An Exploratory Clinical Study on fMRI-Based Evaluation of Intervention Targets for Auricular Acupuncture Therapy
Phase: NA
Conditions: Healthy Participants
Sponsor: Jiangsu Taizhou People's Hospital

Clinical Study
Version: V5.0
December 28, 2025
An Exploratory Clinical Study on
fMRI-Based Evaluation of Intervention
Targets for Auricular Acupuncture
Therapy
Study Protocol
Version: V5.0
Date: December 28, 2025

Clinical Study
Version: V5.0
December 28, 2025
Table of Contents
一、Study Background..........................................................................1
二、Study Objectives............................................................................1
三、Study Design..................................................................................3
3.1 Design Type................................................................................. 3
3.2 Study Population and Recruitment...............................................3
3.3 Sample Size................................................................................. 4
3.4 Study Population and Criteria...................................................... 4
3.5 Randomization and Blinding........................................................4
3.6 Interventions................................................................................ 5
3.7 Observational Indicators.............................................................. 7
3.8 Functional Image Processing and Statistical Analysis.................. 8
四、Ethics and Participant Protection..................................................11
五、References................................................................................... 11

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一、Study Background
Auricular Vagus Nerve Stimulation (aVNS) regulates central nervous system function
and influences neural activity in the brain by stimulating the vagus nerve branch in the concha
region, showing broad application prospects in the field of neuropsychiatric diseases in recent
years [1]. The intervention methods for aVNS include electrical stimulation of the
transcutaneous auricular vagus nerve and stimulation of the auricular acupoint vagus nerve.
Auricular acupuncture, a microsystem therapy, is theoretically based on the reflective
correspondence between specific auricular points and system-level functional areas. This
method simulates the effects of conventional invasive vagus nerve stimulation by applying
needles to the auricle, thereby providing new avenues for treating conditions such as epilepsy,
depression, pain, and cardiac diseases. The existing literature shows that auricular
acupuncture vagus nerve stimulation has been investigated in several diseases, demonstrating
its efficacy for depression [2], migraine [3, 4], and other conditions. Clinically, auricular
acupuncture has proven to be significantly effective for anxiety and pain. Similar to body
acupuncture, auricular acupuncture reduces preoperative anxiety, indicating comparable
efficacy to traditional body acupuncture in anxiety relief [5]. Furthermore, auricular
acupuncture has an analgesic effect on acute pain (such as sore throat), reducing pain scores
and the need for analgesic medications [6]. In temporomandibular joint pain, auricular
acupuncture, as an adjuvant therapy, can synergistically enhance the effects of conventional
treatment, further supporting its ability to modulate pain pathways [7]. As anxiety and pain
are common triggers of sleep disorders, auricular acupuncture may indirectly improve sleep
quality by alleviating anxiety. These improvements may be related to the upregulation of
plasma Brain-Derived Neurotrophic Factor (BDNF) levels [8]. Although these findings
confirm the clinical efficacy of aVNS, the central nervous system mechanism of action has
not been fully elucidated.
Studies suggest that aVNS may affect brain function through multiple pathways,
including the modulation of neurotransmitter release, neurotrophic factor expression, and
neuroinflammatory responses [8]. In particular, BDNF, as an important neurotrophic factor, is
not only related to the efficacy of aVNS but may also participate in neuroplastic processes,
affecting brain network functional reorganization. However, current research on how aVNS

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modulates the functional connectivity of specific brain networks—especially neural networks
closely related to cognition and emotion regulation, such as those involving the insula and
medial prefrontal cortex—remains insufficient.
Resting-state functional magnetic resonance imaging (rs-fMRI) provides a key tool for
investigating the modulation effects of Vagus Nerve Stimulation (VNS) on brain functional
connectivity (FC) by measuring the blood-oxygen-level–dependent (BOLD) signal arising
from spontaneous neural activity in the absence of tasks. Functional connectivity (FC), which
refers to temporal correlations in spontaneous neural activity between distinct brain regions, is
known to be aberrant in various neurological disorders [8-9]. Specific studies have
demonstrated that aVNS can significantly modulate FC in multiple brain networks. Studies
have indicated that aVNS attenuates negative FC between the insula and the medial prefrontal
cortex (mPFC), a mechanism that may enhance the balance between interoceptive awareness
and cognitive experience [1]. In patients with primary insomnia, rs-fMRI analyses revealed
that taVNS reduced FC between the mPFC and dorsal anterior cingulate cortex (dACC, part
of the salience network), as well as between the mPFC and occipital cortex. This reduction in
FC was positively correlated with improvements in the Pittsburgh Sleep Quality Index (PSQI)
scores, suggesting that taVNS may improve sleep by modulating interactions between the
default mode network (DMN), salience network, and the visual cortex [8]. Furthermore, in
migraine research, repeated taVNS treatment modulated resting-state FC (rsFC) between
brainstem areas (e.g., NTS, RN), the limbic system (bilateral hippocampus), pain-processing
regions (bilateral postcentral gyri, thalamus, and mPFC), and basal ganglia (putamen/caudate).
Notably, changes in rsFC between the RN and putamen were significantly correlated with a
reduction in monthly migraine days, underscoring the role of taVNS in pain modulation
pathways [9]. Another study on primary insomnia found that taVNS reduced FC between the
basal forebrain (BF) and the visual cortex (e.g., middle occipital gyrus, fusiform gyrus),
sensorimotor cortex (supplementary motor area), and mPFC. Weakening of the connection
between the basal forebrain and middle occipital gyrus was associated with improvements in
the
Insomnia
Severity
Index
(ISI).
More
importantly,
baseline
FC
within
the
basal-forebrain–visual circuit predicts therapeutic response to taVNS, supporting its potential

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as a biomarker for personalized treatment [10].Recent research has shown that transcranial
auricular
vagus
nerve
stimulation
(taVNS)
effectively
reduces
negative
functional
connectivity between the insula and the medial prefrontal cortex (MPFC)[1]. This modulation
of neural circuitry is linked to improvements in both proprioceptive awareness and emotional
cognition, offering key insights into the therapeutic mechanisms of taVNS.
This
study
systematically
investigates
whether
auricular
acupuncture
elicits
neuromodulatory effects comparable to taVNS on functional connectivity between the insula
and the medial prefrontal cortex (mPFC) in healthy subjects using resting-state functional
magnetic resonance imaging (rs-fMRI). By characterizing the baseline neurophysiological
effects of auricular acupuncture, the work clarifies its core regulatory mechanisms in
cognition and mood. These findings establish key theoretical and evaluative frameworks for
subsequent clinical translation in insomnia, depression, and related disorders.
二、Study Objectives
Primary Objective: This study investigates whether auricular acupuncture therapy can
modulate the functional connectivity between the insula and medial prefrontal cortex (MPFC)
via resting-state functional magnetic resonance imaging (rs-fMRI), similar to transcranial
vagus nerve stimulation (taVNS), and analyzes its association with improvements in
proprioceptive function.
Mechanistic Objective: To investigate how aVNS influences the integration of
interoceptive processing (bodily signal processing) and cognitive function (cerebral cognitive
processing) by examining its regulatory impact on this specific neural circuit.
三、Study Design
3.1 Design Type
This was a parallel-group, randomized, sham-controlled clinical trial.
The study was conducted in accordance with the Consolidated Standards of Reporting
Trials (CONSORT) guidelines and the Standards for Reporting Interventions guidelines. All
participants provided written informed consent prior to participation and received incentives
or compensation for participating in the study.
3.2 Study Population and Recruitment
Healthy adults were recruited from Taizhou People's Hospital through hospital outpatient

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clinics and promotional posters on the hospital's official WeChat accounts. The study adhered
to the principle of voluntary participation, and all subjects were required to sign an informed
consent form before enrollment. All participants were assessed and screened by a physician.
3.3 Sample Size
The sample size estimation for functional MRI (fMRI) studies was based on Desmond’s
research findings. When analyzing fMRI data using a liberal threshold of 0.05, at least 12
subjects are required to achieve 80% or higher activation intensity at the single-voxel level
[11]. This study randomly recruited 32 subjects, with 16 subjects per group, to systematically
analyze changes in brain function before and after auricular acupuncture intervention,
combined with its efficacy.
3.4 Study Population and Criteria
Study Subjects
Inclusion Criteria:
（1）Age 18–30 years;
（2）Basically normal diet and sleep;
（3）No history of mental illness;
（4）No MRI contraindications (e.g., metal implants or pacemakers) or claustrophobia；
（5）Willing to participate in this study and sign the informed consent form.
Exclusion Criteria:
（1）Presence of auricular skin lesions or allergy to adhesive ear patches;
（2）Currently receiving regular acupuncture treatment;
（3）History of bleeding disorders or anticoagulant use (increased bleeding risk);
（4）Previous history of syncope during acupuncture.
3.5 Randomization and Blinding
The enrolled 32 subjects were randomly assigned in a 1:1 ratio to two groups: the real
auricular acupuncture vagus nerve stimulation group (AA) and the sham auricular
acupuncture control group (SA).
This study employed a double-blind design, meaning that neither the subjects in each
group nor the acupuncturist knew whether they belonged to the AA group or the SA group.

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The sham stimulation group used press needles without tips at the same acupoints, preventing
the subjects from distinguishing whether they were receiving real or sham stimulation,
thereby reducing expectancy effects. Randomization and allocation concealment were
performed by an independent statistician, ensuring that neither the study participants nor the
researchers could influence the assignment. The success of participant blinding was assessed
using the Bang Blinding Index (BBI); blinding was considered successful when the BBI value
was between -0.2 and 0.2.
3.6 Interventions
3.6.1Auricular Press Needle Acupuncture
Auricular Points: Heart, Kidney, Shenmen, Subcortex
①Before the application, the participants rested for 5 min, followed by baseline fMRI
examination (including T1-weighted and T2-weighted images).
②After the examination, auricular press needles were inserted. The physician's hands
and local acupoint areas were routinely disinfected. The experimental group used Dingshi
brand disposable sterile press needles (with release paper, Medical Device Registration No.:
Su Xie Zhu Zhun 20212200040, specification 0.20×1.3 mm). Disposable, sterile press needles
without tips were used in the control group. The needle body was inserted at the
aforementioned points, the release paper was removed, and the adhesive tape was gently
pressed to ensure tight adhesion. Both ears were treated at each time-point.
③After application, each acupoint was pressed 20 times. Pressing all eight points twice
took approximately 2 min to complete. Rest for 10 min. A second pressing session was
performed for 2 min. Rest for 10 min. A third pressing session was performed for 2 min, and
the press needles were removed. (Total duration: approximately 26 min).
④Immediately an fMRI examination (T2-weighted images only) was performed to
observe changes in brain regions.
The intervention involved applying auricular press needles (a type of intradermal
embedding needle) to specific acupoints.

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Figure 1. Localization of selected auricular points.
Table 1. Common names, locations, and indications of auricular points.
Common
Name
Acupuncture
Point
Anatomical Location
Indications Based on TCM
and Scientific Research
Mental Health and
Depression Indications
Shenmen
TF4
The upper portion
of the apex of the
triangular fossa
iscloser to the upper
branch of the antihelix.
Analgesic and
anti inflammatory action is
the most critical anesthesia
point on the ear.
Sedative point.
Calms the mind.
Heart
CO15
In the center of the
cavum conchae
Used for cardiac
complaints such as
angina, cardiac rhythm
disorders
Indicated for anxiety,
depression, distress,
mental agitation, and
insomnia.
Kidney
CO10
In the cymba
conchae, in a
groove below the start
of the inferior crus.
Used for issues in the
urinary and genital
systems. Assists with
tinnitus, auditory
conditions, and sleep
disorders.
Indicated for fear,
insecurity, and lack of
willpower.
Subcortex
AT4
Inner and anterior
faces of the
antitragus
Rebalances nervous,
digestive, and
cardiovascular
functions
Harmonizes cortical
excitation and inhibition
Subcorte
Heart
Kidney
Shenmen

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3.6.2 ECG Patch
The ECG patch operation was divided into three stages: from the start of auricular press
needle treatment to the end of the second resting-state fMRI scan.
Operation method:
①Baseline ECG data collection: After the subject rested for 5 min, the patch was attached to
the corresponding chest wall position for 5 min of ECG monitoring, after which the ECG
patch was removed.
②Treatment-period ECG data collection: After the baseline fMRI examination, the ECG
patch was attached. Monitoring continued throughout the auricular acupuncture treatment
period. The ECG patch was removed after treatment completion.
③Post-treatment ECG data collection: After the second fMRI examination, the ECG patch
was attached, 5 min of ECG monitoring was performed, and then the ECG patch was
removed.
The ECG data recorded by the device were transmitted to a computer or cloud,
automatically analyzed using system software, or reviewed by a physician.
3.6.3 Imaging Examination
All participants underwent magnetic resonance imaging on the same day as the auricular
acupuncture treatment using a Siemens Skyra 3T scanner equipped with a 16-channel head
coil.
The
imaging
protocols
included
conventional
T1-weighted
imaging
(T1WI),
T2-weighted
imaging
(T2WI),
T2-fluid-attenuated
inversion
recovery
(T2-FLAIR),
high-resolution 3D-T1WI, and resting-state functional MRI (rs-fMRI). Prior to scanning, the
participants were fitted with noise-canceling headphones and placed in a supine position.
Head motion was minimized using foam padding. Participants were instructed to keep their
eyes closed, maintain a wakeful state, and avoid engaging in any specific cognitive tasks.
High-resolution T1-weighted structural scanning parameters were as follows: TR = 2300 ms,
TE = 2.98 ms, FA = 9 degrees, FOV = 256 mm × 256 mm, voxel size = 1 mm × 1 mm × 1
mm, slice number = 176, thickness = 1 mm; for rs-fMRI, TR = 2160 ms, TE = 30 ms, FA = 90
degrees, FOV = 256 mm × 256 mm, matrix = 64 × 64, number of excitations (NEX) = 1,
voxel size = 4 mm × 4 mm × 3 mm, thickness = 3 mm, slice gap = 0.75 mm, 40 slices, total

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acquisition time = 518 s.
3.7 Observational Indicators
Resting-state functional magnetic resonance imaging was used to conduct pre- and
post-intervention comparative analyses in participants receiving auricular acupuncture.
Data collection is divided into two stages:
Baseline: Collection of demographic data, baseline fMRI scan, and clinical assessment (T0).
post-intervention:fMRI scan and clinical assessment immediately after the intervention (T1).
Primary Outcome Measure
Functional connectivity strength between the insula and the medial prefrontal cortex
(MPFC)(T1-T0)
This connectivity strength serves as a proxy for the efficiency of information exchange
between the “interoceptive hub” and “the self-referential/emotion-regulatory hub”. An
increase following auricular acupuncture may reflect enhanced psychosomatic integration and
top-down emotional regulation, suggesting a functional optimization of this circuit.
Conversely, a decrease could indicate a disruption of pathological hyper-connectivity related
to emotional constraint or an upregulation of control—mechanisms consistent with the
observed alleviation of emotional symptoms
Secondary Outcome Measures
①Local
brain
activity
indicators:regional
homogeneity
(ReHo),
amplitude
of
low-frequency fluctuation (ALFF), fractional ALFF (fALFF)(T1-T0);
ReHo captures the temporal synchrony of neural activity within local brain regions.
Following auricular acupuncture, increased ReHo in regions such as the insula and prefrontal
cortex may reflect improved neural coordination and functional integration. Conversely,
decreased ReHo in specific areas could indicate a modulatory effect on abnormally elevated
local synchrony.
ALFF reflects the intensity of spontaneous neural activity in the brain. An increase in
ALFF within emotion- and cognition-related regions following auricular acupuncture may
indicate an elevation in baseline neural activity or metabolic activity in these areas.
Conversely, a decrease in ALFF in regions such as the limbic system could reflect an

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inhibitory modulatory effect of the intervention on hyperactivity, potentially contributing to
emotional stabilization.
fALFF: Changes in fALFF following the intervention allow a more specific assessment
of auricular acupuncture’s regulatory effects on intrinsic neural oscillations related to
cognition and emotion. When interpreted in conjunction with ALFF changes, the direction of
fALFF alteration can help elucidate the specific mechanisms through which auricular
acupuncture modulates neural activity patterns.
②Interhemispheric
coordination
index:
voxel-mirrored
homotopic
connectivity
(VMHC)
The VMHC value quantifies the functional coordination between homotopic regions
across the two cerebral hemispheres. Following auricular acupuncture, enhanced VMHC in
regions associated with emotional regulation and cognitive control, such as the prefrontal
cortex and anterior cingulate cortex, may suggest improved interhemispheric balance and
integration of emotional processing. Conversely, changes in VMHC within specific regions
could
help
clarify
how
the
intervention
modulates
functional
imbalances
between
hemispheres, particularly those related to constrained emotional states.
3.8Functional Image Processing and Statistical Analysis
3.8.1 Resting-state fMRI Data Processing
All functional imaging data were processed and analyzed using MATLAB R2022b
(MathWorks) with the SPM12 toolkit, RESTplus v1.25, and DPABI v7.0 software packages.
The preprocessing steps included: ①data format conversion; ②removal of the first 10 time
points to eliminate unstable initial signals; ③head motion realignment and slice-timing
correction. Individuals with head movements exceeding 2 mm or 2 degrees were removed;
④spatial normalization to the Montreal Neurological Institute template using the DARTEL
registration algorithm, followed by resampling to an isotropic voxel size of 3 mm × 3 mm × 3
mm；⑤spatial smoothing using a 6-mm Gaussian kernel; ⑥linear detrending; ⑦regression of
nuisance covariates, including 24-head motion parameters, white-matter signal, and
cerebrospinal fluid signal; ⑧application of a 0.01 to 0.08 Hz bandpass filter.
Mean Regional Homogeneity (mReHo): ReHo maps were generated by calculating

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Kendall's coefficient of concordance (KCC) using RESTplus V1.25 software. To minimize
individual differences, the ReHo value of each voxel was normalized by dividing it by the
global mean ReHo value of the entire brain, resulting in mReHo.
Mean Amplitude of Low-Frequency Fluctuation (mALFF) and its derived metric, mean
fractional ALFF (mfALFF): The time series of each voxel was transformed into the frequency
domain using a Fast Fourier Transform (FFT) to obtain the power spectrum. ALFF was
calculated as the square root of the power spectrum. fALFF was defined as the ratio of the
ALFF value in the low-frequency range (0.01–0.08 Hz) to the ALFF value across the entire
frequency range (0–0.25 Hz). To minimize individual variability, the fALFF value of each
voxel was normalized by dividing it by the global mean fALFF value of the whole brain,
resulting in the mfALFF.
Functional images were transformed into a mirrored space after preprocessing. The mean
BOLD time series for each voxel was extracted. Pearson’s correlation coefficients were
computed for mirrored voxels from the bilateral hemispheres to produce VMHC maps. The
Fisher-z transform was used to enable subsequent group-level comparisons.
Z-score normalized Functional Connectivity (zFC) values: Brain regions exhibiting
significant between-group differences in functional metrics were defined as regions of interest
(ROIs). Functional connectivity between each ROI and the entire brain was computed using
the RESTplus software. The resulting correlation coefficients were subsequently standardized
using Fisher's r-to-z transformation to obtain zFC values.
3.8.2 Clinical Data
Datasets: Intent-to-Treat (ITT) and Per-Protocol (PP) analyses were used.
Normally distributed continuous data were described as mean ± standard deviation, and
inter-group comparisons for normally distributed variables used independent-samples t-tests.
Non-normally distributed scale data will be described as medians and interquartile ranges, and
the Mann–Whitney U non-parametric test will be used for statistical analysis. Categorical data
were analyzed using the Chi-square test.
All statistical analyses were performed using SPSS or R software. Statistical significance
was set at P < 0.05.

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四、Ethics and Participant Protection
Before the study begins, the study protocol (including questionnaires and the informed
consent form) will be submitted for review and approval to the Ethics Committee of Taizhou
People's Hospital Affiliated to Nanjing Medical University (ethics number: LSKY
2025-175-01). Any amendments to the study protocol will be submitted to the Ethics
Committee for approval and record-keeping. All participants will be fully informed about the
study purpose, procedures, potential risks, and benefits, and will provide written informed
consent.
五、References
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[2] de Oliveira Rodrigues DM, Menezes PR, Machado Ribeiro Silotto AE, et al. Efficacy and Safety of
Auricular Acupuncture for Depression: A Randomized Clinical Trial [J]. JAMA Netw Open.
2023;6(11):e2345138.
[3] Natbony LR, Zhang N. Acupuncture for Migraine: a Review of the Data and Clinical Insights [J].
Curr Pain Headache Rep. 2020;24(7):32.
[4]
Uddin N, Levine DL. Battlefield Acupuncture for the Treatment of Chronic Migraines [J]. Cureus.
2024;16(5):e60369.
[5]
Wu S, Liang J, Zhu X, et al. Comparing the treatment effectiveness of body acupuncture and
auricular acupuncture in preoperative anxiety treatment[J]. J Res Med Sci. 2011;16(1):39-42.
[6] Moss DA, Crawford P. Ear Acupuncture for Acute Sore Throat: A Randomized Controlled Trial[J].
J Am Board Fam Med. 2015;28(6):697-705.
[7] Ferreira LA, Grossmann E, Januzzi E, et al. Ear Acupuncture Therapy for Masticatory Myofascial
and Temporomandibular Pain: A Controlled Clinical Trial[J]. Evid Based Complement Alternat Med.
2015;2015:342507.
[8] Zhang S, He JK, Meng H, et al. Effects of transcutaneous auricular vagus nerve stimulation on
brain functional connectivity of medial prefrontal cortex in patients with primary insomnia. Anat Rec
(Hoboken). 2021;304(11):2426-2435.
[9] Huang Y, Zhang Y, Hodges S, et al. The modulation effects of repeated transcutaneous auricular
vagus nerve stimulation on the functional connectivity of key brainstem regions along the vagus nerve
pathway in migraine patients. Front Mol Neurosci. 2023;16:1160006.
[10] Qi M, Huang Y, Mai R, et al. Baseline functional connectivity of the basal forebrain-cortical
circuit predict taVNS treatment response in primary insomnia: a randomized controlled trial and fMRI
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[11] Desmond JE, Glover GH. Estimating sample size in functional MRI (fMRI) neuroimaging studies:

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statistical power analyses. J Neurosci Methods. 2002 Aug 30;118(2):115–28.