min BL, Stress and Recovery intervals). Noise detection was left at the standard settings, and unclear beats were removed manually. Segments with more than 5% of beats in need of correction were excluded from the analysis. For the present analysis we extracted heart rate (HR) in beats per minute, root mean square of successive differences between normal heartbeats (RMSSD) in ms, the absolute power of high frequency heartrate variability (HF)-HRV, and low frequency (LF)-HRV both fast Fourier transformed. We opted for these measures given our interest in CPA, which can be measured by RMSSD. For the sake of comparability with other articles reporting frequency-domain rather than time-domain measures, we also report the HF-HRV. Furthermore, 29 for completeness, we also report LF-HRV, and heart rate was selected as a general index of cardiac output and physiological homeostasis 25 . Stratification based on Cardiac Parasympathetic Activity After conducting the analysis separated by diagnosis (MDD vs HC), we performed a post-hoc stratification based on the biological parameter “cardiac parasympathetic activity” (CPA). To stratify the participants, we used a median split for RMSSD in milliseconds (ms) across all participants to obtain basal CPA. We used the baseline of the sham condition for stratification purposes. We opted for a median split because we believe that it has clinical utility, but also performed analyses with RMSSD as a continuous predictor, which yielded comparable and highly significant results, reported in the supplementary material . If no data was present for this timepoint, missing values for RMSSD (n=11) were imputed using all ECG data with chained random forests as implemented in the missRanger package 26 in R. We opted for RMSSD instead of HF-HRV or Respiratory sinus arrhythmia because it is less dependent on respiration 27 and respiration was not measured in this study. Given the known influence of tricyclic antidepressants (TCA) on HRV, we excluded three participants on TCA in the analysis stratified by CPA. The results in- and excluding participants on TCA for both stratification approaches (1. stratification based on diagnosis, and 2. stratification based on CPA) are provided in the supplementary information. Blood Sample Preprocessing and Analysis Blood samples of the fasted participants were collected by venipuncture upon arrival and 45 Minutes after the stress task. The blood draw was performed in a fasted state to reduce variability for the inflammatory protein measures, given that postprandial levels of proteins can differ and such that metabolic states are standardized between testing days 28,29 . Blood was collected in K3EDTA Monvettes (Sarstedt) and immediately transferred to cooling containers. It was then spun down (2000g, 10 minutes) in a precooled centrifuge (4°C) no later than 30 minutes after blood sampling. The resulting plasma was then aliquoted into Eppendorf tubes and stored at -70°C until further analysis. To obtain levels of high sensitive (hs) IL-6 and hsTNF-α, we used enzyme-linked immunosorbent assay with precoated kits (IL-6: Human IL-6 High Sensitivity ELISA, Art.Nr.: BMS213HS; TNF-α: Human TNF-α High Sensitivity ELISA Kit, Art.Nr.: BMS223HS and BMS223-2HS both, Invitrogen/ThermoFisher Scientific) as per manufacturer’s instructions. Samples were measured in duplicate, blank values were subtracted, and a standard curve was obtained for each plate using Microsoft Excel, optical densities were converted to concentrations in pg/mL. If the sample duplicate’s coefficient of variation deviated more than 15%, they were repeated. Values below the detection level were replaced by the LOD divided by 2. For TNF-α analytical sensitivity was 0.13 pg/mL, for IL-6 0.03pg/ml. For TNF-α, of the 361 measurements, 47 were below level of detection and were replaced by the level of detection divided by 2 (i.e., 0.13/2). No levels were below the detection level for IL-6. Concentrations ranged from 0.35pg/mL-55.70pg/mL for IL6 and from 0 (undetectable levels)-8.37pg/mL for TNF-α. Sample size The sample size was determined with a predefined power calculation, for a repeated measures Analysis of Variance (ANOVA) with a within and between effects interaction a sample size of 76 was determined, given an assumed effect size of 0.25, alpha 0.05, and a reasonable power of 0.95, correlation among measures 0.3, 2 groups and 2 measurements (i.e., taVNS/sham) (calculated with G*Power 3.1.9.6). Given the potential dropout effects, and assuming that around 20% of values detected with ELISAs are below the threshold of detection, we aimed to recruit 100 participants at T1 (with 1:1 group distributions). A simulation analysis showed that with 100 participants, a smaller effect size of just below f=0.152 would still be detected with 80% power (see Supplementary Figure 1) . Randomization Participants were randomized to receive either sham stimulation or verum stimulation based on a pre-established list, composed by a random sequence in Excel. Participant IDs were linked to the allocation, and this could not be changed. The list was assembled in three blocks of 50 participants. Researchers were not blinded to the allocation, but participants were blinded, i.e., they were not informed of which intervention they received on each day. The random allocation sequence was generated by the senior scientists, while allocation was performed by the study staff. The procedure of sham stimulation and taVNS were identical, except for the location of stimulation. Care was taken to spend an equal amount of time on the establishment of the stimulation intensity thresholds and experimental procedures in both conditions. Statistical methods Demographic variables between groups were compared using t-tests, the Wilcoxon test, or the Chi-square(χ 2) test for proportions. Variables were transformed as indicated. To assess the VAS score, a composite score was calculated for a negative and a positive component, by summing up relevant items. To assess our main hypothesis, we fitted a linear mixed model (estimated using REML and if necessary nloptwrap optimizer) to predict variables of interest with Intervention, Cardiac parasympathetic Activity, Timepoint, Group, Sex, Age, Testday and Stimulation Intensity ( formula: DV~ Intervention * Group * Timepoint + Group + Sex + Age + Testday + Stimulation Intensity ). The model included ID as a random effect ( formula : ~1 | ID ). Models were fitted using lme4 30 and lmerTest packages 31 . A type III anova table was computed for F and p values for fixed effects using Satterthwaite's method for the approximation to degrees of freedom. We repeated the same model for post-hoc stratified analyses, where instead of diagnostic group, we used CPA. The insignificant 3-way interaction for RMSSD was compared with post-hoc tests, given the specific interest to our study design. Where reported, post-hoc tests were computed as contrasts with the emmeans package 32 and if applicable to the contrasts, the standard inbuilt Tukey method for comparing a family of tests of the emmeans package was automatically applied. Additionally, we also investigated antidepressant medication status yes/no (here, most people were taking SSRIs or SNRIs). Results did not change, and antidepressant medication was not a significant predictor for HRV, therefore the models are not reported. All statistical analyses were performed with RStudio (Version 4.3.1.) and graphical representations were made in Graphpad Prism, based on summary data extracted from R 33 . Questionnaires Diagnosis or absence thereof was ascertained with the Mini-Neuropsychiatric International Interview (MINI, version 7.0.2 for DSM-5) 34 . The MINI is a short, well-validated, semi-structured diagnostic interview for major psychiatric disorders. Items refer to the last month and/or the entire life and are rated on a yes/no basis, allowing us to assess the absence or presence of psychiatric disorders, based on DSM-5 criteria. The Montgomery Asberg Depression Rating Scale (MADRS) is a well-validated 10-item clinical interview assessing symptoms of depression in the past 7 days, with a cumulative score indicative of the presence of no, light, moderate, or severe depression 35 . Proposed cut-offs are 0-6 (absence of depression), 7-19 (mild depression), 20-34 (moderate depression) and 35-60 (severe depression). In addition, the self-rated Beck’s Depression Inventory (BDI-II) and the Perceived Stress Scale (PSS) 36 were administered. The BDI-II is a 21-item, self-rated scale for depressive symptoms with good sensitivity and specificity 37 . The PSS-10 is a commonly used 10-item scale to assess perceived stress in the past 4 weeks. Scores range from 0 (no stress) to 40 (high stress). The Childhood Trauma Questionnaire (CTQ) was collected to assess childhood trauma 38 . Visual Analogue Scales During each baseline, after stress and after recovery phases, participants rated eleven items on a 100-mm Visual Analogue Scale (VAS=). Two composite scores were calculated for the items of the VAS scale (one containing negative emotions, the other one positive emotions, consisting of the sum of the items (positive: energetic, motivated, happy, in control; negative: scared, alert, stressed, powerless, tense. Additionally, given the experimental stress exposure, we explored the answers on the VAS item “stressed” separately, hereafter named “stress perception”. Experimental stress task To induce stress, we used an adapted, customized, and computerized variation of the mental arithmetic stress task similar to the Montreal Stress Imaging task (MIST) 39 . The task includes a 5-minute baseline, a 1-minute-control/demonstration task (i.e., a mental arithmetic without stressful elements), a 5-minute mental arithmetic task with stressful elements (stress phase), and a 5-min recovery phase. In the current version, the following elements are included to induce stress: social comparison (e.g., playing against an “imaginative peer” whose progress is monitored by a progress bar on the right side of the screen), adaptive time pressure (i.e., if the participants answered correctly, they would receive less time to answer the next question; if they answered wrong several times, the time limit was increased) and feedback by the researcher (i.e., two times verbal feedback to “answer faster” or “try to answer correctly”).This version is a computerized task, which requires participants to select the right answer on the computer screen without requirement for verbalization and participants were instructed not to talk in order not to disrupt breathing patterns. RESULTS In the period from May 2021 to August 2023, 146 people were assessed for eligibility, of which 26 were excluded or piloted with a different study design, 120 were randomized, 110 completed their first intervention and 101 participants completed their second cross-over phase (Fig. 2 ). The trial was ended after the target of included participants was reached. [Figure 2 here] Baseline data For all presented analyses, we compared participants with MDD versus healthy controls (HC) (Table 1 ). Participants with MDD were older compared to the control group, but had a similar BMI, sex, and smoker distribution. There was no difference between education level or contraception intake between groups. All questionnaires (BDI-II, MADRS CTQ, PSS) differed significantly. Table 1 Demographic Characteristics of Participants by Diagnosis. CTRL = Control group, MDD = Major Depressive Disorder group. Bold values indicate significant results.nr = not reported. N CTRL, N = 51 1 MDD, N = 59 1 p-value 2 Sample characteristics Age 109 30.24 (10.78) 38.79 (13.29) < 0.001 Sex 110 0.8 Female 33 (65%) 37 (63%) Male 18 (35%) 22 (37%) BMI 109 23.57 (2.59) 24.32 (3.82) 0.2 Smoker 110 0.5 No 43 (84%) 44 (75%) Occasional 4 (7.8%) 5 (8.5%) Yes 4 (7.8%) 9 (15%) Unknown 0 (0%) 1 (1.7%) Contraception 109 0.2 no 19 (37%) 23 (40%) yes 11 (22%) 5 (8.6%) postmenopausal 1 (2.0%) 6 (10%) male 18 (35%) 22 (38%) unknown 2 (3.9%) 2 (3.4%) Education 110 0.5 0- no degree 0 (0%) 2 (3.4%) 1- primary school 0 (0%) 1 (1.7%) 2- secondary school 20 (39%) 16 (27%) 3- secondary education 6 (12%) 7 (12%) 4- university degree 24 (47%) 32 (54%) 5- doctoral degree 1 (2.0%) 0 (0%) unknown 0 (0%) 1 (1.7%) Unmedicated 109 < 0.001 no 3 (5.9%) 38 (66%) yes 48 (94%) 17 (29%) nr 0 (0%) 3 (5.2%) Antidepressants 109 < 0.001 no 51 (100%) 20 (34%) yes 0 (0%) 34 (59%) nr 0 (0%) 4 (6.9%) Questionnaires BDI-II 109 1.31 (2.16) 24.86 (10.28) < 0.001 MADRS 110 1.20 (2.18) 25.66 (8.66) < 0.001 CTQ Sum Score 108 34.20 (10.51) 51.19 (18.31) < 0.001 PSS-10 109 10.37 (4.69) 25.03 (5.62) < 0.001 1 Mean (SD); n (%) 2 Wilcoxon rank sum test; Pearson’s Chi-squared test; Fisher’s exact test [Table 1 here] Demographics in groups based on CPA When split by CPA, 55 participants were in the high and 54 in the low CPA group. In the low CPA group, 39 of 54 (72%) were MDD participants, in the high CPA group, 19 of 55 (35%) were MDD participants (see Supplementary Table 1A for demographics by CPA). The low CPA group had higher depression scores and higher chronic stress levels, which are likely attributable to the higher percentage of participants with MDD in the low CPA group ( Supplementary Table 1B and C ), but childhood trauma was also higher in the low CPA control group (P = 0.033), see Supplementary Table 1B . Results of Blinding Most participants in both conditions (sham and taVNS) believed that they were receiving the taVNS stimulation (84% of all cases, 81% during sham, 86% during taVNS X 2 = 1.00, p = 0.317) and stimulation amplitude was comparable for both interventions (sham: median: 850, tVNS median: 800, W = 5957.5, p = 0.094). Planned analysis No effect of diagnosis for taVNS First, we assessed our primary analysis, to see if the efficacy of taVNS on our outcomes was modified by diagnosis (MDD or HC). In all analyses except for the inflammatory proteins, we did find a significant main effect of diagnosis and timepoint, but there was no main effect for intervention and no interaction effect of diagnosis and intervention (taVNS vs. sham). This was true for emotions, for HRV and for inflammatory proteins (see Table 2 for the main effect of intervention and the intervention*Group interactions). Full models can be found in Supplementary Table 2. Due to the absence of intervention effects, in the following, we decided to apply a post-hoc stratification based on CPA instead of diagnosis, to assess whether those with lower CPA would profit more from taVNS than those with higher CPA, regardless of diagnosis. Table 2 Results of Planned analysis comparing MDD and HC. No significant effects of intervention, or intervention*group were found. Variables were transformed using log or Tukey’s ladder of power transformations and models included covariates as well as timepoint and interaction effects. Full models and models excluding those taking TCA can be found in the supplementary material table 2. Intervention Group* Intervention F p F p Emotions Positive [Composite score] 1.74 0.187 0.09 0.768 Negative 1.18 0.278 0.34 0.561 [Composite score] Stress [%] 0.62 0.431 1.26 0.263 HRV Heart Rate 0.35 0.552 0.45 0.501 RMSSD 1.75 0.187 0.06 0.809 HF-HRV 0.59 0.444 0.05 0.819 LF-HRV 1.28 0.259 1.44 0.231 LF/HF ratio 0.19 0.66 1.98 0.16 Inflammation IL-6 in pg/ml 1.12 0.291 3.04 0.082 TNF-α in pg/mL 0.57 0.45 0.01 0.922 [Table 2 here] Post-hoc stratified analysis The impact of taVNS on stress perception depends on CPA Timepoint had a significant effect (p < 0.001) on subjective stress levels, which increased during the stress task and reduced following recovery. Participants with MDD reported higher levels of stress compared to healthy controls at all time points (main impact of group p < 0.001). The interaction of intervention (taVNS vs. sham) and CPA was significant (p = 0.024). Follow-up tests showed that for those with high CPA, taVNS did not affect stress levels (t = 0.06, p = 0.549), but significantly increased stress perception in those with low CPA (t=-2.63, p = 0.009). No other interactions were significant. For negative but not positive emotions, timepoint had a significant main effect (p < 0.001 and p = 0.676) and the group was significant in both models(both p < 0.001) but we did not find an effect of intervention (see Supplementary Table 3A for fixed effect estimates of the full models and Fig. 3 A-I. for graphical representations). Analyses with CPA as a continuous score yielded similar results ( Supplementary Table 3B ). [Figure 3 here] The impact of taVNS on heart rate variability depends on CPA To assess the impact of CPA and taVNS on the autonomic stress response, we investigated RMSSD and other measures of heart rate variability (HR, HF-HRV, LF-HRV and LF/HF ratio). Main effects for timepoint and CPA were significant (all p < 0.05) for all measures, only for the LF-HF ratio, timepoint was not significant (p = 0.149), see Supplementary Table 4A . We found an interaction of CPA and intervention for RMSSD (p < 0.001), HF-HRV (P = 0.002), LF-HRV (P = 0.026), but not HR (P = 0.121) or LF/HF ratio (p = 0.127) (see Supplementary Table 4B for post-hoc comparisons of interactions). Follow-up tests showed that RMSSD and HF-HRV numerically increased in the low CPA group during taVNS (sham-taVNS; RMSSD: t=-1.96, P = 0.050, HF-HRV t=-1.84, P = 0.067), but taVNS reduced RMSSD and HF-HRV in the high CPA group (sham-taVNS; RMSSD t = 3.58, p < 0.001; HF-HRV: t = 2.79, P = 0.006), see Fig. 4 B. Furthermore, for all variables, except LF-HRV and LF/HF ratio, a significant CPA*timepoint interaction occurred (all P < 0.05, see Supplementary Table 4A and 4B ). Post-hoc tests for RMSSD showed that people in the low CPA group did not react to stress (baseline-stress: RMSSD: t = 2.23, P = 0.067), compared to strong reactivity in the high CPA group (baseline-stress: RMSSD: t = 7.08, P < 0.001), while both had significant recovery, albeit numerically stronger in the high CPA group (high: t=-5.15 vs. low: t=-2.98), see Fig. 4 A. Results with CPA used as a continuous predictor were comparable to the analysis using a factor for HF-HRV and RMSSD (see Supplementary Table 4C for statistics). Exploratory post-hoc analyses 3-way interaction for RMSSD shows recovery of blunted stress response Given our interest in the treatment effect, we also conducted exploratory contrasts for the non-significant three-way interaction of RMSSD. Contrasts showed that while during sham stimulation, there was indeed a blunted reactivity and recovery for RMSSD in the low CPA group (sham; baseline-stress: t = 0.19, P = 0.981, stress-recovery: t=-0.82, P = 0.692), this was restored during taVNS (baseline-stress: t = 2.96, P = 0.009, stress-recovery: t=-3.39, P = 0.002). In the high CPA group both sham and taVNS showed significant reactivity and recovery during sham (sham; baseline-stress: t = 4.79, p < 0.001, stress-recovery: t=-3.52, P = 0.001) and taVNS (baseline-stress: t = 5.25, p < 0.001, stress-recovery: t=-4.29, p < 0.001), see Fig. 4 C. Effect of taVNS on Inflammatory Proteins is opposite dependent on CPA Overall, as expected in a population without overt inflammatory conditions, levels of IL-6 and TNF-α were low. No main effects of CPA, or timepoint occurred for IL-6 or TNF-α (see Supplementary Table 5A for fixed effects). For TNF-α, an intervention*CPA interaction occurred (P = 0.038). Post-hoc comparisons showed that regardless of group or timepoint, in those with higher CPA, TNF-α was significantly higher during taVNS than during sham stimulation (sham-taVNS: t=-2.09, P = 0.038) while in those with low CPA, TNF-α was not significantly altered (sham-taVNS: t = 0.70, P = 0.483). See Fig. 5 for an overview. If RMSSD (sham, baseline) was used as a continuous variable, the interaction of CPA*Intervention was not significant for TNF-α (p = 0.232), see Supplementary Table 5B . Discussion In this counter-balanced, randomized-controlled study, we aimed to assess whether taVNS restored the acute stress response in people with MDD by improving subjective stress perception, enhancing cardiac reactivity and reducing inflammation compared to healthy controls. Contrary to our hypothesis, we found no effect of taVNS on these outcomes, nor any interaction of taVNS and the diagnostic group. In other words, taVNS had no significant impact on our outcomes, with no difference between MDD patients and HC. Subsequently, we decided to use post-hoc stratification by a biological marker of VN activity - the cardiac parasympathetic activity (CPA) (using the median RMSSD as a surrogate marker). We reasoned that people with low CPA, indicating low VN activity, were most likely to profit from taVNS. In the low CPA group (regardless of diagnosis), taVNS recovered the previously absent cardiac vagal reactivity to stress, increased the perception of stress, but only had a minor, non-significant reductive effect on TNF-α. In contrast, and unexpectedly, if CPA was high at baseline, taVNS dampened heart rate variability (HRV), in creased HR and significantly in creased levels of TNF-α. Our findings clearly suggest that the effects of taVNS depend on baseline CPA, suggesting that consideration of CPA is essential for future studies. taVNS: No effect on biological outcomes in MDD? An adequate response to acute stress is fundamental for survival and is tightly regulated by the autonomic nervous system. Persisting exposure to physical or emotional stress, however, can disrupt homeostasis, mitigate basal cardiac parasympathetic activity (CPA), and impair the physiological ability to face sudden challenges, as often observed in patients suffering from MDD. Vagus nerve stimulation provides a targeted approach to directly stimulate the underlying biological pathways that are impaired by chronic stress exposure. Surprisingly, against our hypotheses, there was an absence of a main and/or interaction effects for the intervention in our primary analyses comparing people with MDD and controls. This could indicate that the non-invasive form of taVNS does not have any acute effects, even in a relatively large sample such as ours. However, our post-hoc stratification revealed a critical feature linked to the response of taVNS: the individual CPA at baseline. Only in the low CPA group, taVNS improved stress perception and recovered cardiac reactivity. Interestingly, while we expected the strongest effects of taVNS for those with low CPA, and somewhat attenuated effects in those with high CPA, this was not the case. Contrary to our expectations, we did not find weaker effects in those with high CPA, but instead, taVNS had stronger and opposed effects to the anti-inflammatory, HRV-enhancing effects we expected from the literature. This is a crucial insight, as effects in opposed directions in those with high and low CPA may lead to null findings for the (ta)VNS, if baseline CPA is disregarded. The absence of a significant modifying effect of MDD on the intervention may be partly attributed to the diagnostic criteria for depression, which currently rely solely on symptomatology and likely encompass various disorders with distinct pathophysiological mechanisms. This may explain why only some, but not all, MDD patients exhibit low CPA. Consequently, CPA could serve as a valuable tool for selecting patients based on a biological stratification marker for VNS treatment. Chronic stress is linked to impaired CPA In line with previous research 40 , we found that chronic stress and childhood trauma were associated with low CPA in our sample. This association has been predominantly reported in clinical populations suffering from chronic stress conditions (i.e., Posttraumatic Stress Disorder, Anxiety Disorders, MDD) 41 . The well-orchestrated response to acute stress starts with an increase in sympathetic activity, catecholamine and cortisol production, energy supply through gluconeogenesis and lipolysis, and cardiac (e.g. increased heart rate and blood pressure) and immune arousal, but is temporally limited and terminated by parasympathetic activation. Yet, sustained stress and the lack of habituation to stressors can lead to deficient CPA (i.e., 42 ), failing to regulate the aforementioned systems and therefore leading to a chronic elevation of sympathetic activity, catecholamine, and cortisol production and reduced cardiac vagal modulation. As in previous studies 5 , 43 , 44 , we here observed the biological effects of chronic stress often described in the literature: We found that in the low CPA group the baseline level of HR was higher, cardiac stress reactivity was blunted, and baseline levels of perceived stress and depressive symptoms were higher. Differential effects of taVNS on cardiac reactivity dependent on CPA Intriguingly, taVNS restored cardiac reactivity and recovery for those with low CPA. Stimulation of the VN’s sensory afferents at the outer ear is considered to activate the dorsomotor nucleus. This activation relayed through the afferent nucleus (NTS) 45 , modulates the parasympathetic projections directed towards the sinoatrial and atrioventricular nodal cells as well as the atrial myocytes 46 , 47 . It is thus possible that taVNS can counteract the effects of chronic stress by activation of parasympathetic projections. The opposed effects in the high CPA group suggest that this mechanism may differ in this group, either by engagement of a different mechanism, overactivation of the projections and/or by activation of the sympathetic rather than parasympathetic nervous system. Indeed, previous studies have discussed the possibility of low-level sympatho-excitation by vagal afferents due to bioelectric stimulation as applied during taVNS 48 . This could provide a potential explanation for the inconsistency reported regarding (ta)VNS effects on heart rate variability, which have shown reductions 49 – 51 , increases 52 – 55 , or no distinguishable effect 56 – 59 , summing up to a lack of a consistent effect on HRV 60 . No acute effects of taVNS on emotional states Furthermore, as a treatment for depression, the integration between the autonomic state, interoceptive signals, and emotional states is crucial and is mediated by highly interconnected brain stem nuclei and higher cortical regions that receive vagal afferents. Stimulation of the vagus nerve (direct and/or via taVNS) provides an intriguing option to investigate the effect of stimulation on this integrative process since it is capable of stimulating the nucleus tractus solitarii , and the interconnected limbic and cortical areas that belong to the central autonomic network in both rodents and humans 10 – 14 . Chronic VNS has been shown to improve negative mood states in humans 2 , 3 , 61 and low CPA has been linked to negative emotional states, in line with our data. A recent rodent study elegantly demonstrated that reduced CPA due to prolonged stress resulted in alterations in the connection between the prefrontal cortex and amygdala, leading to anxious behavior similar to that seen in vagotomy, and chronic VNS restored brain signals and the anxious phenotype 42 . In our sample, however, no intervention effect was found for negative emotions after the short intervention period. Future studies in humans need to shed more light on this process. Differential effects of taVNS on inflammatory cytokines depending on CPA The seminal discovery by Borovikova et al. (2000) highlighted the relevance of the VN for regulating inflammatory responses through the cholinergic anti-inflammatory pathway. Since then, several studies - also in humans – have shown that low cardiac parasympathetic activity is associated with increased cytokines at baseline and after immunological challenges 62 , 63 . Selective stimulation of the VN -both invasive and transcutaneous- suppresses circulating cytokines, but most of the significant effects were only applicable to subgroups and in conditions of acute immunological events 22 . Our results showing differential effects of taVNS depending on baseline CPA raise new questions: first, our results suggest that taVNS could have modest anti-inflammatory potential if baseline CPA is low, whereas if the CPA is already high, taVNS increases TNF-⍺. Second, we did not find a change in IL-6 after taVNS, which is in line with preclinical literature and suggests a rather specific effect of (ta)VNS on TNF-⍺. Analog to the findings in HRV, this might explain the rise in cytokines reported following taVNS in healthy human participants (e.g., 64 , 65 ), who were not recruited based on baseline CPA and might have had high CPA. We are intrigued whether similar findings can be found in studies using longer durations and different pathologies. Inverse effects of taVNS with higher CPA? A call for stratification in existing cohorts using (ta)VNS Although there is a general correlation between high CPA, favourable health outcomes and a well-regulated autonomic nervous system, taken together our results suggest that CPA beyond a certain threshold triggers an alternative mechanism (potentially SNS rather than PNS activation) in response to taVNS. Our results align with prior research that documented decreased heart rate variability following (ta)VNS 50 , 51 and increases in cytokines in healthy subjects 64 . Most of these results involved youthful, healthy subjects with probably high CPA at baseline, who may have surpassed a threshold with (ta)VNS. In the clinical context, vagal hyperactivity (which we don’t see in our participants) has been linked to bradycardia, syncopes, hypotension, and excessive gastric acid secretion 66 – 68 . Here, we used post-hoc stratification with a median split for RMSSD as an index of cardiac vagal activation, due to the sample size constraints, but the exploratory analyses using continuous CPA produced similar results. It is thus possible that a dose-response relationship is present, which should be explored in larger sample sizes. We identified one small clinical trial which also used stratification by baseline CPA after a long-term intervention with invasive VNS in patients suffering from Crohn’s disease 69 . Intriguingly, the effect of VNS on heart rate variability was restorative in 4 of 5 patients with low CPA but decreased in the one person with high CPA at baseline. Others have reported that responders to VNS (as defined by more than 20% decrease in LF/HF), had significantly lower basal CPA than non-responders 55 . Although it is obvious, that VNS should be particularly beneficial if CPA is compromised, the majority of studies so far have not used CPA for stratification. If HRV data is available, we, therefore, call to researchers for a secondary analysis of (ta)VNS datasets, which could offer more insight, particularly for studies assessing long-term effects and antidepressant response. Methodological considerations A distinctive aspect of our study lies in our choice to stimulate the right outer ear. This decision was informed by rodent studies, that demonstrated a closer link between the right VN and limbic areas critical for reward behaviour and emotional processing. Rodent investigations have demonstrated that both right and left VNS impact cardiac reactivity, while effects were more often investigated on the right side 70 – 72 . Because of the greater innervation of the sinus-atrial node by the right VN, invasive and the majority of taVNS applications have avoided this stimulation side fearing bradycardia. Nevertheless, there is currently no data suggesting safety concerns with transcutaneous VNS administered to the right side or bilaterally 73 and increases in HRV through VNS have been demonstrated for both right 53 , 54 , 74 and left VNS 52 , 55 , 75 . In addition, it was shown that specific stimulation parameters elicit an increase rather than a decrease of cytokines 76 indicating that a systematic investigation of stimulation parameters is necessary in future studies to further explore this dynamic in humans. Limitations While our study is highly novel, several limitations need to be mentioned. First, we used transcutaneous, not invasive VNS. We suggested the observed effects to be mediated through the activation of specific brain regions triggered by afferent fibre signaling. Although this signaling pathway lacks the precision of direct vagal fibre stimulation, recent rodent studies suggest it may achieve comparable outcomes 45 . We also did not measure respiration, but CPA is only reliably measured when the respiration rates are in the normal range (e.g., between 7.2–24 breaths/minute) 15 , which could affect the results if breathing occurred outside of these ranges. Finally, the effects we observed (notably for inflammation) are small and observed during acute stimulation. It remains to be seen whether taVNS or invasive VNS will also have this effect during longer stimulations and in clinical populations with higher levels of inflammation. Conclusion and outlook In conclusion, our findings indicate that baseline CPA matters, at least for the biological outcomes of taVNS. A compromised CPA is associated with impaired cardiac and inflammatory response to acute stress exposition and can be restored by acute taVNS. However, people with high CPA may react with the opposite pattern- which could lead to null findings, as we observed in our analysis using diagnostic groups (e.g., MDD vs. controls). We call to all researchers who measured an ECG at baseline in studies using (ta)VNS to perform a secondary analysis. If our findings are replicated, this may help to find a stratification marker for successful taVNS for MDD, and other diagnoses. Declarations Author contributions CS: Conceptualization, Data Collection, Data Processing, Formal Analysis, Writing-Original Draft, Writing- Review& Editing, Visualization, MA: Data Collection, Data Processing, EB: Data Collection, Data Processing, MS: Data Collection, Data Processing, Formal Analysis, KM: Data Collection, Data Processing, TH: Data Collection, Data Processing, CU: Data Collection, Data Processing, KA: Data Collection, Data Processing, AP: Data Collection, Data Processing, MQ: Data Collection, Data Processing, GB: Data Collection, Data Processing, TZ: Data Collection, Data Processing, AB: Data Collection, Data Processing, RH: Writing-Original Draft, Writing- Review& Editing, TH: Writing-Original Draft, Writing- Review& Editing, JR: Writing-Original Draft, Writing- Review& Editing, SM: Writing-Original Draft, Writing- Review& Editing, AR: Writing-Original Draft, Writing- Review& Editing, SET: Conceptualization, Data Collection, Formal Analysis, Writing-Original Draft, Writing- Review& Editing, Visualization, Supervision, Project administration Acknowledgment The authors would like to thank Sabine Stanzel for her exceptional technical assistance throughout the study and Florian Freudenberg for the consultation on the preprocessing of blood samples. 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Qubad","email":"","orcid":"https://orcid.org/0000-0002-9291-5739","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mishal","middleName":"","lastName":"Qubad","suffix":""},{"id":423311000,"identity":"ec225ff2-5b22-4a9b-a473-624c27960051","order_by":5,"name":"Carmen Uckermark","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Uckermark","suffix":""},{"id":423311001,"identity":"717c4a37-5793-42e9-86b2-6865d8cc03f8","order_by":6,"name":"Moritz Schneider","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Moritz","middleName":"","lastName":"Schneider","suffix":""},{"id":423311002,"identity":"57c1df7d-194a-435e-96f0-4dc5f6bcef15","order_by":7,"name":"Konrad Meyer","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Konrad","middleName":"","lastName":"Meyer","suffix":""},{"id":423311003,"identity":"28851a48-0c60-4955-8ac6-9e911cf177ae","order_by":8,"name":"Tirage Hamzehloiya","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tirage","middleName":"","lastName":"Hamzehloiya","suffix":""},{"id":423311004,"identity":"0ef519f6-fd0d-4f56-b34b-5c983eb206a7","order_by":9,"name":"Leona Jacobsen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Leona","middleName":"","lastName":"Jacobsen","suffix":""},{"id":423311005,"identity":"314cf28d-3219-449f-bd0b-87ecce285376","order_by":10,"name":"Kevin Amaral","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Amaral","suffix":""},{"id":423311006,"identity":"afc9bc04-ba52-42dc-b9b3-2490326f30c7","order_by":11,"name":"Joyce Auer","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Joyce","middleName":"","lastName":"Auer","suffix":""},{"id":423311007,"identity":"4d878040-cf64-45b3-9423-e69142955b60","order_by":12,"name":"Gianluca Bruno","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Gianluca","middleName":"","lastName":"Bruno","suffix":""},{"id":423311008,"identity":"08f090f7-bfd1-4b04-b9b4-ca7aeb305574","order_by":13,"name":"Tong Zhao","email":"","orcid":"https://orcid.org/0000-0001-9292-5286","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Zhao","suffix":""},{"id":423311009,"identity":"f48b49d5-e9fc-494b-b383-891cd5687cf3","order_by":14,"name":"Aicha Bouzouina","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Aicha","middleName":"","lastName":"Bouzouina","suffix":""},{"id":423311010,"identity":"816fa292-0422-43ec-8528-9d8a522d7c9e","order_by":15,"name":"Susanne Schillo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Susanne","middleName":"","lastName":"Schillo","suffix":""},{"id":423311011,"identity":"ebb3fe30-1625-4734-9141-db3097820405","order_by":16,"name":"Ruth Hanssen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ruth","middleName":"","lastName":"Hanssen","suffix":""},{"id":423311012,"identity":"58ef1d50-6802-4bb6-8053-72899519fc73","order_by":17,"name":"Tim Hahn","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tim","middleName":"","lastName":"Hahn","suffix":""},{"id":423311013,"identity":"67573660-2848-4355-b3c4-a41ebc579e27","order_by":18,"name":"Jonathan Repple","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"","lastName":"Repple","suffix":""},{"id":423311014,"identity":"dd74de53-daa4-40de-970c-37fec586d98a","order_by":19,"name":"Silke Matura","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Silke","middleName":"","lastName":"Matura","suffix":""},{"id":423311015,"identity":"0b67275f-f4ff-4e9c-93ff-20e4b639361c","order_by":20,"name":"Jonathan Kingslake","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"","lastName":"Kingslake","suffix":""},{"id":423311016,"identity":"98ca5ef6-642c-4212-b6e4-b700edcca104","order_by":21,"name":"Andreas Reif","email":"","orcid":"https://orcid.org/0000-0002-0992-634X","institution":"University Hospital Frankfurt, Germany","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Reif","suffix":""}],"badges":[],"createdAt":"2024-10-31 10:30:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5366818/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5366818/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77765209,"identity":"d650f790-5642-4750-af63-d3ae4897ced8","added_by":"auto","created_at":"2025-03-05 09:57:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":222733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy Design.\u003c/strong\u003e Left panel: \u003cstrong\u003eA.\u003c/strong\u003e Sham stimulation at the earlobe. \u003cstrong\u003eB.\u003c/strong\u003e Verum stimulation at the right cymba conchae. Right panel: Illustration of cross-over, single-blind, randomized, sham-controlled trial. Stimulation was started after the blood sample (-60min) and ended after the stress task. VAS scales were collected after the baseline (0min), after stress exposure (5min) and after recovery (5min). A final blood sample was collected 40 min after stress exposure. Timescales are approximate and did vary according to time needed for completing experimental procedures such as VAS scales.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5366818/v1/0f10f765ce48b6998cb9e66b.png"},{"id":77763299,"identity":"8314a70b-956c-4d0c-8383-616a1dea7273","added_by":"auto","created_at":"2025-03-05 09:49:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":336741,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConsort Flow Diagram of the MODULATE study.\u003c/strong\u003e Ninety-four percent (61/65) of the randomized participants completed a first test day (T1) with taVNS versus 89 percent (49/55) with a sham stimulation; the difference was not significant (P= 0.543). At the second test day (T2) 84.61 (55/65) of randomized participants received the planned sham stimulation and 83.64 % (46/55) received the planned verum stimulation (p\u0026gt;0.999). In total, 51 controls and 59 participants with MDD were randomized to the intervention and completed the first session (T1) and were thus used for analysis.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5366818/v1/a10a29d21cc659e9da51599f.png"},{"id":77761969,"identity":"8aff0458-bf56-49de-862d-750337deacd8","added_by":"auto","created_at":"2025-03-05 09:33:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":252435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of acute stress and taVNS on emotional reactivity.\u003c/strong\u003e \u003cstrong\u003eA-C\u003c/strong\u003e \u003cstrong\u003eEffects on Stress Perception.\u003c/strong\u003e All values were transformed with Tukey ladder of power transformation for statistical analyses but are presented as untransformed scores. From left to right: \u003cstrong\u003eA.\u003c/strong\u003e Main effect of acute stress, \u003cstrong\u003eB.\u003c/strong\u003eMain effect of Group, \u003cstrong\u003eC.\u003c/strong\u003e 2-way interaction effect of intervention and Cardiac Parasympathetic Activity. \u003cstrong\u003eD-F\u003c/strong\u003e \u003cstrong\u003eEffects on Negative Emotions \u003c/strong\u003e(Composite Score). From left to right: \u003cstrong\u003eD.\u003c/strong\u003e Main effect of acute stress, \u003cstrong\u003eE.\u003c/strong\u003eMain effect of Group, \u003cstrong\u003eF.\u003c/strong\u003e 2-way interaction effect of intervention and Cardiac Parasympathetic Activity, \u003cstrong\u003eG-I\u003c/strong\u003e \u003cstrong\u003eEffects on Positive Emotions\u003c/strong\u003e(Composite Score). From left to right: \u003cstrong\u003eG.\u003c/strong\u003e Main effect of acute stress, \u003cstrong\u003eH.\u003c/strong\u003eMain effect of Group, \u003cstrong\u003eI.\u003c/strong\u003e 2-way interaction effect of intervention and Cardiac Parasympathetic Activity, Significance:***:p\u0026lt;0.001, ** p\u0026lt;0.01, *p\u0026lt;0.05, #p\u0026lt;0.1, ns:p\u0026gt;0.1.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5366818/v1/4405de63d6a9540154fe4847.png"},{"id":77761704,"identity":"c584b30f-0d1c-494a-8872-24eb191ed586","added_by":"auto","created_at":"2025-03-05 09:25:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":141992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of acute stress and taVNS on heart rate variabitlity (RMSSD in ms)\u003c/strong\u003e. Results are presented as raw values (in ms). \u003cstrong\u003eA.\u003c/strong\u003e Interaction of Stress and CPA; \u003cstrong\u003eB.\u003c/strong\u003e Interaction of intervention with CPA. \u003cstrong\u003eC.\u003c/strong\u003e 3-way interaction for Stress, Intervention and CPA. Note that scales differ between A,B and C, to be able to display all error bars appropriately. Significance:***:p\u0026lt;0.001, ** p\u0026lt;0.01, *p\u0026lt;0.05, #p\u0026lt;0.1, ns:p\u0026gt;0.1.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5366818/v1/f8589b6cd8318c99bf712d90.png"},{"id":77762880,"identity":"7df8bf43-c4eb-454b-9350-cb3863481edd","added_by":"auto","created_at":"2025-03-05 09:41:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLevels of TNF-α and IL-6 according to intervention. \u003c/strong\u003eValues are back-transformed and were measured in pg/mL. They represent\u003cstrong\u003e \u003c/strong\u003ethe averaged effect of CPA*Intervention regardless of condition (BL/STR). Significance: ***:p\u0026lt;0.001, ** p\u0026lt;0.01, *p\u0026lt;0.05, #p\u0026lt;0.1, ns:p\u0026gt;0.1.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5366818/v1/10308a6fe787cebccb120037.png"},{"id":77765211,"identity":"226001b0-489c-4987-b7eb-14842a872af4","added_by":"auto","created_at":"2025-03-05 09:57:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2882790,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5366818/v1/49a89e57-8994-4269-86b8-50654d628333.pdf"},{"id":77761694,"identity":"3257db66-5232-483e-a173-2ad8415eec28","added_by":"auto","created_at":"2025-03-05 09:25:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":114334,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Tables\u003c/p\u003e","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5366818/v1/c3d745fb64a8f0c1f15b83be.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e\nCS and SET received intramural funding (Fokus Grant A|B) to investigate Vagus nerve stimulation. SET was funded by the Else-Kröner Frisenius Stiftung (2021.EKMS_04), the Leistungszentrum Innovative Therapeutics (TheraNova) funded by the Fraunhofer Society and the Hessian Ministry of Science and Art and the Bundesministerium für Bildung und Forschung (BMBF, Federal Ministry of Education)- 01EO2102 INITIALISE Advanced Clinician Scientist Program.","formattedTitle":"The Heart Knows Best: Baseline Cardiac Parasympathetic Activity as Guide to Transcutaneous Auricular Vagus Nerve Stimulation in Depression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMajor depressive disorder (MDD) is a highly prevalent and debilitating condition. Still, 30% of MDD patients do not respond to treatment, highlighting the need for novel treatment options. In the search for new treatment modalities, the conceptualization of MDD as a condition of chronic stress is of interest \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Specifically, people with MDD report excessive subjective levels of chronic stress and show corresponding physiological alterations, including autonomic dysregulation. This is evident in reduced heart rate variability and impaired responsivity to acute stress on both emotional and physiological measures (e.g., resistance, higher heart rate, blunted autonomic reactivity) \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Hence, specifically targeting biological pathways affected by chronic stress may offer new avenues for MDD treatment.\u003c/p\u003e \u003cp\u003eThe vagus nerve (VN) - the principal parasympathetic component of the autonomous nervous system - is a key player in stress response and recovery. Connecting the brain stem and the inner organs, the VN facilitates the bidirectional communication necessary for rapid adaptations after acute stress exposure. VN efferents influence cardiac reactivity and inflammatory responses via multiple pathways including the cholinergic anti-inflammatory reflex \u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Conversely, interoception and the emotional (and attentional) responses are integrated through strong projections that exist between the VN, brain stem nuclei (nucleus tractus solitarii and dorsal-motor nucleus), the hypothalamus, mesolimbic regions, and higher cortical areas including the insular and ventromedial prefrontal cortices \u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Hence, impaired vagus function, indicated by low cardiac parasympathetic activity (CPA)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e significantly affects the stress response and homeostasis.\u003c/p\u003e \u003cp\u003eGiven the VN\u0026rsquo;s key role in stress response and recovery, it is noteworthy that (invasive) vagus nerve stimulation is an approved treatment for treatment-resistant cases of MDD. Indeed, VN stimulation has shown promising results in reducing depressive symptoms, but the underlying mechanisms for this effect are not understood. With the introduction of non-invasive techniques, evidence on the physiological mechanisms is accumulating. For instance, non-invasive stimulation of sensory vagal afferents via transcutaneous auricular VNS (taVNS) has been shown to 1) elicit vagal brain stem nuclei and the downstream projections in the brain \u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, 2) influence behaviour \u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and 3) exert anti-inflammatory effects under very specific conditions \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. While generally positive effects of taVNS on depressive symptoms have been reported \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, studies (on the working mechanism) in MDD remain scarce.\u003c/p\u003e \u003cp\u003eConsidering that (1) MDD is a chronic stress condition with impaired CPA and reduced autonomic responses to acute stress and (2) taVNS can selectively activate vagal afferents and modulate the central autonomic network, we hypothesized that taVNS would increase acute stress reactivity, restore blunted HRV, and reduce inflammatory response to acute stress in patients with MDD and healthy controls. After analyzing the groups by diagnosis, we performed a stratification of participants by CPA \u0026ndash; a biological marker of vagal impairment - and hypothesized that particularly participants with low CPA will benefit from taVNS compared to those with high CPA.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval and Pre-registration and Trial design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe single-blind randomized controlled trial \u0026ldquo;MODULATE\u0026rdquo; was approved by the Ethical Committee of the Goethe University Frankfurt (Approval number 2021-48) and pre-registered at the German Clinical Trials Register (Identifier:\u0026nbsp;DRKS00024823) and can be retrieved in the WHO Clinical Trial Registration Platform.\u003c/p\u003e\n\u003cp\u003eAll participants underwent a screening visit, and two test-days, where sham stimulation or transcutaneous auricular vagus nerve stimulation (taVNS) were administered in a counter-balanced order (see \u003cstrong\u003eFigure 1.\u003c/strong\u003e). On the screening day, participants were assessed for eligibility using the Mini-Neuropsychiatric International Interview (MINI 7.0.2 for DSM-5)\u0026nbsp;and the Montgomery Asberg Depression Rating Scale (MADRS), completed various questionnaires (see below for details) and registered onto the electronic patient-reported outcome system (P1vital\u0026reg; ePRO system). If eligible, participants were invited to two identical test days, which took place in the morning between 07.00 am and 12.00 am and lasted approximately 2 hours. Participants were required to fast overnight (drinking water was permitted). On arrival of the test day, participants provided a blood sample, filled out questionnaires, and were then asked to complete three tasks on the computer: two reward learning tasks (not reported here) and a modified version of the Montreal Imaging Stress Task (MIST). During the MIST, an ECG was recorded. After the stress task, another blood sample was taken, approximately 45 minutes after the stress task onset. In addition, before, shortly after, and 20 minutes after completion of the stress task, cortisol samples were obtained using\u0026nbsp;Salivette\u003csup\u003e\u0026reg;\u003c/sup\u003e (Sarstedt).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParticipants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants were included at the Department of Psychiatry, Psychotherapy and Psychosomatics at the University Hospital Frankfurt between 24/05/2021 and 27/07/2023. Diagnoses of MDD and potential comorbid diagnoses were assessed with the MINI Diagnostic Interview according to DSM-5 criteria and assessed by trained raters. All participants in the healthy control group had to have an inconspicuous MINI result. All participants were between 18-65 years old. They were excluded if they satisfied the criteria for a bipolar disorder, schizophrenic psychosis, substance abuse disorder or dementia, neurological, autoimmune, rheumatological, neoplastic, dermatologic, or metabolic disorders and for healthy controls only, if they had a previous or current diagnosis of depression. Furthermore, participants with substance-abuse-induced depression were excluded. Both participants and controls were included if they had a BMI below 35, and were excluded if they were pregnant or lactating, or had an acute infection as evidenced by CRP levels above 10 mg/L on testday 1 or 2. In case of any respiratory infection, all procedures were postponed until the participant was symptom-free for at least 48 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntervention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscutaneous Vagus Nerve and Sham Stimulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVagus nerve stimulation was applied at the cymba conchae of the right ear\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003ewhile sham stimulation was applied with identical parameters at the earlobe\u003cstrong\u003e\u0026nbsp;(see Figure 1).\u0026nbsp;\u003c/strong\u003eAll participants received both interventions (e.g., taVNS either at day 1 or 2) in a randomized order.The NEMOS\u003csup\u003e\u0026reg;\u003c/sup\u003e device for research use (tVNS Technologies, Erlangen) was used, with an Impulse duration of 250\u0026micro;s, an impulse frequency of 25Hz, and a stimulation cycle duration of 32 seconds on and 28 seconds off. The stimulation intensity was minimally 100\u0026micro;A and maximally 5000\u0026micro;A and was ramped up during the 32-second stimulation cycle. Stimulation intensity was individually determined for each participant and each test day, by increasing the intensity until the participant reported an uncomfortable sensation, and then brought down in 100\u0026micro;A steps until the participant reported a tingling sensation that was not painful. Individual thresholds were recorded. The stimulation started after the blood sample was taken and the baseline assessments (e.g., BDI, demographic questionnaires) had taken place, approximately 10 minutes before the first reward learning task was initiated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e[Figure 1 here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eECG recording and processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn electrocardiogram (ECG) was recorded using a 10-channel isolated bioamplifier (NeXus-10 MKII, Mind Media BV, Netherlands). Three disposable electrodes (Kendall\u0026trade;, H66LG electrodes) in a modified lead II configuration were placed on the chest to record the ECG at a sampling frequency of 1,024Hz. BioTrace+ Software (Mind Media BV, Netherlands) was used to check the signal and export the data. ECG signal processing was performed with Kubios Pro Scientific Analysis software\u003csup\u003e24\u003c/sup\u003e, which has an inbuilt R peak detection and inbuilt artifact and noise correction. A visual inspection of raw data was performed per segment of interest (the 5-min BL, Stress and Recovery intervals). Noise detection was left at the standard settings, and unclear beats were removed manually. Segments with more than 5% of beats in need of correction were excluded from the analysis. For the present analysis we extracted heart rate (HR) in beats per minute, root mean square of successive differences between normal heartbeats (RMSSD) in ms, the absolute power of high frequency heartrate variability (HF)-HRV, and low frequency (LF)-HRV both fast Fourier transformed. We opted for these measures given our interest in CPA, which can be measured by RMSSD. For the sake of comparability with other articles reporting frequency-domain rather than time-domain measures, we also report the HF-HRV. Furthermore,\u0026nbsp;\u003csup\u003e29\u003c/sup\u003e for completeness, we also report LF-HRV, and heart rate was selected as a general index of cardiac output and physiological homeostasis\u0026nbsp;\u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStratification based on Cardiac Parasympathetic Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter conducting the analysis separated by diagnosis (MDD vs HC), we performed a post-hoc stratification based on the biological parameter \u0026ldquo;cardiac parasympathetic activity\u0026rdquo; (CPA). To stratify the participants, we used a median split for RMSSD in milliseconds (ms) across all participants to obtain basal CPA. We used the baseline of the sham condition for stratification purposes. We opted for a median split because we believe that it has clinical utility, but also performed analyses with RMSSD as a continuous predictor, which yielded comparable and highly significant results, reported in the supplementary material\u003cstrong\u003e.\u003c/strong\u003e If no data was present for this timepoint, missing values for RMSSD (n=11) were imputed using all ECG data with chained random forests as implemented in the \u003cem\u003emissRanger\u003c/em\u003e package\u0026nbsp;\u003csup\u003e26\u003c/sup\u003e in R. We opted for RMSSD instead of HF-HRV or Respiratory sinus arrhythmia because it is less dependent on respiration\u0026nbsp;\u003csup\u003e27\u003c/sup\u003e and respiration was not measured in this study. Given the known influence of tricyclic antidepressants (TCA) on HRV, we excluded three participants on TCA in the analysis stratified by CPA. The results in- and excluding participants on TCA for both stratification approaches (1. stratification based on diagnosis, and 2. stratification based on CPA) are provided in the supplementary information.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood Sample Preprocessing and Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples of the fasted participants were collected by venipuncture upon arrival and 45 Minutes after the stress task.\u0026nbsp;The blood draw was performed in a fasted state to reduce variability for the inflammatory protein measures, given that postprandial levels of proteins can differ and such that metabolic states are standardized between testing days\u0026nbsp;\u003csup\u003e28,29\u003c/sup\u003e. Blood was collected in K3EDTA Monvettes (Sarstedt) and immediately transferred to cooling containers. It was then spun down (2000g, 10 minutes) in a precooled centrifuge (4\u0026deg;C) no later than 30 minutes after blood sampling. The resulting plasma was then aliquoted into Eppendorf tubes and stored at -70\u0026deg;C until further analysis. To obtain levels of high sensitive (hs) IL-6 and hsTNF-\u0026alpha;, we used enzyme-linked immunosorbent assay with precoated kits (IL-6: Human IL-6 High Sensitivity ELISA, Art.Nr.: BMS213HS; TNF-\u0026alpha;: Human TNF-\u0026alpha; High Sensitivity ELISA Kit, Art.Nr.: BMS223HS and BMS223-2HS both, Invitrogen/ThermoFisher Scientific) as per manufacturer\u0026rsquo;s instructions. Samples were measured in duplicate, blank values were subtracted, and a standard curve was obtained for each plate using Microsoft Excel, optical densities were converted to concentrations in pg/mL. If the sample duplicate\u0026rsquo;s coefficient of variation deviated more than 15%, they were repeated. Values below the detection level were replaced by the LOD divided by 2. For TNF-\u0026alpha; analytical sensitivity was 0.13 pg/mL, for IL-6 0.03pg/ml. For TNF-\u0026alpha;, of the 361 measurements, 47 were below level of detection and were replaced by the level of detection divided by 2 (i.e., 0.13/2). No levels were below the detection level for IL-6.\u0026nbsp;Concentrations ranged from 0.35pg/mL-55.70pg/mL for IL6 and from 0 (undetectable levels)-8.37pg/mL for TNF-\u0026alpha;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample size\u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sample size was determined with a predefined power calculation, for a repeated measures Analysis of Variance (ANOVA) with a within and between effects interaction a sample size of 76 was determined, given an assumed effect size of 0.25, alpha 0.05, and a reasonable power of 0.95, correlation among measures 0.3, 2 groups and 2 measurements (i.e., taVNS/sham) (calculated with G*Power 3.1.9.6). Given the potential dropout effects, and assuming that around 20% of values detected with ELISAs are below the threshold of detection, we aimed to recruit 100 participants at T1 (with 1:1 group distributions). A simulation analysis showed that with 100 participants, a smaller effect size of just below f=0.152 would still be detected with 80% power (see \u003cstrong\u003eSupplementary Figure 1)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRandomization\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants were randomized to receive either sham stimulation or verum stimulation based on a pre-established list, composed by a random sequence in Excel. Participant IDs were linked to the allocation, and this could not be changed. The list was assembled in three blocks of 50 participants. Researchers were not blinded to the allocation, but participants were blinded, i.e., they were not informed of which intervention they received on each day. The random allocation sequence was generated by the senior scientists, while allocation was performed by the study staff. The procedure of sham stimulation and taVNS were identical, except for the location of stimulation. Care was taken to spend an equal amount of time on the establishment of the stimulation intensity thresholds and experimental procedures in both conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical methods\u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDemographic variables between groups were compared using t-tests, the Wilcoxon test, or the Chi-square(\u0026chi;\u003csup\u003e2)\u003c/sup\u003e test for proportions. Variables were transformed as indicated. To assess the VAS score, a composite score was calculated for a negative and a positive component, by summing up relevant items. To assess our main hypothesis, we fitted a linear mixed model (estimated using REML and if necessary nloptwrap optimizer) to predict variables of interest with Intervention, Cardiac parasympathetic Activity, Timepoint, Group, Sex, Age, Testday and Stimulation Intensity (\u003cem\u003eformula: DV~ Intervention * Group * Timepoint + Group + Sex + Age + Testday + Stimulation Intensity\u003c/em\u003e). The model included ID as a random effect (\u003cem\u003eformula\u003c/em\u003e: \u003cem\u003e~1 | ID\u003c/em\u003e). Models were fitted using \u003cem\u003elme4\u0026nbsp;\u003c/em\u003e\u003csup\u003e30\u003c/sup\u003e and \u003cem\u003elmerTest\u003c/em\u003e packages\u0026nbsp;\u003csup\u003e31\u003c/sup\u003e. A type III anova table was computed for F and p values for fixed effects using Satterthwaite\u0026apos;s method for the approximation to degrees of freedom. We repeated the same model for post-hoc stratified analyses, where instead of diagnostic group, we used CPA. The insignificant 3-way interaction for RMSSD was compared with post-hoc tests, given the specific interest to our study design. Where reported, post-hoc tests were computed as contrasts with the \u003cem\u003eemmeans\u003c/em\u003e package\u0026nbsp;\u003csup\u003e32\u003c/sup\u003e and if applicable to the contrasts, the standard\u0026nbsp;inbuilt Tukey method for comparing a family of tests of the \u003cem\u003eemmeans\u003c/em\u003e package was automatically applied.\u0026nbsp;Additionally, we also investigated antidepressant medication status yes/no (here, most people were taking SSRIs or SNRIs). Results did not change, and antidepressant medication was not a significant predictor for HRV, therefore the models are not reported.\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed with RStudio (Version 4.3.1.) and graphical representations were made in Graphpad Prism, based on summary data extracted from R\u0026nbsp;\u003csup\u003e33\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuestionnaires\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiagnosis or absence thereof was ascertained with the Mini-Neuropsychiatric International Interview (MINI, version\u0026nbsp;7.0.2 for DSM-5)\u0026nbsp;\u003csup\u003e34\u003c/sup\u003e. The MINI is a short, well-validated, semi-structured diagnostic interview for major psychiatric disorders. Items refer to the last month and/or the entire life and are rated on a yes/no basis, allowing us to assess the absence or presence of psychiatric disorders, based on DSM-5 criteria. The Montgomery Asberg Depression Rating Scale (MADRS) is a well-validated 10-item clinical interview assessing symptoms of depression in the past 7 days, with a cumulative score indicative of the presence of no, light, moderate, or severe depression\u0026nbsp;\u003csup\u003e35\u003c/sup\u003e. Proposed cut-offs are 0-6 (absence of depression), 7-19 (mild\u0026nbsp;depression), 20-34 (moderate depression) and 35-60 (severe\u0026nbsp;depression). In addition, the self-rated Beck\u0026rsquo;s Depression Inventory (BDI-II) and the Perceived Stress Scale (PSS)\u003csup\u003e36\u003c/sup\u003e were administered. The BDI-II is a 21-item, self-rated scale for depressive symptoms with good sensitivity and specificity\u0026nbsp;\u003csup\u003e37\u003c/sup\u003e. The PSS-10 is a commonly used 10-item scale to assess perceived stress in the past 4 weeks. Scores range from 0 (no stress) to 40 (high stress). The Childhood Trauma Questionnaire (CTQ) was collected to assess childhood trauma\u0026nbsp;\u003csup\u003e38\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisual Analogue Scales\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring each baseline, after stress and after recovery phases, participants rated eleven items on a 100-mm Visual Analogue Scale (VAS=). Two composite scores were calculated for the items of the VAS scale (one containing negative emotions, the other one positive emotions, consisting of the sum of the items (positive: energetic, motivated, happy, in control; negative: scared, alert, stressed, powerless, tense. Additionally, given the experimental stress exposure, we explored the answers on the VAS item \u0026ldquo;stressed\u0026rdquo; separately, hereafter named \u0026ldquo;stress perception\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental stress task\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo induce stress, we used an adapted, customized, and computerized variation of the mental arithmetic stress task similar to the Montreal Stress Imaging task (MIST) \u003csup\u003e39\u003c/sup\u003e. The task includes a 5-minute baseline, a 1-minute-control/demonstration task (i.e., a mental arithmetic without stressful elements), a 5-minute mental arithmetic task with stressful elements (stress phase), and a 5-min recovery phase. In the current version, the following elements are included to induce stress: social comparison (e.g., playing against an \u0026ldquo;imaginative peer\u0026rdquo; whose progress is monitored by a progress bar on the right side of the screen), adaptive time pressure (i.e., if the participants answered correctly, they would receive less time to answer the next question; if they answered wrong several times, the time limit was increased) and feedback by the researcher (i.e., two times verbal feedback to \u0026ldquo;answer faster\u0026rdquo; or \u0026ldquo;try to answer correctly\u0026rdquo;).This version is a computerized task, which requires participants to select the right answer on the computer screen without requirement for verbalization and participants were instructed not to talk in order not to disrupt breathing patterns.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eIn the period from May 2021 to August 2023, 146 people were assessed for eligibility, of which 26 were excluded or piloted with a different study design, 120 were randomized, 110 completed their first intervention and 101 participants completed their second cross-over phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The trial was ended after the target of included participants was reached.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e[Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e here]\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003eBaseline data\u003c/h2\u003e \u003cp\u003eFor all presented analyses, we compared participants with MDD versus healthy controls (HC) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Participants with MDD were older compared to the control group, but had a similar BMI, sex, and smoker distribution. There was no difference between education level or contraception intake between groups. All questionnaires (BDI-II, MADRS CTQ, PSS) differed significantly.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic Characteristics of Participants by Diagnosis. CTRL\u0026thinsp;=\u0026thinsp;Control group, MDD\u0026thinsp;=\u0026thinsp;Major Depressive Disorder group. Bold values indicate significant results.nr\u0026thinsp;=\u0026thinsp;not reported.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTRL, N\u0026thinsp;=\u0026thinsp;51\u003csup\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMDD, N\u0026thinsp;=\u0026thinsp;59\u003csup\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eSample characteristics\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.24 (10.78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.79 (13.29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSex\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFemale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33 (65%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37 (63%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 (35%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22 (37%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBMI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.57 (2.59)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.32 (3.82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSmoker\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNo\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43 (84%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44 (75%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOccasional\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (7.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (8.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eYes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (7.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUnknown\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (1.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eContraception\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eno\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19 (37%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23 (40%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eyes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (22%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (8.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003epostmenopausal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003emale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 (35%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22 (38%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eunknown\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (3.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (3.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEducation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0- no degree\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (3.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1- primary school\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (1.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2- secondary school\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 (39%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (27%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3- secondary education\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 (12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4- university degree\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 (47%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32 (54%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5- doctoral degree\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eunknown\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (1.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUnmedicated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eno\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (5.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38 (66%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eyes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48 (94%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17 (29%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003enr\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (5.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAntidepressants\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eno\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20 (34%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eyes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34 (59%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003enr\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (6.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eQuestionnaires\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBDI-II\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.31 (2.16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.86 (10.28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMADRS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.20 (2.18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.66 (8.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCTQ Sum Score\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.20 (10.51)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.19 (18.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePSS-10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.37 (4.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.03 (5.62)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e\u0026nbsp;Mean (SD); n (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e\u0026nbsp;Wilcoxon rank sum test; Pearson\u0026rsquo;s Chi-squared test; Fisher\u0026rsquo;s exact test\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e[Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e here]\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003eDemographics in groups based on CPA\u003c/h2\u003e \u003cp\u003eWhen split by CPA, 55 participants were in the high and 54 in the low CPA group. In the low CPA group, 39 of 54 (72%) were MDD participants, in the high CPA group, 19 of 55 (35%) were MDD participants (see \u003cb\u003eSupplementary Table\u0026nbsp;1A\u003c/b\u003e for demographics by CPA). The low CPA group had higher depression scores and higher chronic stress levels, which are likely attributable to the higher percentage of participants with MDD in the low CPA group (\u003cb\u003eSupplementary Table\u0026nbsp;1B and C\u003c/b\u003e), but childhood trauma was also higher in the low CPA control group (P\u0026thinsp;=\u0026thinsp;0.033), see \u003cb\u003eSupplementary Table\u0026nbsp;1B\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eResults of Blinding\u003c/h2\u003e \u003cp\u003eMost participants in both conditions (sham and taVNS) believed that they were receiving the taVNS stimulation (84% of all cases, 81% during sham, 86% during taVNS X\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;1.00, p\u0026thinsp;=\u0026thinsp;0.317) and stimulation amplitude was comparable for both interventions (sham: median: 850, tVNS median: 800, W\u0026thinsp;=\u0026thinsp;5957.5, p\u0026thinsp;=\u0026thinsp;0.094).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePlanned analysis\u003c/h2\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eNo effect of diagnosis for taVNS\u003c/h2\u003e \u003cp\u003eFirst, we assessed our primary analysis, to see if the efficacy of taVNS on our outcomes was modified by diagnosis (MDD or HC). In all analyses except for the inflammatory proteins, we did find a significant main effect of diagnosis and timepoint, but there was no main effect for intervention and no interaction effect of diagnosis and intervention (taVNS vs. sham). This was true for emotions, for HRV and for inflammatory proteins (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e for the main effect of intervention and the intervention*Group interactions). Full models can be found in \u003cb\u003eSupplementary Table\u0026nbsp;2.\u003c/b\u003e Due to the absence of intervention effects, in the following, we decided to apply a post-hoc stratification based on CPA instead of diagnosis, to assess whether those with lower CPA would profit more from taVNS than those with higher CPA, regardless of diagnosis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of Planned analysis comparing MDD and HC. No significant effects of intervention, or intervention*group were found.\u0026nbsp;Variables were transformed using log or Tukey\u0026rsquo;s ladder of power transformations and models included covariates as well as timepoint and interaction effects. Full models and models excluding those taking TCA can be found in the supplementary material table 2.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eIntervention\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eGroup* Intervention\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003eEmotions\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive [Composite score]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.768\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.278\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.561\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[Composite score]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStress [%]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.431\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.263\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eHRV\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHeart Rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.552\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.501\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRMSSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.809\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHF-HRV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.444\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.819\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLF-HRV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.259\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.231\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLF/HF ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eInflammation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIL-6 in pg/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.291\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.082\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTNF-α in pg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.922\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e[Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e here]\u003c/h2\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003ePost-hoc stratified analysis\u003c/h2\u003e \u003cdiv id=\"Sec26\" class=\"Section4\"\u003e \u003ch2\u003eThe impact of taVNS on stress perception depends on CPA\u003c/h2\u003e \u003cp\u003eTimepoint had a significant effect (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) on subjective stress levels, which increased during the stress task and reduced following recovery. Participants with MDD reported higher levels of stress compared to healthy controls at all time points (main impact of group p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The interaction of intervention (taVNS vs. sham) and CPA was significant (p\u0026thinsp;=\u0026thinsp;0.024). Follow-up tests showed that for those with high CPA, taVNS did not affect stress levels (t\u0026thinsp;=\u0026thinsp;0.06, p\u0026thinsp;=\u0026thinsp;0.549), but significantly increased stress perception in those with low CPA (t=-2.63, p\u0026thinsp;=\u0026thinsp;0.009). No other interactions were significant.\u003c/p\u003e \u003cp\u003eFor negative but not positive emotions, timepoint had a significant main effect (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and p\u0026thinsp;=\u0026thinsp;0.676) and the group was significant in both models(both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but we did not find an effect of intervention (see \u003cb\u003eSupplementary Table\u0026nbsp;3A\u003c/b\u003e for fixed effect estimates of the full models and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-I. for graphical representations). Analyses with CPA as a continuous score yielded similar results (\u003cb\u003eSupplementary Table\u0026nbsp;3B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e[Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e here]\u003c/h2\u003e \u003cdiv id=\"Sec28\" class=\"Section4\"\u003e \u003ch2\u003eThe impact of taVNS on heart rate variability depends on CPA\u003c/h2\u003e \u003cp\u003eTo assess the impact of CPA and taVNS on the autonomic stress response, we investigated RMSSD and other measures of heart rate variability (HR, HF-HRV, LF-HRV and LF/HF ratio).\u003c/p\u003e \u003cp\u003eMain effects for timepoint and CPA were significant (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for all measures, only for the LF-HF ratio, timepoint was not significant (p\u0026thinsp;=\u0026thinsp;0.149), see \u003cb\u003eSupplementary Table\u0026nbsp;4A\u003c/b\u003e. We found an interaction of CPA and intervention for RMSSD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), HF-HRV (P\u0026thinsp;=\u0026thinsp;0.002), LF-HRV (P\u0026thinsp;=\u0026thinsp;0.026), but not HR (P\u0026thinsp;=\u0026thinsp;0.121) or LF/HF ratio (p\u0026thinsp;=\u0026thinsp;0.127) (see \u003cb\u003eSupplementary Table\u0026nbsp;4B\u003c/b\u003e for post-hoc comparisons of interactions).\u003c/p\u003e \u003cp\u003eFollow-up tests showed that RMSSD and HF-HRV numerically increased in the low CPA group during taVNS (sham-taVNS; RMSSD: t=-1.96, P\u0026thinsp;=\u0026thinsp;0.050, HF-HRV t=-1.84, P\u0026thinsp;=\u0026thinsp;0.067), but taVNS reduced RMSSD and HF-HRV in the high CPA group (sham-taVNS; RMSSD t\u0026thinsp;=\u0026thinsp;3.58, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; HF-HRV: t\u0026thinsp;=\u0026thinsp;2.79, P\u0026thinsp;=\u0026thinsp;0.006), see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. Furthermore, for all variables, except LF-HRV and LF/HF ratio, a significant CPA*timepoint interaction occurred (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, see \u003cb\u003eSupplementary Table\u0026nbsp;4A and 4B\u003c/b\u003e). Post-hoc tests for RMSSD showed that people in the low CPA group did not react to stress (baseline-stress: RMSSD: t\u0026thinsp;=\u0026thinsp;2.23, P\u0026thinsp;=\u0026thinsp;0.067), compared to strong reactivity in the high CPA group (baseline-stress: RMSSD: t\u0026thinsp;=\u0026thinsp;7.08, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while both had significant recovery, albeit numerically stronger in the high CPA group (high: t=-5.15 vs. low: t=-2.98), see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. Results with CPA used as a continuous predictor were comparable to the analysis using a factor for HF-HRV and RMSSD (see \u003cb\u003eSupplementary Table\u0026nbsp;4C\u003c/b\u003e for statistics).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eExploratory post-hoc analyses 3-way interaction for RMSSD shows recovery of blunted stress response\u003c/h2\u003e \u003cp\u003eGiven our interest in the treatment effect, we also conducted exploratory contrasts for the non-significant three-way interaction of RMSSD. Contrasts showed that while during sham stimulation, there was indeed a blunted reactivity and recovery for RMSSD in the low CPA group (sham; baseline-stress: t\u0026thinsp;=\u0026thinsp;0.19, P\u0026thinsp;=\u0026thinsp;0.981, stress-recovery: t=-0.82, P\u0026thinsp;=\u0026thinsp;0.692), this was restored during taVNS (baseline-stress: t\u0026thinsp;=\u0026thinsp;2.96, P\u0026thinsp;=\u0026thinsp;0.009, stress-recovery: t=-3.39, P\u0026thinsp;=\u0026thinsp;0.002). In the high CPA group both sham and taVNS showed significant reactivity and recovery during sham (sham; baseline-stress: t\u0026thinsp;=\u0026thinsp;4.79, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, stress-recovery: t=-3.52, P\u0026thinsp;=\u0026thinsp;0.001) and taVNS (baseline-stress: t\u0026thinsp;=\u0026thinsp;5.25, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, stress-recovery: t=-4.29, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEffect of taVNS on Inflammatory Proteins is opposite dependent on CPA\u003c/h3\u003e\n\u003cp\u003eOverall, as expected in a population without overt inflammatory conditions, levels of IL-6 and TNF-α were low. No main effects of CPA, or timepoint occurred for IL-6 or TNF-α (see \u003cb\u003eSupplementary Table\u0026nbsp;5A\u003c/b\u003e for fixed effects). For TNF-α, an intervention*CPA interaction occurred (P\u0026thinsp;=\u0026thinsp;0.038). Post-hoc comparisons showed that regardless of group or timepoint, in those with higher CPA, TNF-α was significantly \u003cem\u003ehigher\u003c/em\u003e during taVNS than during sham stimulation (sham-taVNS: t=-2.09, P\u0026thinsp;=\u0026thinsp;0.038) while in those with low CPA, TNF-α was not significantly altered (sham-taVNS: t\u0026thinsp;=\u0026thinsp;0.70, P\u0026thinsp;=\u0026thinsp;0.483). See Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e for an overview. If RMSSD (sham, baseline) was used as a continuous variable, the interaction of CPA*Intervention was not significant for TNF-α (p\u0026thinsp;=\u0026thinsp;0.232), see \u003cb\u003eSupplementary Table\u0026nbsp;5B\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this counter-balanced, randomized-controlled study, we aimed to assess whether taVNS restored the acute stress response in people with MDD by improving subjective stress perception, enhancing cardiac reactivity and reducing inflammation compared to healthy controls. Contrary to our hypothesis, we found no effect of taVNS on these outcomes, nor any interaction of taVNS and the diagnostic group. In other words, taVNS had no significant impact on our outcomes, with no difference between MDD patients and HC.\u003c/p\u003e\n\u003cp\u003eSubsequently, we decided to use post-hoc stratification by a biological marker of VN activity - the cardiac parasympathetic activity (CPA) (using the median RMSSD as a surrogate marker). We reasoned that people with low CPA, indicating low VN activity, were most likely to profit from taVNS. In the low CPA group (regardless of diagnosis), taVNS recovered the previously absent cardiac vagal reactivity to stress, increased the perception of stress, but only had a minor, non-significant reductive effect on TNF-\u0026alpha;. In contrast, and unexpectedly, if CPA was high at baseline, taVNS \u003cem\u003edampened\u003c/em\u003e heart rate variability (HRV), \u003cem\u003ein\u003c/em\u003ecreased HR and significantly \u003cem\u003ein\u003c/em\u003ecreased levels of TNF-\u0026alpha;. Our findings clearly suggest that the effects of taVNS depend on baseline CPA, suggesting that consideration of CPA is essential for future studies.\u003c/p\u003e\n\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\n \u003ch2\u003etaVNS: No effect on biological outcomes in MDD?\u003c/h2\u003e\n \u003cp\u003eAn adequate response to acute stress is fundamental for survival and is tightly regulated by the autonomic nervous system. Persisting exposure to physical or emotional stress, however, can disrupt homeostasis, mitigate basal cardiac parasympathetic activity (CPA), and impair the physiological ability to face sudden challenges, as often observed in patients suffering from MDD. Vagus nerve stimulation provides a targeted approach to directly stimulate the underlying biological pathways that are impaired by chronic stress exposure. Surprisingly, against our hypotheses, there was an absence of a main and/or interaction effects for the intervention in our primary analyses comparing people with MDD and controls. This could indicate that the non-invasive form of taVNS does not have any acute effects, even in a relatively large sample such as ours. However, our post-hoc stratification revealed a critical feature linked to the response of taVNS: the individual CPA at baseline. Only in the low CPA group, taVNS improved stress perception and recovered cardiac reactivity. Interestingly, while we expected the strongest effects of taVNS for those with low CPA, and somewhat attenuated effects in those with high CPA, this was not the case. Contrary to our expectations, we did not find weaker effects in those with high CPA, but instead, taVNS had stronger and \u003cem\u003eopposed\u003c/em\u003e effects to the anti-inflammatory, HRV-enhancing effects we expected from the literature. This is a crucial insight, as effects in opposed directions in those with high and low CPA may lead to null findings for the (ta)VNS, if baseline CPA is disregarded.\u003c/p\u003e\n \u003cp\u003eThe absence of a significant modifying effect of MDD on the intervention may be partly attributed to the diagnostic criteria for depression, which currently rely solely on symptomatology and likely encompass various disorders with distinct pathophysiological mechanisms. This may explain why only some, but not all, MDD patients exhibit low CPA. Consequently, CPA could serve as a valuable tool for selecting patients based on a biological stratification marker for VNS treatment.\u003c/p\u003e\n \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\n \u003ch2\u003eChronic stress is linked to impaired CPA\u003c/h2\u003e\n \u003cp\u003eIn line with previous research \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, we found that chronic stress and childhood trauma were associated with low CPA in our sample. This association has been predominantly reported in clinical populations suffering from chronic stress conditions (i.e., Posttraumatic Stress Disorder, Anxiety Disorders, MDD) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The well-orchestrated response to acute stress starts with an increase in sympathetic activity, catecholamine and cortisol production, energy supply through gluconeogenesis and lipolysis, and cardiac (e.g. increased heart rate and blood pressure) and immune arousal, but is temporally limited and terminated by parasympathetic activation. Yet, sustained stress and the lack of habituation to stressors can lead to deficient CPA (i.e., \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e), failing to regulate the aforementioned systems and therefore leading to a chronic elevation of sympathetic activity, catecholamine, and cortisol production and reduced cardiac vagal modulation. As in previous studies \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, we here observed the biological effects of chronic stress often described in the literature: We found that in the low CPA group the baseline level of HR was higher, cardiac stress reactivity was blunted, and baseline levels of perceived stress and depressive symptoms were higher.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\n \u003ch2\u003eDifferential effects of taVNS on cardiac reactivity dependent on CPA\u003c/h2\u003e\n \u003cp\u003eIntriguingly, taVNS restored cardiac reactivity and recovery for those with low CPA. Stimulation of the VN\u0026rsquo;s sensory afferents at the outer ear is considered to activate the dorsomotor nucleus. This activation relayed through the afferent nucleus (NTS) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, modulates the parasympathetic projections directed towards the sinoatrial and atrioventricular nodal cells as well as the atrial myocytes \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. It is thus possible that taVNS can counteract the effects of chronic stress by activation of parasympathetic projections. The opposed effects in the high CPA group suggest that this mechanism may differ in this group, either by engagement of a different mechanism, overactivation of the projections and/or by activation of the sympathetic rather than parasympathetic nervous system. Indeed, previous studies have discussed the possibility of low-level sympatho-excitation by vagal afferents due to bioelectric stimulation as applied during taVNS \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This could provide a potential explanation for the inconsistency reported regarding (ta)VNS effects on heart rate variability, which have shown reductions\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, increases \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, or no distinguishable effect \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, summing up to a lack of a consistent effect on HRV \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eNo acute effects of taVNS on emotional states\u003c/h3\u003e\n\u003cp\u003eFurthermore, as a treatment for depression, the integration between the autonomic state, interoceptive signals, and emotional states is crucial and is mediated by highly interconnected brain stem nuclei and higher cortical regions that receive vagal afferents. Stimulation of the vagus nerve (direct and/or via taVNS) provides an intriguing option to investigate the effect of stimulation on this integrative process since it is capable of stimulating the \u003cem\u003enucleus tractus solitarii\u003c/em\u003e, and the interconnected limbic and cortical areas that belong to the central autonomic network in both rodents and humans\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Chronic VNS has been shown to improve negative mood states in humans \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e and low CPA has been linked to negative emotional states, in line with our data. A recent rodent study elegantly demonstrated that reduced CPA due to prolonged stress resulted in alterations in the connection between the prefrontal cortex and amygdala, leading to anxious behavior similar to that seen in vagotomy, and chronic VNS restored brain signals and the anxious phenotype \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In our sample, however, no intervention effect was found for negative emotions after the short intervention period. Future studies in humans need to shed more light on this process.\u003c/p\u003e\n\u003ch3\u003eDifferential effects of taVNS on inflammatory cytokines depending on CPA\u003c/h3\u003e\n\u003cp\u003eThe seminal discovery by Borovikova et al. (2000) highlighted the relevance of the VN for regulating inflammatory responses through the cholinergic anti-inflammatory pathway. Since then, several studies - also in humans \u0026ndash; have shown that low cardiac parasympathetic activity is associated with increased cytokines at baseline and after immunological challenges \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Selective stimulation of the VN -both invasive and transcutaneous- suppresses circulating cytokines, but most of the significant effects were only applicable to subgroups and in conditions of acute immunological events \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Our results showing differential effects of taVNS depending on baseline CPA raise new questions: first, our results suggest that taVNS could have modest anti-inflammatory potential \u003cem\u003eif\u003c/em\u003e baseline CPA is low, whereas if the CPA is already high, taVNS increases TNF-⍺. Second, we did not find a change in IL-6 after taVNS, which is in line with preclinical literature and suggests a rather specific effect of (ta)VNS on TNF-⍺. Analog to the findings in HRV, this might explain the rise in cytokines reported following taVNS in healthy human participants (e.g., \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e), who were not recruited based on baseline CPA and might have had high CPA. We are intrigued whether similar findings can be found in studies using longer durations and different pathologies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInverse effects of taVNS with higher CPA? A call for stratification in existing cohorts using (ta)VNS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough there is a general correlation between high CPA, favourable health outcomes and a well-regulated autonomic nervous system, taken together our results suggest that CPA beyond a certain threshold triggers an alternative mechanism (potentially SNS rather than PNS activation) in response to taVNS. Our results align with prior research that documented decreased heart rate variability following (ta)VNS \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e and increases in cytokines in healthy subjects \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Most of these results involved youthful, healthy subjects with probably high CPA at baseline, who may have surpassed a threshold with (ta)VNS. In the clinical context, vagal hyperactivity (which we don\u0026rsquo;t see in our participants) has been linked to bradycardia, syncopes, hypotension, and excessive gastric acid secretion \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Here, we used post-hoc stratification with a median split for RMSSD as an index of cardiac vagal activation, due to the sample size constraints, but the exploratory analyses using continuous CPA produced similar results. It is thus possible that a dose-response relationship is present, which should be explored in larger sample sizes.\u003c/p\u003e\n\u003cp\u003eWe identified one small clinical trial which also used stratification by baseline CPA after a long-term intervention with invasive VNS in patients suffering from Crohn\u0026rsquo;s disease \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Intriguingly, the effect of VNS on heart rate variability was restorative in 4 of 5 patients with low CPA but decreased in the one person with high CPA at baseline. Others have reported that responders to VNS (as defined by more than 20% decrease in LF/HF), had significantly lower basal CPA than non-responders \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Although it is obvious, that VNS should be particularly beneficial if CPA is compromised, the majority of studies so far have not used CPA for stratification. If HRV data is available, we, therefore, call to researchers for a secondary analysis of (ta)VNS datasets, which could offer more insight, particularly for studies assessing long-term effects and antidepressant response.\u003c/p\u003e\n\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\n \u003ch2\u003eMethodological considerations\u003c/h2\u003e\n \u003cp\u003eA distinctive aspect of our study lies in our choice to stimulate the right outer ear. This decision was informed by rodent studies, that demonstrated a closer link between the right VN and limbic areas critical for reward behaviour and emotional processing. Rodent investigations have demonstrated that both right and left VNS impact cardiac reactivity, while effects were more often investigated on the right side \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Because of the greater innervation of the sinus-atrial node by the right VN, invasive and the majority of taVNS applications have avoided this stimulation side fearing bradycardia. Nevertheless, there is currently no data suggesting safety concerns with transcutaneous VNS administered to the right side or bilaterally \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e and increases in HRV through VNS have been demonstrated for both right \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e and left VNS \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eIn addition, it was shown that specific stimulation parameters elicit an increase rather than a decrease of cytokines \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e indicating that a systematic investigation of stimulation parameters is necessary in future studies to further explore this dynamic in humans.\u003c/p\u003e\n \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e\n \u003ch2\u003eLimitations\u003c/h2\u003e\n \u003cp\u003eWhile our study is highly novel, several limitations need to be mentioned. First, we used transcutaneous, not invasive VNS. We suggested the observed effects to be mediated through the activation of specific brain regions triggered by afferent fibre signaling. Although this signaling pathway lacks the precision of direct vagal fibre stimulation, recent rodent studies suggest it may achieve comparable outcomes \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. We also did not measure respiration, but CPA is only reliably measured when the respiration rates are in the normal range (e.g., between 7.2\u0026ndash;24 breaths/minute)\u003csup\u003e15\u003c/sup\u003e, which could affect the results if breathing occurred outside of these ranges. Finally, the effects we observed (notably for inflammation) are small and observed during acute stimulation. It remains to be seen whether taVNS or invasive VNS will also have this effect during longer stimulations and in clinical populations with higher levels of inflammation.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Conclusion and outlook","content":"\u003cp\u003eIn conclusion, our findings indicate that baseline CPA matters, at least for the biological outcomes of taVNS. A compromised CPA is associated with impaired cardiac and inflammatory response to acute stress exposition and can be restored by acute taVNS. However, people with high CPA may react with the opposite pattern- which could lead to null findings, as we observed in our analysis using diagnostic groups (e.g., MDD vs. controls). We call to all researchers who measured an ECG at baseline in studies using (ta)VNS to perform a secondary analysis. If our findings are replicated, this may help to find a stratification marker for successful taVNS for MDD, and other diagnoses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCS: Conceptualization, Data Collection, Data Processing, Formal Analysis, Writing-Original Draft, Writing- Review\u0026amp; Editing, Visualization, MA: Data Collection, Data Processing, EB: Data Collection, Data Processing, MS: Data Collection, Data Processing, Formal Analysis, KM: Data Collection, Data Processing, TH: Data Collection, Data Processing, CU: Data Collection, Data Processing, KA: Data Collection, Data Processing, AP: Data Collection, Data Processing, MQ: Data Collection, Data Processing, GB: Data Collection, Data Processing, TZ: Data Collection, Data Processing, AB: Data Collection, Data Processing, RH: Writing-Original Draft, Writing- Review\u0026amp; Editing, TH: Writing-Original Draft, Writing- Review\u0026amp; Editing, JR: Writing-Original Draft, Writing- Review\u0026amp; Editing, SM: Writing-Original Draft, Writing- Review\u0026amp; Editing, AR: Writing-Original Draft, Writing- Review\u0026amp; Editing, SET: Conceptualization, Data Collection, Formal Analysis, Writing-Original Draft, Writing- Review\u0026amp; Editing, Visualization, Supervision, Project administration\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Sabine Stanzel for her exceptional technical assistance throughout the study and Florian Freudenberg for the consultation on the preprocessing of blood samples. The authors would like to thank Greg Dunphy from P1Vital for his immense support with the electronic study-reported outcome system (P1vital® ePRO system).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding/Conflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCS and SET received intramural funding (Fokus Grant A|B) to investigate Vagus nerve stimulation. SET was funded by the Else-Kröner Frisenius Stiftung (2021.EKMS_04), the Leistungszentrum Innovative Therapeutics (TheraNova) funded by the Fraunhofer Society and the Hessian Ministry of Science and Art and the Bundesministerium für Bildung und Forschung (BMBF, Federal Ministry of Education)- 01EO2102 INITIALISE Advanced Clinician Scientist Program.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang, Z. \u003cem\u003eet al.\u003c/em\u003e Heart rate variability in generalized anxiety disorder, major depressive disorder and panic disorder: A network meta-analysis and systematic review. \u003cem\u003eJ Affect Disord\u003c/em\u003e \u003cstrong\u003e330\u003c/strong\u003e, 259\u0026ndash;266 (2023).\u003c/li\u003e\n\u003cli\u003ePereira, V. H., Campos, I. \u0026amp; Sousa, N. 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Transcutaneous vagus nerve Stimulation (taVNS) offers a non-invasive method to stimulate the vagus nerve \u0026ndash; the key component of the parasympathetic system \u0026ndash; to restore autonomic balance. Here, we examined whether changes in emotional, cardiac, and inflammatory reactivity are differentially influenced by taVNS in participants with MDD and controls. Additionally, we performed a post-hoc analysis with participants stratified by baseline cardiac parasympathetic activity (CPA) \u0026ndash; as a proxy for vagus nerve activity - to evaluate the utility of biological stratification over diagnostic criteria.\u003c/p\u003e \u003cp\u003eTo assess the effect of chronic stress we conducted a single-blinded, cross-over, randomized controlled trial with 110 participants (51 controls and 59 MDD patients). For the analysis stratified by CPA, we grouped participants into low (n\u0026thinsp;=\u0026thinsp;54) vs. high (n\u0026thinsp;=\u0026thinsp;55) CPA regardless of diagnosis. All participants were subjected to an acute stress paradigm, both with taVNS and sham stimulation on two separate days, in a counter-balanced order. There was no difference in any of the outcomes regarding the effect of taVNS in participants with MDD and controls. Analyses split by CPA, however, showed that for those with low CPA, taVNS restored the blunted cardiac stress response and numerically decreased TNF-α levels. Unexpectedly, in people with high CPA, the opposite pattern was observed: heart rate and TNF-α were significantly increased, and vagally mediated heart rate variability was significantly decreased under taVNS compared to sham stimulation. Analyses using CPA as continuous predictors yielded similar results.\u003c/p\u003e \u003cp\u003eOur findings suggest that CPA-based stratification may a useful stratification marker for (ta)VNS treatment. We encourage researchers with HRV data to re-evaluate their findings through CPA stratification.\u003c/p\u003e","manuscriptTitle":"The Heart Knows Best: Baseline Cardiac Parasympathetic Activity as Guide to Transcutaneous Auricular Vagus Nerve Stimulation in Depression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-05 09:25:39","doi":"10.21203/rs.3.rs-5366818/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a8bd7067-65b4-4dcc-a837-6701552256ab","owner":[],"postedDate":"March 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45111587,"name":"Biological sciences/Neuroscience"},{"id":45111588,"name":"Health sciences/Biomarkers/Prognostic markers"},{"id":45111589,"name":"Health sciences/Diseases/Psychiatric disorders/Depression"}],"tags":[],"updatedAt":"2025-03-05T09:25:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-05 09:25:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5366818","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5366818","identity":"rs-5366818","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}