Fibroblast-derived IL-33 exacerbates orofacial neuropathic pain via the activation of TRPA1 in trigeminal ganglion neurons.

Damage to the peripheral nerves of trigeminal ganglion (TG) neurons leads to intractable orofacial neuropathic pain through the induction of neuroinflammation. However, the details of this process are not yet fully understood. Here, we found that fibroblast-derived interleukin (IL)-33 was required for the development of mechanical allodynia in whisker pad skin following infraorbital nerve injury (IONI). The amount of IL-33 in the TG increased after IONI when the mice exhibited mechanical allodynia. Neutralization of IL-33 in the TG inhibited the development of IONI-induced mechanical allodynia. Conversely, intra-TG administration of recombinant human IL-33 (rhIL-33) elicited mechanical allodynia in naïve mice. IL-33 and its receptor were exclusively expressed in fibroblasts and neurons, respectively, in the TG. Fibroblast ablation caused the loss of IL-33 in the TG and delayed the development of mechanical allodynia after IONI. rhIL-33 elicited an increase in intracellular Ca2+ concentration and subsequent enhancement of Ca2+ influx via transient receptor potential ankyrin 1 (TRPA1) in primary cultured TG neurons. Additionally, rhIL-33 facilitated membrane translocation of TRPA1 in the TG. Mechanical allodynia caused by intra-TG administration of rhIL-33 was significantly inhibited by pharmacological blockade or gene silencing of TRPA1 in the TG. Inhibition of protein kinase A abrogated TRPA1 membrane translocation and delayed mechanical allodynia after IONI. Substance P stimulation caused upregulation of IL-33 expression in primary cultured fibroblasts. Preemptive administration of a neurokinin-1 receptor antagonist in the TG attenuated mechanical allodynia and IL-33 expression following IONI. Taken together, these results indicate that fibroblast-derived IL-33 exacerbates TG neuronal excitability via suppression of tumorigenicity 2 (ST2)-TRPA1 signaling, ultimately leading to orofacial neuropathic pain.

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.

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.

Involvement of oxidative stress in orofacial mechanical pain hypersensitivity following neonatal maternal separation in rats.

Patients with persistent pain have sometimes history of physical abuse or neglect during infancy. However, the pathogenic mechanisms underlying orofacial pain hypersensitivity associated with early-life stress remain unclear. The present study focused on oxidative stress and investigated its role in pain hypersensitivity in adulthood following early-life stress. To establish an early-life stress model, neonatal pups were separated with their mother in isolated cages for 2 weeks. The mechanical head-withdrawal threshold (MHWT) in the whisker pad skin of rats received maternal separation (MS) was lower than that of non-MS rats at postnatal week 7. In MS rats, the expression of 8-hydroxy-deoxyguanosine, a marker of DNA oxidative damage, was enhanced, and plasma antioxidant capacity, but not mitochondrial complex I activity, decreased compared with that in non-MS rats. Reactive oxygen species (ROS) inactivation and ROS-sensitive transient receptor potential ankyrin 1 (TRPA1) antagonism in the whisker pad skin at week 7 suppressed the decrease of MHWT. Corticosterone levels on day 14 increased in MS rats. Corticosterone receptor antagonism during MS periods suppressed the reduction in antioxidant capacity and MHWT. The findings suggest that early-life stress potentially induces orofacial mechanical pain hypersensitivity via peripheral nociceptor TRPA1 hyperactivation induced by oxidative stress in the orofacial region.

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.

PAR2-dependent phosphorylation of TRPV4 at the trigeminal nerve terminals contributes to tongue cancer pain.

OBJECTIVE: This study aimed to clarify the interactions between the tongue and primary afferent fibers in tongue cancer pain. METHODS: A pharmacological analysis was conducted to evaluate mechanical hypersensitivity of the tongues of rats with squamous cell carcinoma (SCC). Changes in trigeminal ganglion (TG) neurons projecting to the tongue were analyzed using immunohistochemistry and western blotting. RESULTS: SCC inoculation of the tongue caused persistent mechanical sensitization and tumor formation. Trypsin expression was significantly upregulated in cancer lesions. Continuous trypsin inhibition or protease-activated receptor 2 (PAR2) antagonism in the tongue significantly inhibited SCC-induced mechanical sensitization. No changes were observed in PAR2 and transient receptor potential vanilloid 4 (TRPV4) levels in the TG or the number of PAR2-and TRPV4-expressing TG neurons after SCC inoculation. In contrast, the relative amount of phosphorylated TRPV4 in the TG was significantly increased after SCC inoculation and abrogated by PAR2 antagonism in the tongue. TRPV4 antagonism in the tongue significantly ameliorated the mechanical sensitization caused by SCC inoculation. CONCLUSIONS: Our findings indicate that tumor-derived trypsin sensitizes primary afferent fibers by PAR2 stimulation and subsequent TRPV4 phosphorylation, resulting in severe tongue pain.

Microglia cause structural remodeling of noradrenergic axon in the trigeminal spinal subnucleus caudalis after infraorbital nerve injury in rats.

The dysfunction of descending noradrenergic (NAergic) modulation in second-order neurons has long been observed in neuropathic pain. In clinical practice, antidepressants that increase noradrenaline levels in the synaptic cleft are used as first-line agents, although adequate analgesia has not been occasionally achieved. One of the hallmarks of neuropathic pain in the orofacial regions is microglial abnormalities in the trigeminal spinal subnucleus caudalis (Vc). However, until now, the direct interaction between descending NAergic system and Vc microglia in orofacial neuropathic pain has not been explored. We found that reactive microglia ingested the dopamine-ß-hydroxylase (DßH)-positive fraction, NAergic fibers, in the Vc after infraorbital nerve injury (IONI). Major histocompatibility complex class I (MHC-I) was upregulated in Vc microglia after IONI. Interferon-γ (IFNγ) was de novo induced in trigeminal ganglion (TG) neurons following IONI, especially in C-fiber neurons, which conveyed to the central terminal of TG neurons. Gene silencing of IFNγ in the TG reduced MHC-I expression in the Vc after IONI. Intracisternal administration of exosomes from IFNγ-stimulated microglia elicited mechanical allodynia and a decrease in DßH in the Vc, which did not occur when exosomal MHC-I was knocked down. Similarly, in vivo MHC-I knockdown in Vc microglia attenuated the development of mechanical allodynia and a decrease in DßH in the Vc after IONI. These results show that microglia-derived MHC-I causes a decrease in NAergic fibers, culminating in orofacial neuropathic pain.

Neonatal Injury Modulates Incisional Pain Sensitivity in Adulthood: An Animal Study.

Neonatal pain experiences including traumatic injury influence negatively on development of nociceptive circuits, resulting in persistent pain hypersensitivity in adults. However, the detailed mechanism is not yet well understood. In the present study, to clarify the pathogenesis of orofacial pain hypersensitivity associated with neonatal injury, the involvement of the voltage-gated sodium channel (Nav) 1.8 and the C-C chemokine ligand 2 (CCL2)/C-C chemokine receptor 2 (CCR2) signaling in the trigeminal ganglion (TG) in facial skin incisional pain hypersensitivity was examined in 190 neonatal facial-injured and sham male rats. The whisker pad skin was incised on postnatal day 4 and week 7 (Incision-Incision group). Compared to the group without neonatal incision (Sham-Incision group), mechanical hypersensitivity in the whisker pad skin was enhanced in Incision-Incision group. The number of Nav1.8-immunoreactive TG neurons and the amount of CCL2 expressed in the macrophages and satellite glial cells in the TG were increased on day 14 after re-incision in the Incision-Incision group, compared with Sham-Incision group. Blockages of Nav1.8 in the incised region and CCR2 in the TG suppressed the enhancement of mechanical hypersensitivity in the Incision-Incision group. Administration of CCL2 into the TG enhanced mechanical hypersensitivity in the Sham-Sham, Incision-Sham and Sham-Incision group. Our results suggest that neonatal facial injury accelerates the TG neuronal hyperexcitability following orofacial skin injury in adult in association with Nav1.8 overexpression via CCL2 signaling, resulting in the enhancement of orofacial incisional pain hypersensitivity in the adulthood.

Astrocytic and microglial interleukin-1ß mediates complement C1q-triggered orofacial mechanical allodynia.

Glial cells, such as microglia and astrocytes, in the trigeminal spinal subnucleus caudalis (Vc) are activated after trigeminal nerve injury and interact with Vc neurons to contribute to orofacial neuropathic pain. Complement C1q released from microglia has been reported to activate astrocytes and causes orofacial mechanical allodynia. However, how C1q-induced phenotypic alterations in Vc astrocytes are involved in orofacial pain remains to be elucidated. Intracisternal administration of C1q caused mechanical allodynia in the whisker pad skin and concurrent significant upregulation of glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 in the Vc. Immunohistochemical analyses clarified that C1q induces a significant increase in the cytokine interleukin (IL)-1ß, predominantly in Vc astrocytes and partially in Vc microglia. The number of c-Fos-positive neurons in the Vc increased significantly in response to C1q. IL-1 receptor antagonist (IL-1Ra) was used to analyze the involvement of IL-1ß in C1q-induced mechanical allodynia. Intracisternal administration of IL-1Ra ameliorated C1q-induced orofacial mechanical allodynia. The present findings suggest that IL-1ß released from activated astrocytes and microglia in the Vc mediates C1q-induced orofacial pain.

Decreased PPARgamma in the trigeminal spinal subnucleus caudalis due to neonatal injury contributes to incision-induced mechanical allodynia in female rats.

Whisker pad skin incision in infancy causes the prolongation of mechanical allodynia after re-incision in adulthood. A recent study also proposed the importance of sex differences in pain signaling in the spinal cord. However, the sex difference in re-incision-induced mechanical allodynia in the orofacial region is not fully understood. In the rats that experienced neonatal injury in the whisker pad skin, the mechanical allodynia in the whisker pad was significantly prolonged after re-incision in adulthood compared to sham injury in infancy. No significant sex differences were observed in the duration of mechanical allodynia. The duration of mechanical allodynia in male rats was shortened by intracisternal administration of minocycline. However, minocycline had no effects on the duration of mechanical allodynia in female rats. In contrast, intracisternal administration of pioglitazone markedly suppressed mechanical allodynia in female rats after re-incision. Following re-incision, the number of peroxisome proliferator-activated receptor gamma (PPARgamma)-positive cells were reduced in the trigeminal spinal subnucleus caudalis (Vc) in female rats that experienced neonatal injury. Immunohistochemical analyses revealed that PPARgamma was predominantly expressed in Vc neurons. Pioglitazone increased the number of PPARgamma-positive Vc neurons in female rats whose whisker pad skin was incised in both infancy and adulthood stages. Pioglitazone also upregulated heme oxygenase 1 and downregulated NR1 subunit in the Vc in female rats after re-incision. Together, PPARgamma signaling in Vc neurons is a female-specific pathway for whisker pad skin incision-induced mechanical allodynia.

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.

Butyric acid modulates periodontal nociception in Porphyromonas gingivalis-induced periodontitis.

PURPOSE: Periodontitis progresses with chronic inflammation, without periodontal pain. However, the underlying mechanisms are not well known. Here, the involvement of butyric acid (BA) in periodontal pain sensitivity in Porphyromonas gingivalis (P. gingivalis)-induced periodontitis was examined. METHODS: P. gingivalis was inoculated into the ligature which was tied around the molar (P. gingivalis-L) and the gingival mechanical head withdrawal threshold (MHWT) was measured. Following P. gingivalis-L, the expressions of orphan G protein-coupled receptor 41 (GPR41) in trigeminal ganglion (TG) neurons were examined. The amount of gingival BA was analyzed following the P. gingivalis-L and the changes in the MHWT in complete Freund's adjuvant (CFA)-injected gingival tissue by gingival BA were examined. The changes in the MHWT following P. gingivalis-L by gingival GPR41 antagonist (HA) were examined. RESULTS: No change in the MHWT was observed, GPR41-immunoreactive TG neurons were increased following P. gingivalis-L. The gingival BA amount increased following P. gingivalis-L, and the gingival BA suppressed the decrease in MHWT following CFA. HA decreased MHWT following P. gingivalis-L. CONCLUSION: Gingival BA modulates periodontal mechanical nociception via GPR41 signaling in P. gingivalis-L-induced periodontitis.

Periodontal acidification contributes to tooth pain hypersensitivity during orthodontic tooth movement.

Tooth movements associated with orthodontic treatment often cause tooth pain. However, the detailed mechanism remains unclear. Here, we examined the involvement of periodontal acidification caused by tooth movement in mechanical tooth pain hypersensitivity. Elastics were inserted between the first and second molars to move the teeth in Sprague-Dawley rats. Mechanical head-withdrawal reflex threshold to first molar stimulation and the pH of the gingival sulcus around the tooth were measured. The expression of acid-sensing ion channel 3 (ASIC3) in trigeminal ganglion neurons and phosphorylation of ASIC3 in the periodontal tissue were analyzed. The mechanical head-withdrawal reflex threshold to first molar stimulation and pH in the gingival sulcus decreased on day 1 after the elastic insertion. These decreases recovered to the sham level by buffering periodontal acidification. Periodontal inhibition of ASIC3 channel activity reversed the decreased mechanical head-withdrawal reflex threshold to first molar stimulation. On day 1 after elastic insertion, the tooth movement did not change the number of ASIC3 immunoreactive trigeminal ganglion neurons innervating the periodontal tissue but increased phosphorylated-ASIC3 levels in the periodontal tissue. Periodontal acidification induced by tooth movement causes phosphorylation of ASIC3, resulting in mechanical pain hypersensitivity in mechanically forced tooth.

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.

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.

Rapamycin Accelerates Axon Regeneration Through Schwann Cell-mediated Autophagy Following Inferior Alveolar Nerve Transection in Rats.

Sensory disturbance in the orofacial region owing to trigeminal nerve injury is caused by dental treatment or accident. Commercially available therapeutics are ineffective for the treatment of sensory disturbance. Additionally, the therapeutic effects of rapamycin, an allosteric inhibitor of mammalian target of rapamycin (mTOR), which negatively regulates autophagy, on the sensory disturbance are not fully investigated. Thus, we investigated the therapeutic effects of rapamycin on the sensory disturbance in the mandibular region caused by inferior alveolar nerve (IAN) transection (IANX) in rats. The expression levels of the phosphorylated p70S6K, a downstream molecule of mTOR, in the proximal and distal stumps of the transected IAN were significantly reduced by rapamycin administration to the injured site. Conversely, the increments of both Beclin 1 and microtubule-associated protein-1 light chain 3-II protein levels in the proximal and distal stumps of the transected IAN was induced by rapamycin administration. Immunohistochemical analyses revealed that Beclin 1 was located in Schwann cells in the proximal stump of the IAN. Accumulation of myelin protein zero and myelin basic protein in the proximal and distal stumps of the IAN was significantly reduced by rapamycin administration. Rapamycin administration facilitated axon regeneration after IANX and increased the number of brain-derived neurotrophic factor positive neurons in the trigeminal ganglion. Thus, recovery from sensory disturbance in the lower lip caused by IANX was markedly facilitated by rapamycin. These findings suggest that rapamycin administration is a promising treatment for the sensory disturbance caused by IANX.

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.

Microglial activation in the trigeminal spinal subnucleus interpolaris/caudalis modulates orofacial incisional mechanical pain hypersensitivity associated with orofacial injury in infancy.

PURPOSE: Infantile tissue injury induces sensory deficits in adulthood. Infantile facial incision (IFI) was reported to cause an enhancement of incision-induced mechanical hypersensitivity in adulthood due to acceleration of the trigeminal ganglion neuronal excitability. However, the effects of IFI on activation of microglia in the spinal trigeminal nucleus and its involvement in facial pain sensitivity is not well known. METHODS: A facial skin incision was made in the left whisker pad in infant (IFI) and/or adult rats (AFI). Mechanical head withdrawal threshold and microglial activation in the trigeminal spinal nucleus were analyzed. RESULTS: Mechanical pain hypersensitivity induced by AFI was significantly exacerbated and prolonged by IFI. The number of Iba1-immunoreactive cells in the trigeminal spinal nucleus following AFI was increased by IFI, suggesting that IFI facilitates microglial hyperactivation following AFI. Intraperitoneal administration of minocycline, a microglial activation inhibitor, suppressed the facial incision-induced microglial hyperactivation in the trigeminal spinal nucleus and the exacerbation of the facial mechanical pain hypersensitivity induced by IFI. CONCLUSION: These results suggest that facial trauma in infants causes hyperactivation of microglia in the trigeminal spinal nucleus following AFI, leading to the prolongation of the facial mechanical pain hypersensitivity.

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.

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.