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The present study investigated the participation of D-serine and NR2 in antinociception produced by blockade of central erythropoietin-producing hepatocellular carcinoma (Eph) A4 (EphA4) signaling in rats with trigeminal neuropathic pain. Trigeminal neuropathic pain was modeled in male Sprague-Dawley rats using mal-positioned dental implants. The left mandibular second molar was extracted under anesthesia, and a miniature dental implant was placed to induce injury to the inferior alveolar nerve. Our current findings showed that nerve injury induced by malpositioned dental implants significantly produced mechanical allodynia; additionally, the inferior alveolar nerve injury increased the expression of D-serine and NR2 subunits in the ipsilateral medullary dorsal horn (trigeminal subnucleus caudalis). Intracisternal administration of EphA4-Fc, an EphA4 inhibitor, inhibited nerve injury-induced mechanical allodynia and upregulated the expression of D-serine and NR2 subunits. Moreover, intracisternal administration of D-amino acids oxidase, a D-serine inhibitor, inhibited trigeminal mechanical allodynia. These results show that D-serine and NR2 subunit pathways participate in central EphA4 signaling after an inferior alveolar nerve injury. Therefore, blockade of D-serine and NR2 subunit pathways in central EphA4 signaling provides a new therapeutic target for the treatment of trigeminal neuropathic pain.

Background Although the Eph receptor plays an important role in the development of neuropathic pain following nerve injury, there has been no evidence of the participation of the ephrin A4 receptor (EphA4) in the development of trigeminal neuropathic pain. The present study investigated the role of EphA4 in central nociceptive processing in rats with inferior alveolar nerve injury. Materials and methods Male Sprague-Dawley rats were used in all our experiments. A rat model for trigeminal neuropathic pain was produced using malpositioned dental implants. The left mandibular second molar was extracted under anesthesia, followed by the placement of a miniature dental implant to injure the inferior alveolar nerve. Results Our current findings show that nerve injury induced by malpositioned dental implants evokes significant mechanical allodynia and up-regulation of EphA4 expression in the ipsilateral trigeminal subnucleus caudalis. Although daily treatment with EphA4-Fc, an EphA4 antagonist, did not produce prolonged anti-allodynic effects after the chronic neuropathic pain had been already established, an early treatment protocol with repeated EphA4-Fc administration significantly attenuated mechanical allodynia before initiation of chronic neuropathic pain. Finally, we confirmed the participation of the central EphA4 pathway in the development of trigeminal neuropathic pain by reducing EphA4 expression using EphA4 siRNA. This suppression of EphA4 produced significantly prolonged anti-allodynic effects. Conclusion These results suggest that early blockade of central EphA4 signaling provides a new therapeutic target for the treatment of trigeminal neuropathic pain.

Recent studies implicate glutamate release from satellite glial cells (SGCs) surrounding the primary sensory neurons in the mechanisms of pathologic pain. However, little is known about the population of SGCs in the trigeminal ganglion that is involved in glutamate signaling associated with craniofacial neuropathic pain. To address this issue, we used quantitative analysis of electron microscopic immunogold labeling to investigate the changes in glutamate levels in trigeminal neurons and their enveloping SGCs in a rat model of craniofacial neuropathic pain, chronic constriction injury of inferior alveolar nerve (CCI-ION). The density of immunogold, a measure for glutamate concentration, in the neuronal cell bodies of all sizes, and in the SGCs surrounding them, was significantly higher in rats with CCI-ION than in sham-operated rats. This effect was more pronounced for the small neurons (2.2 times higher) and their SGCs (1.8 times higher) than for the medium and the large neurons and their SGCs, respectively. These findings suggest that each populations of SGCs and their surrounding trigeminal neurons of different type are involved in the glutamate signaling associated with neuropathic pain at a different level.

In this retrospective multicentre cohort study that included 27 COVID-19 patients who underwent tracheostomy, the mean time between intubation and tracheostomy was 15.8 days and the negative conversion time of COVID-19 was 43.1 days. Eleven patients (40.7%) died of COVID-19 and the use of percutaneous dilatation tracheostomy was significantly associated with in-hospital death. Timely tracheostomy could be performed in COVID-19 patients, regardless of duration of intubation or positivity of COVID-19 test, with an open surgical tracheostomy as a preferable technique.

Mannitol has recently been reported to be effective in enhancing the antinociceptive efficacy of lidocaine. No single study to date, however, has compared diphenhydramine with and without mannitol for nociceptive processing as an alternative local anesthetic. In this study, we examined the antinociceptive efficacy enhancements of diphenhydramine when combined with mannitol. Male Sprague-Dawley rats weighing 230–260 g were used in a hot plate test to evaluate the antinociceptive effects of diphenhydramine. All chemicals were dissolved in isotonic normal saline and administered subcutaneously into the plantar surface of the right hind paw at 10 min before the hot plate test. A subcutaneous injection of 0.5% or 1% diphenhydramine produced significant inhibition of the withdrawal latency time compared with the vehicle treatment. Antinociceptive effects appeared 10 min after the diphenhydramine injections and persisted for over 30 min. The antinociceptive effects of 1% diphenhydramine were not statistically different from those of 1% lidocaine. Although a subcutaneous injection of a 0.5 M mannitol solution alone did not affect the withdrawal latency time, 1% diphenhydramine with 0.5 M mannitol significantly enhanced antinociception. A subcutaneous injection of 1% diphenhydramine with epinephrine (1 : 100,000) solution did not increase the antinociceptive effect of the diphenhydramine. These results suggest that diphenhydramine with mannitol can be used as an alternative local anesthetic.

Parvalbumin (PV), calretinin (CR), calbindin D-28k (CB), stage specific embryonic antigen-4 (SSEA4), and phosphorylated neurofilament 200 (pNF200) have been commonly used as markers for primary afferent neurons with large myelinated (A) fibers but detailed information on the expression of these markers in specific primary afferent fiber types is still lacking. We here examined the fibers that express PV, CR, CB, SSEA4, and pNF200 in the trigeminal ganglion and its peripheral sensory root by light- and electron-microscopic immunohistochemistry and quantitative analysis. We found that all CR-immunopositive (+), CB+, and SSEA4+ fibers and virtually all (98.8%) PV+ fibers were myelinated, most CR+ fibers were large myelinated, whereas most CB+ and SSEA4+ fibers were small myelinated. One half of the PV+ fibers were small myelinated and the other half were large myelinated. Of all pNF200+ fibers, about a third each were small myelinated, large myelinated, and unmyelinated. These findings suggest that PV, CR, CB, and SSEA4 can be used as specific markers for primary afferent neurons with myelinated fibers, but that pNF200 is not suitable as a specific marker for primary afferent neurons with myelinated fibers, and also raise the possibility that PV, CR, CB, and SSEA4 may be expressed in both mechanoreceptive and nociceptive neurons.

The aim of the present study was to examine the effects of preemptive analgesia on the development of trigeminal neuropathic pain. For this purpose, mechanical allodynia was evaluated in male Sprague-Dawley rats using chronic constriction injury of the infraorbital nerve (CCI-ION) and perineural application of 2% QX-314 to the infraorbital nerve. CCI-ION produced severe mechanical allodynia, which was maintained until postoperative day (POD) 30. An immediate single application of 2% QX-314 to the infraorbital nerve following CCI-ION significantly reduced neuropathic mechanical allodynia. Immediate double application of QX-314 produced a greater attenuation of mechanical allodynia than a single application of QX-314. Immediate double application of 2% QX-314 reduced the CCI-ION-induced upregulation of GFAP and p-p38 expression in the trigeminal ganglion. The upregulated p-p38 expression was co-localized with NeuN, a neuronal cell marker. We also investigated the role of voltage-gated sodium channels (Navs) in the antinociception produced by preemptive application of QX-314 through analysis of the changes in Nav expression in the trigeminal ganglion following CCI-ION. Preemptive application of QX-314 significantly reduced the upregulation of Nav1.3, 1.7, and 1.9 produced by CCI-ION. These results suggest that long-lasting blockade of the transmission of pain signaling inhibits the development of neuropathic pain through the regulation of Nav isoform expression in the trigeminal ganglion. Importantly, these results provide a potential preemptive therapeutic strategy for the treatment of neuropathic pain after nerve injury.

The purinergic receptor P2X3, expressed in the central terminals of primary nociceptive neurons in the brainstem, plays an important role in pathological pain. However, little is known about expression of P2X3 in the brainstem astrocytes and its involvement in craniofacial pathologic pain. To address this issue, we investigated the expression of P2X3 in astrocytes in the trigeminal caudal nucleus (Vc) in a rat model of craniofacial neuropathic pain, chronic constriction injury of infraorbital nerve (CCI-ION). We found that 1) P2X3-immunoreactivity is observed in the brainstem astrocytes, preferentially in their fine processes, 2) the number of P2X3-positive fine astrocytic processes and the density of P2X3 in these processes were increased significantly in CCI-ION rats, compared to control rats, and 3) administration of MPEP, a specific mGluR5 antagonist, alleviated the mechanical allodynia and abolished the increase in density of P2X3 in fine astrocytic processes caused by CCI-ION. These findings reveal preferential expression of P2X3 in the fine astrocytic processes in the brainstem, propose a novel role of P2X3 in the fine astrocytic process in the mechanism of craniofacial neuropathic pain, and suggest that the expression of astrocytic P2X3 may be regulated by astrocytic mGluR5.

The purinergic receptor P2X3, expressed in the central terminals of primary nociceptive neurons in the brainstem, plays an important role in pathological pain. However, little is known about expression of P2X3 in the brainstem astrocytes and its involvement in craniofacial pathologic pain. To address this issue, we investigated the expression of P2X3 in astrocytes in the trigeminal caudal nucleus (Vc) in a rat model of craniofacial neuropathic pain, chronic constriction injury of infraorbital nerve (CCI-ION). We found that 1) P2X3-immunoreactivity is observed in the brainstem astrocytes, preferentially in their fine processes, 2) the number of P2X3-positive fine astrocytic processes and the density of P2X3 in these processes were increased significantly in CCI-ION rats, compared to control rats, and 3) administration of MPEP, a specific mGluR5 antagonist, alleviated the mechanical allodynia and abolished the increase in density of P2X3 in fine astrocytic processes caused by CCI-ION. These findings reveal preferential expression of P2X3 in the fine astrocytic processes in the brainstem, propose a novel role of P2X3 in the fine astrocytic process in the mechanism of craniofacial neuropathic pain, and suggest that the expression of astrocytic P2X3 may be regulated by astrocytic mGluR5.

Previous studies have demonstrated that botulinum toxin type A (BoNT-A) attenuates orofacial nociception. However, there has been no evidence of the participation of the voltage-gated sodium channels (Navs) in the antinociceptive mechanisms of BoNT-A. This study investigated the cellular mechanisms underlying the antinociceptive effects of BoNT-A in a male Sprague-Dawley rat model of trigeminal neuropathic pain produced by malpositioned dental implants. The left mandibular second molar was extracted under anesthesia, followed by a miniature dental implant placement to induce injury to the inferior alveolar nerve. Mechanical allodynia was monitored after subcutaneous injection of BoNT-A at 3, 7, or 12 d after malpositioned dental implant surgery. Subcutaneous injections of 1 or 3 U/kg of BoNT-A on postoperative day 3 significantly attenuated mechanical allodynia, although 0.3 U/kg of BoNT-A did not affect the air-puff threshold. A single injection of 3 U/kg of BoNT-A produced prolonged antiallodynic effects over the entire experimental period. Treatment with BoNT-A on postoperative days 7 and 12, when pain had already been established, also produced prolonged antiallodynic effects. Double treatments with 1 U/kg of BoNT-A produced prolonged, more antiallodynic effects as compared with single treatments. Subcutaneous administration of 3 U/kg of BoNT-A significantly inhibited the upregulation of Nav isoform 1.7 (Nav1.7) expression in the trigeminal ganglion in the nerve-injured animals. These results suggest that antinociceptive effects of BoNT-A are mediated by an inhibition of upregulated Nav1.7 expression in the trigeminal ganglion. BoNT-A is therefore a potential new therapeutic agent for chronic pain control, including neuropathic pain.

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) receives nociceptive afferent inputs from thin-myelinated A[Formula: see text] fibers and unmyelinated C fibers and has been shown to be involved in the processing of orofacial nociceptive information. Scutellaria baicalensis Georgi (Huang-Qin, SbG), one of the 50 fundamental herbs of Chinese herbology, has been used historically as anti-inflammatory and antineoplastic medicine. Baicalin, one of the major compounds of SbG, has been reported to have neuroprotective, anti-inflammatory and analgesic effects. However, the receptor type activated by baicalin and its precise action mechanism on the SG neurons of Vc have not yet been studied. The whole-cell patch clamp technique was performed to examine the ion channels activated by baicalin on the SG neurons of Vc. In high Cl[Formula: see text] pipette solution, the baicalin (300[Formula: see text][Formula: see text]M) induced repeatable inward currents ([Formula: see text][Formula: see text]pA, [Formula: see text]) without desensitization on all the SG neurons tested. Further, the inward currents showed a concentration (0.1–3[Formula: see text]mM) dependent pattern. The inward current was sustained in the presence of tetrodotoxin (0.5[Formula: see text][Formula: see text]M), a voltage sensitive Na[Formula: see text] channel blocker. In addition, baicalin-induced inward currents were reduced in the presence of picrotoxin (50[Formula: see text][Formula: see text]M), a GABAAreceptor antagonist, flumazenil (100[Formula: see text][Formula: see text]M), a benzodiazepine-sensitive GABAAreceptor antagonist, and strychnine (2[Formula: see text][Formula: see text]M), a glycine receptor antagonist, respectively. These results indicate that baicalin has inhibitory effects on the SG neurons of the Vc, which are due to the activation of GABAAand/or the glycine receptor. Our results suggest that baicalin may be a potential target for orofacial pain modulation.

IL-32 is a newly discovered cytokine. Recently, various reports suggest that it plays a role as a proinflammatory mediator and may be involved in several cancer carcinogenesis. However, IL-32 expression in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated the expression and role of IL-32α in hepatocellular carcinoma, because IL-32 was identified as an upregulated gene in hepatocellular carcinoma tissues compared to nontumorous regions using DNA microarray. IL-32α was overexpressed in tissue and serum from patients with HCC and localized in the cytoplasm and nucleus of hepatocellular carcinoma tumor cells. Moreover, secreted IL-32α concentration in the serum of patients with hepatocellular carcinoma was elevated as compared with those in the normal serum using a developed sandwich ELISA. Furthermore, IL-32α suppression in hepatocellular carcinoma decreased expression of phospho-p38 MAPK, NF-κB, and antiapoptotic protein Bcl-2 and induced expression of proapoptotic proteins as well as p53 and PUMA resulting in the suppression of cell growth and induction of intrinsic apoptosis. Based on our results, we suggest that IL-32α is involved in the progression of hepatocellular carcinoma and may be a useful biomarker for diagnosis and therapeutic target of hepatocellular carcinoma.

Inhibitory and excitatory synaptic inputs onto trigeminal motoneurons play an important role in coordinating jaw movements. Previously, we reported that the phenotype of the inhibitory boutons apposing the somata of jaw-closing (JC) motoneurons changes from γ-aminobutyric acid (GABA)-positive (GABA+) to predominantly glycine-positive (Gly+) during development. In the present study, we investigated the development of inhibitory and excitatory boutons apposing antagonistic jaw-opening (JO) motoneurons (anterior digastric motoneurons) at postnatal day 2 (P2), P11, and P31 in the rat. JO motoneurons were retrogradely labeled with horseradish peroxidase. Postembedding immunogold staining with antisera against GABA, Gly, and glutamate (Glut) was performed and followed by quantitative ultrastructural analysis. The size of both small and large JO motoneurons increased during development. The number of excitatory (Glut+) and inhibitory (GABA+, Gly+, and GABA+/Gly+) boutons per JO motoneuron increased significantly from P2 to P11 and then remained unchanged until P31. The time course of inhibitory synapse formation differed between JO and JC motoneurons, whereas that of excitatory synapse formation was similar between the two neuronal populations. The fraction of GABA+ boutons decreased by 86% and the fraction of GABA+/Gly+ boutons increased by 200% from P11 to P31, suggesting a switch from GABA+ to GABA+/Gly+ phenotype. The fraction of Gly+ boutons remained unchanged. These results indicate that inhibitory synapses onto somata of JO motoneurons exhibit a developmental pattern distinct from that of synapses onto JC motoneurons, which may reflect distinctive maturation of oral motor system. J. Comp. Neurol. 520:1212–1226, 2012. © 2011 Wiley Periodicals, Inc.

Ultrastructural parameters related to synaptic release and their correlation with synaptic connectivity were analyzed in the low-threshold mechanoreceptive vibrissa afferent boutons in laminae III and IV of the trigeminal caudal nucleus (Vc). Rapidly adapting vibrissa afferents were intra-axonally labeled, and quantitative ultrastructural analyses with serial sections were performed on the labeled boutons and their presynaptic endings (p-endings). The volume of the labeled boutons was widely distributed from small to large ones (0.8~12.3 µm(3)), whereas the p-endings were small and uniform in size. The volume of the labeled boutons was positively correlated with the ultrastructural parameters such as mitochondrial volume (correlation coefficient, r=0.96), active zone area (r=0.82) and apposed surface area (r=0.79). Vesicle density (r=-0.18) showed little correlation to the volume of labeled boutons, suggesting that the total vesicle number of a bouton is proportional to its volume. In addition, the bouton volume was positively correlated with the number of p-endings (r=0.52) and with the number of dendrites postsynaptic to the labeled bouton (r=0.83). These findings suggest that low-threshold mechanoreception conveyed through vibrissa afferents is processed in a bouton size-dependent manner in the Vc, which may contribute to the sensory-motor function of laminae III/IV in Vc.

Introduction Previous studies have suggested that myelinated axons lose their myelin and become thinner in their peripheral course to the target organ. In this study, we investigated the morphologic changes of pulpal myelinated axons between their root portion (radicular pulp) and their terminal area (peripheral pulp). Methods Sections of pulp of the rat upper molar teeth were immunostained for the marker of myelinated axons neurofilament (NF) 200. The proportion of NF200+ myelinated and unmyelinated fibers and their sizes were analyzed by using quantitative electron microscopy. Results The axon area, myelin thickness, and fraction of NF200+ myelinated axons of all NF200+ axons were significantly lower in peripheral than in radicular pulp. In addition, large unmyelinated axons were frequently observed in peripheral pulp. Conclusions These results suggest that pulpal innervation originates predominantly from myelinated axons, and the myelinated axons undergo extensive morphologic changes during their course from the radicular to the peripheral pulp.

Botulinum toxin type A (BoNT/A) has been used therapeutically for various conditions including dystonia, cerebral palsy, wrinkle, hyperhidrosis and pain control. The substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc) receive orofacial nociceptive information from primary afferents and transmit the information to higher brain center. Although many studies have shown the analgesic effects of BoNT/A, the effects of BoNT/A at the central nervous system and the action mechanism are not well understood. Therefore, the effects of BoNT/A on the spontaneous postsynaptic currents (sPSCs) in the SG neurons were investigated. In whole cell voltage clamp mode, the frequency of sPSCs was increased in 18 (37.5%) neurons, decreased in 5 (10.4%) neurons and not affected in 25 (52.1%) of 48 neurons tested by BoNT/A (3 nM). Similar proportions of frequency variation of sPSCs were observed in 1 and 10 nM BoNT/A and no significant differences were observed in the relative mean frequencies of sPSCs among 1–10 nM BoNT/A. BoNT/A-induced frequency increase of sPSCs was not affected by pretreated tetrodotoxin (0.5 µM). In addition, the frequency of sIPSCs in the presence of CNQX (10 µM) and AP5 (20 µM) was increased in 10 (53%) neurons, decreased in 1 (5%) neuron and not affected in 8 (42%) of 19 neurons tested by BoNT/A (3 nM). These results demonstrate that BoNT/A increases the frequency of sIPSCs on SG neurons of the Vc at least partly and can provide an evidence for rapid action of BoNT/A at the central nervous system.

Transient receptor potential ankyrin 1 (TRPA1), responding to noxious cold and pungent compounds, is implicated in the mediation of nociception, but little is known about the processing of the TRPA1-mediated nociceptive information within the trigeminal sensory nuclei (TSN) and the spinal dorsal horn (DH). To address this issue, we characterized the TRPA1-positive (+) neurons in the trigeminal ganglion (TG) and investigated the distribution of TRPA1(+) afferent fibers and their synaptic connectivity within the rat TSN and DH by using light and electron microscopic immunohistochemistry. In the TG, TRPA1 was expressed in unmyelinated and small myelinated axons and also occasionally in large myelinated axons. Many TRPA1(+) neurons costained for the marker for peptidergic neurons substance P (26.8%) or the marker for nonpeptidergic neurons IB4 (44.5%). In the CNS, small numbers of axons and terminals were immunopositive for TRPA1 throughout the rostral TSN, in contrast to the dense network of positive fibers and terminals in the superficial laminae of the trigeminal caudal nucleus (Vc) and DH. The TRPA1(+) terminals contained clear round vesicles, were presynaptic to one or two dendrites, and rarely participated in axoaxonic contacts, suggesting involvement in relatively simple synaptic circuitry with a small degree of synaptic divergence and little presynaptic modulation. Immunoreactivity for TRPA1 was also occasionally observed in postsynaptic dendrites. These results suggest that TRPA1-dependent orofacial and spinal nociceptive input is processed mainly in the superficial laminae of the Vc and DH in a specific manner and may be processed differently between the rostral TSN and Vc.

Trigeminal primary afferents expressing P2X3 receptor are involved in the transmission of orofacial nociceptive information. However, little is known about their central projection pattern and ultrastructural features within the trigeminal brainstem sensory nuclei (TBSN). Here we use multiple immunofluorescence and electron microscopy to characterize the P2X3-immunopositive (+) neurons in the trigeminal ganglion and describe the distribution and synaptic organization of their central terminals within the rat TBSN, including nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc). In the trigeminal ganglion, P2X3 immunoreactivity was mainly in small and medium-sized somata, but also frequently in large somata. Although most P2X3+ somata costained for the nonpeptidergic marker IB4, few costained for the peptidergic marker substance P. Most P2X3+ fibers in the sensory root of trigeminal ganglion (92.9%) were unmyelinated, whereas the rest were small myelinated. In the TBSN, P2X3 immunoreactivity was dispersed in the rostral TBSN but was dense in the superficial laminae of Vc, especially in the inner lamina II. The P2X3+ terminals contained numerous clear, round vesicles and sparse large, dense-core vesicles. Typically, they were presynaptic to one or two dendritic shafts and also frequently postsynaptic to axonal endings, containing pleomorphic vesicles. Such P2X3+ terminals, showing glomerular shape and complex synaptic relationships, and those exhibiting axoaxonic contacts, were more frequently seen in Vp than in any other TBSN. These results suggest that orofacial nociceptive information may be transmitted via P2X3+ afferents to all TBSN and that it may be processed differently in different TBSN. J. Comp. Neurol. 506:627–639, 2008. © 2007 Wiley-Liss, Inc.

We have previously described the distribution pattern of inhibitory synapses on rat jaw-closing (JC) alpha- and gamma-motoneurons. In the present study, we investigated developmental changes in inhibitory synapses on JC motoneurons. We performed a quantitative ultrastructural analysis of putative inhibitory synaptic boutons on JC motoneuron somata by using postembedding immunogold labeling for GABA and glycine. In total, 206, 350, and 497 boutons contacting JC motoneuron somata were analyzed at postnatal days 2 (P2), 11 (P11) and 31 (P31), respectively. The size of the somata increased significantly during postnatal development. The size distribution was bimodal at P31. Mean length of the boutons and percentage of synaptic covering also increased during postnatal development, whereas bouton density did not differ significantly among the three age groups. Synaptic boutons on the somata of JC alpha-motoneurons could be classified into four types: boutons immunoreactive for 1) GABA only, 2) glycine only, 3) both GABA and glycine, and 4) neither GABA nor glycine. There was no developmental change in the proportion of putative inhibitory boutons to the total number of studied boutons. However, the glycine-only boutons increased significantly (15.1% to 27.3%), and the GABA-only boutons decreased significantly (17.7% to 2.6%) during the period from P11 to P31. Our ultrastructural data indicate that the inhibitory synaptic input to JC motoneurons is developmentally regulated and that there is a postnatal switch from GABA to glycine. The postnatal changes revealed in the present study could play an important role in the maturation of the oral motor system.

We examined the effects of peripherally or centrally administered botulinum neurotoxin type A (BoNT-A) on orofacial inflammatory pain to evaluate the antinociceptive effect of BoNT-A and its underlying mechanisms. The experiments were carried out on male Sprague-Dawley rats. Subcutaneous (3 U/kg) or intracisternal (0.3 or 1 U/kg) administration of BoNT-A significantly inhibited the formalin-induced nociceptive response in the second phase. Both subcutaneous (1 or 3 U/kg) and intracisternal (0.3 or 1 U/kg) injection of BoNT-A increased the latency of head withdrawal response in the complete Freund's adjuvant (CFA)-treated rats. Intracisternal administration of N-methyl-D-aspartate (NMDA) evoked nociceptive behavior via the activation of trigeminal neurons, which was attenuated by the subcutaneous or intracisternal injection of BoNT-A. Intracisternal injection of NMDA up-regulated c-Fos expression in the trigeminal neurons of the medullary dorsal horn. Subcutaneous (3 U/kg) or intracisternal (1 U/kg) administration of BoNT-A significantly reduced the number of c-Fos immunoreactive neurons in the NMDA-treated rats. These results suggest that the central antinociceptive effects the peripherally or centrally administered BoNT-A are mediated by transcytosed BoNT-A or direct inhibition of trigeminal neurons. Our data suggest that central targets of BoNT-A might provide a new therapeutic tool for the treatment of orofacial chronic pain conditions.

The present study investigated the role of peripheral group I metabotropic glutamate receptors (mGluRs) in MO-induced nociceptive behaviour and inflammation in the masseter muscles of lightly anesthetized rats. Experiments were carried out on male Sprague-Dawley rats weighing 300-400 g. After initial anesthesia with sodium pentobarbital (40 mg/kg, i.p.), one femoral vein was cannulated and connected to an infusion pump for intravenous infusion of sodium pentobarbital. The rate of infusion was adjusted to provide a constant level of anesthesia. Mustard oil (MO, 30 microl) was injected into the mid-region of the left masseter muscle via a 30-gauge needle over 10s. After 30 microl injection of 5, 10, 15, or 20% MO into the masseter muscle, the total number of hindpaw shaking behaviour and extravasated Evans' blue dye concentration in the masseter muscle were significantly higher in the MO-treated group in a dose-dependent manner compared with the vehicle (mineral oil)-treated group. Intramuscular pretreatment with 3 or 5% lidocaine reduced MO-induced hindpaw shaking behaviour and increases in extravasated Evans' blue dye concentration. Intramuscular pretreatment with 5 mM MCPG, non-selective group I/II mGluR antagonist, or MPEP, a selective group I mGluR5 antagonist, produced a significant attenuation of MO-induced hindpaw shaking behaviour and increases in extravasated Evans' blue dye concentration in the masseter muscle while LY367385, a selective group I mGluR1 antagonist, did not affect MO-induced nociceptive behaviour and inflammation in the masseter muscle. These results indicate that peripheral mGluR5 plays important role in mediating MO-induced nociceptive behaviour and inflammation in the craniofacial muscle.

This study examined the ultrastructures of neuronal elements within trigeminal mesencephalic nucleus by labeling masseteric mesencephalic neurons and masseter motoneurons with injection of horseradish peroxidase into masseteric muscle. Of eight horseradish peroxidase-labeled muscle spindle afferents examined, four terminals showed synaptic contact with labeled dendrites of masseteric motoneurons, two with labeled somata, and the remaining two with unlabeled dendrites. A few of the labeled dendrites showed intimate contact with the somata of the trigeminal mesencephalic nucleus neurons. These results provide morphological evidence of synaptic contact of recurring masseteric muscle spindle afferents with the trigeminal mesencephalic nucleus somata and also suggest the presence of electrical synapses between the somata of the trigeminal mesencephalic nucleus neurons and dendrites of jaw-closing motoneurons.

The influence of NK1 receptor antagonists applied iontophoretically on pulpal blood flow (PBF) was investigated. Along with substance P (SP, 0.8 approximately 20.0 ng/kg) administration to the canine pulp through the catheterized lingual artery, two NK1 receptor antagonists, [D-Pro2,D-Trp7,9]-SP and [D-Pro2,D-Phe7,D-Trp9]-SP (0.2 approximately 3.4 mM) were applied iontophoretically (cathodal current, 0.02 approximately 0.1 mA, 1 min) to the prepared class V dentinal cavity of ipsilateral teeth in 11 generally anesthetized cats. A paired t-test showed that SP administration caused significant increases of PBF (p < 0.05) without changing systemic blood pressure, and that SP and SP antagonist administration caused significantly less increase of PBF than in control of SP and 0.9% saline administration (p < 0.05). These data provide evidence that the iontophoretic application of NK1 receptor antagonists effectively attenuates SP-induced vasodilatation and show the possibility of their use in the control of neurogenic inflammation in the dental pulp.

The present study investigated the effects of intracisternal administration of MCP-1, Rantes or IL-8 on pain transmission in the orofacial area. We also investigated mechanisms of hyperalgesic responses produced by intracisternal administration of IL-8. An orofacial formalin test was employed to assess the effects of chemokines on nociceptive processing. For each animal, the number of behavioral responses and the time spent grooming, rubbing and/or scratching the facial region proximal to the formalin injection site was recorded for nine successive 5-min intervals. Intracisternal administration of MCP-1, Rantes or IL-8 significantly increased formalin-induced scratching behavioral responses in the orofacial area. Intracisternal pretreatment with indomethacin, a non-selective cyclooxygenase inhibitor, did not block IL-8-induced hyperalgesia. Pretreatment with 100 microg propranolol, a non-selective beta-adrenergic receptor antagonist and 50 microg atenolol, a selective beta(1)-adrenergic receptor antagonist, inhibited the number of scratches and the duration of scratching produced by 1 ng of IL-8 injected intracisternally. These results indicate that intracisternal administration of chemokines produce a hyperalgesic response with an orofacial inflammatory pain model and that the IL-8-induced hyperalgesia is mediated by central beta(1)-adrenergic receptor.

The present study was performed to examine peripheral cytokine-induced mechanical allodynia in the orofacial area and to investigate whether peripheral excitatory amino acids participate in the cytokine-induced mechanical allodynia. Experiments were carried out on male Sprague-Dawley rats. After interleukin-1beta (IL-1beta) was applied subcutaneously to the orofacial area, we examined withdrawal responses produced by air puffs applied to the IL-1beta injection site. The threshold of air puffs that produced withdrawal behavioral responses decreased significantly in a dose-dependent manner after injection of IL-1beta. Pretreatment with an IL-1 receptor antagonist abolished the decrease in the threshold of air puffs. Pretreatment with dl-2-amino-5-phosphonvaleric acid, an N-methyl-d-aspartic acid (NMDA) receptor antagonist, did not affect IL-1beta-induced mechanical allodynia. However, pretreatment with 6,7-dinitroquinoxaline-2,3-dione, a non-NMDA receptor antagonist, abolished the decrease in the threshold of air puffs. These results suggest that peripheral cytokine can produce mechanical allodynia in the orofacial area and that excitatory amino acids can modulate IL-1beta-induced mechanical allodynia via non-NMDA receptors.

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.

The present study was performed to investigate the effects of central cytokines on the modulation of nociception in the orofacial area. A nociceptive jaw-opening reflex (JOR) and an orofacial formalin test were monitored after intracisternal administration of tumor necrosis factor (TNF)-alpha in freely moving rats. Experiments were carried out on 83 male rats weighing 300-350 g and surgical procedures were performed under pentobarbital sodium. After intracisternal injection of Tnf-alpha, digastric electromyogram (dEMG) and noxious behavioral responses were monitored. In the nociceptive JOR, dEMG was not significantly changed after intracisternal injection of 200 pg and 2 ng Tnf-alpha. However, 20 ng Tnf-alpha suppressed dEMG to 72+/-6% of the control values. The orofacial formalin responses showed two distinct phases separated by a time of relative inactivity with an early short-lasting response (0-9 min, first phase) and a continuous prolonged response (10-45 min, second phase). In the inflammatory orofacial formalin test, intracisternal injection of 20 pg Tnf-alpha did not change the number of noxious behavioral responses produced by formalin injection. However, 200 pg Tnf-alpha injected intracisternally significantly increased the number of noxious behavioral responses produced by formalin injection in both the early and late phases, and 2 ng Tnf-alpha increased formalin induced noxious behavioral responses in only the late phase. A higher dose of 20 ng Tnf-alpha did not change the number of noxious behavioral responses produced by formalin injection. The hyperalgesic action of Tnf-alpha injected intracisternally was blocked by pretreatment with the interleukin-1 (IL-1) receptor antagonist. These results suggest that central Tnf-alpha modulates the transmission of nociceptive information in the orofacial area. However, the hypo/hyperalgesic response of central Tnf-alpha seems to depend on the orofacial pain model or in a dose-related manner. The hyperalgesic response of central Tnf-alpha seems to be mediated by the IL-1 receptor.

Here we investigated the central processing mechanisms of mechanical allodynia and found a direct excitatory link with low-threshold input to nociceptive neurons. Experiments were performed on male Sprague-Dawley rats weighing 230-280 g. Subcutaneous injection of interleukin 1 beta (IL-1β) (1 ng/10 µL) was used to produce mechanical allodynia and thermal hyperalgesia. Intracisternal administration of bicuculline, a gamma aminobutyric acid A (GABAA) receptor antagonist, produced mechanical allodynia in the orofacial area under normal conditions. However, intracisternal administration of bicuculline (50 ng) produced a paradoxical anti-allodynic effect under inflammatory pain conditions. Pretreatment with resiniferatoxin (RTX), which depletes capsaicin receptor protein in primary afferent fibers, did not alter the paradoxical anti-allodynic effects produced by the intracisternal injection of bicuculline. Intracisternal injection of bumetanide, an Na-K-Cl cotransporter (NKCC 1) inhibitor, reversed the IL-1β-induced mechanical allodynia. In the control group, application of GABA (100 µM) or muscimol (3 µM) led to membrane hyperpolarization in gramicidin perforated current clamp mode. However, in some neurons, application of GABA or muscimol led to membrane depolarization in the IL-1β-treated rats. These results suggest that some large myelinated Aβ fibers gain access to the nociceptive system and elicit pain sensation via GABAA receptors under inflammatory pain conditions.

Transient receptor potential melastatin 8 (TRPM8) ion channels mediate the detection of noxious and innocuous cold and are expressed by primary sensory neurons, but little is known about the processing of the TRPM8-mediated cold information within the trigeminal sensory nuclei (TSN) and the spinal dorsal horn (DH). To address this issue, we characterized TRPM8-positive (+) neurons in the trigeminal ganglion and investigated the distribution of TRPM8+ axons and terminals, and their synaptic organization in the TSN and in the DH using light and electron microscopic immunohistochemistry in transgenic mice expressing a genetically encoded axonal tracer in TRPM8+ neurons. TRPM8 was expressed in a fraction of small myelinated primary afferent fibers (23.7%) and unmyelinated fibers (76.3%), suggesting that TRPM8-mediated cold is conveyed via C and Aδ afferents. TRPM8+ axons were observed in all TSN, but at different densities in the dorsal and ventral areas of the rostral TSN, which dominantly receive sensory afferents from intra- and peri-oral structures and from the face, respectively. While synaptic boutons arising from Aδ and non-peptidergic C afferents usually receive many axoaxonic contacts and form complex synaptic arrangements, TRPM8+ boutons arising from afferents of the same classes of fibers showed a unique synaptic connectivity; simple synapses with one or two dendrites and sparse axoaxonic contacts. These findings suggest that TRPM8-mediated cold is conveyed via a specific subset of C and Aδ afferent neurons and is processed in a unique manner and differently in the TSN and DH.

The present study was performed to examine the central effects of antidepressants on nociceptive jaw opening reflex after intracisternal injection. we also investigated the mechanisms of central antinociceptive action of intracisternal antidepressants. We recorded the jaw opening reflex in freely moving rats and chose to administer antidepressants intracisternally in order to eliminate the effects of anesthetic agents on the pain assessment and evaluate the importance of the spinal site of action of antidepressants. After intracisternal injection of 15 microg imipramine, digastric electromyogram (dEMG) was decreased to 76+/-6% of the control. Intracisternal administration of 30 microg desipramine, nortriptyline or imipramine suppressed dEMG remarkably to 48+/-2, 27+/-8, or 25+/-5% of the control, respectively. The suppression of dEMG was maintained for 50 min. L-NG-Nitroarginine methyl ester (NAME) blocked the suppression of dEMG from 32+/-2 to 81+/-5% of the control. These results indicate that antidepressants produce antinociception through central mechanisms in the orofacial area. The central NO pathway seems to be involved in the antinociception of intracisternal antidepressants at supraspinal sites.

The role of brain angiotensin II (ANG II) in mediating cardiovascular, vasopressin, and renin responses to hemorrhage was assessed in conscious spontaneously hypertensive rats (SHR) and in normotensive Wistar-Kyoto (WKY) and Wistar rats. Intracerebroventricular administration of losartan (10 micrograms) and saralasin (1 microgram.microliter-1.min-1) produced a markedly greater fall in blood pressure and a reduced tachycardia during and after hemorrhage (15 ml/kg) compared with the artificial cerebrospinal fluid control in SHR and Wistar rats but not in WKY rats. Vasopressin release after hemorrhage was also impaired, but renin release was enhanced by intracerebroventricular ANG II antagonists in SHR and Wistar rats but not in WKY rats. Losartan and saralasin produced remarkably similar effects on the cardiovascular, vasopressin, and renin responses to hemorrhage. These data suggest that brain ANG II acting through AT1 receptors plays an important physiological role in mediating rapid cardiovascular regulation and vasopressin release in response to hemorrhage. The relative importance of brain angiotensin system may vary in different strains of rate.

The transient receptor potential ankyrin 1 (TRPA1) is implicated in the mechanical and cold hyperalgesia following inflammation and nerve injury. Its expression has been presumed to be confined to primary afferent terminals. Here, we show that TRPA1 is expressed in astrocytes in the superficial laminae of the rat trigeminal caudal nucleus by use of electron microscopic immunoperoxidase and immunogold labeling techniques. Immunoreactivity for TRPA1 was consistently observed in somata and process of astrocytes and was weaker than that in presumed nociceptive primary afferent terminals, but increased significantly in the fine process of astrocyte in rats with experimental inflammation of the temporomandibular joint. Thus, we provide ultrastructural evidence that TRPA1 is expressed in astrocytes in the brain stem and propose a novel pathway of its involvement in the central mechanism of inflammatory hyperalgesia.

In our previous study, we reported that endothelial cell specific molecule-1 (ESM-1) was increased in tissue and serum from colorectal cancer patients and suggested that ESM-1 can be used as a potential serum marker for early detection of colorectal cancer. The aim of this study was to evaluate the role of ESM-1 as an intracellular molecule in colorectal cancer. ESM-1 expression was knocked down by small interfering RNA (siRNA) in colorectal cancer cells. Expression of ESM-1 siRNA decreased cell survival through the Akt-dependent inhibition of NF-κB/IκB pathway and an interconnected reduction in phospho-Akt, -p38, -ERK1, -RSK1, -GSK-3α/β and -HSP27, as determined by a phospho-MAPK array. ESM-1 silencing induced G 1 phase cell cycle arrest by induction of PTEN, resulting in the inhibition of cyclin D1 and inhibited cell migration and invasion of COLO205 cells. Consistently, ESM-1 overexpression in HCT-116 cells enhanced cell proliferation through the Akt-dependent activation of NF-κB pathway. In addition, ESM-1 interacted with NF-κB and activated NF-κB promoter. This study demonstrates that ESM-1 is involved in cell survival, cell cycle progression, migration, invasion and EMT during tumor invasion in colorectal cancer. Based on our results, ESM-1 may be a useful therapeutic target for colorectal cancer.