Trigeminal Nociceptive Neurons Research Articles

Effect of theanine on the hyperexcitability of trigeminal secondary nociceptive neurons following orofacial inflammation in rats.

The present in vivo study investigated whether systemic administration of theanine attenuates the inflammation-induced hyperexcitability of trigeminal spinal nucleus caudalis (SpVc) neurons associated with hyperalgesia. Complete Freund's adjuvant (CFA) was injected into the whisker pads of 24 rats to induce inflammation, and then mechanical stimulation was applied to the orofacial area to assess the threshold of escape. The mechanical threshold was statistically significantly lower in CFA-inflamed rats compared to uninjected naïve rats, and this lowered threshold returned to control levels after 2 days of theanine administration. The mean discharge frequency of SpVc wide-dynamic range (WDR) neurons to mechanical stimuli in anesthetized CFA-inflamed rats was statistically significantly lower after two days of theanine administration. In addition, the increased mean spontaneous discharge of SpVc WDR neurons in CFA-inflamed rats statistically significantly decreased after theanine administration. Similarly, theanine restored the expanded mean receptive field size in CFA-inflamed rats to control levels. Taken together, these results suggest that administration of theanine attenuates inflammatory hyperalgesia associated with hyperexcitability of nociceptive SpVc WDR neurons. These findings support the potential of theanine as a therapeutic agent in complementary alternative medicine strategies to prevent inflammatory hyperalgesia.

Meningeal P2X7 Signaling Mediates Migraine-Related Intracranial Mechanical Hypersensitivity.

Cortical spreading depolarization (CSD) is a key pathophysiological event that underlies visual and sensory auras in migraine. CSD is also thought to drive the headache phase in migraine by promoting the activation and mechanical sensitization of trigeminal primary afferent nociceptive neurons that innervate the cranial meninges. The factors underlying meningeal nociception in the wake of CSD remain poorly understood but potentially involve the parenchymal release of algesic mediators and damage-associated molecular patterns, particularly ATP. Here, we explored the role of ATP-P2X purinergic receptor signaling in mediating CSD-evoked meningeal afferent activation and mechanical sensitization. Male rats were subjected to a single CSD episode. In vivo, extracellular single-unit recording was used to measure meningeal afferent ongoing activity changes. Quantitative mechanical stimuli using a servomotor force-controlled stimulator assessed changes in the afferent's mechanosensitivity. Manipulation of meningeal P2X receptors was achieved via local administration of pharmacological agents. Broad-spectrum P2X receptor inhibition, selective blockade of the P2X7 receptor, and its related Pannexin 1 channel suppressed CSD-evoked afferent mechanical sensitization but did not affect the accompanying afferent activation response. Surprisingly, inhibition of the pronociceptive P2X2/3 receptor did not affect the activation or sensitization of meningeal afferents post-CSD. P2X7 signaling underlying afferent mechanosensitization was localized to the meninges and did not affect CSD susceptibility. We propose that meningeal P2X7 and Pannexin 1 signaling, potentially in meningeal macrophages or neutrophils, mediates the mechanical sensitization of meningeal afferents, which contributes to migraine pain by exacerbating the headache during normally innocuous physical activities.SIGNIFICANCE STATEMENT Activation and sensitization of meningeal afferents play a key role in migraine headache, but the underlying mechanisms remain unclear. Here, using a rat model of migraine with aura involving cortical spreading depolarization (CSD), we demonstrate that meningeal purinergic P2X7 signaling and its related Pannexin 1 pore, but not nociceptive P2X2/3 receptors, mediate prolonged meningeal afferent sensitization. Additionally, we show that meningeal P2X signaling does not contribute to the increased afferent ongoing activity in the wake of CSD. Our finding points to meningeal P2X7 signaling as a critical mechanism underlying meningeal nociception in migraine, the presence of distinct mechanisms underlying the activation and sensitization of meningeal afferents in migraine, and highlight the need to target both processes for effective migraine therapy.

Aura phenomena do not initiate migraine attacks-Findings from neuroimaging.

As cortical spreading depolarization (CSD) has been suggested to be the cause of migraine aura and as CSD can activate trigeminal nociceptive neurons in animals, it has been suggested that CSD may be the cause of migraine attacks. This raises the question of how migraine pain is generated in migraine attacks without aura and has led to the hypothesis that CSD may also occur in subcortical regions in the form of "silent" CSDs, and accordingly "silent auras". In this case study, we provide evidence for common neuronal alterations preceding headache attacks with and without aura in a male patient with migraine, who underwent daily event-correlated functional magnetic resonance imaging of trigeminal nociception for a period of 30 days. During these days the man experienced migraine attacks with and without aura. Comparing the preictal phases between both attack types revealed a common hyperactivation of the hypothalamus (p < 0.01), which was already present 2 days before the actual attack. The time frame of the central pathophysiological orchestration of migraine attacks, irrelevant of the presence of later aura, strongly suggests that the aura is an epiphenomenon that is unrelated and does not initiate headache attacks.

FM1-43 Dye Memorizes Piezo1 Activation in the Trigeminal Nociceptive System Implicated in Migraine Pain.

It has been proposed that mechanosensitive Piezo1 channels trigger migraine pain in trigeminal nociceptive neurons, but the mechanosensitivity of satellite glial cells (SGCs) supporting neuronal sensitization has not been tested before. Moreover, tools to monitor previous Piezo1 activation are not available. Therefore, by using live calcium imaging with Fluo-4 AM and labeling with FM1-43 dye, we explored a new strategy to identify Piezo channels' activity in mouse trigeminal neurons, SGCs, and isolated meninges. The specific Piezo1 agonist Yoda1 induced calcium transients in both neurons and SGCs, suggesting the functional expression of Piezo1 channels in both types of cells. In Piezo1-transfected HEK cells, FM1-43 produced only a transient fluorescent response, whereas co-application with Yoda1 provided higher transient signals and a remarkable long-lasting FM1-43 'tail response'. A similar Piezo1-related FM1-43 trapping was observed in neurons and SGCs. The non-specific Piezo channel blocker, Gadolinium, inhibited the transient peak, confirming the involvement of Piezo1 receptors. Finally, FM1-43 labeling demonstrated previous activity in meningeal tissues 3.5 h after Yoda1 washout. Our data indicated that trigeminal neurons and SGCs express functional Piezo channels, and their activation provides sustained labeling with FM1-43. This long-lasting labelling can be used to monitor the ongoing and previous activation of Piezo1 channels in the trigeminal nociceptive system, which is implicated in migraine pain.

KCl-induced repetitive cortical spreading depression inhibiting trigeminal neuronal firing is mediated by 5-HT1B/1D and opioid receptors.

We aimed to examine the effects of repetitive cortical spreading depression on the responses of nociceptive trigeminal neurons with dural afferents and characterize the role of 5-HT1B/1D and opioid receptors. Trigeminocervical complex neurons (n = 53) responsive to nociceptive activation of the dura mater were studied in rats using electrophysiological techniques. A sub-population (n = 32) showed an average inhibition of dural-evoked responses of 65 ± 14% from baseline with cortical spreading depression. This response was reversed by the selective 5-HT1B/1D receptor antagonist, GR127935 (3 mg/kg; n = 6, iv), and a non-selective opioid receptor antagonist, naloxone (1.5 mg/kg; n = 6, iv), five minutes after injection. To determine the role of the nucleus raphe magnus in the trigeminocervical complex inhibitory effect, microinjection of lidocaine (2%, n = 6) or muscimol (100 mM, n = 5) into the nucleus raphe magnus was performed. There was no effect on cortical spreading depression-induced inhibition of neuronal firing in trigeminocervical complex by either. The data demonstrate that repetitive cortical spreading depression inhibits a subpopulation of dural nociceptive trigeminocervical neurons, an effect mediated by serotonin and opioid receptors. This inhibition does not involve modulation of nucleus raphe magnus neurons.

Phytochemical quercetin alleviates hyperexcitability of trigeminal nociceptive neurons associated with inflammatory hyperalgesia comparable to NSAIDs.

Quercetin is a flavonoid that is widely found in fruits and vegetables. Quercetin inhibits cyclooxygenase-2 and modulates voltage-gated ion channels, however, its effect on nociceptive neuron-associated inflammatory hyperalgesia remains unknown. The present study investigated under in vivo conditions whether systemic administration of quercetin attenuates the inflammation-induced hyperexcitability of trigeminal spinal nucleus caudalis (SpVc) neurons associated with mechanical hyperalgesia and compared its effect to the non-steroidal anti-inflammatory drug, diclofenac. Complete Freund’s adjuvant was injected into the whisker pads of rats to induce inflammation, and then mechanical stimulation was applied to the orofacial area to assess the threshold of escape. The mechanical threshold was significantly lower in inflamed rats compared to uninjected naïve rats, and this lowered threshold returned to control levels 2 days after administration of quercetin or diclofenac. The mean discharge frequency of SpVc wide-dynamic range (WDR) neurons to both non-noxious and noxious mechanical stimuli in inflamed rats was significantly decreased after quercetin or diclofenac administration under combination of three anesthetic agents (medetomidine, midazolam and butorphanol). In addition, the increased mean spontaneous discharge of SpVc WDR neurons in inflamed rats significantly decreased after quercetin or diclofenac administration. Similarly, quercetin or diclofenac restored the expanded mean receptive field size in inflamed rats to control levels. In this study, the combination of three anesthetic agents did not result in any obvious “noxious pinch-evoked after discharges” in CFA inflamed day 2 rat as described previously in pentobarbital-anesthetized rats. Together, these results suggest that administration of quercetin attenuates inflammatory hyperalgesia associated with hyperexcitability of nociceptive SpVc WDR neurons via inhibition of the peripheral cyclooxygenase-2 signaling cascade and voltage-gated ion channels. These findings support the proposed potential of quercetin as a therapeutic agent in complementary alternative medicine strategies for preventing trigeminal inflammatory mechanical hyperalgesia.

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

ABSTRACT 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.

Electronic Cigarette Liquid Constituents Induce Nasal and Tracheal Sensory Irritation in Mice in Regionally Dependent Fashion.

IntroductionElectronic cigarettes (e-cigs) are currently used by millions of adults and adolescents worldwide. Major respiratory symptoms, such as coughing reported by e-cig users, including patients with e-cig, or vaping, product use-associated lung injury (EVALI), indicate e-cig constituent-induced sensory irritation. However, e-cig constituent-induced nociceptive activity in nasal and tracheal respiratory epithelia (RE) and neuronal activation in the trigeminal ganglia and brainstem nuclei, which receive airway chemosensory inputs have not been examined and compared. Comparisons of physiological responses between freebase nicotine and nicotine salts are also missing.Aims and MethodsEvent-related potential (ERP) was recorded electrophysiologically to assess mouse nasal and tracheal RE chemosensory responses to various flavorings, nicotine, including freebase and nicotine salts, e-liquid mixtures, and tussigenic stimuli. Also, mice were subjected to inhalation exposure to aerosol of a vanilla-flavored e-liquid or air (control), and the activated-trigeminal nociceptive neurons and brainstem neurons were examined using immunohistochemistry.ResultsIndividual constituents and mixtures of e-liquids, capsaicin, and citric and acetic acids evoked significantly larger ERP in the nose than in the trachea with the exception of menthol. ERP responses to freebase nicotine were significantly larger than protonated nicotine. Four nicotine salts (benzoate, lactate, levulinate, and salicylate) induced similar responses. Compared with air-exposed mice, e-liquid aerosol-exposed mice showed a significant increase in numbers of activated trigeminal nociceptive neurons and brainstem neurons in the spinal trigeminal nucleus, paratrigeminal nucleus, and nucleus tractus solitarius.ConclusionsE-liquid constituents region-dependently stimulate airway nociceptive chemosensory systems, and freebase nicotine is more potent than protonated nicotine.ImplicationsNeural abnormalities have been implicated in the development of nasal and respiratory illnesses. The higher sensitivity of the nasal nociceptive chemosensory system to nicotine and flavorings may indicate a health risk for e-liquid aerosol-induced upper airway illnesses via neurogenic alteration and warrants further investigation.

Searching for Predictors of Migraine Chronification: a Pilot Study of 1911A>G Polymorphism of TRPV1 Gene in Episodic Versus Chronic Migraine.

Transient receptor potential vanilloid type 1 (TRPV1) receptors activated by heat and capsaicin are expressed in trigeminal nociceptive neurons and implicated in the generation of migraine pain. Genetic studies suggested that single-nucleotide polymorphism (SNP) 1911A>G (rs8065080), leading to amino acid substitution Ile585Val, in the TRPV1 gene affects functional activity of TRPV1 receptors and is involved in different pain conditions. However, this polymorphism has not been tested in migraine patients. The objective of this pilot study was to investigate genetic factors of migraine susceptibility. We evaluated frequency distribution of AA, AG, and GG variants of SNP 1911A>G in the TRPV1 gene in patients with episodic and chronic migraine compared with healthy individuals. The study included 46 patients diagnosed with migraine (27 episodic and 19 chronic) and 50 healthy individuals as a control group. DNA from peripheral blood was used to test TRPV1 SNP using allele-specific PCR combined with gel electrophoresis. The genotype frequency distribution in episodic migraine was comparable with that in controls (AA 33%, AG 56%, GG 11% and AA 34%, AG 46%, GG 20%, respectively). On the contrary, in chronic migraine, the distribution differed significantly (p < 0.05) (AA 68%, AG 32%, GG 0%). This are first indications for a distinctive genotype frequency distribution of TRPV1 1911A>G in chronic migraine patients compared with episodic migraine patients and controls. Our data confirm a different predisposition to chronic pain in migraine and give a prerequisite for a new look at the nature of chronification of migraine, proposing that the absence of GG genotype may be considered as possible risk biomarker of episodic migraine evolution to chronic form.

Modulatory mechanism underlying how dietary constituents attenuate orofacial pain

Physiological pain protects the body and its systems from damage, but pathological pain has no obvious biological role. Complementary alternative medicine (CAM) agents are being increasingly studied in the treatment of clinical pain, and some dietary constituents (polyphenol, carotenoids, and fatty acids) and supplements may modify pain pathways. Because these substances modulate neuronal excitability-including the trigeminal pain pathway via various voltage-gated ionic channels and transient receptor potential and ligand-gated channels, dietary constituents could contribute to CAM as therapeutic agents for attenuating orofacial noxious sensory information. This review summarizes the current understanding of the mechanisms by which dietary constituents might attenuate excitability of trigeminal nociceptive neurons implicated in blocking pain, particularly in relation to the authors' recent experimental data, and discusses the development of functional foods and the contribution of dietary constituents in the relief of clinical dental pain without the side effects of nonsteroidal anti-inflammatory drugs.

Resolvin D1 suppresses inflammation-induced hyperexcitability of nociceptive trigeminal neurons associated with mechanical hyperalgesia

7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid (resolvin D1 [RvD1]) is biosynthesized from docosahexaenoic acid (DHA), and belongs to a novel family of lipid mediators showing remarkable anti-inflammatory effects; however, the effect of RvD1 on inflammation-induced hyperexcitability of nociceptive neurons under in vivo conditions remains to be determined. The present study, therefore, investigated whether under in vivo conditions, systemic administration of RvD1 could attenuate the inflammation-induced hyperexcitability of spinal trigeminal nucleus caudalis (SpVc) wide-dynamic range (WDR) neurons associated with hyperalgesia in rats.The threshold of escape from mechanical stimulation applied to the orofacial area in rats with complete Freund’s adjuvant-induced inflammation was significantly lower than in naïve rats. The lowered mechanical threshold in rats with inflammation was returned to control levels following administration of RvD1 (3 ng/kg, i.p.) for 3 days. The mean discharge frequency of SpVc WDR neurons in rats with inflammation was significantly decreased after RvD1 administration for both non-noxious and noxious mechanical stimuli. Increased spontaneous discharge of SpVc WDR neurons in rats with inflammation was also significantly decreased after RvD1 administration. Noxious pinch-evoked afterdischarge frequency and occurrence in rats with inflammation was significantly diminished after RvD1 administration. Expansion of the receptive field in rats with inflammation also returned to control levels after RvD1 administration.These results suggest that administration of RvD1 attenuates inflammation-induced hyperexcitability of SpVc WDR neurons associated with inflammatory hyperalgesia. These findings support the idea that RvD1, derived from DHA, as well as DHA itself, are potential complementary or alternative therapeutic agents for the alleviation of inflammatory hyperalgesia.

Dietary constituent genistein inhibits the hyperexcitability of trigeminal nociceptive neurons associated with mechanical hyperalgesia following orofacial inflammation

ObjectivesGenistein, a dietary constituent, modulates voltage-dependent and ligand-gated ionic channels, suggesting that it could also attenuate inflammatory hyperalgesia. However, the mechanism underlying how genistein affects inflammation-induced hyperexcitability of nociceptive neurons in vivo remains to be determined. The present study therefore investigated whether administration of genistein could attenuate the inflammation-induced hyperexcitability of trigeminal spinal nucleus caudalis (SpVc) neurons associated with mechanical hyperalgesia in vivo. MethodsInflammation was induced by injection of complete Freund's adjuvant into the whisker pad. The mechanical thresholds for escape behavior and electrophysiological single-unit recording of SpVc neurons responding to mechanical stimulation were then conducted in naïve rats, inflamed rats, and inflamed rats with genistein administered intraperitoneally. ResultsThe lowered mechanical threshold in the inflamed rats was returned to control level following administration of genistein for 2 days. The mean number of discharge frequencies of SpVc neurons in inflamed rats was significantly decreased after genistein administration with both non-noxious and noxious mechanical stimuli. The increased spontaneous discharges of SpVc neurons in inflamed rats were significantly decreased after genistein administration. Noxious pinch-evoked after-discharge frequency and occurrence in inflamed rats was also significantly diminished after genistein administration, and expansion of the receptive field was significantly returned to control levels in inflamed rats. ConclusionHerein, we present the first evidence that genistein attenuates hyperexcitability of SpVc neurons associated with inflammatory mechanical hyperalgesia. These findings suggest that genistein could be a potential therapeutic agent in complementary alternative medicine for the prevention of trigeminal inflammatory hyperalgesia.

Suppression of hyperexcitability of trigeminal nociceptive neurons associated with inflammatory hyperalgesia following systemic administration of lutein via inhibition of cyclooxygenase-2 cascade signaling

IntroductionLutein is a dietary constituent known to inhibit inflammation; however, its effect on nociceptive neuron-associated hyperalgesia remains to be determined. The present study therefore investigated under in vivo conditions whether administration of lutein attenuates the inflammation-induced hyperexcitability of trigeminal spinal nucleus caudalis (SpVc) neurons that is associated with mechanical hyperalgesia.ResultsComplete Freund’s adjuvant (CFA) was injected into the whisker pads of rats to induce inflammation, and then mechanical stimulation was applied to the orofacial area to assess the threshold of escape. The mechanical threshold was significantly lower in inflamed rats compared to uninjected naïve rats, and this lowered threshold was returned to control levels by 3 days after administration of lutein (10 mg/Kg, i.p.) Also the lutein administration, inflammation-induced thickness of edema was returned to control levels. The mean increased number of cyclooxygenase-2 (Cox-2)-immunoreactive cells in the whisker pads of inflamed rats was also returned to control levels by administration with lutein. The mean discharge frequency of SpVc wide-dynamic range (WDR) neurons to both nonnoxious and noxious mechanical stimuli in inflamed rats was significantly decreased after lutein administration. In addition, the increased mean spontaneous discharge of SpVc WDR in inflamed rats was significantly decreased after lutein administration. Similarly, lutein significantly diminished noxious pinch-evoked mean after discharge frequency and occurrence in inflamed rats. Finally, lutein restored the expanded mean size of the receptive field in inflamed rats to control levels.ConclusionThese results together suggest that administration of lutein attenuates inflammatory hyperalgesia associated with hyperexcitability of nociceptive SpVc WDR neurons via inhibition of the peripheral Cox-2 signaling cascade. These findings support the proposed potential of lutein as a therapeutic agent in complementary alternative medicine strategies for preventing inflammatory mechanical hyperalgesia.

Secondary traumatic stress increases expression of proteins implicated in peripheral and central sensitization of trigeminal neurons

The pathology of migraine, a common neurological disease, involves sensitization and activation of trigeminal nociceptive neurons to promote hyperalgesia and allodynia during an attack. Migraineurs often exhibit characteristics of a hyperexcitable or hypervigilant nervous system. One of the primary reported risk factors for development of a hyperexcitable trigeminal system is chronic, unmanaged stress and anxiety. While primary traumatic stress is a commonly cited risk factor for many pain conditions, exposure to secondary traumatic stress early in life is also thought to be a contributing risk factor. The goal of this study was to investigate cellular changes within the spinal trigeminal nucleus and trigeminal ganglion mediated by secondary traumatic stress. Male Sprague Dawley rats (sender) were subjected to forced swim testing (primary traumatic stress) and were then housed in close visual, olfactory, and auditory proximity to the breeding male and female rats, pregnant female rats, or female rats and their nursing offspring (all receivers). In response to secondary stress, levels of calcitonin gene-related peptide, active forms of the mitogen activated protein kinases ERK, JNK, and p38, and astrocyte expression of glial fibrillary acidic protein were significantly elevated in the spinal trigeminal nucleus in day 45 offspring when compared to naïve offspring. In addition, increased nuclear expression of ERK and p38 was observed in trigeminal ganglion neurons. Our results demonstrate that secondary traumatic stress promotes cellular events associated with prolonged trigeminal sensitization in the offspring, and provides a mechanism of how early life stress may function as a risk factor for migraine.

CD73 Controls Extracellular Adenosine Generation in the Trigeminal Nociceptive Nerves

Purinergic signaling is involved in pain generation and modulation in the nociceptive sensory nervous system. Adenosine triphosphate (ATP) induces pain via activation of ionotropic P2X receptors while adenosine mediates analgesia via activation of metabotropic P1 receptors. These purinergic signaling are determined by ecto-nucleotidases that control ATP degradation and adenosine generation. Using enzymatic histochemistry, we detected ecto-AMPase activity in dental pulp, trigeminal ganglia (TG) neurons, and their nerve fibers. Using immunofluorescence staining, we confirmed the expression of ecto-5′-nucleotidase (CD73) in trigeminal nociceptive neurons and their axonal fibers, including the nociceptive nerve fibers projecting into the brainstem. In addition, we detected the existence of CD73 and ecto-AMPase activity in the nociceptive lamina of the trigeminal subnucleus caudalis (TSNC) in the brainstem. Furthermore, we demonstrated that incubation with specific anti-CD73 serum significantly reduced the ecto-AMPase activity in the nociceptive lamina in the brainstem. Our results indicate that CD73 might participate in nociceptive modulation by affecting extracellular adenosine generation in the trigeminal nociceptive pathway. Disruption of TG neuronal ecto-nucleotidase expression and axonal terminal localization under certain circumstances such as chronic inflammation, oxidant stress, local constriction, and injury in trigeminal nerves may contribute to the pathogenesis of orofacial neuropathic pain.

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

ObjectiveElevated 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. DesignMale 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. ResultsTNF-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. ConclusionsOur 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.

Neuronal NTPDase3 Mediates Extracellular ATP Degradation in Trigeminal Nociceptive Pathway.

ATP induces pain via activation of purinergic receptors in nociceptive sensory nerves. ATP signaling is terminated by ATP hydrolysis mediated by cell surface-localized ecto-nucleotidases. Using enzymatic histochemical staining, we show that ecto-ATPase activity is present in mouse trigeminal nerves. Using immunofluorescence staining, we found that ecto-NTPDase3 is expressed in trigeminal nociceptive neurons and their projections to the brainstem. In addition, ecto-ATPase activity and ecto-NTPDase3 are also detected in the nociceptive outermost layer of the trigeminal subnucleus caudalis. Furthermore, we demonstrate that incubation with anti-NTPDase3 serum reduces extracellular ATP degradation in the nociceptive lamina of both the trigeminal subnucleus caudalis and the spinal cord dorsal horn. These results are consistent with neuronal NTPDase3 activity modulating pain signal transduction and transmission by affecting extracellular ATP hydrolysis within the trigeminal nociceptive pathway. Thus, disruption of trigeminal neuronal NTPDase3 expression and localization to presynaptic terminals during chronic inflammation, local constriction and injury may contribute to the pathogenesis of orofacial neuropathic pain.

ERK-GluR1 phosphorylation in trigeminal spinal subnucleus caudalis neurons is involved in pain associated with dry tongue.

Background Dry mouth is known to cause severe pain in the intraoral structures, and many dry mouth patients have been suffering from intraoral pain. In development of an appropriate treatment, it is crucial to study the mechanisms underlying intraoral pain associated with dry mouth, yet the detailed mechanisms are not fully understood. To evaluate the mechanisms underlying pain related to dry mouth, the dry-tongue rat model was developed. Hence, the mechanical or heat nocifensive reflex, the phosphorylated extracellular signal-regulated kinase and phosphorylated GluR1-IR immunohistochemistries, and the single neuronal activity were examined in the trigeminal spinal subnucleus caudalis of dry-tongue rats.Results The head-withdrawal reflex threshold to mechanical, but not heat, stimulation of the tongue was significantly decreased on day 7 after tongue drying. The mechanical, but not heat, responses of trigeminal spinal subnucleus caudalis nociceptive neurons were significantly enhanced in dry-tongue rats compared to sham rats on day 7. The number of phosphorylated extracellular signal-regulated kinase-immunoreactive cells was also significantly increased in the trigeminal spinal subnucleus caudalis following noxious stimulation of the tongue in dry-tongue rats compared to sham rats on day 7. The decrement of the mechanical head-withdrawal reflex threshold (HWT) was reversed during intracisternal administration of the mitogen-activated protein kinase kinase 1 inhibitor, PD98059. The trigeminal spinal subnucleus caudalis neuronal activities and the number of phosphorylated extracellular signal-regulated kinase-immunoreactive cells following noxious mechanical stimulation of dried tongue were also significantly decreased following intracisternal administration of PD98059 compared to vehicle-administrated rats. Increased number of the phosphorylated GluR1-IR cells was observed in the trigeminal spinal subnucleus caudalis of dry-tongue rats, and the number of phosphorylated GluR1-IR cells was significantly reduced in PD98059-administrated rats compared to the vehicle-administrated tongue-dry rats.Conclusions These findings suggest that the pERK-pGluR1 cascade is involved in central sensitization of trigeminal spinal subnucleus caudalis nociceptive neurons, thus resulting in tongue mechanical hyperalgesia associated with tongue drying.

Two TRPV1 receptor antagonists are effective in two different experimental models of migraine.

BackgroundThe capsaicin and heat responsive ion channel TRPV1 is expressed on trigeminal nociceptive neurons and has been implicated in the pathophysiology of migraine attacks. Here we investigate the efficacy of two TRPV1 channel antagonists in blocking trigeminal activation using two in vivo models of migraine.MethodsMale Sprague–Dawley rats were used to study the effects of the TRPV1 antagonists JNJ-38893777 and JNJ-17203212 on trigeminal activation. Expression of the immediate early gene c-fos was measured following intracisternal application of inflammatory soup. In a second model, CGRP release into the external jugular vein was determined following injection of capsaicin into the carotid artery.ResultsInflammatory up-regulation of c-fos in the trigeminal brain stem complex was dose-dependently and significantly reduced by both TRPV1 antagonists. Capsaicin-induced CGRP release was attenuated by JNJ-38893777 only in higher dosage. JNJ-17203212 was effective in all doses and fully abolished CGRP release in a time and dose-dependent manner.ConclusionOur results describe two TRPV1 antagonists that are effective in two in vivo models of migraine. These results suggest that TRPV1 may play a role in the pathophysiological mechanisms, which are relevant to migraine.