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Short-term plastic changes of the human nociceptive system following acute pain induced by capsaicin
Clinical Neurophysiology 114 (2003) 1879–1890 www.elsevier.com/locate/clinph
Short-term plastic changes of the human nociceptive system following acute pain induced by capsaicin Massimiliano Valeriania,b,*, Lars Arendt-Nielsenc, Domenica Le Peraa,d, Domenico Restucciaa, Tiziana Rossoe, Liala De Armasa,d, Toni Maiesea, Antonio Fiaschie, Pietro Tonalia, Michele Tinazzie a
Istituto di Neurologia, Universita` Cattolica del Sacro Cuore, L.go A. Gemelli 8, 00168 Rome, Italy b Divisione di Neurologia, Ospedale Pediatrico Bambino Gesu`, IRCCS, Rome, Italy c Center for Sensory-Motor Interaction, Laboratory for Experimental Pain Research, Aalborg University, Aalborg, Denmark d Casa di Cura San Raffaele Pisana, Tosinvest Sanita`, Rome, Italy e Dipartimento di Scienze Neurologiche e della Visione, Sezione di Neurologia Riabilitativa, Universita` di Verona, Verona, Italy Accepted 8 May 2003
Abstract Objective: To investigate possible neuroplastic changes induced by pain in cerebral areas devoted to nociceptive input processing. Methods: CO2 laser-evoked potentials (LEPs) were recorded from 10 healthy subjects after stimulation of the right and left hand dorsum. Acute pain was obtained by topical application of capsaicin on the skin of right hand dorsum. LEPs were recorded after right and left hand stimulation before capsaicin, at the peak pain and 10 – 20 min after capsaicin removal. Right hand LEPs were evoked by laser stimuli delivered over the zone of secondary hyperalgesia during capsaicin and on both the zones of primary and secondary hyperalgesia after capsaicin removal. Results: After right hand stimulation, the vertex LEPs, which are generated in the cingulate cortex, were significantly decreased in amplitude during capsaicin application and after capsaicin removal. Moreover, the topography of these potentials was modified after capsaicin removal, shifting from the central toward the parietal region. Dipolar modelling showed that the dipolar source in the anterior cingulate cortex moved backward after capsaicin removal. All these changes were not observed after stimulation of the left hand, contralateral to the application of capsaicin, thus suggesting that functional changes are selective for the painful skin and the adjacent territories. Conclusions: Our results suggest that acute cutaneous pain may inhibit the neural activity in regions of central nervous system processing nociceptive inputs and cortical representation of these inputs can be rapidly modified in presence of acute pain. q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Pain; Plasticity; Central nervous system; Dipolar modelling; Hyperalgesia; Human brain
1. Introduction Research in animals and humans shows that acute or chronic deprivation of somatic input originating from a given part of the body causes major neuroplastic changes in dedicated subcortical or cortical structures which typically influence somatic territories adjacent to the deprived regions and this effect takes place in the developing brain, as well as in the brain of adults (Merzenich et al., 1983a,b; Pons et al., * Corresponding author. Tel.: þ 390630154435; fax: þ390635501909. E-mail address: [email protected] (M. Valeriani).
1991; Elbert et al., 1994; Yang et al., 1994; Rossini et al., 1994; Florence and Kaas, 1995; Valeriani et al., 1996b; Faggin et al., 1997; Tinazzi et al., 1997, 1998; Jones, 2000). The possible role of pain in promoting neuroplastic changes within the somatosensory system has been largely demonstrated in animals (Perl et al., 1976; Price et al., 1978; Kenshalo et al., 1979, 1982; Lamour et al., 1983; McMahon and Wall, 1984; Guilbaud et al., 1986; Woolf and King, 1990; Simone et al., 1991; Dougherty and Willis, 1992; Coderre et al., 1993). In recent times, it has been suggested that pain may promote cortical reorganization in human patients also (Flor et al., 1995, 1997; Birbaumer et al., 1997;
1388-2457/03/$30.00 q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S1388-2457(03)00180-9
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Tinazzi et al., 2000) and in healthy subjects submitted to experimental pain (Soros et al., 2001). All these observations show that the modulation induced by pain within the lemniscal pathway involves the representation of both the painful skin territory and the contiguous cutaneous areas. However, only a few neuroimaging and neurophysiological studies investigated possible neuroplastic changes caused by pain within the nociceptive pathway (Iadarola et al., 1998; Dostrovsky, 1999; Lenz et al., 2000; Olausson et al., 2001). The main issue dealt with by this paper is whether acute cutaneous pain may lead to functional changes of the nociceptive system after stimulation of: (i) the painful cutaneous territory and (ii) the cutaneous areas represented adjacently. We tested healthy subjects in whom acute cutaneous pain was induced experimentally by topical application of capsaicin. Capsaicin is the pungent component of hot peppers and, when applied on the skin, it causes pain mostly by stimulation of C nociceptors (Baumann et al., 1991). The cutaneous area where capsaicin is applied and the one onto which capsaicin diffuses are characterised by the presence of reduced painful threshold (PTh) (hyperalgesia) for both mechanical and heat stimuli (zone of primary hyperalgesia); this zone is surrounded by a zone of secondary hyperalgesia, where hyperalgesia can be demonstrated for mechanical but not for heat stimuli and stimuli which do not normally provoke pain are felt as painful (allodynia) (Treede et al., 1992). The physiologic properties of the nociceptive system before, during and after capsaicin application were investigated by recording the CO2 laser-evoked potentials (LEPs). The study of the scalp LEPs offers a unique opportunity to explore non-invasively the brain areas responding to nociceptive inputs. Indeed, microneurographic studies demonstrated that CO2 laser pulses delivered on the hairy skin activate specifically the thin nociceptive Ad and C-fibers, without any concurrent stimulation of the non-nociceptive Ab afferents (Bromm and Treede, 1984). In particular, LEPs obtained after painful stimulation of the hand skin show a latency range of 150– 450 ms and are generated by Ad-fiber inputs (Bromm and Lorenz, 1998).
2. Materials and methods 2.1. Subjects Ten healthy right-handed subjects (two male, 8 female, mean age 28.9 ^ 5.2), who gave their informed consent, took part in our study. 2.2. CO2 laser stimulation and LEP recording During LEP recording, the subjects lay on a couch in a warm and semi-dark room. Cutaneous heat stimuli were
delivered by a CO2 laser (10.6 mm wave length, 2 mm beam diameter, 10 ms pulse duration—ELEN, Florence, Italy) on the dorsum of the right and left hands. The stimulation site was visualized by a He – Ne laser beam. The location of the impact on the skin was slightly shifted between two successive stimuli, to avoid the sensitization of the nociceptors. CO2 laser stimuli were fixed at the PTh, defined as the lowest intensity that caused subject’s perception of the stimulus as a painful pin-prick. The mean stimulus intensity was 11.1 ^ 2.1 and 10.8 ^ 1.9 W for right and left hand stimulations, respectively. Before the recording session, the individual PTh was tested on both the right and the left hands by using a 10-point visual analogue scale (VAS) in which ‘0’ corresponds to no sensation, ‘4’ to the PTh and ‘10’ to intolerable pain. The interstimulus interval varied randomly between 9 and 11 s. Averaged LEPs resulted from 25– 30 CO2 laser stimuli. LEPs were obtained using 20 recording electrodes, 19 of which placed according to the positions of the 10– 20 international system (excluding Fpz and Oz) and the remaining one above the right eyebrow for electrooculogram (EOG) recording. The reference was at the nose and the ground at Fpz. The electroencephalographic signal (EEG) was amplified and filtered (bandpass 0.3 –70 Hz). The analysis time was 1000 ms with a bin width of 2 ms (500 Hz sampling rate). An automatic artefact rejection algorithm excluded from the average, all runs containing transient exceeding ^ 65 mV at any recording channel including the EOG. 2.3. LEP analysis In this study, the LEP components evoked by painful stimulation of the skin showed a latency consistent with their generation by Ad-fiber inputs (Bromm and Lorenz, 1998). LEP components were identified on the basis of their latency and polarity. They were labelled according to Valeriani et al. (1996a). LEP amplitudes were measured from the baseline. Baseline was measured automatically by our engine (Micromed Brain Quick, Mogliano Veneto, Italy) by calculating the average signal on the whole sweep and subtracting it from the trace. Also, the peak-to-peak amplitude was taken into consideration for the vertex biphasic LEP component (N2a – P2). For the analysis of LEP distribution, colour maps calculated by spline interpolation (Perrin et al., 1987) were used. Grand-averages of LEPs recorded in the different conditions after both right and left hand stimulations were obtained for demonstrative purposes. 2.4. Dipolar source modelling Dipolar source modelling was performed by using the brain electrical source analysis (BESA, Scherg, 1990). BESA calculates potential distributions over the scalp from preset voltage dipoles within the brain and then it evaluates
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the agreement between the recorded and calculated field distributions. The percentage of data that cannot be explained by the model is expressed as residual variance (RV). BESA uses a spherical 3-shell model with an 85 mm radius and assumes that the brain surface is at 70 mm from the centre of the sphere. The spatial position of each dipole is described on the basis of 3 axes: (1) the line drawn through T3 and T4 (x axis); (2) the line drawn through Fpz and Oz (y axis) and (3) the line drawn through Cz (z axis). The 3 axes have their intersection point at the centre of the sphere. The spatial orientation of the dipoles is described by two angles: (1) phi is the angle in the xy plane measured anticlockwise from the nearest x axis and (2) theta is the vertical angle that is measured from z axis and is positive for the right hemisphere. The model calculated by BESA is hypothetic and does not exclude other solutions; nevertheless, it can be validated when consistent with the anatomical and physiological knowledge of the identified source areas (Valeriani et al., 2001). 2.5. Experimental pain Pain was induced by 1 ml of 3% capsaicin in a cream base (Teofarma) which was applied topically by means of a cotton swab on the skin of the right hand dorsum (radial territory), within an area of 4 cm2. The area of application was standardized by using an empty form, which was filled by capsaicin. In all subjects, the area where capsaicin was applied was surrounded by an outer halo of reactive hyperaemia (flare). The site of capsaicin application was considered as the zone of primary hyperalgesia. This definition was conventional, since the ‘real’ zone of primary hyperalgesia was larger than the one we considered, as capsaicin is absorbed by the skin. The skin sites out of the zone of primary hyperalgesia in which a clear hyperalgesia for mechanical stimuli was found, but the PTh for CO2 laser pulses (heat stimuli) was not different from the one before capsaicin application were considered as the zone of secondary hyperalgesia (Treede et al., 1992). The mean (^ standard deviation) radius of the zone of secondary hyperalgesia was 1.4 cm (^ 0.3 cm) from the edge of the capsaicin application area. Capsaicin was removed after 1 h by means of a wet cotton swab. 2.6. Experimental procedure 2.6.1. LEP recording LEPs were recorded after both right and left hand stimulations at 3 times: (1) before capsaicin application (pre-capsaicin recording), (2) 20 –30 min after capsaicin application (capsaicin recording) and (3) 10 –20 min after capsaicin removal (post-capsaicin recording). As for right hand stimulation, in pre-capsaicin recording CO2 laser pulses were delivered over the skin which was expected to correspond to the zones of primary and secondary hyperalgesia after capsaicin application. In capsaicin
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recording, the zone of secondary hyperalgesia was stimulated. In post-capsaicin recording, both the zones of secondary (post-capsaicin area II recording) and primary (post-capsaicin area I recording) hyperalgesia were stimulated. In each of the 3 phases of the experiment, the order of the different recordings was randomly changed across the subjects. 2.6.2. Psychophysics Pain rating was performed by using a 100-point VAS, in which ‘0’ corresponds to no pain and ‘100’ to the worst pain one may conceive. After each LEP recording, the subject was asked to rate the pain induced by CO2 laser pulses (laser pain). Spontaneous pain induced by capsaicin was tested at intervals of 10 min until the end of the experiment. To test hyperalgesia for mechanical stimuli, a needle producing a pin-prick pain was used and the subject was asked to rate this pain. To test allodynia for mechanical stimuli, the subject was asked to rate the pain that a smooth pen-point stroking the skin may have produced. Both hyperalgesia and allodynia were tested at intervals of 10 min. Hyperalgesia and allodynia in the zone of primary hyperalgesia were tested only after capsaicin removal. 2.7. Statistical analysis For psychophysical tests, the pain ratings were compared by one-way ANOVA, by considering the rating time as the variable. If statistical significance was reached, post hoc analysis was performed by paired t test. The LEP latencies across the different times were compared by two-way ANOVA, by considering the recording time and the recording electrode as the variables. For LEP amplitude comparison, LEP amplitudes obtained in capsaicin and post-capsaicin recordings were expressed as percentages of the amplitudes of the corresponding LEP components obtained from pre-capsaicin recording on the same side, which were assumed as 100%. After this normalization, amplitudes were compared by two-way ANOVA, by considering the recording time and the recording electrode as the variables. If statistical significance was reached, post hoc analysis was performed by paired t test. Dipolar locations, which are expressed as x, y and z coordinates were compared across the different times by one-way ANOVA, by considering the recording time as the variable. Paired t test was used for post hoc analysis, when the significance was reached.
3. Results 3.1. Psychophysics Spontaneous pain following capsaicin application was described by all subjects as a burning sensation (Fig. 1). It
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Fig. 1. Top, the graphic shows the mean VAS values of capsaicin-induced spontaneous pain during capsaicin application and after capsaicin removal. Bottom, the histograms show the mean VAS values of laser-evoked pain before, during and after capsaicin. No difference reached the statistic significance. Vertical lines show standard deviation.
reached its peak 20 –30 min after capsaicin (one-way ANOVA: F ¼ 4:37, P , 0:01; t test: P , 0:05), and persisted after capsaicin removal also (in 9 subjects up to 10 min, and in 6 subjects up to 40 min). In the secondary zone, hyperalgesia for pin-prick stimuli reached its maximum 20 –50 min after capsaicin application (one-way ANOVA: F ¼ 2:1, P ¼ 0:04; t test: P , 0:05). In the primary zone, hyperalgesia for pin-prick stimuli peaked 10 min after capsaicin removal (one-way ANOVA: F ¼ 3:87, P ¼ 0:03; t test: P , 0:01). All subjects showed allodynia in both the zones of primary and secondary hyperalgesia. Laser-evoked pain rating (Fig. 1) did not significantly change across the whole experiment, after the stimulation of both the right (one-way ANOVA: F ¼ 0:53, P ¼ 0:66) and left hands (one-way ANOVA: F ¼ 0:02, P ¼ 0:98). 3.2. CO2 laser-evoked potentials In all our subjects, it was possible to recognize the negative N1 response in the temporal region contralateral to the stimulation and at about the same latency, the positive P1 potential in the frontal region. Since, in labelling N1 and P1 a certain difficulty may be caused by noise, it has been suggested to calculate the N1/P1
amplitude off-line (Fig. 2) by referring the contralateral temporal electrode (T3 or T4) to the Fz lead (Kunde and Treede, 1993). The mean latencies of the N1/P1 wave were 143.1 ms (^ 17.2 ms) and 142.6 ms (^ 20.5 ms) after right and left hand stimulations, respectively; the mean N1/P1 amplitudes were 4 mV (^ 1.5 mV) and 4.5 mV (^ 1.3 mV) after right and left hand stimulations, respectively. The main LEP components are represented by the negative N2a potential and by the positive P2 response at Cz vertex (Fig. 2). The mean N2a latencies were 192.3 ms (^ 23.6 ms) and 203.6 ms (^ 19.5 ms) after right and left hand stimulations, respectively; the mean P2 latencies were 303.8 ms (^ 25.3 ms) and 318 ms (^ 44.8 ms) after right and left hand stimulations, respectively. The mean peak-to-peak amplitudes of the N2a –P2 wave were 16.7 mV (^ 9 mV) and 16.6 mV (^ 12.4 mV) after right and left hand stimulations, respectively. A negative LEP component in the Fz traces followed the N2a potential 10 –30 ms later; however, since it was only inconsistently identified in our subjects, it was not taken into consideration for the statistical analysis. Spline maps show a typical dipolar field distribution at the N1/P1 latency with the negative pole in the temporal region and the positive counterpart in the frontal cortex
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Fig. 2. Grand-average traces of pre-capsaicin, capsaicin and post-capsaicin recordings obtained after right and left hand stimulations. N1/P1, N2a and P2 LEP components are shown. Notice the strong reduction of right hand LEP amplitude during and after capsaicin. Left hand LEPs are slightly reduced in amplitude during capsaicin.
(Fig. 3). Later, both the N2a and P2 potentials are widely distributed and reach their maximal amplitude at Cz vertex. 3.3. Effect of capsaicin on LEPs 3.3.1. LEP latencies A significant effect of capsaicin application on both the N2a (F ¼ 21:77, P , 0:01) and P2 (F ¼ 21:77, P , 0:01) latencies was obtained after right hand stimulation. The post hoc analysis showed that the N2a and P2 latencies were significantly delayed in the post-capsaicin area I recording compared with other recording conditions ðP , 0:05Þ. Capsaicin did not change the latencies of the N2a (F ¼ 0:44, P ¼ 0:64) and P2 (F ¼ 0:2, P ¼ 0:82) potentials after stimulation of the left hand, contralateral to capsaicin application. Although there was a tendency for the N1/P1 latency to be later after capsaicin removal, the statistical significance was not reached after both right (F ¼ 2:47, P ¼ 0:08) and left (F ¼ 3:06, P ¼ 0:06) hand stimulations. 3.3.2. LEP amplitudes and topography A strong effect induced by capsaicin was observed on the N2a – P2 amplitude evoked by stimulation of the right hand, ipsilateral to capsaicin application (F ¼ 15:8, P , 0:01). Also, the interaction between recording time and recording electrode reached the statistical significance (F ¼ 1:97,
P ¼ 0:04), thus suggesting a capsaicin-related modification of the N2a –P2 topography. As shown in Fig. 4, the N2a – P2 amplitude was significantly lower (around 50%) in capsaicin recording than in pre-capsaicin recording at Fz, Cz, C3 and C4 electrodes ðP , 0:01Þ, later recovering after capsaicin removal, though still remaining significantly reduced compared with the baseline ðP , 0:05Þ. In Pz traces, there was no N2a – P2 amplitude reduction during capsaicin application (P ¼ 0:34; Fig. 4). We calculated the Cz/Pz ratio of the N2a –P2 amplitude, which decreased significantly (F ¼ 6, P , 0:01) after capsaicin removal (from 1.57 ^ 0.4 in the pre-capsaicin recording to 1.1 ^ 0.36 in the post-capsaicin area II recording and 0.81 ^ 0.42 in the post-capsaicin area I recording). This reduction confirmed the topographic change of the vertex LEP components after capsaicin removal. Spline maps of right hand LEPs show the topographic change of the vertex LEP component which moved from the central region (precapsaicin and capsaicin recordings) towards the parietal region in post-capsaicin area II and mostly, area I recordings (Fig. 3). After left hand stimulation, capsaicin caused no N2a – P2 amplitude changes (F ¼ 0:96, P ¼ 0:39; Fig. 4). No interaction between recording time and recording electrode was found (F ¼ 1, P ¼ 0:43), thus no topographic modification of left hand LEPs was observed (Fig. 3). There was
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Fig. 3. Spline maps calculated at N1/P1 and P2 latencies in pre-capsaicin, capsaicin and post-capsaicin recordings. Negative potentials are represented in red colour, while positive potentials are shown in blue colour. After right hand stimulation, the P2 scalp distribution is focused on the vertex before and during capsaicin application, while it moves toward the parietal region in post-capsaicin recordings from area II and, mostly, from area I. The topography of left hand LEPs is not changed by capsaicin.
no effect of capsaicin application on N1/P1 amplitude after the stimulation of both the right (ANOVA F ¼ 0:44, P ¼ 0:72) and left hands (F ¼ 0:33, P ¼ 0:72). 3.4. Effect of capsaicin on LEP sources A previously built LEP dipolar model (Valeriani et al., 1996a, 2000) was applied to the grand-average traces recorded before capsaicin application after right hand stimulation. This model includes two dipoles, bilaterally located in the opercular region (dipoles 1 and 2), one dipole on the midline (dipole 3) and two dipoles bilaterally located
in the mesial hemispheric surface (dipoles 4 and 5). Garcia-Larrea (1998) projected the source coordinates of this model onto the Talairach and Tournoux stereotactic brain atlas, by using a simplified system of projection of dipole sources onto brain anatomy (Merlet et al., 1999). After anatomical projection, the bilateral opercular source and the midline dipole were found to correspond to the second somatosensory (SII) area and anterior cingulate gyrus, respectively; it was suggested that at least one of the two mesial dipoles may correspond to the insular cortex. Therefore, according to this model, the N1/P1 potential reflects the activity of the SII area contralateral to
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Fig. 4. Upper histogram: modifications of mean N2a–P2 amplitude values in pre-capsaicin, capsaicin and post-capsaicin recordings at different scalp electrodes, after right hand stimulation. Note that the N2a–P2 amplitude measured at Pz electrode is slightly reduced during capsaicin application, while it increases after capsaicin. On the contrary, the N2a–P2 amplitude is strongly reduced during and after capsaicin at Fz, Cz, C3 and C4 recording sites. Lower histogram: modifications of mean N2a–P2 amplitude values in pre-capsaicin, capsaicin and post-capsaicin recordings at different scalp electrodes after left hand stimulation. The asterisks show the significant differences.
stimulation, while the vertex N2a –P2 LEP component is mainly generated in the anterior cingulate gyrus (Tarkka and Treede, 1993; Bromm and Chen, 1995; Valeriani et al., 1996a, 2000). Dipolar modelling cannot discriminate between very close sources showing the same orientation, thus it is not possible to conclude whether the midline dipole represents only the activity of the cingulate gyrus contralateral to the stimulation or it is the result of a bilateral cingulate source. When we applied this dipole model to precapsaicin LEPs, a satisfactory RV (3.4%) was obtained.
The main contribution was given by the anterior cingulate dipole (62.2% of the variance), while the bilateral SII dipole and the bilateral insular dipole contributed for 16.6 and 17.8% of the variance, respectively. Then, the model was applied to capsaicin and post-capsaicin recordings, letting the dipoles’ locations free to change (Table 1). While the bilateral SII and insular dipoles did not change their location during and after capsaicin application, the anterior cingulate dipole moved backward in post-capsaicin area II and, mostly, in post-capsaicin area I recordings (Fig. 5). In order to validate
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Table 1 Coordinates of dipoles Dipole 1
Grand-average Pre-capsaicin Capsaicin Post-capsaicin area II Post-capsaicin area I Individual subjects Pre-capsaicin Mean SD Capsaicin Mean SD Post-capsaicin area II Mean SD Post-capsaicin area I Mean SD
Dipole 2
Dipole 3
Dipole 4
Dipole 5
x
y
z
x
y
z
x
y
z
x
y
z
x
y
z
253 253 253 253
226 226 226 226
20.6 20.6 20.6 20.6
53 53 53 53
226 226 226 226
20.6 20.6 20.6 20.6
27.3 27.3 24.6 25.5
14 14 1 27
33.8 33.8 36.6 34.7
229 229 229 229
21 21 21 21
5.5 5.5 5.5 5.5
27.4 27.4 27.4 27.4
7 7 7 7
4.5 4.5 4.5 4.5
252 3.16
225.9 3.14
20.8 2.82
53 3.37
225.4 2.22
21 3.32
25.44 2.3
14.7 1.83
33 2.6
227.4 3.89
21.6 3.24
4.25 3.06
27.7 7.52
8.1 6.12
6.44 3.61
251.7 3.77
225.7 3.47
20.5 2.88
52.8 3.19
225.4 2.76
21.4 2.96
25.4 2.76
14.7 2.41
33.1 2.52
226.8 3.62
0.9 4.51
5.25 3.88
27.6 7.5
7 5.23
6 4
251.7 4.03
226.1 4.33
20.7 2.83
51.5 2.51
225.5 2.22
21.1 4.23
24.67 1.66
20.1 3.07
33 3.08
227.2 4.66
22.9 4.33
5 5.24
29.4 5.6
6.8 4.26
6 4
251.4 4.43
226.5 4.55
21.1 2.18
53 3.37
225.4 2.22
22.1 2.71
25.33 2.83
28.6 1.84
33 3.04
226.8 4.26
21.1 4.15
3.63 3.54
29 6.38
6.9 5.11
7.1 4.73
Talairach and Tournoux coordinates.
this result, we applied the model issued from the grandaverage traces to individual recordings (Table 1). It was found that the locations of the SII and insular dipoles were not changed during and after capsaicin ðP . 0:05Þ, while the cingulate dipole had a more posterior location in postcapsaicin area II and, mostly, area I recordings compared to pre-capsaicin and capsaicin recordings (y coordinate, ANOVA: F ¼ 242:44, P , 0:01; post hoc test: P , 0:01).
4. Discussion The main result of the present study is that acute cutaneous pain induced by capsaicin may lead to rapid changes in cortical representation of nociceptive inputs coming from the painful territory and from adjacent areas, as suggested by the modification of LEP topography after capsaicin removal. Moreover, the amplitudes of vertex
Fig. 5. Projection of the dipoles’ coordinates onto the Talairach and Tournoux stereotactic brain atlas. Dipoles in the SII area (left) and insular dipoles (right) are represented in red colour; they did not change their cerebral location during and after capsaicin. On the contrary, the anterior cingulate dipole moved progressively backward, from pre-capsaicin and capsaicin recordings (blue) to post-capsaicin recordings obtained after stimulation of the secondary (green) and primary (yellow) zones.
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LEPs obtained by stimulation of both the zones of primary and secondary hyperalgesia were decreased during capsaicin application and after capsaicin removal. All LEP changes were directly related to capsaicin application on the right hand, since they occurred after right hand stimulation, but not stimulating the left hand. 4.1. Short-term cortical inhibition of nociceptive inputs induced by acute cutaneous pain In animals, neuroplastic changes induced by pain have been demonstrated at multiple levels of the central nervous system, such as the dorsal horn (Perl et al., 1976; Price et al., 1978; Kenshalo et al., 1979; McMahon and Wall, 1984; Simone et al., 1991; Woolf and King, 1990; Dougherty and Willis, 1992; Coderre et al., 1993), the thalamus (Guilbaud et al., 1986) and the cerebral cortex (Lamour et al., 1983). Some aspects of these plastic changes have also been demonstrated in humans, showing that pain may lead to a modulation of the cortical representation and neural reactivity to somatic inputs in patients with chronic pain and in healthy subjects submitted to experimental pain (Flor et al., 1995, 1997; Birbaumer et al., 1997; Tinazzi et al., 2000; Soros et al., 2001; Juottonen et al., 2002). An important result of the present study is that acute cutaneous pain induced by topical application of capsaicin determines a transient inhibition of the neural responsiveness to nociceptive inputs delivered on both the painful area (primary hyperalgesia) and the adjacent cutaneous territory (secondary hyperalgesia). In the zone of secondary hyperalgesia, we observed that vertex LEPs were strongly reduced in amplitude, mainly during capsaicin application, but also after its removal. One main element supports the hypothesis that the vertex LEP amplitude decrease is due to an inhibition occurring in the central nervous system, rather than at peripheral level. Indeed, while a peripheral mechanism would be expected to cause a reduction of both the middle-latency (N1/P1) and the vertex LEP amplitudes, the observed amplitude reduction concerned the vertex N2a –P2 potentials, but not the N1/P1 response. The fact that low-amplitude LEP components evoked by stimulation of the secondary zone were not related to any significant change of pain sensation evoked by CO2 laser pulses may appear puzzling. This lack of relationship suggests that in the presence of pain near the stimulated area, the correlation between the amplitude of the vertex LEP components and the subjective intensity of CO2 laser stimuli, demonstrated in the absence of pain (Bjerring and Arendt-Nielsen, 1988; Weiss et al., 1997), may be lost. In patients with central pain, Casey et al. (1996) demonstrated that the vertex LEP amplitude is reduced after stimulation of the painful skin, although the rating of laser pulse intensity is not decreased in the affected side compared to the nonaffected one. This result was attributed to a deficit in spinothalamic tract function in these patients. From this point of view, the clear LEP amplitude reduction in spite of
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an unmodified laser pain rating in our subjects can be due to spinothalamic tract inhibition. This hypothesis is consistent with the observation that in monkeys, the response of spinal sensory tract neurones in the dorsal horn to thermal stimuli is transiently inhibited after intradermal capsaicin injection (Dougherty et al., 1998). Hyperalgesia to heat in the secondary zone has been demonstrated by some investigators (Hardy et al., 1950; Arendt-Nielsen et al., 1996; Serra et al., 1998). However, the disagreement between our results and previous studies can be explained by the different quantity of capsaicin used and by the different technique of delivering the heat stimulation. Indeed, we used a much lower amount of capsaicin than the one in experiments showing heat sensitization in the zone of secondary hyperalgesia. Moreover, while these authors used intradermal injections of capsaicin, we applied it on the skin, thus further reducing the nociceptor stimulation. Lastly, CO2 laser pulses stimulate a smaller cutaneous area than the Peltier thermode stimuli used in previous studies. It is interesting that Ali et al. (1996) who tested the heat sensation in the secondary zone by laser stimuli, did not find hyperalgesia. In the zone of primary hyperalgesia, the vertex LEP amplitude was significantly lower in post-capsaicin than pre-capsaicin recordings. The reduction of the vertex LEP amplitude might be due to an inhibition of the Ad-fiber inputs generating LEPs by the CMH nociceptors, which are sensitized by capsaicin (Treede et al., 1992), at a certain level of the central nervous system. However, a summation of nociceptive activity from Ad and C-fibers occurs at the spinal level, as demonstrated by the increase of the nociceptive withdrawal reflex evoked by Ad-fiber electric stimulation during on-going burning pain, which activates the C nociceptors (Andersen et al., 1994). Therefore, the inhibition of the Ad-fiber inputs that generate LEPs by the sensitized C-fiber inputs should occur at supra-spinal level. This hypothesis explains also the significant delay in latency of the vertex LEP components when CO2 laser stimuli were delivered in the primary zone. As it has already been discussed for the stimulation of the secondary zone, in postcapsaicin area I recording also, the amplitude decrease of the vertex LEP was not paralleled by a reduction in rating of laser pulse intensity. The mechanism underlying this lack of relationship is probably related to a deficit in central pain pathway function (see above). Our findings suggest that acute cutaneous pain induces a short-term inhibition of the cingulate cortex, which generates the N2a – P2 potential. This is of particular interest if compared with recent results obtained by neuroimaging studies. While painful stimuli delivered on the normal side of the body entail an activation of the anterior cingulate gyrus, in patients with Wallenberg syndrome, stimuli delivered within hyperalgesic skin lead to a decrease of cerebral blood flow (CBF) in the anterior cingulate cortex (Peyron et al., 1998). A lack of cingulate cortex activation has been shown by functional magnetic
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resonance imaging (fMRI) also during experimental capsaicin-induced pain (Baron et al., 1999). The cingulate cortex is part of the limbic system and it is important for the emotional component of pain sensation (Derbyshire et al., 1997; Casey, 1999; Peyron et al., 1999; Buchel et al., 2002). Therefore, the role of the cingulate cortex inhibition due to acute pain in our subjects could be addressed to reduce the emotional and affective aspects of noxious stimuli delivered within or near a painful skin area. From this point of view, it is remarkable that the middle-latency N1/P1 wave, which might represent the neurophysiological correlate of the sensory-discriminative aspect of pain (Garcia-Larrea et al., 1997), was not significantly affected by capsaicin. The decreased LEP amplitude during capsaicin application and after its removal might be also due to desynchronization of the neural activity in the regions of central nervous system processing nociceptive inputs. This desynchronization possibly results from increased afferent inputs in presence of acute pain. In a recent article, LEP amplitude after stimulation of both right and left perioral regions was reduced by capsaicin application on the skin of the left cheek (Romaniello et al., 2002). The bilateral LEP amplitude decrease was explained by the so-called diffuse noxious inhibitory control (DNIC; Le Bars et al., 1979a,b; Chen et al., 1985; Arendt-Nielsen and Gotliebsen, 1992; Plaghi et al., 1994; Kakigi and Watanabe, 1996; Svensson et al., 1999; Reinert et al., 2000). DNIC is an electrophysiological phenomenon demonstrated in animals, in which the activity of convergent dorsal horn neurones is inhibited by noxious stimulation applied to various sites of the body (Le Bars et al., 1979a,b). Our findings of a decreased LEP amplitude to right hand stimulation after capsaicin application are unlikely related to a ‘pain inhibits pain’ effect, since LEPs obtained after left hand stimulation were not significantly decreased in amplitude compared to baseline. Moreover, we never observed a significant reduction of pain sensation due to CO2 laser pulses across the whole experiment. In our subjects, the lack of a major DNIC effect on left hand LEPs may be explained by the lower capsaicin concentration (3%) compared to the study by Romaniello et al. (2002) (10%). The difference between our and Romaniello et al.’s study in demonstrating a bilateral effect of acute pain on LEPs might also be due to the greater bilateral representation of face compared with hand (Manger et al., 1996). One may wonder why in our subjects hyperalgesia for pin-prick but not for laser stimuli was found, since both stimuli are expected to activate Ad-fibers. However, according to a model for cutaneous hyperalgesia, capsaicin probably entails facilitation of A-fiber high threshold mechanoreceptors and A-fiber nociceptors with high heat thresholds (AMHs type I). On the contrary, A-fibers that normally signal first pain to heat and carry inputs generating scalp LEPs (AMHs type II) are not facilitated (Ziegler et al., 1999).
4.2. Short-term topographic changes of the nociceptive cerebral cortex induced by acute cutaneous pain Another important result of the present study is that our subjects showed a change in the scalp distribution of the vertex LEP components in post-capsaicin recordings compared to baseline. While before and during capsaicin application the N2a – P2 potential was largely diffused over the scalp and reached its maximal amplitude at Cz vertex, after capsaicin removal, the N2a –P2 topography indeed moved towards the parietal region. This topography modification was recognizable in post-capsaicin traces after the stimulation of the zones of secondary and, mostly, primary hyperalgesia. Dipolar source modelling of LEP scalp distribution showed that the LEP topographic change is due to a backward shift of the dipole in the anterior cingulate cortex. Different subcortical areas within the anterior cingulate cortex are probably related to different aspects of pain sensation. In particular, the activity of the frontal part of the anterior cingulate gyrus reflects the PTh coding, given that subjects with high PTh show a high-level activation of the frontal part of the anterior cingulate gyrus. Instead, the posterior region of the anterior cingulate cortex encodes pain unpleasantness (To¨lle et al., 1999). In our subjects, the backward movement of the anterior cingulate source may be due to a modification of the relative contribution by different regions within the anterior cingulate cortex to the EEG signal evoked by CO2 laser pulses. This modification can result from two different situations: (i) the posterior part of the anterior cingulate cortex, which encodes pain unpleasantness (To¨lle et al., 1999), may be activated or may become hyperactive or (ii) the activity of the anterior part of the anterior cingulated cortex may be reduced. In other words, since a dipolar source represents a ‘centre-of-mass’ of electrical activities coming from one cerebral structure or from several brain areas, we cannot decide whether the observed change in the anterior cingulate dipole location is caused by ex novo activation or by increase of activity of a source in the posterior part of anterior cingulate gyrus or by reduced activity of a generator in the anterior part of this cerebral structure.
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Short-term plastic changes of the human nociceptive system following acute pain induced by capsaicin Massimiliano Valeriania,b,*, Lars Arendt-Nielsenc, Domenica Le Peraa,d, Domenico Restucciaa, Tiziana Rossoe, Liala De Armasa,d, Toni Maiesea, Antonio Fiaschie, Pietro Tonalia, Michele Tinazzie a
Istituto di Neurologia, Universita` Cattolica del Sacro Cuore, L.go A. Gemelli 8, 00168 Rome, Italy b Divisione di Neurologia, Ospedale Pediatrico Bambino Gesu`, IRCCS, Rome, Italy c Center for Sensory-Motor Interaction, Laboratory for Experimental Pain Research, Aalborg University, Aalborg, Denmark d Casa di Cura San Raffaele Pisana, Tosinvest Sanita`, Rome, Italy e Dipartimento di Scienze Neurologiche e della Visione, Sezione di Neurologia Riabilitativa, Universita` di Verona, Verona, Italy Accepted 8 May 2003
Abstract Objective: To investigate possible neuroplastic changes induced by pain in cerebral areas devoted to nociceptive input processing. Methods: CO2 laser-evoked potentials (LEPs) were recorded from 10 healthy subjects after stimulation of the right and left hand dorsum. Acute pain was obtained by topical application of capsaicin on the skin of right hand dorsum. LEPs were recorded after right and left hand stimulation before capsaicin, at the peak pain and 10 – 20 min after capsaicin removal. Right hand LEPs were evoked by laser stimuli delivered over the zone of secondary hyperalgesia during capsaicin and on both the zones of primary and secondary hyperalgesia after capsaicin removal. Results: After right hand stimulation, the vertex LEPs, which are generated in the cingulate cortex, were significantly decreased in amplitude during capsaicin application and after capsaicin removal. Moreover, the topography of these potentials was modified after capsaicin removal, shifting from the central toward the parietal region. Dipolar modelling showed that the dipolar source in the anterior cingulate cortex moved backward after capsaicin removal. All these changes were not observed after stimulation of the left hand, contralateral to the application of capsaicin, thus suggesting that functional changes are selective for the painful skin and the adjacent territories. Conclusions: Our results suggest that acute cutaneous pain may inhibit the neural activity in regions of central nervous system processing nociceptive inputs and cortical representation of these inputs can be rapidly modified in presence of acute pain. q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Pain; Plasticity; Central nervous system; Dipolar modelling; Hyperalgesia; Human brain
1. Introduction Research in animals and humans shows that acute or chronic deprivation of somatic input originating from a given part of the body causes major neuroplastic changes in dedicated subcortical or cortical structures which typically influence somatic territories adjacent to the deprived regions and this effect takes place in the developing brain, as well as in the brain of adults (Merzenich et al., 1983a,b; Pons et al., * Corresponding author. Tel.: þ 390630154435; fax: þ390635501909. E-mail address: [email protected] (M. Valeriani).
1991; Elbert et al., 1994; Yang et al., 1994; Rossini et al., 1994; Florence and Kaas, 1995; Valeriani et al., 1996b; Faggin et al., 1997; Tinazzi et al., 1997, 1998; Jones, 2000). The possible role of pain in promoting neuroplastic changes within the somatosensory system has been largely demonstrated in animals (Perl et al., 1976; Price et al., 1978; Kenshalo et al., 1979, 1982; Lamour et al., 1983; McMahon and Wall, 1984; Guilbaud et al., 1986; Woolf and King, 1990; Simone et al., 1991; Dougherty and Willis, 1992; Coderre et al., 1993). In recent times, it has been suggested that pain may promote cortical reorganization in human patients also (Flor et al., 1995, 1997; Birbaumer et al., 1997;
1388-2457/03/$30.00 q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S1388-2457(03)00180-9
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Tinazzi et al., 2000) and in healthy subjects submitted to experimental pain (Soros et al., 2001). All these observations show that the modulation induced by pain within the lemniscal pathway involves the representation of both the painful skin territory and the contiguous cutaneous areas. However, only a few neuroimaging and neurophysiological studies investigated possible neuroplastic changes caused by pain within the nociceptive pathway (Iadarola et al., 1998; Dostrovsky, 1999; Lenz et al., 2000; Olausson et al., 2001). The main issue dealt with by this paper is whether acute cutaneous pain may lead to functional changes of the nociceptive system after stimulation of: (i) the painful cutaneous territory and (ii) the cutaneous areas represented adjacently. We tested healthy subjects in whom acute cutaneous pain was induced experimentally by topical application of capsaicin. Capsaicin is the pungent component of hot peppers and, when applied on the skin, it causes pain mostly by stimulation of C nociceptors (Baumann et al., 1991). The cutaneous area where capsaicin is applied and the one onto which capsaicin diffuses are characterised by the presence of reduced painful threshold (PTh) (hyperalgesia) for both mechanical and heat stimuli (zone of primary hyperalgesia); this zone is surrounded by a zone of secondary hyperalgesia, where hyperalgesia can be demonstrated for mechanical but not for heat stimuli and stimuli which do not normally provoke pain are felt as painful (allodynia) (Treede et al., 1992). The physiologic properties of the nociceptive system before, during and after capsaicin application were investigated by recording the CO2 laser-evoked potentials (LEPs). The study of the scalp LEPs offers a unique opportunity to explore non-invasively the brain areas responding to nociceptive inputs. Indeed, microneurographic studies demonstrated that CO2 laser pulses delivered on the hairy skin activate specifically the thin nociceptive Ad and C-fibers, without any concurrent stimulation of the non-nociceptive Ab afferents (Bromm and Treede, 1984). In particular, LEPs obtained after painful stimulation of the hand skin show a latency range of 150– 450 ms and are generated by Ad-fiber inputs (Bromm and Lorenz, 1998).
2. Materials and methods 2.1. Subjects Ten healthy right-handed subjects (two male, 8 female, mean age 28.9 ^ 5.2), who gave their informed consent, took part in our study. 2.2. CO2 laser stimulation and LEP recording During LEP recording, the subjects lay on a couch in a warm and semi-dark room. Cutaneous heat stimuli were
delivered by a CO2 laser (10.6 mm wave length, 2 mm beam diameter, 10 ms pulse duration—ELEN, Florence, Italy) on the dorsum of the right and left hands. The stimulation site was visualized by a He – Ne laser beam. The location of the impact on the skin was slightly shifted between two successive stimuli, to avoid the sensitization of the nociceptors. CO2 laser stimuli were fixed at the PTh, defined as the lowest intensity that caused subject’s perception of the stimulus as a painful pin-prick. The mean stimulus intensity was 11.1 ^ 2.1 and 10.8 ^ 1.9 W for right and left hand stimulations, respectively. Before the recording session, the individual PTh was tested on both the right and the left hands by using a 10-point visual analogue scale (VAS) in which ‘0’ corresponds to no sensation, ‘4’ to the PTh and ‘10’ to intolerable pain. The interstimulus interval varied randomly between 9 and 11 s. Averaged LEPs resulted from 25– 30 CO2 laser stimuli. LEPs were obtained using 20 recording electrodes, 19 of which placed according to the positions of the 10– 20 international system (excluding Fpz and Oz) and the remaining one above the right eyebrow for electrooculogram (EOG) recording. The reference was at the nose and the ground at Fpz. The electroencephalographic signal (EEG) was amplified and filtered (bandpass 0.3 –70 Hz). The analysis time was 1000 ms with a bin width of 2 ms (500 Hz sampling rate). An automatic artefact rejection algorithm excluded from the average, all runs containing transient exceeding ^ 65 mV at any recording channel including the EOG. 2.3. LEP analysis In this study, the LEP components evoked by painful stimulation of the skin showed a latency consistent with their generation by Ad-fiber inputs (Bromm and Lorenz, 1998). LEP components were identified on the basis of their latency and polarity. They were labelled according to Valeriani et al. (1996a). LEP amplitudes were measured from the baseline. Baseline was measured automatically by our engine (Micromed Brain Quick, Mogliano Veneto, Italy) by calculating the average signal on the whole sweep and subtracting it from the trace. Also, the peak-to-peak amplitude was taken into consideration for the vertex biphasic LEP component (N2a – P2). For the analysis of LEP distribution, colour maps calculated by spline interpolation (Perrin et al., 1987) were used. Grand-averages of LEPs recorded in the different conditions after both right and left hand stimulations were obtained for demonstrative purposes. 2.4. Dipolar source modelling Dipolar source modelling was performed by using the brain electrical source analysis (BESA, Scherg, 1990). BESA calculates potential distributions over the scalp from preset voltage dipoles within the brain and then it evaluates
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the agreement between the recorded and calculated field distributions. The percentage of data that cannot be explained by the model is expressed as residual variance (RV). BESA uses a spherical 3-shell model with an 85 mm radius and assumes that the brain surface is at 70 mm from the centre of the sphere. The spatial position of each dipole is described on the basis of 3 axes: (1) the line drawn through T3 and T4 (x axis); (2) the line drawn through Fpz and Oz (y axis) and (3) the line drawn through Cz (z axis). The 3 axes have their intersection point at the centre of the sphere. The spatial orientation of the dipoles is described by two angles: (1) phi is the angle in the xy plane measured anticlockwise from the nearest x axis and (2) theta is the vertical angle that is measured from z axis and is positive for the right hemisphere. The model calculated by BESA is hypothetic and does not exclude other solutions; nevertheless, it can be validated when consistent with the anatomical and physiological knowledge of the identified source areas (Valeriani et al., 2001). 2.5. Experimental pain Pain was induced by 1 ml of 3% capsaicin in a cream base (Teofarma) which was applied topically by means of a cotton swab on the skin of the right hand dorsum (radial territory), within an area of 4 cm2. The area of application was standardized by using an empty form, which was filled by capsaicin. In all subjects, the area where capsaicin was applied was surrounded by an outer halo of reactive hyperaemia (flare). The site of capsaicin application was considered as the zone of primary hyperalgesia. This definition was conventional, since the ‘real’ zone of primary hyperalgesia was larger than the one we considered, as capsaicin is absorbed by the skin. The skin sites out of the zone of primary hyperalgesia in which a clear hyperalgesia for mechanical stimuli was found, but the PTh for CO2 laser pulses (heat stimuli) was not different from the one before capsaicin application were considered as the zone of secondary hyperalgesia (Treede et al., 1992). The mean (^ standard deviation) radius of the zone of secondary hyperalgesia was 1.4 cm (^ 0.3 cm) from the edge of the capsaicin application area. Capsaicin was removed after 1 h by means of a wet cotton swab. 2.6. Experimental procedure 2.6.1. LEP recording LEPs were recorded after both right and left hand stimulations at 3 times: (1) before capsaicin application (pre-capsaicin recording), (2) 20 –30 min after capsaicin application (capsaicin recording) and (3) 10 –20 min after capsaicin removal (post-capsaicin recording). As for right hand stimulation, in pre-capsaicin recording CO2 laser pulses were delivered over the skin which was expected to correspond to the zones of primary and secondary hyperalgesia after capsaicin application. In capsaicin
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recording, the zone of secondary hyperalgesia was stimulated. In post-capsaicin recording, both the zones of secondary (post-capsaicin area II recording) and primary (post-capsaicin area I recording) hyperalgesia were stimulated. In each of the 3 phases of the experiment, the order of the different recordings was randomly changed across the subjects. 2.6.2. Psychophysics Pain rating was performed by using a 100-point VAS, in which ‘0’ corresponds to no pain and ‘100’ to the worst pain one may conceive. After each LEP recording, the subject was asked to rate the pain induced by CO2 laser pulses (laser pain). Spontaneous pain induced by capsaicin was tested at intervals of 10 min until the end of the experiment. To test hyperalgesia for mechanical stimuli, a needle producing a pin-prick pain was used and the subject was asked to rate this pain. To test allodynia for mechanical stimuli, the subject was asked to rate the pain that a smooth pen-point stroking the skin may have produced. Both hyperalgesia and allodynia were tested at intervals of 10 min. Hyperalgesia and allodynia in the zone of primary hyperalgesia were tested only after capsaicin removal. 2.7. Statistical analysis For psychophysical tests, the pain ratings were compared by one-way ANOVA, by considering the rating time as the variable. If statistical significance was reached, post hoc analysis was performed by paired t test. The LEP latencies across the different times were compared by two-way ANOVA, by considering the recording time and the recording electrode as the variables. For LEP amplitude comparison, LEP amplitudes obtained in capsaicin and post-capsaicin recordings were expressed as percentages of the amplitudes of the corresponding LEP components obtained from pre-capsaicin recording on the same side, which were assumed as 100%. After this normalization, amplitudes were compared by two-way ANOVA, by considering the recording time and the recording electrode as the variables. If statistical significance was reached, post hoc analysis was performed by paired t test. Dipolar locations, which are expressed as x, y and z coordinates were compared across the different times by one-way ANOVA, by considering the recording time as the variable. Paired t test was used for post hoc analysis, when the significance was reached.
3. Results 3.1. Psychophysics Spontaneous pain following capsaicin application was described by all subjects as a burning sensation (Fig. 1). It
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Fig. 1. Top, the graphic shows the mean VAS values of capsaicin-induced spontaneous pain during capsaicin application and after capsaicin removal. Bottom, the histograms show the mean VAS values of laser-evoked pain before, during and after capsaicin. No difference reached the statistic significance. Vertical lines show standard deviation.
reached its peak 20 –30 min after capsaicin (one-way ANOVA: F ¼ 4:37, P , 0:01; t test: P , 0:05), and persisted after capsaicin removal also (in 9 subjects up to 10 min, and in 6 subjects up to 40 min). In the secondary zone, hyperalgesia for pin-prick stimuli reached its maximum 20 –50 min after capsaicin application (one-way ANOVA: F ¼ 2:1, P ¼ 0:04; t test: P , 0:05). In the primary zone, hyperalgesia for pin-prick stimuli peaked 10 min after capsaicin removal (one-way ANOVA: F ¼ 3:87, P ¼ 0:03; t test: P , 0:01). All subjects showed allodynia in both the zones of primary and secondary hyperalgesia. Laser-evoked pain rating (Fig. 1) did not significantly change across the whole experiment, after the stimulation of both the right (one-way ANOVA: F ¼ 0:53, P ¼ 0:66) and left hands (one-way ANOVA: F ¼ 0:02, P ¼ 0:98). 3.2. CO2 laser-evoked potentials In all our subjects, it was possible to recognize the negative N1 response in the temporal region contralateral to the stimulation and at about the same latency, the positive P1 potential in the frontal region. Since, in labelling N1 and P1 a certain difficulty may be caused by noise, it has been suggested to calculate the N1/P1
amplitude off-line (Fig. 2) by referring the contralateral temporal electrode (T3 or T4) to the Fz lead (Kunde and Treede, 1993). The mean latencies of the N1/P1 wave were 143.1 ms (^ 17.2 ms) and 142.6 ms (^ 20.5 ms) after right and left hand stimulations, respectively; the mean N1/P1 amplitudes were 4 mV (^ 1.5 mV) and 4.5 mV (^ 1.3 mV) after right and left hand stimulations, respectively. The main LEP components are represented by the negative N2a potential and by the positive P2 response at Cz vertex (Fig. 2). The mean N2a latencies were 192.3 ms (^ 23.6 ms) and 203.6 ms (^ 19.5 ms) after right and left hand stimulations, respectively; the mean P2 latencies were 303.8 ms (^ 25.3 ms) and 318 ms (^ 44.8 ms) after right and left hand stimulations, respectively. The mean peak-to-peak amplitudes of the N2a –P2 wave were 16.7 mV (^ 9 mV) and 16.6 mV (^ 12.4 mV) after right and left hand stimulations, respectively. A negative LEP component in the Fz traces followed the N2a potential 10 –30 ms later; however, since it was only inconsistently identified in our subjects, it was not taken into consideration for the statistical analysis. Spline maps show a typical dipolar field distribution at the N1/P1 latency with the negative pole in the temporal region and the positive counterpart in the frontal cortex
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Fig. 2. Grand-average traces of pre-capsaicin, capsaicin and post-capsaicin recordings obtained after right and left hand stimulations. N1/P1, N2a and P2 LEP components are shown. Notice the strong reduction of right hand LEP amplitude during and after capsaicin. Left hand LEPs are slightly reduced in amplitude during capsaicin.
(Fig. 3). Later, both the N2a and P2 potentials are widely distributed and reach their maximal amplitude at Cz vertex. 3.3. Effect of capsaicin on LEPs 3.3.1. LEP latencies A significant effect of capsaicin application on both the N2a (F ¼ 21:77, P , 0:01) and P2 (F ¼ 21:77, P , 0:01) latencies was obtained after right hand stimulation. The post hoc analysis showed that the N2a and P2 latencies were significantly delayed in the post-capsaicin area I recording compared with other recording conditions ðP , 0:05Þ. Capsaicin did not change the latencies of the N2a (F ¼ 0:44, P ¼ 0:64) and P2 (F ¼ 0:2, P ¼ 0:82) potentials after stimulation of the left hand, contralateral to capsaicin application. Although there was a tendency for the N1/P1 latency to be later after capsaicin removal, the statistical significance was not reached after both right (F ¼ 2:47, P ¼ 0:08) and left (F ¼ 3:06, P ¼ 0:06) hand stimulations. 3.3.2. LEP amplitudes and topography A strong effect induced by capsaicin was observed on the N2a – P2 amplitude evoked by stimulation of the right hand, ipsilateral to capsaicin application (F ¼ 15:8, P , 0:01). Also, the interaction between recording time and recording electrode reached the statistical significance (F ¼ 1:97,
P ¼ 0:04), thus suggesting a capsaicin-related modification of the N2a –P2 topography. As shown in Fig. 4, the N2a – P2 amplitude was significantly lower (around 50%) in capsaicin recording than in pre-capsaicin recording at Fz, Cz, C3 and C4 electrodes ðP , 0:01Þ, later recovering after capsaicin removal, though still remaining significantly reduced compared with the baseline ðP , 0:05Þ. In Pz traces, there was no N2a – P2 amplitude reduction during capsaicin application (P ¼ 0:34; Fig. 4). We calculated the Cz/Pz ratio of the N2a –P2 amplitude, which decreased significantly (F ¼ 6, P , 0:01) after capsaicin removal (from 1.57 ^ 0.4 in the pre-capsaicin recording to 1.1 ^ 0.36 in the post-capsaicin area II recording and 0.81 ^ 0.42 in the post-capsaicin area I recording). This reduction confirmed the topographic change of the vertex LEP components after capsaicin removal. Spline maps of right hand LEPs show the topographic change of the vertex LEP component which moved from the central region (precapsaicin and capsaicin recordings) towards the parietal region in post-capsaicin area II and mostly, area I recordings (Fig. 3). After left hand stimulation, capsaicin caused no N2a – P2 amplitude changes (F ¼ 0:96, P ¼ 0:39; Fig. 4). No interaction between recording time and recording electrode was found (F ¼ 1, P ¼ 0:43), thus no topographic modification of left hand LEPs was observed (Fig. 3). There was
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Fig. 3. Spline maps calculated at N1/P1 and P2 latencies in pre-capsaicin, capsaicin and post-capsaicin recordings. Negative potentials are represented in red colour, while positive potentials are shown in blue colour. After right hand stimulation, the P2 scalp distribution is focused on the vertex before and during capsaicin application, while it moves toward the parietal region in post-capsaicin recordings from area II and, mostly, from area I. The topography of left hand LEPs is not changed by capsaicin.
no effect of capsaicin application on N1/P1 amplitude after the stimulation of both the right (ANOVA F ¼ 0:44, P ¼ 0:72) and left hands (F ¼ 0:33, P ¼ 0:72). 3.4. Effect of capsaicin on LEP sources A previously built LEP dipolar model (Valeriani et al., 1996a, 2000) was applied to the grand-average traces recorded before capsaicin application after right hand stimulation. This model includes two dipoles, bilaterally located in the opercular region (dipoles 1 and 2), one dipole on the midline (dipole 3) and two dipoles bilaterally located
in the mesial hemispheric surface (dipoles 4 and 5). Garcia-Larrea (1998) projected the source coordinates of this model onto the Talairach and Tournoux stereotactic brain atlas, by using a simplified system of projection of dipole sources onto brain anatomy (Merlet et al., 1999). After anatomical projection, the bilateral opercular source and the midline dipole were found to correspond to the second somatosensory (SII) area and anterior cingulate gyrus, respectively; it was suggested that at least one of the two mesial dipoles may correspond to the insular cortex. Therefore, according to this model, the N1/P1 potential reflects the activity of the SII area contralateral to
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Fig. 4. Upper histogram: modifications of mean N2a–P2 amplitude values in pre-capsaicin, capsaicin and post-capsaicin recordings at different scalp electrodes, after right hand stimulation. Note that the N2a–P2 amplitude measured at Pz electrode is slightly reduced during capsaicin application, while it increases after capsaicin. On the contrary, the N2a–P2 amplitude is strongly reduced during and after capsaicin at Fz, Cz, C3 and C4 recording sites. Lower histogram: modifications of mean N2a–P2 amplitude values in pre-capsaicin, capsaicin and post-capsaicin recordings at different scalp electrodes after left hand stimulation. The asterisks show the significant differences.
stimulation, while the vertex N2a –P2 LEP component is mainly generated in the anterior cingulate gyrus (Tarkka and Treede, 1993; Bromm and Chen, 1995; Valeriani et al., 1996a, 2000). Dipolar modelling cannot discriminate between very close sources showing the same orientation, thus it is not possible to conclude whether the midline dipole represents only the activity of the cingulate gyrus contralateral to the stimulation or it is the result of a bilateral cingulate source. When we applied this dipole model to precapsaicin LEPs, a satisfactory RV (3.4%) was obtained.
The main contribution was given by the anterior cingulate dipole (62.2% of the variance), while the bilateral SII dipole and the bilateral insular dipole contributed for 16.6 and 17.8% of the variance, respectively. Then, the model was applied to capsaicin and post-capsaicin recordings, letting the dipoles’ locations free to change (Table 1). While the bilateral SII and insular dipoles did not change their location during and after capsaicin application, the anterior cingulate dipole moved backward in post-capsaicin area II and, mostly, in post-capsaicin area I recordings (Fig. 5). In order to validate
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Table 1 Coordinates of dipoles Dipole 1
Grand-average Pre-capsaicin Capsaicin Post-capsaicin area II Post-capsaicin area I Individual subjects Pre-capsaicin Mean SD Capsaicin Mean SD Post-capsaicin area II Mean SD Post-capsaicin area I Mean SD
Dipole 2
Dipole 3
Dipole 4
Dipole 5
x
y
z
x
y
z
x
y
z
x
y
z
x
y
z
253 253 253 253
226 226 226 226
20.6 20.6 20.6 20.6
53 53 53 53
226 226 226 226
20.6 20.6 20.6 20.6
27.3 27.3 24.6 25.5
14 14 1 27
33.8 33.8 36.6 34.7
229 229 229 229
21 21 21 21
5.5 5.5 5.5 5.5
27.4 27.4 27.4 27.4
7 7 7 7
4.5 4.5 4.5 4.5
252 3.16
225.9 3.14
20.8 2.82
53 3.37
225.4 2.22
21 3.32
25.44 2.3
14.7 1.83
33 2.6
227.4 3.89
21.6 3.24
4.25 3.06
27.7 7.52
8.1 6.12
6.44 3.61
251.7 3.77
225.7 3.47
20.5 2.88
52.8 3.19
225.4 2.76
21.4 2.96
25.4 2.76
14.7 2.41
33.1 2.52
226.8 3.62
0.9 4.51
5.25 3.88
27.6 7.5
7 5.23
6 4
251.7 4.03
226.1 4.33
20.7 2.83
51.5 2.51
225.5 2.22
21.1 4.23
24.67 1.66
20.1 3.07
33 3.08
227.2 4.66
22.9 4.33
5 5.24
29.4 5.6
6.8 4.26
6 4
251.4 4.43
226.5 4.55
21.1 2.18
53 3.37
225.4 2.22
22.1 2.71
25.33 2.83
28.6 1.84
33 3.04
226.8 4.26
21.1 4.15
3.63 3.54
29 6.38
6.9 5.11
7.1 4.73
Talairach and Tournoux coordinates.
this result, we applied the model issued from the grandaverage traces to individual recordings (Table 1). It was found that the locations of the SII and insular dipoles were not changed during and after capsaicin ðP . 0:05Þ, while the cingulate dipole had a more posterior location in postcapsaicin area II and, mostly, area I recordings compared to pre-capsaicin and capsaicin recordings (y coordinate, ANOVA: F ¼ 242:44, P , 0:01; post hoc test: P , 0:01).
4. Discussion The main result of the present study is that acute cutaneous pain induced by capsaicin may lead to rapid changes in cortical representation of nociceptive inputs coming from the painful territory and from adjacent areas, as suggested by the modification of LEP topography after capsaicin removal. Moreover, the amplitudes of vertex
Fig. 5. Projection of the dipoles’ coordinates onto the Talairach and Tournoux stereotactic brain atlas. Dipoles in the SII area (left) and insular dipoles (right) are represented in red colour; they did not change their cerebral location during and after capsaicin. On the contrary, the anterior cingulate dipole moved progressively backward, from pre-capsaicin and capsaicin recordings (blue) to post-capsaicin recordings obtained after stimulation of the secondary (green) and primary (yellow) zones.
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LEPs obtained by stimulation of both the zones of primary and secondary hyperalgesia were decreased during capsaicin application and after capsaicin removal. All LEP changes were directly related to capsaicin application on the right hand, since they occurred after right hand stimulation, but not stimulating the left hand. 4.1. Short-term cortical inhibition of nociceptive inputs induced by acute cutaneous pain In animals, neuroplastic changes induced by pain have been demonstrated at multiple levels of the central nervous system, such as the dorsal horn (Perl et al., 1976; Price et al., 1978; Kenshalo et al., 1979; McMahon and Wall, 1984; Simone et al., 1991; Woolf and King, 1990; Dougherty and Willis, 1992; Coderre et al., 1993), the thalamus (Guilbaud et al., 1986) and the cerebral cortex (Lamour et al., 1983). Some aspects of these plastic changes have also been demonstrated in humans, showing that pain may lead to a modulation of the cortical representation and neural reactivity to somatic inputs in patients with chronic pain and in healthy subjects submitted to experimental pain (Flor et al., 1995, 1997; Birbaumer et al., 1997; Tinazzi et al., 2000; Soros et al., 2001; Juottonen et al., 2002). An important result of the present study is that acute cutaneous pain induced by topical application of capsaicin determines a transient inhibition of the neural responsiveness to nociceptive inputs delivered on both the painful area (primary hyperalgesia) and the adjacent cutaneous territory (secondary hyperalgesia). In the zone of secondary hyperalgesia, we observed that vertex LEPs were strongly reduced in amplitude, mainly during capsaicin application, but also after its removal. One main element supports the hypothesis that the vertex LEP amplitude decrease is due to an inhibition occurring in the central nervous system, rather than at peripheral level. Indeed, while a peripheral mechanism would be expected to cause a reduction of both the middle-latency (N1/P1) and the vertex LEP amplitudes, the observed amplitude reduction concerned the vertex N2a –P2 potentials, but not the N1/P1 response. The fact that low-amplitude LEP components evoked by stimulation of the secondary zone were not related to any significant change of pain sensation evoked by CO2 laser pulses may appear puzzling. This lack of relationship suggests that in the presence of pain near the stimulated area, the correlation between the amplitude of the vertex LEP components and the subjective intensity of CO2 laser stimuli, demonstrated in the absence of pain (Bjerring and Arendt-Nielsen, 1988; Weiss et al., 1997), may be lost. In patients with central pain, Casey et al. (1996) demonstrated that the vertex LEP amplitude is reduced after stimulation of the painful skin, although the rating of laser pulse intensity is not decreased in the affected side compared to the nonaffected one. This result was attributed to a deficit in spinothalamic tract function in these patients. From this point of view, the clear LEP amplitude reduction in spite of
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an unmodified laser pain rating in our subjects can be due to spinothalamic tract inhibition. This hypothesis is consistent with the observation that in monkeys, the response of spinal sensory tract neurones in the dorsal horn to thermal stimuli is transiently inhibited after intradermal capsaicin injection (Dougherty et al., 1998). Hyperalgesia to heat in the secondary zone has been demonstrated by some investigators (Hardy et al., 1950; Arendt-Nielsen et al., 1996; Serra et al., 1998). However, the disagreement between our results and previous studies can be explained by the different quantity of capsaicin used and by the different technique of delivering the heat stimulation. Indeed, we used a much lower amount of capsaicin than the one in experiments showing heat sensitization in the zone of secondary hyperalgesia. Moreover, while these authors used intradermal injections of capsaicin, we applied it on the skin, thus further reducing the nociceptor stimulation. Lastly, CO2 laser pulses stimulate a smaller cutaneous area than the Peltier thermode stimuli used in previous studies. It is interesting that Ali et al. (1996) who tested the heat sensation in the secondary zone by laser stimuli, did not find hyperalgesia. In the zone of primary hyperalgesia, the vertex LEP amplitude was significantly lower in post-capsaicin than pre-capsaicin recordings. The reduction of the vertex LEP amplitude might be due to an inhibition of the Ad-fiber inputs generating LEPs by the CMH nociceptors, which are sensitized by capsaicin (Treede et al., 1992), at a certain level of the central nervous system. However, a summation of nociceptive activity from Ad and C-fibers occurs at the spinal level, as demonstrated by the increase of the nociceptive withdrawal reflex evoked by Ad-fiber electric stimulation during on-going burning pain, which activates the C nociceptors (Andersen et al., 1994). Therefore, the inhibition of the Ad-fiber inputs that generate LEPs by the sensitized C-fiber inputs should occur at supra-spinal level. This hypothesis explains also the significant delay in latency of the vertex LEP components when CO2 laser stimuli were delivered in the primary zone. As it has already been discussed for the stimulation of the secondary zone, in postcapsaicin area I recording also, the amplitude decrease of the vertex LEP was not paralleled by a reduction in rating of laser pulse intensity. The mechanism underlying this lack of relationship is probably related to a deficit in central pain pathway function (see above). Our findings suggest that acute cutaneous pain induces a short-term inhibition of the cingulate cortex, which generates the N2a – P2 potential. This is of particular interest if compared with recent results obtained by neuroimaging studies. While painful stimuli delivered on the normal side of the body entail an activation of the anterior cingulate gyrus, in patients with Wallenberg syndrome, stimuli delivered within hyperalgesic skin lead to a decrease of cerebral blood flow (CBF) in the anterior cingulate cortex (Peyron et al., 1998). A lack of cingulate cortex activation has been shown by functional magnetic
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resonance imaging (fMRI) also during experimental capsaicin-induced pain (Baron et al., 1999). The cingulate cortex is part of the limbic system and it is important for the emotional component of pain sensation (Derbyshire et al., 1997; Casey, 1999; Peyron et al., 1999; Buchel et al., 2002). Therefore, the role of the cingulate cortex inhibition due to acute pain in our subjects could be addressed to reduce the emotional and affective aspects of noxious stimuli delivered within or near a painful skin area. From this point of view, it is remarkable that the middle-latency N1/P1 wave, which might represent the neurophysiological correlate of the sensory-discriminative aspect of pain (Garcia-Larrea et al., 1997), was not significantly affected by capsaicin. The decreased LEP amplitude during capsaicin application and after its removal might be also due to desynchronization of the neural activity in the regions of central nervous system processing nociceptive inputs. This desynchronization possibly results from increased afferent inputs in presence of acute pain. In a recent article, LEP amplitude after stimulation of both right and left perioral regions was reduced by capsaicin application on the skin of the left cheek (Romaniello et al., 2002). The bilateral LEP amplitude decrease was explained by the so-called diffuse noxious inhibitory control (DNIC; Le Bars et al., 1979a,b; Chen et al., 1985; Arendt-Nielsen and Gotliebsen, 1992; Plaghi et al., 1994; Kakigi and Watanabe, 1996; Svensson et al., 1999; Reinert et al., 2000). DNIC is an electrophysiological phenomenon demonstrated in animals, in which the activity of convergent dorsal horn neurones is inhibited by noxious stimulation applied to various sites of the body (Le Bars et al., 1979a,b). Our findings of a decreased LEP amplitude to right hand stimulation after capsaicin application are unlikely related to a ‘pain inhibits pain’ effect, since LEPs obtained after left hand stimulation were not significantly decreased in amplitude compared to baseline. Moreover, we never observed a significant reduction of pain sensation due to CO2 laser pulses across the whole experiment. In our subjects, the lack of a major DNIC effect on left hand LEPs may be explained by the lower capsaicin concentration (3%) compared to the study by Romaniello et al. (2002) (10%). The difference between our and Romaniello et al.’s study in demonstrating a bilateral effect of acute pain on LEPs might also be due to the greater bilateral representation of face compared with hand (Manger et al., 1996). One may wonder why in our subjects hyperalgesia for pin-prick but not for laser stimuli was found, since both stimuli are expected to activate Ad-fibers. However, according to a model for cutaneous hyperalgesia, capsaicin probably entails facilitation of A-fiber high threshold mechanoreceptors and A-fiber nociceptors with high heat thresholds (AMHs type I). On the contrary, A-fibers that normally signal first pain to heat and carry inputs generating scalp LEPs (AMHs type II) are not facilitated (Ziegler et al., 1999).
4.2. Short-term topographic changes of the nociceptive cerebral cortex induced by acute cutaneous pain Another important result of the present study is that our subjects showed a change in the scalp distribution of the vertex LEP components in post-capsaicin recordings compared to baseline. While before and during capsaicin application the N2a – P2 potential was largely diffused over the scalp and reached its maximal amplitude at Cz vertex, after capsaicin removal, the N2a –P2 topography indeed moved towards the parietal region. This topography modification was recognizable in post-capsaicin traces after the stimulation of the zones of secondary and, mostly, primary hyperalgesia. Dipolar source modelling of LEP scalp distribution showed that the LEP topographic change is due to a backward shift of the dipole in the anterior cingulate cortex. Different subcortical areas within the anterior cingulate cortex are probably related to different aspects of pain sensation. In particular, the activity of the frontal part of the anterior cingulate gyrus reflects the PTh coding, given that subjects with high PTh show a high-level activation of the frontal part of the anterior cingulate gyrus. Instead, the posterior region of the anterior cingulate cortex encodes pain unpleasantness (To¨lle et al., 1999). In our subjects, the backward movement of the anterior cingulate source may be due to a modification of the relative contribution by different regions within the anterior cingulate cortex to the EEG signal evoked by CO2 laser pulses. This modification can result from two different situations: (i) the posterior part of the anterior cingulate cortex, which encodes pain unpleasantness (To¨lle et al., 1999), may be activated or may become hyperactive or (ii) the activity of the anterior part of the anterior cingulated cortex may be reduced. In other words, since a dipolar source represents a ‘centre-of-mass’ of electrical activities coming from one cerebral structure or from several brain areas, we cannot decide whether the observed change in the anterior cingulate dipole location is caused by ex novo activation or by increase of activity of a source in the posterior part of anterior cingulate gyrus or by reduced activity of a generator in the anterior part of this cerebral structure.
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