Clinical Neurophysiology xxx (2014) xxx–xxx

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Abdominal acupuncture reduces laser-evoked potentials in healthy subjects q C. Pazzaglia a, S. Liguori b, I. Minciotti c, E. Testani c, A.E. Tozzi d, A. Liguori e, F. Petti b, L. Padua a,c, M. Valeriani f,g,⇑ a

Department of Neurology, Don Carlo Gnocchi Onlus Foundation, Milano, Italy Paracelso Institute, Rome, Italy Department of Neuroscience, Catholic University of Sacred Heart, Rome, Italy d Multifactorial Diseases and Complex Phenotypes Research Area, Pediatric Hospital Bambino Gesú, IRCCS, Rome, Italy e Department of Anatomic Histologic, Forensic-medicine and Locomotor-system Sciences, Faculty of Medicine, Sapienza University of Rome, Rome, Italy f Department of Neurology, Pediatric Hospital Bambino Gesù, IRCCS, Rome, Italy g Center for Sensory-Motor Interaction, Aalborg University, Aalborg, Denmark b c

a r t i c l e

i n f o

Article history: Accepted 21 November 2014 Available online xxxx Keywords: Acupuncture Laser-evoked potentials Pain perception

h i g h l i g h t s  Acupuncture is able to reduce pain although the exact mechanism is unknown.  Laser-evoked potential (LEP) is one of the most reliable methods to assess nociceptive pathways.  Abdominal acupuncture is able to modify LEP.

a b s t r a c t Objective: Acupuncture is known to reduce clinical pain, although the exact mechanism is unknown. The aim of the current study was to investigate the effect of acupuncture on laser-evoked potential amplitudes and laser pain perception. Methods: In order to evaluate whether abdominal acupuncture is able to modify pain perception, 10 healthy subjects underwent a protocol in which laser-evoked potentials (LEPs) and laser pain perception were collected before the test (baseline), during abdominal acupuncture, and 15 min after needle removal. The same subjects also underwent a similar protocol in which, however, sham acupuncture without any needle penetration was used. Results: During real acupuncture, both N1 and N2/P2 amplitudes were reduced, as compared to baseline (p < 0.01). The reduction lasted up to 15 min after needle removal. Furthermore, laser pain perception was reduced during real acupuncture, although the difference was marginally significant (p = 0.06). Conclusions: Our results show that abdominal acupuncture reduces LEP amplitude in healthy subjects. Significance: Our results provide a theoretical background for the use of abdominal acupuncture as a therapeutic approach in the treatment of pain conditions. Future studies will have to be conducted in clinical painful syndromes, in order to confirm the analgesic effect of acupuncture in patients suffering from pain. Ó 2014 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction q The study was conducted in the Department of Neurology, Don Carlo Gnocchi Onlus Foundation. ⇑ Corresponding author at: Department of Neurology, Pediatric Hospital Bambino Gesù, Piazza Sant’Onofrio 4, 00165 Rome, Italy. Tel.: +39 0668592289; fax: +39 0668592463. E-mail address: [email protected] (M. Valeriani).

The use of acupuncture, as a complementary and alternative medicine, is increasing, although its clinical efficacy is still a matter of debate (Frass et al., 2012). Although the mechanisms underlying the acupuncture analgesic action are far from being known, several elements support the idea that acupuncture can act at the level of the nociceptive pathways. First, a study comparing the effect of

http://dx.doi.org/10.1016/j.clinph.2014.11.015 1388-2457/Ó 2014 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Pazzaglia C et al. Abdominal acupuncture reduces laser-evoked potentials in healthy subjects. Clin Neurophysiol (2014), http://dx.doi.org/10.1016/j.clinph.2014.11.015

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the stimulation of two different acupoints with the effect of morphine demonstrated that both acupuncture and morphine were able to increase the pain threshold by 80–90%. This effect disappeared after the injection of an anesthetic into the acupoints (Research Group of Acupuncture Anesthesia, 1973). According to this study, the analgesic effect of acupuncture is similar to that of opioid drugs. Second, other studies show that the brain areas involved in acupuncture analgesia belong to the so-called pain matrix, thus suggesting a significant functional overlapping between pain and acupuncture pathways (Treede et al., 1999; Zhao, 2008). In anesthetized rats, Pan and colleagues found that either noxious stimulation or electroacupuncture activate the hypothalamic–pituitary–adrenocortical axis, leading to a marked expression of c-fos in the anterior lobe of the pituitary gland, as well as in the arcuate and some nearby hypothalamic nuclei (Pan et al., 1994). Similar results were achieved by using functional magnetic resonance imaging (fMRI) (Hennig et al., 2000; Hui et al., 2005; Zhang et al., 2003a,b), which showed the important role played by the hypothalamus-limbic system in acupuncture analgesia. Positron emission tomography (PET) studies (Biella et al., 2001; Hsieh et al., 2001; Pariente et al., 2005) showed that acupuncture was able to activate both the hypothalamus and the insula. Third, acupuncture analgesia is possibly mediated by the action of various endogenous neurotransmitters involved in nociception. The effects of acupuncture (and sham acupuncture) on l-opioid receptor binding have been studied in humans (Harris et al., 2009) and the action of acupuncture in modifying the pain threshold in animal models is reversed by naloxone (Pomeranz and Chiu, 1976). The development of tolerance for acupuncture is probably due to desensitization of opioid receptors in the central nervous system (Han et al., 1979, 1981). There are studies that investigated the possible site of action of acupuncture, which, however, remains elusive. When the needle is introduced in the skin, it might stimulate the peripheral fibers, including Ad and C afferents. As these afferents convey the nociceptive input, their stimulation might trigger a conditioning pain modulation (CPM) mechanism, which could reduce the subjective perception of clinical pain (Bing et al., 1990; Fields et al., 2005; Villanueva et al., 1986). However, a recently published study excluded that CPM may explain acupuncture analgesia (Tobbackx et al., 2013). A possible effect of acupuncture at the spinal level is suggested by the segmental specificity of acupoints (Zhang et al., 2003b). The scarce knowledge on the acupuncture mechanisms of action can be due to the almost exclusive use of somatic acupuncture. Indeed, using somatic acupuncture, it is difficult to compare the effects of acupuncture with those obtained in a real sham condition. Sham acupuncture is difficult to obtain, as the needle has to induce a pinprick and/or dull sensation (‘‘De Qi’’ sensation) in order to work. This problem can be solved by using ‘‘abdominal’’ acupuncture (AA). It is an ancient technique based on the stimulation of abdominal points, according to a ‘‘turtle representation’’ of the somatosensory areas (Fig. 1). With respect to ‘‘somatic’’ acupuncture, in AA, the needle is superficially driven and no stimulation is needed. Moreover, while in somatic acupuncture treatment is based on differentiation among syndromes, AA is merely driven by the symptom location, thus allowing the treatment to be more standardized than in somatic acupuncture. The most reliable laboratory tool for assessing nociceptive pathway function is laser-evoked potential (LEP) recording (Haanpää et al., 2011; Valeriani et al., 2012). LEPs are related to the activation of type II AMH mechanothermal nociceptors. The afferent volley is conducted along the small myelinated (Ad) primary sensory neurons and the spinothalamic pathway (Bromm and Treede, 1991). LEPs consist of a temporal lateralized component (N1), with an almost simultaneous frontal positive potential (P1). These are

followed by a larger vertex biphasic potential reaching its maximal amplitude on the Cz vertex (N2/P2). Intracerebral recording studies and dipolar modeling studies agree in suggesting that the N1 and P1 potentials are probably generated in the opercular (SII/insula) area (Valeriani et al., 1996, 2000; Frot et al., 1999). The N2 and P2 potentials have a topographical distribution very similar to the vertex potential obtained in other modalities (Mouraux and Iannetti, 2009), and they probably originate from different sources including the midcingulate cortex and insula (Garcia-Larrea et al., 2003; Dowman et al., 2007; Frot et al., 2008). In order to verify whether AA is able to modify the LEP amplitudes and laser pain perception, we recorded LEPs in healthy subjects during abdominal acupuncture. The results were compared with those obtained during sham acupuncture, in order to separate the possible acupuncture-related analgesic effect from the placebo effect. We expected to find a reduction of both LEP amplitude and laser pain perception during real acupuncture, as compared to the sham condition. 2. Methods The study protocol was approved by the local ethics committee. The study was designed as a single-blind, crossover protocol that allowed us to consider each subject as his/her own control. Ten normal subjects (five male and five female, mean age 38 years, age range 25–49 years) were asked to sign an informed consent form to participate in the study. The subjects were naive to all types of acupuncture. Only volunteers with no historical symptoms nor signs of focal upper limb entrapment, cervicobrachialgia, or polyneuropathy were enrolled. Each subject underwent two separate sessions of real and sham AA. All subjects did not know that there would be a sham acupuncture, and all of them were told that the aim of the study was to investigate the effect of two different kinds of acupuncture on LEPs. Each session included three times: (1) baseline, in which LEPs to stimulation of the skin of both the right and left dorsal wrist were recorded before real or sham AA; (2) acupuncture, in which LEPs were recorded to stimulation of the same sites during real or sham AA; and (3) post, in which LEPs were recorded 15 min after needle removal (Fig. 2). The order of the sessions was randomized across subjects, and the time interval between sessions ranged from 28 to 40 days. We ensured that female subjects underwent both sessions in the same menstrual period, as hormonal changes can influence pain perception (de Tommaso et al., 2009). All subjects were treated by the same acupuncturist (SL). Only the acupuncturist was aware of the type of acupuncture (i.e., real or sham), while all the other experimenters were blind to it. 2.1. Laser stimulation and LEP recording Laser pulses (wavelength, 1.34 lm) were delivered on the dorsum of the right and left wrist separately by a YAP Stimul 1340 (Electronic Engineering, Florence, Italy). The order of side stimulation was counterbalanced across subjects. The laser stimulus intensity was fixed at 38 mJ/mm2, which was perceived by all subjects as a painful pinprick (Cruccu et al., 2003; Valeriani et al., 2002). The interstimulus interval varied randomly between 9 and 11 s. In order to avoid nociceptor fatigue, the laser spot was slightly moved from one stimulus to another. LEPs were recorded from two scalp electrodes placed along the midline (Fz and Cz positions of the 10–20 international system), and one electrode in the left temporal region (T3/T4), contralateral to the stimulation. The reference electrode was placed on the nose, and the ground on the forehead (Fpz). Both vertical and horizontal eye movements and eye blinks were monitored by an electroocu-

Please cite this article in press as: Pazzaglia C et al. Abdominal acupuncture reduces laser-evoked potentials in healthy subjects. Clin Neurophysiol (2014), http://dx.doi.org/10.1016/j.clinph.2014.11.015

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Fig. 1. Localization of acupoints around the umbilicus. They are distributed according to a so-called turtle representation of somatosensory areas. The acupoints selected in our protocol are included in circled boxes.

Fig. 2. Flow diagram of the study protocol.

lographic (EOG) electrode located above the right eyebrow. Signals were amplified and filtered (band pass 0.3–70 Hz). The analysis time was 1000 ms with a bin width of 2 ms. The averages of consecutive 25–30 trials were recorded for each stimulated hand. As the amplitude of the LEP components recorded on the vertex can be reduced by distraction from the stimulus (Garcia-Larrea et al., 2003; Legrain et al., 2002; Lorenz and Garcia-Larrea, 2003), our subjects were asked to count the number of the received laser stimuli silently, in order to ensure that their attention level did not change throughout the entire experiment. The number of averaged trials varied from 25 to 30 to prevent our subjects from learning the number of delivered pulses and keep their attention fixed on the stimulation. Averages with a percentage of mistakes >10% would have been discarded. However, this never happened as all our subjects were extremely cooperative with the procedure. After each LEP recording, the subject was asked to rate laser pain intensity by using a 100-mm visual analog scale (VAS), in which ‘‘0’’ corresponds to no pain and ‘‘100’’ to the worst conceivable pain.

2.2. Abdominal acupuncture protocol As LEPs were recorded to stimulation of the wrist dorsum, the AA protocol was selected accordingly. In particular, the needles were fixed in the areas of the ‘‘turtle representation’’ of the body, corresponding to either the left or the right wrist dorsum (see Fig. 1). More specifically, the acupoints were selected on meridians crossing the abdomen: (1) the Conception Vessel (CV) meridian (also called Ren meridian), (2) the Kidney (K) meridian, and (3) the Stomach (ST) meridian (Fig. 1). In order to determine the exact position of the acupoints, the distances were chosen according to the traditional Chinese medicine system of cun, where 1 cun corresponds to the width of the thumb of the treated subject. On CV meridian, the treated acupoints were: (1) the four CV located 3 cun below the umbilicus, (2) the six CV located 1.5 cun below the umbilicus, and (3) the 12 CV located 4 cun above the umbilicus. On the K meridian, there was one bilaterally treated acupoint: the 17 K located 2 cun above the umbilicus and 0.5 cun lateral to

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the 10 CV (2 cun above the umbilicus). On the ST meridian, we treated the bilateral 24 ST acupoint, located 1 cun above the umbilicus and 2 cun lateral to the nine CV (1 cun above the umbilicus). Moreover, two extra acupoints homolateral to laser stimulation were treated: (1) the AB1 located ½ cun above the nine CV and ½ cun laterally to the 24 ST, and (2) the AB2 located 1 cun laterally to the 24 ST. For both real and sham AA, the needle was inserted in a plastic tuboguide. The tuboguide was rested on a cylindrical plastic support with a hole in the center and an adhesive base. Then, the support and the tuboguide were applied on the abdomen of the subject (Fig. 3). For the real AA, the tuboguide was open and the needle penetrated the skin. The needles remained inserted in the skin for about 10 min. For the sham AA, instead, the tuboguide had a closed sharp base that prevented any needle penetration. Each needle remained in the corresponding tuboguide for about 10 min. In the real AA, most of our subjects did not feel any painful sensation, during both needle insertion and permanence in the skin. Further, during needle insertion, seven subjects perceived a sensation of touch or of a gentle pinprick, while only three out of 10 subjects felt a puncture, described as a weakly painful pinprick. All subjects did not perceive any sensation while the needle was left in the skin. No subject reported any aftersensation after needle removal. For sham AA, when the acupuncturist hit the top of the needle, with the same procedure as the one used in real AA, the subject either felt a touch sensation (three subjects) or perceived a weak pinprick (seven subjects). In order to investigate whether there may have been any difference between the sensation due to needle insertion in real AA and that due to the sharp tip of the tuboguide in sham AA, our subjects were asked to rate this difference in a numerical rating scale (NRS), where 0 corresponded to no sensation difference and 10 corresponded to the largest perceivable difference. The sensation difference was rated as 0 by seven subjects, as 1 by two subjects, and as 2 by the remaining subject.

2.3. LEP analysis and statistics LEP components were identified according to their polarity and distribution. N2 and P2 peak latencies and baseline amplitudes were measured on the Cz electrode. In addition, the peak-to-peak N2/P2 amplitude was calculated. The N1 latency was measured on the temporal trace contralateral to the stimulation, and the N1 amplitude was calculated by referring the temporal electrode contralateral to the stimulation (T3/T4) to the Fz lead off-line (Kunde and Treede, 1993). As LEP data and laser pain ratings collected across successive trials undergo a progressive reduction due to habituation (Valeriani et al., 2003), we normalized the measures in the real session to the corresponding values in the sham session. In each subject, the averages (real and sham) between VAS values and LEP amplitudes and latencies obtained to the right and left wrist stimulation were calculated. For each of the times of the experiment (baseline, acupuncture, and post) in the real condition, we thus analyzed the variations in terms of standard deviations compared with the sham condition. To test the variations across the three times of the experiment (baseline, acupuncture, and post), we used a one-way ANOVA for repeated measures. Statistical significance was set at p < 0.05. 3. Results In both sessions, the temporal N1 response and the vertex N2 and P2 components were recorded in all our subjects at the baseline (Table 1). Fig. 4 shows LEPs in a representative subject. In the real session, both the N1 and N2/P2 amplitudes were reduced during ‘‘acupuncture’’ and ‘‘post’’ times, as compared to baseline. In the sham session, the N2/P2 amplitude was slightly reduced during acupuncture and recovered at post time.

Fig. 3. (A) The needle, the tuboguide, and the plastic support. (B) For real AA, the tuboguide is open and the needle comes out of the plastic support. (C) For sham AA, the closed tuboguide, but not the needle, comes out of the plastic support (sham AA). (D) Needle, tuboguide, and plastic support positioned on the abdomen.

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C. Pazzaglia et al. / Clinical Neurophysiology xxx (2014) xxx–xxx Table 1 Mean values (±SD) of VAS, N1 latency and amplitude, N2 and P2 latency, and N2/P2 amplitude. VAS R

L

N1 latency (ms)

N1 amplitude (lV)

N2 latency (ms)

P2 latency (ms)

N2/P2 amplitude (lV)

R

R

R

R

R

L

L

L

L

L

Real B 41.8 (±18.5) 45.1 (±16.5) 168.7 (±25.7) 160.8 (±11.8) 6.3 (±5.8) 5.9 (±4.2) 215.1 (±34.5) 218.9 (±28.5) 366.4 (±45.2) 365.8 (±47.1) 28.9 (±9.3) 28.0 (±7.6) AA 32.0 (±18.0) 33.5 (±17.1) 169.0 (±19.0) 159.7 (±6.1) 3.4 (±5.0) 4.4 (±4.2) 222.9 (±33.8) 228.1 (±31.9) 351.8 (±39.7) 346.4 (±34.0) 14.8 (±5.9) 15.4 (±8.4) P 35.4 (±20.4) 38.3 (±16.2) 159.4 (±20.3) 168.5 (±15.7) 4.6 (±7.9) 2.6 (±6.3) 213.3 (±25.1) 232.5 (±24.9) 357.6 (±49.6) 348.7 (±43.3) 17.0 (±13.2) 15.5 (±10.8) Sham B 42.1 (±20.4) 41.9 (±20.0) 168.6 (±18.3) 163.9 (±12.8) 6.6 (±6.6) 6.2 (±4.1) 211.6 (±25.8) 212.7 (±23.5) 337 (±48.5) 348.9 (±45.4) 33.7 (±15.2) 30.3 (±10.7) AA 51.4 (±24.3) 46.6 (±26.6) 164.7 (±12.7) 168.3 (±17.1) 7.0 (±5.8) 5.8 (±3.2) 208.3 (±20.5) 218.1 (±31.0) 323.9 (±54.2) 345.9 (±55.2) 27.9 (±13.3) 22.7 (±10.2) P 43.6 (±21.2) 41.0 (±22.5) 161.3 (±10.3) 169.1 (±18.0) 5.5 (±4.9) 5.5 (±5.1) 211.0 (±23.8) 205.9 (±25.5) 337.8 (±53.7) 338.0 (±57.9) 29.7 (±13.0) 29.7 (±9.0) R = right, L = left, B = baseline, AA = acupuncture, P = post.

Fig. 4. LEPs recorded to right-hand stimulation in one representative subject during real (upper) and sham (lower) sessions. While the Cz electrode is referred to nose, the T3 lead is referred to Fz.

Fig. 5. VAS and LEP (latencies and amplitudes) values at baseline, acupuncture, and post times in the real condition are normalized to the sham condition. Negative values represent a decrease with respect to sham, and positive values correspond to an increase. The asterisk indicates the significant differences.

Fig. 5 shows the VAS and LEP values in the real session normalized to the sham session. Negative values represent a decrease with respect to sham, and positive values correspond to an increase.

In the real condition, the VAS values were reduced during acupuncture and post times compared to the sham condition, but the difference was only marginally significant (F(2) = 3.24, p = 0.06).

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A significant effect of time was found on the normalized N1 (F(2) = 6.63, p < 0.01), P2 (F(2) = 9.15, p < 0.01) and N2/P2 (F(2) = 6.38, p < 0.01) amplitudes. In particular, in the real condition, the N1, P2, and N2/P2 amplitudes during acupuncture and post times were lower when compared to the sham condition. No difference across times was found for the normalized latencies (N1: F(2) = 1.08, p = 0.37; N2: F(2) = 0.92, p = 0.43; P2: F(2) = 3.33, p = 0.06), 4. Discussion Our study showed that AA reduces LEP component amplitudes, as compared to the sham condition in which there was no needle penetration into the skin. In addition, laser pain perception was reduced during acupuncture, although the difference was only marginally significant. 4.1. Presumed sites for the AA analgesic effect In our subjects, both the N1 and N2/P2 potentials were reduced in amplitude by AA. When N2 and P2 amplitudes were considered separately, only the second one was reduced during acupuncture. Several previous studies on EEG modeling (Bentley et al., 2003; Garcia-Larrea et al., 2003; Valeriani et al., 1996, 2000, 2002) and on intracranial EEG recording (Frot et al., 1999, 2008; Lenz et al., 1998; Ohara et al., 2006; Dowman et al., 2007; Perchet et al., 2012) showed that the N1 response is probably generated within the SII area contralateral to the stimulation, while the N2/P2 potential is possibly generated by several sources, including the midcingulate cortex and insula. On the grounds of our results, some hypotheses about the AA action site can be made: (1) The reduction of both the N1 and N2/ P2 amplitudes suggests that the AA inhibitory effect on the pain matrix may begin in the SII area, where the early N1 potential is generated. (2) It is also possible that AA may act on the input afferent to the cerebral cortex at a more peripheral level (peripheral fibers? spinal cord?) and that this effect may be mediated by an activation of the descending inhibitory control. As some putative cerebral areas generating LEPs, such as insula and anterior cingulate cortex, are part of the brain opioid system (Petrovic et al., 2002), the descending inhibitory action triggered by AA could be mediated by endogenous opioids. The strict relationship between the analgesic effects of acupuncture and opioids supports this mechanism of action (Research Group of Acupuncture Anesthesia, 1973; Pomeranz and Chiu, 1976; Zhao, 2008; Harris et al., 2009). The opioid system is a major component that regulates the spinal pain transmission, and the opioid receptors, especially l-opioid receptors, are localized in the spinal dorsal horn (Mansour et al., 1995). The l-opioid receptor agonists, including endogenous opioids, stimulate the l-opioid receptors on the terminal of primary afferent neurons at the spinal dorsal horn to cause presynaptic inhibition of neurotransmitter release (Mense, 1983). Moreover, these agonists also stimulate the l-opioid receptors on the cell body of second-order neurons at the spinal dorsal horn to cause postsynaptic hyperpolarization of excitatory neurons (Mizoguchi et al., 2014). Our neurophysiological data fit with the possibility that the AA analgesic effect is mediated by the complex inhibitory action of the endogenous opioids at the spinal cord level. Indeed, in our subjects, both the middle-latency N1 and the late N2/P2 components were dampened by AA, thus suggesting a block at the second-order neuron (spinal cord). 4.2. LEP amplitude change during AA: a possible placebo effect? One of the main problems concerning the research in acupuncture is the choice of the sham procedure. There are three different

methods: (1) The ‘‘wait list’’ consists in not treating the patient as if he/she were in the waiting list for treatment that he/she would receive later. It has been abandoned, as patients cannot be blinded and most studies showed positive results (Haake et al., 2007). (2) The needle-insertion sham consists in a needle penetrating in points that should not be effective. However, there is still a debate whether acupoints have their own specificity (Choi et al., 2012), and a peripheral activation may be caused by needle penetration even in an inert zone, as needle rotation in subcutaneous tissue leads to extensive fibroblast spreading and lamellipodia formation (Langevin et al., 2006). (3) The non-insertion sham procedure seems to be the best control, as the needle does not penetrate the skin. This procedure has been validated (McManus et al., 2007), although there is no uniform agreement (White et al., 2003). In our study, we used this last control method. The support and the tuboguide were used for both sham and real acupuncture, in order to ensure that the subjects were not aware of the different treatments. At the end of the whole experiment, that is, after the second session of stimulation, each subject was explicitly requested to use an NRS to rate any difference he/she may have felt between both types of acupuncture. No major difference was reported and, in particular, all subjects were surprised when they were told that in the sham condition no needle penetration had occurred. One may wonder whether in our subjects also the real AA analgesic effect, in terms of both LEP amplitude and laser pain rating reduction, could be due to a psychological mechanism, in particular to a placebo effect. As placebo analgesia is initiated and maintained by expectations of symptom change and changes in motivation/ emotions (Price et al., 2008), it is conceivable that our subjects, who expected to feel less pain during acupuncture, could undergo a placebo effect. However, in our study, both real and sham AA induced the same expectation of an analgesic effect. In a previous study, we showed that a selective N2/P2 amplitude reduction without any modification of the N1 amplitude and of the laser pain subjective perception can be caused by placebo treatment, when it is based only on a verbal conditioning mechanism (Colloca et al., 2008). In the present study, the verbal conditioning could contribute to the reduced N2/P2 amplitude during both sham and real AA. However, the difference between the effect of real AA and that of sham AA can be considered as the genuine AA analgesic effect. 4.3. Methodological considerations and limitations of the study One of the limitations is represented by the low number of recruited subjects. However, the crossover design, according to which every subject is a control of himself/herself, reduces the negative impact of this problem on our results. In order to evaluate the effectiveness of acupuncture, we compared the N2/P2 reduction in both sessions (real AA and sham AA). To make our results more reliable, we computed the required sample size in order to have a power of the test of 99.9% and an alpha (probability of a type I error) equal to 5%. The sample size was 10. The low number of subjects probably explains why only the neurophysiological result reached the statistical significance, while the AA effect on the laser pain rating was only marginally significant. Indeed, we can suppose that the neurophysiological assessment of the nociceptive pathway is more sensitive to slight pain modifications than the subjective psychophysical rating. This was also found when more standardized analgesic treatments, such as the transcutaneous spinal direct current stimulation, were tested (Truini et al., 2011). It is well known that long latency-evoked potential amplitudes are reduced after repetitive stimulation. This phenomenon, known as habituation, has also been shown for LEPs (Valeriani et al., 2003). With the design of the present study, in which LEPs were recorded at three different times (baseline, AA, and post), habituation might

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bias our results. In order to rule out a possible habituation effect, the data obtained with the real AA were normalized to the sham AA, for which the habituation biasing effect should be the same as for real AA. In order to investigate the acupuncture effect on laser pain and LEP amplitude, we chose AA to minimize the biases of somatic acupuncture, such as: (1) the differentiation of syndromes, which requires a personalized treatment and makes a standardized protocol difficult to establish; and (2) the arrival of the ‘‘De Qi’’ sensation, which makes a convincing sham treatment difficult to obtain. One point of strength of our study is indeed represented by the reliable and well-designed sham treatment. A limitation of our sham treatment is that we could not adopt a double-blind design, as the acupuncturist had to be aware whether the needle was inserted in the skin (real AA) or it was stopped by the closed tuboguide (sham AA). One point that was not investigated in our study concerns the acupoint specificity. Indeed, in our subjects, the possible effect of needles inserted out of the acupoints was not tested. Future studies will have to compare the neurophysiological and psychophysical differences when acupoints and non-acupoints are treated. Lastly, our results demonstrate that AA reduces phasic pain, such as that produced by laser pulses delivered on the skin. It should be underlined that phasic pain is different from the longlasting pain, commonly referred by patients in the clinical practice. Thus, our findings, although providing objective evidence of an AA effect on the nociceptive pathways, cannot guarantee that AA is useful in painful clinical syndromes. 5. Conclusions In conclusion, although research in the field of acupuncture has been growing over the past years, the debate on how and where this technique acts is still a matter of discussion. Our study, which was conducted with a reliable acupuncture protocol, a rigorous sham model, and an objective neurophysiological test to assess the nociceptive pathway, showed that some cerebral areas belonging to the ‘‘pain matrix’’ reduce their activity during AA. This can provide a necessary scientific support for using AA in the clinical routine. Our results were obtained in healthy subjects. Future studies will have to be conducted in clinical painful syndromes, in order to confirm the analgesic effect of AA in patients suffering from pain. Acknowledgments We would like to acknowledge Dr. Mario Sorrentino (Rome), Dr. Sergio Bangrazi (Paracelso Institute, Rome, Italy), and Prof. Bo Zhiyun (China) for their support. Conflict of interest: All authors declare that no funding sources were provided, and they declare no conflicts of interest. References Bentley DE, Derbyshire SW, Youell PD, Jones AK. Caudal cingulate cortex involvement in pain processing: an inter-individual laser-evoked potential source localisation study using realistic head models. Pain 2003;3:265–71. Biella G, Sotgiu ML, Pellegata G, Paulesu E, Castiglioni I, Fazio F. Acupuncture produces central activations in pain regions. Neuroimage 2001;14:60–6. Bing Z, Villanueva L, Le Bars D. Acupuncture and diffuse noxious inhibitory controls: naloxone-reversible depression of activities of trigeminal convergent neurons. Neuroscience 1990;37:809–18. Bromm B, Treede RD. Laser-evoked cerebral potentials in the assessment of cutaneous pain sensitivity in normal subjects and patients. Rev Neurol 1991;10:625–43. Choi EM, Jiang F, Longhurst JC. Point specificity in acupuncture. Chin Med 2012;28:4.

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Please cite this article in press as: Pazzaglia C et al. Abdominal acupuncture reduces laser-evoked potentials in healthy subjects. Clin Neurophysiol (2014), http://dx.doi.org/10.1016/j.clinph.2014.11.015