Event-related potentials evoked by skin puncture reflect activation of Aβ fibers: comparison with intraepidermal and transcutaneous electrical stimulations

Subjects

Eighteen healthy volunteers (13 males and five females; 19–34 years old) participated in this study (eight volunteers in Experiment 1 and 18 in Experiment 2). Their height was 153.0–180.0 cm (169.3 ± 7.1 cm). They were not undergoing any medical treatments and did not take analgesic drugs within the 48 h prior to the experiment. The study was approved by the Ethics Committee of Hiroshima University (approval number: E-2044) and written consent was obtained from all subjects.

Experimental procedure

Three types of stimulation

Click-only.

We used a lancet (BD Microtainer Quikheel™ Lancet 368102, Japan Becton, Dickinson, Japan) that is clinically used for the heel lance of neonates. The lance device produces a click sound when a button on the device is pressed. The click sound was produced four times near the dorsum of the right hand without skin puncture.

Click+SL.

The lancet housed a 2.5 mm spring-loaded blade, which is released by pressing a button on the top of the device. After creating the incision, the blade automatically and immediately retracts back inside the device. The original penetration depth was 1 mm. To minimize the tactile sensation caused by the device touching the skin and the activation of Aβ fibers by the blade reaching the dermis (Munger & Halata, 1983; Mouraux, Iannetti & Plaghki, 2010), we placed a 0.8 mm plate between the device and the skin; we also fixed the device to the plate using a fixing stand (NISSIN SEIKI CO., LTD., Japan), which created a space of approximately 0.1 mm between the plate and the skin surface (Fig. 1). Thus, the blade was inserted to a depth of 0.1 mm into the epidermis, where the free nerve endings of Aδ fibers are located (Novotny & Gommert-Novotny, 1988; Inui et al., 2002). We performed the click+SL procedure four times. The stimulus location was changed for each stimulation to avoid habituation.

WN+SL.

The white noise was presented over headphones. We confirmed that the subjects did not hear the click sound from the lance device. We performed the stimulation at least four times and added the number of times when the peak-to-peak amplitude of the EP was below 20 µV (4–5 times). The stimulus location was changed for each stimulation to avoid habituation.

Sample size calculation.

The sample size was calculated using the following formula: (1)${\displaystyle \begin{array}{c}\hfill \mathrm{n}=\frac{{\lambda }^2{C}^2}{e^2}=17.59\hfill \end{array}}$ where λ is 1.96 (95% confidence interval), C is the coefficient of variance (0.107) obtained from our previous study (Kirimoto et al., 2018), and e is the acceptable error rate of 0.05. Based on this calculation, the sample size was set as 18 in the present study.

Recording EPs

EPs were recorded using an amplifier (FA-DL-160, 4 Assist, Japan) with Cz (International 10–20 system) as the active electrode because the maximum response has previously been recorded from the Cz derivation in both electrical and lance stimulations (Kakigi, Shibasaki & Ikeda, 1989; Slater et al., 2010b; Otsuru et al., 2010). The earlobe (A2) was used as a reference. For the ground electrode, a disposable gel electrode was placed on the right forearm (GE Health Care Japan, Tokyo, Japan). The recording was made at a sampling rate of 4,000 Hz with a bandpass filter of 0.1–50.0 Hz. In Experiment 1, average waveforms were created from at least four artifact-free EPs (4–5 times) for the click-only, click+SL, and WN+SL conditions. In Experiment 2, average waveforms were created from 12 artifact-free EPs for ES and IES, and from at least four artifact-free EPs (4–6 times) for SL and CL. The EPs for the click-only, click+SL, WN+SL, SL, and CL were time-locked to the time at which an accelerometer (FA-DL-111; 4 Assist, Japan) attached to the experimenter’s finger detected the start of lancet button pressing (movement of finger); when the button was pressed, the blade was released from the device to incise the skin. The time lag from when the lancet button started to be pressed until the blade reached the skin was about 2 ms or less as measured by a high-speed camera (1,200 frames/s), which was considered negligible here.

Data and statistical analysis

ES and IES evoked negative–positive waveforms (N2–P2) with different latencies. The peak latencies of N2 and P2 for the ES were measured between 120–180 ms and 180–280 ms, respectively, and these values for the IES were measure between 170–250 ms and 250–350 ms, respectively (Inui et al., 2002; Mouraux, Iannetti & Plaghki, 2010; Otsuru et al., 2010; Kodaira, Inui & Kakigi, 2014; Mouraux, Marot & Legrain, 2014; Omori et al., 2017; Kirimoto et al., 2018). Click-only, click+SL, WN+SL, SL, and CL also evoked N2–P2. For the click-only, click+SL, WN+SL, SL, and CL conditions, we created the grand average of individual ERPs across all subjects and then selected the N2 and P2 peaks in the individual ERP waveforms that were located closest to those in the grand average (Fabrizi et al., 2013).

In Experiment 1, we compared the N2 and P2 latencies and N2–P2 amplitudes among the three stimulation conditions (i.e., click-only, click+SL, and WN+SL) using a one-way repeated-measures ANOVA. In Experiment 2, we compared the N2 and P2 latencies and N2–P2 amplitudes among the four stimulation conditions (i.e., SL, CL, ES, and IES) using a one-way repeated-measures ANOVA. A Bonferroni correction was used for the post hoc analysis. The VAS score was compared among the four stimulation conditions (i.e., SL, CL, ES, and IES) initially with a Friedman test and then with a Wilcoxon signed-rank test for post hoc analysis. The correlations between the VAS score and N2 or P2 latencies and between the VAS score and N2–P2 amplitudes were calculated using Spearman’s rank correlation coefficients. SAS9.4 was used to perform all statistical analyses.

Experiment 1

Figure 2 shows the grand averaged waveforms of potentials evoked by click-only, click+SL, and WN+SL (N2–P2). The mean and SD of the peak latencies for N2 and P2 are shown in Table 1.

The mean and individual latencies of N2 and P2 for each stimulation condition (click-only, click+SL, and WN+SL) are summarized in Fig. 3. A one-way repeated-measures ANOVA showed that the latencies of both N2 and P2 were significantly different among the three stimulation conditions (N2: F_(2,14) = 120.3, P < 0.0001, η²_p = 0.9450; P2: F_(2,14) = 50.89, P < 0.0001, η²_p = 0.8791). Post hoc analyses showed that N2 latencies for the click-only (122 ± 2.9 ms) and click+SL (121 ± 6.5 ms) were significantly shorter than that for the WN+SL (154 ± 7.1 ms: P < 0.0001 for click-only; P < 0.0001 for click+SL). There was no significant difference in N2 latency between the click-only and the click+SL. The P2 latency for the click-only (191 ± 11.3 ms) was significantly shorter than that for the click+SL (249 ± 18.6 ms: P < 0.0001) and that for the WN+SL (253 ± 11.2 ms: P < 0.0001). There was no significant difference in P2 latency between the click+SL and the WN+SL.

A one-way repeated-measures ANOVA showed that the P2 amplitude was also significantly different among the three stimulation conditions (F_(2,14) = 6.32, P = 0.0111, η²_p = 0.4744), but there was no significant difference in N2 amplitude among the three stimulation conditions (Table 1). Post hoc analyses showed that the P2 amplitude for the click-only condition (14.8 ± 5.0 µV) was significantly shorter than those for the click+SL (21.4 ± 4.9 µV: P = 0.0422) and WN+SL (24.7 ± 4.5 µV: P = 0.0257). There was no significant difference in P2 amplitude between the click+SL and the WN+SL.

Experiment 2

Figure 4 shows the grand averaged waveforms of potentials evoked by SL, CL, ES, and IES (N2–P2). The mean and SD of the peak latencies for N2 and P2 are shown in Table 2.

The mean and individual latencies of N2 and P2 for each stimulation condition (SL, CL, ES, and IES) are summarized in Fig. 5. A one-way repeated-measures ANOVA showed that the latencies of both N2 and P2 were significantly different among the four stimulation conditions (N2: F_(3,51) = 65.51, P < 0.0001, η²_p = 0.7940; P2: F_(3,51) = 40.54, P < 0.0001, η²_p = 0.7045). Post hoc analyses showed that N2 latencies for the SL (146 ± 8.3 ms), CL (149 ± 9.9 ms), and ES (148 ± 13.1 ms) were significantly shorter than that for the IES (197 ± 21.2 ms: P < 0.0001 for SL; P < 0.0001 for CL; P < 0.0001 for ES). The P2 latencies for the SL (250 ± 18.2 ms), CL (251 ± 14.1 ms), and ES (237 ± 26.3 ms) were significantly shorter than that for the IES (294 ± 30.0 ms: P < 0.0001 for SL; P < 0.0001 for CL; P < 0.0001 for ES). There was no significant difference in the N2 and P2 latencies between SL, CL, and ES.

A one-way repeated-measures ANOVA showed that the N2–P2 amplitudes were also significantly different among the four stimulation conditions (F_(3,51) = 11.70, P < 0.0001, η²_p = 0.4076) (Table 2). Post hoc analyses showed that N2–P2 amplitudes for the SL (33.0 ± 8.7 µV) and CL (38.1 ± 11.5 µV) were significantly larger than that for the ES (22.6 ± 4.3 µV: P = 0.0025 for SL; P = 0.0001 for CL). There was no significant difference in N2–P2 amplitudes between the SL and the CL.

The Friedman test indicated that the VAS scores differed significantly among the four stimulation conditions (P < 0.0001) (Table 2). Post hoc analyses indicated that the VAS score for the SL (6.4 ± 4.7 points) was significantly smaller than that for the CL (17.9 ± 10.6 points: P < 0.0001) and larger than that for the ES (3.8 ± 3.7 points: P = 0.0348). The VAS score for the CL was significantly larger than those for the ES (P < 0.0001) and IES (8.4 ± 6.6 points: P < 0.0001). In addition, the VAS score for the IES was significantly larger than that for the ES (P = 0.0002). There was no significant difference in the VAS scores between the SL and the IES. There were no correlations between the VAS scores and N2 or P2 latencies, or between the VAS scores and N2–P2 amplitudes, at any stimulation condition.

Effect of the click sound on the ERPs evoked by skin puncture

When a click sound was present (click-only and click+SL), the N2 latencies of ERPs in response to click-only and click+SL and the P2 latencies of ERPs in response to the click-only condition were within the range reported in previous studies investigating ERPs of auditory stimuli (Wolpaw & Penry, 1975; Knight et al., 1980; Carrillo-de-la-Peña & Cadaveira, 2000; Atcherson et al., 2006; Gondan & Röder, 2006; Gondan, Vorberg & Greenlee, 2007; Touge et al., 2008; Sakamoto, Nakata & Kakigi, 2009; Sperdin et al., 2009). N100, which can be observed in response to simple short auditory stimuli, reflects multiple mechanisms and is thought to originate from a wide range of areas (Näätänen & Picton, 1987; Martin, Tremblay & Korczak, 2008; Sakamoto, Nakata & Kakigi, 2009), such as Heschl’s gyrus, the temporal lobe (Okamoto et al., 2007; Altmann et al., 2008), auditory association areas (Knight et al., 1980), and frontal areas (Wolpaw & Penry, 1975; Atcherson et al., 2006). P200, which can be observed following N100, has not been investigated to the same extent as N100, but is also thought to originate from a variety of areas in the cortex (Wolpaw & Penry, 1975; Näätänen & Picton, 1987; Martin, Tremblay & Korczak, 2008). Although the exact mechanisms behind the ERPs evoked by auditory stimuli have not been fully determined, the click sound produced by the lance device appears to generate an auditory evoked potential.

Possibility of multimodal ERPs evoked by lance stimulation

In the present study, the N2 latencies of ERPs generated by click+SL were similar to those of click-only, and the P2 latencies of ERPs in response to click+SL were similar to those of SL (without the click sound), indicating that lance stimulation elicits an ERP composed of multimodal stimuli, namely the click sound and skin puncture. In previous studies on multimodal brain responses, multimodal ERPs were reported to be not simply a sum of the ERPs of each unimodal stimulus (Gondan & Röder, 2006; Gondan, Vorberg & Greenlee, 2007; Touge et al., 2008; Sperdin et al., 2009; Brett-Green et al., 2010; Dionne, Legon & Staines, 2013). Specifically, in these studies, ERPs generated by unimodal stimuli (i.e., auditory (A) or visual (V) alone) and ERPs of bimodal stimuli (i.e., AV) were recorded, and the ERPs of bimodal stimuli were compared with the sum of the two unimodal ERPs (i.e., A + V) (Foxe et al., 2000; Gondan & Röder, 2006; Gondan, Vorberg & Greenlee, 2007; Touge et al., 2008; Brett-Green et al., 2010). Their results revealed that the ERPs of bimodal stimuli did not match the sum of the two unimodal ERPs (i.e., AV ≠ (A + V)) (Foxe et al., 2000; Gondan & Röder, 2006; Gondan, Vorberg & Greenlee, 2007; Touge et al., 2008; Brett-Green et al., 2010), meaning that the ERPs of bimodal stimuli reflect, in addition to the processing associated with the unimodal stimuli, brain activity related to the interaction of two different unimodal stimuli (Gondan & Röder, 2006; Gondan, Vorberg & Greenlee, 2007).

Similarly, previous studies on brain responses to multimodal auditory-somatosensory stimuli have shown that the N2 of ERPs of multimodal auditory-somatosensory stimuli were similar to those of unimodal auditory stimuli (click sound), while the P2 of ERPs of multimodal auditory-somatosensory stimuli were similar to those of unimodal somatosensory stimuli (electrical stimulation) (Touge et al., 2008; Brett-Green et al., 2010). Our finding is in agreement with this result, suggesting the interaction of auditory and somatosensory stimuli (Gobbelé et al., 2003). As the posterior parietal (Gobbelé et al., 2003), auditory (Foxe et al., 2000; Foxe et al., 2002; Gobbelé et al., 2003), and secondary somatosensory (SII) cortices (Lam et al., 1999; Lütkenhöner et al., 2002; Gobbelé et al., 2003) are reported to be involved in auditory-somatosensory information processing, these brain areas may have been activated during the lance stimulation. It can be speculated that lance stimulation (with the click sound) elicits a multimodal ERP of auditory and puncture stimuli, which is different from the sum of two unimodal ERPs and is considered to possibly indicate an interaction of these two stimuli.

Activation of Aβ fibers by SL

The latencies of the ERPs of the ES and IES in this study were within the range reported in previous studies (Inui et al., 2002; Mouraux, Iannetti & Plaghki, 2010; Otsuru et al., 2010; Kodaira, Inui & Kakigi, 2014; Mouraux, Marot & Legrain, 2014; Omori et al., 2017; Kirimoto et al., 2018). The IES produces an electrical current in the epidermis and selectively activates free nerve endings of Aδ fibers when low stimulation current intensities are applied (Mouraux, Iannetti & Plaghki, 2010). In contrast, the electrical current produced by the ES reaches the deeper dermis where receptors of Aβ fibers, which have a lower threshold than Aδ fibers, are located (Mouraux, Iannetti & Plaghki, 2010). These receptors respond to vibration, indentation, and forces applied to the skin (Johnson, 2001; Macefield, 2005). Therefore, the IES and ES used in the present study can be considered to have activated the free nerve endings of Aδ fibers in the epidermis and the receptors of Aβ fibers in the deeper dermis, respectively.

On the other hand, the latencies of the ERPs of the SL in the present study were not within a range reflecting the activation of Aδ fibers, even though the blade of the lance device was inserted to a depth of 0.1 mm into the epidermis where the free nerve endings of Aδ fibers are located, and the effect of click sound was removed. It is likely that the SL generated a penetration force when the blade was inserted into the skin, activating the Aβ fibers. The penetration force has been shown to be the largest force in the process of insertion (Meyer et al., 2014; Leonardi, Viganò & Nicolucci, 2019). For example, a venipuncture needle (27 and 30 gauge (G); 8 mm length) produces a penetration force of 0.23–0.34 N (Egekvist & Arendt-Nielsen, 1999), a hypodermic needle pen (31–34 G; 3.5–8.0 mm length) used for insulin injection produces a penetration force of 0.36–0.73 N (Leonardi, Viganò & Nicolucci, 2019), and a needle used for sclerotomy (23 G; 2.30–3.96 mm length) produces a penetration force of 0.48–2.16 N (Meyer et al., 2014). Although the blade of the SL has a different shape (about 0.1 mm in length and about 0.25 mm wide) from those needles listed above, we speculate that it generates a penetration force. Indeed, the N2 and P2 latencies of the lance ERPs in this study were similar to those of ERPs evoked by lance stimuli in previous studies (N2: 130 ± 40 ms, P2: 258 ± 61 ms (Fabrizi et al., 2013); N2: 102 ms, P2: 249.5 ms (Fabrizi et al., 2016)), and they were also within the range reported in previous studies examining ERPs generated by the stimulation of Aβ fibers (Inui et al., 2002; Mouraux, Iannetti & Plaghki, 2010; Otsuru et al., 2010). As reported in an early study comparing mechanical and transcutaneous ES, in short-latency somatosensory evoked potential components, such as N20, mechanical stimulation with a needle (pain) does not differ significantly from mechanical stimulation with a plastic ball (tactile) and transcutaneous ES (Kakigi & Shibasaki, 1984), supporting our argument. Meanwhile, one may argue that the increased N2–P2 amplitude in the CL and SL as compared to the ES can reflect the activation of Aδ fibers. However, given the latencies of N2 and P2, it is reasonable to assume that the CL and SL activated more cutaneous mechanoreceptors (e.g., vibration receptors in the deeper dermis layer) than the ES. Therefore, it is most likely that the SL activated the Aβ fibers by the penetration force associated with blade insertion, even though the blade was inserted only into the epidermis.

Mismatch between ERPs and pain perception

The pain score on the VAS for the IES was not different from that for the SL, but was smaller than that for the CL. Some previous studies have reported that ERP amplitude is associated with the intensity of pain perception (Beydoun et al., 1993; García-Larrea et al., 1997; Ohara et al., 2004; Iannetti et al., 2005). However, nociceptive brain responses have been observed without pain perception in other studies in which ERP was evoked by the activation of Aδ fibers using IES that did not reach the pain threshold (Hagiwara et al., 2018), by laser stimulation under anesthesia in monkeys (Baumgärtner et al., 2006), and by unperceived painful laser stimulation in humans (Lee, Mouraux & Iannetti, 2009). Additionally, ERPs evoked by electric and laser stimulations are known to be modulated by stimuli expectations (Hird et al., 2018). Indeed, in the present study, we found no correlation between the VAS score and ERP latency or between the VAS score and ERP amplitude under any stimulation conditions. Thus, it seems to be difficult to evaluate pain using ERP amplitude.

Moreover, it has been reported that painful stimuli do not induce nociceptive brain responses when Aβ fibers are activated simultaneously (Torquati et al., 2003; Rustamov et al., 2016). In this case, the ES current reaches the dermis where Aβ fiber receptors are found with lower activation thresholds than those of the free endings of Aδ fibers. Hence, even if the subjects felt pain with the SL, the Aβ fiber receptors could have been activated initially, which may have masked the activation of Aδ fibers.