Increased contact heat pain and shortened latencies of contact heat evoked potentials following capsaicin-induced heat hyperalgesia

Clinical Neurophysiology 123 (2012) 1429–1436

Contents lists available at SciVerse ScienceDirect

Clinical Neurophysiology journal homepage: www.elsevier.com/locate/clinph

Increased contact heat pain and shortened latencies of contact heat evoked potentials following capsaicin-induced heat hyperalgesia C.S. Madsen a,⇑, B. Johnsen b, A. Fuglsang-Frederiksen b, T.S. Jensen a, N.B. Finnerup a a b

Danish Pain Research Center, Aarhus University Hospital, Aarhus, Denmark Department of Clinical Neurophysiology, Aarhus University Hospital, Aarhus, Denmark

a r t i c l e

i n f o

Article history: Accepted 22 November 2011 Available online 20 December 2011 Keywords: Contact heat evoked potentials (CHEPs) Topical capsaicin Sensitization Hyperalgesia Ad and C fibers

h i g h l i g h t s  Contact heat evoked potentials (CHEPs) allow testing of Ad and C fibers.  Topical capsaicin induces burning pain and hyperalgesia to heat stimuli yielding shortened CHEP latencies consistent with sensitization of Ad fibers.  C-fiber related CHEPs were observed following capsaicin application compatible with additional C-fiber sensitization.

a b s t r a c t Objective: To examine changes in contact heat evoked potentials (CHEPs) and perceived pain intensity following acute sensitization with topical capsaicin. Methods: CHEPs were recorded before and after 20 min of topical capsaicin application (200 ll, 5%) during skin warming in 22 healthy subjects. To evaluate the sequence effects and skin warming on CHEPs, 10 of these subjects also participated in a control study. Results: Topical capsaicin yielded an increase in contact heat evoked pain ratings (p < 0.0001) and a shortening in N2 latency from a mean 345.2 ± 37.2 ms to 310.2 ± 38.5 ms recorded from the vertex position (p = 0.003, paired t-test). No difference was found in the N2–P2 peak-to-peak amplitude (p = 0.83). These results were unchanged after controlling for sequence effects and skin warming. Following capsaicin, ultralate CHEPs (N2a latencies 970–1352 ms) were recorded in three subjects. Conclusions: Our study showed a decrease in late CHEPs latencies and appearance of ultralate potentials compatible with sensitization of Ad fibers and C fibers. Significance: Contact heat may be a useful tool to assess sensitization of the pain system. Ó 2011 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction Sustained noxious input or nerve injury may trigger changes in the nervous system with increased responsiveness, decreased thresholds and increased synaptic efficacy of nociceptors and of central connectivity. This change in excitability of the nociceptive system termed sensitization is thought to play a major role in the underlying mechanisms of different pain conditions, and may result in spontaneous pain and sensory hypersensitivity with allodynia and hyperalgesia (Woolf, 2011; Basbaum et al., 2009). Different neurophysiological techniques have been used to assess the function of the peripheral and central nervous system, but

⇑ Corresponding author. Address: Danish Pain Research Center, Aarhus University Hospital, Norrebrogade 44, Building 1A, 1st Floor, DK-8000 Aarhus C, Denmark. Tel.: +45 7846 4139; fax: +45 7846 3269. E-mail address: [email protected] (C.S. Madsen).

to date no technique has been ideal for assessing sensory sensitization. Laser evoked potentials (LEPs) and contact heat evoked potentials (CHEPs) allow testing of small afferent fibers, yielding responses that are time-locked to the stimulus onset. With standard averaging procedures, postsynaptic cerebral potentials can be recorded using surface electrodes placed on the scalp. These responses emerge as deflections in the EEG and are quantified according to polarity (e.g. N2, P2) in latency and amplitude. Radiant and contact heat stimuli have previously been shown to activate thinly myelinated Ad fibers and, to a lesser degree, unmyelinated C fibers, giving rise to late and ultralate responses, respectively (Granovsky et al., 2005; Truini et al., 2007b; Bromm and Treede, 1987b; Bragard et al., 1996; Magerl et al., 1999; Tzabazis et al., 2011). In healthy subjects, the amplitude of EPs is positively correlated with stimulus intensity and pain sensation (Truini et al., 2007b; Carmon et al., 1978; Chen et al., 2001; Granovsky et al., 2008; Roberts et al., 2008; Greffrath et al., 2007; Ohara et al., 2004; Garcia-Larrea et al.,

1388-2457/$36.00 Ó 2011 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2011.11.032

1430

C.S. Madsen et al. / Clinical Neurophysiology 123 (2012) 1429–1436

1997; Romaniello et al., 2002), but pain and amplitude can also be dissociated, and LEPs and CHEPs cannot be used as direct correlates of the neural activity responsible for pain intensity coding in the human cortex (Iannetti et al., 2008). In clinical studies, LEPs and CHEPs have been shown to correlate well with intraepidermal nerve fiber density, showing responses with decreased amplitudes and increased latencies in patients with small fiber loss (Casanova-Molla et al., 2011; Chao et al., 2008). LEPs have, however, been less sensitive in reflecting states of hypersensitivity in patients suffering from chronic pain, and attenuated and delayed LEPs have been demonstrated in neuropathic pain patients with allodynia and hyperalgesia (Wu et al., 1999). Surprisingly, enhanced LEPs have not been demonstrated in patients with allodynia and hyperalgesia, although less attenuation of LEPs has been reported in patients with evoked pain compared with patients with spontaneous pain only (Garcia-Larrea et al., 2002), consistent with the necessity for preserved afferent pathways for the experience of evoked pain. Capsaicin, the pungent ingredient in hot chili peppers, is commonly used to study sensitization (Klein et al., 2005). When applied topically at concentrations of 1–3%, an acute sensation of burning pain usually develops within 30 min (Koltzenburg et al., 1992; Valeriani et al., 2003; de Tommaso et al., 2005, 2007; Roberts et al., 2011) due to activation of transient receptor potential vanilloid (TRPV1) receptors on Ad and C fiber nociceptors (Ringkamp et al., 2001; Baumann et al., 1991; Szolcsanyi et al., 1988). In the primary area, allodynia and hyperalgesia to heat and mechanical stimuli and cold hypoesthesia develop, whereas hyperalgesia to mechanical stimuli develops in the surrounding secondary area (Baumann et al., 1991; LaMotte et al., 1991; Kilo et al., 1994; Treede et al., 1992; Callsen et al., 2008; Ali et al., 1996). The ongoing pain and discharges of the sensitized nociceptors following capsaicin sensitization depend on the skin surface temperature, and mild cooling of the skin results in a reduction of the burning pain sensation, stressing the importance of keeping the skin temperature constant (Koltzenburg et al., 1992; LaMotte et al., 1992). In addition to the initial excitatory action of capsaicin (burning pain and hyperalgesia) examined in this study, prolonged topical application of low-concentration capsaicin has been shown to nearly eliminate epidermal nerve fibers and attenuate heat pain sensitivity (Nolano et al., 1999) and disappearance of LEPs (Beydoun et al., 1996; Mouraux et al., 2010; Rage et al., 2010). In recent LEPs studies, topical capsaicin at doses that produced clinical signs of sensitization with heat hyperalgesia and allodynia either did not change or decreased laser-evoked pain with reduced LEP amplitudes (Romaniello et al., 2002; Valeriani et al., 2003, 2005; de Tommaso et al., 2005, 2007) or did not change or caused a delay in latencies (Valeriani et al., 2003; de Tommaso et al., 2005, 2007). However, Tzabazis et al. (2011) showed that topical capsaicin increased LEP amplitudes related to C-fiber activity, but failed to show enhanced responses in the Ad range following the capsaicin sensitization (Tzabazis et al., 2011). Spatial and temporal summation are mechanisms that may influence the processing of noxious information including evoked potentials (Iannetti et al., 2004). One important difference between radiant and contact heat is that contact heat stimulation activates a larger surface area than radiant heat stimulation elicited by lasers. To our knowledge, there is only one study of CHEPs following topical capsaicin. Despite that topical capsaicin caused a lowering of heat pain threshold, no change in latency or amplitude could be demonstrated, although a trend for a decrease in CHEP amplitude was noted (Roberts et al., 2011). The present study investigates the initial acute sensitizing effect of topical capsaicin (5%) application on the pain sensation as well as CHEP amplitudes and latencies following contact heat stimulation in the primary hyperalgesia area.

2. Methods 2.1. Subjects Twenty-two healthy subjects were included in the study. Ten of these also participated in a control study in order to analyze sequence effects: (1) the effect of 20 min of skin warming using a heating lamp and (2) the effect of a 20-min time delay (sequence effects) on CHEP latencies, amplitudes and pain intensity scores. All subjects were recruited from Aarhus University, Denmark, and were paid for their participation. The protocol was approved by the local ethical committee (No. M-20090060) and the Danish Data Protection Agency (No. 2009-41-3360), and all subjects gave their written informed consent. 2.2. Experimental design The study consisted of two CHEP recording sessions: one at baseline and one after 20 min of topical capsaicin application (capsaicin study). Each session consisted of 20 heat stimuli applied to the subject’s dorsal right hand. The thermode remained in a fixed position in each session, and marking of the skin ensured identical contact areas in the two sessions. During capsaicin application, the skin was warmed using a heating lamp to maintain a constant skin temperature and continuous pain (Fig. 1). In the control study, the subjects completed 2  2 CHEP recording sessions in a balanced order: at baseline and after 20 min of warming the skin on the left or right dorsal hand; and at baseline and after a 20-min delay (without skin warming) on the opposite dorsal hand (control study) (Fig. 1). The recordings were made in a semi-dark room. The subjects lay comfortably on a bed and were told to keep their eyes closed during the recording sessions. Furthermore, it was emphasized that any facial muscular activity should be minimized during the recording session in order to reduce artifacts, and thereby rejections of CHEPs. The subjects rated the intensity of each stimulus on a numeric rating scale (NRS, 0–10), approximately 2 s after the stimulus. Following each recording session, a validated pain questionnaire (Perkins et al., 2004) was used to describe the quality of the contact heat evoked pain. Besides answering the sensory and affective descriptors (yes or no), they were also asked to rate the degree of the sensations (a little or a lot) (Perkins et al., 2004). 2.3. Contact heat stimulation We used a Contact Heat Evoked Potential Stimulator (CHEPS, Medoc Ltd., Ramat Yishai, Israel) to deliver the noxious heat pulses. The thermode, with a contact activation area of 573 mm2, uses a combination of a heating foil and a Peltier element to generate the fast heating and cooling rate. We used the maximum rates available (a heating rate of 70 °C/s and a return rate of 40 °C/s). The stimulus duration was approximately 800 ms (271 ms from baseline to peak temperature and 475 ms to return to baseline) (Roberts et al., 2008). The heat pulses were applied from a baseline temperature of 32 °C to a peak temperature of 51 °C with an interstimulus interval that varied randomly between 32 and 40 s (endto-onset interval). To reduce expectation effects, we applied 1–2 test stimuli prior to the CHEP recording. 2.4. CHEP recording CHEPs were recorded using the 10–20 electrode system with tin cup surface electrodes from the Cz, Fz, C3 and C4 position using A2 as a reference (with Cz-A2, C3-A2, C4-A2, C4-Fz and Cz-Fz montage). The EEG recording (within 0.2–300 Hz bandpass at a sampling rate of 48 kHz and a recording epoch of 4000 ms) was

1431

C.S. Madsen et al. / Clinical Neurophysiology 123 (2012) 1429–1436

Capsaicin study

Baseline Right hand

Topical capsaicin and skin warming 20 min.

After capsaicin and skin warming Right hand

Control study

Baseline Right/left hand

Skin warming 20 min.

After skin warming Right/left hand

Baseline Right/left hand

Time delay (no skin warming) 20 min.

After time delay Right/left hand

Fig. 1. Diagram of the experimental setup for the CHEP recording in the capsaicin (top panel) and control study (middle and lower panel). Top panel: CHEPs were recorded at baseline from stimulation on the dorsal right hand of the subjects (baseline CHEPS). Following topical capsaicin and skin warning for 20 min, CHEPs were recorded from the same stimulation site (after capsaicin and skin warming). Middle and lower panel: in the control study, subjects were stimulated on both hands (2  2 CHEPS) in a balanced order: at baseline and after 20 min of warming the skin on the left or right dorsal hand and at baseline and after a 20-min delay (without skin warming) on the opposite dorsal hand.

triggered at stimulus onset and recorded on a keypoint evoked potential system (Dantec KeypointÒ from NatusÒ). The electrode impedance was kept below 3 kX in the recordings, which was verified prior to the initiation of each session. We only report the recordings from the vertex Cz-A2 montage. 2.5. Induction of experimental pain – capsaicin A 200 ll 5% capsaicin solution (50 mg/ml) was applied to the skin surface of the subject’s dorsum right hand for 20 min. A rubber ring (33 mm in diameter) was fixed to the skin with adhesive material in order to prevent the capsaicin from escaping the examination area. During this period, the skin was warmed with a heating lamp to a skin temperature of approximately 38 °C to maintain a continuous pain. The skin temperature was monitored using a digital infrared temperature scanner (Omega OS90 series, resolution 0.1 °C, Stamford, CT, USA). The size of the rubber ring ensured that the heating foil of the thermode (573 mm2) was fully embedded within the area of topical capsaicin. In the control study, we monitored the skin temperature prior to the two baseline recordings, during the 20 min of skin warming and during the 20-min time delay (continuously), using the same digital infrared laser temperature scanner (average of three measures).

Furthermore, it was ensured that the potentials could be identified in other montages applied (other than the Cz-A2 montage).

2.7. Statistical analysis Statistical analysis was performed using Stata 10. As the differences in latencies, amplitudes and pain intensity scores (NRS, 0– 10) followed a normal distribution (histogram and q–q plot), data were analyzed using a paired t-test. In addition, a variance ratio test (SD-test) of the differences prior to analysis were performed. Differences between interventions, i.e. at baseline and after skin warming (D skin warming) versus baseline and after time delay (D time delay), baseline and after capsaicin and skin warming (D capsaicin) and (D skin warming) were also analyzed using paired design. For the capsaicin and the control data, only subjects who had reproducible scalp potentials in all CHEP recordings sessions were included in the statistical analysis. Data are represented as mean ± SD. Binary paired data obtained from the pain questionnaire were analyzed with McNemar’s test. Differences between the subjects reporting descriptors (total) and the intensity (a lot) were tested. p Values <0.05 are considered to be statistically significant.

3. Results 2.6. Data analysis 3.1. Subjects For the analysis of CHEPs, EEG traces of each subject were averaged separately. Trials contaminated with artifacts exceeding 100 lV were rejected online, and further analysis was performed offline. The N2–P2 complex was identified on the averaged waveform, and the N2 and P2 latencies were measured from stimulus onset to the peak of the responses. The (N2–P2) amplitudes were evaluated as peak-to-peak amplitudes between the negative peak (N2) and the positive peak (P2). Further, the N amplitude of the negative component of the (N2–P2) response was measured from onset-peak to peak of the N wave. To test for their reproducibility, every other of the CHEP waveforms were placed in one buffer (even numbered trials, e.g. 2, 4, 6, etc.) and odd numbered trials in another buffer (odd numbered trials, e.g. 1, 3, 5, etc.). Following a superimposition of the two buffers (odd–even), latencies and amplitudes were measured. A trained neurophysiologist (BJ), who was blinded to all interventions, added the N2 and P2 cursors.

Twenty-two subjects were included (12 males, 10 females; mean age 24.1 ± 2.9 years; range 21–33) in the capsaicin study. Two subjects were excluded due to the lack of reproducible CHEPs in the baseline recording session, and 20 subjects (10 males, 10 females; mean age 24.0 ± 2.9 years; range 21–33) were included in the data analysis. Ten of these subjects participated in the control study (four males, six females; mean age 24.2 ± 3.7 years; range 22–33). Seven subjects participated in the baseline and the 20min delay (i.e. no skin warming) recording sessions (one subject was excluded due to lack of identifiable CHEPs after the 20-min delay) and six subjects (three males, three females; mean age 24.7 ± 4.4 years; range 22–33) were included in the data analysis. Ten subjects participated in the baseline and skin-warming recording sessions (one subject was excluded due to of lack of reproducible CHEPs after skin warming) and nine subjects (four males, five

1432

C.S. Madsen et al. / Clinical Neurophysiology 123 (2012) 1429–1436

Table 1 Neurophysiological data and pain intensity in the capsaicin study and the control study. Capsaicin study

Baseline (n = 20)

Control study

After capsaicin and p⁄ skin warming (n = 20)

N2 latency 345.2 ± 37.2 310.5 ± 38.5 P2 latency 496.5 ± 45.1 468.5 ± 41.3 Amplitude 34.1 ± 13.7 34.8 ± 12.7 Pain 2.4 ± 1.3 4.4 ± 1.7 intensity

Baseline (n = 9)

D skin D capsaicin and skin warming vs. D warming vs. D skin warming time delay After skin warming (n = 9)

p⁄

Baseline (n = 6)

After time delay (n = 6)

p⁄

0.003 331.9 ± 27.0 338.3 ± 30.9 0.23 339.0 ± 31.0 339.7 ± 21.9 0.90 0.013 441.4 ± 54.0 469.3 ± 30.9 0.13 434.0 ± 31.5 461.8 ± 57.7 0.08 0.83 40.7 ± 21.6 37.2 ± 16.9 0.29 36.6 ± 22.8 32.2 ± 16.4 0.30 <0.0001 2.0 ± 1.2 2.0 ± 1.2 0.75 1.7 ± 1.3 1.4 ± 1.0 0.11

p⁄

p⁄

0.33 0.45 0.31 0.14

0.008 0.006 0.47 0.007

Application of 5% capsaicin and 20 min of skin warming caused a significant shortening of N2 latency (34.7 ms) and P2 latency (28 ms) and an increase in heat pain intensity (paired t-test). Capsaicin and skin warming had no significant impact on the amplitudes. In the control study, where each subject received 2  2 CHEPS sessions: at baseline and after 20 min of skin warming and at baseline and after a 20-min delay (with no skin warming), we found no differences in N2 and P2 latencies, N2–P2 amplitude or heat pain intensity (paired t-test). When we controlled for sequence effects (time delay) and skin warming, capsaicin still caused a decrease in peak latencies and pain threshold (paired t-test); D skin warming: after skin warming minus baseline, D time delay: after time delay minus baseline, D capsaicin and skin warning: after capsaicin and skin warming minus after skin warming. Data are presented as mean ± SD. Latencies (ms) are peaks in CHEP recordings and amplitudes are N2-P2 peak-to-peak. Pain intensity scores are ratings on the numeric rating scale (NRS 0–10). ⁄p: statistical significance is <0.05 (bold p-values) on paired design.

females; mean age 24.4 years ± 3.9 years; range 22–33) were included in the data analysis (Table 1). 3.2. Skin temperature measurements During capsaicin administration and skin warming, the skin temperature was kept at approximately 38 °C. In the control study, the skin temperature at the two baseline sessions was 32.7 ± 1.3 °C (n = 6) and 32.3 ± 0.9 °C (n = 9), and the skin temperature after the 20-min delay was 32.5 ± 1.3 °C (n = 6). During the 20 min of skin warming, the mean skin temperature was 37.6 ± 0.3 °C (n = 9). 3.3. Pain intensity ratings In the baseline session, the heat stimuli of 51 °C evoked pain with a mean pain intensity (NRS) of 2.4 ± 1.3 (range 0.7–6.0). After application of topical capsaicin, the pain intensity was significantly higher, with mean pain intensity (NRS) 4.4 ± 1.7 (range 1.5–8.1) (p < 0.001, paired t-test) (Table 1). In the control study, we found no sequence effects (before and after a 20-min delay) (p = 0.11, paired t-test) or effect of skin warming (before and after 20 min of skin warming) (p = 0.75 and p = 0.14 when correcting for sequence effects, paired t-test) on the contact heat-evoked pain intensity (Table 1). When controlling for sequence effects and skin heating, capsaicin still caused an increase in contact heat evoked pain in the subgroup of subjects participating in both the capsaicin and the control sessions (p = 0.007, paired t-test) (Table 1). Following capsaicin application, an increase in the number of subjects reporting shooting–jumping/jolting (p = 0.03) and stinging (p = 0.016) pain was seen (Table 2). Nearly all subjects reported that the contact heat caused a warm/hot-burning sensation at baseline and after capsaicin application (p = 1.00). However, following capsaicin there was an increase in the number of subjects reporting warm/hot-burning sensation as ‘‘a lot’’ (p = 0.008). In addition, the application of capsaicin caused an increase in the affective component annoying/irritating (p = 0.016). No other differences were observed in the sensory or affective descriptors in the capsaicin study. No differences were seen in control study (Table 2). 3.4. CHEPs latencies (late responses) We identified the characteristic biphasic negative–positive potential (N2–P2) at the vertex (Cz) position (Fig. 2). Topical application of capsaicin caused a shortening in N2 latency (34.7 ms) (p = 0.003, paired t-test) and P2 latency (28 ms) (p = 0.01, paired

t-test). In the control study, we found no sequence effects or effect of skin warming in latencies (N2 and P2) (Table 1). When controlled for sequence effects and skin warming, capsaicin still caused a decrease in N2 latency (p = 0.008) and P2 latency (p = 0.006) (Table 1). 3.5. CHEPs amplitudes (late responses) We found no difference in peak-to-peak amplitudes between the recordings at baseline and after capsaicin application (p = 0.83) as well as in the control study between the interventions (Table 1). In addition, we did not find any difference in the N amplitude before (mean 13.4 ± 8.2 lV) and after capsaicin (mean 10.3 ± 8.2 lV) (p = 0.12, paired t-test). 3.6. CHEPs (ultralate responses) After capsaicin application, we could identify (N2a/P2a) responses most likely related to C-fiber activity in three subjects in addition to the late response (Fig. 3). These ultralate responses were in the range of 970–1352 ms (N latency) and 1330– 1589 ms (P latency). The amplitudes were smaller (12.3 ± 4.2 lV) compared with the amplitudes of the late responses. The ultralate responses were only identifiable after capsaicin application, and not visible in the baseline recordings or in any of the recordings in the control study. 4. Discussion The present study demonstrated contact heat evoked pain and CHEPs in terms of shortened latencies following topical capsaicin application. No changes in CHEPS amplitudes were found. These results were robust after correcting for sequence effects and the effect of skin warming. Following capsaicin, contact heat was felt as a more intense warm/hot-burning sensation and more annoying/ irritating, and described as a more shooting–jumping/jolting and stinging sensation than at baseline. These sensory experiences are compatible with small afferent fibers being sensitized by TRPV1 agonists. In addition, after topical capsaicin, CHEPs likely to be related to C-fiber activity were demonstrated in three subjects. These findings suggest that topical capsaicin may sensitize both Ad and C fibers or their central projecting pathways. These results are in line with the clinical expression of heat allodynia and hyperalgesia in the primary hyperalgesic area (Baron, 2006; Woolf and Mannion, 1999; Jensen et al., 2001). Our study thus suggests

1433

C.S. Madsen et al. / Clinical Neurophysiology 123 (2012) 1429–1436 Table 2 Descriptors of heat evoked pain in the capsaicin study and the control study. Capsaicin study

Throbbing-hammering Shooting-jolting/jumping Drilling Pricking Sharp-cutting cramping-tightening Pulling-tugging Stinging Varm/hot-burning Itching Sore-heavy Tender Tiring-exhausting Sickening Fearful Spreading/radiating Punishing annoying/irritating

Control study ⁄

p⁄

p⁄

Baseline (n=20)

After capsaicin and skin warming (n=20)

p

Total/ a lot

Total/ a lot

Total /a lot

Total/ a lot

Total/ a lot

Total/ a lot

Total/ a lot

Total/ a lot

Total/ a lot

3/1 10/4 2/0 14/3 9/1 0/0 3/1 12/3 19/7 3/0 4/2 3/1 3/0 0/0 1/0 2/0 1/0 9/0

7/2 16/7 5/1 15/7 13/4 0/0 4/1 19/9 20/15 5/1 3/1 8/3 2/0 1/0 3/0 5/3 2/0 14/7

0.22/1 0.03/0.25 0.38/1 1/0.22 0.13/0.25 1/1 1/1 0.016/0.11 1.00/0.008 0.50/1 1/1 0.063/0.50 1/1 1/1 0.50/1 0.25/0.25 1/1 0.063/0.016

1/0 6/2 0/0 7/2 4/1 0/0 0/0 6/1 9/1 1/0 1/0 0/0 1/0 0/0 1/0 1/0 1/0 3/0

1/0 4/1 0/0 7/2 5/1 0/0 0/0 6/1 9/1 1/0 0/0 0/0 2/0 0/0 0/0 1/0 0/0 4/0

1/1 0.50/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

0/0 3/1 1/0 5/0 2/0 0/0 0/0 2/2 6/1 0/0 0/0 0/0 0/0 0/0 0/0 1/0 0/0 2/0

0/0 3/0 0/0 5/1 2/0 0/0 0/0 2/2 6/0 0/0 0/0 0/0 0/0 0/0 0/0 1/0 0/0 2/0

1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

Baseline (n=9)

After skin warming (n=9)

Baseline (n=6)

After time delay (n=6)

The subjects rated the descriptors as either ‘‘a little’’ or ‘‘a lot’’. Only those that rated the descriptors ‘‘a lot’’ are presented. After capsaicin and skin warming, the contact heat caused an increase in the number of subjects reporting shooting–jumping/jolting and stinging pain. There was an increase in the number of subjects reporting the contact heat stimuli as being more warm/hot-burning and annoying/irritating after capsaicin and skin warming. No difference was seen in the descriptors in the control study. ⁄p: statistical significance is <0.05 (bold p-values) on paired design (McNemar test).

that CHEPs may be a useful tool for assessing sensitization of the pain system, although CHEPs were not fully reproducible in all subjects. These findings are in contrast to the lack of enhanced evoked pain and attenuation of LEPs found following acute capsaicin application (Valeriani et al., 2003, 2005; de Tommaso et al., 2005). 4.1. Effects of capsaicin on Ad-fiber responses Changes in heat and pain sensation following capsaicin application are dose and time dependent. It is well established that repeated application of a low-concentration (e.g. 0.075%) capsaicin over days to weeks causes reversible nerve fiber degeneration, lowered pain and heat sensitivity (Nolano et al., 1999) and disappearance of LEPs (Beydoun et al., 1996; Mouraux et al., 2010; Rage et al., 2010). This contrasts the acute application of topical capsaicin (5%) examined in this study, which caused sensory hypersensitivity including heat allodynia and hyperalgesia. In the present study, we could not demonstrate increased amplitudes following acute capsaicin application, although there was a tendency towards less reduction in amplitudes following capsaicin compared to the control conditions, where there seemed to be a habituating effect. We did, however, find significantly shortened latencies following acute topical capsaicin. A shortened latency with increased stimulus intensity has been reported (Bjerring and Arendt-Nielsen, 1988; Plaghki et al., 1994), although others found peak latencies unrelated to stimulus intensity (Romaniello et al., 2002; Bromm and Treede, 1987a; Treede et al., 1988). Surprisingly, studies using LEPs have found attenuated rather than facilitated responses using comparable capsaicin doses (1 ml of 3%) (Valeriani et al., 2003; de Tommaso et al., 2005, 2007). Capsaicin caused a burning pain sensation, but radiant heat in the capsaicin-treated area did not cause more pain than the control situation. Also capsaicin caused either a delay in both N2 and P2 latencies (de Tommaso et al., 2005), N2 latency only (de Tommaso et al., 2007) or did not change latency (Valeriani et al., 2003) 20–35 min after capsaicin application. In the latter, Valeriani et al. (2003) found delayed N2 and P2 latencies, but only after removing the capsaicin (approximately 75 min after the application), and the authors speculate that the delay in latency could be due to an

inhibitory action of the sensitized C fibers on the Ad-fiber related response (Valeriani et al., 2003). In these studies, application of topical capsaicin caused a decrease in N2–P2 peak-to-peak amplitude related to the decrease in laser pain ratings, although Valeriani et al. (2003) found only slightly reduced pain ratings. The reason for the differential effect of capsaicin on LEPs and CHEPs is largely unknown. However, some issues may account for these differences. First, capsaicin sensitization results in a decrease in threshold and less contact heat would be needed to activate the peripheral nerve endings. Due to the slow heat ramp of CHEPS; nominal 70 °C/s but even slower at the skin surface (Greffrath et al., 2007), and slow heat transmission from the skin surface to the nociceptors, may suggest that contact heat would be favorable to separate the baseline and capsaicin sensitization condition. We did not record the skin surface temperature under the CHEPS thermode to estimate the threshold change, but Greffrath et al. (2007) measured this to be 42 °C using a peak stimulus temperature of 51 °C. Since the CHEP recording was triggered at stimulus onset, a decrease in the nerve fiber threshold (activation at lower temperature) will result in shorter time to fiber activation, which in turn may result in a shortening of latency due to the capsaicin-evoked sensitization. This decreased latency is compatible with heat allodynia (reduced heat pain threshold) seen in the primary hyperalgesic area (Treede et al., 1992). Second, the ongoing pain and discharges of the sensitized nociceptors following capsaicin sensitization depend on the skin surface temperature (Koltzenburg et al., 1992; LaMotte et al., 1992). In accordance with Koltzenburg et al. (1992), we used an infrared lamp to maintain a constant skin temperature and a substantial, stable level of burning pain at the application site. To our knowledge, this procedure was not applied in the studies by Romaniello et al. (2002), de Tommaso et al. (2005), Roberts et al. (2011), Valeriani et al. (2003). Third, spatial and temporal summation differ between the two methods with larger contact area using CHEPS. Following capsaicin sensitization, a contact heat stimulus may activate a higher number of fibers, and may include Ad fibers with higher conduction velocity, yielding a synchronized response with shortened latency. Peripheral sensitization has been considered to be responsible for heat allodynia and

1434

C.S. Madsen et al. / Clinical Neurophysiology 123 (2012) 1429–1436

Fig. 2. Grand average of CHEPs from capsaicin study (left panel) and control study (middle and right panel). Left panel: application of topical capsaicin (5%) for 20 min caused a shortening of N2 latency (35 ms) and P2 latency (28 ms) (solid line) compared with the baseline recording (dotted line). Middle panel: skin warming for 20 min (solid line) had no effect on N2 or P2 latencies compared with baseline (dotted line) and a 20-min delay (without skin warming) did not change N2 and P2 latencies compared with baseline (right panel). This suggests that the shortening of CHEP latencies were directly related to the capsaicin induced sensitization. No significant changes in amplitudes were observed in the capsaicin or the control study. Grand average CHEPs are from CZ-A2 montage following 20 heat stimuli applied to the dorsum hand of healthy subjects. Arrows indicate the N2 and P2 peaks. Asterisks indicate artifacts from CHEP stimulation.

primary hyperalgesia (Baumann et al., 1991; LaMotte et al., 1991). However, if spatial summation is necessary for a facilitated response, this would suggest that central sensitization is also involved in heat allodynia in the primary hyperalgesic area. Fourth, differences in capsaicin doses and solutions (200 ll 5% capsaicin in 70% ethanol in our study compared with 0.5–1 ml 3% in cream base or an unguent format in the studies by Valeriani et al. (2003), de Tommaso et al. (2005, 2007), Roberts et al. (2011)) may be of importance. It is interesting that Roberts et al. (2011) did not find changes in latencies following 0.5 ml capsaicin (3%) using CHEPS. This may be due to lack of skin warming, necessary to maintain a sufficient capsaicin induced sensitization, the lower capsaicin concentration used, fewer subjects studied or that CHEPs were recorded following only 10 contact heat stimuli. 4.2. Effects of capsaicin on C-fiber responses Noxious heat stimuli co-activates Ad and C nociceptors, and the Ad-fiber afferent volley arrives faster at central projection sites than the slower C-fiber afferent volley due to the differences in conduction velocities. Despite a dual sensation of first and second pain in standard LEP and CHEP recordings, responses appear only in a time window compatible with Ad-fiber conduction velocity. The reason for this effect is not known in detail (Truini et al., 2007a; Mouraux and Iannetti, 2008). C-fiber related ultralate responses are best recorded by avoiding excitation of Ad fibers, either by pressure blockade (Bromm and Treede, 1987b), using controlled temperature stimulation (Magerl et al., 1999; Granovsky et al., 2005), or stimulation with low intensity and long duration laser pulses (Tzabazis et al., 2011). Using CHEPS, Granovsky et al. (2005) could record C-fiber responses with 41 °C contact heat stimuli. This finding, however, could not be replicated (Truini et al., 2007b). It should be emphasized that the ultralate responses related to the C-fiber activity observed in this study were of small amplitude and not easily recognizable (Fig. 3). In addition, they were observed in three subjects only. However, since the ultralate responses were less visible or not visible in any of the baseline recordings, this suggests that the C-fiber responses were a result of the capsaicin induced-sensitization. One possible explanation may be synchronization and recruitment of sensitized C-fibers following capsaicin application. Thus, these results are compatible with C-fiber sensitization or sensitization of related central pain pathways following capsaicin.

Fig. 3. Ultralate CHEPs (C-fiber related responses) recorded from a subject. Following application of topical capsaicin (5%) for 20 min, an ultralate response with latencies of 1352 (N2a wave) and 1589 (P2a wave) appeared in addition to the late response. Note that the ultralate response is not as easily recognizable as the better defined responses related to Ad-fiber activity. In this particular subject, capsaicin sensitization caused a reduction in peak-to-peak amplitude compared with the baseline recording (dotted line). CHEPs are the averaged response following 20 heat stimuli applied to the dorsal hand (Cz-A2 montage). Arrows indicate N2 and P2 peaks (late peaks) and N2a and P2a indicate the ultralate peaks probably related to C-fiber activity.

4.3. Limitations of the study In the present study, the mean latency in the baseline recordings (N2: 332–345 and P2: 434–469 ms) were comparable with other related studies (Roberts et al., 2008; Greffrath et al., 2007; Iannetti et al., 2006; Chen et al., 2006) following contact heat stimulation on the dorsal hand or volar arm, although significantly shorter (Granovsky et al., 2005), and longer latencies (Chen et al., 2001; Warbrick et al., 2009; Valeriani et al., 2002; Le Pera et al., 2002) have been reported and the differences in peak latency among these studies may be related to different stimulus and recording parameters. In our study, the experiments (capsaicin study) were performed on the right hand only. It would have been preferable to have

C.S. Madsen et al. / Clinical Neurophysiology 123 (2012) 1429–1436

performed a parallel recording of both hands to identify possible habituation effects. We used a fixed thermode position, but varying the thermode position after each stimulus will yield larger amplitudes by avoiding nociceptor fatigue and peripheral habituation effects (Greffrath et al., 2007; Warbrick et al., 2009). A positive relationship between stimulus intensity and amplitude using a fixed thermode position has been reported (Granovsky et al., 2005), and in the present study, we used a rather long and non-constant interstimulus interval (varied between 32 and 40 s in a randomized fashion) in order to reduce the response decrement related to this effect (Bromm and Treede, 1987a; Mouraux et al., 2004). Attending to a stimulus is reported to enhance the amplitude, but not the latency of evoked potentials (Garcia-Larrea et al., 1997; Miltner et al., 1989; Bentley et al., 2004; Beydoun et al., 1993; Siedenberg and Treede, 1996). In the present study, subjects were asked to rate every single stimulus as recommended by Treede et al. (2003) in order to keep the same level of attention, but we cannot exclude some effect of attention on our results. In addition, we did not record the early N1 response because of the low SNR and inconsistency of the N1 response using the CHEPS (Greffrath et al., 2007; Iannetti et al., 2006). Since the N1 lateralized component has been shown to be less susceptible to cognitive modulation compared to the vertex response (Garcia-Larrea et al., 1997), the recording of N1 response may have strengthened our study. 5. Conclusion Our study showed increased heat evoked pain and shortened latencies of contact heat evoked potentials following capsaicin sensitization. This suggests that CHEPS heat may be a useful tool to assess sensitization of the pain system, although CHEPs were not fully reproducible in all subjects. The shortened CHEP latencies are compatible with sensitization of peripheral Ad fibers or their central pathways. Thus, it is possible that capsaicin causes a decrease in Ad fiber threshold giving rise to heat allodynia (decreased heat pain threshold) and shortened CHEPs latencies. The fact that CHEPs and not LEPs are facilitated suggests that spatial summation is needed and therefore that central sensitization is also involved in heat allodynia following capsaicin application. In three subjects, ultralate responses were observed following capsaicin application compatible with additional C-fiber sensitization. Conflict of interest None declared. Acknowledgements This study was funded by the Velux Foundation. We thank research secretary Helle Obenhausen Andersen for language revision. References Ali Z, Meyer RA, Campbell JN. Secondary hyperalgesia to mechanical but not heat stimuli following a capsaicin injection in hairy skin. Pain 1996;68:401–11. Baron R. Mechanisms of disease: neuropathic pain – a clinical perspective. Nat Clin Pract Neurol 2006;2:95–106. Basbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and molecular mechanisms of pain. Cell 2009;139:267–84. Baumann TK, Simone DA, Shain CN, LaMotte RH. Neurogenic hyperalgesia: the search for the primary cutaneous afferent fibers that contribute to capsaicininduced pain and hyperalgesia. J Neurophysiol 1991;66:212–27. Bentley DE, Watson A, Treede RD, Barrett G, Youell PD, Kulkarni B, et al. Differential effects on the laser evoked potential of selectively attending to pain localisation versus pain unpleasantness. Clin Neurophysiol 2004;115:1846–56. Beydoun A, Dyke DB, Morrow TJ, Casey KL. Topical capsaicin selectively attenuates heat pain and A delta fiber-mediated laser-evoked potentials. Pain 1996;65:189–96.

1435

Beydoun A, Morrow TJ, Shen JF, Casey KL. Variability of laser-evoked potentials: attention, arousal and lateralized differences. Electroencephalogr Clin Neurophysiol 1993;88:173–81. Bjerring P, Arendt-Nielsen L. Argon laser induced single cortical responses: a new method to quantify pre-pain and pain perceptions. J Neurol Neurosurg Psychiatry 1988;51:43–9. Bragard D, Chen AC, Plaghki L. Direct isolation of ultra-late (C-fibre) evoked brain potentials by CO2 laser stimulation of tiny cutaneous surface areas in man. Neurosci Lett 1996;209:81–4. Bromm B, Treede RD. Human cerebral potentials evoked by CO2 laser stimuli causing pain. Exp Brain Res 1987a;67:153–62. Bromm B, Treede RD. Pain related cerebral potentials: late and ultralate components. Int J Neurosci 1987b;33:15–23. Callsen MG, Moller AT, Sorensen K, Jensen TS, Finnerup NB. Cold hyposensitivity after topical application of capsaicin in humans. Exp Brain Res 2008;191: 447–52. Carmon A, Dotan Y, Sarne Y. Correlation of subjective pain experience with cerebral evoked responses to noxious thermal stimulations. Exp Brain Res 1978;33: 445–53. Casanova-Molla J, Grau-Junyent JM, Morales M, Valls-Sole J. On the relationship between nociceptive evoked potentials and intraepidermal nerve fiber density in painful sensory polyneuropathies. Pain 2011;152:410–8. Chao CC, Hsieh SC, Tseng MT, Chang YC, Hsieh ST. Patterns of contact heat evoked potentials (CHEP) in neuropathy with skin denervation: correlation of CHEP amplitude with intraepidermal nerve fiber density. Clin Neurophysiol 2008;119:653–61. Chen AC, Niddam DM, Arendt-Nielsen L. Contact heat evoked potentials as a valid means to study nociceptive pathways in human subjects. Neurosci Lett 2001;316:79–82. Chen IA, Hung SW, Chen YH, Lim SN, Tsai YT, Hsiao CL, et al. Contact heat evoked potentials in normal subjects. Acta Neurol Taiwan 2006;15:184–91. de Tommaso M, Difruscolo O, Sardaro M, Libro G, Pecoraro C, Serpino C, et al. Effects of remote cutaneous pain on trigeminal laser-evoked potentials in migraine patients. J Headache Pain 2007;8:167–74. de Tommaso M, Losito L, Difruscolo O, Sardaro M, Libro G, Guido M, et al. Capsaicin failed in suppressing cortical processing of CO2 laser pain in migraine patients. Neurosci Lett 2005;384:150–5. Garcia-Larrea L, Convers P, Magnin M, Andre-Obadia N, Peyron R, Laurent B, et al. Laser-evoked potential abnormalities in central pain patients: the influence of spontaneous and provoked pain. Brain 2002;125:2766–81. Garcia-Larrea L, Peyron R, Laurent B, Mauguiere F. Association and dissociation between laser-evoked potentials and pain perception. Neuroreport 1997;8: 3785–9. Granovsky Y, Granot M, Nir RR, Yarnitsky D. Objective correlate of subjective pain perception by contact heat-evoked potentials. J Pain 2008;9:53–63. Granovsky Y, Matre D, Sokolik A, Lorenz J, Casey KL. Thermoreceptive innervation of human glabrous and hairy skin: a contact heat evoked potential analysis. Pain 2005;115:238–47. Greffrath W, Baumgartner U, Treede RD. Peripheral and central components of habituation of heat pain perception and evoked potentials in humans. Pain 2007;132:301–11. Iannetti GD, Hughes NP, Lee MC, Mouraux A. Determinants of laser-evoked EEG responses: pain perception or stimulus saliency? J Neurophysiol 2008;100:815–28. Iannetti GD, Zambreanu L, Tracey I. Similar nociceptive afferents mediate psychophysical and electrophysiological responses to heat stimulation of glabrous and hairy skin in humans. J Physiol 2006;577:235–48. Iannetti GD, Leandri M, Truini A, Zambreanu L, Cruccu G, Tracey I. Adelta nociceptor response to laser stimuli: selective effect of stimulus duration on skin temperature, brain potentials and pain perception. Clin Neurophysiol 2004;115:2629–37. Jensen TS, Gottrup H, Sindrup SH, Bach FW. The clinical picture of neuropathic pain. Eur J Pharmacol 2001;429:1–11. Kilo S, Schmelz M, Koltzenburg M, Handwerker HO. Different patterns of hyperalgesia induced by experimental inflammation in human skin. Brain 1994;117(Pt 2):385–96. Klein T, Magerl W, Rolke R, Treede RD. Human surrogate models of neuropathic pain. Pain 2005;115:227–33. Koltzenburg M, Lundberg LE, Torebjork HE. Dynamic and static components of mechanical hyperalgesia in human hairy skin. Pain 1992;51:207–19. LaMotte RH, Lundberg LE, Torebjork HE. Pain, hyperalgesia and activity in nociceptive C units in humans after intradermal injection of capsaicin. J Physiol 1992;448:749–64. LaMotte RH, Shain CN, Simone DA, Tsai EF. Neurogenic hyperalgesia: psychophysical studies of underlying mechanisms. J Neurophysiol 1991;66: 190–211. Le Pera D, Valeriani M, Niddam D, Chen AC, Arendt-Nielsen L. Contact heat evoked potentials to painful and non-painful stimuli: effect of attention towards stimulus properties. Brain Topogr 2002;15:115–23. Magerl W, Ali Z, Ellrich J, Meyer RA, Treede RD. C- and A delta-fiber components of heat-evoked cerebral potentials in healthy human subjects. Pain 1999;82: 127–37. Miltner W, Johnson Jr R, Braun C, Larbig W. Somatosensory event-related potentials to painful and non-painful stimuli: effects of attention. Pain 1989;38:303–12. Mouraux A, Guerit JM, Plaghki L. Refractoriness cannot explain why C-fiber laserevoked brain potentials are recorded only if concomitant Adelta-fiber activation is avoided. Pain 2004;112:16–26.

1436

C.S. Madsen et al. / Clinical Neurophysiology 123 (2012) 1429–1436

Mouraux A, Iannetti GD. A review of the evidence against the ‘‘first come first served’’ hypothesis. Comment on Truini et al. [Pain 2007;131:43–7]. Pain 2008;136:219–21 [author reply 222–3]. Mouraux A, Iannetti GD, Plaghki L. Low intensity intra-epidermal electrical stimulation can activate Adelta-nociceptors selectively. Pain 2010;150: 199–207. Nolano M, Simone DA, Wendelschafer-Crabb G, Johnson T, Hazen E, Kennedy WR. Topical capsaicin in humans: parallel loss of epidermal nerve fibers and pain sensation. Pain 1999;81:135–45. Ohara S, Crone NE, Weiss N, Treede RD, Lenz FA. Amplitudes of laser evoked potential recorded from primary somatosensory, para sylvian and medial frontal cortex are graded with stimulus intensity. Pain 2004;110:318–28. Perkins FM, Werner MU, Persson F, Holte K, Jensen TS, Kehlet H. Development and validation of a brief, descriptive Danish pain questionnaire (BDDPQ). Acta Anaesthesiol Scand 2004;48:486–90. Plaghki L, Delisle D, Godfraind JM. Heterotopic nociceptive conditioning stimuli and mental task modulate differently the perception and physiological correlates of short CO2 laser stimuli. Pain 1994;57:181–92. Rage M, Van Acker N, Facer P, Shenoy R, Knaapen MW, Timmers M, et al. The time course of CO2 laser-evoked responses and of skin nerve fibre markers after topical capsaicin in human volunteers. Clin Neurophysiol 2010;121:1256–66. Ringkamp M, Peng YB, Wu G, Hartke TV, Campbell JN, Meyer RA. Capsaicin responses in heat-sensitive and heat-insensitive A-fiber nociceptors. J Neurosci 2001;21:4460–8. Roberts K, Papadaki A, Goncalves C, Tighe M, Atherton D, Shenoy R, et al. Contact heat evoked potentials using simultaneous EEG and fMRI and their correlation with evoked pain. BMC Anesthesiol 2008;8:8. Roberts K, Shenoy R, Anand P. A novel human volunteer pain model using contact heat evoked potentials (CHEP) following topical skin application of transient receptor potential agonists capsaicin, menthol and cinnamaldehyde. J Clin Neurosci 2011;7:926–32. Romaniello A, Arendt-Nielsen L, Cruccu G, Svensson P. Modulation of trigeminal laser evoked potentials and laser silent periods by homotopical experimental pain. Pain 2002;98:217–28. Siedenberg R, Treede RD. Laser-evoked potentials: exogenous and endogenous components. Electroencephalogr Clin Neurophysiol 1996;100:240–9.

Szolcsanyi J, Anton F, Reeh PW, Handwerker HO. Selective excitation by capsaicin of mechano-heat sensitive nociceptors in rat skin. Brain Res 1988;446:262–8. Treede RD, Kief S, Holzer T, Bromm B. Late somatosensory evoked cerebral potentials in response to cutaneous heat stimuli. Electroencephalogr Clin Neurophysiol 1988;70:429–41. Treede RD, Lorenz J, Baumgartner U. Clinical usefulness of laser-evoked potentials. Neurophysiol Clin 2003;33:303–14. Treede RD, Meyer RA, Raja SN, Campbell JN. Peripheral and central mechanisms of cutaneous hyperalgesia. Prog Neurobiol 1992;38:397–421. Truini A, Galeotti F, Cruccu G, Garcia-Larrea L. Inhibition of cortical responses to Adelta inputs by a preceding C-related response: testing the ‘‘first come, first served’’ hypothesis of cortical laser evoked potentials. Pain 2007a;131:341–7. Truini A, Galeotti F, Pennisi E, Casa F, Biasiotta A, Cruccu G. Trigeminal small-fibre function assessed with contact heat evoked potentials in humans. Pain 2007b;132:102–7. Tzabazis AZ, Klukinov M, Crottaz-Herbette S, Nemenov MI, Angst MS, Yeomans DC. Selective nociceptor activation in volunteers by infrared diode laser. Mol Pain 2011;7:18. Valeriani M, Le Pera D, Niddam D, Chen AC, Arendt-Nielsen L. Dipolar modelling of the scalp evoked potentials to painful contact heat stimulation of the human skin. Neurosci Lett 2002;318:44–8. Valeriani M, Arendt-Nielsen L, Le Pera D, Restuccia D, Rosso T, De Armas L, et al. Short-term plastic changes of the human nociceptive system following acute pain induced by capsaicin. Clin Neurophysiol 2003;114:1879–90. Valeriani M, Tinazzi M, Le Pera D, Restuccia D, De Armas L, Maiese T, et al. Inhibitory effect of capsaicin evoked trigeminal pain on warmth sensation and warmth evoked potentials. Exp Brain Res 2005;160:29–37. Warbrick T, Derbyshire SW, Bagshaw AP. Optimizing the measurement of contact heat evoked potentials. J Clin Neurophysiol 2009;26:117–22. Woolf CJ. Central sensitization: implications for the diagnosis and treatment of pain. Pain 2011;152:S2–15. Woolf CJ, Mannion RJ. Neuropathic pain: aetiology, symptoms, mechanisms, and management. Lancet 1999;353:1959–64. Wu Q, Garcia-Larrea L, Mertens P, Beschet A, Sindou M, Mauguiere F. Hyperalgesia with reduced laser evoked potentials in neuropathic pain. Pain 1999;80: 209–14.