Youping Zhang

Activation of TRPV1 and TRPA1 leads to muscle nociception and mechanical hyperalgesia

ABSTRACT The involvement of TRPV1 and TRPA1 in mediating craniofacial muscle nociception and mechanical hyperalgesia was investigated in male Sprague–Dawley rats. First, we confirmed the expression of TRPV1 in masseter afferents in rat trigeminal ganglia (TG), and provided new data that TRPA1 is also expressed in primary afferents innervating masticatory muscles in double‐labeling immunohistochemistry experiments. We then examined whether the activation of each TRP channel in the masseter muscle evokes acute nocifensive responses and leads to the development of masseter hypersensitivity to mechanical stimulation using the behavioral models that have been specifically designed and validated for the craniofacial system. Intramuscular injections with specific agonists for TRPV1 and TRPA1, capsaicin and mustard oil (MO), respectively, produced immediate nocifensive hindpaw responses followed by prolonged mechanical hyperalgesia in a concentration‐dependent manner. Pretreatment of the muscle with a TRPV1 antagonist, capsazepine, effectively attenuated the capsaicin‐induced muscle nociception and mechanical hyperalgesia. Similarly, pretreatment of the muscle with a selective TRPA1 antagonist, AP18, significantly blocked the MO‐induced muscle nociception and mechanical hyperalgesia. We confirmed these data with another set of selective antagonist for TRPV1 and TRPA1, AMG9810 and HC030031, respectively. Collectively, these results provide compelling evidence that TRPV1 and TRPA1 can functionally contribute to muscle nociception and hyperalgesia, and suggest that TRP channels expressed in muscle afferents can engage in the development of pathologic muscle pain conditions.

Role of peripheral μ-opioid receptors in inflammatory orofacial muscle pain

The aims of this project were to investigate whether inflammation in the orofacial muscle alters mu opioid receptor (MOR) mRNA and protein expressions in trigeminal ganglia (TG), and to assess the contribution of peripheral MORs under acute and inflammatory muscle pain conditions. mRNA and protein levels for MOR were quantified by reverse-transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively, from the TG of naïve rats, and compared with those from the rats treated with complete Freunds adjuvant (CFA) in the masseter. TG was found to express mRNA and protein for MOR, and CFA significantly up-regulated both MOR mRNA and protein by 3 days following the inflammation. The MOR protein up-regulation persisted to day 7 and returned to the baseline level by day 14. We then investigated whether peripheral application of a MOR agonist, D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin acetate salt (DAMGO), attenuates masseter nociception induced by masseteric infusion of hypertonic saline (HS) in lightly anesthetized rats. DAMGO (1, 5, 10 microg) or vehicle was administered directly into the masseter 5-10 min prior to the HS infusion. The DAMGO effects were assessed on mean peak counts (MPC) and overall magnitude as calculated by the area under the curve (AUC) of the HS-evoked behavioral responses. Under this condition, only the highest dose of DAMGO (10 microg) significantly reduced MPC, which was prevented when H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), a selective MOR antagonist, was co-administered. DAMGO pre-treatment in the contralateral masseter did not attenuate MPC. The same doses of DAMGO administered into CFA-inflamed rats, however, produced a greater attenuation of both MPC and AUC of HS-evoked nocifensive responses. These results demonstrated that activation of peripheral MOR provides greater anti-nociception in inflamed muscle, and that the enhanced MOR effect can be partly explained by significant up-regulation of MOR expression in TG.

Effects of gonadal hormones on the peripheral cannabinoid receptor 1 (CB1R) system under a myositis condition in rats

Summary Testosterone is required for the regulation of cannabinoid receptors in trigeminal ganglia under inflammatory conditions, which explains sex differences in the antihyperalgesic effects of peripherally administered cannabinoids. ABSTRACT In this study, we assessed the effects of peripherally administered cannabinoids in an orofacial myositis model, and the role of sex hormones in cannabinoid receptor (CBR) expression in trigeminal ganglia (TG). Peripherally administered arachidonylcyclopropylamide (ACPA), a specific CB1R agonist, significantly attenuated complete Freund’s adjuvant (CFA)‐induced mechanical hypersensitivity in the masseter muscle in male rats. The ACPA effect was blocked by a local administration of AM251, a specific CB1R antagonist, but not by AM630, a specific CB2R antagonist. In female rats, a 30‐fold higher dose of ACPA was required to produce a moderate reduction in mechanical hypersensitivity. CFA injected in masseter muscle significantly upregulated CB1R mRNA expression in TG in male, but not in female, rats. There was a close correlation between the CB1R mRNA levels in TG and the antihyperalgesic effect of ACPA. Interleukin (IL)‐1β and IL‐6, which are elevated in the muscle tissue following CFA treatment, induced a significant upregulation of CB1R mRNA expression in TG from male rats. The upregulation of CB1R was prevented in TG cultures from orchidectomized male rats, which was restored by the application of testosterone. The cytokines did not alter the CB1R mRNA level in TG from intact as well as ovariectomized female rats. Neither estradiol supplement nor estrogen receptor blockade had any effects on CB1R expression. These data indicate that testosterone, but not estradiol, is required for the regulation of CB1Rs in TG under inflammatory conditions, which provide explanations for the sex differences in the antihyperalgesic effects of peripherally administered cannabinoids.

Involvement of neuronal, inducible and endothelial nitric oxide synthases in capsaicin-induced muscle hypersensitivity

Nitric oxide, which has been implicated in the development of hyperalgesia in the spinal system, has not been systematically studied in the trigeminal system, especially in the context of inflammatory muscle pain condition. In this study, we investigated the functional role of centrally released nitric oxide in the pathogenesis of orofacial muscle pain. Specifically, we examined the contribution of neuronal, inducible and endothelial nitric oxide synthases, nNOS, iNOS and eNOS, respectively, in mediating masseter hypersensitivity under acute inflammatory condition. Time‐dependent changes in nNOS, iNOS and eNOS protein expression in the subnucleus caudalis (Vc) were assessed following capsaicin injection in the masseter muscle of male Sprague Dawley rats. The expression of all three nitric oxide synthases was significantly up‐regulated 30–60min following capsaicin stimulation, which paralleled the time course of the development of capsaicin‐induced masseter hypersensitivity. Pretreatment with each NOS inhibitor significantly attenuated the masseter hypersensitivity. These data showed that all three NOS in the Vc are functionally important for the development of craniofacial muscle hyperalgesia and suggest that the three NOS are closely orchestrated to regulate the level of nitric oxide under normal and pathologic conditions.

Role of soluble guanylate cyclase in the trigeminal subnucleus caudalis in capsaicin-induced muscle hypersensitivity.

Nitric oxide (NO) produces its effects by activating soluble guanylate cyclase (sGC). In the present study, we investigated the potential role of sGC in the subnucleus caudalis (Vc) in mediating masseter hypersensitivity under acute inflammatory condition in male Sprague-Dawley rats. First, our Western blot analysis revealed that sGC protein is reliably detected in the Vc. Subsequent immunohistochemical studies demonstrated that neuronal cell bodies in the superficial laminae of the Vc positively stained for sGC. Astrocytes in deeper lamina of the Vc also showed sGC immunoreactivity. We then tested whether intrathecal administration of sGC inhibitors, methylene blue (MB), and ODQ, in the Vc, attenuates masseter hypersensitivity induced by intramuscular injection of capsaicin. Intrathecal MB or ODQ significantly blocked the capsaicin-induced reduction of mechanical threshold to noxious stimulation of the masseter. These data indicate that the NO-sGC pathway in the Vc is involved in mediating orofacial muscle hypersensitivity under acute inflammatory conditions.

The role of TRPA1 in muscle pain and mechanical hypersensitivity under inflammatory conditions in rats.

Transient receptor potential cation channel, subfamily A, member 1 (TRPA1) is expressed in muscle afferents and direct activation of these receptors induces acute mechanical hypersensitivity. However, the functional role of TRPA1 under pathological muscle pain conditions and mechanisms by which TRPA1 mediate muscle pain and hyperalgesia are not clearly understood. Two rodent behavioral models validated to assess craniofacial muscle pain conditions were used to study ATP- and N-Methyl-D-aspartate (NMDA)-induced acute mechanical hypersensitivity and complete Freunds adjuvant (CFA)-induced persistent mechanical hypersensitivity. The rat grimace scale (RGS) was utilized to assess inflammation-induced spontaneous muscle pain. Behavioral pharmacology experiments were performed to assess the effects of AP18, a selective TRPA1 antagonist under these conditions. TRPA1 expression levels in trigeminal ganglia (TG) were examined before and after CFA treatment in the rat masseter muscle. Pre-treatment of the muscle with AP18 dose-dependently blocked the development of acute mechanical hypersensitivity induced by NMDA and α,β-methylene adenosine triphosphate (αβmeATP), a specific agonist for NMDA and P2X3 receptor, respectively. CFA-induced mechanical hypersensitivity and spontaneous muscle pain responses were significantly reversed by post-treatment of the muscle with AP18 when CFA effects were most prominent. CFA-induced myositis was accompanied by significant up-regulation of TRPA1 expression in TG. Our findings showed that TRPA1 in muscle afferents plays an important role in the development of acute mechanical hypersensitivity and in the maintenance of persistent muscle pain and hypersensitivity. Our data suggested that TRPA1 may serve as a downstream target of pro-nociceptive ion channels, such as P2X3 and NMDA receptors in masseter afferents, and that increased TRPA1 expression under inflammatory conditions may contribute to the maintenance of persistent muscle pain and mechanical hyperalgesia. Mechanistic studies elucidating transcriptional or post-translational regulation of TRPA1 expression under pathological pain conditions should provide important basic information to further advance the treatment of craniofacial muscle pain conditions.

Sex differences in μ-opioid receptor expression in trigeminal ganglia under a myositis condition in rats.

Peripheral opioid receptor expression is up‐regulated under inflammatory conditions, which leads to the increased efficacy of peripherally administered opioids. Sex differences in the effects of inflammation, cytokines and gonadal hormones on μ‐opioid receptor (MOR) expression in trigeminal ganglia (TG) are not well understood.

Peripheral AMPA receptors contribute to muscle nociception and c-fos activation

In this study, involvement of peripheral AMPA receptors in mediating craniofacial muscle pain was investigated. AMPA receptor subunits, GluR1 and GluR2, were predominantly expressed in small to medium size neurons but more GluR2 positive labeling were encountered in trigeminal ganglia (TG) of male Sprague Dawley rats. A greater prevalence of GluR2 is reflected by the significantly higher percentage of GluR2 than GluR1 positive masseter afferents. Nocifensive behavior and c-fos immunoreactivity were assessed from the same animals that received intramuscular mustard oil (MO) with or without NBQX, a potent AMPA/KA receptor antagonist. Masseteric MO produced nocifensive hindpaw shaking responses that peaked in the first 30s and gradually diminished over a few minutes. There was a significant difference in both peak and overall MO-induced nocifensive responses between NBQX and vehicle pre-treated rats. Subsequent Fos studies also showed that peripheral NBQX pre-treatment effectively reduced the MO-induced neuronal activation in the subnucleus caudalis of the trigeminal nerve (Vc). These combined results provide compelling evidence that acute muscle nociception is mediated, in part, by peripherally located AMPA/KA receptors, and that blockade of multiple peripheral glutamate receptor subtypes may provide a more effective means of reducing muscular pain and central neuronal activation.

The role of peripheral N-methyl-D-aspartate receptors in muscle hyperalgesia.

The present study demonstrates that intramuscular administration of N-methyl-D-aspartate receptor antagonist AP5 dose-dependently attenuates complete Freunds adjuvant (CFA) induced muscular hyperalgesia. CFA significantly reduced mean bite force and success rate in the rats trained to produce a specific bite force. Pretreatment with AP5 reversed the overall magnitude of reduction in mean bite force and success rate in CFA inflamed rats, and significantly facilitated the recovery of these measures to pre-injection level. AP5 treatment 1 day after the CFA injection had little effect on CFA-mediated changes in bite force measurements. These data suggest that peripheral N-methyl-D-aspartate receptors play a critical role in the development of persistent muscle hyperalgesia, and provide important new insights for therapeutic alternatives that can be directed at the periphery.

The role of androgen receptor in transcriptional modulation of cannabinoid receptor type 1 gene in rat trigeminal ganglia

We have previously shown that anti-hyperalgesic effects of cannabinoid agonists under inflammatory condition are much greater in male than female, and that inflammatory cytokines upregulate cannabinoid receptor type 1 (CB1) expression in male, but not female, trigeminal ganglia (TG) in a testosterone-dependent manner. In this study, we investigated the mechanisms underlying the testosterone-mediated regulation of peripheral CB1 expression. We hypothesized that testosterone upregulates CB1 through transcriptional modulation by androgen receptor (AR). Interleukin-1 beta (IL-1β), a pro-inflammatory cytokine, upregulated CB1 mRNA expression in TG of male rats. The cytokine-induced upregulation was prevented by the pretreatment with flutamide, a specific antagonist for AR, but not by ICI 182,780, a specific antagonist for estrogen receptor, suggesting that the effects of testosterone are not mediated by estradiol, a testosterone metabolite. The expression levels of AR and IL-1β receptors were comparable between male and female TG, suggesting that the male specific IL-1β effects on CB1 upregulation occurs downstream to these receptors. The chromatin immunoprecipitation assay showed AR binding to the CB1 promoter in the rat TG. Furthermore, luciferase reporter assay revealed that AR activated the CB1 gene in response to testosterone or dihydrotestosterone treatment. These experiments provided compelling evidence that testosterone regulates CB1 gene transcription in TG through AR following cytokine stimulation. These results should provide mechanistic bases for understanding cytokine-hormone-neuron interactions in peripheral cannabinoid systems, and have important clinical implications for pain patients in whom testosterone level is naturally low, gradually declining or pharmacologically compromised.