Single-cell analysis of dorsal root ganglia reveals metalloproteinase signaling in satellite glial cells and pain.

Satellite glial cells (SGCs) are among the most abundant non-neuronal cells in dorsal root ganglia (DRGs) and closely envelop sensory neurons that detect painful stimuli. However, little is still known about their homeostatic activities and their contribution to pain. Using single-cell RNA sequencing (scRNA-seq), we were able to obtain a unique transcriptional profile for SGCs. We found enriched expression of the tissue inhibitor metalloproteinase 3 (TIMP3) and other metalloproteinases in SGCs. Small interfering RNA and neutralizing antibody experiments revealed that TIMP3 modulates somatosensory stimuli. TIMP3 expression decreased after paclitaxel treatment, and its rescue by delivery of a recombinant TIMP3 protein reversed and prevented paclitaxel-induced pain. We also established that paclitaxel directly impacts metalloproteinase signaling in cultured SGCs, which may be used to identify potential new treatments for pain. Therefore, our results reveal a metalloproteinase signaling pathway in SGCs for proper processing of somatosensory stimuli and potential discovery of novel pain treatments.

Major depression-related factor NEGR1 controls salivary secretion in mouse submandibular glands.

Salivary gland cells, which secrete water in response to neuronal stimulation, are closely connected to other neurons. Transcriptomic studies show that salivary glands also express some proteins responsible for neuronal function. However, the physiological functions of these common neuro-exocrine factors in salivary glands are largely unknown. Here, we studied the function of Neuronal growth regulator 1 (NEGR1) in the salivary gland cells. NEGR1 was also expressed in mouse and human salivary glands. The structure of salivary glands of Negr1 knockout (KO) mice was normal. Negr1 KO mice showed tempered carbachol- or thapsigargin-induced intracellular Ca2+ increases and store-operated Ca2+ entry. Of interest, the activity of the large-conductance Ca2+-activated K+ channel (BK channel) was increased, whereas Ca2+-activated Cl- channel ANO1 channel activity was not altered in Negr1 KO mice. Pilocarpine- and carbachol-induced salivation was decreased in Negr1 KO mice. These results suggest that NEGR1 influence salivary secretion though the muscarinic Ca2+ signaling.

Circulation Time-Optimized Albumin Nanoplatform for Quantitative Visualization of Lung Metastasis via Targeting of Macrophages.

The development of molecular imaging probes to identify key cellular changes within lung metastases may lead to noninvasive detection of metastatic lesions in the lung. In this study, we constructed a macrophage-targeted clickable albumin nanoplatform (CAN) decorated with mannose as the targeting ligand using a click reaction to maintain the intrinsic properties of albumin in vivo. We also modified the number of mannose molecules on the CAN and found that mannosylated serum albumin (MSA) harboring six molecules of mannose displayed favorable pharmacokinetics that allowed high-contrast imaging of the lung, rendering it suitable for in vivo visualization of lung metastases. Due to the optimized control of functionalization and surface modification, MSA enhanced blood circulation time and active/passive targeting abilities and was specifically incorporated by mannose receptor (CD206)-expressing macrophages in the metastatic lung. Moreover, extensive in vivo imaging studies using single-photon emission computed tomography (SPECT)/CT and positron emission tomography (PET) revealed that blood circulation of time-optimized MSA can be used to discern metastatic lesions, with a strong correlation between its signal and metastatic burden in the lung.

Novel regulation of the transcription factor ZHX2 by N-terminal methylation.

N-terminal methylation (Nα-methylation) by the methyltransferase NRMT1 is an important post-translational modification that regulates protein-DNA interactions. Accordingly, its loss impairs functions that are reliant on such interactions, including DNA repair and transcriptional regulation. The global loss of Nα-methylation results in severe developmental and premature aging phenotypes, but given over 300 predicted substrates, it is hard to discern which physiological substrates contribute to each phenotype. One of the most striking phenotypes in NRMT1 knockout (Nrmt1-/-) mice is early liver degeneration. To identify the disrupted signaling pathways leading to this phenotype and the NRMT1 substrates involved, we performed RNA-sequencing analysis of control and Nrmt1-/- adult mouse livers. We found both a significant upregulation of transcripts in the cytochrome P450 (CYP) family and downregulation of transcripts in the major urinary protein (MUP) family. Interestingly, transcription of both families is inversely regulated by the transcription factor zinc fingers and homeoboxes 2 (ZHX2). ZHX2 contains a non-canonical NRMT1 consensus sequence, indicating that its function could be directly regulated by Nα-methylation. We confirmed misregulation of CYP and MUP mRNA and protein levels in Nrmt1-/- livers and verified NRMT1 can methylate ZHX2 in vitro. In addition, we used a mutant of ZHX2 that cannot be methylated to directly demonstrate Nα-methylation promotes ZHX2 transcription factor activity and target promoter occupancy. Finally, we show Nrmt1-/- mice also exhibit early postnatal de-repression of ZHX2 targets involved in fetal liver development. Taken together, these data implicate ZHX2 misregulation as a driving force behind the liver phenotype seen in Nrmt1-/- mice.

Comparative analysis of the oral microbiome of burning mouth syndrome patients.

Burning mouth syndrome (BMS) is a chronic pain condition accompanied by unpleasant burning sensations of the oral mucosa. While multiple factors were proposed for the etiology, evidence suggested a neuropathic pain origin while others suspected the use of antibiotics as the underlying cause. Interestingly, several reports demonstrated the intimate interaction of the nervous system and the microbiome. The current study aims to elucidate the correlation of the oral microbiome with the pathophysiology of the primary BMS. Microbiome samples obtained from the unstimulated whole saliva of 19 primary BMS patients and 22 healthy controls were sequenced and analyzed of the V3-V4 region of 16S rRNA gene. There was a distinct difference in the microbial composition between the BMS and the control groups at all taxonomic levels. Alpha diversity indexes of the oral microbiome were significantly lower in the BMS group. The samples were readily distinguished by multidimensional scaling analysis and linear discriminant analysis effect size. Streptococcus, Rothia, Bergeyella, and Granulicatella genus were dominant in the BMS group, while Prevotella, Haemophilus, Fusobacterium, Campylobacter, and Allorevotella genus were more abundant in the healthy group. Distinct microbiome signatures of BMS patients suggested a diagnostic value and a potential role in the pathogenesis of BMS.

Estrogen-dependent regulation of transient receptor potential vanilloid 1 (TRPV1) and P2X purinoceptor 3 (P2X3): Implication in burning mouth syndrome.

Sex differences in the nervous system have gained recent academic interest. While the prominent differences are observed in mood and anxiety disorders, growing number of evidences also suggest sex difference in pain perception. This review focuses on estrogen as the key molecule underlying such difference, because estrogen plays many functions in the nervous system, including modulation of transient receptor potential vanilloid 1 (TRPV1) and P2X purinoceptor 3 (P2X3), two important nociceptive receptors. Estrogen was shown in various studies to up-regulate TRPV1 expression through two distinct pathways, resulting in pro-nociceptive effect. However, estrogen alleviated pain in other studies, by down-regulating nerve growth factor (NGF)-activated pathways and TRPV1. Estrogen may also attenuate nociception by inhibiting P2X3 receptors and ATP-signaling. Understanding the mechanism underlying the pro- and anti-nociceptive effect of estrogen might be crucial to understand pathophysiology of the burning mouth syndrome (BMS), a common chronic orofacial pain disorder in menopausal women. The involvement of TRPV1 is strongly suspected because of burning sensation. Reduced estrogen level of the BMS patient might have caused increased activity of P2X3 receptors. Interestingly, the increased expression of TRPV1 and P2X3 in oral mucosa of BMS patients was reported. The combinational impact of differential modulation of TRPV1/P2X3 during menopause might be an important contributing factor of etiology of BMS. Understanding the estrogen-dependent regulation of nociceptive receptors may provide a valuable insight toward the peripheral mechanism of sex-difference in pain perception.

Long-term efficacy and patient satisfaction of pulsed radiofrequency therapy in temporomandibular disorders: A randomized controlled trial.

BACKGROUND: Pulse radiofrequency (PRF) therapy is one of effective physical therapy modalities for treat temporomandibular disorders (TMD). This prospective randomized controlled trial aimed to evaluate the long-term treatment efficacy and patient satisfaction with PRF therapy in TMD. METHODS: Eighty-six female patients with TMD were randomly assigned to either pulsed radiofrequency or placebo therapy in combination with other conventional treatments once a week for 12 weeks. A final analysis was performed 12 weeks after the completion of treatment. Clinical parameters and patient satisfaction were analyzed at baseline, 4, 8, and 12 weeks of intervention and at 24 weeks from baseline. RESULTS: Pain intensity, comfortable and maximum mouth opening, and pain on capsule and masticatory muscle palpation were significantly improved after treatment in both groups. Notably, the pulsed radiofrequency group showed a significantly lower pain intensity at the final evaluation performed 3 months after the completion of treatment. Significantly more patients reported subjective pain improvement and satisfaction with treatment following intervention at baseline in the PRF group. Most patients did not report any discomfort following treatment in either group. However, significantly more patients in the PRF group reported a burning sensation with intervention. CONCLUSION: Long-term regular pulsed radiofrequency therapy was effective in significantly reducing TMD pain, and the effect was long-lasting following treatment completion. Pulsed radiofrequency therapy should be considered as a supportive physical therapy modality for TMD.

Functional Importance of Transient Receptor Potential (TRP) Channels in Neurological Disorders.

Transient receptor potential (TRP) channels are transmembrane protein complexes that play important roles in the physiology and pathophysiology of both the central nervous system (CNS) and the peripheral nerve system (PNS). TRP channels function as non-selective cation channels that are activated by several chemical, mechanical, and thermal stimuli as well as by pH, osmolarity, and several endogenous or exogenous ligands, second messengers, and signaling molecules. On the pathophysiological side, these channels have been shown to play essential roles in the reproductive system, kidney, pancreas, lung, bone, intestine, as well as in neuropathic pain in both the CNS and PNS. In this context, TRP channels have been implicated in several neurological disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and epilepsy. Herein, we focus on the latest involvement of TRP channels, with a special emphasis on the recently identified functional roles of TRP channels in neurological disorders related to the disruption in calcium ion homeostasis.

Dissolving Microneedles for Rapid and Painless Local Anesthesia.

Microneedles are emerging drug delivery methods for painless treatment. The current study tested dissolving microneedles containing lidocaine (Li-DMN) for use in local anesthesia. An Li-DMN patch was fabricated by centrifugal lithography with carboxymethyl cellulose as a structural polymer and assessed for physical properties by optical microscopy and a fracture force analyzer. The biocompatibility was evaluated by a histology section in vitro and by ear thickness in vivo. The efficacy of the Li-DMN patch was assessed by electrophysiological recordings in primary cultured sensory neurons in vitro and a von Frey test on rats' hind paws in vivo. The physical properties of the microneedle showed enough rigidity for transdermal penetration. The maximal capacity of lidocaine-HCl in the Li-DMN patch was 331.20 ± 6.30 µg. The cytotoxicity of the dissolving microneedle to neuronal cells was negligible under an effective dose of lidocaine for 18 h. Electrophysiological recordings verified the inhibitory effect of the voltage-gated sodium channel current by the Li-DMN patch in vitro. A skin reaction to the edema test and histologic analysis of the rats' ears after application of the Li-DMN patch were negligible. Also, the application of the Li-DMN patch reduced the nocifensive behavior of the rats almost immediately. In conclusion, the dissolving microneedle patch with carboxymethyl cellulose is a promising candidate method for the painless delivery of lidocaine-HCl.

Dexmedetomidine modulates transient receptor potential vanilloid subtype 1.

Dexmedetomidine, a highly selective alpha-2 adrenergic receptor agonist and novel sedative drug with minimal respiratory suppression, have shown anti-nociceptive activity in various pain models by poorly understood mechanisms. Because alpha-2 adrenergic receptor is co-localized with TRPV1 polymodal nociceptive receptor in dorsal root ganglion neurons and up-regulated in neuropathic pain animal models, the analgesic activity might be mediated through inhibition of TRPV1 in the peripheral nervous system. In an effort to elucidate whether modulatory effect of dexmedetomidine on TRPV1 activity could be the potential peripheral mechanism underlying the antinociceptive effect of dexmedetomidine, intracellular calcium concentration after capsaicin application was investigated in mice dorsal root ganglion (DRG) neurons, with and without pretreatment of dexmedetomidine. Dexmedetomidine (10 µM) reduced capsaicin-induced calcium responses by 29.7 ± 7.39% (n = 34, p < 0.0001), in dose-dependent manner. Higher level of inhibition was observed with increased dose of dexmedetomidine (50 µM, 45.1 ± 8.58%, n = 15, p = 0.0002), and lower inhibition by decreased dose (1 µM, 18.8 ± 1.48%, n = 148, p = 0.004). RT-PCR analysis revealed expression of TRPV1 and alpha-2A, alpha-2B and alpha-2C subtypes of adrenergic receptor in mice DRG neurons, and immunocytochemical analysis revealed co-expression of TRPV1 and alpha-2A receptors in primary cultured DRG neurons. In summary, these results suggested the inhibition of TRPV1 expressed in the primary sensory neurons as a potential mechanism that contributes to the anti-nociceptive action of dexmedetomidine.

The Role of Maresins in Inflammatory Pain: Function of Macrophages in Wound Regeneration.

Although acute inflammatory responses are host-protective and generally self-limited, unresolved and delayed resolution of acute inflammation can lead to further tissue damage and chronic inflammation. The mechanism of pain induction under inflammatory conditions has been studied extensively; however, the mechanism of pain resolution is not fully understood. The resolution of inflammation is a biosynthetically active process, involving specialized pro-resolving mediators (SPMs). In particular, maresins (MaRs) are synthesized from docosahexaenoic acid (DHA) by macrophages and have anti-inflammatory and pro-resolving capacities as well as tissue regenerating and pain-relieving properties. A new class of macrophage-derived molecules-MaR conjugates in tissue regeneration (MCTRs)-has been reported to regulate phagocytosis and the repair and regeneration of damaged tissue. Macrophages not only participate in the biosynthesis of SPMs, but also play an important role in phagocytosis. They exhibit different phenotypes categorized as proinflammatory M1-like phenotypes and anti-inflammatory M2 phenotypes that mediate both harmful and protective functions, respectively. However, the signaling mechanisms underlying macrophage functions and phenotypic changes have not yet been fully established. Recent studies report that MaRs help resolve inflammatory pain by enhancing macrophage phagocytosis and shifting cytokine release to the anti-inflammatory M2 phenotypes. Consequently, this review elucidated the characteristics of MaRs and macrophages, focusing on the potent action of MaRs to enhance the M2 macrophage phenotype profiles that possess the ability to alleviate inflammatory pain.

Ion Channels Involved in Tooth Pain.

The tooth has an unusual sensory system that converts external stimuli predominantly into pain, yet its sensory afferents in teeth demonstrate cytochemical properties of non-nociceptive neurons. This review summarizes the recent knowledge underlying this paradoxical nociception, with a focus on the ion channels involved in tooth pain. The expression of temperature-sensitive ion channels has been extensively investigated because thermal stimulation often evokes tooth pain. However, temperature-sensitive ion channels cannot explain the sudden intense tooth pain evoked by innocuous temperatures or light air puffs, leading to the hydrodynamic theory emphasizing the microfluidic movement within the dentinal tubules for detection by mechanosensitive ion channels. Several mechanosensitive ion channels expressed in dental sensory systems have been suggested as key players in the hydrodynamic theory, and TRPM7, which is abundant in the odontoblasts, and recently discovered PIEZO receptors are promising candidates. Several ligand-gated ion channels and voltage-gated ion channels expressed in dental primary afferent neurons have been discussed in relation to their potential contribution to tooth pain. In addition, in recent years, there has been growing interest in the potential sensory role of odontoblasts; thus, the expression of ion channels in odontoblasts and their potential relation to tooth pain is also reviewed.

Critical Role of ATP-P2X7 Axis in UV-Induced Melanogenesis.

Purinergic signaling participates in skin physiology and pathology, such as hair growth, wound healing, inflammation, pain, and skin cancer. However, few studies have investigated the involvement of purinergic signaling in skin pigmentation. This study demonstrated that extracellular adenosine 5'-triphosphate (ATP) released from keratinocytes by UVB radiation promotes melanin production in primary human epidermal melanocytes and ex vivo skin cultures. Intracellular calcium ion and protein kinase C/CREB signaling contributed to ATP-mediated melanogenesis. Also, P2X7 receptor was proven to play a pivotal role in ATP-mediated melanogenesis because P2X7 receptor blockade abrogated ATP-induced melanin production. In addition, MNT1 cells with P2X7 receptor knockout using CRISPR/Cas9 system did not show any increase in MITF expression when co-cultured with UV-irradiated keratinocytes compared to MNT1 cells with intact P2X7 receptor, which showed increased expression of MITF. In conclusion, our results indicate that the extracellular ATP-P2X7 signaling axis is an adjunctive mechanism in UV-induced melanogenesis. Furthermore, ATP-induced purinergic signaling in melanocytes may alter skin pigmentation.

Human salivary gland cells express bradykinin receptors that modulate the expression of proinflammatory cytokines.

Bradykinin is an important peptide modulator that affects the function of neurons and immune cells. However, there is no evidence of the bradykinin receptors and their functions in human salivary glands. Here we have identified and characterized bradykinin receptors on human submandibular gland cells. Both bradykinin B1 and B2 receptors are expressed on human submandibular gland cells, A253 cells, and HSG cells. Bradykinin increased the intracellular Ca2+ concentration ([Ca2+ ]i ) in a concentration-dependent manner. Interestingly, a specific agonist of the B1 receptor did not have any effect on [Ca2+ ]i in HSG cells, whereas specific agonists of the B2 receptor had a Ca2+ mobilizing effect. Furthermore, application of the B1 receptor antagonist, R715, did not alter the bradykinin-mediated increase in cytosolic Ca2+ , whereas the B2 receptor antagonist, HOE140, showed a strong inhibitory effect, which implies that bradykinin B2 receptors are functional in modulating the concentration of cytosolic Ca2+ . Bradykinin did not affect a carbachol-induced rise of [Ca2+ ]i and did not modulate translocation of aquaporin-5. However, bradykinin did promote the expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), implying the role of bradykinin in salivary gland inflammation. These data suggest that bradykinin receptors are involved in Ca2+ signaling in human submandibular gland cells and serve a unique role, which is separate from that of other salivary gland G protein-coupled receptors.

Generation of resonance-dependent oscillation by mGluR-I activation switches single spiking to bursting in mesencephalic trigeminal sensory neurons.

The primary sensory neurons supplying muscle spindles of jaw-closing muscles are unique in that they have their somata in the mesencephalic trigeminal nucleus (MTN) in the brainstem, thereby receiving various synaptic inputs. MTN neurons display bursting upon activation of glutamatergic synaptic inputs while they faithfully relay respective impulses arising from peripheral sensory organs. The persistent sodium current (IN aP ) is reported to be responsible for both the generation of bursts and the relay of impulses. We addressed how IN aP is controlled either to trigger bursts or to relay respective impulses as single spikes in MTN neurons. Protein kinase C (PKC) activation enhanced IN aP only at low voltages. Spike generation was facilitated by PKC activation at membrane potentials more depolarized than the resting potential. By injection of a ramp current pulse, a burst of spikes was triggered from a depolarized membrane potential whereas its instantaneous spike frequency remained almost constant despite the ramp increases in the current intensity beyond the threshold. A puff application of glutamate preceding the ramp pulse lowered the threshold for evoking bursts by ramp pulses while chelerythrine abolished such effects of glutamate. Dihydroxyphenylglycine, an agonist of mGluR1/5, also caused similar effects, and increased both the frequency and impedance of membrane resonance. Immunohistochemistry revealed that glutamatergic synapses are made onto the stem axons, and that mGluR1/5 and Nav1.6 are co-localized in the stem axon. Taken together, glutamatergic synaptic inputs onto the stem axon may be able to switch the relaying to the bursting mode.

Low levels of methyl ß-cyclodextrin disrupt GluA1-dependent synaptic potentiation but not synaptic depression.

Methyl-ß-cyclodextrin (MßCD) is a reagent that depletes cholesterol and disrupts lipid rafts, a type of cholesterol-enriched cell membrane microdomain. Lipid rafts are essential for neuronal functions such as synaptic transmission and plasticity, which are sensitive to even low doses of MßCD. However, how MßCD changes synaptic function, such as N-methyl-d-aspartate receptor (NMDA-R) activity, remains unclear. We monitored changes in synaptic transmission and plasticity after disrupting lipid rafts with MßCD. At low concentrations (0.5 mg/mL), MßCD decreased basal synaptic transmission and miniature excitatory post-synaptic current without changing NMDA-R-mediated synaptic transmission and the paired-pulse facilitation ratio. Interestingly, low doses of MßCD failed to deplete cholesterol or affect α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-R) and NMDA-R levels, while clearly reducing GluA1 levels selectively in the synaptosomal fraction. Low doses of MßCD decreased the inhibitory effects of NASPM, an inhibitor for GluA2-lacking AMPA-R. MßCD successfully decreased NMDA-R-mediated long-term potentiation but did not affect the formation of either NMDA-R-mediated or group I metabotropic glutamate receptor-dependent long-term depression. MßCD inhibited de-depression without affecting de-potentiation. These results suggest that MßCD regulates GluA1-dependent synaptic potentiation but not synaptic depression in a cholesterol-independent manner.

Single-cell RT-PCR and immunocytochemical detection of mechanosensitive transient receptor potential channels in acutely isolated rat odontoblasts.

OBJECTIVE: Hydrostatic force applied to tooth pulp has long been suspected to be the direct cause of dental pain. However, the molecular and cellular identity of the transducer of the mechanical force in teeth is not clear. Growing number of literatures suggested that odontoblasts, secondary to its primary role as formation of tooth structure, might function as a cellular mechanical transducer in teeth. DESIGN: In order to determine whether odontoblasts could play a crucial role in transduction of hydrostatic force applied to dental pulp into electrical impulses, current study investigated the expression of stretch-activated transient receptor potential (TRP) channels in acutely isolated odontoblasts from adult rats by single cell reverse transcriptase polymerase chain reaction and immunocytochemical analysis. RESULTS: As the result, expression of TRPM7 (melastatin 7) was observed in majority (87%) of odontoblasts while mRNAs for TRPC1 (canonical 1), TRPC6 (canonical 6) and TRPV4 (vanilloid 4) were detected in small subpopulations of odontoblasts. TRPM3 (melastatin 3) was not detected in our experimental set-up. Immunocytochemical analysis further revealed TRPM7 expression at protein level. CONCLUSION: Expression of the mechanosensitive TRP channels provides additional evidence that supports the sensory roles of odontoblasts. Given that TRPM7 is a mechanosensitive ion channel with a kinase activity that plays a role in Mg(2+) homeostasis, it is possible that TRPM7 expressed in odontoblasts might play a central role in mineralization during dentin formation.

A Novel Carbamoyloxy Arylalkanoyl Arylpiperazine Compound (SKL-NP) Inhibits Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channel Currents in Rat Dorsal Root Ganglion Neurons.

In this study, we determined mode of action of a novel carbamoyloxy arylalkanoyl arylpiperazine compound (SKL-NP) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents (I(h)) that plays important roles in neuropathic pain. In small or medium-sized dorsal root ganglion (DRG) neurons (<40 µm in diameter) exhibiting tonic firing and prominent I(h), SKL-NP inhibited I(h) and spike firings in a concentration dependent manner (IC(50)=7.85 µM). SKL-NP-induced inhibition of I(h) was blocked by pretreatment of pertussis toxin (PTX) and N-ethylmaleimide (NEM) as well as 8-Br-cAMP, a membrane permeable cAMP analogue. These results suggest that SKL-NP modulates I(h) in indirect manner by the activation of a Gi-protein coupled receptor that decreases intracellular cAMP concentration. Taken together, SKL-NP has the inhibitory effect on HCN channel currents (I(h)) in DRG neurons of rats.

Neurochemical properties of dental primary afferent neurons.

The long belief that dental primary afferent (DPA) neurons are entirely composed of nociceptive neurons has been challenged by several anatomical and functional investigations. In order to characterize non-nociceptivepopulation among DPA neurons, retrograde transport fluorescent dye was placed in upper molars of rats and immunohistochemical detection of peripherin and neurofilament 200 in the labeled trigeminal ganglia was performed. As the results, majority ofDPA neurons were peripherin-expressing small-sized neurons, showing characteristic ofnociceptive C-fibers. However, 25.7% of DPA were stained with antibody against neurofilament 200, indicating significant portion of DPA neurons are related to large myelinated Aß fibers. There were a small number of neurons thatexpressed both peripherin and neurofilament 200, suggestive of Aδ fibers. The possible transition of neurochemical properties by neuronal injury induced by retrograde labeling technique was ruled out by detection of minimal expression of neuronal injury marker, ATF-3. These results suggest that in addition to the large population of C-fiber-related nociceptive neurons, a subset of DPA neurons is myelinated large neurons, which is related to low-threshold mechanosensitive Aß fibers. We suggest that these Aß fiber-related neurons might play a role as mechanotransducers of fluid movement within dentinal tubules.

Eugenol reverses mechanical allodynia after peripheral nerve injury by inhibiting hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

Mechanical allodynia is a common symptom found in neuropathic patients. Hyperpolarization-activated cyclic nucleotide-gated channels and their current, I(h), have been suggested to play an important role in neuropathic pain, especially in mechanical allodynia and spontaneous pain, by involvement in spontaneous ectopic discharges after peripheral nerve injury. Thus, I(h) blockers may hold therapeutic potential for the intervention of mechanical allodynia under diverse neuropathic conditions. Here we show that eugenol blocks I(h) and abolishes mechanical allodynia in the trigeminal system. Eugenol produced robust inhibition of I(h) with IC(50) of 157 µM in trigeminal ganglion (TG) neurons, which is lower than the dose of eugenol that inhibits voltage-gated Na channels. Eugenol-induced I(h) inhibition was not mediated by G(i/o)-protein activation, but was gradually diminished by an increase in intracellular cAMP concentration. Eugenol also inhibited I(h) from injured TG neurons which were identified by retrograde labeling with DiI and reversed mechanical allodynia in the orofacial area after chronic constriction injury of infraorbital nerve. We propose that eugenol could be potentially useful for reversing mechanical allodynia in neuropathic pain patients.