Glial interleukin-1ß upregulates neuronal sodium channel 1.7 in trigeminal ganglion contributing to temporomandibular joint inflammatory hypernociception in rats.

BACKGROUND: The proinflammatory cytokine interleukin-1ß (IL-1ß) drives pain by inducing the expression of inflammatory mediators; however, its ability to regulate sodium channel 1.7 (Nav1.7), a key driver of temporomandibular joint (TMJ) hypernociception, remains unknown. IL-1ß induces cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). We previously showed that PGE2 upregulated trigeminal ganglionic Nav1.7 expression. Satellite glial cells (SGCs) involve in inflammatory pain through glial cytokines. Therefore, we explored here in the trigeminal ganglion (TG) whether IL-1ß upregulated Nav1.7 expression and whether the IL-1ß located in the SGCs upregulated Nav1.7 expression in the neurons contributing to TMJ inflammatory hypernociception. METHODS: We treated rat TG explants with IL-1ß with or without inhibitors, including NS398 for COX-2, PF-04418948 for EP2, and H89 and PKI-(6-22)-amide for protein kinase A (PKA), or with adenylate cyclase agonist forskolin, and used real-time PCR, Western blot, and immunohistofluorescence to determine the expressions or locations of Nav1.7, COX-2, cAMP response element-binding protein (CREB) phosphorylation, and IL-1ß. We used chromatin immunoprecipitation to examine CREB binding to the Nav1.7 promoter. Finally, we microinjected IL-1ß into the TGs or injected complete Freund's adjuvant into TMJs with or without previous microinjection of fluorocitrate, an inhibitor of SGCs activation, into the TGs, and evaluated nociception and gene expressions. Differences between groups were examined by one-way analysis of variance (ANOVA) or independent samples t test. RESULTS: IL-1ß upregulated Nav1.7 mRNA and protein expressions in the TG explants, whereas NS398, PF-04418948, H89, or PKI-(6-22)-amide could all block this upregulation, and forskolin could also upregulate Nav1.7 mRNA and protein expressions. IL-1ß enhanced CREB binding to the Nav1.7 promoter. Microinjection of IL-1ß into the TGs or TMJ inflammation both induced hypernociception of TMJ region and correspondingly upregulated COX-2, phospho-CREB, and Nav1.7 expressions in the TGs. Moreover, microinjection of fluorocitrate into the TGs completely blocked TMJ inflammation-induced activation of SGCs and the upregulation of IL-1ß and COX-2 in the SGCs, and phospho-CREB and Nav1.7 in the neurons and alleviated inflammation-induced TMJ hypernociception. CONCLUSIONS: Glial IL-1ß upregulated neuronal Nav1.7 expression via the crosstalk between signaling pathways of the glial IL-1ß/COX-2/PGE2 and the neuronal EP2/PKA/CREB/Nav1.7 in TG contributing to TMJ inflammatory hypernociception.

Veratridine modifies the gating of human voltage-gated sodium channel Nav1.7.

Veratridine is a lipid-soluble neurotoxin derived from plants in the family Liliaceae. It has been broadly investigated for its action as a sodium channel agonist. However, the effects of veratridine on subtypes of sodium channels, especially Nav1.7, remain to be studied. Here, we investigated the effects of veratridine on human Nav1.7 ectopically expressed in HEK293A cells and recorded Nav1.7 currents from the cells using whole-cell patch clamp technique. We found that veratridine exerted a dose-dependent inhibitory effect on the peak current of Nav1.7, with the half-maximal inhibition concentration (IC50) of 18.39 µM. Meanwhile, veratridine also elicited tail current (linearly) and sustained current [half-maximal concentration (EC50): 9.53 µM], also in a dose-dependent manner. Veratridine (75 µM) shifted the half-maximal activation voltage of the Nav1.7 activation curve in the hyperpolarized direction, from -21.64 ± 0.75 mV to -28.14 ± 0.66 mV, and shifted the half-inactivation voltage of the steady-state inactivation curve from -59.39 ± 0.39 mV to -73.78 ± 0.5 mV. An increased frequency of stimulation decreased the peak and tail currents of Nav1.7 for each pulse along with pulse number, and increased the accumulated tail current at the end of train stimulation. These findings reveal the different modulatory effects of veratridine on the Nav1.7 peak current and tail current.

17ß-estradiol aggravates temporomandibular joint inflammation through the NF-κB pathway in ovariectomized rats.

OBJECTIVE: Women of childbearing age are more likely than men to experience temporomandibular disorders, with pain as the main symptom. Temporomandibular joint (TMJ) inflammation is believed to be a major reason for TMJ pain. Whether sex hormones are involved in the sexual dimorphism of TMJ inflammation and pain remains to be elucidated. The aim of this study was to examine whether estrogen affects TMJ inflammation and pain via the NF-κB pathway. METHODS: Female rats were divided into 6 groups: the control group, the sham-ovariectomized group, and 4 groups of ovariectomized rats treated with 17ß-estradiol at a dosage of 0 µg/day, 20 µg/day, 80 µg/day, and 200 µg/day, respectively, for 10 days and then injected intraarticularly with Freund's complete adjuvant to induce TMJ inflammation. The behavior-related and histologic effects of 17ß-estradiol were evaluated 24 hours after the induction of TMJ inflammation. NF-κB activity in the synovial membrane was examined by electrophoretic mobility shift assay. The expression of the NF-κB target genes tumor necrosis factor α, interleukin-1ß (IL-1ß), IL-6, cyclooxygenase 2, and inducible nitric oxide synthase in the synovial membrane was examined by real-time polymerase chain reaction. RESULTS: Treatment with estradiol potentiated TMJ inflammation in a dose-dependent manner and also exacerbated the inflammation-induced decrease in food intake. Correspondingly, estradiol potentiated the DNA-binding activity of NF-κB and the transcription of its target genes in the synovial membrane. Furthermore, the estrogen receptor antagonist ICI 182780 or the NF-κB inhibitor pyrrolidine dithiocarbamate partially blocked the effects of estradiol on TMJ inflammation and pain and the NF-κB pathway. CONCLUSION: These results suggest that estradiol aggravates TMJ inflammation through the NF-κB pathway, leading to the induction of proinflammatory cytokines.

Progesterone Attenuates Allodynia of Inflamed Temporomandibular Joint through Modulating Voltage-Gated Sodium Channel 1.7 in Trigeminal Ganglion.

Background: Women with temporomandibular disorders (TMDs) experience some amelioration of pain during pregnancy. Progesterone increases dramatically and steadily during pregnancy. Sodium channel 1.7 (Nav1.7) plays a prominent role in pain perceptions, as evidenced by deletion of Nav1.7 alone leading to a complete loss of pain. In a previous study, we showed that Nav1.7 in trigeminal ganglion (TG) is involved in allodynia of inflamed temporomandibular joint (TMJ). Whether progesterone modulates allodynia of inflamed TMJ through Nav1.7 in TG remains to be investigated. Methods: The effects of progesterone on sodium currents of freshly isolated TG neurons were examined using whole-cell recording. Female rats were ovariectomized and treated with increasing doses of progesterone for 10 days. Complete Freund's adjuvant was administered intra-articularly to induce TMJ inflammation. TMJ nociceptive responses were evaluated by head withdrawal thresholds. Real-time PCR and Western blotting were used to examine Nav1.7 mRNA and protein expression in TG. Immunohistofluorescence was used to examine the colocalization of progesterone receptors (PRα/ß) and Nav1.7 in TG. Results: Whole-cell recording showed that progesterone could attenuate sodium currents. Moreover, progesterone dose-dependently downregulated Nav1.7 mRNA expression and reduced the sensitivity of TMJ nociception in ovariectomized rats. Furthermore, treatment with progesterone attenuated allodynia of inflamed TMJ in a dose-dependent manner and repressed inflammation-induced Nav1.7 mRNA and protein expression in ovariectomized rats. The progesterone receptor antagonist, RU-486, partially reversed the effect of progesterone on allodynia of inflamed TMJ and TMJ inflammation-induced Nav1.7 mRNA and protein expression. Conclusion: Progesterone, by modulating trigeminal ganglionic Nav1.7, may represent a promising agent to prevent allodynia of inflamed TMJ.

Genistein Antagonizes 17ß-Estradiol Effects on Glutamate-Evoked Masseter Muscle Hypernociception in Rats.

Temporomandibular disorders (TMDs) predominantly affect women of reproductive ages, with pain as the main symptom. The aim of the present study was to examine the effects of 17ß-estradiol (E2) on glutamate-evoked hypernociception of masseter muscle and to examine whether genistein could antagonize the effects of E2 in female rats. Injection of glutamate into the masseter muscle dose-dependently decreased head withdrawal thresholds, a parameter for mechanical hypernociception. Head withdrawal thresholds in ovariectomized rats also decreased with increasing doses of E2 replacement, and were further aggravated by injection of glutamate (1M, 40µL) into the masseters. Genistein at doses of 7.5 and 15 mg/kg antagonized E2-induced hypernociception of masseter muscle, and at doses of 7.5, 15, and 30 mg/kg also antagonized E2 potentiation of glutamate-evoked hypernociception of masseter muscle. Genistein produced optimal antagonistic effects of E2 on nociception behavior at a dose of 15 mg/kg. On the molecular level, tyrosine phosphorylation of the NR2B subunit of the N-methyl-D-aspartate receptor (pNR2B) and phosphorylated mitogen-activated protein kinase (pERK1/2) were significantly upregulated in the hippocampus following glutamate injection and were further potentiated by E2 replacement. Genistein at dose of 15 mg/kg partially reversed E2-potentiated glutamate-evoked upregulation of pNR2B and pERK1/2 expression in the hippocampus. These results indicated that moderate doses of genistein could antagonize E2 enhanced glutamate-evoked hypernociception of masseter muscle possibly via N-methyl-D-aspartate receptor and ERK1/2 signaling pathways in the hippocampus.

Estradiol upregulates voltage-gated sodium channel 1.7 in trigeminal ganglion contributing to hyperalgesia of inflamed TMJ.

BACKGROUND: Temporomandibular disorders (TMDs) have the highest prevalence in women of reproductive age. The role of estrogen in TMDs and especially in TMDs related pain is not fully elucidated. Voltage-gated sodium channel 1.7 (Nav1.7) plays a prominent role in pain perception and Nav1.7 in trigeminal ganglion (TG) is involved in the hyperalgesia of inflamed Temporomandibular joint (TMJ). Whether estrogen could upregulate trigeminal ganglionic Nav1.7 expression to enhance hyperalgesia of inflamed TMJ remains to be explored. METHODS: Estrous cycle and plasma levels of 17ß-estradiol in female rats were evaluated with vaginal smear and enzyme linked immunosorbent assay, respectively. Female rats were ovariectomized and treated with 17ß-estradiol at 0 µg, 20 µg and 80 µg, respectively, for 10 days. TMJ inflammation was induced using complete Freund's adjuvant. Head withdrawal thresholds and food intake were measured to evaluate the TMJ nociceptive responses. The expression of Nav1.7 in TG was examined using real-time PCR and western blot. The activity of Nav1.7 promoter was examined using luciferase reporter assay. The locations of estrogen receptors (ERα and ERß), the G protein coupled estrogen receptor (GPR30), and Nav1.7 in TG were examined using immunohistofluorescence. RESULTS: Upregulation of Nav1.7 in TG and decrease in head withdrawal threshold were observed with the highest plasma 17ß-estradiol in the proestrus of female rats. Ovariectomized rats treated with 80 µg 17ß-estradiol showed upregulation of Nav1.7 in TG and decrease in head withdrawal threshold as compared with that of the control or ovariectomized rats treated with 0 µg or 20 µg. Moreover, 17ß-estradiol dose-dependently potentiated TMJ inflammation-induced upregulation of Nav1.7 in TG and also enhanced TMJ inflammation-induced decrease of head withdrawal threshold in ovariectomized rats. In addition, the estrogen receptor antagonist, ICI 182,780, partially blocked the 17ß-estradiol effect on Nav1.7 expression and head withdrawal threshold in ovariectomized rats. ERα and ERß, but not GPR30, were mostly co-localized with Nav1.7 in neurons in TG. In the nerve growth factor-induced and ERα-transfected PC12 cells, 17ß-estradiol dose-dependently enhanced Nav1.7 promoter activity, whereas mutations of the estrogen response element at -1269/-1282 and -1214/-1227 in the promoter completely abolished its effect on the promoter activity. CONCLUSION: Estradiol could upregulate trigeminal ganglionic Nav1.7 expression to contribute to hyperalgesia of inflamed TMJ.

Progesterone attenuates temporomandibular joint inflammation through inhibition of NF-κB pathway in ovariectomized rats.

Sex hormones may contribute to the symptomatology of female-predominant temporomandibular disorders (TMDs) inflammatory pain. Pregnant women show less symptoms of TMDs than that of non-pregnant women. Whether progesterone (P4), one of the dominant sex hormones that regulates multiple biological functions, is involved in symptoms of TMDs remains to be explored. Freund's complete adjuvant were used to induce joint inflammation. We evaluated the behavior-related and histologic effects of P4 and the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in the synovial membrane. Primary TMJ synoviocytes were treated with TNF-α or IL-1ß with the combination of P4. Progesterone receptor antagonist RU-486 were further applied. We found that P4 replacement attenuated TMJ inflammation and the nociceptive responses in a dose-dependent manner in the ovariectomized rats. Correspondingly, P4 diminished the DNA-binding activity of NF-κB and the transcription of its target genes in a dose-dependent manner in the synovial membrane of TMJ. Furthermore, P4 treatment showed decreased mRNA expression of proinflammatory cytokines, and partially reversed TNF-α and IL-1ß induced transcription of proinflammatory cytokines in the primary synoviocytes. Moreover, progesterone receptor antagonist RU-486 partially reversed the effects of P4 on NF-κB pathway. In conclusion, progesterone ameliorated TMJ inflammation through inhibition of NF-κB pathway.

Non-steroidal Anti-inflammatory Drugs Attenuate Hyperalgesia and Block Upregulation of Trigeminal Ganglionic Sodium Channel 1.7 after Induction of Temporomandibular Joint Inflammation in Rats.

OBJECTIVE: To investigate the association between the analgesic effect of non-steroidal antiinflammatory drugs (NSAIDs) and sodium channel 1.7 (Nav1.7) expression in the trigeminal ganglion (TG). METHODS: Temporomandibular joint (TMJ) inflammation was induced by complete Freund's adjuvant (CFA) in female rats. Ibuprofen, diclofenac sodium and meloxicam were given intragastrically before induction of TMJ inflammation. Histopathological evaluation and scoring of TMJ inflammation was used to evaluate the level of inflammation. The head withdrawal threshold and food intake were measured to evaluate TMJ nociceptive responses. The mRNA and protein expression of trigeminal ganglionic Nav1.7 was examined using real-time polymerase chain reaction and western blot. RESULTS: Twenty-four hours after the injection of CFA into the TMJs, NSAIDs attenuated hyperalgesia of inflamed TMJ and simultaneously blocked inflammation-induced upregulation of Nav1.7 mRNA and protein expression in the TG. However, ibuprofen and diclofenac sodium slightly attenuated TMJ inflammation and meloxicam did not affect TMJ inflammation. CONCLUSION: Attenuation of hyperalgesia of inflamed TMJ by NSAIDs might be associated with their role in blocking upregulation of trigeminal ganglionic Nav1.7.

A new hypothesis of sex-differences in temporomandibular disorders: estrogen enhances hyperalgesia of inflamed TMJ through modulating voltage-gated sodium channel 1.7 in trigeminal ganglion?

OBJECTIVE: Temporomandibular disorders (TMD) are an assorted set of clinical conditions characterized mainly by pain in the temporomandibular joint (TMJ). TMJ inflammation or synovitis is frequently observed in TMD patients and is the major reason for TMD pain. TMD is prevalent in women of childbearing age, at least twice than in men, implying that estrogen may be involved in TMD pain processing. Estrogen affects a cell mainly through the estrogen receptors (ER). The estrogen-ER complex binds to estrogen response element sequences (ERE) in the promoter region of specific genes and then exerts its regulatory potential. The voltage-gated sodium channel 1.7 (Nav1.7), whose single disruption leads to a complete loss of pain, amplifies weak stimuli in the neurons and acts as the threshold channel for firing action potentials and plays a prominent role in pain perception, including inflammatory pain. Furthermore, our previous study showed that trigeminal ganglionic Nav1.7 was involved in the hyperalgesia of the inflamed TMJ. We propose that estrogen may enhance hyperalgesia of inflamed TMJ through decrease nociceptive threshold of TMJ or inflamed TMJ by modulating both expression and channel threshold of Nav1.7 in trigeminal ganglion.

Stereotaxis of mandibular nerve initial point of trigeminal ganglion in rats.

OBJECTIVE: To acquire parameters for stereotaxis of the mandibular nerve initial point of the trigemenial ganglion (TG) and to test the accuracy of the acquired parameters for microinjection into the mandibular nerve initial point of TG in adult rats. METHODS: Sprague-Dawley rats (260-270 g) were mounted onto a stereotaxic frame. The bregma was set as an anchor point and the three-dimensional parameters between the mandibular nerve initial point of the bilateral TGs and the bregma were measured in 25 rats. The accuracy of these parameters was tested using microinjection of Evans blue dye into the mandibular nerve initial point of the bilateral TGs in 30 rats and the injection sites were evaluated by dissection. RESULTS: The three-dimensional parameters of the mandibular nerve initial point of the bilateral TGs were 3.5 ± 0.1 mm posterior and 3.6 ± 0.2 mm lateral to the bregma, and 12.0 ± 0.2 mm inferior to the skull surface. Accuracy for the microinjection of Evans blue dye into the mandibular nerve initial point of the bilateral TGs was 86.7% (52/60). CONCLUSION: The acquired parameters served well for stereotaxis and microinjection of reagents into the mandibular nerve initial point of TG.

Estradiol-potentiated cadherin-11 in synovial membrane involves in temporomandibular joint inflammation in rats.

Estrogen is involved in inflammation/pain of temporomandibular joint (TMJ), but the underlying mechanisms are largely unknown. Cadherin-11 plays an essential role in synovial inflammation. This study examined whether estrogen could potentiate cadherin-11 in synoviocytes and contribute to TMJ inflammatory pain. Female rats were ovariectomized, treated with increasing doses of 17ß-estradiol for 10 days, and injected intra-articularly with complete Freund's adjuvant to induce TMJ inflammation. The expression of cadherin-11 in synovial membrane was evaluated. TMJ pain was blocked with intra-articular injection of anti-cadherin-11 antibody and evaluated by head withdrawal threshold. Primary TMJ synoviocytes were treated with estradiol and tumor necrosis factor (TNF)-α or blocked with anti-cadherin-11 antibody to assess the expression of cadherin-11, interleukin (IL)-6, cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS). We observed that estradiol potentiated the inflammation-induced expression of cadherin-11 in the synoviocytes of synovial membrane from inflamed TMJ. Estradiol induced cadherin-11 expression in a dose- and time-dependent manner in primary synoviocytes and further potentiated the induction of cadherin-11 by TNF-α in synoviocytes. Furthermore, an estrogen receptor antagonist or a NF-κB inhibitor partially blocked the effects of estradiol on cadherin-11 induction in the synovial membrane. Blocking cadherin-11 partially reversed the TMJ inflammatory pain and estradiol-potentiated proliferation of synovial lining cells accompanied with iNOS expression. In addition, blocking cadherin-11 reversed TNF-α-induced and estradiol-potentiated transcription of IL-6, COX-2, and iNOS in primary synoviocytes. These results suggest that estrogen aggravated TMJ inflammatory pain partially through cadherin-11-mediated release of proinflammatory cytokines and enzymes in the synoviocytes.

Hippocampal nerve growth factor potentiated by 17ß-estradiol and involved in allodynia of inflamed TMJ in rat.

UNLABELLED: The hippocampus is believed to play an important role in sex-based differences of pain perception. Whether estrogen potentiates allodynia in the inflamed temporomandibular joint (TMJ) through affecting the expressions of pain-related genes in the hippocampus remains largely unknown. Because the nerve growth factor (NGF) is an important gene related to inflammatory pain, we tested whether hippocampal NGF may be involved in TMJ inflammatory pain. Here we showed that the rat hippocampal NGF was upregulated by TMJ inflammation induced by complete Freund adjuvant. NGF upregulation was further potentiated by estradiol in a dose-dependent manner. In contrast, NGF transcription in the amygdala, prefrontal cortex, and thalamus was not affected by TMJ inflammation and estradiol. An intrahippocampal injection of NGF antibody or NGF receptor inhibitor K252a (inhibitor for tropomyosin receptor kinase A, TrkA) reduced the allodynia of inflamed TMJ in proestrous rats. Our data suggest that the hippocampal NGF is involved in estradiol-sensitized allodynia of inflammatory TMJ pain. PERSPECTIVE: We report that complete Freund adjuvant-induced temporomandibular joint (TMJ) inflammation upregulated hippocampal nerve growth factor (NGF) expression, and estradiol replacement potentiated this upregulation. These results propose that estradiol could modulate TMJ pain through the NGF signaling pathway in the hippocampus to exacerbate TMJ pain and offer a possible mechanism of sexual dimorphism of temporomandibular disorder pain.

Progression of cartilage degradation, bone resorption and pain in rat temporomandibular joint osteoarthritis induced by injection of iodoacetate.

BACKGROUND: Osteoarthritis (OA) is an important subtype of temporomandibular disorders. A simple and reproducible animal model that mimics the histopathologic changes, both in the cartilage and subchondral bone, and clinical symptoms of temporomandibular joint osteoarthritis (TMJOA) would help in our understanding of its process and underlying mechanism. OBJECTIVE: To explore whether injection of monosodium iodoacetate (MIA) into the upper compartment of rat TMJ could induce OA-like lesions. METHODS: Female rats were injected with varied doses of MIA into the upper compartment and observed for up to 12 weeks. Histologic, radiographic, behavioral, and molecular changes in the TMJ were evaluated by light and electron microscopy, MicroCT scanning, head withdrawal threshold test, real-time PCR, immunohistochemistry, and TUNEL assay. RESULTS: The intermediate zone of the disc loosened by 1 day post-MIA injection and thinned thereafter. Injection of an MIA dose of 0.5 mg or higher induced typical OA-like lesions in the TMJ within 4 weeks. Condylar destruction presented in a time-dependent manner, including chondrocyte apoptosis in the early stages, subsequent cartilage matrix disorganization and subchondral bone erosion, fibrosis, subchondral bone sclerosis, and osteophyte formation in the late stages. Nociceptive responses increased in the early stages, corresponding to severe synovitis. Furthermore, chondrocyte apoptosis and an imbalance between anabolism and catabolism of cartilage and subchondral bone might account for the condylar destruction. CONCLUSIONS: Multi-level data demonstrated a reliable and convenient rat model of TMJOA could be induced by MIA injection into the upper compartment. The model might facilitate TMJOA related researches.