Behavioral and Electrophysiological Study of Thermal and Mechanical Pain Modulation by TRP Channel Agonists
Transient receptor potential channels (TRP) have been extensively investigated over the past few years. Recent findings in the field of pain have established a family of six thermoTRP channels (TRPA1, TRPM8, TRPV1, TRPV2, TRPV3, and TRPV4) that exhibits sensitivity to increases or decreases in te...
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Інститут фізіології ім. О.О. Богомольця НАН України
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irk-123456789-1481262019-02-19T01:24:52Z Behavioral and Electrophysiological Study of Thermal and Mechanical Pain Modulation by TRP Channel Agonists Tsagareli, M.G. Nozadze, I.R. Gurtskaia, G.P. Carstens, M.I. Tsiklauri, N.J. Carstens, E.E. Transient receptor potential channels (TRP) have been extensively investigated over the past few years. Recent findings in the field of pain have established a family of six thermoTRP channels (TRPA1, TRPM8, TRPV1, TRPV2, TRPV3, and TRPV4) that exhibits sensitivity to increases or decreases in temperature, as well as to chemical substances eliciting the respective hot or cold sensations. Such irritants include menthol, cinnamaldehyde, gingerol, mustard oil, capsaicin, camphor, eugenol, and others. In this study, we used behavioral and electrophysiological methods to investigate if mustard oil (allyl isothiocyanate, AITC) and capsaicin affect the sensitivity to thermal, innocuous cold, and mechanical stimuli in male rats. Unilateral intraplantar injection of AITC and capsaicin induced significant decreases in the latency for ipsilateral paw withdrawal from a noxious heat stimulus, i.e., heat hyperalgesia. These agents also significantly reduced the mechanical withdrawal thresholds of the injected paw, i.e., mechanical allodynia. Bilateral intraplantar injections of AITC resulted in a two-phase effect on cold avoidance (thermal preference test). A low concentration of AITC (5%) did not change cold avoidance similarly to the vehicle control, while higher AITC concentrations (10 and 15%) significantly reduced cold avoidance, i.e., induced cold hypoalgesia. Capsaicin acted in almost the same manner. These results indicate that TRPA1 channels are clearly involved in pain reactions, and the TRPA1 agonist AITC enhances the heat pain sensitivity, possibly by indirectly modulating TRPV1 channels, which are co-expressed in nociceptors with TRPA1s. In electrophysiological experiments, neuronal responses to electrical and graded mechanical and noxious thermal stimulations were tested before and after cutaneous application of AITC. Repetitive application of AITC initially increased the firing rate of spinal wide-dynamic range neurons; this was followed by rapid desensitization that persisted when AITC application was reapplied 30 min later. The responses to noxious thermal (but not to mechanical) stimuli were significantly enhanced irrespective of whether the neuron was directly activated by AITC. These findings indicate that AITC produced peripheral sensitization of heat nociceptors. Overall, our data support the role of hermosensitive TRPA1 and TRPV1 channels in pain modulation and show that these thermoTRP channels are promising targets for the development of a new group of analgesic drugs. Канали транзієнтного рецепторного потенціалу (transient receptor potential channels, TRP) були відносно докладно вивчені протягом останніх п’яти років. Згідно з результатами сучасних досліджень у галузі болю існує сім’я канальних структур, що складається з шести видів термо-TRP-каналів (TRPA1, TRPM8, TRPV1, TRPV2, TRPV3 та TRPV4). Такі канали виявляють чутливість до збільшення або зменшення температури, а також до хімічних речовин, котрі викликають відповідне відчуття гарячого або холодного. До подібних агентів належать ментол, коричний альдегід, гінгерол, гірчична олія, капсаїцин, камфора, евгенол та ін. У нашому дослідженні ми використовували поведінкові та електрофізіологічні методи, щоб з’ясувати, чи впливають гірчична олія (аліл ізотіоціанат, АІТЦ) та капсаїцин на чутливість самців щурів до термальних, нешкідливих холодових або механічних стимулів. Унілатеральні внутрішньопідошовні ін’єкції АІТЦ та капсаїцину зумовлювали істотні скорочення латентного періоду відсмикування іпсілатеральної кінцівки від шкідливого гарячого стимулу, тобто теплову гіпералгезію. Ці агенти також істотно зменшували пороги відсмикування кінцівки при дії механічного стимулу на ін’єковану кінцівку, тобто викликали механічну алодінію. Білатеральні внутрішньопідошовні ін’iєкції АІТЦ приводили до двофазного впливу на реакцію уникання від холоду (тест термальної преференції). АІТЦ у низькій концентрації (5%) не змінював холодового уникання порівняно з контролем, тоді як у вищих концентраціях (10 та 15 %) істотно пригнічував реакцію уникання на холод, тобто індукував холодову гіпоалгезію. Капсаїцин діяв майже таким самим чином. Ці результати вказують на те, що TRPA1-канали безперечно залучені у больові реакції і що АІТЦ (агоніст TRPA1) посилює чутливість до теплового болю (можливо, через непряме модулювання TRPV1-каналів, котрі коекспресуються у ноцицепторах з TRPA1s). У електрофізіологічних експериментах нейронні відповіді на електричні та ступінчасті механічні та шкідливі термальні стимули тестувалися перед шкірною аплікацією АІТЦ та після такої дії. Повторна аплікація АІТЦ спочатку збільшувала частоту розрядів спінальних нейронів з широким динамічним діапазоном; це спричиняло швидку десенситизацію, котра утримувалася після аплікації АІТЦ, застосованої за 30 хв. Відповіді на шкідливу термальну (але не на механічну) стимуляцію істотно збільшувалися незалежно від того, чи активувався нейрон АІТЦ безпосередньо. Ці результати вказують на те, що АІТЦ спричинює периферичну сенситизацію теплових рецепторів. У цілому наші результати говорять на користь ролі термосенситивних TRPA1- та TRPV1-каналів у больовій модуляції та свідчать про те, що дані термо-ТRP-канали є перспективними мішенями для розвитку нової групи аналгетиків. 2013 Article Behavioral and Electrophysiological Study of Thermal and Mechanical Pain Modulation by TRP Channel Agonists / M. G. Tsagareli, I. R. Nozadze, G. P. Gurtskaia, M. I. Carstens, N. J. Tsiklauri, E. E. Carstens // Нейрофизиология. — 2013. — Т. 45, № 4. — С. 369-379. — Бібліогр.: 41 назв. — англ. 0028-2561 http://dspace.nbuv.gov.ua/handle/123456789/148126 612.88.577.25 en Нейрофизиология Інститут фізіології ім. О.О. Богомольця НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| description |
Transient receptor potential channels (TRP) have been extensively investigated over the past
few years. Recent findings in the field of pain have established a family of six thermoTRP
channels (TRPA1, TRPM8, TRPV1, TRPV2, TRPV3, and TRPV4) that exhibits sensitivity
to increases or decreases in temperature, as well as to chemical substances eliciting the
respective hot or cold sensations. Such irritants include menthol, cinnamaldehyde, gingerol,
mustard oil, capsaicin, camphor, eugenol, and others. In this study, we used behavioral
and electrophysiological methods to investigate if mustard oil (allyl isothiocyanate, AITC)
and capsaicin affect the sensitivity to thermal, innocuous cold, and mechanical stimuli in
male rats. Unilateral intraplantar injection of AITC and capsaicin induced significant
decreases in the latency for ipsilateral paw withdrawal from a noxious heat stimulus, i.e.,
heat hyperalgesia. These agents also significantly reduced the mechanical withdrawal
thresholds of the injected paw, i.e., mechanical allodynia. Bilateral intraplantar injections
of AITC resulted in a two-phase effect on cold avoidance (thermal preference test). A low
concentration of AITC (5%) did not change cold avoidance similarly to the vehicle control,
while higher AITC concentrations (10 and 15%) significantly reduced cold avoidance, i.e.,
induced cold hypoalgesia. Capsaicin acted in almost the same manner. These results indicate
that TRPA1 channels are clearly involved in pain reactions, and the TRPA1 agonist AITC
enhances the heat pain sensitivity, possibly by indirectly modulating TRPV1 channels,
which are co-expressed in nociceptors with TRPA1s. In electrophysiological experiments,
neuronal responses to electrical and graded mechanical and noxious thermal stimulations
were tested before and after cutaneous application of AITC. Repetitive application of AITC
initially increased the firing rate of spinal wide-dynamic range neurons; this was followed by
rapid desensitization that persisted when AITC application was reapplied 30 min later. The
responses to noxious thermal (but not to mechanical) stimuli were significantly enhanced
irrespective of whether the neuron was directly activated by AITC. These findings indicate
that AITC produced peripheral sensitization of heat nociceptors. Overall, our data support the
role of hermosensitive TRPA1 and TRPV1 channels in pain modulation and show that these
thermoTRP channels are promising targets for the development of a new group of analgesic
drugs. |
| format |
Article |
| author |
Tsagareli, M.G. Nozadze, I.R. Gurtskaia, G.P. Carstens, M.I. Tsiklauri, N.J. Carstens, E.E. |
| spellingShingle |
Tsagareli, M.G. Nozadze, I.R. Gurtskaia, G.P. Carstens, M.I. Tsiklauri, N.J. Carstens, E.E. Behavioral and Electrophysiological Study of Thermal and Mechanical Pain Modulation by TRP Channel Agonists Нейрофизиология |
| author_facet |
Tsagareli, M.G. Nozadze, I.R. Gurtskaia, G.P. Carstens, M.I. Tsiklauri, N.J. Carstens, E.E. |
| author_sort |
Tsagareli, M.G. |
| title |
Behavioral and Electrophysiological Study of Thermal and Mechanical Pain Modulation by TRP Channel Agonists |
| title_short |
Behavioral and Electrophysiological Study of Thermal and Mechanical Pain Modulation by TRP Channel Agonists |
| title_full |
Behavioral and Electrophysiological Study of Thermal and Mechanical Pain Modulation by TRP Channel Agonists |
| title_fullStr |
Behavioral and Electrophysiological Study of Thermal and Mechanical Pain Modulation by TRP Channel Agonists |
| title_full_unstemmed |
Behavioral and Electrophysiological Study of Thermal and Mechanical Pain Modulation by TRP Channel Agonists |
| title_sort |
behavioral and electrophysiological study of thermal and mechanical pain modulation by trp channel agonists |
| publisher |
Інститут фізіології ім. О.О. Богомольця НАН України |
| publishDate |
2013 |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/148126 |
| citation_txt |
Behavioral and Electrophysiological Study of Thermal and Mechanical Pain Modulation by TRP Channel Agonists / M. G. Tsagareli, I. R. Nozadze, G. P. Gurtskaia, M. I. Carstens, N. J. Tsiklauri, E. E. Carstens // Нейрофизиология. — 2013. — Т. 45, № 4. — С. 369-379. — Бібліогр.: 41 назв. — англ. |
| series |
Нейрофизиология |
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NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2013.—T. 45, № 4 369
UDC 612.88.577.25
M. G. TSAGARELI1, I. R. NOZADZE1, G. P. GURTSKAIA1, M. I. CARSTENS2, N. J. TSIKLAURI1, and E. E. CARSTENS2
BEHAVIORAL AND ELECTROPHYSIOLOGICAL STUDY OF THERMAL
AND MECHANICAL PAIN MODULATION BY TRP CHANNEL AGONISTS
Received December 21, 2012.
Transient receptor potential channels (TRP) have been extensively investigated over the past
few years. Recent findings in the field of pain have established a family of six thermoTRP
channels (TRPA1, TRPM8, TRPV1, TRPV2, TRPV3, and TRPV4) that exhibits sensitivity
to increases or decreases in temperature, as well as to chemical substances eliciting the
respective hot or cold sensations. Such irritants include menthol, cinnamaldehyde, gingerol,
mustard oil, capsaicin, camphor, eugenol, and others. In this study, we used behavioral
and electrophysiological methods to investigate if mustard oil (allyl isothiocyanate, AITC)
and capsaicin affect the sensitivity to thermal, innocuous cold, and mechanical stimuli in
male rats. Unilateral intraplantar injection of AITC and capsaicin induced significant
decreases in the latency for ipsilateral paw withdrawal from a noxious heat stimulus, i.e.,
heat hyperalgesia. These agents also significantly reduced the mechanical withdrawal
thresholds of the injected paw, i.e., mechanical allodynia. Bilateral intraplantar injections
of AITC resulted in a two-phase effect on cold avoidance (thermal preference test). A low
concentration of AITC (5%) did not change cold avoidance similarly to the vehicle control,
while higher AITC concentrations (10 and 15%) significantly reduced cold avoidance, i.e.,
induced cold hypoalgesia. Capsaicin acted in almost the same manner. These results indicate
that TRPA1 channels are clearly involved in pain reactions, and the TRPA1 agonist AITC
enhances the heat pain sensitivity, possibly by indirectly modulating TRPV1 channels,
which are co-expressed in nociceptors with TRPA1s. In electrophysiological experiments,
neuronal responses to electrical and graded mechanical and noxious thermal stimulations
were tested before and after cutaneous application of AITC. Repetitive application of AITC
initially increased the firing rate of spinal wide-dynamic range neurons; this was followed by
rapid desensitization that persisted when AITC application was reapplied 30 min later. The
responses to noxious thermal (but not to mechanical) stimuli were significantly enhanced
irrespective of whether the neuron was directly activated by AITC. These findings indicate
that AITC produced peripheral sensitization of heat nociceptors. Overall, our data support the
role of hermosensitive TRPA1 and TRPV1 channels in pain modulation and show that these
thermoTRP channels are promising targets for the development of a new group of analgesic
drugs.
Keywords: heat pain, hyperalgesia, mechanical allodynia, mustard oil, capsaicin,
nociception, thermal preference.
1 Ivane Beritashvili Experimental Biomedicine Center, Tbilisi, Georgia.
2 University of California at Davis, Davis, California, USA.
Correspondence should be addressed to M. G. Tsagareli
(e-mail: tsagareli@biphysiol.ge or merab.tsagareli@caucasus.net).
INTRODUCTION
Recent findings in the field of pain have established a
subset of transient receptor potential (TRP) channels
that are activated by temperature (the so-called
thermoTRP channels) and are capable of initiating
sensory nerve impulses in the course of detection of
thermal, mechanical, and chemical stimuli [1, 2]. A
family of six thermoTRP channels (TRPA1, TRPM8,
TRPV1, TRPV2, TRPV3, and TRPV4) exhibits
sensitivity to increases or decreases in temperature, as
well as to the action of chemical substances eliciting
the respective hot or cold sensations. Such irritants
include menthol (from mint), cinnamaldehyde,
gingerol, capsaicin (from chili peppers), mustard oil,
camphor, eugenol (from cloves), and others [2-4]. Our
recent studies indicated that unilateral intraplantar
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2013.—T. 45, № 4370
Article
injection of cinnamaldehyde (CA) induced a
significant concentration-dependent reduction in the
latency of the ipsilateral paw withdrawal reflex from a
noxious heat stimulus. This agent significantly reduced
mechanical withdrawal thresholds of the injected paw
too. Bilateral intraplantar injections of CA resulted
in significant cold hyperalgesia (cold-plate test) and
a mild enhancement of innocuous cold avoidance
(thermal preference test) [4-6]. Menthol, an agonist of
cold-sensitive TRPM8 channels, reduced heat pain and
exerted a two-phase effect on cold sensation, reducing
cold sensitivity at high concentrations and increasing
cold sensitivity at low concentrations [4, 5, 7].
In this study, we examined whether mustard oil
(MO) [allyl isothiocyanate (AITC)] and capsaicin
affect sensitivity to thermal and mechanical stimuli
in male rats. Isothiocyanate compounds constitute the
pungent ingredients in horseradish, onion, wasabi,
and other mustard extracts. Topical application of
AITC has, for a long time, been known to activate
somatosensory neurons, resulting in acute pain
and neurogenic inflammation through peripheral
release of neuropeptides from the primary afferent
nerve terminals. This, in turn, produces robust
hypersensitivity to thermal and mechanical stimuli
[2]. In the search for the MO receptor, Jordt et
al. discovered that a wide range of isothiocyanate
compounds activate TRPA1 channels [8, 9]. When
applied to the skin, AITC elicits burning pain, thermal
hyperalgesia, and mechanical allodynia [10]. When
acting on the oral or nasal mucosa, AITC causes
burning irritation, which decreases (desensitization)
across trials of repeated application [11, 12], as well
as heat hyperalgesia [13]. Lingual application of AITC
excites neurons in the trigeminal subnucleus caudalis
(Vc) [14-16]. In addition to a behavioral study, here
we tested whether spinal wide-dynamic range (WDR)
dorsal horn neuronal responses to repeated cutaneous
application of AITC similarly exhibit a desensitizing
pattern, and whether their responses to mechanical and
noxious thermal stimuli are enhanced after application
of this chemical.
The capsaicin TRP channels were the first to be
discovered in the mammalian sensory system. Julius,
Caterina, et al. [17] used an expression cloning strategy
to functionally search for capsaicin receptors. Using
this approach, they identified the TRPV1 channel as
a capsaicin receptor. Analyses of TRPV1-deficient
animals have revealed a key role for TRPV1s in both
acute heat detection and thermal hypersensitivity
following injury and inflammation [18, 19].
In this study, we hypothesize that intraplantar
injection of various concentrations of AITC and
capsaicin would induce hyper- or hyposensitivity to
30 and 15°C temperatures (thermal preference test).
METHODS
Animals. Behavioral studies using adult male Wistar
and Sprague–Dawley rats (350-500 g) were singly
housed and given rodent chow and water ad libitum.
Application of Chemicals. AITC at doses of 5,
10, and 15%, capsaicin at concentrations of 0.1, 0.2
and 0.4% (Sigma-Aldrich, USA), or vehicle control
(mineral oil or Tween 80, Fisher Scientific, USA) were
injected intraplantarly using 30-gauge needles.
Behavioral Tests. For behavioral testing, thermal
(Hargreaves method, 390, IITC, USA) and mechanical
(Von Frey method, IITC, USA) paw withdrawal
tests were carried out using the techniques described
previously [5, 7]. After baseline testing, the rat
received unilaterally intraplantar injections of AITC,
capsaicin, or vehicle, and the withdrawal latencies
for both paws were measured at 5, 15, 30, 45, 60, 90,
and 120 min post-injection. In the mechanosensitivity
tests, we used an electronic von Frey filament (2390,
IITC, USA) that was pressed against the ventral paw
surface from below, through a mesh stand the animal
stood on. The device monitored the force (g, ~0.01 N)
at the moment of initiation of the paw withdrawal
reflex.
For thermal preference tests, the rat was placed onto
a surface consisting of two adjacent thermoelectric
plates (AHP-1200DCP, Teca Thermoelectric, USA)
that could be independently heated or cooled to a
preset temperature (–5°C to >50°C). One plate was set
at 30°C, and the other at 15°C in a counterbalanced
design. AITC, or capsaicin, or vehicle, was bilaterally
injected intraplantarly, and the animal was placed onto
one of the plates in a matched block design alternating
the initial position and temperature.
Electrophysiological Testing
Surgery. Rats were anesthetized with sodium
pentobarbital (65 mg/kg, i.p.), and supplemented as
needed so that a strong tail and paw pinch failed to
evoke a withdrawal response. A tracheostomy tube was
inserted, the jugular vein was cannulated with PE-50
tubing for the maintenance of pentobarbital anesthesia,
and wound clips were used to close the incision.
The core body temperature was monitored rectally
using a BAT-12 thermometer (Physitemp, USA) and
maintained at 37 ± 0.2°C with a heating pad. During
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2013.—T. 45, № 4 371
Article
recording, the anesthesia level was maintained by
i.v. pump infusion (10-20 mg/kg⋅h) of pentobarbital.
The posterior superior iliac spine and a midline skin
incision were made from the approximately L6 to T11
spinous processes. The L1 and T13 spinous processes
were cut and removed, and a bilateral laminectomy was
performed at both levels under a dissection microscope
with micro-rongeurs. The dura was removed, and warm
agar was poured over the spinal cord. Vertebral clamps
on the transverse processes of T12 and L2 were used
to stabilize the animal in a stereotaxic frame (Kopf
Instruments, USA). After placement in the stereotaxic
frame, CO2 was monitored by a gas analyzer (Datex-
Ohmeda, USA) and maintained between 3.0 and 4.0%
by adjustment of the tidal volume and/or respiratory
rate.
Stimulation and Recording. A Teflon-coated
tungsten microelectrode (8-11 MΩ) was advanced into
the dorsal horn of the spinal cord using a hydraulic
microdrive to record single unit activity of dorsal
horn neurons. Units isolated for study were always at
depths ~1 mm. Action potentials (APs) were amplified
and displayed by conventional means and sent to a
computer for storage and analysis using a PowerLab
interface and Chart 5.0 software (AD Instruments,
USA). Only units that responded to graded innocuous
(brushing, 4-12 g von Frey) and noxious (76 g von
Frey, pinch) mechanical and noxious thermal (42,
46, and 50°C) stimuli were considered for further
study. Ten minutes after completion of the mechanical
and thermal stimulation series, 60-sec-long baseline
activity was recorded before application of either AITC
or vehicle control (mineral oil). AITC or mineral oil
was then topically applied to the center of the plantar
receptive field area with 1-min-long intervals for
10 min using a Hamilton microsyringe attached to a
PE-50 tubing. Application of AITC was repeated after
a 10-min wait period. Thus, 30 min had passed between
the last AITC application of trial 1 and the first AITC
of trial 2. On completion of the experiment, animals
were euthanized by an overdose of pentobarbital, i.v.
Data Analysis. The latencies of thermal and
mechanical paw withdrawal responses and those in
the thermal preference test were normalized with
respect to the baseline averages and subjected to one-
way repeated-measure analysis of variance (ANOVA)
using InStat 3.05 (GraphPad Software Inc., USA). A
95% confidence interval was used for all statistical
comparisons; standard errors of the mean are shown.
The spontaneous electrical firing rate was calculated
as the sum of the total number of APs generated
for 30 or 60 sec before each stimulus. Responses to
mechanical and thermal stimuli were quantified by
summing the total number of APs recorded during
the 10-sec-long stimulation period and subtracting
the spontaneous firing rate per 10 sec (30 sec
total/3). The afterdischarge was quantified as the
total number of APs during 30 sec after the offset of
the stimulus. Spontaneous firing, evoked responses,
and afterdischarges to each mechanical and thermal
stimulus were compared pre- vs post-treatment for
each treated group (AITC and mineral oil) using
the paired t-test. Responses to AITC and mineral
oil were quantified by summing the total spiking
during a 60-sec-long interval after each application.
Each sum was compared with the sum of APs during
60 sec preceding the first application (baseline)
using univariate ANOVA with the post-hoc Dunnett’s
two-sided t-test. A P value of <0.05 was taken to be
significant. Statistical analyses were performed using
SPSS 9.0 software.
RESULTS
Behavioral Data
AITC Application
Thermal (Hargreaves) Paw Withdrawal Test.
Application of AITC resulted in a significant dose-
dependent reduction in the ipsilateral thermal paw
withdrawal latency. Figure 1A shows the mean
withdrawal latencies of the injected paw vs time relative
to injection of vehicle or AITC at each concentration
tested. There was a dose-dependent reduction in the
latency, with the 15% AITC concentration significantly
different from vehicle and 5% AITC treatments. The
highest dose resulted in a mean reduction to 73.7% of
the pre-injection baseline value by 30 min with partial
recovery at 120 min. For the contralateral paw (B),
there was an overall significant effect of treatment,
with the 15% group being significantly different from
saline.
Mechanical (von Frey) Paw Withdrawal Test.
Mechanically evoked withdrawal thresholds are
plotted vs time for the treated paw in Fig. 1C. At each
AITC concentration, the thresholds were significantly
different from those at vehicle, but not from each other.
The mean withdrawal thresholds for the contralateral
paw (D) were not affected significantly at any AITC
concentration.
Two-Temperature Preference Test. On 30ºC vs
15ºC plates, rats treated with higher (10 and 15%)
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F i g. 1. Dynamics of the normalized thermal paw withdrawal latency (%) vs time (min). A) Following ipsilateral intraplantar injection of
vehicle (control, 1) and allyl isothiocyanate (AITC) at each concentration used (1-3, respectively, 5, 10, and 15% mustard oil, MO). There
were significant effects in AITC groups vs the vehicle group (P < 0.001). B) The same as in A but for the contralateral to AITC injection paw.
There were no significant effects of any AITC concentration vs the vehicle group. C) The same as in A for von Frey mechanically evoked
withdrawal of the injected paw. There were significant effects of all AITC concentrations vs the vehicle group (P < 0.001). D) The same as
in C but for the contralateral paw without significant effects of any AITC concentrations. BL) Pre-injection baseline.
Р и с. 1. Залежність нормованих латентних періодів відсмикування кінцівки при тепловій стимуляції (%) від часу (хв).
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0
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%
Vehicle
(Control)
5% 10% 15%
*** ***
*
**
MO
colder (15ºC) plate compared to vehicle-treated rats
(P < 0.001), i.e., the animals significantly avoided
the warmer 30ºC plate (Fig. 4). Another group of
rats treated with the highest (0.4%) concentration
demonstrated an opposite effect, a significant
preference for the warmer (30ºC) plate (P < 0.001).
Two groups of rats treated with lower (0.1 and 0.2%)
concentrations of capsaicin exhibited no significant
preference for the warmer or colder plates, though the
lowest (0.1%) concentration-treated rats had a strong
preference for the colder (15ºC) plate compared to
vehicle-treated rats (Fig. 4).
Electrophysiological Data. Figure 5 shows
an example of WDR neuronal responses to AITC
applicat ions and to thermal and mechanical
stimulation. Buildup of firing to the initial AITC
stimuli is shown in Fig. 5G. All WDR neuronal units
were also tested for responses to graded (46 and
50°C) heat before and after AITC. Figure 5 shows
responses of a unit before AITC (B and C) and their
clearly visible post-AITC enhancement (H and I). The
WDR units typically exhibited graded responses to
increasing bending forces of the punctuate von Frey
stimuli (D-F), which were minimally affected post-
AITC (J-L).
Figure 6 shows the average responses of WDR
units excited by repeated application of AITC
(Fig. 6, left, filled peristimulus time histograms,
PSTHs) and the responses unaffected by AITC (gray
PSTHs). For the responsive units, the mean firing
rate during the first three stimulus applications was
significantly greater compared to that within pre-AITC
baseline. This index, however, declined to a level
that was not significantly different from the baseline
(Fig. 6, left). After a 30-min-long resting period, AITC
application was repeated in the same manner. Although
there was a trend toward increase in the firing rate,
this effect did not reach statistical significance relative
to the pre-AITC baseline (Fig. 6, right) and, in any
case, was lower compared with that in the first trial.
DISCUSSION
The data presented above provide a comprehensive
view of the effects of intraplantar injections of
AITC and capsaicin on the thermal and mechanical
sensitivity. These influences induced a dose-dependent
heat hyperalgesia and mechanical allodynia lasting
more than 2 h. The AITC-induced enhancement of
heat sensitivity is consistent with findings of our
concentrations of AITC exhibited a significant
preference for the colder 15ºC plate compared to the
values at vehicle- and 5% concentration-treated rats
(Fig. 2), i.e., the animals significantly avoided the
warmer 30ºC plate.
Capsaicin Application
Thermal (Hargreaves) Paw Withdrawal Test. The
hind-paw injected capsaicin provided a concentration-
dependent decrease in the withdrawal latency
(Fig. 3A). The 0.1, 0.2, 0.3, and 0.4% capsaicin-treated
groups were significantly different from the vehicle
group (P < 0.001) but not from each other. There was
some mirror-image effect for the contralateral hindpaw
without significant differences for all concentrations
but significantly different from the vehicle group
(P < 0.01; B).
Mechanical (von Frey) Paw Withdrawal Test.
For the ipsilateral (treated) hindpaw, the 0.1-0.4%
capsaicin groups were significantly different from the
vehicle group (indicating allodynia, Fig. 3C) but not
from each other. For the contralateral hindpaw, there
were some mirror-image effects, especially for the
0.4% capsaicin concentration (P < 0.01; D).
Two-Temperature Preference Test. On 30ºC vs
15ºC plates, rats treated with a relatively high (0.3%)
concentration exhibited significant preference for the
F i g. 2. Biphasic effects of AITC on thermal preference (rats
stood on the 30°C vs 15°C plate). At high (10 and 15%) AITC
concentrations, rats spent significantly more time on the 15°C plate
(gray bars) compared to the 30°C plate (filled bars) (P < 0.05 and
P < 0.01, respectively), indicating cold hyposensitivity. At the lower
(5%, and vehicle) concentration, rats spent significantly more time
on the 30°C plate (P < 0.001), indicating cold hypersensitivity.
Vertical scale) Time spent on the 30°C plate, %. MO) Mustard oil.
Р и с. 2. Двофазний вплив алілу ізотіоціанату на термальну
преференцію (щури стояли на пластинках при різних
температурах, 30° проти 15 °C).
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А
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B
F i g. 3. Dynamics of the normalized thermal paw withdrawal latency (%) vs time (min). A) Following ipsilateral intraplantar injection of
vehicle (control, 1) and capsaicin at each concentration used (0.1, 0.2, 0.3, and 0.4%, respectively 2-5). There were significant effect in
capsaicin groups vs the vehicle group (P < 0.001). B) The same as in A but for the paw contralateral to capsaicin injection. C) The same as in
A for von Frey mechanically evoked withdrawal of the injected paw. There were significant effects of all capsaicin concentrations vs vehicle
(P < 0.001). D) The same as in C for the contralateral paw. Note that there are some mirror effects of capsaicin injections in the thermal (B)
and mechanical withdrawal (D) tests (P < 0.01). BL) Pre-injection baseline.
Р и с. 3. Залежність нормованих латентних періодів відсмикування кінцівки при тепловій стимуляції (%) від часу (хв).
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previous studies with CA injections in rats [4-6] and
applications of this agent in human subjects [10]. Other
investigators found that topical application of AITC
produces neurogenic inflammation and, concurrently,
heat and mechanical hyperalgesia, presumably via
a centrally mediated sensitization process, and that
these effects are TRPA1-mediated [3, 8, 9, 20-23].
Dose-dependent increases in the magnitude and
duration of heat hyperalgesia induced by AITC and CA
were similar to that induced by intraplantar capsaicin
herein. Since TRPA1s are co-expressed in sensory
neurons expressing TRPV1s [24], heat hyperalgesia
induced by AITC and CA might involve activation
of these receptors (sensory intradermal terminals
of nociceptor nerve endings) in an intracellular
mechanism leading to enhanced heat sensitivity of
TRPV1s. Alternatively, AITC and CA may cause
intradermal release of inflammatory mediators,
which lower the heat threshold of TRPV1s [7, 18].
Capsaicin in higher concentrations may also trigger
central sensitization, leading to the observed reduction
in the withdrawal latency for the contralateral paw
(Fig. 3B, D).
Long-lasting enhancement of mechanosensitivity
(i.e., allodynia) following AITC and capsaicin
applications (Figs. 1C and 3C) is consistent with
previous studies that showed a prolonged decrease in
the mechanical withdrawal threshold in mice following
intraplantar injection of a TRPA1 agonist, bradykinin
[25, 26], and with allodynia induced in the human
skin by topical application of AITC [13]. The role of
TRPA1s in mechanical allodynia is further supported
by reports that a TRPA1 antagonist, 4-hydroxynonenal,
attenuated inflammation- or nerve injury-induced
decreases in the mechanical paw withdrawal thresholds
and decreased mechanically evoked responses in C
fibers in mice [27]. In addition, AITC and CA were
shown to induce mechanical allodynia in humans [28].
However, TRPA1 as a ligand-gated ion channel in
sensory neurons was initially reported to be activated
by cold temperatures (below 18ºC) [10, 13, 19],
although this opinion has been disputed [3, 9]. TRPA1
knockout mice exhibited either normal cold sensitivity
[3], or mild [13], or severe deficits [10]. Just recently,
Pertovaara et al. [29] showed that TRPA1 channels in
the skin contribute to sustained, as well as to noxious
mechanical stimulus-evoked, postoperative pain, while
spinal TRPA1 channels contribute predominantly to
innocuous mechanical stimulus-evoked postoperative
pain [30]. Furthermore, spinal TRPA1s are responsible
for central pain hypersensitivity under various
pathophysiological conditions, such as inflammatory
and neuropathic pain [30-33].
However, our previous behavioral data support the
role of TRPA1s in cold detection, since intraplantar
injection of CA in rats resulted in enhanced avoidance
from a cold surface (temperature preference test) and
significantly lowered the withdrawal threshold in 0ºC
and +5ºC (cold-plate test), phenomena indicative of
cold hyperalgesia [4, 5, 7]. These results are consistent
with the possibility that TRPA1 agonists can enhance
cold-evoked gating of TRPA1 channels to increase
their cold sensitivity [34, 35].
Concerning capsaicin-induced effects in previous
human experiments, applications of capsaicin on
the tongue significantly enhanced heat pain but not
cold pain [13]. This finding is consistent with prior
psychophysical studies showing that intradermal
capsaicin enhanced the heat pain intensity within a
small region around the injection site for up to 2 h
[36-38]. The TRPV1 channels sensitive to capsaicin
respond to temperatures above the pain threshold
[19]. The results presented, might thus be explained
0
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80
100
%
Vehicle
(Control)
1 2 3 4
** ***
**
F i g. 4. Biphasic effects of capsaicin (1-4, respectively, 0.1, 0.2,
0.3, and 0.4%) on thermal preference (rats stood on the 30°C vs
15°C plate). At the realitively high (0.3%) capsaicin concentration,
rats spent significantly more time on the 15°C plate (gray bars)
compared to the 30°C plate (filled bar) (P < 0.001), indicating
cold hyposensitivity. However, at the highest (0.4%) capsaicin
concentration, rats spent significantly more time on the 30°C
plate (filled bar) compared to the 15°C plate (gray bar) (P < 0.01),
indicating cold hypersensitivity. At a lower (0.2%) concentration,
there was no significant difference between the warm (30°C, filled
bar) and cold (15°C, gray bar) plates. Vertical scale) Time spent on
the 30°C plate, %.
Р и с. 4. Двофазний вплив капсаїцину (1-4 відповідно, 0.1,
0.2, 0.3 і 0.4 %) на термальну преференцію (щури стояли на
пластинках при різних температурах, 30° проти 15 °C).
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AITC 2 µl AITC 2 µl AITC 2 µl AITC 2 µl AITC 2 µl
46 °C pre-AITC 46 °C post-AITC
50 °C post-AITC
4 g post-AITC
12 g post-AITC
76 g post-AITC
50 °C pre-AITC
4 g pre-AITC
12 g pre-AITC
76 g pre-AITC
F i g. 5. Example of an AITC-sensitive lumbar spinal wide-dynamic range (WDR) neuron. A) Shaded area shows extent of the
mechanosensitive receptive field on the lateral hindpaw. Arrow indicates the site of AITC application. B and C) Raw spike traces of
responses to 46 and 50°C heat stimuli before AITC application. D-F) Responses to graded mechanical stimulation at indicated von Frey
bending forces. G) Spike trace of activity during repeated applications of AITC at 1-min-long intervals (arrows). Note progressive increase
in firing rate with desensitization after the 5th stimulus. H-L) Spike traces of responses to graded noxious heat (H, I) and mechanical
stimulation (J-L) after sequential application of AITC. Note increased responses to heat but not to mechanical stimulation.
Р и с. 5. Приклад активності люмбального спінального нейрона з великим динамічним діапазоном, чутливого до алілу ізотіоціанату.
А G
H
I
J
K
L
B
C
D
E
F
60 sec
by a capsaicin-induced enhancement of thermal gating
of TRPV1s expressed in polymodal nociceptors
mediating thermal pain sensation [13, 39].
A main finding of our electrophysiological study
is that AITC sensitizes dorsal horn wide-dynamic
range (WDR) neuronal responses to noxious heat,
while not significantly affecting responses of these
units to mechanical stimuli. This heat sensitization
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was observed irrespective of whether AITC directly
excited the neuron. The desensitizing response pattern
observed presently was similar to that of responses
of neurons in trigeminal subnucleus caudalis (Vc)
to lingual application of AITC [15, 16], as well
as to a desensitizing temporal pattern of irritancy
ratings elicited by lingual AITC in humans [12]. On
reapplication, AITC evoked no significant increase
in WDR neuronal firing, which is consistent with the
self-desensitization reported for AITC irritancy on the
tongue that lasts less than 10 min in humans [12].
The mechan i sm unde r ly ing AITC se l f -
desensitization could involve a peripheral or central
site of action. Peripherally, repeated application
of AITC may lead to desensitization of TRPA1s
expressed in nociceptive endings. AITC-related self-
desensitization was recently reported to occur via a
calcium- and calcineurin-independent mechanism in
an in vitro assay of peptide release from skin–nerve
biopsies [40]. Alternatively (or in addition), central
inhibition might contribute to the reduced response of
WDR neurons to repeated application of AITC. This is
supported by the previous observation that a windup
elicited by electrical stimulation of the sciatic nerve
significantly attenuated post-AITC. However, such a
central inhibitory effect proposed was insufficient to
prevent AITC and CA-related enhancements of WDR
neuronal responses to noxious heat [41]. Thus, these
results strongly indicate that AITC produces peripheral
sensitization of heat nociceptors and/or central
sensitization at both behavioral and neuronal levels.
Thus, our findings indicate that thermosensitive
ion channels are capable of signaling temperature
changes across the range normally encountered in the
environment. We have a particular interest in the ability
of TRP channel agonists to modulate the temperature
sensitivity with important ramifications for pain
sensation. The TRPV1, being heat sensors, respond
to their agonist capsaicin, which elicits corresponding
heating sensations. Capsaicin is known to lower the
threshold and enhance heat-evoked gating of TRPV1s.
The TRPA1 is an exception, since when it is stimulated
by various agonists (e.g., MO, CA, etc.), the resultant
sensation is one of burning pain rather than of cold.
AITC AITCAITC AITCAITC AITCAITC AITCAITC AITCAITC AITCAITC AITCAITC AITCAITC AITCAITC AITC
30min
Trial 1
A
*** ** *
Trial 2
Bimp./sec
40
35
30
25
20
25
10
5
0
10 4 5 6 7 8 9 10 11 39 40 41 42 43 44 45 46 47 48 49 5032 min
F i g. 6. Desensitization of responses to repeated application of AITC. Shown are averaged peristimulus time histograms (PSTHs, 1-sec
bin width) of unit firing during repeated applications of AITC at 1-min-long intervals (arrows) for 10 min to the center of the receptive
field area on the ipsilateral hindpaw. Filled PSTHs) 14 WDR units that exhibited increased firing during the initial application of AITC.
Gray PSTHs) 13 units unresponsive to AITC. Error bars are omitted for clarity. PSTHs in A) Responses to the first trial of sequential AITC
application. PSTHs in B) Responses to second trial of sequential AITC application starting 30 min after the end of the first trial. One, two,
and three asterisks indicate significant differences compared with the initial 60-sec-long baseline period before the first application of AITC
(P < 0.05, P < 0.01, P < 0.001, respectively).
Р и с. 6. Десенситизація відповідей на повторну аплікацію алілу ізотіоціанату.
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However, the role of TRPA1s in cold reception and
cold pain sensitivity remains controversial. Our recent
data support the role of TRPA1 in cold detection, as the
TRPA1 agonist CA enhanced cold sensitivity in two
behavioral assays. The TRPA1 is, no doubt, involved
in pain, and TRPA1 agonists enhance the heat pain
sensitivity, possibly by indirectly modulating TRPV1s,
which are co-expressed with TRPA1s in nociceptors.
The thermosensitive TRP channels thus represent an
important set of new targets for the development of
analgesic drugs.
The Beritashvili EBMC and UC Davis Animal Care and Use
Committees approved the study protocols.
The authors, M. G. Tsagareli, I. R. Nozadze, G. P. Gurtskaia,
M. I. Carstens, N. J. Tsiklauri, and E. E. Carstens, proclame that
they have no conflict of interests.
The work was supported by the grant from Shota Rustaveli
National Science Foundation of Georgia (#1-6/27) and National
Institute of Health (DEO13685).
М. Г. Цагарелі1, І. Р. Нозадзе1, Г. П. Гурцкая1,
М. І. Карстенс2, Т. Дж. Циклаурі1, Е. Е. Карстенс2
ПОВЕДІНКОВЕ ТА ЕЛЕКТРОФІЗІОЛОГІЧНЕ ТЕСТУ-
ВАННЯ ТЕРМАЛЬНОЇ ТА МЕХАНІЧНОЇ МОДУЛЯЦІЇ
БОЛЮ З ВИКОРИСТАННЯМ АГОНІСТІВ TRP-КАНАЛІВ
1 Експериментальний біомедичний центр ім. І. Беріташвілі,
Тбілісі (Грузія).
2 Каліфорнійський університет, Девіс (США).
Р е з ю м е
Канали транзієнтного рецепторного потенціалу (transient
receptor potential channels, TRP) були відносно докладно ви-
вчені протягом останніх п’яти років. Згідно з результатами
сучасних досліджень у галузі болю існує сім’я канальних
структур, що складається з шести видів термо-TRP-каналів
(TRPA1, TRPM8, TRPV1, TRPV2, TRPV3 та TRPV4). Такі
канали виявляють чутливість до збільшення або зменшення
температури, а також до хімічних речовин, котрі виклика-
ють відповідне відчуття гарячого або холодного. До подіб-
них агентів належать ментол, коричний альдегід, гінгерол,
гірчична олія, капсаїцин, камфора, евгенол та ін. У нашо-
му дослідженні ми використовували поведінкові та електро-
фізіологічні методи, щоб з’ясувати, чи впливають гірчич-
на олія (аліл ізотіоціанат, АІТЦ) та капсаїцин на чутливість
самців щурів до термальних, нешкідливих холодових або
механічних стимулів. Унілатеральні внутрішньопідошов-
ні ін’єкції АІТЦ та капсаїцину зумовлювали істотні скоро-
чення латентного періоду відсмикування іпсілатеральної
кінцівки від шкідливого гарячого стимулу, тобто теплову
гіпералгезію. Ці агенти також істотно зменшували поро-
ги відсмикування кінцівки при дії механічного стимулу на
ін’єковану кінцівку, тобто викликали механічну алодінію.
Білатеральні внутрішньопідошовні ін’iєкції АІТЦ приво-
дили до двофазного впливу на реакцію уникання від холо-
ду (тест термальної преференції). АІТЦ у низькій концен-
трації (5%) не змінював холодового уникання порівняно з
контро лем, тоді як у вищих концентраціях (10 та 15 %) іс-
тотно пригнічував реакцію уникання на холод, тобто інду-
кував холодову гіпоалгезію. Капсаїцин діяв майже таким са-
мим чином. Ці результати вказують на те, що TRPA1-канали
безперечно залучені у больові реакції і що АІТЦ (агоніст
TRPA1) посилює чутливість до теплового болю (можливо,
через непряме модулювання TRPV1-каналів, котрі коекспре-
суються у ноцицепторах з TRPA1s). У електрофізіологічних
експериментах нейронні відповіді на електричні та ступін-
часті механічні та шкідливі термальні стимули тестувалися
перед шкірною аплікацією АІТЦ та після такої дії. Повтор-
на аплікація АІТЦ спочатку збільшувала частоту розрядів
спінальних нейронів з широким динамічним діапазоном;
це спричиняло швидку десенситизацію, котра утримувала-
ся після аплікації АІТЦ, застосованої за 30 хв. Відповіді
на шкідливу термальну (але не на механічну) стимуляцію
істотно збільшувалися незалежно від того, чи активувався
нейрон АІТЦ безпосередньо. Ці результати вказують на те,
що АІТЦ спричинює периферичну сенситизацію теплових
рецепторів. У цілому наші результати говорять на користь
ролі термосенситивних TRPA1- та TRPV1-каналів у больо-
вій модуляції та свідчать про те, що дані термо-ТRP-канали
є перспективними мішенями для розвитку нової групи анал-
гетиків.
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