Spinal transient receptor potential ankyrin 1 channel contributes to central pain hypersensitivity in various pathophysiological conditions in the rat

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PAIN 152 (2011) 582–591

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Spinal transient receptor potential ankyrin 1 channel contributes to central pain hypersensitivity in various pathophysiological conditions in the rat Hong Wei a,c, Ari Koivisto b, Marja Saarnilehto b, Hugh Chapman b, Katja Kuokkanen b, Bin Hao c, Jin-Lu Huang c, Yong-Xiang Wang c, Antti Pertovaara a,⇑ a b c

Institute of Biomedicine/Physiology, University of Helsinki, Helsinki, Finland Orion Corporation, OrionPharma, R&D, Turku, Finland King’s Lab, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, PR China

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

a r t i c l e

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Article history: Received 12 October 2010 Accepted 29 November 2010

Keywords: Cholecystokinin Cinnamon aldehyde Pain hypersensitivity Rat Spinal cord Tactile allodynia Transient receptor potential ankyrin 1 channel

a b s t r a c t The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed on nociceptive primary afferent neurons. On the proximal nerve ending within the spinal dorsal horn, TRPA1 regulates transmission to spinal interneurons, and thereby pain hypersensitivity. Here we assessed whether the contribution of the spinal TRPA1 channel to pain hypersensitivity varies with the experimental pain model, properties of test stimulation or the behavioral pain response. The antihypersensitivity effect of intrathecally (i.t.) administered Chembridge-5861528 (CHEM; a selective TRPA1 channel antagonist; 5–10 lg) was determined in various experimental models of pain hypersensitivity in the rat. In spinal nerve ligation and rapid eye movement (REM) sleep deprivation models, i.t. CHEM attenuated mechanical hypersensitivity. Capsaicin-induced secondary (central) but not primary (peripheral) mechanical hypersensitivity was also reduced by i.t. administration of CHEM or A-967079, another TRPA1 channel antagonist. Formalininduced secondary mechanical hypersensitivity, but not spontaneous pain, was suppressed by i.t. CHEM. Moreover, mechanical hypersensitivity induced by cholekystokinin in the rostroventromedial medulla was attenuated by i.t. pretreatment with CHEM. Independent of the model, the antihypersensitivity effect induced by i.t. CHEM was predominant on responses evoked by low-intensity stimuli (66 g). CHEM (10 lg i.t.) failed to attenuate pain behavior in healthy controls or mechanical hypersensitivities induced by i.t. administrations of a GABAA receptor antagonist, or NMDA or 5-HT3 receptor agonists. Conversely, i.t. administration of a TRPA1 channel agonist, cinnamon aldehyde, induced mechanical hypersensitivity. The results indicate that the spinal TRPA1 channel exerts an important role in secondary (central) pain hypersensitivity to low-intensity mechanical stimulation in various pain hypersensitivity conditions. Ó 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

1. Introduction Transient receptor potential ankyrin 1 (TRPA1) is a nonselective cation channel that is located on peptidergic primary afferent nociceptive nerve fibers [29,38]. On the distal ending of primary afferent nerve fibers, the TRPA1 ion channel contributes to transduction of harmful stimuli into electric signals. Although the TRPA1 channel was originally identified as a cold-activated channel [37], its role in transduction of cold is still not quite clear [29,38]. There is, however, abundant evidence indicating that the TRPA1 channel is involved in sensing chemical irritants [2,3,11,16,20,24,42] and noxious mechanical stimuli [4,7,12,16,17,31,45]. ⇑ Corresponding author. Address: Institute of Biomedicine/Physiology, P.O. Box 63, University of Helsinki, 00014 Helsinki, Finland. Tel.: +358 9 191 25280; fax: +358 9 191 25302. E-mail address: antti.pertovaara@helsinki.fi (A. Pertovaara).

On the central ending of the nociceptive primary afferent nerve fiber, the TRPA1 ion channel is involved in facilitation of glutamatergic transmission between the primary afferent terminal and the central neuron in the spinal dorsal horn [14,43,51]. Recent behavioral studies suggest that the TRPA1 ion channel on the central terminal of the primary afferent ending may have an important role in the maintenance of pain hypersensitivity. This is indicated by the findings that intrathecal (i.t.) administration of a low dose of a TRPA1 channel antagonist attenuated cold and mechanical hyperalgesia in animals with a Complete Freund’s Adjuvant-induced inflammation [4] and had a mechanical antihypersensitivity effect in diabetic or topical mustard oil-treated animals [46]. The finding that the spinal TRPA1 channel contributed to pain hypersensitivity in three different experimental pain models raises the question of whether the contribution of spinal TRPA1 channel is restricted to these particular conditions, or whether the spinal TRPA1 channel has a more general role in regulation of central pain hypersensitiv-

0304-3959/$36.00 Ó 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2010.11.031

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H. Wei et al. / PAIN 152 (2011) 582–591

ity, independent of the pathophysiological mechanism inducing hypersensitivity. Moreover, previous results still leave open whether the role of the spinal TRPA1 in central pain hypersensitivity varies depending on the test stimulus properties (such as stimulus intensity), type of pain behavior (such as evoked versus spontaneous pain behavior), and whether there are differences in modulation of pain behavior by cutaneous versus spinal TRPA1 channels. In the present study, we determined the influence of an i.t. administered TRPA1 channel antagonist on pain behavior after rapid eye movement (REM) sleep deprivation [25,44], spinal nerve ligation [13], intraplantar (i.pl.) formalin (a TRPA1 channel agonist [24]), or i.pl. capsaicin (a TRPV1 channel agonist [49]). Moreover, we assessed whether i.t. pretreatment with a TRPA1 channel antagonist influences descending pain facilitation induced by cholecystokinin (CCK) in the rostroventromedial medulla (RVM [10,52]) or that induced by activation of the spinal 5-HT3 receptor [5,27,39]). When appropriate, stimulus-evoked and spontaneous pain behaviors were assessed separately. With stimulus-evoked pain, the focus was on mechanically induced pain behavior, as mechanical hypersensitivity, particularly in an uninjured skin area, is typically caused by central mechanisms [41]. Moreover, to assess further whether a TRPA1 channel antagonist has only pre- or also postsynaptic action in the spinal dorsal horn, we determined the influence of a TRPA1 channel antagonist on pain hypersensitivity induced by direct chemical activation or disinhibition of central pain-relay neurons. For comparison, we determined pain behavior after blocking of the cutaneous TRPA1 channels or after spinal administration of cinnamon aldehyde, a selective TRPA1 channel agonist [3]. 2. Methods 2.1. Experimental animals The experiments were performed in adult male Hannover-Wistar (HW) rats (weight: 160–240 g; CAS, Shanghai, China and Harlan, Horst, the Netherlands). All experiments were approved by the local ethics committees at the University of Helsinki, Finland, and at the Shanghai Jiao Tong University, China, and followed the guidelines of the Ethics Standards of the International Association for the Study of Pain. All efforts were made to minimize animal suffering, to use only the number of animals necessary to produce reliable scientific data, and to use alternatives to in vivo techniques, if available. The sleep deprivation study was performed at the Shanghai Jiao Tong University, whereas other in vivo experiments were performed at the University of Helsinki. 2.2. Chembridge-5861528, a TRPA1 channel antagonist Chembridge-5861528 (CHEM; a derivative of HC-030031), which was synthesized by ChemBridge Corporation (San Diego, CA) was used as a TRPA1 channel antagonist [45]. Calcium imaging results in human TRPA1- and TRPV1-transfected HEK cells showed that when mustard oil or 4-hydroxynonenal (4-HNE) was used as a TRPA1 channel agonist, IC50 value of CHEM was 14.3 ± 0.7 lmol/L or 18.7 ± 0.3 lmol/L, respectively [45]. Moreover, CHEM showed no TRPA1 or TRPV1 channel agonism and no TRPV1 channel antagonism up to a dose of 100 lmol/L [46]. Patch clamp recordings in rat TRPA1-transfected HEK cells indicated that CHEM is a reversible rat TRPA1 channel antagonist with an IC50 of 230 nmol/L and a Hill coefficient of 0.6 [46]. In each experiment, CHEM was dissolved in physiological saline immediately before its intrathecal (i.t.) or intraplantar (i.pl.) administration, or in 0.5% methylcellulose before its intraperitoneal (i.p.) administration. In one control experiment, A-967079 [23], a TRPA1 channel antagonist with a

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structure different from that of CHEM, was used to exclude the possibility that the CHEM-induced effects were due to action not related to the TRPA1 channel. 2.3. Preparations for i.t. drug administrations For i.t. drug injections, a catheter (Intramedic PE-10, Becton Dickinson and Company, Sparks, MD) was administered into the lumbar level of the spinal cord under pentobarbital anesthesia (50 mg/kg i.p.) as described in detail elsewhere [36]. After recovery from anesthesia, the correct placing of the catheter was verified by administering lidocaine (4%, 7–10 ll followed by a 15 ll of saline for flushing) with a 50-ll 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 after i.t. administration of lidocaine were studied further. For i.t. administration, the drugs were microinjected with a 50-ll Hamilton microsyringe in a volume of 5 ll followed by a saline flush in a volume of 15 ll. The installation of the i.t. catheter was performed approximately 1 week before the start of the actual experiments. I.pl. administrations of CHEM were performed at a volume of 50 ll using a 50-ll Hamilton syringe connected via polyethylene tubing to a 27-gauge hypodermic needle. 2.4. Assessment of pain-related behavior All animals were habituated to pain testing procedures at least 1 to 2 h per day for 2 days before assessing drug effects on pain behavior. To assess tactile allodynia-like 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, Morgan Hill, CA) was applied in ascending order 5 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. In some of the experiments, mechanical nociception and hyperalgesia was assessed using the paw pressure test. Test device was a Basile Analgesy-meter (Ugo Basile, Varese, Italy). Force was applied at a rate of 32 g/s to the plantar skin of the paw until limb withdrawal or the cut-off force of 250 g. At each time point, two threshold determinations were performed at 1-min intervals. A decrease in the paw pressure threshold represented mechanical hyperalgesia. In some of the experiments, heat nociception or hyperalgesia 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 [9]). The cut-off point was set at 15 s. Because 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) 1 min before application of each heat stimulation to the paw. 2.5. Influence by i.t. administration of CHEM on pain behavior in healthy controls In healthy controls, pain behavior was determined before and at various time points (15, 30, 60, and 120 min) after i.t. administration of CHEM (5 or 10 lg) or vehicle. At each time point, the monofilament test was performed first and was followed by the paw pressure test, skin temperature measurement, and heat-induced paw flick test. Each animal participated in 3 testing sessions at an interval of 2 to 3 days. The order of testing different drug conditions was counterbalanced between the animals.

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2.6. Sleep deprivation-induced hypersensitivity The pedestal-over-water technique of rapid eye movement sleep deprivation (REMSD) was modified from a method described earlier [25]. Briefly, the rat was placed on top of a platform surrounded by water. The platform was 7.5 cm in diameter and 7.5 cm high. The base of the cage was submerged in 4 cm of water. REMSD was performed for 2 days (48 h). During REMSD, the animals had free access to water and food. REMSD of 48 h duration induces mechanical hypersensitivity [44]. In the present study, influence of a TRPA1 antagonist on REMSD-induced hypersensitivity was assessed 48 h after REMSD. Immediately after REMSD, the animals were administered i.t. CHEM (5 or 10 lg) or saline and the monofilament test was performed 15, 30, 60, and 120 min after i.t. administration of CHEM/vehicle. After REMSD and monofilament testing, the animals were allowed to recover at least for 1 week before new testing session. The order of testing different drug conditions was counterbalanced between the animals. 2.7. Peripheral nerve injury-induced hypersensitivity

administration of CHEM or vehicle). At each time point, monofilament test was performed first at the site of primary and then secondary hypersensitivity. The order of testing was the same in CHEM- and vehicle-treated animals. When testing the same animal more than once, the interval between testing sessions was 3 to 7 days, and the second application of capsaicin was contralateral to the first application. Moreover, it was verified before second testing session that the hypersensitivity had disappeared, as indicated by a baseline response that was not different from that in the earlier session. Instead of CHEM, 1 group of animals was pretreated with A967079, a TRPA1 antagonist with a structure different from that of CHEM [23], when performing the capsaicin test. Because our preliminary results indicated that the potency of A-967079 is stronger than that of CHEM, it was administered i.t. at the dose of 3 lg, whereas the effect of CHEM in the capsaicin test was assessed using a dose of 10 lg. 2.9. Formalin-induced spontaneous pain and secondary hypersensitivity

Spinal nerve ligation was used to induce a peripheral neuropathy and neuropathic hypersensitivity. The unilateral ligation of 2 spinal nerves (L5 and L6) was performed under pentobarbitone anesthesia (50 mg/kg i.p.) as described in detail earlier [13]. Briefly, the left L5 and L6 spinal nerves were isolated and tightly ligated with 6–0 silk thread. After ligation, the wound was sutured and the rats were allowed to recover. Of the nerve-ligated animals, only those with marked tactile allodynia-like symptoms (monofilament-induced limb withdrawal threshold 133, P < .0001; not shown). The i.t. pretreatment with 10 lg of CHEM failed to influence development of the capsaicin-induced primary hypersensitivity (F1,30 = 4.1; Fig. 4A), whereas it significantly suppressed development of the capsaicin-induced secondary hypersensitivity (F1,30 = 12.6, P = .0013; Fig. 4B). Interestingly, the CHEM-induced antihypersensitivity effect at the site of secondary hypersensitivity was significant only at low (