Chinese Medical Journal 2012;125(9):1627-1632 1627

Original article
Regional homogeneity analysis on acupoint specificity with
resting-state functional magnetic resonance imaging
REN Xiu-jun, CHEN Hong-yan, WANG Bao-guo, ZHAO Bai-xiao, LI Shao-wu, ZHANG Lei, DAI Jian-ping,
LIU Xiao-yuan and LUO Fang

Keywords: acupoint; functional magnetic resonance imaging; transcutaneous electric nerve stimulation
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Background The mechanism of acupuncture analgesia in craniotomy has been widely studied. However, the
theoretical basis for selection of acupoints has not been examined. In this study, we used the regional homogeneity
method blood oxygen level-dependent (BOLD) signals to determine changes in brain activity in response to
transcutaneous electrical stimulation on acupoints and non-acupoints in resting state functional magnetic resonance
imaging (fMRI).
Methods Twelve healthy volunteers were enrolled in this study. BOLD fMRI scanning of the brain was performed for
306 seconds before and 30 minutes after transcutaneous electrical stimulation on acupoints UB63 (Jinmen), LV3 (Tai
chong), ST36 (Zusanli), and GB40 (Qiuxu). The procedure was repeated after one week with stimulation on
non-acupoints (one was 9 above BL67, the second was 12 above BL67 (Kunlun), the third was 7 above KI3, and the
fourth was 10 above KI3 (Taixi)).
Results The regional homogeneity in the acupoint group was increased in the left thalamus, caudate, putamen,
lentiform nucleus (BA19, 30, 39), postcentral gyrus, precentral gyrus (BA3, 4, 30, 32), calcarine fissure, middle temporal
gyrus (BA30), right superior temporal gyrus, inferior temporal gyrus (BA38), cuneus, and precuneus (BA7, 19) when
compared to the non-acupoint group. The regional homogeneity of the acupoint group was decreased in the left
cerebellum posterior lobe, middle frontal gyrus (BA10), double-side precuneus (BA7), and the postcentral gyrus (BA40).
Conclusions The brain region activated following acupoint stimulation is the ipsilateral pain-related brain region, which
may relate to the therapeutic effect of acupuncture on pain relief. Further acupoint stimulation causes different central
nervous responses compared to non-acupoint stimulation.
Chin Med J 2012;125(9):1627-1632

A cupuncture is an important part of traditional

Chinese medicine.1 Although the mechanisms of
acupuncture remain unclear, acupuncture is one of the
most popular alternative and complementary medical
treatment modalities used for treatment of pain.
Acupuncture therapy is also one of the most scientifically
researched therapies.2 The functions of acupuncture differ
with the specificity of acupoints; i.e. acupoint,
non-acupoint, and other acupoint sites.3 The traditional
system of acupuncture indicates that individual point has
identifiable and reproducible clinical effects, although
some practitioners of modern (western) medical
acupuncture suggest that there is little difference in
exactly where the needles are inserted. The specificity of
acupuncture needling sites remains uncertain.4
Recently, functional magnetic resonance imaging (fMRI)
has been used to investigate the neurobiological
mechanisms underlying acupuncture needle
manipulation.5-7 Acupoint specificity had been studied
with fMRI.5,8-10 However, there are contrasting data with
reports of both acupoint specificity8 and non-acupoint
specific effects.10 Experimental design is the most
important aspect of fMRI brain imaging studies. Previous
acupuncture fMRI studies were largely based on a block
paradigm design that detected acupuncture effects
according to a presumable temporal pattern of brain
activation induced by acupuncture administration.5,10,11
This method uses an “R-A-R-A-R” design, where “R”
represents rest and “A” represents acupuncture
stimulation. The stimulation time ranges from several
seconds8,12 to minutes.13,14 The common basic
assumptions of block design are that brain responses to
various stimuli are restored to pre-stimulus steady state
DOI: 10.3760/cma.j.issn.0366-6999.2012.09.019
School of Acupuncture-moxibustion and Tuina, Beijing University
of Chinese Medicine, Beijing 100029, China (Ren XJ and Zhao
BX)
Beijing Tiantan Hospital, Capital Medical University, Beijing
100050, China (Chen HY, Wang BG, Dai JP, Liu XY and Luo F)
Beijing Sanbo Brain Hospital, Capital Medical University, Beijing
100093, China (Wang BG)
Beijing Neurosurgical Institute, Beijing 100050, China (Li SW and
Zhang L)
Correspondence to: WANG Bao-guo, Beijing Sanbo Brain
Hospital, Capital Medical University, Beijing 100093, China (Tel:
86-10-62856766. Fax: 86-10-62856902. Email: wbgttyy@
sina.com); ZHAO Bai-xiao, School of Acupuncture-moxibustion
and Tuina, Beijing University of Chinese Medicine, Beijing
100029, China (Email: baixiao100@yahoo.com.cn)
This work was supported by the grants from the National Key
Basic Research and Development Program “973” Project (No.
2007CB512503), and the China Postdoctoral Science Foundation
(No. 20070420403).
1628
prior to subsequent stimulus.15 However, acupuncture can
produce time-dependent effects. Other limitations of
block design studies include design complexity, as well as
difficulties for acupuncture operation and patient
cooperation.
Resting-state fMRI (Rest-fMRI) is a promising technique
for measuring brain activity during rest, as it is starting to
be used in the study of acupuncture. Rest-fMRI is simple,
and is easier for a doctor to operate and for a patient to
cooperate compared to the classic design, making it very
suitable for clinical research. The resting state is defined
as the period of no prescribed cognitive tasks during an
fMRI scan. Participants are instructed to simply remain
motionless, keep their eyes closed, and not to think of
anything in particular.16-18 Resting state fMRI studies
have no task design or stimulating states, and statistical
parametric mapping (SPM)19 or analysis of functional
neuroimages (AFNI)20 can not be used for data analysis.
Recently, a new method, regional homogeneity (ReHo),
has been developed to analyze the blood oxygen
level-dependent (BOLD) signal of the brain at resting
state.18 ReHo measures the regional homogeneity (i.e.
similarity). This method is based on observations that
meaningful fMRI activity is more likely to occur in
clusters of several spatially contiguous voxels than in a
single voxel. ReHo assumes that within a functional
cluster, the hemodynamic characteristics of every voxel
are similar or synchronous with those of its neighbors,
and that such similarity can be changed or modulated by
different conditions.
UB63 (Jinmen), LV3 (Taichong), ST36 (Zusanli), and
GB40 (Qiuxu) are acupoints used for craniotomy in
China. The meridians of the four acupoints go to different
regions of the brain. Thus, needling the four points
together can cure different kinds of headache, and can be
used for different kinds of craniotomy. There are four
non-acupoints located 9 and 12 directly above BL67
(Kunlun) and 7 and 10 directly above KI3 (Taixi). These
are not meridian or extra-meridian points.
Transcutaneous acupoint electrical stimulation (AES) is a
novel treatment involving transcutaneous electrical nerve
stimulation (ENS) combined with acupuncture points.
AES has been confirmed to exhibit similar
electro-acupuncture analgesia, peripheral and central
effects as normal acupuncture, and is non-invasive,
easy-to-use, and patient compliant.21 In animal
experiments, tolerance to the analgesic effects of
electro-acupuncture developed after a repeated
stimulation pattern of alternating frequencies between 2
and 100 Hz that produced maximum analgesic effects in
acute22 and chronic pain conditions, presumably through
the simultaneous release of four different endogenous
opioid peptides.23
In the present study, we examined acupoint specificity in
the resting state by the fMRI-ReHo method via
Chin Med J 2012;125(9):1627-1632
transcutaneous electrical stimulation of acupoints and
non-acupoints with alternating frequencies between 2 and
100 Hz for 30 minutes. We hypothesized that acupoint
stimulation would produce a different fMRI-ReHo pattern
as compared with non-acupoint stimulation.
METHODS
Subjects
Twelve healthy and right-handed volunteers (2 male and
10 female; age (24.25±1.71) years, range 22.54–25.96
years) were enrolled in this study. All subjects randomly
received acupoint stimulation, and after 1-week recovery
received non-acupoint stimulation. No patient had a
history of neurological illness, head injury, substance
abuse, history of chronic pain, or long-term use of
analgesics. All subjects gave written informed consent,
and the study was approved by the Ethical Committee of
Beijing Tiantan Hospital, Capital Medical University.
Experimental protocol
All subjects underwent two scanning sessions (one
electric acupoint stimulation session and one electric
non-acupoint stimulation session, order balanced) 7 days
apart. Each session consisted of two scans (before and
after EAS). The EAS stimulation time was 30 minutes.
The experimental protocol is summarized in Figure 1.
Stimuli
Electrodes with a 3 mm × 3 mm area were placed on the
acupoints and non-acupoints in the left leg. Han’s
acupoint nerve stimulator (HANS LH202H, Huawei Co.
Ltd., Beijing, China) was used to stimulate the points
with a frequency of 2–100 Hz. The stimuli were
habituated in each subject with a test protocol before the
formal experiment. No subject reported any additional
pressure by the thermode other than the light touch
contact. The intensity was adjusted to a maximal but
comfortable level. No noxious or any unpleasant feeling
was allowed. BL63 (or Jinmen, on the lateral aspect of
the foot, directly below the anterior border of the external
malleolus, lateral to the lower border of the cuboid bone)
and LV3 (or Taichong, on the dorsum of the foot, in the
depression proximal to the first metatarsal space) were
stimulated with 3–9 mA ((5.83±2.17) mA) intensity.
ST36 (or Zusanli, on the anterior lateral aspect of the leg,
3 cm below ST35 (Dubi), one finger breadth (middle
finger) from the anterior crest of the tibia) and GB40 (or
Qiuxu, on the foot, anterior and inferior to the external
malleolus, in the depression on the lateral side of the
Figure 1. Experiment paradigm. Rest, the stage before
stimulation. After: the stage after 30 minutes stimulation.
Chinese Medical Journal 2012;125(9):1627-1632
tendon of muscle extensor digitorum longus) were
stimulated with 3–9 mA ((6.25±2.09) mA) intensity.
Sham points 1 and 2 (9 and 12 straight above BL67,
Kunlun) were stimulated with 3–9 mA ((6.25±1.86) mA)
intensity. Sham points 2 and 3 (7 and 10 directly above
KI3, Taixi) were stimulated with 3–9 mA ((5.33±1.83)
mA) intensity. Each subject was stimulated for 30
minutes. The locations of acupoints and non-acupoints
are shown in Figure 2.
Imaging protocol
Each subject received two scans (before electric needle
stimulation (rest) and 30 minutes after electric needle
stimulation (after)). Each BOLD scan lasted 306 seconds.
The stimulation and scan procedures are summarized in
Figure 1.
Apparatus and scanning procedures
Experiments were performed with a 3.0 T whole body
scanner (Magnetom Trio, Siemens, Germany) with a
standard head coil. Subjects were asked to lie in the
supine position on a scanner bed. All subjects were
informed that their brains would be scanned and that they
would receive acupuncture during scanning. However,
patients did not know the exact stimulation time or points.
Subjects were fitted with a headset during the resting
scan. Their eyes were closed and covered with blinders
and their ears were plugged with earplugs. Subjects were
instructed to remain as motionless as possible, and not to
think about anything in particular. The lights in the
scanning room were dimmed and there were no sounds
other than the noise from the scanner.
Functional images were collected axially by using blood
Figure 2. Locations of acupoints and non-acupoints. BL63, LV
3, ST36, and GB40 were left acupoints, and were stimulated
together by transcutaneous AES. Non-acupoints 1–4 were left
non-acupoints and were stimulated together a week later.
1629
oxygen level dependent imaging sequence. The
acquisition parameters were TR/TE=2000/30 ms,
thickness/gap=4.0/0 mm, FOV=256×256 mm2,
resolution=64×64, flip angle=90°, and slice=32. In
addition, a T2-weighted sagittal three-dimensional
MPRAGE sequence was acquired, covering the entire
brain (250 slices, TR=2100 ms, TE=3.25 ms, slice
thickness=1.0 mm, flip angle=10°, TI=1200,
FOV=256×256 mm2, base resolution=256×256). For each
subject, the fMRI scan during the resting state lasted for
306 seconds, and 150 volumes were obtained.
Image preprocessing
Image preprocessing was performed using statistical
parametric mapping (SPM5, Wellcome Department of
Imaging Neuroscience, London, UK). The first 10
volumes of each functional time series were discarded
because of instability of initial magnetic resonance
imaging signal and adaptation of participants to the
circumstance, leaving 140 volumes. The remaining fMRI
images were preprocessed using SPM5 software. Images
were slice-time corrected, aligned to the first image of
each session for motion correction, and then spatially
normalized.
Regional homogeneity analysis
ReHo analysis18 was performed using REST software
(Resting state fMRI data analysis toolkit,
http://sourceforge.net/projects/resting-fMRI). First, linear
regression was used to remove the influence of head
motion, whole brain signals, and linear trends, and the
fMRI data were temporally band-pass filtered (0.01–0.08
Hz) with REST. REST software was used to measure
regional homogeneity, as reported by Zang et al.18 We
used Kendall’s coefficient of concordance (KCC)24 to
measure regional homogeneity of the time series of a
given voxel with its nearest 26-neighbor voxels in a
voxel-wise way:
W= ∑ (R )
i
2
− n( R ) 2
1 2 3
K ( n − n)
12
where W is the KCC among given voxels , ranged from 0
to 1, Ri the sum rank of the ith time point where
R=((n+1)K)/2 is the mean of the Ri’s, K is the number of
time series within a measured cluster (here, K=27, one
given voxel plus the number of its neighbors), and n is the
number of ranks (here n=175). The KCC value was
calculated according to this voxel, and an individual KCC
map was obtained for each subject. This procedure was
implemented by REST software. Subsequently, the
functional images were spatially smoothed with a
Gaussian kernel of 4×4×4 mm3 full-width at half
maximum.
Group statistical analysis
We calculated the ReHo of the “after phase” minus that of
“rest phase” for both the acupoint and non-acupoint
groups. A second-level random-effect two-sample t-test
was then performed. The resulting statistical map was set
1630 Chin Med J 2012;125(9):1627-1632

at a combined threshold of P <0.05 (t >2.2622) and Table 1. ReHo differences in the KCC maps between acupoint
cluster size >1458 mm3, which resulted in a corrected group and non-acupoint group
MNI
threshold of P <0.05 determined by AlphaSim of Monte Cluster t-Score of
Connected regions BA Coordinates of
Carlo25 (http: //afni.nih.gov/afni/docpdf/AlphaSim.pdf). size peak voxel
peak voxel
L: Cerebellum posterior lobe 102 −4.5705 −9 −78 −51
RESULTS R: Superior temporal 38 59 3.5023 27 6 −45
gyrus/inferior temporal gyrus
ReHo results from the acupoint and non-acupoint groups L: Middle frontal gyrus 10 55 −7.2481 −24 66 12
L: Calcarine fissure/middle 30 81 4.5644 −39 −63 12
are shown in Figure 3 and the Table 1. The regional temporal gyrus
homogeneity in the acupoint group was significantly L: Thalamus/caudate/ 19/30/39 72 5.009 −24 3 15
increased in the left thalamus, caudate, putamen, Putamen/lentiform nucleus
lentiform nucleus (BA19, 30, and 39), postcentral gyrus, L: Postcentral gyrus/precentral 3/4/30 57 4.4139 −36 −18 48
precentral gyrus (BA3, 4, 30, and 32), calcarine fissure, gyrus
L: Medial frontal gyrus 32 86 4.6972 −18 18 42
middle temporal gyrus (BA30), right superior temporal
R: Cuneus/precuneus 7/19 58 4.7906 9 −75 36
gyrus, inferior temporal gyrus (BA38), cuneus, and Precuneus 7 70 −6.0372 9 −63 45
precuneus (BA7 and 19) compared to the non-acupoint Postcentral gyrus 40 57 −3.4392 30 −42 60
group (P <0.05). The regional homogeneity of the BA: Brodmann’s area. L: left. R: right.
acupoint group was decreased in the left cerebellum
posterior lobe, middle frontal gyrus (BA10), double-sided is a Xi point of the foot Taiyang urinary bladder meridian
precuneus (BA7), and postcentral gyrus (BA40) that goes up to the back of the head. UB63 can cure the
compared to the non-acupoint group (P <0.05). pain at the back of the head. ST6 is the point of the foot
Yangming stomach meridian that goes up to the front of
DISCUSSION the head. GB40 is the Yuan source point of the foot
Shaoyang gall bladder meridian that goes up to the lateral
In the present study, we investigated the ReHo fMRI side of the head. GB40 can cure migraine headache. LV3
signal changes evoked by transcutaneous AES, and found is the Yuan point of the foot Jueyin liver meridian that
that the acupoint group exhibited a different regional goes to the apex of the head. LV3 can cure the apex pain
homogeneity from that of the non-acupoint group. UB63 of the head. Acupuncture of these four points together can

Figure 3. The differences of ReHo shown

as a comparison of KCC maps between
acupoint group and non-acupoint group.
The left brain hemisphere is displayed in
the right side of the picture. The numbers
at the left side of the images refer to the z
coordinates. The color values on the
lower right corner refer to t. The statistical
threshold was set at t >2.2622 (P <0.05)
and cluster size>1458 mm3, which
corresponded to a corrected P <0.05.
Chinese Medical Journal 2012;125(9):1627-1632
cure headaches. Sham points 1–4 do not belong to the
traditional meridians, and according to traditional theories
they should not produce any treatment function.
ReHo supposes that voxels within a functional brain area
are more temporally homogeneous when this area is
involved in a specific condition.18 ReHo measures the
ReHo of the time series of the regional BOLD signal.
Thus, ReHo reflects the temporal homogeneity of the
regional BOLD signal rather than its density. As the
BOLD signal of fMRI may reflect neural activity,
abnormal ReHo may reflect regional changes in temporal
aspects of neural activity. ReHo may therefore detect
brain regions with abnormal activity. ReHo analysis is
commonly used to study the default network of the brain
in the normal resting state.25,26 It has also been used to
study the default network of the brain in the abnormal
resting state.27 Abnormal ReHo has been observed in
Alzheimer’s disease,28 schizophrenia,29 and attention
deficit hyperactivity disorder30 in the resting state. In the
current study, we only focused on pain related regions.
After 30 minutes of transcutaneous electrical stimulation
we found a higher ReHo at ipsilateral pain-related regions
including the thalamus, caudate, postcentral gyrus, and
precentral gyrus (BA3, 4, 30, and 32) when compared to
that of non-acupoint regions. BA3 belongs to the primary
somatosensory (SI), which is located at the postcentral
gyrus. BA4 is the first body movement cortex.
Some areas of the brain, including the ipsilateral middle
frontal gyrus, middle temporal gyrus, contralateral
superior temporal gyrus, and inferior temporal gyrus, are
involved in major functions other than pain processes,
including cognitive function. In the current study,
however, we only examined changes in homogeneity at
pain related regions. Previous fMRI studies have shown
that acupuncture can activate numerous brain regions
including the precentral gyrus, postcentral gyrus,
thalamus, hypothalamus, and the limbic system.8,14,31,32
fMRI studies of acupoint stimulation have shown
differing results. For example, needling acupoints have
been reported to activate multiple brain regions,33 while
needling non-acupoints can activate different regions, not
activate any regions,34 or produce similar results to
acupoint stimulation.35 Wu et al8 reported that the
hypothalamus-limbic system was significantly modulated
by electrical acupuncture at acupoints rather than at
non-meridian points, while visual and auditory cortical
activation was not a specific effect of treatment of
relevant acupoints.
Pain perception in the human brain is commonly studied
using EEG/MEG brain topography and PET/fMRI
neuroimaging techniques. Numerous brain regions can be
activated by pain including the thalamus and primary
somatic area (SI), secondary somatic area (SII), insular
cortex (IC), prefrontal cortex (PFC), cingulate, parietal
cortices, brainstem, hippocampus, amygdala, and
supplementary motor area (SMA).36 Activation of the
lateral thalamus, SI, SII, and insula are considered to be
1631
related to the sensory-discriminative aspects of pain
processing. The thalamic response is bilateral, likely
reflecting generalized arousal in reaction to pain.37 The
pain impulse transmission at the brainstem, thalamus, SI,
and SII is required for ascending activation in
sensory-discriminative function and descending
regulation. The acute pain network has been shown to
include the thalamus, SI, SII, ACC, IC, and PFC.38 The
caudate nucleus has also been shown to be involved in
pain modulation, while Han et al suggested that
acupuncture signals can enter the cerebral cortex through
the thalamus.39 We speculated that transcutaneous
electrical stimulating acupoints could relieve pain through
an effect on pain-related regions. Transcutaneous
electrical stimulation of acupoints could strengthen the
temporal homogeneity of neural activity in pain related
regions more than the non-acupoint stimulation.
We used the regional homogeneity method to analyze
BOLD signals to determine changes in brain activity in
response to transcutaneous electrical stimulation on
acupoints and non-acupoints in Rest-fMRI. This study
may find some evidence of acupoint specificity, but it still
needs to do more work in clinic.
In conclusion, using the fMRI-ReHo method we found
that acupoint stimulation led to different central nervous
responses compared to those with non-acupoint
stimulation. Transcutaneous EAS may relieve pain via
pain-related regions.
Acknowledgements: The authors are highly grateful to Professor
ZANG Yu-feng (State Key Laboratory of Cognitive Neuroscience
and Learning, Beijing Normal University) for software support.
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(Received May 18, 2011)
Edited by WANG De