Transcutaneous auricular vague nerve stimulation improved brain connection activity on patients of disorders of consciousness:a pilot study

WANG Yifei,YANG Yi,WANG Yu,ZHANG Jinling,ZHAI Weihang,LI Shaoyuan,WU Mozheng,HE Jianghong,RONG Peijing

WANG Yifei,Yu Wang,ZHANG Jinling,ZHAI Weihang,LI Shaoyuan,WU Mozheng,RONG Peijing,Institute of Acupuncture and Moxibustion,China Academy of Chinese Medical Sciences,Beijing 100700,China

YANG Yi,HE Jianghong,Department of Neurosurgery,Beijing Tiantan Hospital,Capital Medical University,100700,Beijing,China

Abstract OBJECTIVE:To evaluate the clinical effect of transcutaneous auricular vagus nerve nerve stimulation(taVNS) on disorders of consciousness (DOC) patients with Coma Recovery Scale-Revised (CRS-R) and cerebral cortex activity by electroencephalogram (EEG)detection.METHODS:Randomized controlled methods were used to evaluate the clinical effect of taVNS on patients with DOC.Twelve patients with initial CRS-R of 6-10 were randomly divided into the treatment group of taVNS and control group of transcutaneous non-auricular vague nerve stimulation (tnVNS).According to clinical diagnosis,the treatment group was divided into vegetative state (VS)group and minimally conscious state (MCS) group.RESULTS:The energy of delta and beta bands is positively correlated with the brain activity of patients.taVNS has different regulatory effects on patients with different conscious States.In taVNS group,the energy of delta band in local brain regions changed significantly.Significant changes in brain connection activity were limited to local brain regions.While in patients with MCS in the taVNS group,delta and beta band energy significantly changed in multiple brain regions and crossbrain connection activity also changed significantly.CONCLUSION:These findings suggest that taVNS may be a related extra method for arousing patients'awakening by improving brain connection activity.And the effect is remarkable in MCS patients.

Keywords:consciousness disorders;electroencephalography;treatment outcome;transcutaneous auricular vagus nerve stimulation;brain connection activity

1.INTRODUCTION

In recent years,advances in medicine and intensive care have guaranteed more and more patients to survive from severe brain injury.1Some patients manifest with very poor prognoses and finally suffer from disorders of consciousness (DOC).DOC refer to the disorders of patients' ability to identify and perceive the surrounding environment and their own states for over 28 d after brain injury.2Neurocognitive impairment is more common in patients with severe brain injury.Those who fail to fully recover consciousness may stay in disorders of consciousness,such as coma,Vegetative State (VS) or Minimally Conscious State (MCS).3VS,also known as unresponsive wakefulness state (UWS),refers to a state of unperceived awakening which coexists in the sleepawakening cycle.4Patients with MCS may show repeatable but unstable signs of consciousness,such as responding to commands or tracking things.5

DOC have become an important public health issue in China due to the limited sources of medical treatment and rehabilitation.There are nearly 50 000-70 000 patients with VS in China.And patients with MCS are 10 times of those with VS.6The annual medical expenditure for DOC in our country amounts to 30-50 billionyuan,which represents a public health concern in terms of loss of or reduction in workforce economic productivity and increase in utilization of healthcare resources in the treatment and rehabilitation.Hence,it is urgent to find more effective and safer treatment strategies for improving the consciousness of patients with DOC.

As a potential restorative therapy for DOC,nerve stimulation has attracted more and more attention.7,8Results of a prospective study showed that vagus nerve stimulation (VNS) ameliorated the sequelae of severe head injury.That study has obtained the laboratory equipment exemption license from U.S.Food &Drug Administration (FDA) and will be an important step in determining that VNS can improve DOC after severe brain injury.9However,VNS still requires surgery,which is expensive and carries risks such as postoperative infection.10

And the vagus nerve distributing area of the auricle has a body surface representative area corresponding to each viscera.11The vagal nerve carries somatic and visceral efferents and afferents distributed throughout the brain,either monosynaptically orviathe nucleus tractus solitary (NTS).The vagal nerve has a branch of afferent projections in the auricular concha and external ear channels.Thus,transcutaneous auricular vagus nerve nerve stimulation (taVNS) was developed based on this anatomical characteristic and was previously found to produce comparable efficacy with classic VNS,which means taVNS is a promising form of classic VNS.Previous studies have shown that after 1month taVNS treatment to patients with DOC caused by cardiopulmonary arrest and hypoxic-ischemic enceph-alopathy,Coma Recovery Scale-Revised (CRS-R) raised from 6 to 13,and the diagnosis changed from VS to MCS,with repeatable compliance exercise.12

taVNS has been recently been used in neurorehabilitation,it is also suggested that taVNS could be a safe and effective tool to facilitate consciousness recovery in DOC Before the French team publication,13,14our team also has a peer-reviewed case report on the beneficial effects of taVNS on the consciousness level of a 73-yearold female single patient who developed into DOC after cardiopulmonary resuscitation for 50 d.After a 4-week taVNS treatment,her CRS-R scores rose from 6 to 13,and her diagnosis was changed from VS to MCS.The BOLD functional magnetic resonance imaging (fMRI)outcomes also showed improved brain functional connectivity (FC).This was the first case of taVNS in a DOC patient and the first report of encouraging results from clinical conditions to brain FC.

The purpose of this study was to explore the effect of taVNS on brain areas related to consciousness in patients with DOC based on EEG.

2.MATERIALS AND METHODS

2.1.Subjects

Due to the particularity of DOC,we followed the same procedure found in previous studies with similar study design.Anticipating possible exclusions due to drop-outs,we recruited 20 participants.Twelve patients with DOC aged 18-53 years (mean age=36.5,SD=10.8) were recruited,including 5 patients with MCS and 7 patients with VS/UWS assessed by neurologists based on the CRS-R.The inclusion criteria were as follows:(a)inability to follow commands;(b) post-comatose patients;(c) patients with intact skin at the stimulation site;(d) patients ＞ 21 d after the acute brain injury.If the total score was ≥ 8,the patient was diagnosed as MCS;if it was ＜ 8,the patient was diagnosed as VS.The exclusion criteria were as follows:(a) pregnant patients;and (b) patients with an active implant (c)patient with premorbid neurology antecedent.The etiology included stroke (8 cases),anoxia (2 cases),and trauma (2 cases).The course of disease was 3-13 months (7.2 ± 3.2).All patients were in stable conditions and were not treated with other therapies that might alter cortical excitability.

All the patients were from the Department of Neurosurgery,PLA Army General Hospital.This study was approved by the Ethics Committee of the Institute of Acupuncture and Moxibustion,China Academy of Chinese Medical Sciences (Approval KYSQ2021-387-01),and the written informed consent of the legal guardian was obtained.There was no statistical difference in age and gender between the two groups (P＞0.05),which indicated that the two groups were comparable.Table 1 summarizes the characteristics of all the patients.

Table 1 Characteristics of patients

2.2.Experimental design

The center randomization method will be adopted in the trial.The random scheme is generated randomly in computer and kept by a special person.Twelve patients are assigned to taVNS or transcutaneous non-auricular vague nerve stimulation (tnVNS) groups.Each stimulation lasted 14 d.The complete medical history,including etiology and course of disease,were recorded before and after each stimulation.EEG was collected before and after treatment and CRS-R was assessed by two neurologists at the same time.All subjects received routine care and the same medical care during the study.We started treatment as soon as possible when the patient's hemodynamics was stable.EEG is primary outcome,and clinical scores Clinical score was regarded as the end point.

In taVNS group,taVNS was applied to the patient's bilateral auricular concha for 30 min in each ear,twice per day for four consecutive weeks with an intensity of 4-6 mA and at a frequency of 20 Hz (less than 1 ms wave width).

In tnVNS group,taVNS was applied to the patient's bilateral auricular non-concha region and the other manipulations were the same as the those in taVNS group.We employed the The Huatuo brand electronic acupuncture instrument (SDZ-Ⅱ B type,Suzhou Medical Products Factory Co.,Ltd.).TaVNS:Two electrodes are placed in the cymba conchae of ears,an area thought to be exclusively innervated by the auricular branch of the vagus nerve (ABVN),in order to electrically stimu-late.TnVNS:the electrodes are placed on the tail of helix,which is thought to be free of vagal innervation these vagal fibers (Figure 1).15

Figure 1 TaVNS and TnVNS

2.3.Measurement

Resting state EEG before and immediately after all treatment course,taVNS was acquired from 62 channels(BrainAmp 64 MRplus,Brain-Products) with positions of the international 10-20 system which were Fp1,FPz,Fp2,AF7,AF3,AF4,AF8,F7,F5,F3,F1,Fz,F2,F4,F6,F8,FT7,FC5,FC3,FC1,FC2,FC4,FC6,FT8,T7,C5,C3,C1,Cz,C2,C4,C6,T8,TP9,TP7,CP5,CP3,CP1,CPz,CP2,CP4,CP6,TP8,TP10,P7,P5,P3,P1,Pz,P2,P4,P6,P8,PO7,PO3,POz,PO4,PO8,O1,Oz,O2 and Lz.The equipment used Ag/AgCl pin electrodes with a band pass filtered at DC set to 1000 Hz in the recorder.The EEG signal was digitized at a sampling rate of 2.5 kHz.During the experiment,skin/electrode impedance was maintained below 5 kV.

Offline analysis was performed with EEGLAB 12.0.2.5b running in a MATLAB environment (Version 2013b,MathWorks Inc.,Natick,MA,USA).The 50 Hz power signal was removed through a notch filter.Independent component analysis was used to identify and remove the artefact-relevant components such as eye movement and muscle activation.The EEG data were downsampled to 500 Hz,average referenced and divided into epochs of 10 s with 50% overlap.Then,all the epochs were visually inspected to identify artefact epochs.In total,at least 30 artefact-free epochs were obtained from each patient (Figure 2).

Figure 2 Electroencephalogram pre-process flow

Figure 3 Consort diagram

Figure 4 Brain local regions

2.4.Method

2.4.1.Relative power

In this study,we calculated EEG power in conventionally used frequency bands of delta (1-4 Hz),theta (4-8 Hz),alpha (8-13 Hz),beta (13-30 Hz) and gamma (30-45 Hz).The power spectral density (PSD) was calculatedviapwelch method,with a window length of 10 seconds (s).The RP of the sub-band was calculated as:

where Power sub-band indicates the absolute power in the sub-band,such as the delta or theta.The Power (1,45)is the sum of the power in the frequency band from 1 to 45 Hz.

2.4.2.Coherence

Coherence was measured with spectral cross-correlation and normalized power spectra of signals obtained from two electrodes as the following equation:

to indicate the cross-power spectral density and respective auto-power spectral densities of the signals.

2.5.Statistical analysis

Off-line analysis was performed with EEGLAB 12.0.2.5b,running in a MATLAB environment (Version 2013b,MathWorks Inc.,Natick,USA).The modulation effect of taVNS and tnVNS was assessed with a Wilcoxon match-paired signed-rank test.Results were considered significant whenP＜ 0.05.Pearson’s linear correlation was employed for correlation analysis between CRS-R values and changes of PeEn in local regions.This analysis explored critical local regions in taVNS and the relation between local responses and consciousness level.

3.RESULTS

All patients received taVNS and tnVNS treatment,and no significant improvement in clinical scores was found in them.We hope to explore the effect of taVNS on the brain activity of DOC through EEG.

First of all,the results of baseline EEG analysis,as shown in Figure 5,show that there are significant differences in the relative energy of Delta and Beta bands between the two groups in the whole brain and regional brain regions.

As shown in Figure 6,the correlation analysis of clinical scores,further illustrates that the relative energy of Delta and Beta bands can represent the brain activity of patients in different states,that is,the level of consciousness.Based on these results,we explored the differences in the regulation of brain activity in different groups of patients with taVNS treatment.

Figure 5 Correlation between EEG and CRS-R

Figure 6 Topography of spectral density

Figure 7 Spectral density

Figure 8 Region -Coherence

4.DISCUSSION

At present,the evaluation of the consciousness in patients with DOC mainly depends on behavior,but the patients need to understand the doctor's instructions and are willing to cooperate.Also,they should have a complete motion projection system to ensure that docking stimuli such as visual,tactile,and auditory stimuli produce motor output.Therefore,patients suffering from DOC with motor and sensory pathway injuries may be misdiagnosed,caused by clinicians’subjective judgments,motor function injuries,and patients’ fluctuating levels of awareness16-18 And up to 40% of patients suffering from DOC with inability to communicate are misdiagnosed as vegetative state,19which has an adverse impact on the treatment that they may receive at a later stage.20,21The 2020 guidelines of the European Society of Neurologyrecommend the use of EEG to assess the prognosis of patients with DOC when it is feasible.22The recording and analysis of EEG in resting state are at relatively low cost,easy to operate and effectively improve the accuracy of predicting consciousness recovery.23,24EEG reflects the electrical activity of cortical neurons and contains information on neuronal population oscillations,information flow pathways and neural activity networks.EEG can quantify the connectivity among brain regions blocked or reduced in patients with DOC.The effective connection in thalamic cortex is the key neurophysiological mechanism of consciousness recovery,and the change of EEG can occur before the change of CRS-R score.25Therefore,the effect of taVNS on the brain function of patients with DOC can be evaluated by the relevant indexes of EEG.

In order to explore the potential regulatory effect of taVNS on neural activity in patients with DOC,we analyzed the resting state EEG power spectrum,functional connection and network before and after taVNS.Studies have shown that taVNS can increase the energy of Delta band and decrease the energy of Beta band in patients with VS.On the contrary,in patients with MCS,taVNS decreases the energy of Delta band and increases the energy of Beta band.It has been previously reported that the spectral structure of EEG measured by spectral entropy increased in patients with MCS,but decreased in patients with VS.26For patients with severe brain injury,a study found that highfrequency brain activity is related to rehabilitation.24The improvement of consciousness in patients with brain injury can be observed when 15-30Hz high-frequency prefrontal cortex EEG gradually replaced low-frequency EEG.27

In addition,other non-invasive neural regulations can also enhance high-frequency rhythms and frontalparietal functional connections in normal subjects,patients with MCS and a small number of patients with VS.28Some studies have proved that the prefrontal lobe,especially the dorsolateral prefrontal lobe,is an important center involved in human advanced cognitive function,29such as the regulation of emotion,the formation of attention and working memory.30More interactions between the occipital cortex and the frontal lobe have been shown to associate with the recovery of consciousness.31

The EEG results of this study showed that taVNS could significantly improve the functional connection between frontal lobe and occipital lobe,especially in MCS group,but there was no significant difference in tnVNS group.In general,DOC can be expressed as disconnection syndrome because there is significant cortical thalamuscortical pathway damage.32In particular,the extensive damage of frontal-parietal functional connection is often an important feature of DOC.33That study found that the high-frequency frontal-parietal junction in patients with MCS can be regulated by simple somatosensory stimulation.While in patients with VS there is no significant change in stimulation,33,34which is consistent with the results of this study.In practice,behavioral changes induced by treatment might be long-lasting accumulated effects that could not be observed immediately.In a large number of EEG active consciousness paradigm studies,although patients with severe brain injury may not be able to show any signs of consciousness,15% of patients with VS can follow orders by changing brain activity.35It indicates that consciousness is also likely to improve even if CRS-R has not changed.Results from current studies suggest that habit stimulation can arouse prefrontal region EEG responses which the detailed neurobiological mechanism could be involved in Dopamine released by the brain’s reward centers.36Dopaminergic neurons are mainly located in the ventral tegmental area,and also have projections into several parts of the brain,including the nucleus accumbens,prefrontal cortex,hippocampi,and amygdala.37Corazzolet al38illustrated that VNS increased activity of the thalamocortical network associated with the level of consciousness in PET and fMRI.Dietrichet al39used blood oxygen leveldependent fMRI to demonstrate that transcutaneous auricular vagal stimulation activates the left blue spot nucleus,thalamus,left prefrontal cortex,postcentral gyrus,cingulate gyrus,and left insula,which are associated with the vagal pathway-related brain regions.Therefore,considering the close relationship between beta functional connection and consciousness rehabilitation and the results of this study,we concluded that the regulation of taVNS on the relative energy and power of frontal-parietal lobe as well as functional connectivity in patients with MCS may be the electrophysiological mechanism of taVNS for consciousness rehabilitation in patients with DOC.

Regarding to brain injuries and disorders of consciousness,every major progress in clinical medicine means a deeper understanding of brain function.At present,although some progress has been made in the study of DOC and its awakening therapy,the overall prognosis still remains poor in the short term.Our study also has some limitations.Firstly,there is a relatively small number of cases and a lack of the exact sample size required by calculation.Therefore,it may deviate from the real curative effect.In future research,the sample size should be further expanded.Secondly,we only evaluated the patients before and after treatment and did not carry out long-term follow-up.The principle of experiencedependent neuroplasticity shows that significant changes in clinical manifestations require time accumulation and intensive repetitive stimulation.Therefore,the efficacy of taVNS may require longer treatment time.40Due to the lack of follow-up data,we failed to determine the longterm impact of taVNS.In future studies,we should strengthen the follow-up of patients to further determine the long-term effect.In addition,some patients with brain injury need surgery,and the uneven distribution of these patients in the sample also has a certain impact on the experimental results.At the same time,due to the particularity of patients with DOC,the duration,frequency and intensity of stimulation in the treatment still need to be further explored.In this study,the total score of CRS-R,as the main index for evaluating the efficacy in patients with DOC,did not change.In the future multicenter large sample study,we can add the comparison of CRS-R subscale.Since the significance of the total score for fine differentiation of consciousness state is limited41and there is a lack of repetitive evaluation of consciousness level in a short term,especially in patients with MCS who respond to the fluctuation of consciousness level and behavior.42,43This also affects the accuracy of the score.The latest European guidelines recommends that Full Outline of Unresponsiveness should be used to evaluate fewer conscious patients with DOC in order to identify improved awareness more accurately.44In this study,we only adopt the CRS-R rating scale with high public recognition.We will be able to evaluate the state of consciousness more accurately by combining a variety of clinical scoring criteria.In the future research,we should also vigorously carry out the related research of comprehensive awakening rehabilitation treatments.

In conclusion,taVNS can affect the brain activity of different groups of patients in various frequency bands,and the brain areas are mainly concentrated in the frontal lobe and parietal lobe.Among them,the EEG results of patients with MCS suggest that the level of consciousness may improve,but it may not improve in patients with VS.

This may be related to cortical function and the degree of network damage.TaVNS can regulate the high frequency activity of patients with MCS.Therefore,it indicates that taVNS can enhance the high-frequency relative power spectrum energy and functional connectivity of patients with MCS.

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