Noninvasive vagus nerve stimulation and morphine transiently inhibit trigeminal pain signaling in a chronic headache model.

INTRODUCTION: Chronic headache conditions are characterized by persistent sensitization of the trigeminal system, which involves dysfunction of descending pain modulation. We previously reported that noninvasive vagus nerve stimulation (nVNS) inhibits trigeminal nociception in models of episodic migraine through a mechanism involving enhanced serotonergic and GABAergic descending pain signaling. OBJECTIVES: The analgesic effectiveness of nVNS and morphine were investigated in an animal model of chronic headache mediated by the combination of the 3 migraine risk factors of neck muscle tension, paradoxical sleep deprivation, and pungent odors. METHODS: Sprague-Dawley rats were injected with complete Freund's adjuvant in the trapezius and sleep deprived for 1 night to promote trigeminal sensitization. After 7 days, animals were exposed to a pungent odor, and mechanical nocifensive head withdrawal responses were determined using von Frey filaments. Beginning on day 3 after odor exposure, animals were treated daily with either nVNS or morphine for 7 days. RESULTS: Exposure of animals sensitized by neck inflammation and sleep deprivation to a pungent odor resulted in a prolonged state of trigeminal nociception. Daily administration of nVNS or morphine significantly repressed the nocifensive response; however, cessation resulted in a return to heightened pretreatment nocifensive levels. CONCLUSIONS: The combination of reported migraine risk factors promotes a state of sustained trigeminal hypersensitivity characteristic of chronic headache. Daily nVNS was similarly effective as morphine in inhibiting nociception and may represent a safer, opioid-sparing therapeutic option for other chronic pain disorders involving sensitization of the trigeminal system by promoting descending pain modulation.

Enriched Chicken Bone Broth as a Dietary Supplement Reduces Nociception and Sensitization Associated with Prolonged Jaw Opening.

AIMS: To test a commercially available enriched chicken bone broth (ECBB) product for its potential anti-inflammatory properties and to evaluate its ability to reduce nociception and expression of protein kinase A (PKA) in a clinically relevant model of temporomandibular disorder (TMD) caused by prolonged jaw opening in rats. METHODS: The potential of the ECBB and of a homemade broth was investigated using the Folin-Ciocalteu reagent and percent inhibition of cyclooxygenase-2 (COX-2) activity, which was determined using a commercially available kit. Additionally, the effect of ECBB and homemade broth on nocifensive head withdrawal responses to mechanical stimulation in male Sprague-Dawley rats subjected to prolonged jaw opening was evaluated. Differences were considered significant at P < .025. Changes in PKA expression in the medullary dorsal horn region of the spinal trigeminal nucleus associated with prolonged jaw opening were assessed using immunofluorescence, and these changes were considered significant at P < .05. Behavioral data were analyzed by using multiple nonparametric tests, and immunohistochemistry data were analyzed by using one-way analysis of variance with Games-Howell post hoc tests in SPSS software. RESULTS: ECBB exhibited greater reducing potential and inhibition of COX-2 activity compared to homemade broth. Near maximal jaw opening was sufficient to induce sustained nocifensive responses to mechanical stimuli for 7 days. This increased sensitivity was correlated with elevated levels of the active form of PKA. Importantly, dietary inclusion of ECBB, but not of homemade broth, for 2 weeks prior to jaw opening was sufficient to reduce nocifensive behaviors and PKA expression. CONCLUSION: Findings from this study provide evidence that ECBB attenuates nociception and expression of the pro-inflammatory protein PKA and thus may be beneficial as a nutraceutical supplement to manage inflammatory pain associated with TMD.

Central Role of Protein Kinase A in Promoting Trigeminal Nociception in an In Vivo Model of Temporomandibular Disorders.

AIMS: To investigate cellular changes in the spinal trigeminal nucleus (STN) and trigeminal ganglion (TG) associated with trigeminal nociception mediated by inflammation in the temporomandibular joint (TMJ). METHODS: Male Sprague-Dawley rats (n = 86) were utilized to investigate cellular and behavioral responses to prolonged TMJ inflammation caused by bilateral injection of Complete Freund's Adjuvant (CFA) in the TMJ capsules. To investigate the cellular effects of protein kinase A (PKA) in the STN, rats were injected intrathecally with the selective PKA inhibitor KT5720 prior to injection of CFA into both TMJ capsules. Levels of calcitonin gene-related peptide (CGRP), active PKA, and ionized calcium-binding adapter molecule 1 (Iba1) in the STN and expression of phosphorylated extracellular regulated kinases (p-ERK) in the TG were determined with immunohistochemistry (n ≥ 3 experiments per test condition). Nocifensive head withdrawal responses to mechanical stimulation of the cutaneous tissue over the TMJ were monitored following CFA injection in the absence or presence of KT5720 (n = 7). Statistical analysis was performed using parametric analysis of variance (ANOVA) tests. RESULTS: Intrathecal injection of KT5720 significantly inhibited the stimulatory effect of CFA on levels of CGRP, PKA, and Iba1 in the STN. In addition, administration of KT5720 decreased the average number of CFA-induced nocifensive withdrawal responses to mechanical stimulation and the CFA-mediated increase in p-ERK expression in the ganglion. CONCLUSION: These findings provide evidence that elevated PKA activity in the STN promotes cellular events temporally associated with trigeminal nociception caused by prolonged TMJ inflammation.

Vagus nerve stimulation inhibits trigeminal nociception in a rodent model of episodic migraine.

INTRODUCTION: Although neck muscle tension is considered a risk factor for migraine, pungent odors can act as a trigger to initiate an attack in sensitized individuals. Although noninvasive vagus nerve stimulation (nVNS) is now an approved treatment for chronic migraine, how it functions to inhibit trigeminal nociception in an episodic migraine model is not known. OBJECTIVES: The objectives of this study were to determine if nVNS could inhibit trigeminal nociception in a novel model of episodic migraine and investigate changes in the expression of proteins implicated in peripheral and central sensitization. METHODS: Sprague-Dawley male rats were injected with an inflammatory agent in the trapezius muscle before exposure to pungent volatile compounds, which was used to initiate trigeminal nociceptor activation. The vagus nerve was stimulated transdermally by a 1-ms pulse of 5 kHz sine waves, repeated at 25 Hz for 2 minutes. Nocifensive head withdrawal response to von Frey filaments was determined and immunoreactive protein levels in the spinal cord and trigeminal ganglion (TG) were investigated. RESULTS: Exposure to the pungent odor significantly increased the number of nocifensive withdrawals in response to mechanical stimulation of sensitized TG neurons mediated by neck muscle inflammation. Noninvasive vagus nerve stimulation inhibited nociception and repressed elevated levels of P-ERK in TG, Iba1 in microglia, and GFAP in astrocytes from sensitized animals exposed to the pungent odor. CONCLUSION: Our findings demonstrate that nVNS inhibits mechanical nociception and represses expression of proteins associated with peripheral and central sensitization of trigeminal neurons in a novel rodent model of episodic migraine.

Tumor necrosis factor-Alpha stimulates cytokine expression and transient sensitization of trigeminal nociceptive neurons.

OBJECTIVE: Elevated levels of tumor necrosis factor- alpha (TNF-α) in the capsule of the temporomandibular joint (TMJ) are implicated in the underlying pathology of temporomandibular disorders (TMD). TMD are a group of conditions that result in pain in the TMJ and/or muscles of mastication, and are associated with significant social and economic burdens. The goal of this study was to investigate the effect of elevated TNF-α levels in the TMJ capsule on nocifensive behavioral response to mechanical stimulation of trigeminal neurons and regulation of cytokines within the trigeminal ganglion. DESIGN: Male Sprague-Dawley rats were injected bilaterally in the TMJ capsule with TNF-α and changes in nocifensive head withdrawal responses to mechanical stimulation of cutaneous tissue directly over the capsule was determined using von Frey filaments. Cytokine levels in trigeminal ganglia were determined by protein array analysis at several time points post injection and correlated to nocifensive behavior. RESULTS: TNF-α caused a significant increase in the average number of nocifensive responses when compared to naive and vehicle treated animals 2h post injection, but levels returned to control levels at 24h. Based on array analysis, the levels of eight cytokines were significantly elevated above vehicle control levels at 2h following TNF-α injection, but all eight had returned to the vehicle control levels after 24h. CONCLUSIONS: Our findings provide evidence that elevated levels of TNF-α in the joint capsule, which is reported to occur in TMD, promotes nociception in trigeminal ganglia neurons via a mechanism that temporally correlates with differential regulation of several cytokines.

Elevated levels of calcitonin gene-related peptide in upper spinal cord promotes sensitization of primary trigeminal nociceptive neurons.

Orofacial pain conditions including temporomandibular disorder (TMD) and migraine are characterized by peripheral and central sensitization of trigeminal nociceptive neurons. The goal of this study was to investigate the role of calcitonin gene-related peptide (CGRP) in promoting bidirectional signaling within the trigeminal system to mediate sensitization of primary nociceptive neurons. Adult male Sprague-Dawley rats were injected intercisternally with CGRP or co-injected with the receptor antagonist CGRP8-37 or KT 5720, a protein kinase A (PKA) inhibitor. Nocifensive head withdrawal response to mechanical stimulation was investigated using von Frey filaments. Expression of PKA, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adapter molecule 1 (Iba1) in the spinal cord and phosphorylated extracellular signal-regulated kinase (P-ERK) in the ganglion was studied using immunohistochemistry. Some animals were co-injected with CGRP and Fast Blue dye and the ganglion was imaged using fluorescent microscopy. CGRP increased nocifensive responses to mechanical stimulation when compared to control. Co-injection of CGRP8-37 or KT 5720 with CGRP inhibited the nocifensive response. CGRP stimulated PKA and GFAP expression in the spinal cord, and P-ERK in ganglion neurons. Seven days post injection, Fast Blue was observed in ganglion neurons and satellite glial cells. Our results demonstrate that elevated levels of CGRP in the upper spinal cord promote sensitization of primary nociceptive neurons via a mechanism that involves activation of PKA centrally and P-ERK in ganglion neurons. Our findings provide evidence of bidirectional signaling within the trigeminal system that facilitate increased neuron-glia communication within the ganglion associated with trigeminal sensitization.

Prolonged Jaw Opening Promotes Nociception and Enhanced Cytokine Expression.

AIMS: To test the hypothesis that prolonged jaw opening, as can occur during routine dental procedures, increases nociceptive sensitivity of the masseter muscle and increases cytokine expression. METHODS: Sprague-Dawley rats were used to investigate behavioral and cellular changes in response to prolonged jaw opening. A surgical retractor was placed around the maxillary and mandibular incisors, and the jaw was held at near maximal opening for 20 minutes. Head-withdrawal responses to mechanical stimuli applied to the facial skin overlying the left and right masseter muscles were determined following jaw opening. Cytokine levels in the upper cervical spinal cord containing the caudal part of the spinal trigeminal nucleus were evaluated using protein antibody microarrays (n = 3). Statistical analysis was performed using a nonparametric Mann-Whitney U test. RESULTS: Prolonged jaw opening significantly increased nocifensive head withdrawal to mechanical stimuli at 2 hours, and days 3 and 7 postinduction (P < .05). The increase in nociceptive response resolved after 14 days. Sustained jaw opening also stimulated differential cytokine expression in the trigeminal ganglion and upper cervical spinal cord that persisted 14 days postprocedure (P < .05). CONCLUSION: These findings provide evidence that near maximal jaw opening can lead to activation and prolonged sensitization of trigeminal neurons that results in nociceptive behavior evoked by stimulation of the masseter muscle, a physiologic event often associated with temporomandibular disorders (TMD). Results from this study may provide a plausible explanation for why some patients develop TMD after routine dental procedures that involve prolonged jaw opening.

Validation of a novel rat-holding device for studying heat- and mechanical-evoked trigeminal nocifensive behavioral responses.

AIMS: To test the reliability and validity of a novel rat-holding device designed to be used in conjunction with the plantar test apparatus for studying nocifensive behavioral responses in an established model of temporomandibular joint (TMJ) pathology. METHODS: Thirty-five young adult male Sprague-Dawley rats were used. Withdrawal latencies in response to infrared 40 heat stimulation of the submandibular region in naïve animals (n = 4) and animals injected with saline or complete Freund's adjuvant (CFA) in the TMJ (n > 9) were measured over a 2-week time period. Nocifensive responses to mechanical stimulation of the cutaneous tissue directly over the TMJ with von Frey filaments were investigated in animals injected with CFA in the TMJ (n = 6). The effect on nocifensive responses to heat and mechanical stimulation of subcutaneous administration of buprenorphine (0.05 mg/kg) into the hindquarter was assessed in CFA and cotreated animals (n = 6). Statistical analysis was performed using a nonparametric Mann-Whitney U test. RESULTS: Under basal conditions, withdrawal latencies to heat stimulation of the orofacial region remained consistently around 15 seconds over 14 days. Unilateral CFA injection in the TMJ significantly decreased heat-withdrawal latencies on days 1, 2, 7, and 14 in the ipsilateral side (P < .05), but not contralateral side, when compared with basal values. CFA also significantly decreased the nocifensive threshold to mechanical stimulation on days 1, 2, and 7 postinjection (P < .05). CFA-mediated changes in heat withdrawal and mechanical thresholds in the orofacial region were significantly suppressed by subcutaneous administration of buprenorphine into the hindquarter (P < .05). CONCLUSION: Findings from this study provide evidence to validate the use of this holding device for studying nocifensive behaviors in the orofacial region of rats in response to heat or mechanical orofacial stimulation.