Xu- Dong

Sex-related differences in NMDA-evoked rat masseter muscle afferent discharge result from estrogen-mediated modulation of peripheral NMDA receptor activity.

In the present study, the hypothesis that sex-related differences in glutamate-evoked rat masseter muscle afferent discharge may result from estrogen-related modulation of peripheral N-methyl-d-aspartate (NMDA) receptor activity and/or expression was tested by examining afferent fiber discharge in response to masseter injection of NMDA and the expression of NR2A/B subunits by masseter ganglion neurons in male and female rats. The results showed that injection of NMDA into the masseter muscle evoked discharges in putative mechanonociceptive afferent fibers and increased blood pressure that was concentration-dependent, however, a systemic action of NMDA appeared responsible for increased blood pressure. NMDA-evoked afferent discharge was significantly greater in female than in male rats, was positively correlated with plasma estrogen levels in females and was significantly greater in ovariectomized female rats treated with a high dose (5 mug/day) compared with a low dose (0.5 mug/day) of estrogen. Pre-treatment of high dose estrogen-treated-ovariectomized female rats with the Src tyrosine kinase inhibitor PP2 did not affect NMDA-evoked afferent discharge. NMDA-evoked afferent discharge was attenuated by the antagonists ketamine and ifenprodil, which is selective for NR2B containing NMDA receptors. Fewer masseter ganglion neurons expressed the NR2A (16%) subunit as compared with the NR2B subunit (38%), which was expressed at higher frequencies in intact female (46%) and high dose estrogen-treated ovariectomized female (60%) rats than in male (31%) rats. Taken together, these results suggest that sex-related differences in NMDA-evoked masseter afferent discharge are due, at least in part, to an estrogen-mediated increase in expression of peripheral NMDA receptors by masseter ganglion neurons in female rats.

Botulinum neurotoxin type A (BoNTA) decreases the mechanical sensitivity of nociceptors and inhibits neurogenic vasodilation in a craniofacial muscle targeted for migraine prophylaxis

&NA; The mechanism by which intramuscular injection of BoNTA into the craniofacial muscles decreases migraine headaches is not known. In a blinded study, the effect of BoNTA on the mechanical and chemical responsiveness of individual temporalis muscle nociceptors and muscle neurogenic vasodilation was investigated in female rats. Mechanical threshold was measured for 3 h following intramuscular injection of BoNTA or vehicle, and for 10 min after a subsequent injection of the algogen glutamate. Injection of BoNTA significantly increased the mechanical threshold of muscle nociceptors without altering the muscle surface temperature and blocked glutamate‐induced mechanical sensitization and neurogenic vasodilation. None of these effects were reproduced by pancuronium‐induced muscle paralysis. Western blot analysis of temporalis muscles indicated that BoNTA significantly decreased SNAP‐25. Measurement of interstitial glutamate concentration with a glutamate biosensor indicated that BoNTA significantly reduced glutamate concentrations. The mechanical sensitivity of muscle nociceptors is modulated by glutamate concentration through activation of peripheral NMDA receptors. Immunohistochemical experiments were conducted and they indicated that half of the NMDA‐expressing temporalis nerve fibers co‐expressed substance P or CGRP. Additional electrophysiology experiments examined the effect of antagonists for NMDA, CGRP and NK1 receptors on glutamate‐induced effects. Glutamate‐induced mechanical sensitization was only blocked by the NMDA receptor antagonist, but muscle neurogenic vasodilation was attenuated by NMDA or CGRP receptor antagonists. These data suggest that injection of BoNTA into craniofacial muscles acts to decrease migraine headaches by rapidly decreasing the mechanical sensitivity of temporalis muscle nociceptors through inhibition of glutamate release and by attenuating the provoked release of CGRP from muscle nociceptors.

Systemic Administration of Monosodium Glutamate Elevates Intramuscular Glutamate Levels and Sensitizes Rat Masseter Muscle Afferent Fibers

Abstract There is evidence that elevated tissue concentrations of glutamate may contribute to pain and sensitivity in certain musculoskeletal pain conditions. In the present study, the food additive monosodium glutamate (MSG) was injected intravenously into rats to determine whether it could significantly elevate interstitial concentrations of glutamate in the masseter muscle and whether MSG administration could excite and/or sensitize slowly conducting masseter afferent fibers through N‐methyl‐d‐aspartate (NMDA) receptor activation. The interstitial concentration of glutamate after systemic injection of isotonic phosphate‐buffered saline (control) or MSG (10 and 50 mg/kg) was measured with a glutamate‐selective biosensor. The pre‐injection baseline interstitial concentration of glutamate in the rat masseter muscle was 24 ± 11 μM. Peak interstitial concentration after injection of 50 mg/kg MSG was 63 ± 18 μM and remained elevated above baseline for ∼18 min. In vivo single unit recording experiments were undertaken to assess the effect of MSG (50 mg/kg) on masseter afferent fibers. Injection of MSG evoked a brief discharge in one afferent fiber, and significantly decreased (∼25%) the average afferent mechanical threshold (n = 10) during the first 5 min after injection of MSG. Intravenous injection of ketamine (1 mg/kg), 5 min prior to MSG, prevented the MSG‐induced decreases in the mechanical threshold of masseter afferent fibers. The present results indicate that a 2‐ to 3‐fold elevation in interstitial glutamate levels in the masseter muscle is sufficient to excite and induce afferent mechanical sensitization through NMDA receptor activation. These findings suggest that modest elevations of interstitial glutamate concentration could alter musculoskeletal pain sensitivity in humans.

Sensitivity of rat temporalis muscle afferent fibers to peripheral N-methyl-D-aspartate receptor activation

The temporalis muscle is a common source of pain in headache and chronic craniofacial pain conditions such as temporomandibular disorders, which have an increased prevalence in women. The characteristics of slowly conducting temporalis afferent fibers have not been investigated. Therefore, the aim of the present study was to examine the characteristics of slowly conducting temporalis muscle afferent fibers and to determine whether these fibers are excited by activation of peripheral N-methyl-D-aspartate receptors. The response properties of a total of 117 temporalis afferent fibers were assessed in male and female rats. A majority of these fibers had high mechanical thresholds and slow conduction velocities (<10 m/s). The mechanical threshold of the temporalis afferent fibers was inversely correlated with afferent conduction velocity, however, no sex-related differences in mechanical threshold were identified. There were also no sex-related differences in N-methyl-D-aspartate-evoked afferent discharge. Indeed, injection of a high concentration (1600 mM) of N-methyl-D-aspartate into the temporalis muscle was necessary to evoke significant afferent discharge. Thirty minutes after the initial injection of N-methyl-D-aspartate into the temporalis muscle, a second injection of N-methyl-D-aspartate produced a response only about 50% as large as the initial injection. Co-injection of ketamine (20 mM) with the second injection of N-methyl-D-aspartate significantly decreased N-methyl-D-aspartate-evoked afferent discharge in both sexes. This concentration of ketamine is greater than that needed to attenuate afferent discharge evoked by injection of glutamate into the masseter muscle. These results suggest that unlike masseter afferent fibers, temporalis afferent fibers are relatively insensitive to peripheral N-methyl-D-aspartate receptor activation.

Serotonin (5-HT) excites rat masticatory muscle afferent fibers through activation of peripheral 5-HT3 receptors

&NA; In the present study, we combined immunohistochemical experiments with in vivo single unit recordings to examine whether 5‐HT3 receptors are expressed by masticatory (masseter and temporalis) sensory ganglion neurons and to investigate the effects of intramuscular injection of 5‐HT on the excitability and mechanical threshold of rat masticatory muscle afferent fibers. The expression of 5‐HT3 receptors by masticatory ganglion neurons was examined using immunohistochemical techniques. In vivo extracellular single unit recording techniques were used to assess changes in the excitability of individual masticatory muscle afferent fibers. Immunohistochemical experiments detected a relatively high frequency (52%) of 5‐HT3 receptor expression by masticatory ganglion neurons. Injection of 5‐HT (10−4, 10−3, 10−2 M) evoked concentration‐related increases in the magnitude of afferent discharge, but did not significantly sensitize muscle afferent fibers to mechanical stimuli. No significant sex‐related differences in 5‐HT‐evoked afferent discharge were identified. Afferent discharge evoked by 5‐HT was significantly attenuated by co‐injection with the selective 5‐HT3 receptor antagonist tropisetron (10−3 M). Afferent discharge was also evoked by the selective 5‐HT3 receptor agonist 2‐methyl‐5‐HT. Unexpectedly, a significant concentration‐related decrease in median blood pressure in response to 5‐HT injection was found. This 5‐HT‐induced decrease in blood pressure was not antagonized by tropisetron or mimicked by 2‐methyl‐5‐HT, indicating that the drop in blood pressure was not 5‐HT3 receptor‐mediated. The present results indicate that 5‐HT excites slowly conducting masticatory muscle afferent fibers through activation of peripheral 5‐HT3 receptors, and suggest that similar mechanisms may contribute to 5‐HT‐evoked muscle pain in human subjects.

Glutamate dysregulation in the trigeminal ganglion: A novel mechanism for peripheral sensitization of the craniofacial region

In the trigeminal ganglion (TG), satellite glial cells (SGCs) form a functional unit with neurons. It has been proposed that SGCs participate in regulating extracellular glutamate levels and that dysfunction of this SGC capacity can impact nociceptive transmission in craniofacial pain conditions. This study investigated whether SGCs release glutamate and whether elevation of TG glutamate concentration alters response properties of trigeminal afferent fibers. Immunohistochemistry was used to assess glutamate content and the expression of excitatory amino acid transporter (EAAT)1 and EAAT2 in TG sections. SGCs contained glutamate and expressed EAAT1 and EAAT2. Potassium chloride (10 mM) was used to evoke glutamate release from cultured rat SGCs treated with the EAAT1/2 inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)ben zoyl]amino]phenyl]methoxy]-L-aspartic acid (TFB-TBOA) or control. Treatment with TFB-TBOA (1 and 10 μM) significantly reduced the glutamate concentration from 10.6 ± 1.1 to 5.8 ± 1.4 μM and 3.0 ± 0.8 μM, respectively (p<0.05). Electrophysiology experiments were conducted in anaesthetized rats to determine the effect of intraganglionic injections of glutamate on the response properties of ganglion neurons that innervated either the temporalis or masseter muscle. Intraganglionic injection of glutamate (500 mM, 3 μl) evoked afferent discharge and significantly reduced muscle afferent mechanical threshold. Glutamate-evoked discharge was attenuated bythe N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovalerate (APV) and increased by TFB-TBOA, whereas mechanical sensitization was only sensitive to APV. Antidromic invasion of muscle afferent fibers by electrical stimulation of the caudal brainstem (10 Hz) or local anesthesia of the brainstem with lidocaine did not alter glutamate-induced mechanical sensitization. These findings provide a novel mechanism whereby dysfunctional trigeminal SGCs could contribute to cranial muscle tenderness in craniofacial pain conditions such as migraine headache.

The analgesic action of topical diclofenac may be mediated through peripheral NMDA receptor antagonism

ABSTRACT The analgesic mechanism underlying the efficacy of topical diclofenac in the treatment of musculoskeletal pain is incompletely understood. The present study investigated whether intramuscular injection of diclofenac (0.1 mg/ml, ∼340 μM) could attenuate jaw‐closer muscle nociceptor discharge and mechanical sensitization induced by activation of peripheral 5‐hydroxytryptamine (serotonin) or excitatory amino acid receptors in anesthetized Sprague–Dawley rats. Diclofenac inhibited nociceptor discharge evoked by NMDA, but had no effect on nociceptor discharge evoked by 5‐hydroxytryptamine or AMPA. Subsequent experiments revealed that diclofenac‐mediated inhibition of NMDA‐evoked nociceptor discharge was competitive. Intramuscular injection of 5‐hydroxytryptamine, NMDA and AMPA also decreased nociceptor mechanical threshold, however, only the mechanical sensitization produced by NMDA was reversed by diclofenac. Co‐administration of the proinflammatory prostaglandin PGE2 did not alter the ability of diclofenac to significantly attenuate NMDA‐evoked nociceptor discharge or NMDA‐induced mechanical sensitization. Intramuscular injection of either diclofenac or the competitive NMDA receptor antagonist dl‐2‐amino‐5‐phosphonovalerate (50 mM) alone could elevate nociceptor mechanical threshold for a 30 min period post‐injection. The present study indicates that in vivo, diclofenac can exert a selective, competitive inhibition of peripheral NMDA receptors at muscle concentrations achievable after topical administration of diclofenac containing preparations. This property may contribute to the analgesic effect of topical diclofenac when used for muscle pain.

TNFα Mechanically Sensitizes Masseter Muscle Afferent Fibers of Male Rats

Behavioral evidence in rats indicates that injection of tumor necrosis factor alpha (TNFalpha) into skeletal muscle results in a prolonged mechanical sensitization without gross inflammation. To investigate whether a peripheral mechanism could underlie this effect, in the present study, TNFalpha (1 or 0.1 microg) was injected into the rat masseter muscle to assess its effect on the excitability and mechanical threshold (MT) of muscle nociceptors as well as on inflammation. Expression of TNFR1 (P55 receptors) and TNFR2 (P75 receptors) by the masseter muscle and trigeminal ganglion neurons that innervate that muscle was determined by Western blot and immunohistochemistry, respectively. The Evans blue dye technique was used at the end of the TNFalpha experiments to assess for plasma protein extravasation. In subsequent experiments to confirm the involvement of receptor activation in TNFalpha-induced effects, P55 or P75 receptor antibody was co-injected with TNFalpha. Intramuscular injection of 1 microg TNFalpha did not excite nociceptors but did significantly decrease MT compared with vehicle control. There was no evidence of gross inflammation 3 h after injection of TNFalpha. Co-injection of TNFalpha with P55 or P75 receptor antibodies attenuated TNFalpha-induced mechanical sensitization. P55 and P75 receptors were expressed by 29 and 62% of masseter nociceptors, respectively. These findings indicate that TNFalpha induces mechanical sensitization of masseter nociceptors that is mediated through activation of peripheral P55 and P75 receptors. These results support the hypothesis that a peripheral receptor mechanism could contribute to TNFalpha-induced noninflammatory mechanical sensitization of skeletal muscle previously reported in behaving rats.

Human nerve growth factor sensitizes masseter muscle nociceptors in female rats

&NA; Injection of nerve growth factor (NGF) into the masseter muscle is not painful but does induce a localized, quick onset (˜1 h) and long‐lasting mechanical sensitization in healthy human subjects. We tested the hypothesis that human NGF mechanically sensitizes masseter muscle nociceptors by increasing the sensitivity of peripheral N‐methyl‐d‐aspartate (NMDA) receptors. Co‐expression of the NR2B subunit of the NMDA receptor with P75 and TrkA NGF receptors by trigeminal ganglion neurons that innervate the masseter muscle was investigated immunohistochemically. Nociceptor activity was recorded extracellularly from the trigeminal ganglion of anaesthetized female rats. Nociceptor mechanical threshold was assessed before and every 30 min for 3 h after injection of human NGF (25 &mgr;g/ml, 10 &mgr;l, n = 12), and in subsequent experiments NGF with TrkA (n = 12) or P75 (n = 11) receptor antibodies. Glutamate (1 M, 10 &mgr;l) was injected at the end of each experiment. Approximately 85% of NR2B positive masseter ganglion neurons co‐expressed P75 or TrkA receptors, suggesting the potential for interaction. When compared with the vehicle control, it was found that injection of NGF into the masseter muscle did not evoke significant nociceptor discharge but did significantly reduce nociceptor mechanical threshold (˜30%). There was no effect of NGF on glutamate‐evoked nociceptor discharge or glutamate‐induced mechanical sensitization. Additional experiments indicated that NGF‐induced mechanical sensitization could be partially attenuated with TrkA receptor antibodies, but not P75 receptor antibodies. These findings indicate that human NGF‐induced sensitization of masseter nociceptors results, in part, from the activation of TrkA receptors, but does not appear to be mediated through enhanced peripheral NMDA receptor activity.

The Role of Peripheral Glutamate and Glutamate Receptors in Muscle Pain

Objectives: To review evidence that associates elevated muscle interstitial glutamate concentration and the activation of peripheral glutamate receptors with the development and maintenance of muscle pain in conditions such as myofascial temporomandibular disorders [TMDs]. Findings: In certain noninflammatory musculoskeletal pain conditions, an association between pain, elevated glutamate concentrations and the expression of peripheral N-methyl-d-aspartate receptors [NRs] has been made. Myofascial TMDs are a noninflammatory muscle pain disorder characterized by pain and localized mechanical sensitivity in the masticatory muscle and a greater prevalence in young women than in men. Artificial elevation of glutamate concentrations in the masticatory muscles of healthy subjects evokes intense, short duration pain that is significantly greater in woman than in men and induces a more prolonged, localized mechanical sensitization that is similar in both sexes. Glutamate-evoked pain and sensitization may be attenuated by coinjection of the NR antagonist ketamine, which suggests that these effects are mediated, in part, through activation of peripheral NRs. In male and female animals, a two- to three-fold elevation of muscle interstitial glutamate concentrations appears sufficient to both excite and sensitize masticatory muscle nociceptors through activation of peripheral NRs. The magnitude of nociceptor discharge in response to peripheral NR activation is greater in females than in males, and this sex-related difference appears to be due, in part, to an estrogen-mediated enhancement of peripheral NR expression. Conclusions: These findings suggest that modest elevations of interstitial glutamate concentration could alter musculoskeletal pain sensitivity in a sex-related manner through activation of peripheral NR receptors.