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Objective: The efficacy of melatonin as an analgesic agent has been well documented in animals and humans. However, the underlying mechanisms by which melatonin exerts antinociceptive effects on inflammatory pain are poorly understood. Here, we investigated the potential of melatonin to ameliorate inflammatory pain., Materials and Methods: In vitro, ND7/23 neurons were treated with capsaicin. We used PCR and Western blot analyses to detect the expression of neuronal nitric oxide synthase (nNOS) in response to melatonin. Orofacial inflammatory pain was induced by 4% formalin administration on the right whisker pad of Sprague Dawley (SD) rats. The analgesic effect of melatonin was evaluated using mechanical threshold analyses. The expression level of nNOS in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (Vc) neurons was assessed by RNAscope and immunohistochemistry., Results: In vitro, capsaicin upregulated the expression of nNOS, which was dose-dependently reversed by melatonin pretreatment (p < 0.001). In a rat model of orofacial inflammatory pain, melatonin pretreatment significantly attenuated mechanical allodynia in both the acute and chronic phases (p < 0.05). Furthermore, melatonin decreased the formalin-evoked elevated nNOS mRNA and protein levels in the TG and Vc neurons in the acute and chronic phases (p < 0.05)., Conclusions: Taken together, these results suggest that nNOS may play an active role in both peripheral and central processing of nociceptive information following orofacial inflammatory pain induction. The regulatory effect of melatonin on nNOS in inflammatory pain may have potential implications for the development of novel analgesic strategies.

Hydrogen sulfide (H 2 S) is an endogenous neuromodulator produced mainly by the enzyme cystathionine gamma-lyase (CSE) in peripheral tissues. A pronociceptive role of endogenously produced H 2 S has been previously reported by our group in a model of orofacial inflammatory pain. Using the established persistent orofacial pain rat model induced by complete Freund's adjuvant (CFA) injection into temporomandibular joint (TMJ), we have now investigated the putative role of endogenous H 2 S modulating hypernociceptive responses. Additionally, plasmatic extravasation on TMJ was measured following different treatments by Evans blue dye quantification. Thus, rats were submitted to Von Frey and Formalin tests in orofacial region before and after pharmacological inhibition of the CSE-H 2 S system combined or not with CFA-induced TMJ inflammation. Pretreatment with CSE inhibitor, propargylglycine (PAG; 88.4 μmol/kg) reduced temporomandibular inflammatory pain when injected locally as well as systemically. In particular, local PAG injection seems to be more effective for hypernociceptive responses in orofacial persistent inflammation since its action is evidenced in the majority analyzed periods of the inflammatory process compared to its systemic use. Moreover, local injection seems to act on temporomandibular vascular permeability, evidenced by decreased plasmatic extravasation induced by local PAG administration. Our data are consistent with the notion that the endogenous synthetized gas H 2 S modulates persistent orofacial pain responses revealing the pharmacological importance of the CSE inhibitor as a possible therapeutic target for their control., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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

There is evidence that low-grade inflammation may be responsible for pain and development of degenerative changes in temporomandibular joint internal derangement. This article reviews the current knowledge of the molecular mechanisms behind TMJ internal derangements. A non-systematic search was carried out in PubMed, Embase and the Cochrane library for studies regarding pathophysiological mechanisms behind internal derangements focusing on pain-mediating inflammatory and cartilage-degrading molecules. Recent data suggest that release of cytokines may be the key event for pain and cartilage destruction in TMJ internal derangements. Cytokines promote the release of matrix metalloproteinases (MMPs), and due to hypoxia, vascular endothelial growth factor (VEGF) is released. This activates chondrocytes to produce MMPs and reduce their tissue inhibitors (TIMPs) as well as the recruitment of osteoclasts, ultimately leading to cartilage and bone resorption. Also, proteoglycans have an important role in this process. Several cytokines, MMPs, TIMPs and VEGF have been identified in higher concentrations in the TMJ synovial fluid of patients with painful internal derangements and shown to be associated with the degree of degeneration. Other molecules that show elevated levels include hyaluronic acid synthase, disintegrin and metalloproteinase with thrombospondin motifs (ADAMTs), aggrecan, fibromodulin, biglycan and lumican. Taken together, more or less pronounced inflammation of TMJ structures with release of cytokines, MMPs and other molecular markers that interact in a complex manner may be responsible for tissue degeneration in internal derangements. As internal derangements may be symptom-free, the degree of inflammation, but also other mechanisms, may be important for pain development., (© 2017 John Wiley & Sons Ltd.)

This study was to investigate the role of p38 activation via ERK1/2 phosphorylation in neurons and microglia of the spinal trigeminal subnucleus caudalis (Vc) in the promotion of orofacial hyperalgesia induced by unilateral anterior crossbite (UAC) traumatic occlusion in adult rats. U0126, a p-ERK1/2 inhibitor, was injected intracisternally before UAC implant. The effects of the U0126 injection were compared to those following the injection of SB203580, a p-p38 inhibitor. Mechanical hyperalgesia was evaluated via pressure pain threshold measurements. Brain stem tissues were processed for a Western blot analysis to evaluate the activation of ERK1/2 and p38. Double immunofluorescence was also performed to observe the expression of p-ERK1/2 and p-p38 in neurons (labeled by NeuN) and microglia (labeled by OX42). The data showed that UAC caused orofacial hyperalgia ipsilaterally on d1 to d7, peaking on d3 (P<0.05). An upregulation of p-ERK1/2 was observed in the ipsilateral Vc on d1 to d3, peaking on d1. An upregulation of p-p38 was also observed on d1 to d7, peaking on d3 (P<0.05). p-ERK1/2 primarily co-localized with NeuN and, to a lesser extent, with OX42, while p-p38 co-localized with both NeuN and OX42. Pretreatment with U0126 prevented the upregulation of both p-ERK1/2 and p-p38. Similarly to an intracisternal injection of SB203580, U0126 pretreatment attenuated the UAC-induced orofacial hyperalgesia. These data indicate that UAC caused orofacial hyperalgesia by inducing central sensitization via the activation of ERK1/2 and p38 in both neurons and microglia in the Vc, potentially impacting the effects of p-ERK1/2 during p38 activation., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Microglia are the resident immune cells in the spinal cord and brain. Mounting evidence suggests that activation of microglia plays an important role in the pathogenesis of chronic pain, including chronic orofacial pain. In particular, microglia contribute to the transition from acute pain to chronic pain, as inhibition of microglial signaling reduces pathologic pain after inflammation, nerve injury, and cancer but not baseline pain. As compared with inflammation, nerve injury induces much more robust morphologic activation of microglia, termed microgliosis, as shown by increased expression of microglial markers, such as CD11b and IBA1. However, microglial signaling inhibitors effectively reduce inflammatory pain and neuropathic pain, arguing against the importance of morphologic activation of microglia in chronic pain sensitization. Importantly, microglia enhance pain states via secretion of proinflammatory and pronociceptive mediators, such as tumor necrosis factor α, interleukins 1β and 18, and brain-derived growth factor. Mechanistically, these mediators have been shown to enhance excitatory synaptic transmission and suppress inhibitory synaptic transmission in the pain circuits. While early studies suggested a predominant role of microglia in the induction of chronic pain, further studies have supported a role of microglia in the maintenance of chronic pain. Intriguingly, recent studies show male-dominant microglial signaling in some neuropathic pain and inflammatory pain states, although both sexes show identical morphologic activation of microglia after nerve injury. In this critical review, we provide evidence to show that caspase 6-a secreted protease that is expressed in primary afferent axonal terminals surrounding microglia-is a robust activator of microglia and induces profound release of tumor necrosis factor α from microglia via activation of p38 MAP kinase. The authors also show that microglial caspase 6/p38 signaling is male dominant in some inflammatory and neuropathic pain conditions. Finally, the authors discuss the relevance of microglial signaling in chronic trigeminal and orofacial pain., Competing Interests: The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article., (© International & American Associations for Dental Research 2016.)

Aims: To explore the possible relationship between protein kinase C gamma (PKCγ) and phosphorylated forms of extracellular signal-regulated kinases 1/2 (pERK1/2) in the rat medullary dorsal horn and the facial hypersensitivity indicative of dynamic mechanical allodynia (DMA) following chronic constriction of the infraorbital nerve (CCI-IoN)., Methods: A well-established rat model of trigeminal neuropathic pain involving CCI-IoN was used. Facial mechanical hypersensitivity was tested with non-noxious dynamic mechanical stimulation (air-puff), and the medullary dorsal horn was examined immunohistochemically using PKCγ and pERK1/2 as pain markers. Statistical analysis was performed using Student t test or one-way analysis of variance (ANOVA)., Results: Increased PKCγ and pERK1/2 expressions within the medullary dorsal horn were associated with DMA following CCI-IoN. A segmental network composed of PKCγ-positive cells located in medullary dorsal horn laminae II/III, contacting more superficially located pERK1/2-expressing cells, was identified. Ultrastructural analysis confirmed the presence of PKCγ to pERK1/2-positive cells. Moreover, intracisternal administration of the selective PKCγ inhibitor KIG31-I blocked both the DMA and pERK1/2 expression in a dose-dependent manner. Although the number of pERK1/2-positive cells was significantly elevated with air-puff stimulation, DMA rats not receiving air-puff stimulation showed significant pERK1/2 expression, suggesting they were experiencing spontaneous pain., Conclusion: PKCγ cells in the medullary dorsal horn may be involved in DMA following CCI-IoN through the activation of pERK1/2-expressing cells, which then may relay non-nociceptive information to lamina I cells in the medullary dorsal horn.

Background: Orofacial inflammatory pain is likely to accompany referred pain in uninflamed orofacial structures. The ectopic pain precludes precise diagnosis and makes treatment problematic, because the underlying mechanism is not well understood. Using the established ectopic orofacial pain model induced by complete Freund's adjuvant (CFA) injection into trapezius muscle, we analyzed the possible role of p38 phosphorylation in activated microglia in ectopic orofacial pain., Results: Mechanical allodynia in the lateral facial skin was induced following trapezius muscle inflammation, which accompanied microglial activation with p38 phosphorylation and hyperexcitability of wide dynamic range (WDR) neurons in the trigeminal spinal subnucleus caudalis (Vc). Intra-cisterna successive administration of a p38 mitogen-activated protein kinase selective inhibitor, SB203580, suppressed microglial activation and its phosphorylation of p38. Moreover, SB203580 administration completely suppressed mechanical allodynia in the lateral facial skin and enhanced WDR neuronal excitability in Vc. Microglial interleukin-1β over-expression in Vc was induced by trapezius muscle inflammation, which was significantly suppressed by SB203580 administration., Conclusions: These findings indicate that microglia, activated via p38 phosphorylation, play a pivotal role in WDR neuronal hyperexcitability, which accounts for the mechanical hypersensitivity in the lateral facial skin associated with trapezius muscle inflammation.

Orofacial inflammatory pain is a difficult clinical problem, and the specific molecular mechanisms for this pain remain largely unexplained. The present study aimed to determine the differential expression of microRNAs (miRNAs) and disclose the underlying role of miR-125a-3p in orofacial inflammatory pain induced by complete Freund's adjuvant (CFA). Thirty-two differentially expressed miRNAs were first screened using a microarray chip in ipsilateral trigeminal ganglions (TGs) following CFA injection into the orofacial skin innervated by trigeminal nerve, and a portion of them, including miR-23a*, -24-2*, -26a, -92a, -125a-3p, -183 and -299 were subsequently selected and validated by qPCR. The target genes were predicted based on the miRWalk website and were further analyzed by gene ontology (GO). Further studies revealed miR-125a-3p expression was down-regulated, whereas both the expression of p38 MAPK (mitogen-activated protein kinase) alpha and CGRP (calcitonin gene-related peptide) were up-regulated in ipsilateral TGs at different time points after CFA injection compared with control. Furthermore, mechanistic study revealed that miR-125a-3p negatively regulates p38 alpha gene expression and is positively correlated with the head withdrawal threshold reflecting pain. Luciferase assay showed that binding of miR-125a-3p to the 3'UTR of p38 alpha gene suppressed the transcriptional activity, and overexpression of miR-125a-3p significantly inhibited the p38 alpha mRNA level in ND8/34 cells. Taken together, our results show that miR-125a-3p is negatively correlated with the development and maintenance of orofacial inflammatory pain via regulating p38 MAPK.

Background: Cyclin-dependent kinase 5 (Cdk5) is a unique member of the serine/threonine kinase family. This kinase plays an important role in neuronal development, and deregulation of its activity leads to neurodegenerative disorders. Cdk5 also serves an important function in the regulation of nociceptive signaling. Our previous studies revealed that the expression of Cdk5 and its activator, p35, is upregulated in nociceptive neurons during peripheral inflammation. The aim of the present study was to characterize the involvement of Cdk5 in orofacial pain. Since mechanical hyperalgesia is the distinctive sign of many orofacial pain conditions, we adapted an existing orofacial stimulation test to assess the behavioral responses to mechanical stimulation in the trigeminal region of the transgenic mice with either reduced or increased Cdk5 activity., Results: Mice overexpressing or lacking p35, an activator of Cdk5, showed altered phenotype in response to noxious mechanical stimulation in the trigeminal area. Mice with increased Cdk5 activity displayed aversive behavior to mechanical stimulation as indicated by a significant decrease in reward licking events and licking time. The number of reward licking/facial contact events was significantly decreased in these mice as the mechanical intensity increased. By contrast, mice deficient in Cdk5 activity displayed mechanical hypoalgesia., Conclusions: Collectively, our findings demonstrate for the first time the important role of Cdk5 in orofacial mechanical nociception. Modulation of Cdk5 activity in primary sensory neurons makes it an attractive potential target for the development of novel analgesics that could be used to treat multiple orofacial pain conditions.

This study examined the participation of central mitogen-activated protein kinases (MAPKs) in the central sensitization produced by a subcutaneous injection of interleukin-1β (IL-1β) in male Sprague-Dawley rats. Formalin-induced responses were evaluated 24h after an IL-1β injection. A subcutaneous injection of 10ng of IL-1β elevated the formalin-induced scratching response significantly in the second phase compared to the vehicle-treated group. Pretreatment with an IL-1 receptor antagonist reduced the IL-1β-induced sensitization. Pretreatment with IL-1β increased the p-ERK and p-p38 expression induced by the formalin injection. Double immunofluorescence data revealed increases in phospho-extracellular signal-regulated kinase (p-ERK) immunoreactive cells that co-localize with neuronal nuclei (NeuN), a neuronal marker, and in phospho-p38 (p-p38) immunoreactive cells that co-localize with NeuN and OX42, a microglia marker. The intracisternal administration of minocycline (50μg), a microglia inhibitor, attenuated the increased formalin-induced scratching responses in the IL-1β-treated rats. The intracisternal administration of PD98059 (1, 10μg), a MEK inhibitor, and SB203580 (1, 5μg), a p38 inhibitor, also attenuated the number of formalin-induced scratches in the second phase in the IL-1β-treated rats. These results suggest that the IL-1β-induced central sensitization of nociception is mediated by the central MAPK pathways, which are activated differentially in the neurons and microglia under inflammatory pain conditions. Therefore, blockade of the MAPK pathways can be as a potential therapeutic target for the central sensitization of inflammatory pain., (© 2013 Elsevier Inc. All rights reserved.)

Unlabelled: We previously developed a rat dental occlusal interference model of facial pain that was produced by bonding a crown onto the right maxillary first molar and was reflected in sustained facial hypersensitivity that was suggestive of the involvement of central sensitization mechanisms. The aim of the present study was to investigate potential central mechanisms involved in the occlusal interference-induced facial hypersensitivity. A combination of behavioral, immunohistochemical, Western blot, and electrophysiological recording procedures was used in 98 male adult Sprague Dawley rats that either received the occlusal interference or were sham-operated or naive rats. Immunohistochemically labeled astrocytes and microglia in trigeminal subnucleus caudalis (Vc) showed morphological changes indicative of astrocyte and microglial activation after the occlusal interference. Prolonged upregulation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) was also documented in Vc after placement of the occlusal interference and was expressed in both neurons and glial cells at time points when rats showed peak mechanical facial hypersensitivity. The intrathecal administration of the p38 MAPK inhibitor SB203580 to the medulla significantly inhibited the occlusal interference-induced hypersensitivity, and the ERK inhibitor PD98059 produced an even stronger effect. Central sensitization of functionally identified Vc nociceptive neurons following placement of the occlusal interference was also documented by extracellular electrophysiological recordings, and intrathecal administration of PD98059 could reverse the neuronal central sensitization. These novel findings suggest that central mechanisms including central sensitization of trigeminal nociceptive neurons and non-neuronal processes involving MAPKs play significant roles in the production of occlusal interference-induced facial pain., Perspective: Central mechanisms including trigeminal nociceptive neuronal sensitization, non-neuronal processes involving glial activation, and MAPKs play significant roles in occlusal interference-induced facial pain. These mechanisms may be involved in clinical manifestations of facial pain that have been reported in patients with an occlusal interference., (Copyright © 2013 American Pain Society. Published by Elsevier Inc. All rights reserved.)

The aim of the present study was to clarify the involvement of protein kinase Cγ (PKCγ) in the facial neuropathic pain following infraorbital nerve injury. We analyzed the change in PKCγ expression in the trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1/C2) following chronic constriction injury of the infraorbital nerve (ION-CCI). We also studied ION-CCI-mediated mechanical nocifensive behavior in rats. The mechanical head-withdrawal threshold significantly decreased 1 to 14 days after ION-CCI compared with that before ION-CCI and in sham rats. The expression of PKCγ was significantly larger in the ipsilateral Vc compared with the contralateral side in ION-CCI rats 3, 7, and 14 days after ION-CCI. Intrathecal (i.t.) administration of the PKCγ inhibitor chelerythrine prevented an increase in the PKCγ expression in the ipsilateral Vc. Moreover, i.t. administration of chelerythrine annulled ION-CCI-mediated reduction in the head-withdrawal threshold. Taken together, these findings suggest that PKCγ expression in the Vc played an important role in the mechanism of orofacial static mechanical allodynia following trigeminal nerve injury.

Pain is among the major problems during orthodontic treatment. Recent studies have shown that central Cyclooxygenase2 (COX2) pathway was involved in several pain models. The present study investigated whether inducible COX2 within the trigeminal nucleus caudalis (Vc) contributed to experimental tooth movement pain in freely moving rats. Elastic rubber bands were inserted between the first and second maxillary molars bilaterally to establish tooth movement model. The directed mouth wiping behavior was used to evaluate the pain during tooth movement. COX2 distribution in Vc was studied by immunohistochemistry and the changes of COX2 expression were detected by Western blot at different time point after rubber band insertion. Our results showed that tooth movement significantly increased COX2 expression in Vc and the time spent on mouth wiping, reaching a maximum at 1 day and then decreasing gradually. Furthermore, the rhythm change of COX2 expression in Vc and the mouth wiping behavior were much correlative with each other. All of the COX2-immunoreactive structures in Vc exhibited NeuN-immunopositive staining and most of these COX2-immunoreactive neurons were Fos-immunopositive. Importantly, the mouth wiping behavior could be attenuated by intracisternal injection of NS-398 (a selective COX2 inhibitor) but not by periodontal administration of NS-398. All these results suggested that increased COX2 in Vc was involved in tooth movement pain and thus may be a central target for orthodontic pain treatment.

The present study was carried out, using inhibitors to secretory phospholipase A2 (sPLA2, 12-epi-scalaradial), cytosolic phospholipase A2 (cPLA2, AACOCF3), or calcium-independent phospholipase A2 (iPLA2, bromoenol lactone), to compare possible contributions of central nervous PLA2 isoforms to the development of allodynia after facial carrageenan injection in mice. C57BL/6J (B6) mice showed increased responses to facial stimulation using a von Frey hair (1 g force), at 8 h, 1 day, and 3 days after facial carrageenan injection. On the other hand, BALB/c mice did not show increased responses at any of the time points. In both B6 and BALB/c mice, intracerebroventricular injection of inhibitors to each of the three PLA2 isoforms significantly reduced responses to von Frey hair stimulation at 8 h and 1 day after facial carrageenan injection, but at 3 days after injection, only the sPLA2 inhibitor had an effect. Since BALB/c mice did not show increased responses after facial carrageenan injection, the reduction in responses actually indicates that there is loss of normal sensitivity to von Frey hair stimulation after intracerebroventricular injection of each of these inhibitors, in this strain of mice. The effects of PLA2 inhibitors are unlikely to be due simply to inhibition of arachidonic acid generation, since intracerebroventricular injection of arachidonic acid also had an anti-nociceptive effect. The above results support an important role of central nervous PLA2s in neurotransmission and pain transmission.

The present study was performed to investigate effects of central cyclooxygenase (COX) on interleukin (IL)-1beta-induced hyperalgesia in the orofacial area. Experiments were carried out on 72 male Sprague-Dawley rats weighing 220-280 g. Surgical procedures were performed under pentobarbital sodium. We examined noxious behavioral scratching responses induced by 50 microl of 5% formalin injected subcutaneously into the vibrissa pad without any restraints. The orofacial formalin responses exhibited two distinct phases with early responses (0-10 min) and continuous prolonged responses (11-45 min). Intracisternal injection of 100 pg IL-1beta significantly increased noxious behavioral responses. Pretreatment with indomethacin, a non-selective COX inhibitor, or NS-398, a selective COX-2 inhibitor, blocked IL-1beta-induced hyperalgesic responses. However, pretreatment with SC-560, a selective COX-1 inhibitor, did not change hyperalgesic response to IL-1beta. These data suggest that central IL-1beta modulates the transmission of nociceptive information in the orofacial area and that central COX-2 plays an important role in IL-1beta-induced hyperalgesia.

We measured matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP) in temporomandibular joint (TMJ) disorders. All patients were fully investigated, by visual analogue pain scores, plain radiographs and double-contrast arthrotomograms. The patients were grouped according to whether or not they were in pain. There were 35 patients with painful joints; 16 painless crepitating joints and 10 with chronic closed locked joints. A further group of 9 volunteers with no symptoms of TMJ abnormalities were used as controls. We found that synovial fluid concentration of MMP-3 was significantly increased (1117.2 (164.0) ng/ml) (P< 0.05) in the painful group compared with controls (436.2 (94.8) ng/ml) and with the two groups with painless TMJ (475.0 (113. 0) ng/ml/crepitation, and 516.0 (115.1) ng/ml/closed locked joints). MMP-1 and TIMP-1 were not recordable in most joints, and the serum concentrations of MMP-1, MMP-3, and TIMP-1 were similar to those in controls in all groups. There was no correlationship between MMP-3 concentration and joint morphology as shown by plain radiographs and double contrast arthrotomograms. These findings indicate that the synovia of painful joints are inflamed., (Copyright 2000 The British Association of Oral and Maxillofacial Surgeons.)