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Research Report

Electroacupuncture modulates cortical activities evoked bynoxious somatosensory stimulations in human

Yan Zenga, Xun-chang Lianga, Jia-pei Daia, Yun Wanga, Zhong-le Yangb, Man Lia,Guang-ying Huanga, Jing Shia,⁎aDepartment of Neurobiology, Tongji Medical College, Huazhong University of Science and Technology, No. 13, Hangkong Rd.,430030 Wuhan, PR ChinabCognitive Science Laboratory, Central-South-China University for Nationalities, Wuhan 430050, PR China

A R T I C L E I N F O

⁎ Corresponding author. Fax: +86 027 8686293E-mail address: sj@mails.tjmu.edu.cn (J. S

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.03.123

A B S T R A C T

Article history:Accepted 15 March 2006Available online 21 June 2006

A noninvasive high-resolution imaging technique of cerebral electric activities has beendeveloped to directly link scalp potential measurement with the magnetic resonanceimages of the subjects, which is very helpful for the elucidation of the cortical processingfollowing various stimulations. Here, we used a 64-channel Neuroscan ESI-128 system toexplore the specific cortical activities elicited by electroacupuncture (EA) acupoint in normalvolunteers and the modulatory effect of EA on cortical activities evoked by noxioussomatosensory stimulation. A specific later-latency somatosensory-evoked potential (SEP,P150) located in bilateral anterior cingulated cortex was observed after EA acupoint but notnon-acupoint. Two pain-specific SEP components (P170 and N280), located in bilateralsuprasylvian operculum and anterior cingulated cortex respectively were observedfollowing painful median nerve stimulation. Binding EA acupoint with painful mediannerve stimulation, the amplitudes of P170 and N280 appeared to be attenuated significantly,2D topography exhibited tremendous decrease of cortical activation between 120 ms and296 ms in latency, and visual analogue scale (VAS) changes also showed a similar pattern tothe change of amplitude. The bilateral anterior cingulated cortex recruited followingacupoint stimuli might, to some extent, suggest that EA has the specific physiologicaleffects. Decrease of pain-induced cortical activation by EA acupoint was considered to bemainly due to an interaction of the signals in anterior cingulated cortex ascending from thepain stimulation and EA.

© 2006 Elsevier B.V. All rights reserved.

Keywords:ElectroacupunctureAcupointNon-acupointNoxious stimulationSomatosensory-evoked potentialsMRI

Abbreviations:EA, electroacupunctureSEPs, somatosensory-evokedpotentialsMRI, magnetic resonance imagingVAS, visual analogue scaleEEG, electroencephalogramPAG, periaqueductal grayPET, positron emission tomographyANOA, analysis of varianceGOF, goodness of fitfMRI, functional magnetic resonanceimaging

7.hi).

er B.V. All rights reserved.

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1. Introduction

Acupuncture originated from ancient China has been usedin the orient to manage various clinical disorders forthousands of years. Among its multiple clinical applica-tions, acupuncture analgesia is one of the earliest and sofar most widely accepted treatment (He et al., 2004; Zhanget al., 2003; Cho et al., 2002; Han, 2003; Huang et al., 2002).Possible mechanisms for pain relief by acupuncture havebeen extensively studied in animal experiments (Huang etal., 2002; Peets and Pomeranz, 1978; Wang et al., 1990; Jin etal., 1986). However, the underlying mechanisms of acu-puncture analgesia in human remain largely unknown.Recently, some achievements have been obtained by usingfunctional neuroimaging combining with acupuncture stim-ulation in humans (Biella et al., 2001; Chiu et al., 2001; Choet al., 1998; Cao, 2002; Fang et al., 2004; Wei et al., 2000; Wuet al., 2002). The findings suggest that manual acupunctureor electroacupuncture (EA) produces signal activation insubcortical gray structures (the pontine raphe area, thethalamus) and cortical areas (the primary somatosensoryand motor cortical areas, the superior frontal gyrus andinsulae, the frontal areas and anterior cingulus, and thecerebellum). While a variety of imaging studies also havebeen published that look at the whole mechanism behindpain processing (Kakigi et al., 2004). A number of corticalareas have been shown to be involved in pain processing,these include the primary somatosensory cortex, thesecondary somatosensory cortex, the insula, the anteriorcingulated cortex and the prefrontal cortex (Treede et al.,1999) as well as the hypothalamus and periaqueductal gray(PAG). It appears that there are substantial overlapsbetween the pain matrix that have already been identifiedwithin the brain and its response to acupuncture and pain.Therefore, studies argue that acupuncture designed toprovide analgesia involves the pain-related neuromatrix.But most methods of needle stimulation using in imagingstudies are limited to see if different areas were activatedor deactivated in response to the nature of the acupunctureitself. The methods to observe any interaction betweenpain inputs and acupuncture afferents as well as themodulation of pain with acupuncture during EA analgesiaare still lack.

Fig. 1 – Bar graph shows averaged psychophysical responses revariable on the horizontal axis. (a) Pain scores, *significantly diffP < 0.005), #significantly different from painful median nerve stimAcupuncture sensation scores, *significantly different from acupexpressed as mean ± SE, n = 24.

Multi-channels electroencephalogram (EEG), anotherpowerful tool for investigating cortical responses to variousstimuli, has an advantage over imaging modalities in that itcan provide temporal information on the activity inaddition to its location. Numerous EEG studies previouslyreported in literature showed that the late component ofsomatosensory-evoked potential (SEP) elicited by high-intensity stimulation potentially provides a reliable indica-tor of analgesic efficacy since it reflects the level ofactivation of the nociceptive system. It can be modified invarious interfering conditions including skin, muscle andjoint stimulations using electric, mechanic and vibrationstimuli (Hoshiyama and Kakigi, 2000) and reflect occlusionof afferent input transmission at subcortical or corticallevels. Effects of sleep and distraction on late component inhumans have been investigated (Wang et al., 2003; Yama-saki et al., 2000), which suggested late components aremuch affected by consciousness. There is no EEG reportthat late components following painful stimuli were affect-ed by traditional EA specific to acupoints. Imaging studieshave showed that there were specific cortical activitieswhen considering the traditional Chinese functions attrib-utable to certain specific acupoints (George et al., 2005). Thepresent study was conducted, therefore, to detect thetemporal behavior of acupoint stimuli-produced corticalactivities using EEG and to determine whether the latecomponent of SEP induced by noxious stimulation and painintensity are inhibited by electrical stimuli specific to theacupoint.

2. Results

2.1. Assessment of pain intensity and acupuncturesensations

The psychophysical assessments showed that painful mediannerve stimulation elicited severe pain responses in subjects,and pain scores was 68.62 ± 8.23 in session B (Fig. 1). Aftercombining with the interference stimulations from acupointand non-acupoint, the pain scores were decreased to24.34 ± 4.67 (n = 24, P = 0.004) and 49.56 ± 7.34 (n = 24,P = 0.03) in session C and session D, respectively. Low-

ported by the subjects, who used a score of 0–100 for eacherent from painful median nerve stimulations (session B,ulation + non-acupoint stimulations (session D, P < 0.005). (b)oint stimulations alone (session E, P < 0.005). Data were

Fig. 2 – Grand mean wave forms of somatosensory-evoked potentials across 24 subjects elicited by painful median nervestimulation at 64 electrodes according 10–20 system. The highest potential was localized in FCZ electrode position, at 170 ms,cortical activation both at left electrodes position and right electrodes position were observed, but amplitudes of latecomponents P170 and N280 were larger at right electrodes.

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frequency (2 Hz) EA at both acupoint and non-acupointproduced behavioral analgesia, but EA at acupoint appearedto attenuate pain perception more significantly than at non-acupoint (n = 24, P = 0.04). The intensity of electricalstimulation must be sufficient to cause muscle contractionin order to obtain EA analgesia. In all subjects, the afferents ofLI 4 stimulations elicited clear non-painful acupuncturesensations of heaviness, fullness and numbness in the areasurrounding the needle, whereas we found significant differ-ences between acupoint (73.65 ± 9.54) and non-acupoint(19.56 ± 2.46) (n = 24, P = 0.0006) (Fig. 1). The non-acupointstimulations elicited uncomfortable and moderately painfulfeeling. The comparison showed that EA at acupoint resultedin significantly higher degrees of acupuncture sensation thanat non-acupoint.

Table 1 – Comparisons between the amplitude of SEPs acrossfollowing painful median nerve stimulation

N20 P40

Left recording electrodes 0.66 ± 0.11 0.47 ± 0.06Right recording electrodes 0.93 ± 0.27* 0.87 ± 0.08*

Mean ± SE. *Significant difference, P < 0.05. Versus left side, n = 24, μV.

2.2. SEPs mapping

2.2.1. Painful and non-painful median nerve stimulationGrandmean wave forms of evoked potentials obtained followingthepainful electricalmediannerve stimulation (Fig. 2) showed thetypical recordings of 64 channels SEPs. The highest potential waslocalized in FCZ electrode position, from 170 ms to 175 ms, andcortical activation both at left and right recording electrodes wereobserved, but amplitudes were larger at right electrodes (Table 1).Non-painful electrical stimulation evoked only three early- andmiddle-latency components, N20, P40, and N80, whereas painfulelectrical stimulation evoked additional pain-specific later-laten-cy components, P170 and N280, except for N20, P40, and N80 (Fig.3). Raw data were further submitted to brain electrical sourceanalysis. First, a latency range of about 10–290 ms was chosen as

the left recording electrodes and right recording electrodes

N80 P170 N280

2.33 ± 0.57 8.64 ± 3.13 7.27 ± 2.672.48 ± 0.76 12.48 ± 4.13* 10.77 ± 3.67

Fig. 3 – Grand mean somatosensory-evoked potentialwaveform to painful electrical median nerve stimulation(solid line) and non-painful electrical median nervestimulation (dash line) recorded at FCZ across 24 subjects.Data are presented for 100 ms before and 400 ms followingstimulus onset, N20 first negative deflection of SEPwave; P40second positive deflection of SEP wave; N80 third negativedeflection of SEP wave; P170 fourth positive deflection of SEPwave; N280 fifth negative deflection of SEP wave. P170 andN280 are uniquely to painful stimuli.

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an analysis period for painful evoked electric field.We started theanalysis with one source placed around the Sylvian fissure sincethis area has been reported as a major source responsible topainful stimulation (Kakigi et al., 1995). Themost effective sourcewas usually localized to the upper bank of the Sylvian fissure

Fig. 4 – Schematic projections of the locations of the equivalent dipthe image. First cortical somatosensory response (N20) to painful apostcentral gyrus (S area, green dot) onto a sagittal MR slice and corcomponent), localizes to the suprasylvian operculum to a coronal slcomponent, localizes to the anterior cingulate cortex to a sagittal slof electric. (For interpretationof the references to colour in this figur

supporting previous reports. However, this source could notexplain the electric fields during the period of analysis (meanGOF = 62.5%). Therefore, we next tested a pair of sources placedaround the Sylvian fissure that explained the electric field mosteffectively. Then the period of analysis was expanded to 10–400 ms, and two sources were added to obtain a GOF larger than90%. The four-source model provided a GOF value of more than90% in all the subjects, and the location and orientation of thesources were fixed. The same procedure was applied to theanalysis of SEPs from the acupoint stimuli. Location andorientation of these fields concurred with the expected evokedcompound action currents along the course of the nerve fibers.The N20 in response to median nerve stimulation was explainedby a single dipole (Fig. 4, green dot) and fitted around 20ms, afterregistered to MRI scan, a source in the contralateral post-centralgyrus could be seen. The N80 in the epoch (80ms–87ms), P170 inthe epoch (170 ms–175 ms), was explained by two dipoles,registered to MRI scan, two contralateral suprasylvian operculumdipoles probably increased significantly their moments when thesubjects with the sensation of pain (Fig. 4, white dot and red dot).Another late component N280 in the epoch (275 ms–296 ms)explained by a dipole in the bilateral anterior cingulate cortex (Fig.4, yellow dot).

2.2.2. Acupoint and non-acupoint stimulationMean wave forms of evoked potentials following acupointstimulation and non-acupoint stimulation were showed inFig. 5. The first major positive deflection P40 in latency 40 msand the first negative deflection N80 in latency 78 ms wereevoked by non-acupoint stimulation, explained by two

oles of a single case SEPs. Left part of the brain is on the left ofnd non-painful left median stimulation, localizes to theonal slice of an individual subjects. N80 and P170 (pain-relatedice (S area,white dot and red dot). N280, another painful relatedice and horizontal slice. Tails represent the direction of currente legend, the reader is referred to theweb versionof this article.)

Fig. 5 – Comparisons of the somatosensory-evoked potentials in responses to acupoint stimuli and non-acupoint stimuli.Mean across 24 subjects of cortical surface SEPs recorded at sites FZ, FCZ, PZ, OZ. P40 first positive deflection of SEP wave, N80first negative deflection, and P150 second broad positive deflection, specific to acupoint stimuli.

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dipoles, located to the contralateral post-central gyrus andsuprasylvian operculum, respectively (Fig. 6). The two-sourcemodel provided a GOF value of more than 90% in all thesubjects. Acupoint stimulation elicited not only P40–N80 butalso the third broad positive potential P150, not describedbefore in literatures, in a latency range from 140ms to 170 ms,

Fig. 6 – Schematic projection of locations of the equivalent dipolesof the image. First cortical somatosensory response (P40) to acupoonto a sagittal MR slice of an individual subjects. N80 localizes to thP150, an acupoint-specific component, localizes to the anterior cinof current of electric. (For interpretation of the references to colourof this article.)

best explained by a single dipole, fitted around 145ms, locatedto the anterior cingulated cortex (Fig. 6). The amplitude ofacupoint-specific component P150 increased significantlywith an increased acupuncture sensation scores acrosssubjects. There was no difference in the amplitude of P40between acupoint and non-acupoint, as seen from Fig. 5 and

of SEPs from single subject. Left part of the brain is on the leftint stimulation localizes to the postcentral gyrus (green dot)e suprasylvian operculum to a coronal slice (S area, white dot).gulated cortex to a sagittal slice. Tails represent the directionin this figure legend, the reader is referred to the web version

Fig. 7 – Comparisons of amplitudes of SEPs from acupointstimuli and non-acupoint stimuli. The amplitude ofN80 on theacupoint stimuli is significantly higher than that on the non-acupoint stimuli, amplitude of P40 appeared no differencebetween acupoint stimuli and non-acupoint stimuli, *P < 0.05,versus non-acupoint. Data were expressed as mean ± SE.

Fig. 9 – Comparison of mean amplitudes of SEPs in sessionsB, C, and D. No significant changes were observed in N20 andP40 in sessions B, C, and D. But significant attenuation wasobserved inN80 (P < 0.05), P170 (P < 0.01), andN280 (P < 0.01) insessionCandsessionD. Significant differences alsowerealsoobserved between session C and session D, they appeared tosession B > session D > session C. *Significant difference(P < 0.05), versus session B. #Significant difference (P < 0.05),versus session D. The data were expressed as mean ± SE.

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Fig. 7, however, the amplitude of N80 on the acupoint stimuliwas significantly higher than that on the non-acupoint stimuli(n = 12, P = 0.016), and increased with acupuncture sensations.A polarity reversal of SEPs was observed between the fronto-central recording electrodes and parietal–occipital recordingelectrodes following acupoint stimuli (Fig. 5).

2.2.3. Effects of EA on SEPsThe effects of EA on SEPs in response to painful median nervestimulation were investigated. Fig. 8 showed the wave forms,latencies, and amplitudes of the SEPs from part electrodes.There were no significant differences in the waveform, thelatency, and the amplitudes of N20 and P40 among differentsessions.TheamplitudesofN80,P170, andN280weredecreased

Fig. 8 – Comparisons of SEPs across 24 subjects recorded at sitesdifferences in SEP waveform were shown in different sessions. Tintroduction of acupoint stimuli and non-acupoint stimuli. In secomparison with that in session D.

significantly after introduction of acupoint interfering stimuliand non-acupoint interfering stimuli, but these componentswere attenuated more significantly in acupoint interferingstimuli condition than in non-acupoint interfering stimulicondition (n = 24, P = 0.044, P = 0.001, P = 0.035, versus sessionD) (Fig. 9). The result showed a remarkable agreement betweensubjective ratings of pain and reduction of late SEP amplitudes

FZ, CZ, PZ, OZ, C3, C4, P3, and P4 from sessions A–D. Nohe amplitudes of N80, P170, and N280 decreased withssion C amplitude level of three components were higher in

Table 2 – Comparison of amplitudes (base-to-peak, peak-to-peak) derived from CZ electrode (mean ± SE. n = 24, μV)

N20 P40 N80 P170 N280 N20-P40 N80-P170 P170-N280

Session B 0.82 ± 0.13 0.57 ± 0.08 2.83 ± 0.56 11.68 ± 2.13 9.27 ± 1.67 1.57 ± 0.89 14.41 ± 2.98 21.57 ± 4.86Session C 0.83 ± 0.12 0.51 ± 0.08 1.58 ± 0.5 ⁎, ⁎⁎ 2.47 ± 0.9 ⁎, ⁎⁎ 2.80 ± 1.0 ⁎, ⁎⁎ 1.63 ± 0.9 ⁎, ⁎⁎ 3.95 ± 1.4 ⁎, ⁎⁎ 5.28 ± 1.9 ⁎, ⁎⁎Session D 0.82 ± 0.16 0.56 ± 0.09 2.19 ± 0.61 ⁎⁎ 6.44 ± 1.33 ⁎⁎ 4.85 ± 1.36 ⁎⁎ 1.66 ± 0.87 ⁎⁎ 8.36 ± 2.15 ⁎⁎ 11.29 ± 2.65 ⁎⁎

⁎ P < 0.05, versus session D.⁎⁎ P < 0.05, versus session B.

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in response to noxious stimulation. Table 2 showed the detaildataof electrodeCZobtained fromsessionsA, B,C, andD. Fig. 10showed the dynamic two-dimension topography of cerebralpotentials evoked by painful electrical stimulation at leftmedian nerve (left side) and comparison of cerebral electricalactivation among sessions B, C, and D (right side). The electricalactivation following painful median nerve stimulationappearedat 20ms, disappearedat 316ms, the strongest positivepotential was at 170 ms; the strongest negative potential wasbetween 275 ms and 296 ms, and the electrical activationsbetween 129 ms and 316 ms were significantly attenuated insession C, moderately attenuated in session D.

3. Discussion

3.1. P170 and N280 were related to pain perception

In the present study, we observed an early latency SEPN20 andtwo middle latency components P40 and N80 both in non-

Fig. 10 – Topography of mean SEP across 24 subjects induced bytopography among sessions B, session C, and session D (right). Linterval between two maps is about 20 ms. Right side comprises+316 ms. Potentials scale is −17 μV to +17 μV, red expresses posinterpretation of the references to colour in this figure legend, th

painful and painful stimulation. We also found two latercomponents P170 and N280, which were specific to painfulstimulation, and their amplitudes were strongly correlatedwith the subjective experience of pain intensity. It has alreadybeen reported that the amplitude of late component withlatency between 150 and 250–300 ms is most closely associ-ated with the pain intensity (Inui et al., 2003; Chudler andDong, 1983). With regard to the location of such SEPcomponents in the human brain, N20 was located in thecontralateral postcentral gyrus, N80 and P170 in the contra-lateral suprasylvian operculum. fMRI and EEG studies con-stantly found that bilateral suprasylvian operculum are thoseresponding with the shortest latency to peripheral pain inputs(Umino et al., 1995; Frot and Mauguiere, 1999), and multiplesensation such as taste and olfactory (Frot and Mauguiere,2003), tactile afferent (Faurion et al., 1999). Our results supportprevious findings and suggest that both non-painful andpainful stimulation activated contralateral primary sensorycortex and suprasylvian operculum. Another late componentN280 was also located in the bilateral anterior cingulated

painful median nerve stimuli (left), and comparison ofeft side comprises of 25 maps from −100 ms to +400 ms, theof three groups (session B−D) between +129.20 ms anditive potentials, and blue expresses negative potentials. (Fore reader is referred to the web version of this article.)

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cortex in this study. We considered that anterior cingulatedcortex activities were related to pain sensation and perceptionprocessing. Recent imaging studies have found that cingu-lated cortex is highly related to pain perception and sensation(Faurion et al., 1999; Baron et al., 1999; Devinsky et al., 1995;Qiu et al., 2004; Bantick et al., 2002; Price et al., 2002), paincognition (Ploner and Schnitzler, 2004), pain inhibition (Pet-rovic and Ingvar, 2002), and response selection processes(Dowman, 2002). Anterior cingulated cortex activities emergedapparently later than S and suprasylvian operculum activities,and its amplitude was related to pain intensity. Our late SEPpeaks polarities are the opposite of those reported by others.Some investigators reported a pain-related negative peakaround 150 ms and a pain-related positive peak at about 250–300 ms (Inui et al., 2002). These differences could be due to thedifferences of the individual laboratory, the nature of thepainful stimulation used, stimulus duration, calibrationtechniques and recording techniques, resistance of arm,stimulating electrode. Therefore, our late component funda-mentally could be regard as the same component with thosepreviously reported by others.

3.2. P150 was specific to acupoint stimulation and mightrelated to acupuncture sensations

Although there are many imaging studies that showed thespecific locations of cortical activation during acupointstimulations, this is the first report to reveal the temporalprocessing of acupoint stimulation in details and to comparethe temporal behavior of cortical activations between acu-point and non-acupoint stimulations. The data showed thatthe temporal behavior of cortical responses was different inthat P150 was specific to acupoint stimulations.

Neurophysiological studies indicated that acupuncture/EAneedle manipulation may cause stimulation of a wide varietyof sensory mechanoreceptors and/or nociceptors, and theperipheral neural pathway of acupuncture signals involves inboth A-beta fibers and A-delta fibers, even C fibers (Cho et al.,2002; Andersson and Holmgren, 1975; Kawakita and Funa-koshi, 1982; Liu et al., 1990). In our data, both acupoint andnon-acupoint stimuli elicited a positive–negative potentialP40–N80 located in primary somatosensory cortex and supra-sylvian operculum, mostly assumed to represent cutaneousrather than muscle afferents through A-beta fibers (Allison etal., 1991; Kunesch et al., 1995). In addition, P40–N80may reflectthe first and second phases of primary somatosensory-evokedpotentials, which often elicited by median nerve stimulation,mediated by the dorsal column/medial lemniscal system(Kany and Treede, 1997). However, P150 located in anteriorcingulated cortex was speculated to represent muscle affer-ents ascending through A-delta fibers and closely related toadvanced brain function. Moreover, P150 was much depen-dent on acupuncture sensation. Acupuncture sensations havebeen widely observed clinically and experimentally. Theyhave been hypothesized to be triggered by sufficient activationof characteristic acupuncture afferent pathways (Kaptchuk,2002; Park et al., 2002; Ulett et al., 1998). It is generally acceptedthat acupuncture sensations are the “sine qua non” ofacupuncture for the achievement of clinical therapeuticeffects (Wang et al., 1990; Liu et al., 1990; Leem et al., 1994).

Generation of acupuncture sensations that subjects experi-enced is closely associated with nerves (Hou, 1986) anddepends on amounts and types of sensory receptors evokedby stimulation of acupoint. In the present study, acupoint LI4was selected as EA point, and anatomical studies show thatthere are superficial and deep radial nerves, and a largeamount of nerve endings and muscle spindles in LI4, whereasonly superficial and deep radial nerves, and less amounts ofnerve endings in non-acupoint (Kakigi et al., 1991). Thedifference in the anatomical structure of acupoint and non-acupoint, especially abundant nerve endings and musclespindles distributed in acupoint, may explain the differenceof acupuncture sensations. Thus, we suggest that acupunc-ture sensation-dependent functional activation of anteriorcingulated cortex represented an acupoint–brain correlation,and P150 may be a characteristic activation in response toacupoint afferent.

3.3. Integration of EA and pain inputs in cerebral cortex

We found that painful related SEP components P170 and N280were significantly inhibited by EA accompanied with theobvious analgesic effects as evaluated by pain perceptionscores. The analgesic effects in acupoint appeared to be moreeffective than that in non-acupoint. Stimulations of theacupuncture point were achieved in inducing not only P150but also the state of acupuncture sensations. In addition, bothpainful related SEP components P170 and N280, and EA-dependent SEP component P150 were located in the supra-sylvian operculum area and anterior cingulated cortex. Thisindicates that both the EA afferents and pain inputs arrive atthe same cortical areas, activate the cerebral neurons, andresult in an integration effects. Studies on the mechanisms ofEA action have also revealed possibility of chemical (neuro-transmitters, neuromodulators) effect. Han (2003) reportedthat endogenous opioid peptides in the central nervoussystem play an essential role in mediating the analgesiceffect of EA. Further studies have shown that different kindsof neuropeptides are released by EA with different frequen-cies. For example, EA of 2 Hz accelerates the release ofenkephalin, beta-endorphin and endomorphin, while that of100 Hz selectively increases the release of dynorphin. Acombination of the two frequencies produces a simultaneousrelease of all four opioid peptides, resulting in a maximaltherapeutic effect.

It has been demonstrated for long time that non-painfuland painful afferent information are conducted via differentneuronal pathways and velocities, the formermainly in dorsalcolumns, at a velocity approximately 50–70 m/s, appear not tobe affected easily, and the latter mainly through spino-thalamic tract, at a velocity of 10 m/s (Kakigi et al., 1982,1991), is susceptive to other interference. Electrical stimula-tion of acupoint at the high threshold provides various typesof peripheral nerve afferents (Wei et al., 2000). We speculatethat the peripheral inputs from the acupoint stimuli maymainly affect pain cortical process mediated in suprasylvianoperculum area and anterior cingulated cortex.

Our studies have not been completely blinded becausevolunteers in these studies were awake. Therefore, otherunspecific effects could have contributed to the reported

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results. The results might have been affected by changes ofattention, judgment, and other cognitive activities. Neverthe-less, this study demonstrates that EA significantly decreasesthe magnitudes of late SEP amplitudes during electricalnoxious stimulation in normal subjects.

In conclusions, we evaluated the effects of EA on painstimulation in human cortical activities by using SEPs andfound that suprasylvian operculum cortex and anteriorcingulated cortex may be important areas in humans tomediate and integrate the painful stimulation and EAsensation and result in analgesia. Our findings provide abasis for future comprehensive investigations of EA analgesiamodulation circuits in the human brain and also suggest thatSEPs mapping technique is useful to directly observe modu-lated cortical activities following pain stimulation after theadministration of EA.

4. Experimental procedures

4.1. Subjects preparation

24 healthy and right-handed paid volunteers (12 males, 12females, aged 20–32 years) were recruited in the experiment.All participants gave written informed consent, comfortablyseated in a reclining chair with their forearms on armrests,in a noise and light reduced room, which had a temperatureof 24 °C and was electrically shielded. The subjects wereblinded about the experimental design and order of stimula-tions and were instructed that all stimulations would beendurable.

4.2. Assessment of pain and acupuncture sensation

Pain perception at the end of each stimulation session and theacupuncture sensation effect after completing 30-s acupunc-ture induction were recorded by using a 100-mm visualanalogue scale (VAS). A score of 0 represented no intensityat all, and a score of 100 represented the strongest imaginableintensity. Tominimize the effect of anticipation, subjectswereinstructed that any combination of different or same types ofstimulation may be performed in a session.

4.3. Experimental design

Six stimulation sessions were nested within the experimentin a balanced, randomized order as follows: (1) non-painfulelectrical median nerve stimuli at left wrist (session A)—theelectrical stimuli that caused a faint sensation, VAS < 25; (2)painful electrical median nerve stimuli (session B)—theelectrical stimuli that caused the strongest pain bearable,VAS between 60 and 75; (3) left LI4 acupoint electricalstimuli, an interfering stimulation for 30 s alone, thentogether with painful electrical median nerve stimuli(session C); (4) non-acupoint stimuli at a non-meridianfocus near LI4 for 30 s alone, then together with painfulelectrical median nerve stimuli (session D); (5) left LI4acupoint stimuli alone (session E); (6) non-acupoint stimulialone (session F). Each session lasted 20 min with a 2- to 3-min break, in which two stimulation sessions were inter-

posed within a 30-min rest period. An experienced acu-puncturist, blinded to the SEPs recording team, performedall stimulation sessions.

4.4. Stimulation

The skin was cleaned with alcohol. For painful mediannerve stimulation, the anode was inserted just proximal tothe left palmar crease, and the cathode was insertedbetween the tendons of the palmaris longus muscle(1.25 ± 0.25 mm) in depth and (3 ± 0.25 cm) proximal tothe anode. The biggest advantage of this method is thatsignals ascending through both A-beta fibers relating totouch and A-delta fibers relating to pain can be recordedsimultaneously (Wang et al., 2003; Yamasaki et al., 2000).For interfering stimulation, two stainless steel needles0.25 mm in diameter and 3 cm in length were inserted inacupoint and non-acupoint, respectively (1.25 ± 0.45 cm) indepth perpendicular to the skin surface, one at the LI4acupoint (Hegu), over the dorsum of the hand, between thefirst and second metacarpal bones, in the middle of thesecond metacarpal bone, on the protuberance of the muscleon the radial side, and the other one at the non-acupoint,approximately 5–6 mm distant laterally to the LI4. Theseelectrodes were connected to the output of a nervestimulator (Singapore, LY257) and transmitted consistentsquare wave. The stimulation frequency of pulses applied tomedian nerve, acupoint and non-acupoint electrodes was2 Hz, and the corresponding pulse width was 0.6 ms. Thestimulation intensity was 3.3 mA ∼4.2 mA for non-painfulmedian nerve stimuli (mean perceptive threshold 2.8 mA–3.7 mA); 10.6 mA–13.7 mA for painful median nerve stimuli(mean pain threshold 9.1 mA ± 1.97 mA); 6.6 mA–7.5 mA foracupoint stimuli or non-acupoint stimuli (acupuncturepractices are administered clinically to relief pain withparameters of high intensity-low frequency).

4.5. SEPs recording

Somatosensory-evoked potentials (SEPs) recordings weremade by using a 64-channel Quikcaps (NeuroScan ESI-128system) from 64 scalp positions evenly positioned over bothhemispheres according to the 10–20 system. Electrode imped-ance was kept below 5 KΩ. Both earlobe electrodes were usedas references, the forehead electrode served as a ground. Theelectrical potentials (band-pass filtered between 1 Hz and1000 Hz, sampling rate of 2000 Hz) were recorded in epochsfrom 100ms before to 400ms after the stimulus. A total of 2400stimuli-related epochs were recorded for each subject in eachsession.

4.6. Electrodes and head 3D-position digitizing

Individual head shapes were co-registered with the sensorcoordinate system by digitizing (Polhemus Fastrack Digitizer)skull landmarks. The y-axis connects the nasion and the inion,and the x-axis is perpendicular to the y-axis at its midpointand is parallel to the line that connects the right and leftauditory meatus, the z-axis through their intersection point.These landmarks enabled registration of SEPs activity with

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individual anatomical magnetic resonance scans that wereused to help constrain realistic source reconstruction.

4.7. MRI

Subjects were scanned with a neurooptimized GE SignaLX 1.5-T system (General Electric). In a structural session,whole-head T1-weighted images (spatial resolution,1.0 × 1.0 × 1.5 mm; in-plane matrix, 256 × 256; 124 slices, nogap) were acquired with a quadrature head coil by using athree-dimensional spoiled gradient echo sequence (repetitiontime/echo time/flip angle = 24 ms/8 ms/24°).

4.8. Data evaluation

After registration, the 2400 epochs were digitally filtered(band-pass filter 1–100 Hz, 24 dB/Oct), averaged by using theNeuroscan software (SCAN 4.1). Further off-line analysis wasdone; the amplitudes (base-to-peak, peak-to-peak) and laten-cies of the evoked potentials were determined. Based SEPsdata, the corresponding sources were localized on theanatomy by using multimodal neuroimaging softwareCURRY 4.0. SEPs surface field distributions were fitted inconjunction to obtain maximal localization power. Thelocation of dipoles were calculated by an iterative least-squares fit. The goodness of fit (GOF) indicated the percent-age of data that can be explained by the model. We used theGOF value to determine whether or not the model was anappropriate one. GOF values larger than 90% were consideredto indicate a good model. To search for the best location of asource, movement was made in steps of 0.5–2.0 mm, and theGOF was calculated at each location. We repeated thisprocedure until the largest GOF was obtained. All datawere expressed as mean ± SE and analyzed by using two-way repeated ANOVA or Student's paired t test. Statisticalsignificance was set at the level of P < 0.05.

Acknowledgments

The work was supported by the National Natural ScienceFoundation of China (nos. 90209009) and the Natural ScienceFoundation of Huazhong University of Science and Technology.

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