Involvement of interferon gamma signaling in spinal trigeminal caudal subnucleus astrocyte in orofacial neuropathic pain in rats with infraorbital nerve injury.

Background: Trigeminal nerve injury causes orofacial pain that can interfere with activities of daily life. However, the underlying mechanism remains unknown, and the appropriate treatment has not been established yet. This study aimed to examine the involvement of interferon gamma (IFN-γ) signaling in the spinal trigeminal caudal subnucleus (Vc) in orofacial neuropathic pain. Methods: Infraorbital nerve (ION) injury (IONI) was performed in rats by partial ION ligation. The head-withdrawal reflex threshold (HWT) to mechanical stimulation of the whisker pad skin was measured in IONI or sham rats, as well as following a continuous intracisterna magna administration of IFN-γ and a mixture of IFN-γ and fluorocitrate (inhibitor of astrocytes activation) in naïve rats, or an IFN-γ antagonist in IONI rats. The IFN-γ receptor immunohistochemistry and IFN-γ Western blotting were analyzed in the Vc after IONI or sham treatment. The glial fibrillary acid protein (GFAP) immunohistochemistry and Western blotting were also analyzed after administration of IFN-γ and the mixture of IFN-γ and fluorocitrate. Moreover, the change in single neuronal activity in the Vc was examined in the IONI, sham, and IONI group administered IFN-γ antagonist. Results: The HWT decreased after IONI. The IFN-γ and IFN-γ receptor were upregulated after IONI, and the IFN-γ receptor was expressed in Vc astrocytes. IFN-γ administration decreased the HWT, whereas the mixture of IFN-γ and fluorocitrate recovered the decrement of HWT. IFN-γ administration upregulated GFAP expression, while the mixture of IFN-γ and fluorocitrate recovered the upregulation of GFAP expression. IONI significantly enhanced the neuronal activity of the mechanical-evoked responses, and administration of an IFN-γ antagonist significantly inhibited these enhancements. Conclusions: IFN-γ signaling through the receptor in astrocytes is a key mechanism underlying orofacial neuropathic pain associated with trigeminal nerve injury. These findings will aid in the development of therapeutics for orofacial neuropathic pain.

M2 macrophage-derived cathepsin S promotes peripheral nerve regeneration via fibroblast-Schwann cell-signaling relay.

BACKGROUND: Although peripheral nerves have an intrinsic self-repair capacity following damage, functional recovery is limited in patients. It is a well-established fact that macrophages accumulate at the site of injury. Numerous studies indicate that the phenotypic shift from M1 macrophage to M2 macrophage plays a crucial role in the process of axon regeneration. This polarity change is observed exclusively in peripheral macrophages but not in microglia and CNS macrophages. However, the molecular basis of axonal regeneration by M2 macrophage is not yet fully understood. Herein, we aimed to identify the M2 macrophage-derived axon regeneration factor. METHODS: We established a peripheral nerve injury model by transection of the inferior alveolar nerve (IANX) in Sprague-Dawley rats. Transcriptome analysis was performed on the injured nerve. Recovery from sensory deficits in the mandibular region and histological reconnection of IAN after IANX were assessed in rats with macrophage depletion by clodronate. We investigated the effects of adoptive transfer of M2 macrophages or M2-derived cathepsin S (CTSS) on the sensory deficit. CTSS initiating signaling was explored by western blot analysis in IANX rats and immunohistochemistry in co-culture of primary fibroblasts and Schwann cells (SCs). RESULTS: Transcriptome analysis revealed that CTSS, a macrophage-selective lysosomal protease, was upregulated in the IAN after its injury. Spontaneous but partial recovery from a sensory deficit in the mandibular region after IANX was abrogated by macrophage ablation at the injured site. In addition, a robust induction of c-Jun, a marker of the repair-supportive phenotype of SCs, after IANX was abolished by macrophage ablation. As in transcriptome analysis, CTSS was upregulated at the injured IAN than in the intact IAN. Endogenous recovery from hypoesthesia was facilitated by supplementation of CTSS but delayed by pharmacological inhibition or genetic silencing of CTSS at the injured site. Adoptive transfer of M2-polarized macrophages at this site facilitated sensory recovery dependent on CTSS in macrophages. Post-IANX, CTSS caused the cleavage of Ephrin-B2 in fibroblasts, which, in turn, bound EphB2 in SCs. CTSS-induced Ephrin-B2 cleavage was also observed in human sensory nerves. Inhibition of CTSS-induced Ephrin-B2 signaling suppressed c-Jun induction in SCs and sensory recovery. CONCLUSIONS: These results suggest that M2 macrophage-derived CTSS contributes to axon regeneration by activating SCs via Ephrin-B2 shedding from fibroblasts.

Pannexin 1 role in the trigeminal ganglion in infraorbital nerve injury-induced mechanical allodynia.

OBJECTIVES: The detailed pathological mechanism of orofacial neuropathic pain remains unknown. We aimed to examine the pannexin 1 (Panx1) signaling in the trigeminal ganglion (TG) involvement in infraorbital nerve injury (IONI)-induced orofacial neuropathic pain. MATERIALS AND METHODS: Mechanical head-withdrawal threshold (MHWT) was measured in IONI-treated rats receiving intra-TG Panx1 inhibitor or metabotropic glutamate receptor 5 (mGluR5) antagonist administration and MHWTs in naive rats receiving intra-TG mGluR5 agonist administration post-IONI. Glutamate and Panx1 in the TG were measured post-IONI. Panx1, mGluR5, and glutamine synthetase expression in TG were immunohistochemically identified, and changes in the number of mGluR5-P2X3 -expressed TG neurons were examined. RESULTS: MHWT was significantly decreased post-IONI, and this decrease was reversed by Panx1 inhibition or mGluR5 antagonism. mGluR5 agonism induced a decrease in the MHWT. IONI increased extracellular glutamate in TG. Panx1 was expressed in satellite glial cells and TG neurons, and intra-TG mGluR5 antagonism decreased the number of mGluR5 and P2X3 positive TG neurons post-IONI. CONCLUSIONS: IONI facilitates glutamate release via Panx1 that activates mGluR5 which was expressed in the nociceptive TG neurons innervating the orofacial region. In turn, P2X3 receptor-expressed TG neurons are enhanced via mGluR5 signaling, resulting in orofacial neuropathic pain.

IL-33 induces orofacial neuropathic pain through Fyn-dependent phosphorylation of GluN2B in the trigeminal spinal subnucleus caudalis.

Orofacial neuropathic pain can cause considerable disruptions in patients' daily lives, especially because of a lack of effective medications as its underlying causative mechanisms are not fully understood. Here, we found neuron-specific expression of the interleukin (IL)-33 receptor in the trigeminal spinal subnucleus caudalis (Vc), distinct from the spinal dorsal horn. Reduction in head withdrawal threshold in response to von Frey filament stimulation of the whisker pad skin was inversely correlated with the upregulation of IL-33 in the Vc after infraorbital nerve injury (IONI). Neutralization of IL-33 in the Vc alleviated mechanical allodynia in the whisker pad skin after IONI; conversely, intracisternal administration of IL-33 elicited mechanical allodynia in the whisker pad skin, which was relieved by GluN2B antagonism. Moreover, IL-33 triggered the potentiation of GluN2B-containing N-methyl-D-aspartate receptor-mediated synaptic currents and phosphorylation of synaptosomal GluN2B in the Vc, whereas IONI-induced GluN2B phosphorylation was inhibited by neutralization of IL-33 in the Vc. IL-33-induced GluN2B phosphorylation was mediated by phosphorylation of Fyn kinase, and inhibition of the Fyn kinase pathway prevented the development of IL-33-induced mechanical allodynia. Our findings provide insights into a new mechanism by which IL-33 directly regulates synaptic transmission and suggest that IL-33 signaling could be a candidate target for therapeutic interventions for orofacial neuropathic pain.

Age-Related Differences in Transient Receptor Potential Vanilloid 1 and 2 Expression Patterns in the Trigeminal Ganglion Neurons Contribute to Changes in the Palatal Mucosal Heat Pain Sensitivity.

Aging affects various sensory functions of the body. However, the effect on the oral mucosal nociception has remain unclear, so this elucidation is very important. Therefore, this study aimed to evaluate the effect of age-related changes in transient receptor potential vanilloid 1 (TRPV1) and TRPV2 expression in the trigeminal ganglion (TG) neurons on intraoral mucosal heat sensitivity in the senescence-accelerated mouse prone 8 (SAMP8) model. We used 23-week-old (aged) and 7-week-old (young) SAMP8 mice. Heat stimulation was applied to the palatal mucosa under light anesthesia; moreover, the heat head withdrawal threshold (HHWT) was measured. We counted the number of TRPV1-immunoreactive (IR) and TRPV2-IR TG neurons innervating the palatal mucosa. Additionally, we investigated changes in HHWT when TRPV1 or TRPV2 antagonists (SB366791 or Tranilast) were administered to the palatal mucosa. Aged SAMP8 mice showed a higher HHWT than young SAMP8 mice. Compared with the aged SAMP8 mice, young SAMP8 mice showed a larger number of TRPV1-IR small-diameter neurons and a smaller number of TRPV2-IR medium-sized neurons innervating the palatal mucosa. SB366791 administration increased the HHWT in young, but not aged SAMP8 mice. Contrastingly, Tranilast administration increased the HHWT in aged, but not young SAMP8 mice. These results suggest that the modulation of heat pain sensitivity in the oral mucosa due to aging is dependent on changes in the TRPV1 and TRPV2 expression patterns in the TG neurons innervating the palatal mucosa.

Effect of low-intensity pulsed ultrasound on orofacial sensory disturbance following inferior alveolar nerve injury: Role of neurotrophin-3 signaling.

Percutaneous treatment of low-intensity pulsed ultrasound (LIPUS) to the site of inferior alveolar nerve (IAN) transection promotes functional regeneration, but the detailed mechanism is unknown. We examined the involvement of neurotrophin-3 (NT-3), which primarily binds with tropomyosin receptor kinase C (TrkC), in functional transected IAN regeneration following LIPUS treatment in rats. Daily LIPUS treatment to the transected IAN was performed, and the mechanical sensitivity of the facial skin was measured for 14 d. On day 5 after IAN transection, the expression of NT-3 in the transected IAN and TrkC-positive trigeminal ganglion neurons were immunohistochemically examined. Further, the effect of TrkC neutralization on the acceleration of facial mechanosensory disturbance restoration due to LIPUS treatment was analyzed. LIPUS treatment to the site of IAN transection significantly facilitated functional recovery from sensory disturbance on facial skin. Schwann cells in the transected IAN expressed NT-3, and LIPUS treatment increased the amount of NT-3. The facilitated recovery from the mechanosensory disturbance by continuous LIPUS treatment was inhibited by the ongoing TrkC neutralization at the IAN transection site. These results suggest that LIPUS treatment accelerates the recovery of orofacial mechanosensory function following IAN transection through the enhancement of NT-3 signaling in the transected IAN.

Pannexin 1-Mediated ATP Signaling in the Trigeminal Spinal Subnucleus Caudalis Is Involved in Tongue Cancer Pain.

Pain is one of the most severe concerns in tongue cancer patients. However, the underlying mechanisms of tongue cancer pain are not fully understood. We investigated the molecular mechanisms of tongue cancer-induced mechanical allodynia in the tongue by squamous cell carcinoma (SCC) inoculation in rats. The head-withdrawal threshold of mechanical stimulation (MHWT) to the tongue was reduced following SCC inoculation, which was inhibited by intracisternal administration of 10Panx, an inhibitory peptide for pannexin 1 (PANX1) channels. Immunohistochemical analyses revealed that the expression of PANX1 was upregulated in the trigeminal spinal subnucleus caudalis (Vc) following SCC inoculation. The majority of PANX1 immunofluorescence was merged with ionized calcium-binding adapter molecule 1 (Iba1) fluorescence and a part of it was merged with glial fibrillary acidic protein (GFAP) fluorescence. Spike frequencies of Vc nociceptive neurons to noxious mechanical stimulation were significantly enhanced in SCC-inoculated rats, which was suppressed by intracisternal 10Panx administration. Phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive (IR) neurons increased significantly in the Vc after SCC inoculation, which was inhibited by intracisternal 10Panx administration. SCC inoculation-induced MHWT reduction and increased pERK-IR Vc neuron numbers were inhibited by P2X7 purinoceptor (P2X7R) antagonism. Conversely, these effects were observed in the presence of P2X7R agonist in SCC-inoculated rats with PANX1 inhibition. SCC inoculation-induced MHWT reduction was significantly recovered by intracisternal interleukin-1 receptor antagonist administration. These observations suggest that SCC inoculation causes PANX1 upregulation in Vc microglia and adenosine triphosphate released through PANX1 sensitizes nociceptive neurons in the Vc, resulting in tongue cancer pain.

Analgesic Mechanisms of Steroid Ointment against Oral Ulcerative Mucositis in a Rat Model.

Despite the long history of use of steroid ointments for oral mucositis, the analgesic mechanism has not been fully elucidated. In this study, we examined the effects of triamcinolone acetonide (Tmc) on oral ulcerative mucositis-induced pain in conscious rats by our proprietary assay system. Based on evaluations of the physical properties and retention periods in the oral mucosa of human volunteers and rats, we selected TRAFUL® ointment as a long-lasting base. In oral ulcerative mucositis model rats, TRAFUL® with Tmc suppressed cyclooxygenase-dependent inflammatory responses with upregulations of glucocorticoid receptor-induced anti-inflammatory genes and inhibited spontaneous nociceptive behavior. When an ointment with a shorter residual period was used, the effects of Tmc were not elicited or were induced to a lesser extent. Importantly, TRAFUL® with Tmc also improved oral ulcerative mucositis-induced mechanical allodynia, which has been reported to be independent of cyclooxygenase. Ca2+ imaging in dissociated trigeminal ganglion neurons showed that long-term preincubation with Tmc inhibited the hypertonic stimulation-induced Ca2+ response. These results suggest that the representative steroid Tmc suppresses oral ulcerative mucositis-induced pain by general anti-inflammatory actions and inhibits mechanical sensitivity in peripheral nerves. For drug delivery, long-lasting ointments such as TRAFUL® are needed to sufficiently induce the therapeutic effects.

P2X3 receptor upregulation in trigeminal ganglion neurons through TNFα production in macrophages contributes to trigeminal neuropathic pain in rats.

BACKGROUND: Trigeminal neuralgia is a characteristic disease that manifests as orofacial phasic or continuous severe pain triggered by innocuous orofacial stimulation; its mechanisms are not fully understood. In this study, we established a new animal model of trigeminal neuralgia and investigated the role of P2X3 receptor (P2X3R) alteration in the trigeminal ganglion (TG) via tumor necrosis factor alpha (TNFα) signaling in persistent orofacial pain. METHODS: Trigeminal nerve root compression (TNC) was performed in male Sprague-Dawley rats. Changes in the mechanical sensitivity of whisker pad skin, amount of TNFα in the TG, and number of P2X3R and TNF receptor-2 (TNFR2)-positive TG neurons were assessed following TNC. The effects of TNFR2 antagonism in TG and subcutaneous P2X3R antagonism on mechanical hypersensitivity following TNC were examined. RESULTS: TNC induced unilateral continuous orofacial mechanical allodynia, which was depressed by carbamazepine. The accumulation of macrophages showing amoeboid-like morphological changes and expression of TNFα in the TG was remarkably increased following TNC treatment. The number of P2X3R- and TNFR2-positive TG neurons innervating the orofacial skin was significantly increased following TNC. TNFα was released from activated macrophages that occurred in the TG following TNC, and TNFR2 antagonism in the TG significantly diminished the TNC-induced increase in P2X3R-immunoreactive TG neurons. Moreover, subcutaneous P2X3R antagonism in the whisker pad skin significantly depressed TNC-induced mechanical allodynia. CONCLUSIONS: Therefore, it can be concluded that the signaling of TNFα released from activated macrophages in the TG induces the upregulation of P2X3R expression in TG neurons innervating the orofacial region, resulting in orofacial mechanical allodynia following TNC.

Microglia-Astrocyte Communication via C1q Contributes to Orofacial Neuropathic Pain Associated with Infraorbital Nerve Injury.

Trigeminal nerve injury causes a distinct time window of glial activation in the trigeminal spinal subnucleus caudalis (Vc), which are involved in the initiation and maintenance phases of orofacial neuropathic pain. Microglia-derived factors enable the activation of astrocytes. The complement component C1q, which promotes the activation of astrocytes, is known to be synthesized in microglia. However, it is unclear whether microglia-astrocyte communication via C1q is involved in orofacial neuropathic pain. Here, we analyzed microglia-astrocyte communication in a rat model with infraorbital nerve injury (IONI). The orofacial mechanical hypersensitivity induced by IONI was significantly attenuated by preemptive treatment with minocycline. Immunohistochemical analyses revealed that minocycline inhibited the increase in c-Fos immune-reactive (IR) cells and the fluorescence intensity of both Iba1 and glial fibrillary acidic protein (GFAP) in the Vc following IONI. Intracisternal administration of C1q caused orofacial mechanical hypersensitivity and an increase in the number of c-Fos-IR cells and fluorescence intensity of GFAP. C1q-induced orofacial mechanical hypersensitivity was completely abrogated by intracisternal administration of fluorocitrate. The present findings suggest that the enhancement in the excitability of Vc nociceptive neurons is produced by astrocytic activation via the signaling of C1q released from activated microglia in the Vc following IONI, resulting in persistent orofacial neuropathic pain.

Aging-Related Phenotypic Conversion of Medullary Microglia Enhances Intraoral Incisional Pain Sensitivity.

Activated microglia involved in the development of orofacial pain hypersensitivity have two major polarization states. The aim of this study was to assess the involvement of the aging-related phenotypic conversion of medullary microglia in the enhancement of intraoral pain sensitivity using senescence-accelerated mice (SAM)-prone/8 (SAMP8) and SAM-resistant/1 (SAMR1) mice. Mechanical head-withdrawal threshold (MHWT) was measured for 21 days post palatal mucosal incision. The number of CD11c-immunoreactive (IR) cells [affective microglia (M1)] and CD163-IR cells [protective microglia (M2)], and tumor-necrosis-factor-α (TNF-α)-IR M1 and interleukin (IL)-10-IR M2 were analyzed via immunohistochemistry on days 3 and 11 following incision. The decrease in MHWT observed following incision was enhanced in SAMP8 mice. M1 levels and the number of TNF-α-IR M1 were increased on day 3 in SAMP8 mice compared with those in SAMR1 mice. On day 11, M1 and M2 activation was observed in both groups, whereas IL-10-IR M2 levels were attenuated in SAMP8 mice, and the number of TNF-α-IR M1 cells increased, compared to those in SAMR1 mice. These results suggest that the mechanical allodynia observed following intraoral injury is potentiated and sustained in SAMP8 mice due to enhancement of TNF-α signaling, M1 activation, and an attenuation of M2 activation accompanying IL-10 release.

Oxytocin-Dependent Regulation of TRPs Expression in Trigeminal Ganglion Neurons Attenuates Orofacial Neuropathic Pain Following Infraorbital Nerve Injury in Rats.

We evaluated the mechanisms underlying the oxytocin (OXT)-induced analgesic effect on orofacial neuropathic pain following infraorbital nerve injury (IONI). IONI was established through tight ligation of one-third of the infraorbital nerve thickness. Subsequently, the head withdrawal threshold for mechanical stimulation (MHWT) of the whisker pad skin was measured using a von Frey filament. Trigeminal ganglion (TG) neurons innervating the whisker pad skin were identified using a retrograde labeling technique. OXT receptor-immunoreactive (IR), transient receptor potential vanilloid 1 (TRPV1)-IR, and TRPV4-IR TG neurons innervating the whisker pad skin were examined on post-IONI day 5. The MHWT remarkably decreased from post-IONI day 1 onward. OXT application to the nerve-injured site attenuated the decrease in MHWT from day 5 onward. TRPV1 or TRPV4 antagonism significantly suppressed the decrement of MHWT following IONI. OXT receptors were expressed in the uninjured and Fluoro-Gold (FG)-labeled TG neurons. Furthermore, there was an increase in the number of FG-labeled TRPV1-IR and TRPV4-IR TG neurons, which was inhibited by administering OXT. This inhibition was suppressed by co-administration with an OXT receptor antagonist. These findings suggest that OXT application inhibits the increase in TRPV1-IR and TRPV4-IR TG neurons innervating the whisker pad skin, which attenuates post-IONI orofacial mechanical allodynia.

The transcriptional co-repressor TLE3 regulates myogenic differentiation by repressing the activity of the MyoD transcription factor.

Satellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity.

Prostanoid-dependent spontaneous pain and PAR2-dependent mechanical allodynia following oral mucosal trauma: involvement of TRPV1, TRPA1 and TRPV4.

Abstract: During dental treatments, intraoral appliances frequently induce traumatic ulcers in the oral mucosa. Such mucosal injury-induced mucositis leads to severe pain, resulting in poor quality of life and decreased cooperation in the therapy. To elucidate mucosal pain mechanisms, we developed a new rat model of intraoral wire-induced mucositis and investigated pain mechanisms using our proprietary assay system for conscious rats. A thick metal wire was installed in the rats between the inferior incisors for one day. In the mucosa of the mandibular labial fornix region, which was touched with a free end of the wire, traumatic ulcer and submucosal abscess were induced on day 1. The ulcer was quickly cured until next day and abscess formation was gradually disappeared until five days. Spontaneous nociceptive behavior was induced on day 1 only, and mechanical allodynia persisted over day 3. Antibiotic pretreatment did not affect pain induction. Spontaneous nociceptive behavior was sensitive to indomethacin (cyclooxygenase inhibitor), ONO-8711 (prostanoid receptor EP1 antagonist), SB-366791, and HC-030031 (TRPV1 and TRPA1 antagonists, respectively). Prostaglandin E2 and 15-deoxyΔ12,14-prostaglandin J2 were upregulated only on day 1. In contrast, mechanical allodynia was sensitive to FSLLRY-NH2 (protease-activated receptor PAR2 antagonist) and RN-1734 (TRPV4 antagonist). Neutrophil elastase, which is known as a biased agonist for PAR2, was upregulated on days 1 to 2. These results suggest that prostanoids and PAR2 activation elicit TRPV1- and TRPA1-mediated spontaneous pain and TRPV4-mediated mechanical allodynia, respectively, independently of bacterial infection, following oral mucosal trauma. The pathophysiological pain mechanism suggests effective analgesic approaches for dental patients suffering from mucosal trauma-induced pain.

Activation of dura-sensitive trigeminal neurons and increased c-Fos protein induced by morphine withdrawal in the rostral ventromedial medulla.

Aims Overuse of medications used to treat migraine headache can increase the frequency of headaches. Sudden abstinence from migraine medication can also lead to a period of withdrawal-induced headaches. The aim of this study was to examine the effect of morphine withdrawal localized to the rostral ventromedial medulla (RVM) on the activity of dura-sensitive spinal trigeminal nucleus caudalis (Vc) neurons. Methods Rats were implanted with either morphine or placebo pellets for six to seven days before the microinjection of naloxone methiodide or phosphate-buffered saline into the RVM in urethane-anesthetized animals. Dura-sensitive neurons were recorded in the Vc and the production of c-Fos-like immunoreactivity was quantified. Results In chronic morphine-treated animals, naloxone methiodide microinjections produced a significant increase both in ongoing and facial heat-evoked activity and an increase in Fos-positive neurons in the Vc and in the nucleus reticularis dorsalis, a brainstem region involved in diffuse noxious inhibitory controls. Conclusions These results indicate that activation of pronociceptive neurons in the RVM under conditions of morphine withdrawal can increase the activity of neurons that transmit headache pain. Modulation of the subnucleus reticularis dorsalis by the RVM may explain the attenuation of conditioned pain modulation in patients with chronic headache.

[6]-gingerol and [6]-shogaol, active ingredients of the traditional Japanese medicine hangeshashinto, relief oral ulcerative mucositis-induced pain via action on Na+ channels.

The traditional Japanese herbal medicine hangeshashinto (HST) has beneficial effects for the treatment of oral ulcerative mucositis (OUM) in cancer patients. However, the ingredient-based mechanism that underlies its pain-relieving activity remains unknown. In the present study, to clarify the analgesic mechanism of HST on OUM-induced pain, we investigated putative HST ingredients showing antagonistic effects on Na+ channels in vitro and in vivo. A screen of 21 major ingredients using automated patch-clamp recordings in channel-expressing cells showed that [6]-gingerol and [6]-shogaol, two components of a Processed Ginger extract, considerably inhibited voltage-activated Na+ currents. These two ingredients inhibited the stimulant-induced release of substance P and action potential generation in cultured rat sensory neurons. A submucosal injection of a mixture of [6]-gingerol and [6]-shogaol increased the mechanical withdrawal threshold in healthy rats. In a rat OUM model, OUM-induced mechanical pain was alleviated 30min after the swab application of HST despite the absence of anti-bacterial and anti-inflammatory actions in the OUM area. A swab application of a mixture of [6]-gingerol and [6]-shogaol induced sufficient analgesia of OUM-induced mechanical or spontaneous pain when co-applied with a Ginseng extract containing abundant saponin. The Ginseng extract demonstrated an acceleration of substance permeability into the oral ulcer tissue without an analgesic effect. These findings suggest that Na+ channel blockage by gingerol/shogaol plays an essential role in HST-associated analgesia of OUM-induced pain. This pharmacological mechanism provides scientific evidence supporting the use of this herbal medicine in patients suffering from OUM-induced pain.

Muscle regulatory factors regulate T1R3 taste receptor expression.

T1R3 is a T1R class of G protein-coupled receptors, composing subunit of the umami taste receptor when complexed with T1R1. T1R3 was originally discovered in gustatory tissue but is now known to be expressed in a wide variety of tissues and cell types such the intestine, pancreatic ß-cells, skeletal muscle, and heart. In addition to taste recognition, the T1R1/T1R3 complex functions as an amino acid sensor and has been proposed to be a control mechanism for the secretion of hormones, such as cholecystokinin, insulin, and duodenal HCO3(-) and activates the mammalian rapamycin complex 1 (MTORC1) to inhibit autophagy. T1R3 knockout mice have increased rate of autophagy in the heart, skeletal muscle and liver. Thus, T1R3 has multiple physiological functions and is widely expressed in vivo. However, the exact mechanisms regulating T1R3 expression are largely unknown. Here, we used comparative genomics and functional analyses to characterize the genomic region upstream of the annotated transcriptional start of human T1R3. This revealed that the T1R3 promoter in human and mouse resides in an evolutionary conserved region (ECR). We also identified a repressive element located upstream of the human T1R3 promoter that has relatively high degree of conservation with rhesus macaque. Additionally, the muscle regulatory factors MyoD and Myogenin regulate T1R3 expression and T1R3 expression increases with skeletal muscle differentiation of murine myoblast C2C12 cells. Taken together, our study raises the possibility that MyoD and Myogenin might control skeletal muscle metabolism and homeostasis through the regulation of T1R3 promoter activity.

Analgesic effect of linalool odor on oral ulcerative mucositis-induced pain in rats.

Oral ulcerative mucositis (OUM) induces severe pain, leading to a low quality of life. Linalool odor exposure has recently been reported to suppress inflammatory pain in the hind paws. However, the analgesic effect of linalool odor on orofacial pain remains unclear. In this study, we examined the mechanism underlying the analgesic effect of linalool odor on oral pain caused by OUM using nocifensive behavioral and immunohistochemical analyses in rats. OUM was developed by treating the labial fornix region of the inferior incisors with acetic acid. Linalool at 1% was exposed for 5 min at 30 min before nocifensive behavioral measurements. OUM induced spontaneous pain and mechanical allodynia, which were suppressed by the linalool odor. Mechanical allodynia in the hind paw following the injection of complete Freund's adjuvant was also suppressed by linalool odor. Application of lidocaine to the olfactory bulb attenuated the inhibition of spontaneous pain and hyperactivation of trigeminal spinal nucleus caudalis neurons in OUM model rats. Linalool odor exposure-induced neuronal activation in the locus coeruleus (LC) of OUM model rats was decreased by lidocaine application to the olfactory bulb. The decrease in neuronal activation in the LC was attenuated by the administration of orexin 1 receptor (OX-1) antagonist to the LC. These results suggest that linalool odor stimulation through the olfactory pathway activates LC neurons via OX-1 signaling, leading to the suppression of OUM-induced oral pain.

Association between central sensitivity syndrome and psychophysical factors in patients with masticatory myofascial pain.

PURPOSE: This study explored the relationship between central sensitization symptoms, assessed using the Central Sensitization Inventory (CSI), and psychophysical factors in patients with chronic masticatory myofascial pain (MMP) transitioning from the acute to chronic stages. METHODS: In this study, 23 patients with MMP and 22 healthy volunteers were assessed using psychophysical tests, including measurements of pressure pain threshold (PPT) and temporal summation of pain (TSP). Additionally, CSI scores were recorded to evaluate central sensitization symptoms. RESULTS: Patients with chronic MMP showed significantly lower PPT in all masticatory muscles and extratrigeminal areas compared with controls. However, there was no significant correlation between CSI scores and psychophysical test results in patients with MMP. CONCLUSION: The significant enhancement of TSP in patients with subchronic MMP suggests a potential role in the onset of myofascial pain. The main finding suggests that sub-chronic symptom patients show higher CSI scores despite no sensory testing changes, indicating that central sensitization possibly precedes observable symptoms.

Recent advances in the treatment of oral ulcerative mucositis from clinical and basic perspectives.

BACKGROUND: Oral ulcerative mucositis (OUM) is common in patients with cancer, particularly in those undergoing chemoradiation therapy. The effective management of OUM is crucial for continuous cancer care and patient well-being. Recent studies have advanced our understanding of the causes, leading to clinical trials toward novel treatments. This review focuses on the contemporary therapeutic landscape, and provides the latest insights into the mechanisms of mucosal healing and pain. HIGHLIGHTS: Management strategies for oral ulcerative mucositis in patients with cancer include maintaining good oral hygiene, reducing mucosal irritation against radiation, and using various topical analgesic treatments, including herbal medicines. However, the current management practices have limitations that necessitate the development of more efficacious and novel treatments. Molecular research on transient receptor potential (TRP) channels in the oral mucosa is crucial for understanding the mechanisms of wound healing and pain in patients with OUM. Targeting TRP subfamily V member 3 (V3) and TRPV4 can enhance wound healing through re-epithelialization. The suppression of TRPV1, TRPA1, and TRPV4 may be effective in alleviating OUM-induced pain. CONCLUSION: Research advancements have improved our understanding and potentially led to novel treatments that offer symptomatic relief. This progress highlights the importance of collaborations between clinical researchers and scientists in the development of innovative therapies.