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cyclic guanosine monophosphate signaling pathway
Neuroscience 164 (2009) 724 –732
PERIPHERAL ESTRADIOL INDUCES TEMPOROMANDIBULAR JOINT ANTINOCICEPTION IN RATS BY ACTIVATING THE NITRIC OXIDE/CYCLIC GUANOSINE MONOPHOSPHATE SIGNALING PATHWAY N. C. FÁVARO-MOREIRA,a K. E. TORRES-CHÁVEZ,a L. FISCHERb AND C. H. TAMBELIa*
Key words: estradiol, nociception, temporomandibular joint pain, nitric oxide, cGMP, formalin.
a Laboratory of Orofacial Pain, Department of Physiology, Faculty of Dentistry of Piracicaba, State University of Campinas-UNICAMP, Avenue Limeira 901, CEP 13414-900, Piracicaba, SP, Brazil
The majority of chronic pain conditions (Unruh, 1996) such as temporomandibular dysfunctions (TMDs) (Dworkin et al., 1990) are more prevalent and severe in women than in men. These findings may apparently suggest a pronociceptive effect of estradiol, as supported by some animal studies (Bereiter, 2001; Cairns et al., 2002; Okamoto et al., 2003). However, our findings that formalin or glutamateinduced temporomandibular joint (TMJ) nociception in rats (Fischer et al., 2008) and those that TMD pain in women (LeResche et al., 2003) is higher during low estradiol times of the reproductive cycle suggest that estradiol attenuates TMJ pain. This suggestion is also supported by other studies in humans (Smith et al., 2006) and in animals (Gaumond et al., 2002; Ceccarelli et al., 2003; Pajot et al., 2003; Kuba et al., 2006; Mannino et al., 2007) that use other pain models. Based on our previous animal studies, we suggest that the lower prevalence of TMJ pain in men may result from a protective effect of testosterone that reduces the risk of males developing TMJ pain (Fischer et al., 2007) and the higher severity of TMJ pain in women may result from the estrogen fluctuation during reproductive cycle, in that TMJ pain is increased during low serum estradiol levels (Fischer et al., 2008). Although the mechanisms underlying the antinociceptive effect of estradiol are presently unknown, estradiol might induce antinociception, at least in part, by a peripheral non-genomic mechanism. This idea is supported by “in vitro” studies showing that estradiol inhibits calcium channel currents in neurons of the dorsal root ganglia via activation of membrane estrogen receptors (Lee et al., 2002; Chaban et al., 2003). In contrast to estrogen receptors located in the cytosol, those located on the membrane mediate the rapid non-genomic effects of estradiol through the activation of second messenger cascades (Evrard and Balthazart, 2004), including the L-arginine–nitric oxide (NO)– cyclic guanosine mono-phosphate (cGMP) pathway (L-arg–NO– cGMP), as demonstrated in endothelial cells (Stefano et al., 2000). This pathway involves the synthesis of NO from L-arginine and the subsequent activation of the guanylate cyclase, leading to increased levels of cGMP, a second messenger that has been associated with peripheral antinociception, including that mediated by peripheral opioids (Durate et al., 1990; Pol, 2007). Estradiol via activation of non-genomic mechanisms might induce antinociception by increasing the release of endogenous opioids
b
Laboratory of Pain Physiology, Division of Biological Sciences, Department of Physiology, Federal University of Parana, Curitiba, Parana, Brazil
Abstract—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. © 2009 Published by Elsevier Ltd on behalf of IBRO. *Corresponding author. Tel: ⫹55-19-2106-5305; fax: ⫹55-19-2106-5212. E-mail address: [email protected] (C. H. Tambeli). Abbreviations: cGMP, cyclic guanosine mono-phosphate; DMSO, dimethyl sulfoxide; E–BSA, estradiol coupled to bovine serum albumin; L-NNA, nitro-L-arginine; NO, nitric oxide; ODQ, 1H-(1,2,4)-oxadiasolo (4,2-a) quinoxalin-1-one; OVX, ovariectomized; TMJ, temporomandibular joint.
0306-4522/09 $ - see front matter © 2009 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2009.08.012
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by opioid peptide-containing immune cells (Rittner et al., 2008) and/or by interacting with opioid receptor-coupled second messenger systems (Lagrange et al., 1995; Brown et al., 2007). In fact, peripheral opioid mechanisms mediate the antinociceptive effect induced by pregnancy hormones in the rat’s TMJ (Arthuri et al., 2005). Therefore, in this study we have used the TMJ formalin model 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 NO– cGMP signaling pathway and of opioid receptors.
EXPERIMENTAL PROCEDURES
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OVX females and the most effective dose was selected for further experiments.
Estrous phase determination Estrous phase was determined by daily microscope examination of vaginal smears between 7 and 8 AM. In the day of the experiment, estrous phase was confirmed before and immediately after each experiment to ensure that the rats remained in the same phase. Proestrus phase and the initial phase of diestrus (first 4 h) were identified by the predominance (⬎70%) of nucleated epithelial cells and leukocytes, respectively (Butcher et al., 1974) in rats with at least two consecutive regular 4 –5 day cycles. These phases were chosen because they represent phases of high and low ovarian hormonal level, respectively (Butcher et al., 1974).
Animals
Gonadectomy
This study was carried out in 200 –300 g ovariectomized (OVX, n⫽223), diestrus (n⫽128) and proestrus (n⫽43) female and in male (n⫽41) Wistar rats. All efforts were made to minimize the number of animals used and their suffering. The animals were used in experiments when they were 60 –75 days old; the mean weights of OVX females (266.2⫾7.8) were significantly greater (t-test, P⫽⬍0.05) than that of intact proestrus and diestrus females (225.0⫾5.8). To investigate a peripheral estradiol-mediated effect we firstly used OVX females because the depletion of serum estradiol level possibly facilitates its assessment. Then we included diestrus and proestrus females because they represent a physiological condition of low and high serum estradiol level, respectively. Male rats were also included because the antinociceptive effect of systemic estradiol is sex specific (Fischer et al., 2007; 2008) and therefore we asked if the peripheral effect might also be. All animal experimental procedures and protocols were approved by the Committee on Animal Research of the University of Campinas and are in accordance with IASP guidelines for the study of pain in animals (Zimmermann, 1983). The animals were maintained on a temperature-controlled room (⫾23 °C) and were housed in plastic cages (45⫻30⫻15 cm3) with soft bedding (five/ cage) on a 12:12 light cycle (lights on at 6:00 AM) with food and water available ad libitum.
Ovariectomy (45 days old females; (Gordon and Soliman, 1994)) was performed through bilateral upper flank incisions. The ovarian bundles were tied off with 4-O silk sutures and the ovaries removed. The fascia and the skin were closed with 4-O silk sutures. Rats of two experimental groups were sham operated and underwent a surgical procedure similar to that of OVX animals, except that the ovaries were not removed. These groups were used to demonstrate that the surgical procedure does not affect the nociceptive behavior response in diestrus and proestrus females (naïve vs. sham). The procedures were carried out under anesthesia induced by an i.m. injection of a mixture of ketamine (55 mg/kg) and xylazine (5.5 mg/kg). An s.c. injection of ketoprofen (5 mg/kg) was used for post-operative analgesia (Roughan and Flecknell, 2000). OVX and sham-operated rats were used in experiments when they were 3 months of age. The efficacy of ovariectomy was confirmed by the absence of estrous cycle determined by observation of vaginal smears during 10 days.
Drugs Formalin was prepared from commercially available stock formalin (an aqueous solution of 37% of formaldehyde) further diluted in 0.9% NaCl to a concentration of 1.5% (Roveroni et al., 2001); estradiol (17-estradiol, 0.4, 1.2 g; (Ceccarelli et al., 2004) and 3.6 g) was dissolved in propylene glycol; estradiol coupled to bovine serum albumin (E–BSA, 1.2 g of estradiol plus BSA); the NO synthase inhibitor nitro-L-arginine (L-NNA 22 g; (Toda et al., 1993)) and the opioid receptor antagonist naloxone (10 g; (Eisenberg et al., 1996), and 30 g) were dissolved in 0.9% NaCl. The selective estrogen receptor antagonist ICI 182780 (0.16, 1 and 6 g; (Ceccarelli et al., 2004) and the guanylate cyclase inhibitor 1H-(1,2,4)-oxadiasolo (4,2-a) quinoxalin-1-one (ODQ 0.8 and 8 g; (Cunha et al., 1999) were dissolved in dimethyl sulfoxide (DMSO)). Formalin, estradiol, E–BSA, naloxone and L-NNA were purchased from Sigma Aldrich, St. Louis, MO, USA; ODQ and ICI182780 were purchased from Tocris Bioscience, St. Louis, MO, USA. Steroid hormones conjugated with bovine serum albumin have been extensively used to assess their non-genomic effects (Kelly and Levin, 2001). However, it was suggested that E–BSA has biological activity not observed with estradiol. For this reason, the experiments were performed using both 17-estradiol and E–BSA. The dose–response curves of all drugs were performed in
TMJ injections The animals were briefly anesthetized by inhalation of halothane to allow the TMJ injection; each animal regained consciousness approximately 30 s after discontinuing the anesthetic. The TMJ injection was performed with a 30-gauge needle introduced into the TMJ at the moment of injection. The contralateral TMJ injections, when necessary, were performed immediately after the ipsilateral injections. A cannula consisting of a polyethylene tube was connected to the needle and also to a Hamilton syringe (50 l) (Roveroni et al., 2001). The total volume injection varied between 30 and 45 l, when two or three drugs were injected in the same TMJ, they were administered at a volume of 15 l per drug in a unique injection. At the conclusion of the behavior test, each animal was anesthetized by an i.p. injection of a mixture of urethane (1 g/kg) and ␣-chloralose (50 mg/kg). To confirm the correct site of injection, the Evans Blue dye (5 mg/kg) was systemically injected and 15 min later the animals were submitted to cardiac perfusion with normal saline. Since this dye binds to plasma protein, the correct site of injection was indicated by the observation that the plasma extravasation induced by the TMJ injection of formalin was restricted to the TMJ region (Haas et al., 1992).
Testing procedure for TMJ pain Behavior test was performed during light phase (between 9:00 AM and 5:00 PM) in a quiet room maintained at ⫾23 °C (Rosland, 1991). Rats did not have access to food or water during the test and each animal was used once. The nociceptive response was assessed by an observer blinded to the experimental manipulation. Before the experiments, each animal was manipulated for 7 days in the test room (handled for approximately 1 min) to be habituated to the experimental manipulation. On the day of the
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The nociceptive behavior score, obtained by summing the flinching and rubbing behaviors recorded during the entire duration of the experiment was used in statistical analysis in Figs. 1–5. The concentration of the Evan’s Blue dye extravasated per gram weight of TMJ tissue was used in statistical analysis in Fig. 6. To determine if there were significant differences among treatment groups one-way ANOVA was performed. If there was a significant between-subjects main effect of treatment group after one-way ANOVA, post hoc contrasts, using the Tukey test, were performed to determine the basis of the significant difference. A minimum of four subjects in each group is sufficient to detect a significant difference at a power of 0.8. The level for statistical significance was P⬍0.05. Data are expressed in figures as means⫾SEM.
TMJ and by physiological estradiol are not additive. To investigate whether they are on the same pathway we verified if the co-administration of the estrogen receptor antagonist ICI 182780 with formalin into the TMJ affects the antinociceptive effect induced by high serum estradiol level in proestrus females. As demonstrated in Fig. 1C, the co-administration of the estrogen receptor antagonist ICI 182780 into the ipsilateral, but not into the contralateral TMJ of proestrus females significantly increased their nociceptive behavior. Formalin-induced TMJ nociceptive behavior in proestrus females receiving ICI 182780 into the ipsilateral TMJ was similar to that of diestrus and OVX females receiving only formalin or formalin plus ICI 182780 (one way ANOVA, data not shown). Co-administration of estradiol with formalin into the TMJ did not affect nociception in males, even at a dose of estradiol three times higher than that used in females and even using an equi-nociceptive concentration of formalin (5%) (Fig. 1D). To discard that the antinociceptive effect induced by the TMJ injection of estradiol could derivate from a nonspecific action, we evaluated the involvement of estrogen receptors in estradiol-induced antinociception using the estrogen receptor antagonist ICI 182780. The blockade of TMJ estrogen receptors by the administration of ICI 182780 into the ipsilateral, but not into the contralateral TMJ blocked the antinociceptive effect of estradiol and of E–BSA in OVX (Fig. 2A, B, respectively) and of estradiol in diestrus females (Fig. 2C). The blockade of NO synthesis and the inhibition of the guanylate cyclase activity by the administration of L-NNA or of ODQ, respectively, into the ipsilateral, but not into the contralateral TMJ blocked the antinociceptive effect of estradiol (Figs. 3A, 4A, respectively) and of E–BSA (Figs. 3B, 4B, respectively) in OVX and of estradiol in diestrus females (Figs. 3C, 4C, respectively). The blockade of opioid receptors by the administration of naloxone in the TMJ did not affect estradiol-induced antinociception in OVX (Fig. 5A) and in diestrus (Fig. 5B) females. Formalin-induced plasma extravasation was not affected by the administration of estradiol into the TMJ of OVX (Fig. 6A) and of diestrus (Fig. 6B) females. Formalin-induced TMJ nociception was similar (t-test, P⬎0.05) between intact (422.5⫾15.5) and sham-operated diestrus females (415.8⫾17.6) and between intact (254.0⫾ 24.2) and sham-operated proestrus females (236.83⫾15.2).
RESULTS
DISCUSSION
The co-administration of estradiol or E–BSA into the ipsilateral, but not into the contralateral TMJ significantly decreased 1.5% formalin-induced TMJ nociception in OVX (Fig. 1A) and in diestrus (Fig. 1B) females. However, the co-administration of estradiol did not affect formalin-induced nociception in proestrus females (Fig. 1C). These findings together with our previous findings that the high serum estradiol level of proestrus females decreases TMJ nociception, indicate that the antinociceptive effects induced by the administration of estradiol into the
This study demonstrated that estradiol decreases TMJ nociception in female rats through a peripheral nongenomic mechanism. This was evidenced by the finding that estradiol administration into the ipisilateral TMJ significantly decreased formalin-induced nociception in OVX and in diestrus females and by the finding that the membrane impermeable compound E–BSA mimicked the antinociceptive effect induced by estradiol. The effect of E–BSA is compatible with a non-genomic mechanism because its action is restricted to membrane estrogen recep-
experiment, each animal was individually placed in a test chamber (30⫻30⫻30 cm3 mirrored-wood chamber with a glass at the front side) for a 15 min habituation period to minimize stress. After the TMJ injection, the animal was returned to the test chamber for counting two types of nociceptive behavior, rubbing the orofacial region asymmetrically with the ipsilateral fore or hind paw and flinching the head in an intermittent and reflexive way characterized by high frequency shakes of the head. These behaviors were quantified in blocks of 5 min for 45 min. For each block of 5 min, the behavior characterized by rubbing the orofacial region was quantified by a chronometer that recorded the amount of time that the animal exhibited it and the behavior characterized by flinching the head was quantified by a hand tally counter that recorded its occurrence. From a theoretical perspective, the occurrence of a given behavior is proportional to the proportion of time that the behavior occupies. Thus, considering that the flinching head behavior followed a uniform pattern of 1 s in duration, each flinching was expressed as 1 s. The TMJ formalin nociceptive behaviors (flinching and rubbing) were summed and expressed in seconds as previously described (Roveroni et al., 2001).
Plasma extravasation measurement Animals were anaesthetized with an i.m. injection of a mixture of ketamine (55 mg/kg) and xylazine (5.5 mg/kg) before the TMJ injection; then the Evans Blue dye (Sigma Chemicals, St. Louis, MO, USA; 30 mg/kg, (Haas et al., 1992)) was injected into the right femoral vein. Forty-five minutes later animals were perfused transcardially with saline (0.9% NaCl) to flush the dye from the vasculature. Joint tissues were dissected to a standardize size (30⫾2 mg) and stored at ⫺30 °C until analysis. The dye was extracted by immersing the samples in 1 ml of formamide at 60 °C for 24 h. The amount of blue dye (g) in the tissue sample was determined using a spectrophotometer that measured the absorbance of the formamide solution (⫽620 nm). The concentration of dye was then calculated per gram weight of tissue and was used as a marker for plasma protein extravasation in the inflammatory site (Haas et al., 1992).
Statistical analysis
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Fig. 1. Effect of intra-articular administration of estradiol on formalin-induced TMJ nociception. 17-estradiol or E–BSA significantly reduced formalin-induced TMJ nociception in OVX (A) and diestrus (B) females. The symbol “⫹” indicates a nociceptive response significantly lower than that induced by the TMJ injection of formalin plus propylene glycol or of formalin into the right and estradiol or E–BSA into the left TMJ (A, B); the symbol “*” indicates a nociceptive response significantly greater than that induced by the TMJ injection of 0.9% NaCl (A, B, C, D) and the symbol “#” indicates a nociceptive response significantly greater than that induced by the TMJ injection of formalin (1.5%) plus propylene glycol or formalin plus estradiol (C, D; Tukey test, P⬍0.05). In this and in subsequent figures, the black bars represent the effect of drug administration in the contralateral TMJ, data are plotted as mean⫾SEM, group sample sizes are shown in parentheses; see Experimental Procedures for additional details regarding data presentation and analysis. Abbreviations: OVX⫽Ovariectomized; E–BSA⫽Estradiol coupled to bovine serum albumin.
tors (Kelly and Levin, 2001). In fact, the antinociceptive effect observed in the current study is incompatible with the classic genomic effects of estradiol that take hours to days to occur (McEwen, 2001). The blockade of this peripheral antinociceptive effect by the estrogen receptor antagonist ICI 182780 confirms the specificity of the estrogen effect. The higher dose of ICI 182780 required to block estradiol induced-antinociception in OVX than in diestrus females might result from an increased estrogen receptor expression in trigeminal primary afferent neurons after ovariectomy, as previously demonstrated in neurons of the trigeminal subnucleus caudalis (Pajot et al., 2003).
Interestingly, local administration of estradiol did not affect TMJ nociception in proestrus females. Considering that the high serum estradiol level of proestrus females decreases TMJ nociception, as we have previously demonstrated (Clemente et al., 2004; Fischer et al., 2008), this finding indicates that a pharmacological dose of estradiol administered into the TMJ does not increase the antinociceptive effect of endogenous estradiol. When two effects are not additive, they might be on the same pathway. This possibility is supported by data showing that the antinociceptive effect observed in proestrus females is blocked by the estrogen receptor antagonist administered into the
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TMJ at the moment of the nociceptive test. This suggests that the physiological effect of estradiol in nociception is mediated, at least in part, by a peripheral non-genomic mechanism, a finding somewhat surprising in view of the widespread action of estradiol in the CNS. Evidently, in physiological conditions the antinociceptive effect of estradiol in proestrus females (Clemente et al., 2004; Fischer et al., 2008) and in women (LeResche et al., 2003) may be mediated by several mechanisms in addition to the peripheral non-genomic one and our present data cannot exclude them. For this reason, further experiments designed to investigate the mechanisms underlying the peripheral non-genomic mechanism of estradiol were carried out in OVX and in diestrus females receiving a TMJ injection of estradiol. In contrast to proestrus females, males have a low serum estradiol level that is quantitatively similar to that of diestrus females (Fischer et al., 2007; 2008). Therefore, the lack of effect of estradiol in males may be explained by a sex specificity of its peripheral antinociceptive effect. This is consistent with our previous data showing that systemic estradiol administration decreases nociception in female (Fischer et al., 2008) but not in male (Fischer et al., 2007) rats. However, because 1.5% TMJ formalin induces lower nociceptive behavior in males than in diestrus and in OVX females (one way ANOVA, data not shown), we matched the nociceptive behaviors using an equi-nociceptive concentration of formalin in males (5%). This procedure allowed a direct comparison of the effect of estradiol between sexes. Estradiol administration into the ipisilateral TMJ did not affect 5% formalin-induced nociception in males, further confirming the sex specificity of the peripheral antinociceptive effect of estradiol. The mechanisms underlying the sex-specificity of estradiol-induced TMJ antinociception are unknown, however, organizational effects of estradiol during female development (Jost, 1983) may contribute to that. The peripheral non-genomic antinociceptive effect of estradiol is mediated by the activation of the NO– cGMP signaling pathway, since it was blocked by the administration of the NO synthase inhibitor, L-NNA or of the guanylate cyclase inhibitor, ODQ in the ipisilateral TMJ. These data are in accordance with findings showing that estradiol increases the activity of the NO synthase (Chen et al., 1999; Simoncini et al., 2000) and guanylate cyclase (Fiorelli et al., 1996) through non-genomic mechanisms. The increased activity of these enzymes results in an increased level of cGMP, which has been associated with peripheral antinociception (Durate et al., 1990; Qian et al., 1996; Almeida and Duarte, 2007). It was demonstrated that the peripheral analgesic effect of opioids is mediated
Fig. 2. Effect of intra-articular administration of the estrogen receptor antagonist ICI 182780 on the antinociceptive effect induced by estradiol and E–BSA. The estrogen receptor antagonist blocked the
antinociceptive effect of estradiol (A) and E–BSA (B) in OVX females and of estradiol in diestrus females (C) The symbol “⫹” indicates a nociceptive response significantly higher than that induced by the TMJ injection of formalin plus estradiol or E–BSA and DMSO and the symbol “ⴱ” indicates a nociceptive response significantly lower than that induced by the TMJ injection of formalin plus vehicle (propylene glycol or 0.9% NaCl; Tukey test, P⬍0.05). Abbreviation: DMSO⫽ dimethylsulfoxide.
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by the activation of NO– cGMP signaling pathway (Granados-Soto et al., 1997; Pol, 2007) and that estradiol modulates the opioid system through non-genomic mechanisms (Lagrange et al., 1995; Brown et al., 2007). However, the opioid receptor antagonist naloxone did not affect the peripheral non-genomic antinociceptive effect of estradiol, suggesting that this effect is independent of peripheral opioid mechanisms. NO is a potent vasodilatator, known to increase plasma extravasation. However, a previous study, that is in accordance with unpublished observations from our laboratory, has indicated that high serum estradiol level is associated with decreased TMJ plasma extravasation (Flake et al., 2006). One of the speculations made in this study is that estrogen may act at the adrenal medulla to alter sympathetic tone, and perhaps also influence the Hypothalamic–Pituitary–Adrenal axis, effects that occur remote from the TMJ but influence the extent of inflammation. Therefore, we investigated the effect of local administration of estradiol on formalin-induced TMJ plasma extravasation and found that it was not affected by ipisilateral estradiol administration. These data suggest that the peripheral non-genomic antinociceptive effect of estradiol is not mediated by a modulation of the inflammatory response and it is in accordance with the idea that the mechanisms underlying the effects of estradiol in inflammation occur remote from the TMJ region. This study demonstrated for the first time to our knowledge that estradiol decreases nociception “in vivo” by a peripheral non-genomic mechanism. This finding corroborates our previous studies showing an antinociceptive effect of high serum estradiol level (Clemente et al., 2004; Fischer et al., 2008). In a first view, both findings are apparently discrepant from clinical data showing a higher prevalence and severity of TMJ pain in women (Dworkin et al., 1990). However, we have previously suggested that the lower prevalence of TMJ pain in men may result from a protective effect of testosterone, since we have demonstrated that a concentration of formalin that does not induce nociception in naïve male rats induces in gonadectomized ones and in females. The higher severity of TMJ pain in women may be a consequence of hormonal fluctuation during reproductive cycle, since we have demonstrated that glutamate or formalin-induced nociception is high during low serum estradiol level of the estrous cycle (Fischer et al., 2008). This last finding parallels clinical findings showing a high TMJ pain in women during low estradiol levels of the menstrual cycle. Similarly, the lower TMJ nociception in pregnant rats (Arthuri et al., 2005) parallels the lower TMJ pain during pregnancy in women (LeResche et al., 2005). However, the literature regarding the role of ovarian hormones in TMJ pain is controversial.
Fig. 3. Effect of intra-articular administration of the NO synthase inhibitor L-NNA on the antinociceptive effect induced by estradiol and
E–BSA. The NO synthase inhibitor blocked the antinociceptive effect of estradiol (A) and E–BSA (B) in OVX females and of estradiol in diestrus females (C) The symbol “⫹” indicates a nociceptive response significantly higher that induced by the TMJ injection of formalin plus estradiol or E–BSA and the symbol “ⴱ” indicates a nociceptive response significantly lower than that induced by the TMJ injection of formalin plus vehicle (propylene glycol or 0.9% NaCl; Tukey test, P⬍0.05).
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Fig. 5. Effect of intra-articular administration of the opioid receptor antagonist naloxone on the antinociceptive effect induced by estradiol. The opioid receptor antagonist did not affect the antinociceptive effect induced by estradiol in OVX and in diestrus females. The symbol “ⴱ” indicates a nociceptive response significantly lower than that induced by the TMJ injection of formalin plus 0.9% NaCl (Tukey test, P⬎0.05).
For example, data obtained by Fos-positive neurons (Bereiter, 2001), assessment of jaw muscle activity (Cairns et al., 2002) and single units recording of neurons of the trigeminal subnucleus caudalis (Okamoto et al., 2003) suggest that estradiol increases TMJ nociception. Although differences in the genomic and non-genomic effects of estradiol cannot explain all discrepancies in the literature in this field, they may contribute to some differences between our findings and those from these previous studies.
Fig. 4. Effect of intra-articular administration of the guanylate cyclase inhibitor ODQ on the antinociceptive effect induced by estradiol and E–BSA. The guanylate cyclase inhibitor blocked the antinociceptive effect of estradiol (A) and E–BSA (B) in OVX females and of estradiol
in diestrus females (C) The symbol “⫹” indicates a nociceptive response significantly higher that induced by the TMJ injection of formalin plus estradiol or E–BSA and DMSO and the symbol “ⴱ” indicates a nociceptive response significantly lower than that induced by the TMJ injection of formalin plus vehicle (propylene glycol or 0.9% NaCl; Tukey test, P⬍0.05).
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of reproductive cycle (LeResche et al., 2003; Fischer et al., 2008), another advantage may be the counteraction of this increased sensitivity without systemic estrogenic effects. Acknowledgments—This work was supported in part by a PhD fellowship to L.F. from CNPq and by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP. N.C. F-M was supported as an undergraduate research fellow by FAPESP. We thank Carlos Alberto Feliciano for excellent technical assistance.
REFERENCES
Fig. 6. Effect of intra-articular administration of estradiol on formalininduced TMJ plasma extravasation. 17-estradiol did not affect formalin-induced TMJ plasma extravasation in OVX and diestrus females (t-test, P⬎0.05). The symbol “ⴱ” indicates a significantly higher plasma extravasation than that induced by the TMJ injection of 0.9% NaCl plus propylene glycol or plus.
CONCLUSION This study presents a new mechanism by which estradiol decreases nociception and provides preliminary evidence that estradiol, in physiological conditions, decreases nociception, at least in part, by a peripheral mechanism. Although further studies are necessary to evaluate the physiological relevance of this effect, the therapeutic potential of the peripheral non-genomic antinociceptive effect of estradiol is evident. Estrogen receptor ligands devoid of classic estrogenic activity have been successfully used at experimental conditions (Keith et al., 2005) and the present data suggest that membrane estrogen receptors and the NO– cGMP signaling pathway may be valuables molecular targets for the development of future drugs of this class. Therapeutic strategies in this field may have the advantage of avoiding side effects related to the other drugs, such as opioids, that also activate the NO– cGMP signaling pathway to induce peripheral antinociception. In addition, taking into account that TMJ pain sensitivity in females appears to be increased during low estradiol level
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(Accepted 5 August 2009) (Available online 11 August 2009)
PERIPHERAL ESTRADIOL INDUCES TEMPOROMANDIBULAR JOINT ANTINOCICEPTION IN RATS BY ACTIVATING THE NITRIC OXIDE/CYCLIC GUANOSINE MONOPHOSPHATE SIGNALING PATHWAY N. C. FÁVARO-MOREIRA,a K. E. TORRES-CHÁVEZ,a L. FISCHERb AND C. H. TAMBELIa*
Key words: estradiol, nociception, temporomandibular joint pain, nitric oxide, cGMP, formalin.
a Laboratory of Orofacial Pain, Department of Physiology, Faculty of Dentistry of Piracicaba, State University of Campinas-UNICAMP, Avenue Limeira 901, CEP 13414-900, Piracicaba, SP, Brazil
The majority of chronic pain conditions (Unruh, 1996) such as temporomandibular dysfunctions (TMDs) (Dworkin et al., 1990) are more prevalent and severe in women than in men. These findings may apparently suggest a pronociceptive effect of estradiol, as supported by some animal studies (Bereiter, 2001; Cairns et al., 2002; Okamoto et al., 2003). However, our findings that formalin or glutamateinduced temporomandibular joint (TMJ) nociception in rats (Fischer et al., 2008) and those that TMD pain in women (LeResche et al., 2003) is higher during low estradiol times of the reproductive cycle suggest that estradiol attenuates TMJ pain. This suggestion is also supported by other studies in humans (Smith et al., 2006) and in animals (Gaumond et al., 2002; Ceccarelli et al., 2003; Pajot et al., 2003; Kuba et al., 2006; Mannino et al., 2007) that use other pain models. Based on our previous animal studies, we suggest that the lower prevalence of TMJ pain in men may result from a protective effect of testosterone that reduces the risk of males developing TMJ pain (Fischer et al., 2007) and the higher severity of TMJ pain in women may result from the estrogen fluctuation during reproductive cycle, in that TMJ pain is increased during low serum estradiol levels (Fischer et al., 2008). Although the mechanisms underlying the antinociceptive effect of estradiol are presently unknown, estradiol might induce antinociception, at least in part, by a peripheral non-genomic mechanism. This idea is supported by “in vitro” studies showing that estradiol inhibits calcium channel currents in neurons of the dorsal root ganglia via activation of membrane estrogen receptors (Lee et al., 2002; Chaban et al., 2003). In contrast to estrogen receptors located in the cytosol, those located on the membrane mediate the rapid non-genomic effects of estradiol through the activation of second messenger cascades (Evrard and Balthazart, 2004), including the L-arginine–nitric oxide (NO)– cyclic guanosine mono-phosphate (cGMP) pathway (L-arg–NO– cGMP), as demonstrated in endothelial cells (Stefano et al., 2000). This pathway involves the synthesis of NO from L-arginine and the subsequent activation of the guanylate cyclase, leading to increased levels of cGMP, a second messenger that has been associated with peripheral antinociception, including that mediated by peripheral opioids (Durate et al., 1990; Pol, 2007). Estradiol via activation of non-genomic mechanisms might induce antinociception by increasing the release of endogenous opioids
b
Laboratory of Pain Physiology, Division of Biological Sciences, Department of Physiology, Federal University of Parana, Curitiba, Parana, Brazil
Abstract—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. © 2009 Published by Elsevier Ltd on behalf of IBRO. *Corresponding author. Tel: ⫹55-19-2106-5305; fax: ⫹55-19-2106-5212. E-mail address: [email protected] (C. H. Tambeli). Abbreviations: cGMP, cyclic guanosine mono-phosphate; DMSO, dimethyl sulfoxide; E–BSA, estradiol coupled to bovine serum albumin; L-NNA, nitro-L-arginine; NO, nitric oxide; ODQ, 1H-(1,2,4)-oxadiasolo (4,2-a) quinoxalin-1-one; OVX, ovariectomized; TMJ, temporomandibular joint.
0306-4522/09 $ - see front matter © 2009 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2009.08.012
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by opioid peptide-containing immune cells (Rittner et al., 2008) and/or by interacting with opioid receptor-coupled second messenger systems (Lagrange et al., 1995; Brown et al., 2007). In fact, peripheral opioid mechanisms mediate the antinociceptive effect induced by pregnancy hormones in the rat’s TMJ (Arthuri et al., 2005). Therefore, in this study we have used the TMJ formalin model 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 NO– cGMP signaling pathway and of opioid receptors.
EXPERIMENTAL PROCEDURES
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OVX females and the most effective dose was selected for further experiments.
Estrous phase determination Estrous phase was determined by daily microscope examination of vaginal smears between 7 and 8 AM. In the day of the experiment, estrous phase was confirmed before and immediately after each experiment to ensure that the rats remained in the same phase. Proestrus phase and the initial phase of diestrus (first 4 h) were identified by the predominance (⬎70%) of nucleated epithelial cells and leukocytes, respectively (Butcher et al., 1974) in rats with at least two consecutive regular 4 –5 day cycles. These phases were chosen because they represent phases of high and low ovarian hormonal level, respectively (Butcher et al., 1974).
Animals
Gonadectomy
This study was carried out in 200 –300 g ovariectomized (OVX, n⫽223), diestrus (n⫽128) and proestrus (n⫽43) female and in male (n⫽41) Wistar rats. All efforts were made to minimize the number of animals used and their suffering. The animals were used in experiments when they were 60 –75 days old; the mean weights of OVX females (266.2⫾7.8) were significantly greater (t-test, P⫽⬍0.05) than that of intact proestrus and diestrus females (225.0⫾5.8). To investigate a peripheral estradiol-mediated effect we firstly used OVX females because the depletion of serum estradiol level possibly facilitates its assessment. Then we included diestrus and proestrus females because they represent a physiological condition of low and high serum estradiol level, respectively. Male rats were also included because the antinociceptive effect of systemic estradiol is sex specific (Fischer et al., 2007; 2008) and therefore we asked if the peripheral effect might also be. All animal experimental procedures and protocols were approved by the Committee on Animal Research of the University of Campinas and are in accordance with IASP guidelines for the study of pain in animals (Zimmermann, 1983). The animals were maintained on a temperature-controlled room (⫾23 °C) and were housed in plastic cages (45⫻30⫻15 cm3) with soft bedding (five/ cage) on a 12:12 light cycle (lights on at 6:00 AM) with food and water available ad libitum.
Ovariectomy (45 days old females; (Gordon and Soliman, 1994)) was performed through bilateral upper flank incisions. The ovarian bundles were tied off with 4-O silk sutures and the ovaries removed. The fascia and the skin were closed with 4-O silk sutures. Rats of two experimental groups were sham operated and underwent a surgical procedure similar to that of OVX animals, except that the ovaries were not removed. These groups were used to demonstrate that the surgical procedure does not affect the nociceptive behavior response in diestrus and proestrus females (naïve vs. sham). The procedures were carried out under anesthesia induced by an i.m. injection of a mixture of ketamine (55 mg/kg) and xylazine (5.5 mg/kg). An s.c. injection of ketoprofen (5 mg/kg) was used for post-operative analgesia (Roughan and Flecknell, 2000). OVX and sham-operated rats were used in experiments when they were 3 months of age. The efficacy of ovariectomy was confirmed by the absence of estrous cycle determined by observation of vaginal smears during 10 days.
Drugs Formalin was prepared from commercially available stock formalin (an aqueous solution of 37% of formaldehyde) further diluted in 0.9% NaCl to a concentration of 1.5% (Roveroni et al., 2001); estradiol (17-estradiol, 0.4, 1.2 g; (Ceccarelli et al., 2004) and 3.6 g) was dissolved in propylene glycol; estradiol coupled to bovine serum albumin (E–BSA, 1.2 g of estradiol plus BSA); the NO synthase inhibitor nitro-L-arginine (L-NNA 22 g; (Toda et al., 1993)) and the opioid receptor antagonist naloxone (10 g; (Eisenberg et al., 1996), and 30 g) were dissolved in 0.9% NaCl. The selective estrogen receptor antagonist ICI 182780 (0.16, 1 and 6 g; (Ceccarelli et al., 2004) and the guanylate cyclase inhibitor 1H-(1,2,4)-oxadiasolo (4,2-a) quinoxalin-1-one (ODQ 0.8 and 8 g; (Cunha et al., 1999) were dissolved in dimethyl sulfoxide (DMSO)). Formalin, estradiol, E–BSA, naloxone and L-NNA were purchased from Sigma Aldrich, St. Louis, MO, USA; ODQ and ICI182780 were purchased from Tocris Bioscience, St. Louis, MO, USA. Steroid hormones conjugated with bovine serum albumin have been extensively used to assess their non-genomic effects (Kelly and Levin, 2001). However, it was suggested that E–BSA has biological activity not observed with estradiol. For this reason, the experiments were performed using both 17-estradiol and E–BSA. The dose–response curves of all drugs were performed in
TMJ injections The animals were briefly anesthetized by inhalation of halothane to allow the TMJ injection; each animal regained consciousness approximately 30 s after discontinuing the anesthetic. The TMJ injection was performed with a 30-gauge needle introduced into the TMJ at the moment of injection. The contralateral TMJ injections, when necessary, were performed immediately after the ipsilateral injections. A cannula consisting of a polyethylene tube was connected to the needle and also to a Hamilton syringe (50 l) (Roveroni et al., 2001). The total volume injection varied between 30 and 45 l, when two or three drugs were injected in the same TMJ, they were administered at a volume of 15 l per drug in a unique injection. At the conclusion of the behavior test, each animal was anesthetized by an i.p. injection of a mixture of urethane (1 g/kg) and ␣-chloralose (50 mg/kg). To confirm the correct site of injection, the Evans Blue dye (5 mg/kg) was systemically injected and 15 min later the animals were submitted to cardiac perfusion with normal saline. Since this dye binds to plasma protein, the correct site of injection was indicated by the observation that the plasma extravasation induced by the TMJ injection of formalin was restricted to the TMJ region (Haas et al., 1992).
Testing procedure for TMJ pain Behavior test was performed during light phase (between 9:00 AM and 5:00 PM) in a quiet room maintained at ⫾23 °C (Rosland, 1991). Rats did not have access to food or water during the test and each animal was used once. The nociceptive response was assessed by an observer blinded to the experimental manipulation. Before the experiments, each animal was manipulated for 7 days in the test room (handled for approximately 1 min) to be habituated to the experimental manipulation. On the day of the
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The nociceptive behavior score, obtained by summing the flinching and rubbing behaviors recorded during the entire duration of the experiment was used in statistical analysis in Figs. 1–5. The concentration of the Evan’s Blue dye extravasated per gram weight of TMJ tissue was used in statistical analysis in Fig. 6. To determine if there were significant differences among treatment groups one-way ANOVA was performed. If there was a significant between-subjects main effect of treatment group after one-way ANOVA, post hoc contrasts, using the Tukey test, were performed to determine the basis of the significant difference. A minimum of four subjects in each group is sufficient to detect a significant difference at a power of 0.8. The level for statistical significance was P⬍0.05. Data are expressed in figures as means⫾SEM.
TMJ and by physiological estradiol are not additive. To investigate whether they are on the same pathway we verified if the co-administration of the estrogen receptor antagonist ICI 182780 with formalin into the TMJ affects the antinociceptive effect induced by high serum estradiol level in proestrus females. As demonstrated in Fig. 1C, the co-administration of the estrogen receptor antagonist ICI 182780 into the ipsilateral, but not into the contralateral TMJ of proestrus females significantly increased their nociceptive behavior. Formalin-induced TMJ nociceptive behavior in proestrus females receiving ICI 182780 into the ipsilateral TMJ was similar to that of diestrus and OVX females receiving only formalin or formalin plus ICI 182780 (one way ANOVA, data not shown). Co-administration of estradiol with formalin into the TMJ did not affect nociception in males, even at a dose of estradiol three times higher than that used in females and even using an equi-nociceptive concentration of formalin (5%) (Fig. 1D). To discard that the antinociceptive effect induced by the TMJ injection of estradiol could derivate from a nonspecific action, we evaluated the involvement of estrogen receptors in estradiol-induced antinociception using the estrogen receptor antagonist ICI 182780. The blockade of TMJ estrogen receptors by the administration of ICI 182780 into the ipsilateral, but not into the contralateral TMJ blocked the antinociceptive effect of estradiol and of E–BSA in OVX (Fig. 2A, B, respectively) and of estradiol in diestrus females (Fig. 2C). The blockade of NO synthesis and the inhibition of the guanylate cyclase activity by the administration of L-NNA or of ODQ, respectively, into the ipsilateral, but not into the contralateral TMJ blocked the antinociceptive effect of estradiol (Figs. 3A, 4A, respectively) and of E–BSA (Figs. 3B, 4B, respectively) in OVX and of estradiol in diestrus females (Figs. 3C, 4C, respectively). The blockade of opioid receptors by the administration of naloxone in the TMJ did not affect estradiol-induced antinociception in OVX (Fig. 5A) and in diestrus (Fig. 5B) females. Formalin-induced plasma extravasation was not affected by the administration of estradiol into the TMJ of OVX (Fig. 6A) and of diestrus (Fig. 6B) females. Formalin-induced TMJ nociception was similar (t-test, P⬎0.05) between intact (422.5⫾15.5) and sham-operated diestrus females (415.8⫾17.6) and between intact (254.0⫾ 24.2) and sham-operated proestrus females (236.83⫾15.2).
RESULTS
DISCUSSION
The co-administration of estradiol or E–BSA into the ipsilateral, but not into the contralateral TMJ significantly decreased 1.5% formalin-induced TMJ nociception in OVX (Fig. 1A) and in diestrus (Fig. 1B) females. However, the co-administration of estradiol did not affect formalin-induced nociception in proestrus females (Fig. 1C). These findings together with our previous findings that the high serum estradiol level of proestrus females decreases TMJ nociception, indicate that the antinociceptive effects induced by the administration of estradiol into the
This study demonstrated that estradiol decreases TMJ nociception in female rats through a peripheral nongenomic mechanism. This was evidenced by the finding that estradiol administration into the ipisilateral TMJ significantly decreased formalin-induced nociception in OVX and in diestrus females and by the finding that the membrane impermeable compound E–BSA mimicked the antinociceptive effect induced by estradiol. The effect of E–BSA is compatible with a non-genomic mechanism because its action is restricted to membrane estrogen recep-
experiment, each animal was individually placed in a test chamber (30⫻30⫻30 cm3 mirrored-wood chamber with a glass at the front side) for a 15 min habituation period to minimize stress. After the TMJ injection, the animal was returned to the test chamber for counting two types of nociceptive behavior, rubbing the orofacial region asymmetrically with the ipsilateral fore or hind paw and flinching the head in an intermittent and reflexive way characterized by high frequency shakes of the head. These behaviors were quantified in blocks of 5 min for 45 min. For each block of 5 min, the behavior characterized by rubbing the orofacial region was quantified by a chronometer that recorded the amount of time that the animal exhibited it and the behavior characterized by flinching the head was quantified by a hand tally counter that recorded its occurrence. From a theoretical perspective, the occurrence of a given behavior is proportional to the proportion of time that the behavior occupies. Thus, considering that the flinching head behavior followed a uniform pattern of 1 s in duration, each flinching was expressed as 1 s. The TMJ formalin nociceptive behaviors (flinching and rubbing) were summed and expressed in seconds as previously described (Roveroni et al., 2001).
Plasma extravasation measurement Animals were anaesthetized with an i.m. injection of a mixture of ketamine (55 mg/kg) and xylazine (5.5 mg/kg) before the TMJ injection; then the Evans Blue dye (Sigma Chemicals, St. Louis, MO, USA; 30 mg/kg, (Haas et al., 1992)) was injected into the right femoral vein. Forty-five minutes later animals were perfused transcardially with saline (0.9% NaCl) to flush the dye from the vasculature. Joint tissues were dissected to a standardize size (30⫾2 mg) and stored at ⫺30 °C until analysis. The dye was extracted by immersing the samples in 1 ml of formamide at 60 °C for 24 h. The amount of blue dye (g) in the tissue sample was determined using a spectrophotometer that measured the absorbance of the formamide solution (⫽620 nm). The concentration of dye was then calculated per gram weight of tissue and was used as a marker for plasma protein extravasation in the inflammatory site (Haas et al., 1992).
Statistical analysis
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Fig. 1. Effect of intra-articular administration of estradiol on formalin-induced TMJ nociception. 17-estradiol or E–BSA significantly reduced formalin-induced TMJ nociception in OVX (A) and diestrus (B) females. The symbol “⫹” indicates a nociceptive response significantly lower than that induced by the TMJ injection of formalin plus propylene glycol or of formalin into the right and estradiol or E–BSA into the left TMJ (A, B); the symbol “*” indicates a nociceptive response significantly greater than that induced by the TMJ injection of 0.9% NaCl (A, B, C, D) and the symbol “#” indicates a nociceptive response significantly greater than that induced by the TMJ injection of formalin (1.5%) plus propylene glycol or formalin plus estradiol (C, D; Tukey test, P⬍0.05). In this and in subsequent figures, the black bars represent the effect of drug administration in the contralateral TMJ, data are plotted as mean⫾SEM, group sample sizes are shown in parentheses; see Experimental Procedures for additional details regarding data presentation and analysis. Abbreviations: OVX⫽Ovariectomized; E–BSA⫽Estradiol coupled to bovine serum albumin.
tors (Kelly and Levin, 2001). In fact, the antinociceptive effect observed in the current study is incompatible with the classic genomic effects of estradiol that take hours to days to occur (McEwen, 2001). The blockade of this peripheral antinociceptive effect by the estrogen receptor antagonist ICI 182780 confirms the specificity of the estrogen effect. The higher dose of ICI 182780 required to block estradiol induced-antinociception in OVX than in diestrus females might result from an increased estrogen receptor expression in trigeminal primary afferent neurons after ovariectomy, as previously demonstrated in neurons of the trigeminal subnucleus caudalis (Pajot et al., 2003).
Interestingly, local administration of estradiol did not affect TMJ nociception in proestrus females. Considering that the high serum estradiol level of proestrus females decreases TMJ nociception, as we have previously demonstrated (Clemente et al., 2004; Fischer et al., 2008), this finding indicates that a pharmacological dose of estradiol administered into the TMJ does not increase the antinociceptive effect of endogenous estradiol. When two effects are not additive, they might be on the same pathway. This possibility is supported by data showing that the antinociceptive effect observed in proestrus females is blocked by the estrogen receptor antagonist administered into the
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TMJ at the moment of the nociceptive test. This suggests that the physiological effect of estradiol in nociception is mediated, at least in part, by a peripheral non-genomic mechanism, a finding somewhat surprising in view of the widespread action of estradiol in the CNS. Evidently, in physiological conditions the antinociceptive effect of estradiol in proestrus females (Clemente et al., 2004; Fischer et al., 2008) and in women (LeResche et al., 2003) may be mediated by several mechanisms in addition to the peripheral non-genomic one and our present data cannot exclude them. For this reason, further experiments designed to investigate the mechanisms underlying the peripheral non-genomic mechanism of estradiol were carried out in OVX and in diestrus females receiving a TMJ injection of estradiol. In contrast to proestrus females, males have a low serum estradiol level that is quantitatively similar to that of diestrus females (Fischer et al., 2007; 2008). Therefore, the lack of effect of estradiol in males may be explained by a sex specificity of its peripheral antinociceptive effect. This is consistent with our previous data showing that systemic estradiol administration decreases nociception in female (Fischer et al., 2008) but not in male (Fischer et al., 2007) rats. However, because 1.5% TMJ formalin induces lower nociceptive behavior in males than in diestrus and in OVX females (one way ANOVA, data not shown), we matched the nociceptive behaviors using an equi-nociceptive concentration of formalin in males (5%). This procedure allowed a direct comparison of the effect of estradiol between sexes. Estradiol administration into the ipisilateral TMJ did not affect 5% formalin-induced nociception in males, further confirming the sex specificity of the peripheral antinociceptive effect of estradiol. The mechanisms underlying the sex-specificity of estradiol-induced TMJ antinociception are unknown, however, organizational effects of estradiol during female development (Jost, 1983) may contribute to that. The peripheral non-genomic antinociceptive effect of estradiol is mediated by the activation of the NO– cGMP signaling pathway, since it was blocked by the administration of the NO synthase inhibitor, L-NNA or of the guanylate cyclase inhibitor, ODQ in the ipisilateral TMJ. These data are in accordance with findings showing that estradiol increases the activity of the NO synthase (Chen et al., 1999; Simoncini et al., 2000) and guanylate cyclase (Fiorelli et al., 1996) through non-genomic mechanisms. The increased activity of these enzymes results in an increased level of cGMP, which has been associated with peripheral antinociception (Durate et al., 1990; Qian et al., 1996; Almeida and Duarte, 2007). It was demonstrated that the peripheral analgesic effect of opioids is mediated
Fig. 2. Effect of intra-articular administration of the estrogen receptor antagonist ICI 182780 on the antinociceptive effect induced by estradiol and E–BSA. The estrogen receptor antagonist blocked the
antinociceptive effect of estradiol (A) and E–BSA (B) in OVX females and of estradiol in diestrus females (C) The symbol “⫹” indicates a nociceptive response significantly higher than that induced by the TMJ injection of formalin plus estradiol or E–BSA and DMSO and the symbol “ⴱ” indicates a nociceptive response significantly lower than that induced by the TMJ injection of formalin plus vehicle (propylene glycol or 0.9% NaCl; Tukey test, P⬍0.05). Abbreviation: DMSO⫽ dimethylsulfoxide.
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by the activation of NO– cGMP signaling pathway (Granados-Soto et al., 1997; Pol, 2007) and that estradiol modulates the opioid system through non-genomic mechanisms (Lagrange et al., 1995; Brown et al., 2007). However, the opioid receptor antagonist naloxone did not affect the peripheral non-genomic antinociceptive effect of estradiol, suggesting that this effect is independent of peripheral opioid mechanisms. NO is a potent vasodilatator, known to increase plasma extravasation. However, a previous study, that is in accordance with unpublished observations from our laboratory, has indicated that high serum estradiol level is associated with decreased TMJ plasma extravasation (Flake et al., 2006). One of the speculations made in this study is that estrogen may act at the adrenal medulla to alter sympathetic tone, and perhaps also influence the Hypothalamic–Pituitary–Adrenal axis, effects that occur remote from the TMJ but influence the extent of inflammation. Therefore, we investigated the effect of local administration of estradiol on formalin-induced TMJ plasma extravasation and found that it was not affected by ipisilateral estradiol administration. These data suggest that the peripheral non-genomic antinociceptive effect of estradiol is not mediated by a modulation of the inflammatory response and it is in accordance with the idea that the mechanisms underlying the effects of estradiol in inflammation occur remote from the TMJ region. This study demonstrated for the first time to our knowledge that estradiol decreases nociception “in vivo” by a peripheral non-genomic mechanism. This finding corroborates our previous studies showing an antinociceptive effect of high serum estradiol level (Clemente et al., 2004; Fischer et al., 2008). In a first view, both findings are apparently discrepant from clinical data showing a higher prevalence and severity of TMJ pain in women (Dworkin et al., 1990). However, we have previously suggested that the lower prevalence of TMJ pain in men may result from a protective effect of testosterone, since we have demonstrated that a concentration of formalin that does not induce nociception in naïve male rats induces in gonadectomized ones and in females. The higher severity of TMJ pain in women may be a consequence of hormonal fluctuation during reproductive cycle, since we have demonstrated that glutamate or formalin-induced nociception is high during low serum estradiol level of the estrous cycle (Fischer et al., 2008). This last finding parallels clinical findings showing a high TMJ pain in women during low estradiol levels of the menstrual cycle. Similarly, the lower TMJ nociception in pregnant rats (Arthuri et al., 2005) parallels the lower TMJ pain during pregnancy in women (LeResche et al., 2005). However, the literature regarding the role of ovarian hormones in TMJ pain is controversial.
Fig. 3. Effect of intra-articular administration of the NO synthase inhibitor L-NNA on the antinociceptive effect induced by estradiol and
E–BSA. The NO synthase inhibitor blocked the antinociceptive effect of estradiol (A) and E–BSA (B) in OVX females and of estradiol in diestrus females (C) The symbol “⫹” indicates a nociceptive response significantly higher that induced by the TMJ injection of formalin plus estradiol or E–BSA and the symbol “ⴱ” indicates a nociceptive response significantly lower than that induced by the TMJ injection of formalin plus vehicle (propylene glycol or 0.9% NaCl; Tukey test, P⬍0.05).
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Fig. 5. Effect of intra-articular administration of the opioid receptor antagonist naloxone on the antinociceptive effect induced by estradiol. The opioid receptor antagonist did not affect the antinociceptive effect induced by estradiol in OVX and in diestrus females. The symbol “ⴱ” indicates a nociceptive response significantly lower than that induced by the TMJ injection of formalin plus 0.9% NaCl (Tukey test, P⬎0.05).
For example, data obtained by Fos-positive neurons (Bereiter, 2001), assessment of jaw muscle activity (Cairns et al., 2002) and single units recording of neurons of the trigeminal subnucleus caudalis (Okamoto et al., 2003) suggest that estradiol increases TMJ nociception. Although differences in the genomic and non-genomic effects of estradiol cannot explain all discrepancies in the literature in this field, they may contribute to some differences between our findings and those from these previous studies.
Fig. 4. Effect of intra-articular administration of the guanylate cyclase inhibitor ODQ on the antinociceptive effect induced by estradiol and E–BSA. The guanylate cyclase inhibitor blocked the antinociceptive effect of estradiol (A) and E–BSA (B) in OVX females and of estradiol
in diestrus females (C) The symbol “⫹” indicates a nociceptive response significantly higher that induced by the TMJ injection of formalin plus estradiol or E–BSA and DMSO and the symbol “ⴱ” indicates a nociceptive response significantly lower than that induced by the TMJ injection of formalin plus vehicle (propylene glycol or 0.9% NaCl; Tukey test, P⬍0.05).
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of reproductive cycle (LeResche et al., 2003; Fischer et al., 2008), another advantage may be the counteraction of this increased sensitivity without systemic estrogenic effects. Acknowledgments—This work was supported in part by a PhD fellowship to L.F. from CNPq and by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP. N.C. F-M was supported as an undergraduate research fellow by FAPESP. We thank Carlos Alberto Feliciano for excellent technical assistance.
REFERENCES
Fig. 6. Effect of intra-articular administration of estradiol on formalininduced TMJ plasma extravasation. 17-estradiol did not affect formalin-induced TMJ plasma extravasation in OVX and diestrus females (t-test, P⬎0.05). The symbol “ⴱ” indicates a significantly higher plasma extravasation than that induced by the TMJ injection of 0.9% NaCl plus propylene glycol or plus.
CONCLUSION This study presents a new mechanism by which estradiol decreases nociception and provides preliminary evidence that estradiol, in physiological conditions, decreases nociception, at least in part, by a peripheral mechanism. Although further studies are necessary to evaluate the physiological relevance of this effect, the therapeutic potential of the peripheral non-genomic antinociceptive effect of estradiol is evident. Estrogen receptor ligands devoid of classic estrogenic activity have been successfully used at experimental conditions (Keith et al., 2005) and the present data suggest that membrane estrogen receptors and the NO– cGMP signaling pathway may be valuables molecular targets for the development of future drugs of this class. Therapeutic strategies in this field may have the advantage of avoiding side effects related to the other drugs, such as opioids, that also activate the NO– cGMP signaling pathway to induce peripheral antinociception. In addition, taking into account that TMJ pain sensitivity in females appears to be increased during low estradiol level
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(Accepted 5 August 2009) (Available online 11 August 2009)