Introduction
Mammals evolved possessing protective mechanisms that facilitated survival in both cold and hot temperatures. A sensitive response of the nervous system to changes in temperature is of predominant importance for homeotherms to maintain a stable body temperature. The thermoregulatory system contains sensory receptors/signal transducers, integrators, and effector organs designed to regulate body temperature within a narrow range (Tipton et al., 2008; Taylor, 2014). A number of temperature-sensitive transient receptor potential (TRP) ion channels have been studied as nociceptors that respond to extreme temperatures and harmful chemicals. Strong activation of these channels in the nervous system elicits pain (Zheng, 2013).
Recent findings have established a family of six thermo-TRP channels (TRPA1, TRPM8, TRPV1, TRPV2, TRPV3, and TRPV4) that exhibit sensitivity to increases or decreases in temperature, as well as to chemical substances eliciting the respective hot or cold sensations. Such irritants include capsaicin (from chili pepper), menthol, cinnamaldehyde, gingerol, mustard oil, camphor, eugenol, and others (Belmonte and Viana, 2008; Basbaum et al., 2009; Gerhold and Bautista, 2009; Carstens et al., 2010; Moran et al., 2011; Tsagareli, 2011, 2013; O’Neill et al., 2012).
The role of the TRP ion channel subtype ankyrin 1 (TRPA1) in thermal and mechanical transduction is controversial. TRPA1 was previously found to be cold sensitive, being activated at temperatures below 17°C (Story et al., 2003; Kwan et al., 2006; Tominaga, 2009). As most of the expression of TRPA1 overlaps with that of TRPV1, it seemed reasonable to link TRPA1 to cold nociception and to consider it responsible for chemical hypersensitivity, chronic cough, and airway inflammation in asthma (Bessac and Jordt, 2008). However, increasing data connect TRPA1 to heat sensitivity. In particular, TRPA1 agonists induce heat and mechanical hyperalgesia and a burning pain sensation, but no cold hyperalgesia (Namer et al., 2005; Albin et al., 2008; Merrill et al., 2008; Zanotto et al., 2008; Sawyer et al., 2009; Carstens et al., 2010; Tsagareli et al., 2010; Tsagareli, 2011, 2013; Hoffmann et al., 2013).
Exposure to capsaicin evokes a painful burning sensation through the vanilloid TRPV1 receptor, which is also activated by noxious thermal stimuli above 43°C or by an acidic environment of pH 5.4 (Caterina, 2007; Tominaga, 2009). In addition to its sensitivity to various pain-inducing stimuli such as capsaicin, heat, and protons, the TRPV1 ion channel has many features that a receptor related to nociception is supposed to have, such as its preferential distribution in the central nervous system within small-sized to medium-sized spinal dorsal root and trigeminal ganglia neurons, which are believed to serve as nociceptive nerve cells (Carstens et al., 2010). Although TRPV1 was shown to be activated by noxious heat, studies in TRPV1-knockout mice have revealed intact or partly reduced heat sensitivity (Caterina et al., 2000; Davis et al., 2000; Zimmermann et al., 2005). This suggests that TRPV1 cannot alone be responsible for heat nociception in the 42–52°C temperature range, and this also applies to all other heat-activated ion channels as far as knockout mice phenotypes are concerned (Hoffmann et al., 2013).
In this study, we used a battery of behavioral tests to examine whether allyl isothiocyanate (AITC), a natural compound of mustard oil, and capsaicin affect sensitivity to thermal and mechanical stimuli in male rats. We hypothesized that intraplantar injection of various concentrations of AITC and capsaicin would induce hypersensitivity or hyposensitivity to 30 and 15°C temperatures (thermal preference test) and to +5, 0, and −5°C temperatures (cold plate test).
Methods
Subjects
Behavioral studies were conducted on adult male Wistar rats (350–450 g) that were singly housed with free access to rodent chow and water. The Beritashvili Experimental BMC Animal Care and Use Committee approved the study protocol. Every effort was made to minimize both the number of animals used and their suffering. Guidelines of the International Association for the Study of Pain with regard to animal experimentation were followed throughout (Zimmermann, 1983).
Application of chemicals
AITC at doses 5, 10, 15, or 20%, capsaicin at concentrations of 0.1, 0.2, 0.3, or 0.4% (Sigma-Aldrich, St. Louis, Missouri, USA), or vehicle control (mineral oil or Tween 80; Fisher Scientific, Pittsburg, Pennsylvania, USA) was injected intraplantarly using 30 G needles. For thermal and mechanical paw withdrawal tests, AITC, capsaicin, or vehicle was applied to one hindpaw. For the thermal preference and cold plate tests, the chemicals or vehicle was applied to both paws. The rationale behind bilateral application was to ensure that at least one hindpaw was in contact with the thermal surface even if the animal guarded the other paw. Different groups of animal (n=6 per group) were used for each experiment, and they were only tested with one concentration of the chemical (AITC or capsaicin) or vehicle and were not repeatedly used.
Behavioral testing
Before formal testing, the baselines were assessed for rats in the experimental and control groups in thermal and mechanical withdrawal tests, averaging multiple (five times) baseline measurements for the left and right hind paws, with 5 min intervals between tests.
Thermal paw withdrawal (Hargreaves) test
Rats were first habituated over three successive daily sessions to stand on a glass surface heated to 30±1°C within a ventilated Plexiglas enclosure. Before formal testing, baseline latencies for paw withdrawals evoked by radiant thermal stimulation were measured at least three times/paw, with at least 5 min elapsing between tests for a given paw. A light beam (Plantar Test 390; IITC, Woodland Hills, California, USA) was focused onto the plantar surface of the hindpaw through the glass plate from below, and the latency from the onset of light application to brisk withdrawal of the stimulated paw was measured. To prevent potential tissue damage, a cutoff time of 20 s was imposed if no paw movement occurred. Withdrawal latencies for both treated and untreated paws were measured 5, 15, 30, 45, 60, 90, and 120 min after application of AITC, capsaicin, or vehicle to the same hindpaw.
Mechanical paw withdrawal (Von Frey) test
Baseline mechanical withdrawal thresholds were assessed using an electronic Von Frey filament (1601C; IITC) pressed against the plantar surface of one hindpaw. This device registered the force (g) at the moment that the hindpaw was withdrawn away from the filament. After application of AITC, capsaicin, or vehicle, the mechanical paw withdrawal thresholds were measured at the same postapplication time points as mentioned above for thermal paw withdrawals.
Two-temperature preference test
The apparatus consisted of two adjacent thermoelectric surfaces (each 13.3×6.37 inches, AHP-1200DCP; Teca Thermoelectric, Chicago, Illinois, USA) that could be independently heated or cooled to a preset temperature (–5°C to >50°C), which was maintained within ±1.0°C. A Plexiglas box enclosed both plates, which were separated by a central partition with an opening in the middle to allow the rat to move freely between the two surfaces. Rats were habituated to the test arena, with both plates set at 30°C. They were videotaped from above for 20 min, and the time the animal spent on each plate was recorded. Preference testing was performed by setting one plate at 30°C and another plate at 15°C in a counterbalanced design. AITC, capsaicin, or vehicle was topically injected bilaterally. The animal was placed onto one of the plates in a matched-block design alternating initial rat position and temperature. The temperature preference test was performed 3–5 min after injection of the irritant.
Cold plate test
To test the sensitivity to cold temperatures, the rat received bilateral intraplantar injections of capsaicin, AITC, or vehicle, and 5 min later, it was placed onto the thermoelectric surface that was set at +5°C, 0°C, or −5°C. The latency for nocifensive behavior (lifting and licking one hindpaw, or jumping) was measured, at which point the rat was immediately removed. All animals were retested at 5, 30, 60, 90, and 120 min after the injections.
Data analysis
Latencies of the thermal and mechanical withdrawal responses, and the time spent on the plate in the temperature preference and cold plate tests were normalized to baseline averages and subjected to one-way repeated-measures analyses of variance with Tukey–Kramer post-hoc tests, using InStat 3.05 software (GraphPad Software Inc., La Jolla, California, USA). A 95% confidence interval was used for all statistical comparisons, and the error reported is the SEM.
Results
Allyl isothiocyanate application
Hargreaves’ 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 versus 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 being significantly different from vehicle and 5% AITC treatments. The highest dose resulted in a mean reduction to 73.7% of the preinjection baseline value by 30 min. For the contralateral paw, there was an overall significant effect of treatment, with the 15% group being significantly different from the vehicle group (Fig. 1b).
Von Frey’s test
Mechanically evoked withdrawal thresholds are plotted versus time for the treated paw in Fig. 1c. At each AITC concentration, with partial recovery at 120 min, the thresholds were significantly different from those with vehicle, but not from each other. The mean withdrawal thresholds for the contralateral paw were not affected significantly at any AITC concentration (Fig. 1d).
Two-temperature preference test
On 30 versus 15°C plates, rats treated with higher (10 and 15%) concentrations of AITC exhibited a significant preference for the colder (15°C) plate (P<0.05 and P<0.01, respectively) compared with vehicle-treated and 5% concentration-treated rats (Fig. 2) – that is, the animals significantly avoided the warmer (30°C) plate.
Cold plate test
Bilateral intraplantar injection of AITC induced a significant reduction in cold plate latency compared with the vehicle control, which we interpret as a cold hyperalgesia. However, there were no antinociceptive effects on latency at the +5, 0 and −5°C temperatures. In the −5°C cold plate test, AITC treatment resulted in a highly significant difference between the vehicle-treated (mineral oil) and AITC-treated groups (P<0.001; Fig. 3). Note that a vehicle solution shows some protective effects against cold temperatures, especially against −5°C in the mineral oil control group, compared with AITC injections (Fig. 3c).
Capsaicin application
Hargreaves’ test
The hindpaw-injected capsaicin yielded a decrease in the withdrawal latency (Fig. 4a). The 0.1, 0.2, 0.3, and 0.4% capsaicin-treated groups were all significantly different from the vehicle-treated group (P<0.001), but not from each other. There were some mirror-image effects on the contralateral hindpaw, which were not significantly different among all concentrations, but were significantly different compared with the vehicle group (P<0.01; Fig. 4b).
Von Frey’s test
For the ipsilateral (treated) hindpaw, the 0.1–0.4% capsaicin groups were significantly different from the vehicle group (indicating allodynia, Fig. 4c), 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; Fig. 4d).
Two-temperature preference test
On 30 versus 15°C plates, rats treated with a relatively high (0.3%) concentration exhibited significant preference for the colder (15°C) plate compared with vehicle-treated rats (P<0.001) – that is, the animals significantly avoided the warmer (30°C) plate (Fig. 5). Another group of rats treated with the highest (0.4%) concentration demonstrated an opposite effect, similar to the effect on the control group – that is, a significant preference for the warmer (30°C) 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 plate, although the lowest (0.1%) concentration produced a strong preference for the colder (15°C) plate, compared with vehicle-treated rats, and this difference across treatments (vehicle vs. 0.1% group to 15°C plate) was significant (P<0.05; Fig. 5).
Cold plate test
We found almost similar results for capsaicin injections and AITC injections, except at −5°C, at which the difference between the vehicle-treated (tween+saline) and capsaicin-treated groups was less significant (Fig. 6c) than with AITC injections (Fig. 3c).
Discussion
The presented data provide a comprehensive view of the effects of intraplantar injections of AITC and capsaicin on thermal and mechanical sensitivities. These influences induced heat hyperalgesia and mechanical allodynia lasting more than 2 h. The AITC-induced enhancement of heat sensitivity is consistent with the findings of our previous studies with cinnamaldehyde (CA) injections in rats (Carstens et al., 2010; Tsagareli et al., 2010; Tsagareli, 2011) and on applications of this agent in human participants. In particular, capsaicin, AITC, and CA enhanced lingual heat pain elicited by a 49°C stimulus. At the same time AITC and CA weakly enhanced lingual cold pain (9.5°C), whereas capsaicin had no effect (Albin et al., 2008). Other investigators found that topical application of AITC produces neurogenic inflammation and, concurrently, heat and mechanical hyperalgesia, presumably through a centrally mediated sensitization process, and that these effects are TRPA1-mediated (Bandell et al., 2004; Macpherson et al., 2005; Bautista et al., 2006; Kwan et al., 2006; Belmonte and Viana, 2008; Bráz and Basbaum, 2010). Dose-dependent increases in the magnitude and duration of heat hyperalgesia induced by AITC and CA were similar to those induced here by intraplantar capsaicin. As TRPA1 is coexpressed in sensory neurons expressing TRPV1 (Kobayashi et al., 2005), heat hyperalgesia induced by AITC and CA might involve activation of these receptors (sensory intradermal terminals of nociceptor nerve endings) through an intracellular mechanism, leading to enhanced heat sensitivity of TRPV1.
Aternatively, AITC and CA may cause intradermal release of inflammatory mediators, which lowers the heat threshold of TRPV1 (Caterina et al., 2000; Klein et al., 2010). Capsaicin at higher concentrations may also trigger central sensitization, leading to the observed reduction in the withdrawal latency for the contralateral paw (Fig. 4b and d). The finding of long-lasting enhancement of mechanosensitivity (i.e. allodynia) following AITC and capsaicin applications (Figs 1c and 4c) is consistent with previous studies that showed a prolonged decrease in the threshold for mechanical withdrawal in mice following intraplantar injection of a TRPA1 agonist, bradykinin (Chuang et al., 2001; Sugiura et al., 2002), and with studies showing the induction of allodynia in human skin by topical application of AITC (Albin et al., 2008). The role of TRPA1 in mechanical allodynia is further supported by reports that a TRPA1 antagonist, 4-hydroxynonenal, attenuated inflammation-induced or nerve injury-induced decreases in mechanical paw withdrawal thresholds and decreased mechanically evoked responses in C-fibers in mice (Trevisani et al., 2007). In addition, AITC and CA were shown to induce mechanical allodynia in humans (Koltzenburg et al., 1992). However, TRPA1, as a ligand-gated ion channel in sensory neurons, was initially reported to be activated by cold temperatures (below 18°C; Namer et al., 2005; Albin et al., 2008; Caterina 2007), although this opinion has been disputed (Jordt et al., 2004; Belmonte and Viana, 2008). TRPA1-knockout mice exhibited either normal cold sensitivity (Belmonte and Viana, 2008) or mild (Albin et al., 2008) or severe deficits (Namer et al., 2005) in cold sensitivity in human participants.
It has been recently shown that TRPA1 channels in the skin contribute to sustained and noxious mechanical stimulus-evoked postoperative pain, whereas spinal TRPA1 channels contribute predominantly to innocuous mechanical stimulus-evoked postoperative pain (Pertovaara, Koivisto, 2011; Wei et al., 2012). Furthermore, spinal TRPA1 receptors are responsible for central pain hypersensitivity under various pathophysiological conditions, such as inflammatory and neuropathic pain (Eid et al., 2008; Kerstein et al., 2009; Wei et al., 2011). However, our previous behavioral data support the role of TRPA1 receptors in cold detection, as intraplantar injection of CA in rats resulted in enhanced avoidance of a cold surface (temperature preference test) and significantly lowered the withdrawal threshold at 0 and +5°C (cold plate test), which are phenomena indicative of cold hyperalgesia (Carstens et al., 2010; Tsagareli et al., 2010; Tsagareli, 2011). Here, in the cold plate test, we revealed cold hyperalgesia in AITC-treated and capsaicin-treated groups and did not observe any antinociceptive effects. These results are consistent with the possibility that TRPA1 agonists can enhance cold-evoked gating of TRPA1 channels to increase their cold sensitivity (Story et al., 2003; Karashima et al., 2009). With regard to capsaicin-induced effects in previous human experiments, application of capsaicin on the tongue significantly enhanced heat pain but not cold pain (Albin et al., 2008). 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 (LaMotte et al., 1991, 1992; Torebjork et al., 1992). The TRPV1 channels that are sensitive to capsaicin respond to temperatures above the pain threshold (Caterina, 2007). The results presented might thus be explained by a capsaicin-induced enhancement of thermal gating of TRPV1 expressed in polymodal nociceptors mediating thermal pain sensation (Carstens et al., 2007; Albin et al., 2008). However, we do not exclude the possibility that, in the two-temperature preference tests, the rats prefer the cool side because they are seeking alleviation of the ‘burning’ pain (De Felice et al., 2013; Navratilova et al., 2012, 2013) that can be specified on the modulation of TRP channel sensitivity.
Hoffmann et al. (2013) have recently shown that TRPA1 and TRPV1 channels contribute to thermal nociception, and that both TRPA1 and TRPV1 null mice presented behavioral deficits in heat sensitivity. Furthermore, TRPV1-knockout mice showed both reduced behavioral heat sensitivity and heat-induced calcitonin gene related peptide release. They confirmed that TRPV1 is not the sole noxious heat sensor and that other contributors must be expressed in the TRPV1 lineage (Mishra et al., 2011; Hoffmann et al., 2013). These authors hypothesized a possible synergistic or conditional relationship between TRPA1 and TRPV1 receptors involved in the response of cutaneous nociceptors to noxious heat, and direct as well as indirect interactions between the two receptor channels can be considered (Hoffmann et al., 2013).
Just recently, the molecular mechanisms underlying TRPV1 and TRPA1 interactions in nociceptive neurons have been elucidated. Particularly, Spahn et al. (2014) found a sensitization of TRPV1 after TRPA1 stimulation with mustard oil in a calcium and cAMP/protein kinase A (PKA)-dependent manner. TRPA1 stimulation enhanced TRPV1 phosphorylation through the putative PKA phosphorylation site, serine 116. They also detected a calcium-sensitive increase in TRPV1 activity after TRPA1 activation in dorsal root ganglion neurons. Overall, this study showed sensitization of TRPV1 through activation of TRPA1, which involves adenylyl cyclase, increased cAMP, subsequent translocation and activation of PKA, and phosphorylation of TRPV1 at PKA phosphorylation residues (Spahn et al., 2014). In other experiments, Fischer et al. (2014) found evidence of a TRPV1–TRPA1 interaction that is predominantly calcium dependent, and it has been suggested that the two proteins might form a heteromeric channel. Thus, these data provide some molecular evidence that supports our behavioral results.
Conclusion
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 temperature sensitivity, with important ramifications for pain sensation. TRPV1, being a heat sensor, responds to its agonist capsaicin, which elicits corresponding heating sensations. Capsaicin is known to lower the threshold and enhance heat-evoked gating of TRPV1. TRPA1 is an exception, as when it is stimulated by various agonists (e.g. AITC, CA, etc.), the resultant sensation is burning pain rather than cold. However, the role of TRPA1 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. TRPA1 is undoubtedly involved in pain, and TRPA1 agonists enhance sensitivity to heat pain, possibly by indirectly modulating TRPV1 coexpressed with TRPA1 in nociceptors.
Acknowledgements
This study was supported by the grants from Shota Rustaveli National Science Foundation of Georgia (#6/27; #31/40).
Conflicts of interest
There are no conflicts of interest.
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