laser-evoked potentials - C-fibers - nociception - pain perception
potenciais evocados a laser - fibras C - nocicepção - percepção dolorosa
The criteria for diagnosis of definite neuropathic pain depend on a plausible neuroanatomic
distribution of the pain, a history suggestive of somatosensory lesion or disease
and at least one objective confirmatory test of the existence of such relevant somatosensory
lesion or disease[1]. Somatosensory evoked potentials (SEPs) and laser evoked potentials (LEPs) are becoming
“standards” to document possible involvement of the neural systems in patients with
neuropathic pain: the sensitivity of LEP to small lesions on the involved pathways
are conferring to it medical legal value in some European countries[2]. Although exquisitely sensitive to spinothalamic impairments, LEPs are considered
as a supportive tool to diagnose small fiber neuropathies (given their poor localization
value)[3]. These observations suggest that the search for alternative stimulus sources to
obtain LEPs, aiming to reduce the risks of complications (e.g. skin burns) and the
costs of the procedure, are important to increase the availability of these exams;
further, the stimulus should be able to allow separate evaluations of Aδ and C fibers.
Laser heat stimulators have been extensively used to study time-locked nociception
responses, as they provide a near-ideal method to selectively activate cutaneous Aδ-fiber
and C-fiber nociceptors. The high power outputs of lasers, which allow fast heat ramps,
concomitantly activate these two systems and produce a dual perception compatible
with conduction in small myelinated Aδ and unmyelinated C fibers[4].
Cortical responses to high intensity stimulation, however, do not reflect such dual
activation. Scalp recordings of LEP show a major negative-positive wave (N2-P2) in
the latency range of 200-400 ms when stimulating the hand dorsum. These results have
been associated with Aδ-fiber activity, with none of the responses at latencies related
to signals ascending through C-fibers[5]
,
[6]. Responses mediated exclusively by C-fibers, an ultra-late LEP (ULEP) at a latency
of about 1000 ms, can be elicited only by special methods that allow their selective
activation, such as low-power heating of the skin below Aδ threshold[7]
,
[8], pressure nerve block[9] or the stimulation of tiny skin surfaces[10]. It is not clear yet why concomitant activation of Aδ- and C-fibers does not allow
the individualization of both late and ultra-late LEPs[6]
,
[11]
,
[12]
,
[13], interestingly, depending on the intensity of stimulation, both responses can occasionally
be observed[7], also related to this issue is the recent suggestion that laser pulses may also
occasionally, in special circumstances, lead to tactile sensations[14].
Among the methods used to selectively activate C-fibers, stimulation of tiny areas
of skin requires few adjustments of the laser stimulator. This process can be implemented
by interposing a thin plate, drilled with one or more small holes, between the stimulus
probe and the skin surface in order to act as a spatial filter for the laser beam[15]
,
[16]
,
[17]. The principle of this method is based on higher innervation density of C-fiber
terminals on the skin (three or four times more numerous than Aδ-fibers in humans),
resulting in a higher probability of stimulating the terminals of C-fibers than those
of Aδ-fibers[18].
Different laser emission sources have been used to elicit evoked responses in clinical
studies, with their practical differences relying on the physical properties of the
different wavelengths: argon (488-515 nm), copper vapor (510-577 nm), neodymium-YAG
(1,064 nm), thulium-YAG (2,000 nm) and diode lasers (700-1,000 nm); the most commonly
used is the infrared CO2 laser, which has been supported by a large number of studies[19]. However, as flexible optical fibers do not conduct the CO2 infrared heat well, the use of extra devices to orient the laser target over the skin
becomes necessary.
Developments in the field of semiconductors have turned diode lasers into a good choice
for pain stimulation in basic and clinical applications. Practical advantages of this
laser include its small size, low price and ability to transmit its output heat pulse
via a flexible quartz fiber. One disadvantage of diode lasers is their sensitivity
to skin pigmentation. Variations in pigmentation alter the penetration depth of the
heat pulse and, consequently, the amount of energy delivered to the receptors[20]
,
[21]. To minimize this variability, some studies have blackened the target area of the
hand dorsum using carbon black with a high emissivity factor, assuring that energy
is absorbed uniformly on the superficial layer of the skin and can reach the nearby
receptors by passive heat conduction[22]
,
[23]. To date, only a few investigators have focused on human LEP elicited by diode lasers,
which have applied high-power intensities to obtain only Aδ responses[24]
,
[25] or have observed simultaneous Aδ- and C-fiber responses that were restricted to
a single subject[26].
Previously we were able to record LEPs related to Aδ fibers using a low power diode
laser after skin blackening[7], In the present study, we investigated the feasibility of selectively activating
C-fiber responses in healthy volunteers using a low-power diode laser applied to tiny
areas of blackened skin.
Subjects
Twenty healthy Caucasians volunteers participated in this study (18 males, 2 females,
26.3 ± 4.5 years old). They were previously screened for medical and neurological
conditions that might affect normal somatosensory perception, as well as for smoking,
alcohol and substance abuse. All subjects gave written informed consent before participating
in this study, which had the approval of the Ethics Committee of the Federal University
of Sao Paulo (UNIFESP-1592/06).
Experimental procedure
Subjects were first presented with the stimulation device and an explanation of the
experimental procedures. During the experiments, they stayed in a quiet room on a
reclining armchair and were instructed to remain awake with their eyes open, staring
at a point on the wall in front of them to minimize eye movements. The right forearm
and hand of the volunteers were immobilized over a table by a specially designed orthosis.
The experiment lasted approximately 30 minutes per subject; during this period, the
temperature of the experimental room was kept constant.
Laser stimulation
The stimulus consisted of short laser pulses of 50 ms duration, applied to the dorsum
of the right hand by a diode laser device of 810 nm wavelength (FTC2000, Opto Laser).
The stimulated area of the skin was previously blackened with water-based ink (high
emissivity factor). To selectively activate the C-fibers, we attached an aluminum
plate that had one thin hole drilled (0.1 mm diameter) at the top of the stimulus
probe. A servomotor device, controlled by a PC running LabView software (National
Instruments, USA), was developed to program the stimulation protocol and to slightly
displace the target site of the collimated beam between trials; the stimulus spot
moved in a circular pathway with a diameter of approximately 6 cm on the dorsum of
the hand to avoid receptors adaptation. The laser output power was rechecked using
an optical meter after the stimulator system was assembled to consider losses imposed
by the transmitting optical fiber, the collimator lens and the aluminum dot plate.
Experimental procedure
Before EEG acquisition, subjects were presented with single laser pulses with increasing
intensities – starting from a very low intensity level (sub-threshold) and steadily
increasing until a warm sensation could be perceived by the volunteer. This intensity
was noted and designated the warmth perception threshold (WPT).
One-hundred laser pulses were automatically delivered to subjects with an inter-stimulus
interval of 5 s. The laser intensity was constant at approximately twice the perception
threshold. After stimulation, volunteers were prompted to rate the intensity of perception
by pointing to a visual analog scale (VAS), ranging from 0 (no sensation) to 10 (maximum
pain). In addition, to focus their attention on the stimulation site, they were asked
to mentally count the perceived number of total laser pulses.
Laser-evoked potential
Brain electrical activity was recorded on Cz referenced to linked earlobes, using
polysomnography equipment (S7000, Embla Systems, USA) at the facilities of the Sleep
Institute. The sampling rate was set to 200 Hz, and the internal analog filtering
of the equipment was set to 0.3-90 Hz, besides the 60 Hz powerline rejection. To monitor
ocular movements and eye-blinks and to discard EEG contaminated trials, electrooculographic
(EOG) signals were simultaneously recorded with surface electrodes. Impedances were
kept bellow 5 kΩ. Signal processing routines were written in Matlab software (Mathworks,
USA) to perform off-line averaging of the EEG sweeps and calculate the laser-evoked
potential.
RESULTS
The WPT was 41 ± 25 mW for the set of volunteers and the mean intensity used for their
stimulation was 70 ± 32 mW. Subjects often described the perception of their stimulus
as a warm sensation or a bearable pain.
Repetitive laser stimulation elicited clear and reproducible ULEPs for all the subjects
([Figure 1]) and was characterized by a negative-positive deflection at the latencies of 806
± 61 ms (N2) and 1033 ± 60 ms (P2). The low inter-individual variability of N2-P2
components resulted in a well-defined grand average ([Figure 2]).
Figure 1 Evoked potentials of the subjects. Evoked potentials following laser stimulation
of the twenty subjects. The ultra-late component of LEP can be consistently observed
at a latency of ~1 s.
Figure 2 Grand average. Grand average of the evoked responses.
The VAS results, which indicate the mean perceived laser intensity perception, varied
by a wide range (2 ± 2), most likely reflecting their subjective nature. The pulse
count was 66 ± 20, corresponding to approximately 66% of the total pulses in each
set (100). These results are summarized in the [Table].
Table
Results obtained after laser stimulation of the subjects’ right hand.
|
Mean ± SD
|
|
WPT (mW)
|
41 ± 25
|
|
VAS (0-10)
|
2 ± 2
|
|
Stimulation intensity (mW)
|
70 ± 32
|
|
Ratio : Stimulation Intensity / WPT
|
1.9 ± 0.2
|
|
Latency N2 (ms)
|
806 ± 61
|
|
Latency P2 (ms)
|
1,033 ± 60
|
|
Pulse count (%)
|
66 ± 20
|
WPT: warm perceptual threshold; VAS: visual analogic scale; SD: standard deviation.
DISCUSSION
Our results indicate that reproducible C-fiber evoked responses can be obtained using
a low-power diode laser applied to tiny areas of blackened skin. Using this stimulation
protocol, the ULEP was consistently elicited with latencies in accordance with those
reported in other studies that used high-power CO2 lasers applied to small areas of natural (non-blackened) skin[10]
,
[27]
,
[28]
,
[29]
,
[30]. The few investigators that have previously employed the darkening procedure reported
only Aδ responses after high-intensity stimulation in three subjects[5] or have not focused on laser-evoked potentials, but on subjective pain and thermal
thresholds[21]
,
[31]. In a previous study we were also able to study Aδ responses using the same methodology
except for a larger area of stimulation[7].
When comparing our data with other ULEP studies, one must be aware of the differences
in the referred stimulus intensities. In the present investigation, laser output power
was rechecked after the aluminum plate was installed, and a calibration curve was
constructed to correct the output power indicated by the equipment. Somewhat surprisingly,
real intensities were approximately 10% of those displayed by the equipment. Most
of the output energy was, in fact, blocked by the small hole in the aluminum plate
– when the plate was removed, the measured power increased to 55% of the equipment’s
indication; the remaining losses were most likely due to the collimator lens and the
optical fiber transmission. Moreover, darkening of the skin is known to drastically
modify the heat pattern on the laser spot. Leandri and colleagues[23] carried out a detailed measurement of the skin temperature during and immediately
after irradiation of white and blackened skin with CO2 and Nd:YAP lasers. Their report indicates that this procedure matched the thermal
effects of both lasers, homogenizing the way receptors at different depths are activated.
When the same stimulus was delivered to both types of skin, temperatures and perceived
pain were consistently higher for the blackened skin. Consequently, lower stimulus
intensities can be employed to elicit C-fiber responses, which reduces the possibility
of damage to the skin. We used a very low intensity range that was near the evoked
response threshold, as suggested by the low power (in the mW range) and confirmed
by the relatively low number of laser pulses counted by the volunteers (66%). With
such approach, none of the subjects in our study developed superficial burns on the
skin and clear evoked reponses were recorded in all of them. It should be pointed
out that even dealing with low power lasers, care need to be exercised since noxious
levels of stimulation are intimately related to potential or actual tissue injury.
In conclusion, the present investigation showed that combining a skin-blackening procedure
with a tiny area of stimulation technique allows the selective activation of C-fiber
evoked responses by the use of a very low-power diode laser. This strategy offers
a non-invasive and easy to implement methodology that minimizes damage to the tissue.
