Method Article

Transauricular Vagus Nerve Stimulation and Electroencephalographic Assessment in Disorders of Consciousness

DOI:

10.3791/68062

⸱

July 11th, 2025

 ,  ,  ,  ,  ,  ,  , 
1NEUROCAP Research Group, Psychology Department and Human Neuroscience Laboratory, Faculty of Psychology and Education, Universidad Loyola Andalucia, Avenida de las Universidades, 2IRENEA, Instituto de Rehabilitación Neurológica, Fundación Hospitales Vithas, València, Spain

* These authors contributed equally

In This Article

Summary

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This protocol aims to provide novel methodological insights into non-invasive neurostimulation, specifically transauricular vagus nerve stimulation (taVNS), in patients with disorders of consciousness. Additionally, a clinical and electroencephalographic analysis protocol is proposed to assess the efficacy of the treatment in this cohort, along with the discussion of relevant ethical considerations.

Abstract

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The technique of transauricular vagus nerve stimulation (taVNS) is methodologically applied by placing electrodes on the auricular region, specifically on the cymba, cavum, or tragus, to stimulate the vagus nerve non-invasively. Additionally, the earlobe, which is not innervated by the vagus nerve, can serve as a placebo site for control stimulation. The stimulation intensity is adjusted between 0.5-3.0 mA, with session durations ranging from 20-60 min, administered either daily or weekly, depending on the study protocol. The total number of sessions varies according to the protocol, extending from a few weeks to several months. Ethically, research involving taVNS must adhere to regulations set by the local Agency for Medicines and Health Products and European Union guidelines. Approval from an Ethical Committee is required for studies involving healthy participants, while a Medical Ethical Committee is necessary for clinical research, ensuring that written information and informed consent are provided. Additionally, rigorous exclusion criteria must be established to safeguard participant safety, excluding pregnant individuals, those with cardiovascular, pulmonary, or metabolic diseases, psychiatric disorders, epilepsy, hypertension, or those using medications affecting the autonomic nervous system. In terms of clinical outcomes, the current study protocol demonstrated promising results with taVNS in patients with Disorders of Consciousness, showing significant improvement in alertness recovery and response to stimuli. Patients treated with taVNS experienced neurophysiological changes compared to the control group. Moreover, electroencephalographic biomarkers can be utilized to evaluate treatment efficacy, even in the absence of observable behavioral indicators. These findings suggest significant clinical potential for taVNS in managing Disorder of Consciousness, although the lack of specific regulations for taVNS underscores the need to adhere to international scientific best practices and seek consensus on European guidelines for its research application.

Introduction

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Disorders of Consciousness (DoCs), such as unresponsive wakefulness syndrome (UWS) and minimally conscious state (MCS), are severe outcomes of brain injury, characterized by impaired arousal and awareness1. Patients with disorders of consciousness face high medical risks and present emotional, ethical, and legal challenges for families and healthcare providers2,3. Ethical dilemmas primarily concern patient best interests, family well-being, and professional decision-making4. Therefore, identifying effective treatments and establishing ethical guidelines is crucial in this context.

Therapeutic options remain highly limited, although electromagnetic brain stimulation techniques have recently been explored for their potential to modulate brain activity and promote cognitive recovery. Among these techniques, deep brain stimulation (DBS) and transcranial magnetic stimulation (TMS) have yielded inconsistent results in enhancing arousal and awareness in patients, whereas transcranial direct current stimulation (tDCS) has shown Class II evidence supporting its efficacy5. Transauricular Vagus Nerve Stimulation (taVNS) is an emerging, non-invasive therapeutic approach for the treatment of disorders of consciousness6. The use of this stimulation technique in this patient cohort is rationally supported by previous anatomical studies, which have suggested that the auricular branch of the vagus nerve innervates specific regions of the ear7. Therefore, stimulation of these areas via taVNS is expected to modulate autonomic and central nervous system activity, engaging serotonin and norepinephrine pathways that connect key brain regions associated with consciousness, such as the ascending reticular activating system, the salience network, the default mode network, and the external network8. In addition, compared to other non-invasive electromagnetic stimulation techniques, such as TMS or tDCS, taVNS appears to more effectively modulate key brain networks associated with consciousness, particularly through its bottom-up neural transmission pathway9. Furthermore, various neurophysiological changes have been detected through electroencephalographic biomarkers following taVNS10, thereby strengthening its potential as a diagnostic tool. Although the safety and feasibility of taVNS have been documented in clinical interventions11, a standardized stimulation protocol for this patient population has not been fully established and widely implemented, with its efficacy still insufficiently explored12.

Some studies have investigated the effects of taVNS on disorders of consciousness, employing various methodologies and stimulation parameters13. Although clinical research has indicated improvements in patients receiving taVNS, as assessed by behavioral scales such as the Coma Recovery Scale-Revised (CRS-R)14, these outcomes remain inconsistent across different studies13. Additionally, recent trials have identified electroencephalographic patterns following taVNS10, suggesting diagnostic and prognostic potential in disorders of consciousness. However, research on the effects of taVNS in this population remains inconclusive13. Lastly, existing protocols have lacked emphasis on ethical considerations. Given the lack of consensus on the parametrization and other ethical or methodological aspects of taVNS in disorders of consciousness, further research is essential to clarify its impact and effectiveness in this patient cohort.

The primary objective of the proposed protocol is to establish a standardized methodological framework for the application of taVNS in disorders of consciousness, emphasizing both ethical and methodological rigor. By outlining stimulation parameters, ethical guidelines, and clinical assessment strategies, this study aims to facilitate reproducibility and strengthen the reliability of taVNS as a potential therapeutic intervention. As a first step, ethical considerations are addressed in accordance with Spanish15,16 and European Union (EU)17,18 regulations, including informed consent requirements for legal representatives and necessary approvals from Ethics Committees and the Spanish Agency for Medicines and Health Products (AEMPS)19. Strict inclusion and exclusion criteria are defined to ensure patient safety. Participants must have a stable disorder of consciousness diagnosis for at least 28 days, whether UWS or MCS, and meet specific age requirements. Exclusion criteria include epilepsy, cardiovascular, metabolic, or autonomic disorders, metal implants, pregnancy, and medications affecting autonomic function, while specific criteria for study withdrawal include uncontrolled seizures or severe infections.

Methodological guidelines for taVNS research in disorders of consciousness are also presented, including specific recommendations. The stimulation device must be in optimal condition, and patients should be comfortably positioned20. Different stimulation targets in the ear, such as the tragus or cymba, are outlined, along with device parameters, including recommendations for frequency, intensity, pulse duration, and the number of sessions13. Similarly, follow-up assessment approaches are proposed to effectively evaluate the impact of taVNS on disorders of consciousness, incorporating both behavioral scales and electroencephalographic methods. Additionally, the main findings from taVNS research in disorders of consciousness are discussed, highlighting recovery in patients as measured by both behavioral scales and electroencephalographic data.

Finally, the standardization of the taVNS protocol in disorders of consciousness could increase its clinical impact, helping to identify the most effective parameters and thereby improving patients' quality of life. Given the limited treatment options available for this pathology, this technique represents a promising avenue for improving patient outcomes, provided that rigorous methodological and ethical guidelines are followed. Additionally, this method is particularly suitable for clinicians and researchers working on neuromodulation in disorders of consciousness, particularly during the chronic and stable stages, as outlined by the inclusion and exclusion criteria mentioned above. It is also relevant for those interested in behavioral and neurophysiological monitoring to assess treatment responses.

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Protocol

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This study protocol was approved by the Regional Ethics Committee (Comité Ético de Investigación Clínica Sevilla Sur, Hospital Universitario Virgen de Valme, Nº 0271-N-23), on 27 February 2024. The study was conducted in accordance with the Declaration of Helsinki (1964) and its subsequent amendments. In addition, informed consent was obtained from the legal representatives of all participating patients. The inclusion criteria required a diagnosis of a disorder of consciousness for at least 28 days, classified as either UWS or MCS, and an age between 18-70 years. Exclusion criteria included the presence of cardiovascular, metabolic, or autonomic disorders, epilepsy, psychiatric conditions, metal implants (e.g., pacemakers, cochlear implants), pregnancy, or the use of medications affecting autonomic function. The reagents and equipment used in this study are listed in the Table of Materials.

1. Ethical issues and preliminary considerations

  1. Consider ethical issues, informed by international regulations and the general guidelines for scientific integrity established by reputable institutions in Europe and Spain15,16.
  2. Obtain approval from a local Ethics Committee or Medical Ethics Committee, and authorization from the AEMPS or local agency. If modifications are necessary, submit a revised version of the research protocol for re-approval.
  3. Disclose all potential conflicts of interest, including financial or personal relationships, patents, and any significant benefits related to the study.
  4. Include all relevant information about the research techniques and procedures in a written informed consent, which participants or their legal representatives must voluntarily accept or withdraw from at any time. Make sure the information is clear, accessible, and comprehensive, covering the nature, purpose, benefits, and risks of the study.
  5. Anonymise data that could identify participants and do not transfer it to other projects or researchers, nor use it for purposes other than those specified in the approved protocol.
  6. If anonymization is not possible for justified reasons, perform pseudonymization procedures to prevent researchers from accessing identifiable personal data.
  7. Inform participants or their legal representatives about the purposes and handling of their personal data, as well as their rights.
  8. Use the standardized case report form (CRF) to systematically collect relevant clinical and demographic data while addressing ethical considerations21.
  9. Organise data files according to the Brain Imaging Data Structure (BIDS)22 and its extensions23,24,25,26,27,28,29, ensuring proper nomenclature, formats, and structured storage.
  10. Use secure repositories, such as Research Electronic Data Capture (REDCap), for the safe storage and sharing of data as necessary.
  11. Design the research protocol as a randomized controlled trial (RCT) and register it in a Clinical Trials Registry, adhering to the guidelines outlined in the Consolidated Standards of Reporting Trials (CONSORT)30 and the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT)31.
  12. Divide patients into distinct groups by aetiology (TBI or non-TBI) and diagnosis (MCS or UWS), including a healthy control group.
  13. Assign patients to experimental and control groups based on whether they receive taVNS treatment or control stimulation, using random assignment whenever possible.
  14. Implement a crossover design in which all patients receive taVNS, initially treating one group while the other serves as the control. After a washout period, switch the roles of the groups, allowing the former control group to receive the treatment (Figure 1).
  15. Conduct a statistical test for carryover effects to assess the validity of the crossover design, helping identify potential biases arising from baseline imbalances, incomplete washout effects, or blinding integrity issues32.
  16. Apply a blinding strategy to prevent any unconscious influence on the results, ideally using a double-blind approach where an organiser assesses the patient groups, while both the experimenter and the patients remain unaware.
  17. Assess blinding effectiveness after the intervention by asking participants, experimenters, or clinicians which type of control or real taVNS treatment they believe was administered. Then, calculate Cohen's κ statistic and the χ2 test to detect potential associations between the actual and perceived stimulation distribution33.
    NOTE: This step can only be followed in responsive patients and healthy participant, as well as experimenters and clinicians, but not in unresponsive patients.
  18. Calculate the required sample size for a repeated measures ANOVA F-family test assessing both within-subject and between-subject interactions, using a priori power analysis with a medium effect size (0.25) and an alpha error probability of 0.05.
    NOTE: An example of software usage is provided in Supplementary Figure 1.
  19. Consider the number of groups, typically two based on diagnosis (MCS or UWS) or aetiology (TBI or No-TBI), and two based on whether they receive stimulation (experimental) or not (control), which leads to four possible group combinations. Additionally, account for the number of measurements, ensuring at least one session before stimulation and another afterward, although additional follow-up measurements are recommended.
  20. Consider certain inclusion criteria before selecting patients, such as a clinical diagnosis of persistent UWS or MCS following an acquired brain injury, with a stable clinical evaluation for four weeks. Adjust these criteria if the study is conducted during the acute phase of disorders of consciousness (<28 days). Additionally, an age range of 18 to 70 years is recommended20, although this is not mandatory and may vary depending on the study focus.
  21. Exclude patients with metal implants, such as pacemakers or cochlear implants, and individuals with a history of cardiovascular disorders, seizures, ear pain, or other contraindications. Also, exclude those who are pregnant, dependent on alcohol or illicit drugs, or taking medications with potential epileptogenic effects20.
  22. Describe where the clinical trial will be conducted, such as at a single hospital, specific associations for disorders of consciousness, or academic centers, with a multicentre collaboration recommended to obtain a more representative sample of patients.

2. Methodological aspect of taVNS in disorders of consciousness

  1. Follow the general recommendations for the application of taVNS in experimentation20.
  2. Position patients comfortably in a chair or on a bed, as dictated by clinical requirements, ensuring their heads are properly supported. Use additional straps or cushions to maintain a stable posture and prevent shifts during stimulation.
  3. Ensure the integrity of the device before initiating any stimulation protocol by inspecting the electrodes for signs of wear and disinfecting them with alcohol.
  4. Select the appropriate region on the ear to stimulate the auricular branch of the vagus nerve in the experimental group, typically placing the anode on the cymba34,35,36.
    NOTE: While many protocols recommend stimulating the left cymba37,38,39,40 for cardiac safety, bilateral stimulation has also been applied, either simultaneously41,42,43,44 or sequentially45. Other protocols suggested targeting the cavum conchae44 or the left tragus46 (Figure 2).
  5. Use the same setup in the control group as in the experimental group, but without electrical stimulation, with very low intensity39,40,41 or targeting the earlobe35,45.
    NOTE: The choice of control stimulation depends on the effect that is desired to be detected. Testing different intensity values in the same location as the experimental group helps determine whether stimulation is necessary to produce the effect, while stimulating the earlobe with the same intensity helps assess whether the auricular branch of the vagus nerve is necessary32.
  6. Clean the targeted ear skin with an alcohol-impregnated wipe to remove oils or debris, and avoid makeup or earrings.
  7. Apply a thin layer of conductive gel or paste before placing the electrodes on the skin, ensuring that excessive pressure is not applied to the stimulated area to avoid discomfort.
  8. Connect the electrodes to the stimulation device, respecting the color code for the anode (red) and cathode (black).
  9. Place the anode on the targeted area, such as de cymba, tragus, or cavum conchae, and the cathode on the earlobe.
  10. Verify that both electrodes remain in place without shifting during stimulation.
  11. Select the appropriate parametrization of the taVNS device (Figure 3).
  12. Determine a session duration of 30 min36,39,41,42,43,46, although durations of 20 min44,45, 45 min35, 60 min38, or even 4 h34,37have also been proposed.
  13. Select a duty cycle of 30 s on and 30 s off34,35,37,38,41, with a sinusoidal39,41,46or quadratic waveform34,38 and a pulse width between 200 µs and 500 µs34,35,36,37,38,39,40,41,46, although other protocols have utilized very short pulses of 30 µs47 or longer pulses of up to 1 ms43,44.
  14. Explore the different stimulation frequencies ranging from 1 Hz to 100 Hz to achieve optimal responses44, with 20 Hz36,39,40,41,42,43,46and 25 Hz34,35,38being the most commonly used.
  15. Use an initial current intensity of 3 mA20, but reduce it if the Nociception Coma Scale-Revised (NCS-R)48 score increases by three points after stimulation, blood oxygenation drops to 95%, or heart rate increases by 20%47. Several studies have established different current intensity ranges, such as 4-6 mA36,42,43, 1-1.5 m44,47, 0.2-1.5 mA34 or 0.5-1 mA47. Additionally, single current intensities of 1 mA38, 1.5 mA46, 2 mA41, or 3 mA35 have been utilized.
    NOTE: An example of taVNS device usage is provided in Supplementary Figure 2.
  16. Re-parameterize or discontinue the stimulation promptly if any side effects are observed, if the patient experiences discomfort, or if the device alerts of suboptimal stimulation.
  17. Remove the electrodes after the stimulation session, clean any residual conductive gel from both ears, and sterilize the device using alcohol.

3. Stimulation timing

  1. Decide the number of taVNS sessions to be applied, from one daily session for 5 days35to 6 months (180 sessions)34.
    NOTE: A common protocol involves 40 sessions, administered once45or twice daily, 5 days per week for 4 weeks38,46, although some protocols have also proposed 48 sessions, twice daily, 6 days a week for 4 weeks39,40,41. Further studies have recommended a total of 56 sessions, either once daily for eight weeks37or twice daily for 4 weeks36,42, with one protocol suggesting up to 100 sessions over 50 days43.
  2. Leave a delay of 12-24 h between taVNS sessions20.

4. Behavioral assessment

  1. Use behavioral scales to assess consciousness in patients with disorders of consciousness, evaluating cognitive and attentional responses to monitor the effectiveness of taVNS during follow-up assessments, where observed changes post-stimulation may indicate treatment efficacy.
  2. Decide which behavioral scale to use, with the Coma Recovery Scale-Revised (CRS-R) being the most widely used tool for assessing consciousness and response capacity in patients, ranging from 0-23 points across six dimensions based on standardized behavioral responses14.
  3. Place patients comfortably in a quiet, well-lit room and free from distractions to ensure accurate evaluation.
  4. Conduct an initial assessment to identify potential spontaneous responses, with each response across scale items being intentional and repeated three times to ensure reproducibility49.
  5. Establish an appropriate interval between scale items, as behavioral responses may be slow49.
  6. Complete the entire protocol fully, even in the absence of apparent responses, with evaluations conducted at different times by various professionals to ensure consistency49.

5. Electroencephalographic assessments

  1. Use electroencephalographic assessments to complement behavioral, identifying variations in power, frequency, amplitude, latency, or topography as indicators of distinct patterns of brain processing in patients following taVNS50.
  2. Follow the guidelines for clinical electroencephalographic research51,52.
  3. Conduct recording sessions in a specialized room with soft, evenly distributed lighting, avoiding direct lights or harsh shadows and maintaining lighting levels between 100-200 lux to ensure comfort and prevent interference with the procedure. Additionally, maintain the room temperature between 20 °C and 25 °C (68 °F and 77 °F) for optimal conditions.
  4. Welcome all patients warmly, providing a complete and concise explanation of the study's objectives, procedures, and detailed instructions.
  5. Place patients in a comfortable chair or recline them on a bed, based on their clinical needs.
  6. Establish a proper montage according to the device manufacturer's specifications, using a recommended interval of 16-64 electrodes according to the 10-20 system.
  7. Position the electrode cap with the Cz channel centered along the ear-to-ear and nasion-to-inion axes, avoiding any discomfort for the patient and considering potential lesions.
  8. Refill electrodes with conductive gel or saline solution, as specified by the manufacturer, and use a toothpick to remove hair to ensure optimal contact with the scalp.
  9. Connect the amplifier to the acquisition computer using Bluetooth, Wi-Fi, or cable.
  10. Maintain the acquisition sampling rate at a minimum of 500 Hz, with impedance kept below 10 kΩ by applying additional gel or removing hair if necessary.
  11. Ensure patients remain as motionless as possible, with their gaze fixed on a specific point on the screen, and apply the arousal facilitation protocol if signs of drowsiness are observed.
  12. Perform paradigm stimulation on a separate computer connected to the recording system or use a single computer with an additional monitor if preferred.
  13. Conduct a resting-state period without controlled stimulation for 3-5 min, based on the visual quality of the recording, preferably with eyes open, though it can also be done with eyes closed.
    NOTE: An example of electroencephalographic recording software usage is provided in Supplementary Figure 3.
  14. Design an oddball perceptual auditory paradigm53 to elicit brain processes in dedicated software, using various stimuli presented through speakers that are time-locked to electroencephalographic recordings with specific triggers.
    NOTE: An example of paradigm design software usage is provided in Supplementary Figure 4.
  15. Present deviant high-pitched tones (1500 Hz) randomly, ideally constituting around 25% of the stimuli, alongside standard low-pitched (1000 Hz) tones that make up the remaining 75%, with an interval of 1500 ms between stimuli and an intensity of 60-70 dB.
  16. Conduct the paradigm initially in a passive condition, requiring only that the participant attends to the paradigm sequence without engaging in any active tasks.
  17. Repeat the paradigm, instructing patients to mentally count the deviant stimuli to engage voluntary cognitive processes as part of an active condition54.
    NOTE: This process must follow a structured protocol that includes clear verbal instructions and specific guidance to maintain patient engagement, such as encouraging mental counting or active focus on the target stimuli.

6. Treatment monitoring

  1. Develop appropriate assessments to evaluate the long-term efficacy of taVNS, considering the availability, circadian rhythms, and arousal levels of patients (Figure 4).
  2. Perform an initial behavioral and electroencephalographic assessment immediately prior to the stimulation to accurately establish the patients' baseline condition, ideally with a 5-day interval before the stimulation procedure.
  3. Carry out additional weekly behavioral assessments throughout the entire duration of stimulation37,39,41,46, and potentially daily assessments34.
  4. Undertake another behavioral and electroencephalographic assessment immediately after the stimulation period ends, preferably 5 days following its completion35.
  5. Optionally, perform assessments immediately before and after both the first and last taVNS sessions to closely monitor the specific effects of stimulation35.
  6. Establish a structured follow-up after the stimulation period to assess the duration of its effects, including weekly assessments during the first46 or second month34, followed by evaluations every 3 months for up to 1 year45.
  7. If patients leave the institution where the research is being conducted, arrange telephone interviews with their relatives to evaluate the patients' functional progress one to three months after the stimulation period35,39,41.

7. Electroencephalographic data processing

  1. Interpolate bad channels topographically, with a recommended maximum of 10% of the channels55. Reject the recording if this proportion is exceeded.
  2. Optionally, downsample the signal to 250 Hz to improve processing speed.
  3. Segment the data into equal-sized epochs, with a 1-s duration for the resting state and from 200 ms before to 1000 ms after the stimuli for the auditory paradigm, though these intervals may be adjusted depending on the paradigm used.
  4. Apply baseline correction to the first 100 ms for the resting state and to the 200 ms pre-stimulus period for the auditory paradigm.
  5. Filter the data sequentially, preferably using a finite impulse response (FIR) Gaussian 50 Hz notch filter, a high-pass filter from 0.1 Hz to 0.5 Hz, and a low-pass filter from 30 Hz to 60 Hz.
  6. Inspect for potential filter-derived artifacts during this step to ensure the signal remains genuine.
  7. Reject artifacts visually and semi-automatically, using a suggested voltage threshold of ±80 µV, and exclude fragments lasting more than 100 ms with amplitudes less than 0.5 µV.
  8. Optionally, re-reference the signal using the average of all electrodes as the new reference.
  9. Conduct Independent Component Analysis to identify potential segments unrelated to brain activity, such as eye blinks or muscle activity, based on component shape, frequency, and topography.
  10. Perform a maximum resolution Fast Fourier Transform on the resting state recording and calculate the average absolute power and relative power in the Delta (0.5-4 Hz), Theta (4-8 Hz), Alpha (8-14 Hz), Beta (14-30 Hz) and Gamma (>30 Hz) frequency bands.
  11. Average the signal across all trials independently for both the deviant and standard stimuli in the auditory paradigm recording, ensuring a minimum of 20 trials for each stimulus type56,57, although a minimum of 30 trials is recommended. Additionally, apply a low-pass filter with a range of 10 Hz to smooth the signal, if desired.
  12. Define the Mismatch Negativity (MMN) component as the most pronounced negative deflection in the averaged signal between 100 ms and 300 ms after stimulus onset58, while defining the P300 component as the most prominent positive deflection occurring between 250 ms and 800 ms following stimulus onset59.
  13. Use the electrode with the highest amplitude for the event-related potential component as the latency reference for the other electrodes, and visually detect the inflection point just prior to the component.
  14. Calculate the peak-to-peak amplitude as the difference between the absolute amplitude of the component and the preceding inflection point60.
  15. Collect the values for latency, topography, absolute amplitude, and peak-to-peak amplitude of the different components for further analysis.
  16. Group the electrode values from the aforementioned analysis, both from the resting state and the auditory paradigm, into clusters corresponding to the fronto-central, parieto-occipital, and left and right regions of interest.
    NOTE: An example of electroencephalography analysis software usage is provided in Supplementary Figure 5.

8. Statistics

  1. Report detailed individual data in tables or scatter plots to ensure transparency and facilitate a more detailed interpretation of variability across participants.
  2. Perform a normality test on the data using the Shapiro-Wilk test if the sample size is less than 50, or the Kolmogorov-Smirnov test if the sample size is greater than 50.
  3. If normality is confirmed, perform a repeated measures ANOVA, treating assessment sessions as within-subject factors and patient groups as between-subject factors, while considering the values of behavioral assessments, resting state recording, or event-related potential characteristics for both deviant and standard stimuli. Then, conduct post-hoc analyses, such as Bonferroni or Tukey, with a significance level set at p < 0.05. If normality is not met, use a Friedman test instead.
    NOTE: An example of statistical analysis software usage is provided in Supplementary Figure 6.
  4. Calculate the effect size and apply additional statistical methods, such as linear mixed models, bootstrap techniques, or Bayesian approaches, if appropriate.
  5. Clearly indicate whether each analysis was conducted in a confirmatory or exploratory manner based on pre-registration status. If an endpoint was pre-registered, report the results as confirmatory. Otherwise, classify them as exploratory to facilitate the interpretation of the results.

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Results

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Behavioral assessments
Several outcomes have been documented following taVNS in individuals with disorders of consciousness following similar protocol, whose feasibility, safety, and efficacy were initially validated in a study conducted by our research group46. This study included six UWS patients and eight MCS patients who underwent taVNS, with CRS-R assessments conducted at baseline, after four weeks of treatment, and at a 4-week follow-up. The UWS patients had a mean basel...

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Discussion

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Transauricular vagus nerve stimulation is a promising non-invasive technique for treating disorders of consciousness. However, the lack of consensus on ethical procedures and protocols complicates efforts to clarify the true effects of this stimulation technique in this patient cohort12.

The care of patients with disorders of consciousness presents several ethical challenges and legal considerations that require careful evaluation64. Decisions r...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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All patients at IRENEA-Vithas Sevilla Aljarafe Hospital, along with their relatives, and all professionals, deserve special recognition for their invaluable support during the completion of this research. Additionally, this study was conducted as part of the Marie Skłodowska-Curie Actions DOC-BOX project (#101131344).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
64-electrode Active TwoBioSemihttps://www.biosemi.com/products.htmElectroencephalography recording device
ActiCHamp Plus / BrainAmp 64 MRplusBrain Productshttps://brainvision.com/products/Electroencephalography recording device
Brain Vision Analyzer 2.0Brain Productshttps://brainvision.com/products/analyzer-2/Electroencephalography analysis software
EASY-Clip / RELIfit-Tragus / RELI-StickSoterix Medicalhttps://soterixmedical.com/research/tavnstaVNS device
Electro GelTelic GroupG-10Electroencephalographic gel
Enobio/ StartimNeuroelectricshttps://www.neuroelectrics.com/Electroencephalography recording device
E-PrimePsychological Software Toolshttps://pstnet.com/products/e-prime/Paradigm design software
G*PowerUniversität Düsseldorfhttps://www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-arbeitspsychologie/gpowerStatistical software
g.NAUTILUSG.Techttps://www.gtec.at/Electroencephalography recording device
JY-VNS-200Jingyi Medical Technologyhttps://www.jingyimed.com/taVNS device
MATLab and EEGLabMathWorkshttps://www.mathworks.com/products/matlab.htmlElectroencephalography analysis software
NemosCerbomed / tVNS Technologieshttps://t-vns.com/taVNS device
Nicolet MonitorNicolet / Natus Medical Incorporatedhttps://natus.com/es/neuro/monitor-nicolet/Electroencephalography recording device
ParasymParasymhttps://www.parasym.co/taVNS device
PythonPython Software Foundationhttps://www.python.org/Electroencephalography analysis software
RR Foundation for Statistical Computinghttps://www.r-project.org/Statistical software
SDZ-IIBSuzhou Medical Appliance Factory / Hwato http://www.hwato-med.com/taVNS device
SPSSIBMhttps://www.ibm.com/es-es/products/spss-statisticsStatistical software
StatisticaStatSofthttps://www.statsoft.de/en/data-science-applications/tibco-statistica/Statistical software
taVNS501Changzhou Rishena Medical Devicehttps://www.rishena.com/?l=entaVNS device
TEN-20BionicD-04-00WVElectroencephalographic conductive paste
TENS-200AEveryway Medical Instruments Co., LtdE200A16A000144taVNS device

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Pistarini, C., Maggioni, G. Disorders of Consciousness. Clinical Pathways in Stroke Rehabilitation. Platz, T. , Springer International Publishing. Cham. 57-70 (2021).
  2. Estraneo, A., Loreto, V., Masotta, O., Pascarella, A., Trojano, L. Do medical complications impact long-term outcomes in prolonged disorders of consciousness. Arch Phys Med Rehabil. 99 (12), 2523-2531 (2018).
  3. Wang, J., Wang, W., Laureys, S., Di, H. Burnout syndrome in healthcare professionals who care for patients with prolonged disorders of consciousness: A cross-sectional survey. BMC Health Serv Res. 20 (1), 841(2020).
  4. Gallagher, A. Ethics and compromised consciousness. Nurs Ethics. 19 (4), 449-450 (2012).
  5. Bourdillon, P., Hermann, B., Sitt, J. D., Naccache, L. Electromagnetic brain stimulation in patients with disorders of consciousness. Front Neurosci. 13, 223(2019).
  6. Wang, Y., et al. Current status and prospect of transcutaneous auricular vagus nerve stimulation for disorders of consciousness. Front Neurosci. 17, 1274432(2024).
  7. Butt, M. F., Albusoda, A., Farmer, A. D., Aziz, Q. The anatomical basis for transcutaneous auricular vagus nerve stimulation. J Anat. 236 (4), 588-611 (2020).
  8. Briand, M. -M., Gosseries, O., Staumont, B., Laureys, S., Thibaut, A. Transcutaneous auricular vagal nerve stimulation and disorders of consciousness: A hypothesis for mechanisms of action. Front Neurol. 11, 933(2020).
  9. Wang, L., et al. Transcutaneous auricular vagus nerve stimulation in the treatment of disorders of consciousness: mechanisms and applications. Front Neurosci. 17, 1286267(2023).
  10. Gianlorenco, A. C. L., et al. Electroencephalographic patterns in taVNS: A systematic review. Biomedicines. 10 (9), 2208(2022).
  11. Kim, A. Y., et al. Safety of transcutaneous auricular vagus nerve stimulation (taVNS): a systematic review and meta-analysis. Sci Rep. 12 (1), 22055(2022).
  12. Jang, S. H., Cho, M. J. Transcutaneous auricular vagus nerve stimulation in disorders of consciousness: A mini-narrative review. Medicine. 101 (50), e31808(2022).
  13. Dong, X., Tang, Y., Zhou, Y., Feng, Z. Stimulation of vagus nerve for patients with disorders of consciousness: a systematic review. Front Neurosci. 17, 1257378(2023).
  14. Kalmar, K., Giacino, J. The JFK coma recovery scale-revised. Neuropsychol Rehabil. 15 (3-4), 454-460 (2005).
  15. Consejo Superior de Investigaciones Científicas (CSIC). Code of good scientific practices of CSIC. , (2025).
  16. Código de buenas prácticas científicas. , Instituto de Salud Carlos III. https://publicaciones.isciii.es/ (2021).
  17. Kondziella, D., et al. European Academy of Neurology guideline on the diagnosis of coma and other disorders of consciousness. Eur J Neurol. 27 (5), 741-756 (2020).
  18. Clinical Trials Regulation. , European Medicine Agency. https://www.ema.europa.eu/en/human-regulatory-overview/research-development/clinical-trials-human-medicines/clinical-trials-regulation#:~:text=The%20Clinical%20Trials%20Regulation%20harmonises,Economic%20Area%20(EEA)%20countries. (2025).
  19. Agencia Española de Medicamentos y Productos Sanitarios (AEMPS). , https://www.aemps.gob.es/ (2025).
  20. Badran, B. W., et al. Laboratory administration of transcutaneous auricular vagus nerve stimulation (taVNS): Technique, targeting, and considerations. J Vis Exp. (143), e58984(2019).
  21. Bellary, S., Krishnankutty, B., Latha, M. Basics of case report form designing in clinical research. Perspect Clin Res. 5 (4), 159-166 (2014).
  22. Gorgolewski, K. J., et al. The brain imaging data structure is a format for organizing and describing the outputs of neuroimaging experiments. Sci Data. 3, 160044(2016).
  23. Bourget, M. -H., et al. Microscopy-BIDS: An extension to the brain imaging data structure for microscopy data. Front Neurosci. 16, 871228(2022).
  24. Holdgraf, C., et al. iEEG-BIDS, extending the brain imaging data structure specification to human intracranial electrophysiology. Sci Data. 6 (1), 102(2019).
  25. Karakuzu, A., et al. qMRI-BIDS: An extension to the brain imaging data structure for quantitative magnetic resonance imaging data. Sci Data. 9 (1), 517(2022).
  26. Moreau, C. A., et al. The genetics-BIDS extension: Easing the search for genetic data associated with human brain imaging. GigaScience. 9 (10), giaa104(2020).
  27. Niso, G., et al. the brain imaging data structure extended to magnetoencephalography. Sci Data. 5, 180110(2018).
  28. Norgaard, M., et al. PET-BIDS, an extension to the brain imaging data structure for positron emission tomography. Sci Data. 9 (1), 65(2022).
  29. Pernet, C. R., et al. EEG-BIDS, an extension to the brain imaging data structure for electroencephalography. Sci Data. 6 (1), 103(2019).
  30. Moher, D., Schulz, K. F., Altman, D. G. The CONSORT statement: revised recommendations for improving the quality of reports of parallel-group randomized trials. Lancet. 357, 1191-1194 (2001).
  31. Chan, A. -W., et al. SPIRIT 2013 explanation and elaboration: guidance for protocols of clinical trials. BMJ. 346, e7586(2013).
  32. Verma, N., et al. Auricular Vagus Neuromodulation-A systematic review on quality of evidence and clinical effects. Front Neurosci. 15, 664740(2021).
  33. Sanchez-Perez, J. A., et al. Transcutaneous auricular vagus nerve stimulation and median nerve stimulation reduce acute stress in young healthy adults: A single-blind sham-controlled crossover study. Front Neurosci. 17, 1213982(2023).
  34. Osińska, A., et al. Non-invasive vagus nerve stimulation in treatment of disorders of consciousness - longitudinal case study. Front Neurosci. 16, 834507(2022).
  35. Vitello, M. M., et al. Transcutaneous vagal nerve stimulation to treat disorders of consciousness: Protocol for a double-blind randomized controlled trial. Int J Clin Health Psychol. 23 (2), 100360(2023).
  36. Yu, Y., et al. Cerebral hemodynamic correlates of transcutaneous auricular vagal nerve stimulation in consciousness restoration: An open-label pilot study. Front Neurol. 12, 684791(2021).
  37. Hakon, J., et al. Transcutaneous vagus nerve stimulation in patients with severe traumatic brain injury: A feasibility trial. Neuromodulation. 23 (6), 859-864 (2020).
  38. Zhou, L. -Y., et al. Transcutaneous auricular vagal nerve stimulation for consciousness recovery in patients with prolonged disorders of consciousness (TAVREC): Study protocol for a multicenter, triple-blind, randomized controlled trial in China. BMJ Open. 14 (5), e083888(2024).
  39. Zhou, Y., et al. The efficacy and safety of transcutaneous auricular vagus nerve stimulation for patients with minimally conscious state: a sham-controlled randomized double-blind clinical trial. Front Neurosci. 17, 1323079(2023).
  40. Zhou, Y. -F., Kang, J. -W., Xiong, Q., Feng, Z., Dong, X. -Y. Transauricular vagus nerve stimulation for patients with disorders of consciousness: A randomized controlled clinical trial. Front Neurol. 14, 1133893(2023).
  41. Wang, Y., et al. The efficacy and safety of bilateral synchronous transcutaneous auricular vagus nerve stimulation for prolonged disorders of consciousness: A multicenter, double-blind, stratified, randomized controlled trial protocol. Front Neurol. 15, 1418937(2024).
  42. Yifei, W., et al. Transcutaneous auricular vague nerve stimulation improved brain connection activity on patients of disorders of consciousness: a pilot study. J Tradit Chin Med. 42 (3), 463-471 (2022).
  43. Yu, Y., et al. Transcutaneous auricular vagus nerve stimulation in disorders of consciousness monitored by fMRI: The first case report. Brain Stimul. 10 (2), 328-330 (2017).
  44. Zhai, W., et al. Optimizing the modulation paradigm of transcutaneous auricular vagus nerve stimulation in patients with disorders of consciousness: A prospective exploratory pilot study protocol. Front Neurosci. 17, 1145699(2023).
  45. Cheng, L., et al. Randomized trial of transcutaneous auricular vagus nerve stimulation on patients with disorders of consciousness: A study protocol. Front Neurol. 14, 1116115(2023).
  46. Noé, E., et al. safety and efficacy of transauricular vagus nerve stimulation in a cohort of patients with disorders of consciousness. Brain Stimul. 13 (2), 427-429 (2020).
  47. Zhuang, Y., et al. Effects of simultaneous transcutaneous auricular vagus nerve stimulation and high-definition transcranial direct current stimulation on disorders of consciousness: A study protocol. Front Neurol. 14, 1165145(2023).
  48. Schnakers, C., et al. The Nociception Coma Scale: A new tool to assess nociception in disorders of consciousness. Pain. 148 (2), 215-219 (2010).
  49. Ferri, J., Noé, E., Lloréns, R. The Spanish version of the coma recovery scale-revised: Events on a correct timeline. Brain Inj. 29 (7-8), 1002-1003 (2015).
  50. Bai, Y., Lin, Y., Ziemann, U. Managing disorders of consciousness: the role of electroencephalography. J Neurol. 268 (11), 4033-4065 (2021).
  51. André-Obadia, N., et al. Recommendations for the use of electroencephalography and evoked potentials in comatose patients. Neurophysiol Clin. 48 (3), 143-169 (2018).
  52. Kappenman, E. S., Luck, S. J. Best practices for event-related potential research in clinical populations. Biol Psychiatry Cogn Neurosci Neuroimaging. 1 (2), 110-115 (2016).
  53. Squires, N. K., Squires, K. C., Hillyard, S. A. Two varieties of long-latency positive waves evoked by unpredictable auditory stimuli in man. Electroencephalogr Clin Neurophysiol. 38 (4), 387-401 (1975).
  54. Schnakers, C., et al. Voluntary brain processing in disorders of consciousness. Neurology. 71 (20), 1614-1620 (2008).
  55. Dong, L., et al. Reference Electrode Standardization Interpolation Technique (RESIT): A Novel Interpolation Method for Scalp EEG. Brain Topogr. 34 (4), 403-414 (2021).
  56. Boudewyn, M. A., Luck, S. J., Farrens, J. L., Kappenman, E. S. How many trials does it take to get a significant ERP effect? It depends. Psychophysiology. 55 (6), e13049(2018).
  57. Cohen, J., Polich, J. On the number of trials needed for P300. Int J Psychophysiol. 25 (3), 249-255 (1997).
  58. Wijnen, V. J. M., van Boxtel, G. J. M., Eilander, H. J., de Gelder, B. Mismatch negativity predicts recovery from the vegetative state. Clin Neurophysiol. 118 (3), 597-605 (2007).
  59. Schorr, B., et al. Stability of auditory event-related potentials in coma research. J Neurol. 262 (2), 307-315 (2015).
  60. Picton, T. W., et al. Guidelines for using human event-related potentials to study cognition: recording standards and publication criteria. Psychophysiology. 37 (2), 127-152 (2000).
  61. López-Rodríguez, S., et al. Do all patients suffering from disorders of consciousness respond in the same way to transauricular vagus nerve stimulation?: An electroencephalographic study in resting state. IBRO Neurosci Rep. 15, S602(2023).
  62. López-Rodríguez, S., et al. Modulación diferencial de la taVNS sobre la amplitud del componente MMN en pacientes en estado de mínima consciencia. 75, XIII Congreso de la Sociedad Española de Psicofisiología y Neurociencia Cognitiva y Afectiva. https://sepneca.es/wp-content/uploads/2023/09/SEPNECA_2023_con_ISBN.pdf (2023).
  63. López-Rodríguez, S., et al. La estimulación transauricular del nervio vago modula el componente P300 en pacientes en estado de mínima consciencia. Neurology Perspectives. 3, https://www.elsevier.es/en-revista-neurology-perspectives-17-congresos-lxxv-reunion-anual-sociedad-espanola-160-sesion-xxi-jornadas-sociedad-espanola-neurorrehabilitacion-7804-comunicacion-la-estimulacion-transauricular-del-nervio-95640 1065(2023).
  64. Young, M. J., et al. The neuroethics of disorders of consciousness: A brief history of evolving ideas. Brain. 144 (11), 3291-3310 (2021).
  65. Fins, J. J., Wright, M. S., Bagenstos, S. R. Disorders of consciousness and disability law. Mayo Clin Proc. 95 (8), 1732-1739 (2020).
  66. Fins, J. J., Bernat, J. L. Ethical, palliative, and policy considerations in disorders of consciousness. Arch Phys Med Rehabil. 99 (9), 1927-1931 (2018).
  67. Kondziella, D., Friberg, C. K., Frokjaer, V. G., Fabricius, M., Møller, K. Preserved consciousness in vegetative and minimal conscious states: systematic review and meta-analysis. J Neurol Neurosurg Psychiatry. 87 (5), 485-492 (2016).
  68. Farmer, A. D., et al. International consensus based review and recommendations for minimum reporting standards in research on transcutaneous vagus nerve stimulation (Version 2020). Front Hum Neurosci. 14, 568051(2021).
  69. Burger, A. M., D'Agostini, M., Verkuil, B., Van Diest, I. Moving beyond belief: A narrative review of potential biomarkers for transcutaneous vagus nerve stimulation. Psychophysiology. 57 (6), e13571(2020).
  70. Zhang, Q., et al. Transcutaneous auricular vagus nerve stimulation for epilepsy. Seizure. 119, 84-91 (2024).
  71. Yap, J. Y. Y., et al. Critical review of transcutaneous vagus nerve stimulation: challenges for translation to clinical practice. Front Neurosci. 14, 284(2020).

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Auricular Vagus StimulationComa Recovery ScaleEEG MonitoringMinimally Conscious StatePlacebo StimulationBrain Network DynamicsNeurophysiological Biomarkers

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