Clinical Neurophysiology 113 (2002) 298–304 www.elsevier.com/locate/clinph Pain-related magnetic fields evoked by intra-epidermal electrical stimulation in humans Koji Inui*, Tuan Diep Tran, Yunhai Qiu, Xiaohong Wang, Minoru Hoshiyama, Ryusuke Kakigi Department of Integrative Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan Accepted 26 November 2001 Abstract Objectives: We recently developed a new method for the preferential stimulation of Ad fibers in humans The aim of the present study was to examine whether this method can serve as an appropriate stimulus in a magnetoencephalographic study Methods: We recorded somatosensory-evoked magnetic fields (SEFs) following intra-epidermal electrical stimulation applied to the hand and elbow Superficial parts of the skin were electrically stimulated through a needle electrode whose tip was inserted in the epidermis Results: In all 13 subjects, the equivalent current dipole was estimated in the secondary somatosensory cortices (SII) In out of 13 subjects, simultaneous activation of the primary somatosensory cortex (SI) in the hemisphere contralateral to the stimulation was identified The mean peak latencies of magnetic fields corresponding to contralateral SI, SII and ipsilateral SII activation following hand stimulation were 162, 158 and 171 ms, respectively The respective latency following elbow stimulation was 137, 139 and 157 ms, respectively Estimated peripheral conduction velocity was 15.6 m/s Conclusions: All the results were consistent with previous findings in pain SEF studies We concluded that our novel intra-epidermal electrical stimulation is useful for pain SEF studies since it does not need special equipment and is easy to control q 2002 Elsevier Science Ireland Ltd All rights reserved Keywords: Ad fibers; Electrical stimulation; Magnetoencephalography; Pain; Somatosensory evoked magnetic fields Introduction The mechanism of pain perception in humans is still largely unknown, although some epoch-making studies have been reported One of the main reasons for this is that selective stimulations of the nociceptive system are technically difficult Painful transcutaneous electrical stimulation, for example, is an easy way to produce painful sensations, however, it activates not only nociceptive Ad and C fibers but also large myelinated fibers A laser beam has been used in pain research (Bromm and Lorenz, 1998; Kakigi et al., 2000), since it can stimulate nociceptive fibers specifically with minimal effects on other fibers (Bromm and Treede, 1984) In addition, a laser beam provides a sufficiently steep stimulus time profile and, therefore, is very useful in stimulus-locked averaging studies including analyses of somatosensory-evoked potentials (SEP) and magnetic fields (SEF), although the method requires a specially made stimulator We recently developed a new method to preferentially activate skin Ad fibers (Inui et al., 2002) Using a pushpin-type needle electrode inserted in the epidermis, we could electrically stimulate nociceptive thin fibers in the epidermis or in the superficial part of the dermis (Kruger et al., 1985; Novotny and Gommert-Novotny, 1988) The intra-epidermal stimulation minimized the activation of other nerve fibers belonging to the tactile system that run deeper than the epidermis (Munger and Halata, 1983) This method does not need any special instruments and is applicable to any area of the body We used the intra-epidermal stimulation and recorded SEFs in this study Magnetoencephalography (MEG) has theoretical advantages for localizing cerebral dipoles over electroencephalography (EEG) due to reduced effects of cerebrospinal fluid, skull and skin The aim of the present study was to determine the sites of the brain where the response following intra-epidermal stimulation occurs and to examine whether our new method can serve as an appropriate stimulus in an SEF study * Corresponding author Tel.: 181-564-55-7769; fax: 181-564-52-7913 E-mail address: inui@nips.ac.jp (K Inui) 1388-2457/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd All rights reserved PII: S13 88- 2457(01)0073 4-9 CLINPH 2001133 K Inui et al / Clinical Neurophysiology 113 (2002) 298–304 Methods The experiment was performed on 13 healthy volunteers, females and 10 males, aged 26–42 years (mean 33.2 ^ 4.0) The study was approved in advance by the Ethical Committee of the National Institute for Physiological Sciences and written consent was obtained from all the subjects 2.1 Painful electrical stimulation To produce a pain test stimulus, a method we recently developed (Inui et al., 2002) was used A disposable pushpin-type needle electrode with a needle tip 0.2 mm in length was used By pressing the electrode plate against the skin gently, the needle tip was inserted in the same layer of nerve endings of the thin myelinated fibers in the epidermis and superficial part of the dermis A surface electrode 1.0 cm in diameter was placed on the skin at a distance of cm from the needle electrode as the anode The electric stimulus was a current constant square wave pulse delivered at a random interval of 0.1–0.3 Hz The stimulus duration was 0.5 ms The current intensity was at the level producing a definite pain sensation in each subject, which was determined prior to the experiment We stimulated distal and proximal sites of the left upper limb The distal site was the dorsum of the left hand between the first and second metacarpal bones The proximal site was the lateral aspect of the elbow joint The mean stimulus intensity was 0.16 ^ 0.09 mA (mean ^ SD) for hand stimulation and 0.23 ^ 0.09 mA for elbow stimulation The insertion of the needle electrode caused no bleeding or visible damage to the skin 2.2 MEG recordings The pain SEF responses were measured with dual 37channel axial-type first-order biomagnetometers (Magnes, Biomagnetic Technologies, San Diego, CA) The detection coils of the biomagnetometers were arranged in a uniformly distributed array in concentric circles over a spherically concave surface Each device was 144 mm in diameter with a radius of 122 mm The outer coils were 72.58 apart Each coil was 20 mm in diameter and the centers of the coils were 22 mm apart Each coil was connected to a superconducting quantum interference device (SQUID) The two probes were centered on the C3 and C4 positions as based on the international 10-20 system This position covered the primary and secondary somatosensory cortices (SI and SII) The magnetic fields were recorded with a filter of 0.1– 200 Hz at a sampling rate of 2083 Hz and then filtered at low pass, 100 Hz The analysis window was 100 ms before and 500 ms after the stimulus, and the prestimulus period was used as the DC baseline Thirty responses were collected and averaged in one trial and trials were obtained with intervals of a few minutes to avoid habituation After 299 the reproducibility had been confirmed, trials were averaged and used for the analysis 2.3 EEG recordings The SEP was recorded simultaneously with the SEF The exploring electrodes were placed at Cz (vertex), C3 (2 cm posterior to C3), and C4 (2 cm posterior to C4) referring to the linked earlobes (A1 A2) of the international 10-20 system C3 and C4 positions were in the hand area of SI in the left and right hemispheres, respectively The impedance of all electrodes was below kV The sampling rate, sampling window and filters were the same as those for the SEF recordings 2.4 Data analysis A source analysis based on a single moving equivalent current dipole (ECD) model (Sarvas, 1987) in a spherical conductor, was applied to the magnetic field distribution The location, orientation and amplitude of a single ECD were estimated at the peak response The correlation coefficient (Corr.) between the theoretical field generated by the model and the measured field was calculated Since the measured fields in several subjects were considered to contain two or more temporally overlapping sources as will be described in Section 3, the single ECD model was inappropriate in such cases Therefore, we used the brain electric source analysis (BESA, Scherg, 2000) software package (NeuroScan, Inc, Mclean, VA) for the analysis of theoretical multiple source generators The goodness-of-fit (GOF) indicated the percentage of the data that can be explained by the model We used the GOF value to determine whether or not the model was an appropriate one In all subjects, both the single ECD model analysis and BESA were performed Because the main purpose of this study was to confirm that SEF provoked by our new method using a needle electrode was compatible to that in previous pain studies, only primary components were investigated Magnetic resonance imaging (MRI) scans (Shimadzu Magnes, Kyoto, Japan, 150XT 1.5 T) were obtained from all subjects T1-weighted coronal, axial and sagittal image slices obtained every 1.5 mm were used for superimposition of the MEG source locations Data were expressed as the mean ^ standard deviation (SD) Statistical significance was assessed by a paired t test and P , 0:05 was considered to be significant Results In all subjects, epidermal stimulation elicited weak but well-defined pricking sensations without tactile sensations A clear magnetic field (termed 1M) was identified following stimulation of the hand in the hemisphere contralateral to the stimulation (contralateral hemisphere) in all 13 subjects, and in the hemisphere ipsilateral to the stimulation (ipsilat- 300 K Inui et al / Clinical Neurophysiology 113 (2002) 298–304 Fig Evoked magnetic fields following intra-epidermal electrical stimulation of the dorsum of the left hand (A) Superimposed waveforms recorded from 37 channels at position C3 (ipsilateral) and C4 (contralateral), and evoked potentials recorded in Cz Arrow heads indicate the peak latency of the first component (1M) (B) Isocontour maps at peak latency Thin lines show fields directed out of the head and dotted lines into the head Isocontours are separated by 20 fT (C) Location of source generators overlaid on MRI scans eral hemisphere) in 12 subjects (Fig 1) Its onset and peak latencies were 110.2 ^ 13.8 and 156.2 ^ 10.8 ms, respectively, for the 1M recorded from the contralateral hemisphere, and 129.5 ^ 13.8 and 170.6 ^ 13.0 ms for the 1M from the ipsilateral hemisphere The time difference of the peak latency (13.8 ^ 6.7 ms) between both hemispheres was significant (t ¼ 7:1, P , 0:0001) No clear component before 1M could be identified in either of the hemispheres We separated subjects into two types, depending on whether a multi-dipole model was necessary A single dipole model was enough for subjects (type 1), but a multi-dipole model was necessary for subjects (type 2) Type 1: In the subjects classified into Type 1, isocontour maps of both hemispheres showed a typical one dipole pattern indicating the presence of a single ECD, and the dipole was estimated to lie in the superior bank or bottom of the Sylvian fissure, SII or insula, of both hemispheres (Corr ¼ 97.5 ^ 1.2% for ipsilateral, 98.0 ^ 0.6% for contralateral hemisphere, Fig 1) Because multiple source analysis was necessary to explain the ECDs in the other subjects as described below, BESA was also applied in these subjects to confirm that the single ECD model was actually appropriate in these subjects Since the duration of the 1M component was approximately 80–100 ms (Figs and K Inui et al / Clinical Neurophysiology 113 (2002) 298–304 301 2), a slightly shorter period, 30 ms before and after the peak of each 1M component, was used as the analysis period for BESA The mean GOF value during this 60 ms period was measured for the analysis Although we tried to find sources other than SII by using BESA, GOF was already high with the SII source alone (91.4 ^ 3.3% for ipsilateral and 88.9 ^ 2.6% for contralateral hemisphere) and no significant or consistent source could be additionally identified For example, an additional SI source in the contralateral hemisphere increased GOF only slightly, from 88.9 ^ 2.6 to 92.6 ^ 2.2% (Fig 2) Since the larger the number of sources we placed, the more the GOF value increased, Fig Evoked magnetic fields and potentials due to intra-epidermal stimulation applied to the dorsum of the left hand A representative case with multiple source generators (A) Superimposed waveforms recorded from 37 channels at C3 (ipsilateral) and C4 (contralateral), and evoked potentials recorded at C3 and C4 ; (B) isocontour maps at each peak latency Note that the complex field pattern in the contralateral hemisphere suggests two or more dipoles Fig Multiple source analysis in the same subject in Fig (A,B) temporal profile of source strength (upper trace) and goodness-of-fit (lower trace) ‘GOF’ indicates the mean value of goodness-of-fit during a 60 ms period, 30 ms before and after the peak latency (Aa, Ba) Results of analysis with the secondary somatosensory cortex (SII) source alone; (Ab, Bb) results of analysis with SII and additional primary somatosensory cortex (SI) sources; (Ac, Bc) location of sources such a small improvement of GOF could not be regarded as significant Type 2: A single dipole model could not be applied to the remaining subjects classified into Type due to a poor correlation value and multi-dipole pattern of the isocontour maps (Fig 3) Multiple source analysis of the contralateral hemisphere showed a very low GOF (67.2 ^ 19.2%) with the SII source alone but the GOF value was markedly increased when the SI source was added (92.5 ^ 3.8%, Fig 4) In these subjects, however, the GOF of the ipsilateral hemisphere was 89.5 ^ 4.2% with a single SII source, similar to that in the subjects described above The peak latency of contralateral SI and SII activities in these subjects was 158.6 ^ 14.5 and 161.8 ^ 15.6 ms, respectively The ECD in the SI source showed an anterior–posterior orientation in all subjects The ECDs in the bilateral SII pointed upwards in all subjects Clear magnetic fields were obtained in the contralateral and ipsilateral hemispheres following stimulation of the elbow in 12 and 11 subjects, respectively The onset and peak latencies of the 1M component were 96.9 ^ 11.0 and 136.2 ^ 10.9 ms, respectively, in the contralateral hemisphere, and 117.4 ^ 15.2 and 153.3 ^ 12.2 ms, respectively, in the ipsilateral hemisphere Therefore, magnetic fields appeared approximately 20 ms earlier following stimulation of the elbow than stimulation of the hand The results of source analysis were almost the same as those for 302 K Inui et al / Clinical Neurophysiology 113 (2002) 298–304 ranged from 11.9 to 22.1 m/s, and its mean was 15.6 ^ 3.2 m/s In all 13 subjects, vertex potentials consisting of a small amplitude negativity (N1) and a subsequent larger amplitude positivity (P1) were identified in EEG recordings (Fig 1) The peak latency of N1 was 199.9 ^ 9.9 ms, which was 43 ms later than that of the contralateral 1M The peak latency of P1 was 313.8 ^ 13.3 ms The peak latency of N1 and P1 following stimulation of the elbow was 180.7 ^ 10.6 and 291.8 ^ 16.2 ms, respectively In all subjects, evoked potentials in C3 and C4 were almost identical (Fig 3) and no definite activity exclusive to the contralateral hemisphere was noticed Discussion Fig Multiple source analysis in the same subject in Fig (A,B) temporal profile of source strength (upper trace) and GOF (lower trace); (Aa) results of analysis with the secondary somatosensory cortex (SII) source alone in the ipsilateral hemisphere; (Ba and Bb) results of analysis in the contralateral hemisphere with the SII source alone (Ba) and with an additional primary somatosensory cortex (SI) source (Bb); (Ab, Bc) location of sources Note that the SI source is necessary to explain the field pattern of the contralateral hemisphere in this subject stimulation of the hand In all subjects in whom SI areas in addition to bilateral SII areas were activated following hand stimulation, activation of these cortical areas was identified again following elbow stimulation The location of the ECD of SI activity following elbow stimulation was 13 mm medial to that after hand stimulation, but the difference was not significant In the other subjects, the ECD was estimated in the bilateral SII (n ¼ 6) or in the contralateral SII (n ¼ 1) The peak latency of the SI and SII activity is shown in Table Since the contralateral SII response was constant and the earliest response to epidermal stimulation, we calculated the peripheral conduction velocity (CV) responsible for epidermal stimulation-induced magnetic fields by dividing the distance between the two stimulation sites by the time difference of the peak latency of the contralateral SII response in 12 subjects The estimated CV in each subject In a preceding SEP study (Inui et al., 2002), we concluded that our intra-epidermal electrical stimulation could preferentially stimulate Ad fibers with minimal effects on Ab fibers based on the following findings: (1) stimulation induced a well-defined pricking pain sensation without any tactile sensations; (2) the peripheral CV was 15.1 m/s, which was in the range of CV of Ad fibers; (3) the CV correlated with that obtained by CO2 laser stimulation, which is known to selectively stimulate Ad fibers; and (4) epidermal stimulation-induced SEPs did not contain earlier components corresponding to short- and middle latency components elicited by conventional transcutaneous electrical stimulation at a non-painful intensity, N20, P30 and so on In the present study, we evaluated the activated cortical areas following the intra-epidermal painful stimulation using MEG The main ECDs of the early component of magnetic fields (1M) following epidermal stimulation were located in the bilateral SII area Simultaneous activities of the contralateral SI were noticed in out of 13 subjects in addition to the SII activities in good agreement with previous pain SEF studies The peripheral CV responsible for mediating the magnetic fields following epidermal stimulation was 15.6 m/s, which corresponded to that of the Ad fibers being responsible for the sharp pain sensation Therefore, we considered that Ad afferents-related SEFs were successfully recorded by our new stimulus method The main source generators for the 1M component were located in the bilateral upper bank or bottom of the Sylvian fissure thus corresponding to SII-insula in accordance with Table The mean peak latencies of cortical responses to epidermal stimulation SI contralateral SII contralateral SII ipsilateral Hand 161.8 ^ 15.6 ðn ¼ 5ị 158.5 ^ 11.6 n ẳ 13ị 170.6 ^ 13.0 (ms) n ẳ 12ị Elbow 137.2 ^ 17.9 n ẳ 5ị 139.0 ^ 13.2 n ẳ 12ị 156.8 ^ 16.1 n ẳ 11ị K Inui et al / Clinical Neurophysiology 113 (2002) 298–304 previous pain SEF studies using laser stimulation (Kakigi et al., 1995; Watanabe et al., 1998; Ploner et al., 1999; Kanda et al., 2000), stimulation of the nasal mucosa with CO2 gas (Huttunen et al., 1986; Hari et al., 1997), and painful electrical stimulation of the tooth pulp (Hari et al., 1983) The peak latency of SII activity (158 ms) following stimulation of the hand in the present study was shorter than that following laser stimulation; Kakigi et al (1995) (180–210 ms), Ploner et al (1999) (mean: 163 ms), and Kanda et al (2000) (mean: 212 ms) This time difference can be explained by differences in the activation time of nociceptive afferent terminals Skin receptors are activated via temperature conduction by laser beams, whereas electrical stimulation directly stimulates thin fibers Therefore, laser beams take longer to activate nociceptive afferents than does electrical stimulation For CO2 laser stimulation, a microneurographic study by Bromm and Treede (1984) showed that the activation latency of the Ad-nociceptor was 41 ms In this regard, we confirmed in a previous study (Inui et al., 2002) that the SEPs induced by epidermal stimulation appeared 40 ms earlier than those induced by CO2 laser stimulation Therefore, the time difference between the two CO2 laser stimulation studies (Kakigi et al., 1995; Kanda et al., 2000) and ours seems to be reasonable On the other hand, the contralateral SII peak latency reported by Ploner et al (1999) is longer by only ms than that in this study This is probably due to the fact that they used Tm:YAG laser beams of a very short duration (1 ms) and high energy The SII in the ipsilateral hemisphere was always activated later than the contralateral SII, by 12 and 18 ms for hand and elbow stimulation, respectively, confirming the results of previous laser stimulation studies (Yamasaki et al., 1999; Ploner et al., 1999, 2000) This delay between ipsilateral and contralateral activations could be due to a delay of transmission in the callosal fibers or in the ipsilateral pathway from the thalamic nuclei (Forss et al., 1999) The present results also showed the activities in the contralateral SI area confirming recent reports by Ploner et al (1999, 2000) and Kanda et al (2000) In the present study, however, the SI source could be identified in only out of 13 subjects Ploner et al (1999) identified SI activity in 11 out of 12 contralateral hemispheres, and Kanda et al (2000) identified the activity in out of 12 subjects The reason why the SI source could not be estimated in out of 13 subjects in the present study can be explained, at least in part, by possibilities: (1) SI neurons that responded to epidermal stimulation were too small in number to evoke detectable magnetic fields; (2) SI neurons in Brodmann’s area that responded to epidermal stimulation (Kenshalo and Isensee, 1983) mainly generated radially oriented magnetic fields that could not be easily detected by MEG; and therefore (3) SI source was estimated only in the subjects whose SI activities had tangential components of detectable strength The peak latency did not differ between the SI and SII sources in the present study, indicating a 303 simultaneous activation of these two areas, consistent with the reports by Ploner et al (1999) and Kanda et al (2000) In EEG recordings, we confirmed our previous finding that epidermal stimulation applied to the hand evoked a vertex negative component at around 200 ms and a positive component at around 300 ms, that appeared 40 ms earlier than those evoked by laser stimulation The negative component of the vertex potentials always peaked later than the peak of the 1M component The time delay of the N peak was 43.7 ms for hand stimulation and 42.3 ms for elbow stimulation A similar value was reported by Kakigi et al (1995) who showed a 40–60 ms delay in a CO2 laser simulation study Therefore, the temporal relation between evoked fields and potentials was similar between responses evoked by CO2 laser stimulation and by epidermal stimulation Therefore, as previous reports discussed (Kakigi et al., 1995; Watanabe et al., 1998; Yamasaki et al., 1999), the MEG responses seem to reflect the primary activities following painful stimulation while EEG responses reflect not only primary activities but also mainly cognitive functions The result that MEG responses were not affected, but EEG responses were much affected by distraction or attentional change (Yamasaki et al., 1999), supports this view For the reason why such a difference occurs between MEG and EEG responses, we speculated that brain activities corresponding to EEG responses, and therefore cognitive components, originate from deep areas, and their magnetic fields are difficult to detect The estimated peripheral CV responsible for mediating epidermal stimulation-induced magnetic fields in the present study was 15.6 m/s, confirming our previous report (15.1 m/s) in which the CV was calculated from the peak latency of P1 components of evoked potentials The value, 15 m/s, is in the mid-range of the CV of Ad fibers measured by microneurography: 4–30 m/s (Vallbo et al., 1979) and 19.2 ^ 9.4 m/s (Adriaensen et al., 1983) The CV was also similar to the values calculated from the response evoked by CO2 laser stimulation, which stimulates skin nociceptors specifically; 5–16 m/s (Kenton et al., 1980, Bromm and Treede, 1987; Kakigi et al., 1991; Tran et al., 2001) Therefore, we considered that the peripheral signals responsible for magnetic fields evoked by epidermal stimulation were conveyed through Ad fibers In a pain SEF study, the stimulus should be steep and short in order to induce a constant activation time for the stimuluslocked averaging (Bromm and Lorenz, 1998) as well as a nociceptive selective activation In this regard, we consider that our intra-epidermal electrical stimulation method offers a major advantage In laser stimulation, one must move irradiation points for each stimulus to avoid heat burns and habituation, resulting in activated receptors that are different for each stimulus Since laser stimulation has a relatively 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