SPINAL TRANSIENT RECEPTOR POTENTIAL ANKYRIN 1 CHANNEL INDUCES MECHANICAL HYPERSENSITIVITY, INCREASES CUTANEOUS BLOOD FLOW, AND MEDIATES THE PRONOCICEPTIVE ACTION OF DYNORPHIN A

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel that is expressed on a subpopulation of nociceptive primary afferent nerve fibers (1). In the periphery, it contributes to transduction of harmful stimuli to neuronal discharge, whereas on central endings of nociceptive nerve fibers it amplifies glutamatergic transmission to spinal dorsal horn interneurons (2, 3). TRPA1 channel agonists, such as cinnamaldehyde or mustard oil (4, 5), allow studying actions induced by the TRPA1 channel activation in experimental settings. In the skin, cinnamaldehyde and mustard oil induce hypersensitivity to various types of stimuli, most prominently to mechanical stimulation that is accompanied by sustained pain (6-9). Moreover, peripheral administration of cinnamaldehyde induces an axon reflex-mediated cutaneous blood flow increase (10).

There are still only few studies addressing pain behavior induced by spinal or intrathecal administration of TRPA1 channel agonists (11). One study reported antinociception following intrathecal (i.t.) administration of cinnamaldehyde or metabolites of paracetamol that are TRPA1 channel agonists (12). However, others have reported that i.t. administration of cinnamaldehyde produces hypersensitivity to mechanical stimulation (13) or both to mechanical and heat stimulation (14). Hepoxilin A3 (15) and 5,6-EET (16), two endogenous pronociceptive compounds in the spinal dorsal horn, have also proved to increase mechanical pain hypersensitivity through a TRPA1 channel-mediated mechanism.

It is still not quite clear which experimental parameters determine the direction of effect on pain behavior induced by activation of spinal TRPA1 channels or which other endogenous compounds, in addition to hepoxilin A3 and 5,6-EET, promote pain hypersensitivity through action on the spinal TRPA1 channel. Also, it is not yet established whether a selective TRPA1 channel agonist acting on central terminals of nociceptive nerve fibers might induce their antidromic activation, leading to cutaneous release of vasoactive compounds and increased blood flow, and thereby promotion of cutaneous neurogenic inflammation. It may be speculated that the antidromic effect induced by activation of the spinal TRPA1 channel could be a direct one (i.e., the TRPA1 channel induces antidromic activation of the nerve fiber expressing it), synaptic one (i.e., TRPA1 channel-mediated amplification of glutamatergic action on the GABAergic interneuron that induces antidromic action potentials in the adjacent nerve fibers (17)), or both.

To investigate further the spinal TRPA1 channel-induced effects and their underlying mechanisms, we assessed heat nociception, mechanical hypersensitivity and cutaneous blood flow in animals receiving i.t. treatment with cinnamaldehyde. Also, since glial cells of the spinal cord are known to play an important role in pain hypersensitivity (18), we attempted to suppress cinnamaldehyde-induced effects by i.t. pretreatment with compounds that have attenuated pain hypersensitivity induced by activation of astrocytes or microglia. To study role of the spinal TRPA1 channel in pain hypersensitivity induced by three endogenous pronociceptive compounds (dynorphin A (19), cholecystokinin (CCK) (20) and prostaglandin F2α (21)), we attempted to reverse their pain hypersensitivity effect by blocking the spinal TRPA1 channel. Finally, we attempted to determine whether a spinal TRPA1 channel agonist could induce cutaneous neurogenic inflammation due to a direct or synaptic induction of an antidromic impulse discharge in nociceptive primary afferent nerve fibers.

MATERIALS AND METHODS

Experimental animals

The experiments were performed with male Hannover-Wistar rats (220–260 g; Harlan, Horst, The Netherlands) in Biomedicum Helsinki. All experiments were approved by the ethical committee for experimental animals studies of the State Provincial Office of Southern Finland (Hameenlinna, Finland) and the experiments were performed according to the guidelines of European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, if available. The animals were housed in polycarbonate cages with a deep layer of saw dust, one to three animals in each cage, in a thermostatically controlled room at 24.0±0.5°C. The room was artificially illuminated from 8.30 AM to 8.30 PM. The animals received commercial pelleted rat feed (CRM-P pellets, Special Diets Services, Witham, Essex, England) and tap water ad libitum.

Surgical procedures for the installation of intrathecal catheter

For intrathecal drug injections, a catheter (Intramedic PE-10, Becton Dickinson and Company, Sparks, MD, USA) was administered into the lumbar level of the spinal cord under pentobarbital anesthesia (50 mg/kg intraperitoneally) as described in detail elsewhere (22). Following recovery from anesthesia, the correct placing of the catheter was verified by administering lidocaine (4%, 7–10 µl followed by a 15 µl of saline for flushing) with a 50-µl Hamilton syringe (Hamilton Company, Bonaduz, Switzerland). Only those rats that had no motor impairment before lidocaine injection but had a bilateral paralysis of hind limbs following intrathecal administration of lidocaine were studied further. The installation of the intrathecal catheter was performed about one week before the start of the actual experiments. In the actual experiments, the drugs were microinjected i.t. with a 50-µl Hamilton microsyringe in a volume of 5 µl followed by a saline flush in a volume of 15 µl.

Assessment of pain-related behavior evoked by peripheral test stimulation

All animals were habituated to handling and pain testing procedures at least 1-2 h per day for two days before assessing drug effects on pain behavior. To assess mechanically evoked pain behavior, the frequency of withdrawal responses to the application of monofilaments (von Frey hairs) to the hind paw was examined. A series of monofilaments that produced forces varying from 0.4 (or 1) to 15 (or 26) g (North Coast Medical, Inc., Morgan Hill, CA, USA) was applied in ascending order five times to the plantar skin at a frequency of 0.5 Hz. A visible lifting of the stimulated hind limb was considered a withdrawal response. An increase in the response rate represents mechanical hypersensitivity. When comparing effects of multiple compounds on pain behavior, a cumulative response rate to the presentation of all mechanical test stimuli at each time point before and after each treatment was calculated and used for assessing treatment-induced changes in mechanically evoked pain behavior.

Heat nociception was assessed by determining limb withdrawal latency induced by heat applied to the plantar skin using radiant heat equipment (Plantar test model 7370, Ugo Basile, Varese, Italy). The cut-off point was set at 15 s. Since a change in skin temperature is a significant confounding factor when assessing radiant heat-induced response latencies, skin temperature in the hind paw was measured with BAT-12 microprobe thermometer (Physitemp Instruments, Clifton, NJ, USA) one minute before application of each heat stimulation to the paw.

Assessment of cutaneous blood flow

To assess whether the cinnamaldehyde-induced activation of TRPA1 channels in the spinal dorsal horn induces antidromic activation of nociceptive nerve fibers that promotes neurogenic inflammation in the skin (17), we determined cutaneous blood flow following i.t. treatment with cinnamaldehyde. Blood flow in the plantar skin was monitored by Periflux Pf2 laser Doppler flowmeter (Perimed AB, Solna, Sweden). Electrical calibration for zero blood flow was made in all recordings (23). The analogue output of this equipment gives no absolute values but relative changes of cutaneous blood flow. The gain of the flowmeter was kept the same in all experiments. The output signal was sampled using CED micro 1401 (Cambridge Electronic Design, Cambridge, U.K.) and analyzed using Spike 2 software (Cambridge Electronic Design). When measuring the cutaneous blood flow, a probe holder was attached by a double-sided adhesive to the plantar skin of the hind paw.

In blood flow experiments that were performed in session separate from behavioral experiments, animals were under general anesthesia that was induced by intraperitoneal administration of pentobarbitone (50 mg/kg). When needed, further doses of pentobarbitone (15–20 mg/kg) were given to keep the level of anesthesia constant during the experiment, total duration of which was less than one hour. Blood flow values measured in arbitrary units for up to 10 min after cinnamaldehyde injections were considered in the data analysis. Movement-related artifacts (sharp deflections in the blood flow measurement curve) were excluded from the data analysis. In each experiment, the cinnamaldehyde-induced increase in blood flow was determined by subtracting the mean baseline blood flow level measured for 5 min before cinnamaldehyde treatment from that measured for 10 min after cinnamaldehyde treatment. Thus, positive values represent increase of blood flow by cinnamaldehyde treatment. The maximum cinnamaldehyde-induced increase from the baseline blood flow in each condition was used in further calculations.

An in vitro experiment for assessing properties of minocycline as a TRPA1 channel antagonist

An in vitro study was performed to assess whether minocycline-induced effects under in vivo conditions could be explained by a block of the TRPA1 channel. Therefore, properties of minocycline as a TRPA1 channel antagonist were determined in a membrane potential assay as described below.

Cell culture for in vitro study

Rat (HEK-Lacl-rTRPA1 clone 1A4) TRPA1-inducible HEK-293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with newborn-calf serum (10%), 25 mM N-2-hydroxyethylpiperazine-N'-2-ethane (HEPES), 2 mM L-glutamine, 1 mM Na-pyruvate, 100 U ml-1 streptomycin, 20 µg ml-1 hygromycin and 0.5 mg geneticin ml-1. Cells were subcultured twice weekly.

In vitro fluorometric imaging with plate reader

Changes in the membrane potential were measured in rat TRPA1-inducible HEK-cells using the fluorometric imaging plate reader FLIPRtetra™ (Molecular Devices, Sunnyvale, CA, USA). The day before experiments, HEK-cells were plated onto poly-D-lysine-coated, clear bottom black 96-well plates (BIOCAT®, Bedford, UK) at a density of 42,000 cells/well in medium supplemented with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). On the experiment day the growth medium was removed, cells were loaded with the membrane potential assay kit RED (Molecular Devices) reagent. In order to determine antagonism, 100 µl of either minocycline (0.1–300 µM), or A-967079 (0.01–30 µM) diluted in Ringer solution was pipetted onto the cells together with 100 µl dye followed by a 30 minute incubation at 37°C in the dark. Ringer solution was composed of (in mM): NaCl 143, KCl 4, MgCl2 1.2, CaCl2 1.8, Hepes 10, and glucose 5. The pH was adjusted to 7.4, and the osmolarity was adjusted to 298-305 mOsm with Osmostat® OM-6020 osmometer (DIC Kyoto Daiichi Kagagu Co. Ltd, Kyoto, Japan). The agonist, 50 µM cinnamaldehyde (Acros Organics, Geel, Belgium), was added onto the cells by the plate reader. The activation by cinnamaldehyde and the antagonism of the compounds were detected with FLIPRTETRA™ using a five minute protocol.

All experiments were performed at 37°C. Excitation wavelength was 510–545 nm and emission was measured at 565–625 nm. The fluorescence value, maximum minus baseline, was calculated for each well with ScreenWorks 2.0 software (Screen Works Inc., Dayton, OH, USA). The IC50 value was calculated from the dose-response curves. Dose-response curves were constructed from a mean of 4 separate wells at each antagonist concentration. Fitting of antagonist dose-response results were performed with nonlinear regression curve fit in Prism 4.0 (GraphPad Software Inc., La Jolla, CA, USA).

Drugs

Cinnamaldehyde (a TRPA1 channel agonist), carbenoxolone (a gap junction decoupler), minocycline (an inhibitor of microglial activation), bicuculline (a GABAA receptor antagonist), MK-801 (an NMDA receptor antagonist), CCK and dynorphin A were obtained from Sigma-Aldrich (St.Louis, MO, USA). Prostaglandin F2α was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). The doses used were chosen on previous studies showing that i.t. administration of cinnamaldehyde produces mechanical hypersensitivity (13) while that of carbenoxolone (10 µg) (24) or minocycline (100 µg (25)) reduces mechanical hypersensitivity induced by nerve injury or sleep-deprivation. A-967079 (26) or Chembridge-5861528 (27, 28), highly selective TRPA1 channel antagonists, were used to reverse the actions induced by cinnamaldehyde or the studied endogenous pronociceptive compounds at an i.t. dose that has proved effective in attenuating pain hypersensitivity in various pathophysiological conditions (5–10 µg (13, 29)). The i.t. dose of MK-801 used has proved effective in attenuating mechanically induced pain behavior (5 µg) (30). While an earlier study demonstrated that i.t. treatment with 5 µg of bicuculline effectively suppresses the dorsal root reflex (31), due to side-effects (strong muscular contractions) this dose proved to be too high in the unparalyzed animals of the present study. The i.t. dose of bicuculline used in the present study was 1 µg, which has proved effective in disinhibiting GABAergic interneurons (1 µg) (13). The i.t. doses of dynorphin A (15 nmol) (19), cholecystokinin (180 ng) (20) and prostaglandin F2α (10 nmol) (21) have proved effective in producing pain hypersensitivity in earlier studies. Time points for testing drug effects were based on earlier studies. When attempting to attenuate pronociceptive effects of compounds with a short onset and/or duration of action (cinnamaldehyde, CCK or prostaglandin F2α), the i.t. pretreatment with Chembridge-5861528, A-967079, minocycline or carbenoxolone was performed 30 min earlier. The suppression of the pronociceptive action was assessed at the time point in which each pronociceptive compound produced its maximum effect (which time point was determined with administrations of the pronociceptive compound alone). Since the onset of pronociceptive action induced by dynorphin A takes about two days (19), the attempt to attenuate dynorphin A-induced pronociceptive action with Chembridge-5861528 was performed 48 h after i.t. administration of dynorphin A.

Statistical analysis

Data analysis was performed using one- or two-way analysis of variance followed by Tukey's test (comparisons between three or more groups), or with a t-test (comparisons between two groups). P<0.05 was considered to represent a significant difference.

RESULTS

Facilitation of mechanically induced pain behavior by cinnamaldehyde

I.t. treatment with cinnamaldehyde produced a dose-related (0 µg, 3 µg, 10 µg) mechanical hypersensitivity effect (main effect of dose: F2,112 = 54.1, P<0.0001; Fig. 1A). The mechanical hypersensitivity effect was maximal within two min following i.t. administration of cinnamaldehyde, and the effect completely disappeared within two hours (Fig. 1B). Repeated i.t. administration of cinnamaldehyde at a 2 h interval (i.e., after recovery from the hypersensitivity effect induced by the first injection) produced an identical mechanical hypersensitivity effect as the first administration (main effect of repetitive cinnamaldehyde administration: F1,80 = 0.27; Fig. 1C). I.t. pretreatment with 10 µg of A-967079, a selective TRPA1 channel antagonist, significantly attenuated the mechanical hypersensitivity effect induced by 10 µg of cinnamaldehyde (main effect of A-967079 on cinnamaldehyde-induced hypersensitivity: F1,72 = 22.5, P<0.0001; Fig. 1D). Independent of the dose, i.t. treatment with cinnamaldehyde produced no obvious signs of ongoing pain behavior, such as vocalizations.

Fig. 1. Mechanical hypersensitivity effect induced by intrathecal treatment with cinnamaldehyde (CA), a TRPA1 channel agonist. (A) Effect of cinnamaldehyde dose. (B) Time course of the hypersensitivity effect, shown by the response to a monofilament producing a force of 2 g. (C) Reproducibility of the hypersensitivity effect following repeated application of 3 µg of cinnamaldehyde at an interval of 2 h. (D) Attenuation of the cinnamaldehyde-induced hypersensitivity by intrathecal pretreatment with A-967079 (10 µg), a selective TRPA1 channel antagonist. The higher the response rate shown by the Y-axis, the stronger the hypersensitivity. The error bars represent S.E.M. (n=6, except that nCA10=5). CA3 and CA10 = cinnamaldehyde at a dose of 3 µg or 10 µg, Sal=saline control, *P<0.05, **P<0.01, ***P&lt;0.005 (Tukey's test; reference: the corresponding saline value).

Heat nociception following cinnamaldehyde administration

When compared with saline control group, i.t. treatment with 10 µg of cinnamaldehyde failed to produce a significant change in the radiant heat-induced paw flick latency determined 2 min after i.t. treatment (t5 = 1.2; Fig. 2A). Cinnamaldehyde at the i.t. dose of 10 g failed to produce a significant increase in the skin temperature of the hind paw (t5 = 1.1; Fig. 2B).

Fig. 2. Heat nociception in the plantar skin (A) and the plantar skin temperature (B) two minutes following intrathecal administration of 10 µg of cinnamaldehyde (CA), a TRPA1 channel agonist. The error bars represent S.E.M. (n=6). Sal= saline control.

To exclude the possibility that a potential cinnamaldehyde-induced change in heat nociception had an onset latency longer than two min, the radiant heat-induced paw-flick latency and skin temperature of the hind paw were assessed in a separate group of six animals 30 min and 60 min after as well as before i.t. treatment with 10 µg of cinnamaldehyde. Cinnamaldehyde failed to induce any change in the heat-evoked paw-flick latency (main effect of time: F2,5 = 0.2; not shown) or skin temperature (main effect of time: F2,5 = 1.0; not shown) at the studied time points.

Cinnamaldehyde-induced mechanical hypersensitivity effect following pretreatment with carbenoxolone or minocycline

Cinnamaldehyde at the i.t. dose of 3 µg produced a significant mechanical hypersensitivity effect, which was not attenuated by i.t. pretreatment with 10 µg of carbenoxolone, a gap junction blocker used to suppress astrocyte-mediated facilitation (Fig. 3A). In contrast, i.t. pretreatment with 100 µg of minocycline, an inhibitor of microglial activation, significantly attenuated mechanical hypersensitivity effect induced by i.t. administration of 3 µg of cinnamaldehyde (Fig. 3B). Carbenoxolone or minocycline treatments alone at the currently used doses failed to influence mechanically evoked pain behavior (Fig. 3A and 3B, respectively).

Fig. 3. Attempts to suppress mechanical hypersensitivity effect induced by intrathecal administration of 3 µg of cinnamaldehyde, a TRPA1 channel agonist, with intrathecal pretreatments with 10 µg of carbenoxolone (A) or 100 µg of minocycline (B). The Y-axis shows the treatment induced maximum change in the cumulative response rate to series of monofilaments. Response rates >0 Hz represent hypersensitivity effect. The error bars represent S.E.M. (nCA=6, in other groups n=5). ***P<0.005, **P<0.01, *P<0.05 (Tukey's test). CA, cinnamaldehyde; CB, carbenoxolone; MC, minocycline; Sal, saline control.

An in vitro assessment of the properties of minocycline as a TRPA1 channel antagonist

To assess whether the attenuation of the cinnamaldehyde-induced hypersensitivity by minocycline was due to blocking the TRPA1 channel, we determined in the membrane potential assay the IC50 for blocking the membrane potential induced by 50 µM of cinnamaldehyde. Minocycline only poorly blocked the TRPA1 channel as indicated by the finding that the IC50 for minocycline was 212+52 µM (+ S.E.M., n=4), whereas that for A-967079, a selective TRPA1 channel antagonist, was 1.9+0.2 µM (n=4).

Effect by block of the spinal TRPA1 channel on mechanical hypersensitivity effect induced by endogenous pronociceptive compounds dynorphin A, CCK, and prostaglandin F2α

To study the role of the spinal TRPA1 channel in mechanical hypersensitivity induced by endogenous pronociceptive compounds, we assessed the effect of a selective TRPA1 channel antagonist on the mechanical hypersensitivity effects induced by dynorphin A, CCK and prostaglandin F2α.

I.t. administration of dynorphin A at the dose of 15 nmol produced a significant mechanical hypersensitivity effect two days after its administration (Fig. 4A). The mechanical hypersensitivity effect of dynorphin A (15 nmol) was reversed by i.t. treatment with 10 µg of Chembridge-5861528, a selective TRPA1 channel antagonist (Fig. 4A). The role of spinal TRPA1 channels in the hypersensitivity effect induced by dynorphin A (15 nmol) was tested also using another TRPA1 channel antagonist, A-967079. In this separate group of animals, dynorphin A produced in two days an increase of the cumulative response rate by 195±22% (±S.E.M., n=4; not shown) and intrathecally administered A-967079 (5 µg) reduced in 15 min the cumulative response rate by 90±25% (t3=7.0, P=0.006, paired t-test).

Fig. 4. Attempts to suppress mechanical hypersensitivity induced by intrathecal administration of 15 nmol of dynorphin A (A), 180 ng of cholecystokinin (B) or 10 nmol of prostaglandin F2α (C) with 10 µg of Chembridge-5861528, a TRPA1 channel antagonist. The Y-axis shows the treatment induced maximum change in the cumulative response rate to series of monofilaments. Response rates >0 Hz represent hypersensitivity effect. The error bars represent S.E.M. (nSal=5, in other groups n=6). ***P<0.005, **P<0.01, *P<0.05, ns, non-significant (Tukey's test). Dyn, dynorphin A; Ch, Chembridge-5861528; CCK, cholecystokinin; PGF, prostaglandin F2a; Sal, saline control.

I.t. administration of CCK at the dose of 180 ng produced a significant mechanical hypersensitivity effect that reached its peak effect in 5 min (Fig. 4B) and that lasted less than 30 min. I.t. pretreatment with 10 µg of Chembridge-5861528 failed to reduce the mechanical hypersensitivity effect induced by 180 ng of CCK (Fig. 4B).

I.t. administration of prostaglandin F2α at the dose of 10 nmol produced a significant mechanical hypersensitivity that reached its peak effect in 15 min (Fig. 4C) and that lasted less than one hour. I.t. pretreatment with 10 µg of Chembridge-5861528 failed to reduce the mechanical hypersensitivity effect induced by 10 nmol of prostaglandin F2α (Fig. 4C). Chembridge-5861528 alone at the currently used dose (10 µg i.t.) failed to produce a significant change in mechanically induced pain behavior (Fig. 4A-4C).

Spinal cinnamaldehyde-induced cutaneous blood flow response

Blood flow in the hind paw skin was determined following i.t. administration of cinnamaldehyde to assess whether spinal TRPA1 channels can induce cutaneous neurogenic inflammation (Fig. 5A). I.t. treatment with cinnamaldehyde produced a dose-related (3 µg, 10 µg) increase in the cutaneous blood flow (Fig. 5B). The peak blood flow increase occurred within about one min and the blood flow had recovered to the baseline level in <5 minutes (Fig. 5A). The 95% confidence limits of the blood flow response in the hind paw skin were above the baseline blood flow in animals receiving i.t. treatment with cinnamaldehyde at the dose of 10 µg, but not at the dose of 3 µg. I.t. pretreatment with 10 µg of A-967079, a TRPA1 channel antagonist, prevented the cutaneous blood flow increase induced by i.t. administration of 10 µg of cinnamaldehyde (Fig. 5B).

Fig. 5. Cutaneous blood flow response in the plantar skin induced by intrathecal treatment with cinnamaldehyde (CA), a TRPA1 channel agonist. (A) An example of laser Doppler flow measurement of cutaneous blood flow. The arrow indicates the time point of administering 10 µg of cinnamaldehyde. The horizontal calibration bar represents 100 s, and the vertical one 0.5 units. (B) Dose-related blood flow increase and the suppression of cinnamaldehyde-induced blood flow increase by intrathecal pretreatment with 10 µg of A-967079 (A), a selective TRPA1 channel antagonist. (C) An attempt to suppress the cinnamaldehyde-induced blood flow increase with intrathecal pretreatment with 1 µg of bicuculline (BIC), a GABAA receptor antagonist. (D) An attempt to suppress the cinnamaldehyde-induced blood flow increase with intrathecal pretreatment with 5 µg of MK-801 (MK), an NMDA receptor antagonist. In B-D, the error bars represent S.E.M. (nCA3=5, nCA10=10, nMK+CA10=12, in other groups n=6). *P<0.05 (Tukey's test).

Next, we assessed whether cinnamaldehyde acting on central terminals of primary afferent nociceptive nerve fibers increased cutaneous blood flow due to a direct induction of antidromic impulse discharge, amplification of the dorsal root reflex (synaptically mediated induction of antidromic impulse discharge involving glutamatergic activation of GABAergic interneurons that synapse with central terminals of adjacent primary afferent nerve fibers (17)), or both. Therefore, we attempted to reverse the cinnamaldehyde-induced blood flow increase by i.t. pretreatment with bicuculline, a GABAA receptor antagonist, or MK-801, an NMDA receptor antagonist. I.t. pretreatment with 1 µg of bicuculline failed to reduce the plantar skin blood flow increase induced by i.t. administration of 10 µg of cinnamaldehyde (t14=0.7; Fig. 5C). I.t. pretreatment with 5 µg of MK-801 also failed to reduce the cutaneous blood flow increase induced by i.t. administration of 10 µg of cinnamaldehyde (t20=0.6; Fig. 5D).

DISCUSSION

Activation of the spinal TRPA1 channel promotes mechanical hypersensitivity

Spinal administration of cinnamaldehyde, a TRPA1 channel agonist, produced mechanical hypersensitivity. This finding is in line with some (13-16), although not all (12) previous results. Moreover, the present result is in agreement with the aggravation of secondary (central) hypersensitivity to mechanical stimulation in humans with a gain of function mutation in the TRPA1 channel (32) and with the converse finding, the mechanical antihypersensitivity effect induced by blocking the TRPA1 channel in various pathophysiological conditions (13, 28, 29, 33, 34). The TRPA1 channel-mediated amplification of glutamatergic transmission between primary afferent nerve fibers and spinal dorsal horn interneurons (35-37) is likely to play a role in the facilitation of pain behavior by cinnamaldehyde. The mechanical hypersensitivity effect induced by spinally administered cinnamaldehyde had a brief onset of action (≤2 min), which is in line with the rapid onset of amplification of glutamatergic transmission in electrophysiological studies (35-37). At the currently used dose range (up to 10 µg), the duration of the pronociceptive effect induced by spinal cinnamaldehyde was short (less than 15 min), but an increase of the dose is expected to increase the duration of the effect. In line with this, a recent study indicated that a dose of cinnamaldehyde that is about 3–10 times higher than in the present study induced facilitation of pain behavior lasting up to one hour (14).

Paradoxically, previous patch clamp studies in spinal cord slices showed that the TRPA1 channel agonist-induced presynaptic increase in spontaneous glutamatergic transmission was accompanied by a suppression of the peripheral stimulus-evoked excitatory postsynaptic action on spinal dorsal horn neurons (36, 37). This finding raises the hypothesis that the spinal cinnamaldehyde-induced facilitation of the mechanically evoked pain behavior in the present study was due to facilitation of central pain-relay neurons receiving convergent inputs from various types of primary afferent nerve fibers rather than facilitation of the evoked signal carried by the TRPA1 channel-expressing nerve fibers. This hypothesis implies that one of the sources of convergent inputs to central pain-relay neurons is activated by low-intensity mechanical stimulation and does not express the TRPA1 channel, while one of the sources of peripheral inputs expresses the TRPA1 channel and facilitates the central pain-relay neuron due to a TRPA1 channel-mediated amplification of transmitter release from its central ending. In line with this proposal, two mechanically activated primary afferent nerve fiber types that do not express the TRPA1 channel, the mechanoreceptive Aβ-fiber in humans (6) and the low-threshold mechanoreceptive C-fiber in rodents (38), are considered to be mediating tactile allodynia-like symptoms. According to this hypothesis, the spinal cinnamaldehyde-induced presynaptic increase in the sustained release of glutamate from the TRPA1 channel-expressing nerve fiber endings sensitizes the central pain-relay neuron to the mechanoreceptive inputs. This leads to postsynaptic facilitation of mechanoreceptive nerve fiber-mediated afferent barrage to the central pain-relay neuron, independent of the peripheral stimulus-evoked postsynaptic action of the TRPA1 channel-expressing nerve fiber (Fig. 6A).

Fig. 6. Potential circuitries explaining the cutaneous blood flow increase and the mechanical hypersensitivity effect induced by spinal administration of cinnamaldehyde, a TRPA1 channel agonist. In graph A, cinnamaldehyde acting on the TRPA1 channel on the central ending of the nociceptive primary afferent nerve fiber induces directly an antidromic volley that causes a release of vasoactive neuropeptides (such as calcitonin gene-related peptide, CGRP) in the skin and thereby, increased cutaneous blood flow. At the same time, TRPA1 agonist amplifies glutamatergic transmission of nociceptive signals to pain-relay neurons receiving convergent inputs from low-threshold mechanoreceptive (LTM) as well as TRPA1-expressing nociceptive nerve fibers. The TRPA1-mediated presynaptic amplification in transmission increases postsynaptically excitability of convergent pain-relay neurons. Thereby, peripheral signals carried from LTM nerve fibers to pain-relay neurons are expected to be facilitated, which provides a potential mechanism for the mechanical hypersensitivity effect. In graph B, cinnamaldehyde amplifies glutamatergic transmission from the nociceptive primary afferent nerve fiber to a GABAergic interneuron (35) that has efferent projections to the central terminals of adjacent nociceptive primary afferent nerve fibers (17). Due to high intracellular chloride concentration in primary afferent nerve fibers, the activation of chloride channel on primary afferent terminals by increased GABAergic drive is expected to induce synaptic depolarization of terminals. Thereby, an antidromic volley is induced in the nociceptive nerve fibers. This provides a (di)synaptic mechanism (dorsal root reflex, DRR) for increased cutaneous blood flow induced by the spinal TRPA1 channel (47). The DRR-induced cutaneous blood flow increase is expected to be reversed by blocking the spinal glutamatergic and/or GABAergic receptors (17, 31), whereas blocking the spinal TRPA1 channel is expected to reverse the cutaneous blood flow increase induced by the mechanisms shown in graph A as well as B.

The spinal TRPA1 channel agonist-induced mechanical hypersensitivity and spinal glia

There is abundant evidence indicating that coupling of astrocytes and activation of microglia may contribute to pain hypersensitivity (e.g., 25, 39). Recent studies suggest that the TRPA1 channel might play a role in hypersensitivity conditions associated with glial cells. Namely, it has been shown that the TRPA1 channel regulates astrocytes' resting calcium and through it, inhibitory synapse efficacy (40), while microglial activation may promote nociception by induction of endogenous TRPA1 channel agonists (41). In the present study, i.t. pretreatment with an inhibitor of microglial activation, but not with a gap junction decoupler reduced the cinnamaldehyde-induced mechanical hypersensitivity effect. Based on this finding and the fact that the currently used doses of carbenoxolone and minocycline have previously proved to decouple astrocytes and reduce microglial activation, respectively, it might be suggested that microglia rather than astrocytes have a role in the spinal cinnamaldehyde-induced mechanical hypersensitivity. The present in vitro experiment, however, indicates that minocycline only poorly blocks the TRPA1 channel and therefore, the attenuation of the cinnamaldehyde-induced mechanical hypersensitivity by minocycline was not likely to be due to a minocycline-induced block of the TRPA1 channel. Among plausible explanations for the minocycline-induced antihypersensitivity effect in the present study is its action other than inhibition of microglia, such as scavenging of peroxynitrite (42), a compound with pronociceptive properties (43). Indirectly this finding suggests that the cinnamaldehyde-induced hypersensitivity is not only due to action on the TRPA1 channel, which finding is in line with the incomplete reversal of cinnamaldehyde-induced hypersensitivity by a selective TRPA1 channel antagonist (Fig. 1D). Moreover, it should also be noted that interpretations from the current carbenoxolone result need to be considered preliminary ones until the carbenoxolone result is replicated with a more selective inhibitor of astrocytes.

Activation of the spinal TRPA1 channel failed to influence heat nociception

In the present study, spinal cinnamaldehyde at a dose producing mechanical hypersensitivity effect failed to induce heat hypersensitivity. A recent study reported that spinal cinnamaldehyde administered at doses of about 3–10 times higher than in the present study produced both heat and mechanical hypersensitivity (14). Another recent study indicated that a metabolite of spinal 12-lipoxygenase that induced a robust TRPA1 channel-mediated mechanical hypersensitivity had a weak heat hypersensitivity effect that was observed only at a dose exceeding that needed to induce mechanical hypersensitivity (15). Together these results suggest that the spinal TRPA1 channel has a stronger effect on amplification of pain-related signals evoked by mechanical than heat stimulation.

In contrast to studies indicating that the spinal TRPA1 channel has pronociceptive actions, it has been reported that i.t. administration of cinnamaldehyde suppressed pain behavior induced by heat and mechanical stimulation in mice (12). This in vivo finding was accompanied by an in vitro finding demonstrating that a TRPA1 channel agonist inhibited activity of voltage-gated sodium and calcium channels of dorsal root ganglion neurons and thereby reduced their excitability, providing a plausible explanation for the suppression of pain behavior following spinal administration of a TRPA1 channel agonist at a high dose (12). It still remains to be studied which experimental factors are critical in determining whether the behavioral effect induced by a spinally administered TRPA1 channel agonist is pain facilitation or suppression.

Endogenous pronociceptive compounds and the spinal TRPA1 channel

A number of reactive compounds resulting in pathophysiological conditions, such as oxidative stress, are TRPA1 channel agonists (2). In the spinal cord, potential TRPA1 channel agonists with a pronociceptive action may be released e.g. by the spinal microglia (41). Recently, it was shown that two endogenous pronociceptive compounds, hepoxilin A3 (15) and 5,6-EET (16), induce mechanical hypersensitivity through action on the spinal TRPA1 channel. The present results extend these findings by showing that the long-term mechanical hypersensitivity effect induced by dynorphin A, also an endogenous pronociceptive compound (19), is maintained by the spinal TRPA1 channel. Since spinal administration of dynorphin A increases the level of spinal prostaglandin E2 (44) and since prostaglandin E2 is a TRPA1 channel agonist (45), the TRPA1 channel-reversible pronociceptive action of dynorphin A may be mediated by prostaglandin E2.

The mechanical hypersensitivity effects induced by two other endogenous pronociceptive compounds of the spinal cord, CCK (20) or prostaglandin F2α (21), were not attenuated by a TRPA1 channel antagonist. Interestingly, while the spinal CCK-induced hypersensitivity was not reversed by blocking the spinal TRPA1 channel, our earlier results indicate that the spinal TRPA1 channel is involved in mediating the mechanical hypersensitivity effect induced by CCK in the rostral ventromedial medulla (13). Together these findings indicate that the hypersensitivity effects induced by medullary and spinal CCK have different underlying mechanisms. In line with this proposal, it has been shown that medullary CCK activates descending pain-facilitatory pathways that elicit spinal release of prostaglandin E2 (46), a compound that is a TRPA1 channel agonist (45) and that has pronociceptive actions in the spinal cord (21).

Activation of the spinal TRPA1 channel promotes cutaneous blood flow

Spinal administration of cinnamaldehyde at a dose range 3–10 µg produced a short-lasting (<2-3 min) cutaneous blood flow increase that was suppressed by spinal pretreatment with a selective TRPA1 channel antagonist. Activation of the TRPA1 channel on central terminals of nociceptive nerve fibers amplifies glutamatergic transmission to inhibitory as well as excitatory interneurons in the spinal dorsal horn (35). Activation of the inhibitory interneurons by nociceptive afferent barrage is known to induce a dorsal root reflex, a synaptic circuitry that causes increased cutaneous blood flow through antidromic activation of adjacent nociceptive nerve fibers by the inhibitory interneurons (17). A recent study indicated that the spinal TRPA1 channel amplifies the dorsal root reflex (47). Based on these earlier findings, the blood flow increase induced by spinal cinnamaldehyde could be due to central activation of the dorsal root reflex (i.e.; a TRPA1 channel-mediated increase in spinal release of glutamate that leads to glutamatergic activation of GABAergic interneurons which induce an antidromic volley in the adjacent nociceptive nerve fibers (17)), direct antidromic activation of nociceptive nerve fibers, or both. However, the present finding that spinal pretreatment with an antagonist of the glutamate or GABAA receptor failed to reduce the spinal cinnamaldehyde-induced increase of cutaneous blood flow is in line with the hypothesis that the blood flow increase was due to a direct antidromic activation of nociceptive primary afferent nerve fibers (the lower part of Fig. 6A) rather than a synaptic circuitry within the spinal dorsal horn (Fig. 6B).

While the present study focused on cinnamaldehyde-induced changes in the skin, it should be noted that TRPA1 channel agonists have been shown to exert a role also in the pathogenesis of visceral inflammation (48, 49), although some of these effects may, at least partly, be due to mechanisms other than action on the TRPA1 channel (50). It remains to be studied whether TRPA1 channel agonists in the spinal cord have pro-inflammatory actions in the viscera as well as in the skin.

Conclusions

The results of the present study indicate that activation of the spinal TRPA1 channel induces mechanical hypersensitivity and an increase of cutaneous blood flow. Dynorphin A, but not spinal CCK or prostaglandin F2α, is among endogenous pronociceptive compounds that produces pronociception through a mechanism that involves activation of the spinal TRPA1 channel. The attenuation of the TRPA1 channel agonist-induced mechanical hypersensitivity effect by minocycline may be explained by mechanisms other than its direct action on the spinal TRPA1 channel. It should be noted that the cinnamaldehyde-induced mechanical hypersensitivity effect was not completely reversed by a TRPA1 channel antagonist. This finding together with the earlier finding that cinnamaldehyde may act also on other channels (51) indicates that only those cinnamaldehyde-induced effects that were sensitive to blocking the TRPA1 channel with a selective antagonist can be considered to represent TRPA1 channel-mediated functions. The present results add to the evidence indicating that blocking the spinal TRPA1 channel provides a possibility to reduce mechanical hypersensitivity and cutaneous neurogenic inflammation in selected pathophysiological conditions.

Abbreviations: CCK, cholecystokinin; TRPA1, transient receptor potential ankyrin 1;

Acknowledgements: This study was supported by the Sigrid Jusélius Foundation, Helsinki, Finland, the Academy of Finland, Helsinki, Finland, OrionPharma, Orion Corporation, Finland, and the EU Erasmus Exchange Program, Brussels, Belgium.

Conflict of interests: Three of the authors (M.S., L.F. and A.K.) are employees of the pharmaceutical company (OrionPharma, Finland) that supported this study.

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