Forced mouth opening induces post-traumatic hyperalgesia and associated peripheral sensitization after temporomandibular joints injury in mice.

Temporomandibular disorder (TMD) is the most prevalent painful condition in the craniofacial area. The pathophysiology of TMD is not fully understood, and it is necessary to understand pathophysiology underlying painful TMD conditions to develop more effective treatment methods. Recent studies suggested that external or intrinsic trauma to TMJ is associated with chronic TMD in patients. Here, we investigated the effects of the TMJ trauma through forced-mouth opening (FMO) in mice to determine pain behaviors and peripheral sensitization of trigeminal nociceptors. FMO increased mechanical hyperalgesia assessed by von Frey test, spontaneous pain-like behaviors assessed by mouse grimace scale, and anxiety-like behaviors assessed by open-field test. In vivo GCaMP Ca 2+ imaging of intact trigeminal ganglia (TG) showed increased spontaneous Ca 2+ activity and mechanical hypersensitivity of TG neurons in the FMO compared to the sham group. Ca 2+ responses evoked by cold, heat, and capsaicin stimuli were also increased. FMO-induced hyperalgesia and neuronal hyperactivities were not sex dependent. TG neurons sensitized following FMO were primarily small to medium-sized nociceptive afferents. Consistently, most TMJ afferents in the TG were small-sized peptidergic neurons expressing calcitonin gene-related peptides, whereas nonpeptidergic TMJ afferents were relatively low. FMO-induced intraneural inflammation in the surrounding tissues of the TMJ indicates potentially novel mechanisms of peripheral sensitization following TMJ injury. These results suggest that the TMJ injury leads to persistent post-traumatic hyperalgesia associated with peripheral sensitization of trigeminal nociceptors.

Voluntary biting behavior as a functional measure of orofacial pain in mice.

INTRODUCTION: Pain-related behavior secondary to masticatory function can be assessed with the rodent bite force model. A reduction of the bite force has been shown to be related to pain associated with the masseter muscle and jaw activity, while an increase in bite force suggests improvement of muscle function and less pain. To evaluate the usefulness of the bite force measure in studying long-lasting orofacial pain we analyzed biting parameters during prolonged myofascial pain induced by ligation injury of the masseter muscle tendon (TL) in mice. METHODS: C57Bl/6 mice were habituated to bite at a pair of aluminum plates attached to a force displacement transducer. The transduced voltage signals were amplified and converted to force through calibration with a standard weight set. Voluntary biting behavior was recorded for 100 s/session and those with bite forces ≥980 mN were analyzed. Nociception was also verified with von Frey, conditioned place avoidance (CPA) tests and mouse grimace scale. Persistent orofacial pain was induced with unilateral ligation of one tendon of the masseter muscle (TL). RESULTS: To reduce interference of random bites of smaller forces, the top 5 or 15 bite forces (BF5/15) were chosen as a measure of masticatory function and related to pain behavior. Both male and female mice exhibited similar BF5/15. For the first nascent test of all mice, mean bite force was significantly and positively correlated with the body weight. However, this correlation was less clear in the latter tests (2-8 w). TL induced a reduction of BF5/15 that peaked at 1 w and returned to the baseline within 3 w. The von Frey and CPA tests indicated that mechanical allodynia/hyperalgesia persisted at the time when the BF had returned to the pre-injury level. Infusion of pain-relieving bone marrow stromal cells improved biting behavior in both male and female mice as shown by significantly increased BF5/15, compared to vehicle-treated mice. CONCLUSIONS: Mouse voluntary biting behavior can be reliably measured and quantified with a simplified setup. The bite force showed an inverse relationship with the level of pain after TL and was improved by pain-relieving manipulations. However, the injury-induced reduction of bite force peaked early and did not parallel with other measures of nociception in the later phase of hyperalgesia. The results suggest that multiple factors such as the level of habituation, cognitive motive, physical status, and feeding drive may affect random voluntary biting and confound the biting parameters related to maintained hyperalgesia.

Spontaneous and Bite-Evoked Muscle Pain Are Mediated by a Common Nociceptive Pathway With Differential Contribution by TRPV1.

Spontaneous pain and function-associated pain are prevalent symptoms of multiple acute and chronic muscle pathologies. We established mouse models for evaluating spontaneous pain and bite-evoked pain from masseter muscle, and determined the roles of transient receptor potential cation channel subfamily V member 1 (TRPV1) and the contribution of TRPV1- or neurokinin 1 (NK1)-dependent nociceptive pathways. Masseter muscle inflammation increased Mouse Grimace Scale scores and face-wiping behavior, which were attenuated by pharmacological or genetic inhibition of TRPV1. Masseter inflammation led to a significant reduction in bite force. Inhibition of TRPV1 only marginally relieved the inflammation-induced reduction of bite force. These results suggest a differential extent of contribution of TRPV1 to the 2 types of muscle pain. However, chemical ablation of TRPV1-expressing nociceptors or chemogenetic silencing of TRPV1-lineage nerve terminals in masseter muscle attenuated inflammation-induced changes in Mouse Grimace Scale scores as well as bite force. Furthermore, ablation of neurons expressing NK1 receptor in trigeminal subnucleus caudalis also prevented both types of muscle pain. Our results suggest that TRPV1 differentially contributes to spontaneous pain and bite-evoked muscle pain, but TRPV1-expressing afferents and NK1-expressing second-order neurons commonly mediate both types of muscle pain. Therefore, manipulation of the nociceptive circuit may provide a novel approach for management of acute or chronic craniofacial muscle pain. PERSPECTIVE: We report the profound contribution of TRPV1 to spontaneous muscle pain but not to bite-evoked muscle pain. These 2 types of muscle pain are transmitted through a common nociceptive pathway. These results may help to develop new strategies to manage multiple modes of muscle pain simultaneously by manipulating pain circuits.

Classification of muscle spindle afferents innervating the masseter muscle in rats.

Taylor et al. [Taylor, A., Durbaba, R., Rodgers, J.F., 1992a. The classification of afferents from muscle spindles of the jaw-closing muscles of the cat. J Physiol 456, 609-628] developed a method to classify muscle spindle afferents using succinylcholine (Sch) and ramp and hold stretches. They demonstrated that cat jaw muscle spindle afferents show high proportion of intermediate responses to ramp and hold jaw stretch. Together with observations on the responses to Sch their data suggests that the majority of jaw muscle spindle afferents are influenced by a combination of nuclear bag(2) and nuclear chain fibres. Relatively few are influenced solely by nuclear bag(1) fibres. The purpose of this study was to categorize jaw muscle spindle afferent in rodents in response to ramp and hold stretches. Several measures were used to classify spindle afferents including (1) conduction velocity, (2) coefficient of variation (C.V.) of the interspike interval during jaw opening, and (3) the dynamic sensitivity and the initial discharge of spindle afferents before and after succinylcholine infusion (Sch, 100mg/kg, i.v.). Consistent with observations in the cat jaw muscles, the distribution of the conduction velocity and the C.V. of Vmes masseter afferents were unimodal. Therefore, these parameters were of little value in functional classification of spindle innervation. Succinylcholine injection either markedly increased the dynamic sensitivity or produced no change in Vmes afferents. Unlike cat jaw muscle spindle afferents, the effect of Sch on the initial discharge was not clearly separable from those responding or not responding to Sch. These results suggest that rat jaw muscle spindle afferents, have physiological properties that are primarily intermediate in nature and are likely to reflect a predominance of influence from nuclear bag(2) and chain fibres. However, the distinction between bag(2) and chain fibres influences is not as clearly defined in the rat compared to the cat.

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 Freund's 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 effect of experimental muscle pain on the amplitude and velocity sensitivity of jaw closing muscle spindle afferents.

The effect of experimental muscle pain on the amplitude and velocity sensitivity of muscle spindle primary afferent neurons in the trigeminal mesencephalic nucleus (Vmes) was examined. Extracellular recordings were made from 45 neurons designated as spindle primary- or secondary-like on the basis of their response to ramp-and-hold jaw movements. Velocity sensitivity was assessed in spindle primary-like afferents by calculating the mean dynamic index of each unit in response to three different velocities of jaw opening before and after intramuscular injection with hypertonic saline (HS, 5%, 100 microl). The amplitude sensitivity of all jaw muscle spindle afferents was assessed by calculating the mean firing rate of each unit in response to three different amplitudes of jaw openings during both the open and hold phases of the movement and with best-fit lines obtained, using linear regression analysis, before and after HS injection. The variance of the two regression lines obtained for each unit before and after the injection was compared using the coincidence test, and changes in intercept and slope were determined. Seventy-five percent of the primary-like units and 80% of the secondary-like units presented with changes in static behavior after HS injection. Thirty-six percent of the primary-like units showed changes in dynamic behavior. Injection of isotonic saline (control) did not alter the responses of the spindle afferent to jaw opening. Thus, our results demonstrate that the predominant effect of noxious stimulation was a shift in the amplitude sensitivity of both spindle primary-like and secondary-like afferents and, to a lesser extent, the velocity sensitivity of the spindle primary-like unit. In accordance with earlier studies in the cat hindlimb and neck muscles, these results suggest that the activation of masseter muscle nociceptor alters spindle afferent responses to stretch acting primarily through static gamma motor neurons.

Bite force measurement in awake rats: a behavioral model for persistent orofacial muscle pain and hyperalgesia.

AIMS: To test the hypotheses that masseteric inflammation produces a reduction of mean bite force and success rates and that classical anti-inflammatory agents prevent inflammation-induced changes in bite force. METHODS: Rats were initially trained to produce a bite force greater than 400 g. Once the rats attained above 70% of successful responses in a 10-minute test period, the bite force required for reinforcement was increased gradually to the target force of 1.3 kg. Seven trained rats received bilateral masseteric injections of complete Freund's adjuvant (CFA; 50 microL in isotonic saline). The mean number of attempted bites, the percentage of correct responses, and the bite force measured before and 1, 2, 3, 7, 10, and 14 days following the CFA injection were compared. Five additional trained rats were injected with the same volume of vehicle control. Other rats (n = 10) were treated with anti-inflammatory agents before and after the CFA injection. RESULTS: Intramuscular CFA, but not the vehicle, produced a significant reduction of mean bite force and success rate at days 1, 2, and 3. Bite force and success rate gradually increased; they returned to baseline by 14 days. The CFA-mediated reduction of bite force and success rate was prevented in rats treated with anti-inflammatory agents administered intraperitoneally (dexamethasone, n = 5, or indomethacin, n = 5, 4 mg/kg). CONCLUSION: These results provide further evidence that bite force measurements in awake rats can be a useful method for the study of inflammatory muscle hyperalgesia.

Innocuous jaw movements increase c-fos expression in trigeminal sensory nuclei produced by masseter muscle inflammation.

Muscle tenderness and pain during movements are prominent symptoms associated with persistent jaw muscle pain. However, there is virtually no information on how trigeminal neurons respond to jaw movements (JM) or muscle palpation in the presence of muscle tissue injury or myositis. In this study, we investigated the effects of innocuous JM in the presence of acute masseteric inflammation on postsynaptic responses in the trigeminal brainstem nuclei by examining the expression of c-fos. In one group of rats, unilateral injections of an inflammatory substance, mustard oil (MO: 20%, 25 microl) were made into a masseter muscle. In another group, controlled and systematic JM were provided following MO injection. Three additional groups of rats were used to control for anesthetic, JM, and injection procedure. MO injected in the masseter muscle induced a high level of Fos protein expression in four principal trigeminal regions: the subnucleus caudalis (Vc), the ventral and dorsal regions of the Vc/Vi (subnucleus interpolaris) transition zone, and the paratrigeminal nucleus (PTN). Movements following MO injection consistently produced a significantly greater level of Fos expression in all these areas, especially in the Vc/Vi transition region and caudal Vc on the ipsilateral side. Importantly, movements also induced a significantly greater level of Fos expression in the caudal Vc on the contralateral side. The present results provide the first documentation that innocuous JM in the presence of muscle inflammation significantly increase the MO-induced c-fos expression in the trigeminal brainstem nuclei, which may explain the greater pain experienced during movement of inflamed or injured muscles.

Jaw muscle spindle afferents coordinate multiple orofacial motoneurons via common premotor neurons in rats: an electrophysiological and anatomical study.

Jaw muscle spindle afferents (JMSA) in the mesencephalic trigeminal nucleus (Vme) project to the parvocellular reticular nucleus (PCRt) and dorsomedial spinal trigeminal nucleus (dm-Vsp). A number of premotor neurons that project to the trigeminal motor nucleus (Vmo), facial nucleus (VII) and hypoglossal nucleus (XII) are also located in the PCRt and dm-Vsp. In this study, we examined whether these premotor neurons serve as common relay pool for relaying JMSA to multiple orofacial motoneurons. JMSA inputs to the PCRt and dm-Vsp neurons were verified by recording extracellular responses to electrical stimulation of the caudal Vme or masseter nerve, mechanical stimulation of jaw muscles and jaw opening. After recording, biocytin in recording electrode was inotophorized into recording sites. Biocytin-Iabeled fibers traveled to the Vmo, VII, XII, and the nucleus ambiguus (Amb). Labeled boutons were seen in close apposition with Nissl-stained motoneurons in the Vmo, VII, XII and Amb. In addition, an anterograde tracer (biotinylated dextran amine) was iontophorized into the caudal Vme, and a retrograde tracer (Cholera toxin B subunit) was delivered into either the VII or Xll to identify VII and XII premotor neurons that receive JMSA input. Contacts between labeled Vme neuronal boutons and premotor neurons were observed in the PCRt and adjacent dm-Vsp. Confocal microscopic observations confirmed close contacts between Vme boutons and VII and XII premotor neurons. This study provides evidence that JMSA may coordinate activities of multiple orofacial motor nuclei, including Vmo, VII, XII and Amb in the brainstem via a common premotor neuron pool.

Effects of experimental muscle pain on electromyographic activity of masticatory muscles in the rat.

The aim of the present study was to investigate the effects of noxious chemical stimulation of a jaw muscle on postural electromyographic (EMG) activity from several masticatory muscles in lightly anesthetized rats. Unilateral injection of a substance known to induce acute muscle pain (5% NaCl) or longer duration of pain with inflammation (mustard oil) was made into the masseter muscle. The changes in EMG activity following the injection were recorded from the injected and contralateral masseter muscles and the ipsilateral digastric muscle. The algesic chemicals produced a significant but transient increase in EMG activity in all three muscles. The data from the present study and similar observations from clinical and experimental human studies suggest that increased activity from muscle nociceptors is not sufficient to produce a prolonged increase in postural EMG activity. Therefore, the development and maintenance of chronic jaw muscle pain does not appear to result from a feedback cycle mechanism.