Luana Fischer

Titanium and zirconia particle-induced pro-inflammatory gene expression in cultured macrophages and osteolysis, inflammatory hyperalgesia and edema in vivo.

AIMS The biological reaction to wear debris is critical to the osteolysis underlying aseptic loosening of joint prosthetic implants. In an attempt to reduce aseptic loosening, ceramics have been introduced. This study was designed to evaluate, compare and correlate the expression of Toll-like receptors (TLRs), their intracellular adaptors and proinflammatory cytokines in cultured macrophages challenged with titanium or zirconia particles, as well as particle-induced osteolysis in calvaria and hyperalgesia and edema in hind paw. MAIN METHODS TLRs and their adaptors were evaluated at the mRNA level by RT-PCR, and cytokine expression was evaluated at the mRNA and protein levels. Osteolysis and hyperalgesia and edema were evaluated in vivo, in calvaria and hind paw, respectively. KEY FINDINGS Cultured macrophages challenged with zirconia or titanium particles expressed increased mRNA for TLRs 2, 3, 4 and 9, and their adaptors MyD88, TRIF and NF-κB and cytokines TNF-α, IL-1β and IL-6, which were also increased at protein level. Quantitative differences are evident and, in general, zirconia particle-induced pro-inflammatory gene expression was lower than that induced by titanium particles. In in vivo experiments, exposition to titanium or zirconia particles induced osteolysis in calvaria and hyperalgesia and edema in hind paw; however those induced by zirconia particles were significantly lower. There is a strong and positive correlation between the expressions of mRNA for TLR4, NF-κB, TNF-α, IL-1β and IL-6. SIGNIFICANCE Collectively, our data suggest that zirconia ceramic particles are less bioactive than titanium particles.

Effect of gonadal steroid hormones on formalin-induced temporomandibular joint inflammation

We have recently demonstrated that gonadal steroid hormones decrease formalin‐induced temporomandibular joint nociception in rats. Given that the attenuation of inflammation is a potential mechanism underlying this antinociceptive effect, we evaluated the effect of gonadal steroid hormones on formalin‐induced temporomandibular joint inflammation. Plasma extravasation, a major sign of acute inflammation, and neutrophil migration, an important event related to tissue injury, were evaluated. Formalin induced significantly lower temporomandibular joint plasma extravasation and neutrophil migration in proestrus females than in males and in diestrus females. Since estradiol serum level is high in proestrus females and low in diestrus females and in males, these findings suggest that the high physiological level of estradiol decreases temporomandibular joint inflammation. Estradiol but not progesterone administration in ovariectomized females significantly decreased formalin‐induced plasma extravasation and neutrophil migration, an effect that was blocked by the estrogen receptor antagonist ICI 182780. Plasma extravasation and neutrophil migration were not affected by orchiectomy, but testosterone or estradiol administration in orchidectomized males significantly decreased them. The androgen receptor antagonist flutamide blocked the anti‐inflammatory effect of testosterone while ICI 182780 blocked that of estradiol in males. Previous intravenous administration of a nonspecific selectin inhibitor significantly decreased formalin‐induced temporomandibular joint nociception and neutrophil migration in males, revealing a potent and positive correlation between temporomandibular joint nociception and inflammation. Taken together, these findings demonstrate a pronounced anti‐inflammatory effect of estradiol and testosterone in the temporomandibular joint region and suggest that this effect may mediate, at least in part, the antinociceptive effect of these hormones.

The contribution of transient receptor potential ankyrin 1 (TRPA1) to the in vivo nociceptive effects of prostaglandin E2

AIMS Although evidence suggest that TRPA1 mediates some effects of prostaglandins, it is not known whether TRPA1 contributes to the in vivo nociceptive effects of prostaglandin E2 (PGE2), a key mediator of inflammatory pain. MAIN METHODS To address this issue, the effect of the pharmacological blockade of TRPA1 or of its gene silencing on the hyperalgesia induced in the rat paw by PGE2 or its downstream signaling molecules, protein kinase A (PKA) or protein kinase C-epsilon (PKCε), was evaluated. TRPA1 expression on dorsal root ganglia cells was assessed by western blot. KEY FINDINGS The pharmacological blockade of local TRPA1 by its selective antagonist, HC 030031 decreased and reversed PGE2-induced hyperalgesia. The TRPA1 gene silencing induced by intrathecal pre-treatment with antisense oligodeoxynucleotide blocked PGE2-induced hyperalgesia and strongly reduced TRPA1 expression in dorsal root ganglia cells (L5 and L6). PGE2 injection into the hind paw did not significantly increase TRPA1 expression in dorsal root ganglia cells. Treatment with either HC 030031 or antisense oligodeoxynucleotide significantly decreased the hyperalgesia induced by PKA or PKCε. Since both kinases are the major components of PGE2-induced intracellular signal transduction, the modulation of TRPA1 function by PGE2 may be downstream PKA and PKC-epsilon. SIGNIFICANCE These findings show that TRPA1 is essential to the in vivo nociceptive effects induced by one of the most important mediators of inflammatory pain, PGE2. This is one of the crucial findings necessary to support TRPA1 as a promising target for the development of future drugs to pain treatment and control.

Local kappa opioid receptor activation decreases temporomandibular joint inflammation.

In an attempt to decrease central side effects associated with the use of opioids, some strategies have been developed by targeting peripheral opioid receptors. In this context, kappa receptors are of major interest, since, in contrast to other opioid receptors, their activation is not associated with potent peripheral side effects. We have recently demonstrated that local activation of kappa opioid receptors significantly decreases formalin-induced temporomandibular joint nociception; however, whether it also decreases temporomandibular joint inflammation is not known. To address this issue, we evaluated if a specific kappa opioid receptor agonist, U50,488 (trans-(1S,2S)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide hydrochloride hydrate), administered into the temporomandibular joint decreases formalin-induced plasma extravasation and neutrophil migration. Ipsilateral, but not contralateral, administration of U50,488 into the temporomandibular joint blocked formalin-induced plasma extravasation and neutrophil migration in a dose-dependent manner. This anti-inflammatory effect was reversed by the ipsilateral, but not contralateral, administration of the kappa opioid receptor antagonist nor-BNI (nor-binaltorphimine dihydrochloride). This study demonstrates that local activation of kappa opioid receptors decreases two important parameters of temporomandibular joint inflammation, that is, plasma extravasation and neutrophil migration, in a dose-dependent and antagonist-reversible manner. This anti-inflammatory effect taken together with the potent antinociceptive effect, suggests that drugs targeting peripheral kappa opioid receptors are promising for the treatment of inflammatory temporomandibular joint pain and probably, other articular pain conditions with an inflammatory basis.

PERIPHERAL ESTRADIOL INDUCES TEMPOROMANDIBULAR JOINT ANTINOCICEPTION IN RATS BY ACTIVATING THE NITRIC OXIDE/CYCLIC GUANOSINE MONOPHOSPHATE SIGNALING PATHWAY

Recently, we have reported that high physiological estradiol level during the proestrus phase of the estrous cycle or systemic estradiol administration in ovariectomized rats decreases formalin-induced temporomandibular joint nociception. However, the mechanisms underlying the antinociceptive effect of estradiol are presently unknown. In this study, we used the temporomandibular joint formalin model in rats to investigate whether estradiol decreases nociception by a peripheral non-genomic mechanism, and if so, whether this mechanism is mediated by the activation of the nitric oxide-cyclic guanosine monophosphate signaling pathway and of opioid receptors. The administration of estradiol into the ipsilateral, but not into the contralateral temporomandibular joint significantly reduced formalin-induced temporomandibular joint nociception in ovariectomized and diestrus but not in proestrus females. However, the administration of the estrogen receptor antagonist ICI 182780 into the ipsilateral, but not into the contralateral temporomandibular joint blocked the antinociceptive effect of serum estradiol in proestrus females, suggesting that the physiological effect of estradiol in nociception is mediated, at least in part, by a peripheral mechanism. The administration of estradiol into the ipisilateral temporomandibular joint did not affect formalin-induced nociception in male rats. The antinociceptive effect of temporomandibular joint estradiol administration in ovariectomized and diestrus females was mimicked by estradiol conjugated with bovine serum albumin, which does not diffuse through the plasma membrane, and was blocked by the estrogen receptor antagonist ICI 182780. The administration of the nitric oxide synthase inhibitor (nitro-l-arginine) or of a guanylate cyclase inhibitor (1H-(1,2,4)-oxadiasolo (4,2-a) quinoxalin-1-one) into the ipsilateral, but not into the contralateral temporomandibular joint blocked the antinociceptive effect of estradiol and of estradiol conjugated with bovine serum albumin, while the opioid receptor antagonist naloxone had no effect. These findings suggest that estradiol decreases temporomandibular joint nociception in female rats through a peripheral non-genomic activation of the nitric oxide-cyclic guanosine monophosphate signaling pathway.

Mechanisms underlying transient receptor potential ankyrin 1 (TRPA1)-mediated hyperalgesia and edema

The aim of this study was to investigate the mechanisms that contribute to hyperalgesia and edema induced by TRPA1 activation. The injection of allyl isothiocyanate (AITC, 50, 100, or 300 µg/paw) into the rats hind paw induced dose and time‐dependent hyperalgesia and edema, which were blocked by the selective TRPA1 antagonist, HC 030031 (1,200 µg/paw), or by treatment with antisense oligodeoxynucleotide (four daily intrathecal injections of 5 nmol). These results demonstrate that the hyperalgesia and edema induced by AITC depend on TRPA1 activation. AITC‐induced hyperalgesia and edema were significantly reduced by treatment with neurokinin 1 (L‐703,606, 38 µg/paw) or calcitonin gene‐related peptide (CGRP8‐37, 5 µg/paw) receptor antagonists, with a mast cell degranulator (compound 48/80, four daily injections of 1, 3, 10, and 10 µg/paw) or with H1 (pyrilamine, 400 µg/paw), 5‐HT1A (wAy‐100,135, 450 µg/paw) or 5‐HT3 (tropisetron, 450 µg/paw) receptor antagonists. Pre‐treatment with a selectin inhibitor (fucoidan, 20 mg/kg) significantly reduced AITC‐induced hyperalgesia, edema, and neutrophil migration. Finally, a cyclooxygenase inhibitor (indomethacin, 100 µg/paw), a β1 (atenolol, 6 µg/paw) or a β2 (ICI 118, 551, 1.5 µg/paw) adrenoceptor antagonist also significantly reduced AITC‐induced hyperalgesia and edema. Together, these results demonstrate that TRPA1 mediates some of the key inflammatory mechanisms, suggesting a key role of this receptor in pain and inflammation.

Contribution of endogenous opioids to gonadal hormones-induced temporomandibular joint antinociception.

The authors have recently demonstrated that the high serum estradiol level during the proestrus phase of the estrous cycle and that the administration of estradiol or progesterone in ovariectomized female and of testosterone in orchiectomized male rats significantly decrease formalin-induced temporomandibular joint (TMJ) nociception. In this study, the authors investigate the contribution of endogenous opioids to this antinociceptive effect of gonadal hormones in the TMJ formalin test. The opioid receptor antagonist naloxone was administrated either in the surrounding of the trigeminal sensory complex or in the TMJ region. The antinociceptive effect induced by endogenous estradiol in proestrus females and by exogenous estradiol in ovariectomized females was blocked by the administration of naloxone in the surrounding of the trigeminal sensory complex, but not in the TMJ region. The antinociceptive effect induced by the administration of progesterone in ovariectomized females and of testosterone in orchiectomized males was blocked by the administration of naloxone either in the surrounding of the trigeminal sensory complex or in the TMJ region. The authors conclude that central and peripheral opioid mechanisms mediate the antinociceptive effect of progesterone and testosterone, and central opioid mechanisms mediate the antinociceptive effect of estradiol. These findings suggest that the enhanced pain perception during low gonadal hormone periods in women and animals may be mediated by a decrease in endogenous opioid activity. This suggestion helps explain the higher severity of some pain conditions, such as temporomandibular dysfunctions in women than in men, that have no hormonal fluctuations.

Sexual Dimorphism on Cytokines Expression in the Temporomandibular Joint: The Role of Gonadal Steroid Hormones

Temporomandibular joint pain-related conditions are generally characterized by local inflammation; however, little studies have focused on the role of gonadal hormones in the expression of inflammatory mediators, such as cytokines. Therefore, we asked whether gonadal steroid hormones affect formalin-induced cytokines expression in the rat temporomcandibular joint. The expression of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and cytokine-induced neutrophil chemoattractant (CINC)-1 was significantly higher in males than in diestrus and proestrus females and was decreased by orchiectomy and restored by testosterone replacement. The expression of IL-6 was significantly higher in diestrus and proestrus females than in males, and was decreased by ovariectomy and restored by estradiol or progesterone administration. We conclude that testosterone increases the expression of TNF-α, IL-1β and CINC-1, and estradiol and progesterone increase the expression of IL-6. New clinical approaches based on inhibition of pro-inflammatory mediators are starting to supplant traditional immunosuppressive therapies and gonadal hormones may influence their effectiveness or clinical dosage.

The functional role of ascending nociceptive control in defensive behavior.

Ascending nociceptive control is a novel spino-striato-rostral ventral medulla pain modulation pathway that mediates heterosegmental pain-induced analgesia, i.e., noxious stimulus-induced antinociception. In this study, we used the dorsal immobility response in rats as a model of the defensive responses. We demonstrated that the activation of ascending nociceptive control by peripheral noxious stimulation and spinal AMPA and mGluR1 receptor blockade significantly potentiated the duration of the dorsal immobility response in rats via an opioid-dependent mechanism in the nucleus accumbens. These results demonstrated the functional role of ascending nociceptive control in the modulation of defensive responses and spinal glutamatergic receptors in the dorsal immobility response. The immobility response is an antipredator behavior that reflects the underlying state of fear, and ascending nociceptive control may modulate fear.

Nucleus accumbens mediates the pronociceptive effect of sleep deprivation: the role of adenosine A2a and dopamine D2 receptors

Abstract Sleep disorders increase pain sensitivity and the risk of developing painful conditions; however, the underlying mechanisms are poorly understood. It has been suggested that nucleus accumbens (NAc) influences sleep-wake cycle by means of a balance between adenosine activity at A2A receptors and dopamine activity at D2 receptors. Because the NAc also plays an important role in pain modulation, we hypothesized that the NAc and its A2A and D2 receptors mediate the pronociceptive effect of rapid eye movement (REM) sleep deprivation (SD). We found that 24 hours of REM-SD induced an intense pronociceptive effect in Wistar rats, which decreases progressively over a sleep rebound period. Although the level of fecal glucocorticoid metabolites increased with SD within group, it did not differ between sleep-deprived group and control group, indicating a stress response with similar magnitude between groups. The pronociceptive effect of REM-SD was prevented by excitotoxic lesion (N-Methyl-D-aspartate, 5.5 &mgr;g) of NAc and reverted by its acute blockade (Qx-314, 2%). The administration of an A2A receptor antagonist (SCH-58261, 7 ng) or a D2 receptor agonist (piribedil, 6 &mgr;g) into the NAc increased home cage activity and blocked the pronociceptive effect of REM-SD. Complementarily, an A2A receptor agonist (CGS-21680, 24 ng) impaired the reversal of the pronociceptive effect and decreased home cage activity, as it did a D2 receptor antagonist (raclopride, 5 &mgr;g). Rapid eye movement SD did not affect the expression of c-Fos protein in NAc. These data suggest that SD increases pain by increasing NAc adenosinergic A2A activity and by decreasing NAc dopaminergic D2 activity.