δ opioid receptors mediates 15-deoxy-Δ12,14-prostaglandin J2 induced-antinociception in rat temporomandibular joint

Neuroscience 163 (2009) 1211–1219

ACTIVATION OF PERIPHERAL ␬/␦ OPIOID RECEPTORS MEDIATES 15-DEOXY-⌬12,14-PROSTAGLANDIN J2 INDUCED-ANTINOCICEPTION IN RAT TEMPOROMANDIBULAR JOINT activation of the intracellular L-arginine/NO/cGMP/KⴙATP channel pathway. The pharmacological properties of the peripheral administration of 15d-PGJ2 highlight the potential use of this PPAR-␥ agonist on TMJ inflammatory pain conditions. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

D. R. PENA-DOS-SANTOS,a F. P. SEVERINO,a S. A. L. PEREIRA,a D. B. R. RODRIGUES,a F. Q. CUNHA,b S. M. VIEIRA,b,c M. H. NAPIMOGAa AND J. T. CLEMENTE-NAPIMOGAa* a Laboratory of Biopathology and Molecular Biology, University of Uberaba, Uberaba, Brazil b

Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, São Paulo, Brazil

Key words: TMJ, pain, PGJ2, 15d-PGJ2, PPAR-gamma, opioid receptors.

c Laboratory of Pharmacology, National Institute for Research in the Amazon, Manaus, Brazil

There is consensus in the literature that inflammatory hypernociception occurs, at least in part, as a consequence of the sensitization of primary afferent nociceptors. This phenomenon has been attributed to the direct action of hypernociceptive inflammatory mediators (mainly prostaglandins and sympathetic amine) on their receptors present in the nociceptor membrane (Khasar et al., 1999). Temporomandibular joint (TMJ) pain is a significant part of symptoms in patients with temporomandibular disorders (TMD) and a common source of chronic orofacial pain (Alstergren et al., 1999). A considerable amount of evidence suggests that TMJ pain may result from an inflammatory episode (Kopp, 2001). Although the mechanism underlying TMJ pain conditions is not completely known, it has been shown that inflammatory mediators such as prostaglandin E2, 5-HT and pro-inflammatory cytokines (tumoral necrosis factor alpha [TNF-␣], interleukin-1␤ [IL1␤]) are highly present in the synovial fluid of patients with TMD (Kopp, 2001). In addition, recently it has been demonstrated that sympathomimetic amines also contribute to the inflammatory TMJ hyperalgesia by activating ␤2-adrenoceptors (Rodrigues et al., 2006). Several lines of evidence support the concept that endogenous formation of cyclooxygenase (COX)-derived electrophilic lipid oxidation products may play an anti-inflammatory role (Jiang et al., 1998; Ricote et al., 1998; Zingarelli and Cook, 2005; Napimoga et al., 2008a). The 15-deoxy-⌬12,14-prostaglandin J2 (15d-PGJ2), a potent lipid mediator derived from the prostaglandin D2 in vivo by dehydration (Yu et al., 1995), is a natural ligand for peroxisome proliferator activated receptors (PPAR-␥) (Schoonjans et al., 1997; Ricote et al., 1998). 15d-PGJ2, is abundantly produced by mast cells, platelets, alveolar macrophages and has been proposed as a key immunoregulatory lipid mediator (Straus and Glass, 2001; Zingarelli and Cook, 2005). In addition to its anti-inflammatory effects, we have previously demonstrated for the first time that 15d-PGJ2 inhibits carrageenan-induced mechanical inflammatory hypernociception in rat paws via PPAR-␥

Abstract—This study assessed the effect of the agonist 15dPGJ2 administered into the rat temporomandibular joint (TMJ) on nociceptive behavioral and the anti-inflammatory potential of this prostaglandin on TMJ. It was observed that 15-deoxy-⌬12,14-prostaglandin J2 (15d-PGJ2) significantly reduced formalin-induced nociceptive behavior in a dose dependent manner, however injection of 15d-PGJ2 into the contralateral TMJ failed to reduce such effects. This antinociceptive effect is dependent on peroxisome proliferator-activated receptors-␥ (PPAR-␥) since pre-treatment with GW9662 (PPAR-␥ receptor antagonist) blocked the antinociceptive effect of 15d-PGJ2 in the TMJ. In addition, the antinociceptive effect of 15d-PGJ2 was also blocked by naloxone suggesting the involvement of peripheral opioids in the process. Confirming this hypothesis pre-treatment with ␬, ␦, but not ␮ receptor antagonists significantly reduced the antinociceptive effect of 15d-PGJ2 in the TMJ. Similarly to opioid agonists, the 15d-PGJ2 antinociceptive action depends on the nitric oxide (NO)/guanilate cyclase (cGMP)/ATP-sensitive potassium channel blocker(KⴙATP) channel pathway since it was prevented by the pre-treatment with the inhibitors of nitric oxide synthase (NOS; aminoguanidine), cGMP (ODQ), or the KⴙATP (glibenclamide). In addition, 15d-PGJ2 (100 ng/ TMJ) inhibits 5-HT-induced TMJ hypernociception. Besides, TMJ treated with 15d-PGJ2 showed lower vascular permeability, assessed by Evan’s Blue extravasation, and also lower neutrophil migration induced by carrageenan administration. Taken together, these results demonstrate that 15d-PGJ2 has a potential peripheral antinociceptive and anti-inflammatory effect in the TMJ via PPAR-␥ activation. The results also suggest that 15d-PGJ2 induced-peripheral antinociceptive response in the TMJ is mediated by ␬/␦ opioid receptors by the *Corresponding author. Tel: ⫹55-34-3319-8958; fax: ⫹55-34-3314-8910. E-mail address: [email protected] (J. T. Clemente-Napimoga). Abbreviations: cGMP, guanilate cyclase; CTOP, D-Phe-Cys-Tyr-DTrp-Orn-Thr-Pen-Thr-NH2; GW9662, 2-chloro-5-nitro-N-phenylbenzamide; ICI 174,864, N, N-diallyl-Tyr-Aib-Aib-PheLeu; K⫹ATP, ATP-sensitive potassium channel blocker; MPO, myeloperoxidase; NOS, nitric oxide synthase; Nor-BNI, nor-binaltorphimine dihydrochloride; ODQ, 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxaline-1-one; PPAR-␥, peroxisome proliferators activated receptors-␥; TMD, temporomandibular disorders; TMJ, temporomandibular joint; 15d-PGJ2, 15-deoxy-⌬12,14-prostaglandin J2.

0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.07.052

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activation that is dependent on local macrophages and endogenous opioids production (Napimoga et al., 2008b). However 15d-PGJ2 did not inhibit formalin-induce nociception in the rat paws while it blocked formalin-induced nociception in the TMJ of rats (Napimoga et al., 2008b). These findings led us to investigate the potential antihypernociceptive and anti-inflammatory properties of exogenous administration of these lipids into TMJ joint of rats. The possible cellular mechanisms involved in the antinociceptive effect of 15d-PGJ2 were also addressed.

EXPERIMENTAL PROCEDURES Animals This study was carried out with male Wistar rats (150 –250 g) maintained in a temperature-controlled room (23⫾1 °C) with a 12 h light/dark cycle. All experiments were conducted in accordance to the IASP guidelines on using laboratory animals for investigations of experimental pain in conscious animals (Zimmermann, 1983). All animal experimental procedures and protocols were approved by the Committee on Animal Research of the University of Uberaba. The animals’ suffering and number per group were kept at a minimum and each animal was used once.

Drugs 15d-PGJ2 and 2-chloro-5-nitro-N-phenylbenzamide (GW9662) from Calbiochem, San Diego, CA, USA; naloxone, aminoguanidine, glibenclamide, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), Nor-Binaltorphimine (Nor-BNI) and an aqueous solution of 37% of formaldehyde were obtained from Sigma, St. Louis, MO, USA; 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ) and N, N-diallyl-Tyr-Aib-Aib-Phe-Leu (ICI 174,864) were obtained from Tocris Cookson, Ballwin, MO, USA. Formalin solution were prepared from commercially stock formalin (an aqueous solution of 37% of formaldehyde) and further diluted in 0.9% NaCl. The ATP-sensitive potassium channels blocker glibenclamide was dissolved in 2% Tween 80 and re-suspended in saline. Naloxone and aminoguanidine were dissolved in saline. GW9662 and ODQ were dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) and re-suspended in saline to minimize the final concentration of DMSO (max. 0.5%).

Testing procedure for TMJ pain Testing sessions took place during light phase (between 9:00 AM and 5:00 PM) in a quiet room maintained at 23 °C. Each animal was manipulated for 7 days to be habituated to the experimental manipulation. After this period, the animal was placed in a test chamber (30⫻30⫻30 cm3 mirrored wood chamber with a glass at the front side) for 15 min habituation period to minimize stress. Animals were briefly anesthetized by inhalation of halothane to allow the TMJ injection, which was performed with a 30-gauge needle connected to a 50 ␮l-Hamilton syringe (Roveroni et al., 2001). Each animal regained consciousness approximately 30 s after discontinuing the anesthetic and was returned to the test chamber for counting nociceptive responses. The nociceptive response score was defined as the cumulative total number of seconds that the animal spent rubbing the orofacial region asymmetrically with the ipsilateral fore or hind paw plus the number of head flinches counted during the observation period as described previously. Since head flinches followed a uniform pattern of 1 s of duration, each flinch was expressed as 1 s. Results are expressed as the duration time of nociceptive behavior (Roveroni et al., 2001; Clemente et al., 2004). At the conclusion of the experiment, animals were anesthetized by an i.p. injection of a mixture of ure-

thane (1 g/kg) and ␣-chloralose (50 mg/kg), followed by i.v. administration of Evan’s Blue dye (1%; 5 mg/kg), to visualize formalin-induced plasma extravasation upon post-mortem examination of injected TMJs (Haas et al., 1992). This procedure also confirmed that the plasma extravasation induced by TMJ injection at the correct site was restricted to the immediate TMJ region. A different investigator performed each test, prepared the solution, and administered the TMJ injections. All animals received a final volume of 45 ␮l into TMJ as previously described (Clemente et al., 2004). All experiments were conducted in a double blind fashion in which the person who injected the solutions was different of the one who made the behavioral assessment.

Experimental protocols Effect of 15d-PGJ2 on formalin-induced TMJ nociception. Rats were pretreated (15 min) with an intra-TMJ injection of 15dPGJ2 (1, 10 or 100 ng; n⫽6; 15 ␮l/TMJ; Napimoga et al., 2008b) followed by ipsilateral intra-TMJ injection of 1.5% formalin in a final volume of 45 ␮l. Behavioral nociception response was evaluated for a 45 min observation period. In order to confirm the peripheral 15d-PGJ2-mediated antinociception, the highest dose of 15dPGJ2 was also injected in the contralateral TMJ that received injection of 1.5% formalin. Effect of PPAR-␥ receptor antagonist on 15d-PGJ2-induced antinociception. Rats were pretreated (15 min) with an intraTMJ injection of PPAR-␥ receptor antagonist GW9662 (0.3, 1 or 3 ng/15 ␮l/TMJ; n⫽6; Napimoga et al., 2008b) followed by 15 dPGJ2 (100 ng/15 ␮l/TMJ) 15 min prior ipsilateral intra-TMJ injection of 1.5% formalin (15 ␮l/TMJ). Behavioral nociception response was evaluated for 45 min observation period. In order to confirm the peripheral effect of GW9662, the highest dose of 15d-PGJ2 was also injected into the contralateral TMJ that received pretreatment of 15d-PGJ2 and 1.5% formalin. All animals received a final volume of 45 ␮l of solutions into TMJ. Effect of the nonselective opioid receptor antagonist naloxone on 15d-PGJ2-induced antinociception. Rats were pretreated (15 min) with an intra-TMJ injection of naloxone (10 ␮g/15 ␮l/TMJ; n⫽6; Eisenberg et al., 1996) followed by 15d-PGJ2 (100 ng/15 ␮l/TMJ) 15 min prior ipsilateral intra-TMJ injection of 1.5% formalin (15 ␮l/TMJ). Behavioral nociception response was evaluated for 45 min observation period. All animals received a final volume of 45 ␮l of solutions into TMJ. Role of NO/cGMP/ATP-sensitive potassium channel blocker (KATP⫹) channel pathway on 15d-PGJ2-induced antinociception. Rats were divided in groups of six animals, and each group was pretreated (15 min) with an intra-TMJ injection of a nonselective inhibitor of nitric-oxide synthase (NOS) aminoguanidine (0.1 mol/15 ␮l/TMJ; Sachs et al., 2004) or inhibitor of soluble cGMP enzyme ODQ (8 ␮g/15 ␮l/TMJ; Napimoga et al., 2008b) or the ATP-potassium sensitive channel blocker glibenclamide (160 ␮g/15 ␮l/TMJ; Napimoga et al., 2008b) followed by 15d-PGJ2 (100 ng/15 ␮l/TMJ; n⫽6) 15 min prior ipsilateral intra-TMJ injection of 1.5% formalin (15 ␮l/TMJ). Behavioral nociception response was evaluated for 45 min period observation. All animals received a final volume of 45 ␮l of solutions into TMJ. Role of ␮, ␦ and ␬-opioid receptors on 15d-PGJ2-induced antinociception. Rats were divided in groups of six animals, and each group was pretreated (15 min) with an intra-TMJ injection of a specific inhibitor of ␮-opioid receptor CTOP (20 or 60 ␮g/ 15␮l/TMJ; Picolo et al., 2000) or the inhibitor of ␦-opioid receptor ICI 174,864 (10 or 30 ␮g/15 ␮l/TMJ; Picolo et al., 2000) followed by 15d-PGJ2 (100 ng/15 ␮l/TMJ; n⫽6) 15 min prior ipsilateral intra-TMJ injection of 1.5% formalin (15 ␮l/TMJ). The selective ␬-opioid receptor antagonist Nor-BNI (200 ␮g/15 ␮l/TMJ; Clemente et al., 2004) was injected 24 h prior to ipsilateral 15d-PGJ2

D. R. Pena-dos-Santos et al. / Neuroscience 163 (2009) 1211–1219 (100 ng/15 ␮l/TMJ; n⫽6) and after 15 min it was applied intra-TMJ injection of 1.5% formalin (15 ␮l/TMJ). Behavioral nociception response was evaluated for 45 min period observation. All animals received a final volume of 45 ␮l of solutions into TMJ. Effect of the 15d-PGJ2 on 5-HT induced TMJ nociception. In a previous study it was demonstrated that sympathomimetic amines such as norepinephrine are released at the site of injury where they contribute to the inflammatory TMJ hyperalgesia by acting on ␤2-adrenoreceptors (Rodrigues et al., 2006). Considering the fact that the involvement of sympathomimetics amines in TMJ hyperalgesia may be relevant to clinical TMJ inflammatory pain states less sensitive to non-steroidal anti-inflammatory drugs (NSAIDs), in this study we tested the hypothesis that 15d-PGJ2induced antinociception by inhibiting the sympathetic component of the TMJ nociception. Rats were pretreated (15 min) with an intra-TMJ injection of 15d-PGJ2 (100 ng/15 ␮l/TMJ; n⫽6) followed by ipsilateral intra-TMJ injection of 5-HT (225 ␮g/15 ␮l/TMJ; Rodrigues et al., 2006). As a control, to demonstrate the role of sympathomimetic amines such as norepinephrine which plays an important role in inflammatory hyperalgesia, in a different set of animals, we depleted the peripheral stores of norepinephrine by systemically applying guanethidine (30 mg/kg; s.c. for 3 days; Chang et al., 1965; Rodrigues et al., 2006) before the intra-TMJ injection of 5-HT. Behavioral nociception response was evaluated for 30 min observation period. All animals received a final volume of 45 ␮l of solutions into the TMJ. Effect of 15d-PGJ2 in a plasma-protein extravasation induced by carrageenan. Immediately after carrageenan (300 ␮g/TMJ; n⫽6) injection, the Evan’s Blue dye (1%, 5 mg/kg) was injected into the left femoral vein and 45 min later TMJ inflammation was assessed by the extravasation of Evan’s Blue dye bound to plasma protein (Haas et al., 1992; Fiorentino et al., 1999). Each rat was then sacrificed under deep anesthesia and perfused with saline. Joint tissues were dissected, weighed and stored at ⫺20 °C. Evans’ Blue dye was extracted by immersing the joint tissue in 1 ml of formamide at 60 °C for 24 h. The samples absorbance was then determined in a spectrophotometer (Genesys) at 620 nm, and the Evan’s Blue dye concentration determined by comparison to a standard curve of known amounts of Evan’s Blue dye in extraction solution, which was assessed within the same assay. The amount of Evan’s Blue (micrograms) was then calculated per gram weight of tissue.

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Effect of 15d-PGJ2 on neutrophil migration induced by carrageenan injected in TMJ. Neutrophil migration into the rat TMJ tissues was evaluated by using a myeloperoxidase (MPO) kinetic colorimetric assay, as previously described (Cunha et al., 2008). Samples of TMJ injected with vehicle or carrageenan (300 ␮g) were collected in 50 mM K2HPO4 buffer (pH 6.0) containing 0.5% hexadecyl trimethylammonium bromide and kept at ⫺80 °C until use. Samples were homogenized using a Polytron (PT3100), centrifuged at 15000⫻g for 4 min and the resulting supernatant assayed spectrophotometrically for MPO activity determination at 450 nm (Spectra max), with three readings in 1 min. The MPO activity of samples was compared to a standard curve of neutrophils. Briefly, 10 ml of sample was mixed with 200 ml of 50 mM phosphate buffer pH 6.0, containing 0.167 mg/ml O-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The results were presented as MPO activity (number of neutrophils per mg of tissue).

Statistical analysis To determine if there were significant differences (P⬍0.05) among treatment groups, the data were analyzed using the t-test or one-way ANOVA as appropriate. If there was a significant between-subjects main effect of treatment group following one-way ANOVA, post-hoc contrasts, using the Bonferroni test, were performed to determine the basis of the significant difference. Data are presented in figures as means⫾SEM.

RESULTS 15d-PGJ2 inhibits formalin-induced nociception in the TMJ joint is dependent on PPAR-␥ activation 15d-PGJ2 (1, 10 and 100 ng/TMJ) inhibited in a dose dependent manner the behavioral nociception induced by formalin injected into the TMJ of rats (P⬍0.001). This effect was peripheral since the administration of 15d-PGJ2 into the contralateral TMJ did not inhibit nociception (Fig. 1A). In an attempt to investigate whether the antinocicep-

Fig. 1. Effect of locally administered 15d-PGJ2 on TMJ formalin-induced nociceptive response. (A) 15d-PGJ2 (1, 10 and 100 ng) significantly reduced the magnitude of 1.5% formalin-induced nociceptive responses. Last bar: application of 15d-PGJ2 (100 ng) into contralateral (ct) TMJ did not affect the magnitude of 1.5% formalin induced nociceptive response (P⬎0.05, t-test). (B) Administration of the PPAR-␥ antagonist (GW9662) significantly diminished the 15d-PGJ2 effect in the TMJ. The symbol (*) indicates statistical significance (P⬍0.05, t-test) compared to saline; the symbol (**) indicates statistical significance (P⬍0.05, ANOVA, Bonferroni test) compared to 1.5% formalin group; the symbol (⫹) indicates no statistical significance (P⬎0.05, ANOVA) compared to saline.

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tive action of 15d-PGJ2 depends on PPAR-␥ activation, we tested the effect of pretreatment with selective PPAR-␥ antagonist GW9662 upon 15d-PGJ2-induced antinociception. Injection of rats TMJ with GW9662 (3 ng/TMJ), 15dPGJ2 (100 ng/TMJ) and formalin (1.5%/TMJ), abrogated the antinociceptive activity (P⬍0.001) of 15d-PGJ2 on formalin-induced TMJ nociception (Fig. 1B).

The NO/cGMP/KⴙATP pathway mediates the antinociceptive effect of 15d-PGJ2 Fig. 2 shows that peripheral application of naloxone (10 ␮g/TMJ) blocked (P⬍0.01) the antinociceptive effect of 15d-PGJ2 (100 ng/TMJ). This result suggests that the antinociceptive action of 15d-PGJ2 depends on local opioids receptor activation. Injection with an inhibitor of the enzyme NOS aminoguanidine (0.1 mol/TMJ), the specific inhibitor of the soluble cGMP ODQ (8 ␮g/TMJ), or the K⫹ATP channel blocker glibenclamide (160 ␮g/TMJ), blocked the antinociceptive effect of 15d-PGJ2 (P⬍0.01) upon formalin-induced TMJ nociception (Fig. 2).

The antinociceptive effect of 15d-PGJ2 is mediated by ␦ and ␬-opioid receptors Fig. 3A shows that peripheral application of ␮-opioid receptor CTOP (20, 60 and 120 ␮g/ 15 ␮l/TMJ) did not abrogate the antinociceptive effect of 15d-PGJ2 (100 ng/ TMJ). However, the antagonist of ␦-opioid receptor ICI 174,864 (10 and 30 ␮g/15 ␮l/TMJ) and the antagonist of ␬-opioid receptor Nor-BNI. (200 ␮g/15 ␮l/TMJ) partially blocked (P⬍0.01) the antinociceptive effect of 15d-PGJ2 (Fig. 3B, C). This results suggest that the antinociceptive action of 15d-PGJ2 depends on ␦ (mainly) and ␬-opioid receptors activation. It is also important to note that the highest doses used of all three antagonists did not affect formalin-induced nociceptive response. 15d-PGJ2 inhibits 5-HT-induced nociception in the TMJ joint A previous study has demonstrated that 5-HT injected into the TMJ joint also induced nociception via sympathetic amines release (Rodrigues et al., 2006). To confirm this, our results demonstrated that the intra-TMJ administration

Fig. 2. Inhibition of 15d-PGJ2 antinociception by antagonists of opioid pathway. (A) naloxone (Nlx; 1 ␮g/TMJ) (B) aminoguanidine (AG; 0.1 mol/TMJ); (C) ODQ (8 ␮g/TMJ); (D) glibenclamide (Gbl; 160 ␮g/TMJ) abolished the antinociceptive effect of 15d-PGJ2 (100 ng/TMJ). The symbol (*) indicates statistical significance (P⬍0.05, t-test) compared to saline group; the symbol (**) indicates statistical significance (P⬍0.05, ANOVA, Bonferroni test) compared to formalin control group.

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Fig. 3. Role of ␮, ␦ and ␬-opioid receptors on 15d-PGJ2-induced antinociception. (A) CTOP (20 or 60 ␮g/TMJ) did not affect the antinociceptive effect of 15d-PGJ2 (100 ng/TMJ; P⬎0.05). The symbol (*) indicates statistical significance (P⬍0.05, t-test) compared to saline group; the symbol (**) indicates statistical significance (P⬍0.05, ANOVA, Bonferroni test) compared to formalin control group. (B) Nor-BNI (200 ␮g/TMJ) significantly diminished the antinociceptive effect of 15 d-PGJ2 (100 ng/TMJ). The symbol (*) indicates statistical significance (P⬍0.05, t-test) compared to saline group; the symbol (**) indicates statistical significance (P⬍0.05, ANOVA, Bonferroni test) compared to formalin control group; the symbol (⫹) indicates statistical significance (P⬍0.05, ANOVA, Bonferroni test) compared to formalin plus 15d-PGJ2 (100 ng/TMJ) group. (C) ICI 174,864 (10 or 30 ␮g /TMJ) significantly diminished the antinociceptive effect of 15d-PGJ2 (100 ng/TMJ). The symbol (*) indicates statistical significance (P⬍0.05, t-test) compared to saline group; the symbol (**) indicates statistical significance (P⬍0.05, ANOVA, Bonferroni test) compared to formalin control group; the symbol (⫹) indicates statistical significance (P⬍0.05, ANOVA, Bonferroni test) compared to formalin plus 15d-PGJ2 (100 ng/TMJ) group.

of 5-HT (225 ␮g) induced a nociceptive response (Fig. 4A). Depletion of peripheral stores of norepinephrine by

guanethidine (30 mg/kg; s.c. for 3 days) significantly reduced (P⬍0.001) the nociceptive response induced by

Fig. 4. Effect of locally administered 15d-PGJ2 on TMJ 5-HT-induced nociceptive response. (A) Guanethidine (s.c. 30 mg/kg for 3 days before TMJ injection of 5-HT) significantly reduced 5-HT-induced nociceptive response into TMJ. (B) 15d-PGJ2 (100 ng) significantly reduced the magnitude of 5-HT-induced nociceptive response (5-HT; 250 ␮g). The symbol (*) indicates statistical significance compared to saline group; the symbol (**) indicates statistical significance compared to 5-HT control group. P⬍0.05, one-way ANOVA followed by the Bonferroni test.

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Fig. 5. Effect of 15d-PGJ2 upon vascular permeability and neutrophil migration. Rats were pretreated with 15d-PGJ2 (100 ng/TMJ) or vehicle (saline) 15 min before the intra-TMJ injection of Cg (300 ␮g/TMJ), or saline (negative control). (A) Pretreatment of the TMJ with 15d-PGJ2 (100 ng) significantly diminished plasma extravasation induced by Cg. (B) Pretreatment of the TMJ with 15 d-PGJ2 (100 ng) inhibited neutrophil migration (MPO activity) induced by Cg (300 ␮g). The symbol (*) indicates statistical significance compared to saline group; the symbol (**) indicates statistical significance compared to Cg control group. P⬍0.05, one-way ANOVA followed by the Bonferroni test.

injection of 5-HT into the TMJ (Fig. 4A). Intra-TMJ administration of the 15d-PGJ2 (100 ng/TMJ) with 5-HT significantly decreased nociceptive behavior (P⬍0.05) when compared to 5-HT alone (Fig. 4B), suggesting that 15dPGJ2 is also effective on the sympathetic pathway of the inflammatory nociception. 15d-PGJ2 inhibits vascular permeability and neutrophil recruitment Treatment of rats with 15d-PGJ2 (100 ng/TMJ) significantly inhibited both plasma extravasation (P⬍0.01) measured by Evan’s Blue extravasation and carrageenan-induced neutrophil migration (P⬍0.001) into the TMJ tissue (Fig. 5A, B).

DISCUSSION Recently, there has been a great deal of interest in the involvement of PPAR in inflammation. There are evidences from many in vitro and a few in vivo studies demonstrating that PPAR-␥ agonist may present anti-inflammatory activity. In the present study, we provide further evidence to support this activity by demonstrating that 15d-PGJ2, a PPAR-␥ agonist, has antinociceptive and anti-inflammatory activities in the TMJ of rats. The most relevant finding of the present investigation is that 15dPGJ2 induced-peripheral antinociceptive response in the TMJ, which may result from the co-activation of more than one neuronal receptor since our results suggest the participation of ␬/␦ opioid receptors in this effect. It has been demonstrated that 15d-PGJ2 is able to exert its effects in models of inflammatory diseases dependent on PPAR-␥ activation (Ricote et al., 1998; Heneka et al., 2000; Cuzzocrea et al., 2003; Zingarelli et al., 2003; Napimoga et al., 2008a). In the present study, we demonstrated that GW9662 (PPAR-␥ antagonist) abrogated the antinociceptive effects of 15d-PGJ2. These findings, therefore, suggest that 15d-PGJ2 activates the PPAR-␥ recep-

tor resulting in attenuation of the TMJ nociception, supporting that functional PPAR-␥ receptors are present in the rat TMJ. In agreement with our results Churi et al. (2008) showed PPAR-␥ expression in the spinal cord and reported that activation of these receptors inhibits allodynia in a neuropathic pain model. These receptors when activated suppress formalin-induced TMJ afferent activity, which is in line with previous studies demonstrating PPAR-␥ receptor-mediated antinociception (Napimoga et al., 2008b). Moreover, this effect was arguably the result of activation of PPAR-␥ receptors local to the site of inflammation since 15d-PGJ2 injection into the contralateral joint did not reproduce an antinociceptive effect. These findings support the suggestion that functional PPAR-␥ receptors are present in the rat TMJ. In a previous report we could not inhibit the nociceptive action of formalin in the rat paw or hot plate test with 15d-PGJ2 (Napimoga et al., 2008b); however in this report our results demonstrated an inhibition of formalin-induced nociception in the TMJ. Painful TMD involve deep tissues such as the TMJ and has characteristics different from pain of cutaneous origin (Dubner, 1991; Zhou et al., 1999). Several models have demonstrated the characteristic of orofacial deep-tissue pain is different from pain of cutaneous origin (Yu et al., 1993; Fiorentino et al., 1999; Imbe et al., 2001; Roveroni et al., 2001; Clemente et al., 2004). Previous studies reported that TMJ inflammation produced greater facilitatory effects on medullary dorsal horn nociceptive neurons (Iwata et al., 1999) and enhanced induction of Fos-protein expression (Zhou et al., 1999) compared to cutaneous tissue inflammation. Deep craniofacial inputs evoked the expansion of mechanoreceptive field (Yu et al., 1993) and robust neuronal hyperexcitability in trigeminal nucleus more effectively than cutaneous inputs (Ren and Dubner, 1999). Therefore, we hypothesized that greater inflammatory responses in the TMJ tissue stimulate the endogenous modulatory pain system which in turn increase the antinociceptive action of 15d-PGJ2.

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Otherwise, another relevant fact is that TMJ tissues are more sensitive to sympathomimetic amines and PGE2 than cutaneous tissues (Rodrigues et al., 2006). It is well known that inflammatory pain has also a sympathetic component (Levine et al., 1986; Parada et al., 2001) that may predominate in pain less sensitive to NSAIDs and that TMJ receives a rich sympathetic innervation (Widenfalk and Wiberg, 1990; Yoshino et al., 1998; Kido et al., 2001). In addition, it has been demonstrated that sympathomimetic amines contribute to inflammatory TMJ hyperalgesia (Rodrigues et al., 2006). It may be possible that the TMJ is more sensible to prostaglandins in comparison to paw tissue, which could explain our results. In the present work we have demonstrated that 5-HTinduced pain in the TMJ. 5-HT is an important mediator that has been associated with both “overt pain” and hyperalgesia (Parada et al., 2001; Rodrigues et al., 2006; Tambeli et al., 2006). It contributes to pain in TMJ disorders in humans, since the level of 5-HT is increased in patients with TMJ inflammatory disorders (Kopp, 1998; Alstergren et al., 1999), and the 5-HT3 receptor antagonist granisetron reduces TMJ pain in humans (Voog et al., 2000). Our results demonstrated that sympathetic amines contribute to the TMJ nociception induced by 5-HT and that 15dPGJ2 was able to inhibit such effect. This result is very interesting since we demonstrated that 15d-PGJ2 may be useful as a pharmacological tool to control TMJ-sympathetic pain. Studies provide potentially conflicting evidence that 15d-PGJ2 is an agonist of TRPA1 channels to elicit inflammatory pain in the nervous system (Takahashi et al., 2008; Andersson et al., 2008; Cruz-Orengo et al., 2008). TRPA1 is a member of the transient receptor potential (TRP) family of cation channels that is highly expressed by a subset of C-fiber nociceptors (Story et al., 2003; Kobayashi et al., 2005) that plays an important role in modulating nociceptor excitability and neurogenic inflammation in the setting of tissue injury (Bautista et al., 2006; Kwan et al., 2006). TRPA1 is activated by a number of environmental irritants that cause pain, including formalin (Macpherson et al., 2007; McNamara et al., 2007). Of importance, whereas low concentrations of 15d-PGJ2 can exert an analgesic and anti-inflammatory effect (present data; Napimoga et al., 2008a,b; Landar et al., 2006; Ou et al., 2006), higher concentrations of 15d-PGJ2 can cause tissue damage and pain (Takahashi et al., 2008; Andersson et al., 2008; CruzOrengo et al., 2008; Koh et al., 2005). Thus, these contradictions could be related to the fact that the effect of 15d-PGJ2 is concentration dependent. Previously, we suggested that 15d-PGJ2 promotes peripheral analgesia in the paw of rats by the stimulation of endogenous opioid release (Napimoga et al., 2008b). The antinociceptive effect mediated by 15d-PGJ2 in the formalin-induced nociceptive response in the TMJ of rats seems to involve a peripheral opioid effect since its antinociceptive effect was abrogated by intra-TMJ injection of naloxone, a nonspecific opioid antagonist. In the present work, when specific receptor opioid antagonists were used, instead of naloxone, ICI 164,864 (a selective ␦-opioid recep-

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tor antagonist) and Nor-BNI (a selective ␬-opioid receptor antagonist) but not CTOP (a selective ␮-opioid receptor antagonist) were able to interfere with the action of 15dPGJ2. These findings suggest that 15d-PGJ2 induced-peripheral antinociceptive response in the TMJ may result from the co-activation of more than one neuronal receptor since our results suggest the participation of ␬/␦ opioid receptors in such effect. In our previous work, we observed that 15d-PGJ2 was not able to cause antinociception when administered intraganglionarly (Napimoga et al., 2008b), supporting the suggestion that 15d-PGJ2 is not acting directly on sensitive neurons and pointing to an action through endogenous opioid release by resident cells. However, whether opioid receptors are expressed in the resident cells of the trigeminal ganglia remains to be investigated. Otherwise, co-application of the NOS inhibitor (aminoguanidine) or cGMP inhibitor (ODQ) or K⫹ATP (glibenclamide) abrogated 15d-PGJ2-mediated antinociception in the TMJ, confirming our previous results in which 15dPGJ2 induced antinociception in rat’s hind paw dependent on L-arginine/NO/cGMP similar to opioid-related drugs (Napimoga et al., 2008b). It is well known that the peripheral antinociceptive effects of opioid receptors are mediated by the activation of the intracellular L-arginine/NO/ cGMP pathway (Ferreira et al., 1991; Amarante and Duarte, 2002). The mechanism by which activation of the L-arginine/NO/cGMP pathway by opioid receptors results in analgesia is presently unknown. Data from literature suggest that the peripheral action of morphine (Rodrigues and Duarte, 2000) and of nitric oxide donor sodium nitroprusside (Soares et al., 2000) could potentially involve the opening of ATP-dependent K⫹ channels that, in turn, would result in K⫹ outward currents thereby counteracting the lowering of the nociceptor threshold, which corroborates with our results. Previous studies demonstrate that inflammatory stimuli besides inducing the production of mediators that sensitize the nociceptor also induce the release of mediators, which mediate leukocyte migration and other inflammatory events, such as oedema. In the present study, we demonstrated that 15d-PGJ2 significantly reduced carrageenaninduced plasma extravasation and neutrophil migration in the TMJ. Previously, we also demonstrated that 15d-PGJ2mediated downregulation of neutrophil migration, is related to an increase on NO production which, in turn, suppresses ICAM-1 expression on microvessels (Napimoga et al., 2008a). On the other hand there are evidences in the literature demonstrating that neutrophils are involved in the genesis of inflammatory hypernociception and oedema (Levine et al., 1985; Cunha et al., 2008). Thus, the inhibition of neutrophil might explain in part the 15d-PGJ2 antinociceptive activity.

CONCLUSION We provide evidences that 15d-PGJ2 presents a peripheral antinociceptive effect, which may result from the coactivation of more than one neuronal receptors. 15d-PGJ2

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has a potential peripheral antinociceptive and anti-inflammatory effect in the TMJ via PPAR-␥ with the co-participation of ␬/␦ opioid receptors. These findings taken together with the evidence that TMJ pain is a significant part of symptoms in patients with temporomandibular disorders and a common source of chronic orofacial pain highlight the potential clinical relevance of 15d-PGJ2 in the treatment of TMD. Acknowledgments—This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG # 34/07 and # 40/07) PAPE-UNIUBE (2007/007) and CAPES.

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(Accepted 22 July 2009) (Available online 30 July 2009)