Dietary supplementation with grape seed extract from Vitus vinifera prevents suppression of GABAergic protein expression in female Sprague Dawley trigeminal ganglion in a model of chronic temporomandibular joint disorder.

OBJECTIVE: To investigate cellular changes in protein expression in the trigeminal ganglion in an established preclinical chronic model of temporomandibular joint disorder (TMD) in response to grape seed extract (GSE) supplementation based on its beneficial use in preclinical chronic orofacial pain models. DESIGN: Three experimental conditions included female Sprague-Dawley rats as naïve controls, and animals subjected to neck muscle inflammation and prolonged jaw opening with and without daily supplementation of GSE in the drinking water prior to inflammation. Changes were evaluated in mechanical sensitivity to von Frey filaments and protein expression in the trigeminal ganglion of animals 14 days post jaw opening. RESULTS: Calcitonin-gene related peptide and protein kinase A, proteins positively associated with peripheral sensitization and enhanced nociception, did not show elevated expression at day 14 in the model compared to naïve or GSE supplemented animals. However, neuronal levels of glutamate decarboxylase (GAD) 65/67, which are enzymes responsible for the synthesis of the inhibitory neurotransmitter GABA that functions to suppress neuronal excitability, were significantly decreased on day 14 post jaw opening. Similarly, a significant decrease in neuronal expression of the GABA receptor subunits GABAB1 and GABAB2, but not GABAA, was observed in the TMD model. Importantly, GSE prevented suppression of GAD 65/67 and GABAB subunits, maintaining levels similar to naïve animals. CONCLUSION: Results from our study provide evidence of the downregulation of inhibitory GABAergic proteins in trigeminal ganglion neurons in a preclinical chronic TMD model and the benefits of GSE supplementation in preventing their suppression and maintaining normal levels.

Benefit of Dietary Supplementation of Nutraceuticals as an Integrative Approach for Management of Migraine: Evidence From Preclinical and Clinical Studies.

PURPOSE OF REVIEW: To provide information from preclinical and clinical studies on the biological activity and health benefits of dietary inclusion of nutraceuticals as a safe, effective, non-pharmacological approach for the treatment of migraine. RECENT FINDINGS: There is emerging evidence of the therapeutic benefit of nutraceuticals to inhibit oxidative stress, suppress inflammation, and prevent changes in the normal gut microbiome, which are implicated in migraine pathology. Nutraceuticals can be enriched in polyphenols, which act as molecular scavengers to reduce the harmful effects of reactive oxygen species and phytosterols that suppress inflammation. Nutraceuticals also function to inhibit dysbiosis and to maintain the commensal intestinal bacteria that produce anti-inflammatory molecules including short-chain fatty acids that can act systemically to maintain a healthy nervous system. Dietary inclusion of nutraceuticals that exhibit antioxidant, anti-inflammatory, and anti-nociceptive properties and maintain the gut microbiota provides a complementary and integrative therapeutic strategy for migraine.

Method for cryopreservation of trigeminal ganglion for establishing primary cultures of neurons and glia.

BACKGROUND: Primary neuronal cultures are used to elucidate cellular and molecular mechanisms involved in disease pathology and modulation by pharmaceuticals and nutraceuticals, and to identify novel therapeutic targets. However, preparation of primary neuronal cultures from rodent embryos is labor-intensive, and it can be difficult to produce high-quality consistent cultures. To overcome these issues, cryopreservation can be used to obtain standardized, high-quality stocks of neuronal cultures. NEW METHOD: In this study, we present a simplified cryopreservation method for rodent primary trigeminal ganglion neurons and glia from Sprague-Dawley neonates, using a 90:10 (v/v) fetal bovine serum/dimethyl sulfoxide cell freezing medium. RESULTS: Cryopreserved trigeminal ganglion cells stored for up to one year in liquid nitrogen exhibited similar neuronal and glial cell morphology to fresh cultures and retained high cell viability. Proteins implicated in inflammation and pain signaling were expressed in agreement with the reported subcellular localization. Additionally, both neurons and glial cells exhibited an increase in intracellular calcium levels in response to a depolarizing stimulus. Cryopreserved cells were also transiently transfected with reporter genes. COMPARISON WITH EXISTING METHODS: Our method is simple, does not require special reagents or equipment, will save time and money, increase flexibility in study design, and produce consistent cultures. CONCLUSIONS: This method for the preparation and cryopreservation of trigeminal ganglia results in primary cultures of neurons and glia similar in viability and morphology to fresh preparations that could be utilized for biochemical, cellular, and molecular studies, increase reproducibility, and save laboratory resources.

CGRP, Migraine, and Brain MRI in CADASIL: A Pilot Study.

BACKGROUND: Migraine is associated with neuroimaging differences in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). However, it is unknown if migraine-related disability (MRD) or if calcitonin gene-related peptide (CGRP), a vasoactive peptide important in migraine pathology, have radiographic implications. The aims of this study were to identify whether MRD or interictal serum CGRP levels impacted neuroimaging findings for those with CADASIL. MATERIALS AND METHODS: A cross-sectional analysis was performed. The primary outcomes were neuroimaging differences associated with MRD among those with migraine or interictal serum CGRP levels of those with and without migraine. MRD was defined by 2 migraine disability scales (Migraine Disability Assessment, Headache Impact Test-6). Retrospective brain magnetic resonance imaging was reviewed (average 1.7 ± 2.0 y before enrollment). Rank-sum and χ 2 tests were used. RESULTS: Those with migraine (n=25, vs. n=14 without) were younger [median 49 (25 to 82) y vs. 60 (31 to 82) y, P <0.007], had fewer cerebral microbleeds (0 to 31 vs. 0 to 50, P =0.02) and less frequently had anterior temporal lobe T2 hyperintensities [68% (17/25) vs 100% (14/14), P =0.02]. MRD scale outcomes had no significant radiographic associations. Interictal serum CGRP did not differ (migraine: n=18, 27.0±9.6 pg/mL vs. no migraine: n=10, 26.8±15.7 pg/mL, P =0.965). CONCLUSIONS: Migraine may forestall microangiopathy in CADASIL, though possibly independent of severity as measured by MRD. Interictal serum CGRP did not differ in our cohort suggesting CGRP may not be vital to migraine pathophysiology in CADASIL. Larger studies are needed to account for age differences.

Grape seed extract suppresses calcitonin gene-related peptide secretion and upregulates expression of GAD 65/67 and GABAB receptor in primary trigeminal ganglion cultures.

The trigeminal ganglion is implicated in the underlying pathology of migraine and temporomandibular joint disorders (TMD), which are orofacial pain conditions involving peripheral and central sensitization. The neuropeptide calcitonin gene-related peptide (CGRP) is synthesized in some trigeminal ganglion neurons, and its release promotes inflammation, peripheral and central sensitization, and pain signaling. Recent studies in preclinical migraine and TMD models provide evidence that dietary supplementation with grape seed extract (GSE) inhibits trigeminal pain signaling. The goal of this study was to investigate the cellular mechanisms by which GSE modulates primary trigeminal ganglion cultures. The effect of GSE on CGRP secretion was determined by radioimmunoassay. To determine if GSE effects involved modulation of CGRP or the GABAergic system, expression of CGRP, GAD 65 and 67, GABAA receptor, and GABAB1 and GABAB2 receptor subunits were investigated by immunocytochemistry. GSE significantly inhibited basal CGRP secretion but did not alter neuronal CGRP expression. GAD 65 and 67 expression levels in neurons were significantly increased in response to GSE. While GSE did not cause a change in the neuronal expression of GABAA, GSE significantly increased GABAB1 expression in neurons, satellite glial cells, and Schwann cells. GABAB2 expression was significantly elevated in satellite glia and Schwann cells. These findings support the notion that GSE inhibition of basal CGRP secretion involves increased neuronal GAD 65 and 67 and GABAB receptor expression. GSE repression of CGRP release coupled with increased GABAB1 and GABAB2 glial cell expression would be neuroprotective by suppressing neuronal and glial excitability in the trigeminal ganglion.

Inhibition of Nociception in a Preclinical Episodic Migraine Model by Dietary Supplementation of Grape Seed Extract Involves Activation of Endocannabinoid Receptors.

Migraine is associated with peripheral and central sensitization of the trigeminal system and dysfunction of descending pain modulation pathways. Recently, dietary inclusion of grape seed extract (GSE) was shown to inhibit mechanical nociception in a preclinical model of chronic temporomandibular joint disorder, a condition often comorbid with migraine, with the antinociceptive effect mediated, in part, by activation of 5-HT3/7 and GABAB receptors. This study further investigated the mechanisms by which GSE inhibits mechanical nociception in a preclinical model of episodic migraine. Hyperalgesic priming of female and male Sprague Dawley rats was induced by three consecutive daily two-hour episodes of restraint stress. Seven days after the final restraint stress, rats were exposed to pungent odors from an oil extract that contains the compound umbellulone, which stimulates CGRP release and induces migraine-like pain. Some animals received dietary supplementation of GSE in their drinking water beginning one week prior to restraint stress. Changes in mechanical sensitivity in the orofacial region and hindpaw were determined using von Frey filaments. To investigate the role of the endocannabinoid receptors in the effect of GSE, some animals were injected intracisternally with the CB1 antagonist AM 251 or the CB2 antagonist AM 630 prior to odor inhalation. Changes in CGRP expression in the spinal trigeminal nucleus (STN) in response to stress, odor and GSE supplementation were studied using immunohistochemistry. Exposure of stress-primed animals to the odor caused a significant increase in the average number of withdrawal responses to mechanical stimulation in both the orofacial region and hindpaw, and the effect was significantly suppressed by daily supplementation with GSE. The anti-nociceptive effect of GSE was inhibited by intracisternal administration of antagonists of CB1 and CB2 receptors. GSE supplementation inhibited odor-mediated stimulation of CGRP expression in the STN in sensitized animals. These results demonstrate that GSE supplementation inhibits trigeminal pain signaling in an injury-free model of migraine-like pain via activation of endocannabinoid receptors and repression of CGRP expression centrally. Hence, we propose that GSE may be beneficial as a complementary migraine therapeutic.

5-HT3/7 and GABAB receptors mediate inhibition of trigeminal nociception by dietary supplementation of grape seed extract.

Background: Temporomandibular joint disorder is a prevalent orofacial pain condition involving sensitization and activation of trigeminal nociceptive neurons. Dietary supplementation with a proanthocyanin-enriched grape seed extract (GSE) was found to inhibit trigeminal nociception in a chronic TMD model. In this study, the cellular mechanisms by which GSE inhibits sustained trigeminal nociception in male and female Sprague Dawley rats were investigated.Methods: Some animals were supplemented with 0.5% GSE dissolved in their water one week prior to neck muscle inflammation induced by injection of complete Freund's adjuvant into the trapezius. To investigate the mechanism of GSE, some animals were injected intracisternally with antagonists of 5-HT3, 5-HT7, GABAA, or GABAB, receptor prior to jaw opening.Results: In males and females, trapezius inflammation prior to jaw opening resulted in sustained mechanical hypersensitivity of trigeminal nociceptors that was significantly inhibited by GSE. Further, GSE beginning 14 days post jaw opening also inhibited trigeminal nociception. Intracisternal injection of antagonists of the 5-HT3/7 and GABAB, but not GABAA receptors reduced the anti-nocifensive effect of GSE in both sexes. Neuronal expression of GABAB protein and mRNA in the spinal cord and trigeminal ganglion were detected.Conclusions: The inhibitory effect of GSE is mediated via activation of 5-HT3/7 receptors and GABAB to enhance central descending inhibitory pain pathways and suppress ongoing trigeminal nociception. Further, our findings support the use of GSE as a dietary supplement in the management of pain associated with TMD and other orofacial pain conditions involving central sensitization and dysfunction of descending pain modulation.

Hypervigilance, Allostatic Load, and Migraine Prevention: Antibodies to CGRP or Receptor.

Migraine involves brain hypersensitivity with episodic dysfunction triggered by behavioral or physiological stressors. During an acute migraine attack the trigeminal nerve is activated (peripheral sensitization). This leads to central sensitization with activation of the central pathways including the trigeminal nucleus caudalis, the trigemino-thalamic tract, and the thalamus. In episodic migraine the sensitization process ends with the individual act, but with chronic migraine central sensitization may continue interictally. Increased allostatic load, the consequence of chronic, repeated exposure to stressors, leads to central sensitization, lowering the threshold for future neuronal activation (hypervigilance). Ostensibly innocuous stressors are then sufficient to trigger an attack. Medications that reduce sensitization may help patients who are hypervigilant and help to balance allostatic load. Acute treatments and drugs for migraine prevention have traditionally been used to reduce attack duration and frequency. However, since many patients do not fully respond, an unmet treatment need remains. Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide involved in nociception and in the sensitization of peripheral and central neurons of the trigeminovascular system, which is implicated in migraine pathophysiology. Elevated CGRP levels are associated with dysregulated signaling in the trigeminovascular system, leading to maladaptive responses to behavioral or physiological stressors. CGRP may, therefore, play a key role in the underlying pathophysiology of migraine. Increased understanding of the role of CGRP in migraine led to the development of small-molecule antagonists (gepants) and monoclonal antibodies (mAbs) that target either CGRP or the receptor (CGRP-R) to restore homeostasis, reducing the frequency, duration, and severity of attacks. In clinical trials, US Food and Drug Administration-approved anti-CGRP-R/CGRP mAbs were well tolerated and effective as preventive migraine treatments. Here, we explore the role of CGRP in migraine pathophysiology and the use of gepants or mAbs to suppress CGRP-R signaling via inhibition of the CGRP ligand or receptor.

Migraine is a neurological disease affecting one in eight people. Symptoms include nausea and/or sensitivity to light and sound, and a throbbing headache. Although certain genes may increase the likelihood of migraine, environmental stimuli and molecules that increase the sensitivity of brain blood vessels and their innervations also play a role. During a migraine attack, nerves in the brain are activated, leading to increased sensitivity to stimuli, lowering the future threshold for activation, and making patients hypervigilant. Chronic, repeated exposure to certain stimuli can also lower this activation threshold, such that relatively innocuous stimuli can trigger an attack. Excessive use of certain migraine treatments can increase headache frequency over time and produce unwanted side effects; thus, selective agents are needed that specifically target the systems and pathways affected in migraine pathophysiology. Calcitonin gene-related peptide (CGRP), produced and released by nerve cells, is important in migraine pathophysiology. CGRP binds smooth muscle cell receptors, dilating blood vessels supplying the brain, and also binds to peripheral nerve cells that transmit pain signals to the spinal cord and brain. CGRP levels are elevated during a migraine attack. Medications targeting the CGRP pathway may decrease sensitivity and potentially normalize responses in hypervigilant patients. Two commercially available oral drugs that block CGRP receptors ('gepants') have reduced symptoms during migraine attacks in clinical trials. Four monoclonal antibodies (proteins that bind a specific molecule) have also been developed that target CGRP or the receptor and have been shown to significantly reduce the number of migraine days per month. Role of CGRP in Migraine.

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.

Dietary supplementation with grape seed extract prevents development of trigeminal sensitization and inhibits pain signaling in a preclinical chronic temporomandibular disorder model.

BACKGROUND: The risk factors neck muscle tension, prolonged jaw opening, and female gender are associated with developing temporomandibular disorders (TMD), which are characterized by persistent sensitization of trigeminal neurons and enhanced pain signaling. Dietary supplementation with a grape seed extract (GSE) can modulate expression of proteins that decrease neuronal excitability and trigeminal sensitization. METHODS: Mechanical nocifensive thresholds over the masseter were determined using von Frey filaments in male and female adult Sprague Dawley rats. To promote trigeminal sensitization, animals were injected with complete Freund's adjuvant in the upper trapezius. After 8 days, animals were subjected to near maximal jaw opening and head withdrawal responses were determined for 28 days. Some animals received continuous supplementation with 0.5% GSE in their drinking water two weeks prior to trapezius injections. RESULTS: Prolonged jaw opening increased the average number of nocifensive responses to mechanical stimuli for 14 days in males and females. However, trapezius inflammation prior to jaw opening promoted persistent mechanical sensitivity up to 28 days post-jaw opening in females, while in males nociceptive levels were still elevated at day 21. Supplementation with GSE, which is enriched in polyphenols and exhibits antioxidant and COX-2 activity, inhibited trigeminal nociception in response to jaw opening in both male and female sensitized animals. CONCLUSIONS: Our findings provide evidence that multiple risk factors contribute to the development of a prolonged state of trigeminal sensitization that is more severe in females and provide preclinical evidence that supplementation with GSE could be beneficial in the management of TMD.

Neuroprotective Effect of Enriched Chicken Bone Broth as a Dietary Supplement in a Model of Migraine Mediated by Early Life Stress.

Early life stress is a risk factor for development of migraine, a prevalent painful neurological disease characterized by sensitization and activation of trigeminal neurons. Secondary early life stress was previously shown to cause increased expression of neuronal proteins implicated in peripheral and central sensitization. Recently, dietary supplementation of chicken bone broth was shown to attenuate trigeminal nociception in an orofacial pain model. Accordingly, the focus of this study was to determine the effects of early life stress and dietary inclusion of bone broth on trigeminal nociceptor sensitization and activation in a model of episodic migraine. Adult Sprague-Dawley male sender rats subjected to primary traumatic stress were placed next to breeding or pregnant female rats that served as receiver rats (secondary traumatic stress) and in proximity to the offspring until weaning. Unstressed and stressed young adult offspring were tested for mechanical nocifensive response after exposure to a pungent odor known to be a migraine trigger, and in response to daily supplementation of bone broth. Early life stress promoted a primed state of trigeminal nociceptors that were activated by the pungent odor in both genders. Female animals exhibited a higher basal sensitization level and prolonged nociception compared with males. Supplementation of bone broth beginning at the time of weaning inhibited basal and triggered trigeminal mechanical sensitivity. Early life stress caused development of a sensitized trigeminal system that is implicated in migraine pathology and dietary supplementation with bone broth suppressed trigeminal sensitization, and thus may provide neuroprotective activity for reducing migraine risk.

Inhibition of Trigeminal Nociception by Non-invasive Vagus Nerve Stimulation: Investigating the Role of GABAergic and Serotonergic Pathways in a Model of Episodic Migraine.

Migraine is a prevalent neurological disease that is characterized by unpredictable episodic attacks of intense head pain. The underlying pathology involves sensitization and activation of the trigeminal system. Although non-invasive vagus nerve stimulation (nVNS) is recommended for the treatment of migraine, the abortive mechanism of action is not well-understood. The goal of this study was to compare the ability of nVNS and sumatriptan to inhibit trigeminal activation in two animal models of episodic migraine and to investigate the receptor mechanism of action of nVNS. Nocifensive head withdrawal response was investigated in adult male Sprague Dawley rats using von Frey filaments. To induce trigeminal nociceptor sensitization, complete Freund's adjuvant was injected in the trapezius muscle and trigeminal neurons were activated by exposure to a pungent odor or injection of the nitric oxide donor sodium nitroprusside. Some animals received nVNS or sumatriptan as treatment. Some animals were injected intracisternally with antagonists of GABAA, 5-HT3 or 5-HT7 receptors prior to nVNS since these receptors are implicated in descending modulation. While unsensitized animals exposed to the pungent odor or nitric oxide alone did not exhibit enhanced mechanical nociception, sensitized animals with neck muscle inflammation displayed increased trigeminal nocifensive responses. The enhanced nociceptive response to both stimuli was attenuated by nVNS and sumatriptan. Administration of antagonists of GABAA, 5-HT3, and 5-HT7 receptors in the upper spinal cord suppressed the anti-nocifensive effect of nVNS. Our findings suggest that nVNS inhibits trigeminal activation to a similar degree as sumatriptan in episodic migraine models via involvement of GABAergic and serotonergic signaling to enhance central 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.

Diverse Physiological Roles of Calcitonin Gene-Related Peptide in Migraine Pathology: Modulation of Neuronal-Glial-Immune Cells to Promote Peripheral and Central Sensitization.

The neuropeptide calcitonin gene-related peptide (CGRP) is implicated in the underlying pathology of migraine by promoting the development of a sensitized state of primary and secondary nociceptive neurons. The ability of CGRP to initiate and maintain peripheral and central sensitization is mediated by modulation of neuronal, glial, and immune cells in the trigeminal nociceptive signaling pathway. There is accumulating evidence to support a key role of CGRP in promoting cross excitation within the trigeminal ganglion that may help to explain the high co-morbidity of migraine with rhinosinusitis and temporomandibular joint disorder. In addition, there is emerging evidence that CGRP facilitates and sustains a hyperresponsive neuronal state in migraineurs mediated by reported risk factors such as stress and anxiety. In this review, the significant role of CGRP as a modulator of the trigeminal system will be discussed to provide a better understanding of the underlying pathology associated with the migraine phenotype.

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.

The Role of Salivary Neuropeptides in Pediatrics: Potential Biomarkers for Integrated Therapies.

INTRODUCTION: Objective measures of symptom response to integrated complementary approaches in pediatrics are evolving. The purpose of this study was to document the concentration range of salivary neuropeptides in healthy controls and in children with cancer, to explore correlations between serum and salivary measurements for Calcitonin Gene-Related Peptide (CGRP) and Vasoactive Intestinal Polypeptide (VIP), and to determine whether there is a change in these salivary neuropeptide levels in response to integrated mind-body therapies. METHODS: A non-randomized pragmatic study with three phases: Phase 1- Healthy Control Saliva-10 healthy controls provided saliva samples; Phase 2- Cancer Diagnosis Serum-Saliva- 16 mixed-type cancer patients provided blood and saliva samples; Phase 3- Acute Lymphocytic Leukemia (ALL) Saliva Intervention- 12 patients with ALL provided pre- and post-complementary intervention saliva samples. INTERVENTIONS: 20-minutes of structured touch or scripted relaxation breathing were administered to patients in Phase 3; Phase 1 and 2 patients did not receive this intervention. OUTCOME MEASURES: cortisol, CGRP, VIP, State/Trait Anxiety Scale, visual analogue scale, vital signs. RESULTS: Salivary CGRP and VIP were similar for children in Phases 1 and 2. There was a correlation between serum and salivary VIP in the mixed cancer group, though not between serum and salivary CGRP. In Phase 3 children, following a complementary intervention, salivary CGRP, heart rate, and systolic blood pressure decreased. DISCUSSION/CONCLUSIONS: These data provide evidence of a decrease in sympathetic output after integrative/complementary therapy intervention in children with cancer. The study underscores the potential role of salivary neuropeptides as non-invasive biomarkers for integrated therapies in pediatrics.