Spinal Trigeminal Nucleus Caudalis Research Articles

H3 Receptor Expression in the Spinal Trigeminal Nucleus Caudalis in a Rat Model of Allergic Rhinitis

Background: Allergic rhinitis is one of the most frequently observed allergic diseases. Although antihistamine medication is an effective symptomatic treatment for this disease, it is not suffcient enough to improve quality of life, requiring an additional treatment. Excessive sneezing is a typical symptom of allergic rhinitis in response to immunological release of histamine, and we hypothesized that this could be due to characteristic changes on the reflex pathway, particularly a nucleus receiving nasal trigeminal afferents, the spinal trigeminal nucleus caudalis (Sp5C), via a histamine mechanism(s). In the present study, we created an allergic rhinitis model by conventional methods, harvesting brainstem tissues to perform immunohistochemistry for histamine receptor subtype 3 (H3R) in Sp5C. Methods: Male Fisher 344 rats (8 weeks old) were sensitized and nasally challenged to Ovalbumin. After the Ovalbumin challenge into the nasal cavity, the number of sneezing was visually counted for 13 min and repeated for 7 days. After that, animals were euthanized, brainstems were extracted, and immunohistochemistry were performed with various antibodies, including anti-H3R and -GFAP. In some animals, to identify the subregion within Sp5C where nasal trigeminal afferents are transmitted from the nasal mucosa, capsaicin (0.3mM), a potent nociceptive substance, was administered into the nasal cavity and then immunohistochemistry of c-Fos expression was performed. Quantitative analysis was performed by area of fluorescent staining. Results: Intranasal administration of capsaicin markedly increased c-Fos expression in Sp5C compared to non-administration control, confirming the subregion of Sp5C receiving nasal afferent information. The allergic rhinitis model rats showed a significant increase in sneezing frequency in response to Ovalbumin nasal challenge compared to non-sensitized rats challenged with Ovalbumin or saline (56.8±3.9 vs. 16.6±1.6 vs. 23.7±3.2 [sneezes during a total observation period]; p<0.001). The expression pattern of H3Rs was punctate and co-stained with GFAP in many cases. Both H3 receptor and GFAP expressions were significantly decreased in allergic model rats compared to control rats (control vs. allergic; 21.2±1.2 vs.6.4±0.5 [%] in H3Rs, p<0.001; 16.2±0.5 vs.10.9±0.8 [%] in GFAP, p<0.001). No difference in DAPI staining was observed (14.5±0.8 vs.13.7±0.5 [%], p=0.223), suggesting similar population of cells in data analyzed from both groups. Discussion: These results may suggest that Sp5C is part of the sneezing reflex pathway and that a decrease in H3Rs, including those on astrocytes, is involved in worsening clinical symptoms of allergic rhinitis such as excessive sneezing. This research was supported by KAKENHI, Grant-in-Aid for challenging Exploratory Research # 21K19182. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hypothalamic Paraventricular Stimulation Inhibits Nociceptive Wide Dynamic Range Trigeminocervical Complex Cells via Oxytocinergic Transmission.

Oxytocinergic transmission blocks nociception at the peripheral, spinal, and supraspinal levels through the oxytocin receptor (OTR). Indeed, a neuronal pathway from the hypothalamic paraventricular nucleus (PVN) to the spinal cord and trigeminal nucleus caudalis (Sp5c) has been described. Hence, although the trigeminocervical complex (TCC), an anatomical area spanning the Sp5c, C1, and C2 regions, plays a role in some pain disorders associated with craniofacial structures (e.g., migraine), the role of oxytocinergic transmission in modulating nociception at this level has been poorly explored. Hence, in vivo electrophysiological recordings of TCC wide dynamic range (WDR) cells sensitive to stimulation of the periorbital or meningeal region were performed in male Wistar rats. PVN electrical stimulation diminished the neuronal firing evoked by periorbital or meningeal electrical stimulation; this inhibition was reversed by OTR antagonists administered locally. Accordingly, neuronal projections (using Fluoro-Ruby) from the PVN to the WDR cells filled with Neurobiotin were observed. Moreover, colocalization between OTR and calcitonin gene-related peptide (CGRP) or OTR and GABA was found near Neurobiotin-filled WDR cells. Retrograde neuronal tracers deposited at the meningeal (True-Blue, TB) and infraorbital nerves (Fluoro-Gold, FG) showed that at the trigeminal ganglion (TG), some cells were immunopositive to both fluorophores, suggesting that some TG cells send projections via the V1 and V2 trigeminal branches. Together, these data may imply that endogenous oxytocinergic transmission inhibits the nociceptive activity of second-order neurons via OTR activation in CGRPergic (primary afferent fibers) and GABAergic cells.

Unveiling the therapeutic potential of Dl-3-n-butylphthalide in NTG-induced migraine mouse: activating the Nrf2 pathway to alleviate oxidative stress and neuroinflammation

BackgroundMigraine stands as a prevalent primary headache disorder, with prior research highlighting the significant involvement of oxidative stress and inflammatory pathways in its pathogenesis and chronicity. Existing evidence indicates the capacity of Dl-3-n-butylphthalide (NBP) to mitigate oxidative stress and inflammation, thereby conferring neuroprotective benefits in many central nervous system diseases. However, the specific therapeutic implications of NBP in the context of migraine remain to be elucidated.MethodsWe established a C57BL/6 mouse model of chronic migraine (CM) using recurrent intraperitoneal injections of nitroglycerin (NTG, 10 mg/kg), and prophylactic treatment was simulated by administering NBP (30 mg/kg, 60 mg/kg, 120 mg/kg) by gavage prior to each NTG injection. Mechanical threshold was assessed using von Frey fibers, and photophobia and anxious behaviours were assessed using a light/dark box and elevated plus maze. Expression of c-Fos, calcitonin gene-related peptide (CGRP), Nucleus factor erythroid 2-related factor 2 (Nrf2) and related pathway proteins in the spinal trigeminal nucleus caudalis (SP5C) were detected by Western blotting (WB) or immunofluorescence (IF). The expression of IL-1β, IL-6, TNF-α, Superoxide dismutase (SOD) and malondialdehyde (MDA) in SP5C and CGRP in plasma were detected by ELISA. A reactive oxygen species (ROS) probe was used to detect the expression of ROS in the SP5C.ResultsAt the end of the modelling period, chronic migraine mice showed significantly reduced mechanical nociceptive thresholds, as well as photophobic and anxious behaviours. Pretreatment with NBP attenuated nociceptive sensitization, photophobia, and anxiety in the model mice, reduced expression levels of c-Fos and CGRP in the SP5C and activated Nrf2 and its downstream proteins HO-1 and NQO-1. By measuring the associated cytokines, we also found that NBP reduced levels of oxidative stress and inflammation. Most importantly, the therapeutic effect of NBP was significantly reduced after the administration of ML385 to inhibit Nrf2.ConclusionsOur data suggest that NBP may alleviate migraine by activating the Nrf2 pathway to reduce oxidative stress and inflammation in migraine mouse models, confirming that it may be a potential drug for the treatment of migraine.Graphical

Electroacupuncture regulates the P2X7R-NLRP3 inflammatory cascade to relieve decreased sensation on ocular surface of type 2 diabetic rats with dry eye.

Dry eye (DE) is a prevalent ocular surface disease in patients with type 2 diabetes (T2DM). However, current medications are ineffective against decreased sensation on the ocular surface. While electroacupuncture (EA) effectively alleviates decreased sensation on ocular surface of DE in patients with T2DM, the neuroprotective mechanism remains unclear. This study explored the pathogenesis and therapeutic targets of T2DM-associated DE through bioinformatics analysis. It further investigated the underlying mechanism by which EA improves decreased sensation on the ocular surface of DE in rats with T2DM. Bioinformatic analysis was applied to annotate the potential pathogenesis of T2DM DE. T2DM and DE was induced in male rats. Following treatment with EA and fluorometholone, comprehensive metrics were assessed. Additionally, the expression patterns of key markers were studied. Key targets such as NLRP3, Caspase-1, and NOD-like receptor signaling may be involved in the pathogenesis of T2DM DE. EA treatment improved ocular measures. Furthermore, EA potently downregulated P2X7R, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1 expression within the trigeminal ganglion and spinal trigeminal nucleus caudalis. Targeted P2X7R antagonist (A-438079) and agonist (BzATP) employed as controls to decipher the biochemistry of the therapeutic effects of EA showed an anti-inflammatory effect with A-438079, while BzATP blocked the anti-inflammatory effect of EA. EA relieved DE symptoms and attenuated inflammatory damage to sensory nerve pathways in T2DM rats with DE. These findings suggest a crucial role of EA inhibition of the P2X7R-NLRP3 inflammatory cascade to provide these benefits.

Mitochondria-targeting nanozyme alleviating temporomandibular joint pain by inhibiting the TNFα/NF-κB/NEAT1 pathway.

Inflammatory cytokines that are secreted into the spinal trigeminal nucleus caudalis (Sp5C) may augment inflammation and cause pain associated with temporomandibular joint disorders (TMD). In a two-step process, we attached triphenylphosphonium (TPP) to the surface of a cubic liposome metal-organic framework (MOF) loaded with ruthenium (Ru) nanozyme. The design targeted mitochondria and was designated Mito-Ru MOF. This structure scavenges free radicals and reactive oxygen species (ROS) and alleviates oxidative stress. The present study aimed to investigate the effects and mechanisms by which Mito-Ru MOF ameliorates TMD pain. Intra-temporomandibular joint (TMJ) injections of complete Freund's adjuvant (CFA) induced inflammatory pain for ≥10 d in the skin areas innervated by the trigeminal nerve. Tumor necrosis factor-alpha (TNF-α), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), long non-coding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1), and ROS also have been proved to be significantly upregulated in the Sp5C of TMD mice. Moreover, a single Mito-Ru MOF treatment alleviated TMD pain for 3 d and downregulated TNF-α, NF-κB, lncRNA NEAT1, and ROS. NF-κB knockdown downregulated NEAT1 in the TMD mice. Hence, Mito-Ru MOF inhibited the production of ROS and alleviated CFA-induced TMD pain via the TNF-α/NF-κB/NEAT1 pathway. Therefore, Mito-Ru MOF could effectively treat the pain related to TMD and other conditions associated with severe acute inflammatory activation.

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

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

Electroacupuncture Alleviates Dry Eye Ocular Pain Through TNF-ɑ Mediated ERK1/2/P2X3R Signaling Pathway in SD Rats.

This study aimed to examine electroacupuncture's influence on ocular pain and its potential modulation of the TNF-ɑ mediated ERK1/2/P2X3R signaling pathway in dry eye-induced rat models. Male Sprague-Dawley rats with induced dry eye, achieved through extraorbital lacrimal gland removal, were treated with electroacupuncture. Comprehensive metrics such as the corneal mechanical perception threshold, palpebral fissure height, eyeblink frequency, eye wiping duration, behavioral changes in the open field test, and the forced swimming test were employed. Additionally, morphological changes in microglia and neurons were observed. Expression patterns of key markers, TNF-ɑ, TNFR1, p-ERK1/2, and P2X3R, in the trigeminal ganglion (TG) and spinal trigeminal nucleus caudalis (SpVc) regions, were studied with etanercept serving as a control to decipher the biochemistry of electroacupuncture's therapeutic effects. Electroacupuncture treatment demonstrated a notable decrease in the corneal mechanical perception threshold, improvement in palpebral fissure height, and significant reductions in both eyeblink frequency and eye wiping duration. Moreover, it exhibited a promising role in anxiety alleviation. Notably, the technique effectively diminished ocular pain by curbing microglial and neuronal activation in the TG and SpVc regions. Furthermore, it potently downregulated TNF-ɑ, TNFR1, p-ERK1/2, and P2X3R expression within these regions. Electroacupuncture attenuated damage to sensory nerve pathways, reduced pain, and eased anxiety in dry eye-afflicted rats. The findings suggest a crucial role of TNF-ɑ mediated ERK1/2/P2X3R signaling pathway inhibition by electroacupuncture in these benefits.

TET1-mediated epigenetic regulation of tumor necrosis factor-α in trigeminal ganglia contributes to chronic temporomandibular joint pain

Chronic temporomandibular joint (TMJ) pain profoundly affects patients' quality of life. Trigeminal tumor necrosis factor-α (TNFα) plays a pivotal role in mediating TMJ pain in mice, yet the underlying epigenetic mechanisms remain enigmatic. To unravel these epigenetic intricacies, we employed a multifaceted approach. Hydroxymethylated DNA immunoprecipitation (hMeDIP) and chromatin immunoprecipitation (ChIP) followed by qPCR were employed to investigate the demethylation of TNFα gene (Tnfa) and its regulation by ten-eleven translocation methylcytosine dioxygenase 1 (TET1) in a chronic TMJ pain mouse model. The global levels of 5-hydroxymethylcytosine (5hmc) and percentage of 5hmc at the Tnfa promoter region were measured in the trigeminal ganglia (TG) and spinal trigeminal nucleus caudalis (Sp5C) following complete Freund's adjuvant (CFA) or saline treatment. TET1 knockdown and pain behavioral testing were conducted to ascertain the role of TET1-mediated epigenetic regulation of TNFα in the pathogenesis of chronic TMJ pain. Our finding revealed an increase in 5hmc at the Tnfa promoter region in both TG and Sp5C of CFA-treated mice. TET1 was upregulated in the mouse TG, and the ChIP result showed TET1 direct binding to the Tnfa promoter, with higher efficiency in the CFA-treated group. Immunofluorescence revealed the predominant expression of TET1 in trigeminal neurons. TET1 knockdown in the TG significantly reversed CFA-induced TNFα upregulation and alleviated chronic TMJ pain. In conclusion, our study implicates TET1 as a vital epigenetic regulator contributing to chronic inflammatory TMJ pain via trigeminal TNFα signaling. Targeting TET1 holds promise for epigenetic interventions in TMJ pain management.

Effect of theanine on the hyperexcitability of trigeminal secondary nociceptive neurons following orofacial inflammation in rats.

The present in vivo study investigated whether systemic administration of theanine attenuates the inflammation-induced hyperexcitability of trigeminal spinal nucleus caudalis (SpVc) neurons associated with hyperalgesia. Complete Freund's adjuvant (CFA) was injected into the whisker pads of 24 rats to induce inflammation, and then mechanical stimulation was applied to the orofacial area to assess the threshold of escape. The mechanical threshold was statistically significantly lower in CFA-inflamed rats compared to uninjected naïve rats, and this lowered threshold returned to control levels after 2 days of theanine administration. The mean discharge frequency of SpVc wide-dynamic range (WDR) neurons to mechanical stimuli in anesthetized CFA-inflamed rats was statistically significantly lower after two days of theanine administration. In addition, the increased mean spontaneous discharge of SpVc WDR neurons in CFA-inflamed rats statistically significantly decreased after theanine administration. Similarly, theanine restored the expanded mean receptive field size in CFA-inflamed rats to control levels. Taken together, these results suggest that administration of theanine attenuates inflammatory hyperalgesia associated with hyperexcitability of nociceptive SpVc WDR neurons. These findings support the potential of theanine as a therapeutic agent in complementary alternative medicine strategies to prevent inflammatory hyperalgesia.

Elevated Serotonin in Mouse Spinal Dorsal Horn Is Pronociceptive.

Serotonergic neurons in the rostral ventral medulla (RVM) contribute to bidirectional control of pain through modulation of spinal and trigeminal nociceptive networks. Deficits in this pathway are believed to contribute to pathologic pain states, but whether changes in serotonergic mechanisms are pro- or antinociceptive is debated. We used a combination of optogenetics and fiber photometry to examine these mechanisms more closely. We find that optogenetic activation of RVM serotonergic afferents in the spinal cord of naive mice produces mechanical hypersensitivity and conditioned place aversion (CPA). Neuropathic pain, produced by chronic constriction injury of the infraorbital nerve (CCI-ION), evoked a tonic increase in serotonin (5HT) concentrations within the spinal trigeminal nucleus caudalis (SpVc), measured with liquid chromatography-tandem mass spectroscopy (LC-MS/MS). By contract, CCI-ION had no effect on the phasic serotonin transients in SpVc, evoked by noxious pinch, and measured with fiber photometry of a serotonin sensor. These findings suggest that serotonin release in the spinal cord is pronociceptive and that an increase in sustained serotonin signaling, rather than phasic or event driven increases, potentiate nociception in models of chronic pain.

Annexin A1 exerts analgesic effect in a mouse model of medication overuse headache

Medication overuse headache (MOH) is a serious global condition. The interaction between headache attacks and medication overuse complicates the understanding of its pathophysiology. In this study, we developed a preclinical MOH model that incorporates these two key factors by overusing rizatriptan benzoate (RIZ, 4mg/kg, i.g.) in a glyceryl trinitrate (GTN, 10mg/kg, i.p.) induced chronic migraine mouse model. We observed that RIZ overuse aggravated GTN-induced cutaneous allodynia and caused a prolonged state of latent sensitization. We also detected a significant upregulation of Annexin-A1 (ANXA1), a protein mainly expressed in the microglia of the spinal trigeminal nucleus caudalis (SPVC), in GTN+RIZ mice. Intracerebroventricular injection of ANXA1-derived peptide Ac2-26 trifluoroacetic acid (TFA) (5μg/mouse) inhibited bright light stress (BLS) induced acute allodynia via the formyl peptide receptor (FPR) in GTN+RIZ mice. These results suggest that ANXA1 may have an analgesic effect in triptan-associated MOH and could potentially serve as a therapeutic target.

Optogenetic Inhibition of Glutamatergic Neurons in the Dysgranular Posterior Insular Cortex Modulates Trigeminal Neuropathic Pain in CCI-ION Rat.

In individuals with chronic neuropathic pain, the posterior insular cortex (PIC) has been found to exhibit increased glutamatergic activity, and the dysgranular portion of PIC (DPIC) has been investigated as a novel cortical target for pain modulation. However, the role of DPIC glutamatergic neurons (DPICg) in trigeminal neuropathic pain (TNP) remains unclear. Here, we examined the outcomes of DPICg inhibition in a rat model of chronic constriction injury of the infraorbital nerve (CCI-ION). Animals were randomly divided into TNP, sham, and control groups. TNP animals underwent CCI-ION surgery. Either optogenetic or null viruses were delivered to the contralateral DPICg of TNP and sham animals. In vivo single-unit extracellular recordings from the ipsilateral spinal trigeminal nucleus caudalis (TNC) and contralateral ventral posteromedial (VPM) thalamus were obtained under both "ON" and "OFF" stimulation states. Behavioral responses during the stimulation-OFF and stimulation-ON phases were examined. Expression of c-Fos, pERK, and CREB immunopositive neurons were also observed. Optogenetic inhibition of contralateral DPICg decreased the neural firing rate in both TNC and VPM thalamus, the expression of sensory-responsive cell bodies, and transcriptional factors in the DPIC of TNP group. Improvements in hyperalgesia, allodynia, and anxiety-like responses in TNP animals were also observed during stimulation-ON condition. In fine, descending pain processing is influenced by neuroanatomical projections from the DPIC to the pain matrix areas, and DPICg could play a necessary role in this neural circuitry. Therefore, the antinociceptive effect of DPICg inhibition in this study may provide evidence for the therapeutic potential of DPICg in TNP.

A female-specific role for trigeminal dynorphin in orofacial pain comorbidity.

Migraine is commonly reported in patients with temporomandibular disorders (TMDs), but little is known about the mechanisms underlying the comorbid condition. Here, we prepared a mouse model to investigate this comorbidity, in which masseter muscle tendon ligation (MMTL) was performed to induce a myogenic TMD, and the pre-existing TMD enabled a subthreshold dose of nitroglycerin (NTG) to produce migraine-like pain in mice. RNA sequencing followed by real-time quantitative polymerase chain reaction confirmation showed that MMTL plus NTG treatment increased prodynorphin ( Pdyn ) mRNA expression in the spinal trigeminal nucleus caudalis (Sp5C) of female mice but not in male mice. Chemogenetic inhibition of Pdyn -expressing neurons or microinjection of antidynorphin antiserum in the Sp5C alleviated MMTL-induced masseter hypersensitivity and diminished the MMTL-enabled migraine-like pain in female mice but not in male mice. Moreover, chemogenetic activation of Pdyn -expressing neurons or microinjection of dynorphin A (1-17) peptide in the Sp5C enabled a subthreshold dose of NTG to induce migraine-like pain in female mice but not in male mice. Taken together, our results suggest that trigeminal dynorphin has a female-specific role in the modulation of comorbid TMDs and migraine.

Exercise Improves Orofacial Pain and Modifies Neuropeptide Expression in a Rat Model of Parkinson's Disease.

Pain is a common non-motor symptom of Parkinson's disease (PD), which often occurs in the early disease stages. Despite the high prevalence, it remains inadequately treated. In a hemi-parkinsonian rat model, we aimed to investigate the neurochemical factors involved in orofacial pain development, with a specific focus on pain-related peptides and cannabinoid receptors. We also evaluated whether treadmill exercise could improve orofacial pain and modulate these mechanisms. Rats were unilaterally injected in the striatum with either 6-hydroxydopamine (6-OHDA) or saline. Fifteen days after stereotactic surgery, the animals were submitted to treadmill exercise (EX), or remained sedentary (SED). Pain assessment was performed before the surgical procedure and prior to each training session. Pain-related peptides, substance P (SP), calcitonin gene-related peptide (CGRP), and transient receptor potential vanilloid type 1 (TRPV1) activation and cannabinoid receptor type 1 (CB1) and type 2 (CB2) were evaluated in the trigeminal nucleus. In order to confirm the possible involvement of cannabinoid receptors, we also injected antagonists of CB1 and CB2 receptors. We confirmed the presence of orofacial pain after unilateral 6-OHDA-injection, which improved after aerobic exercise training. We also observed increased pain-related expression of SP, CGRP and TRPV1 and decreased CB1 and CB2 in the trigeminal ganglion and caudal spinal trigeminal nucleus in animals with PD, which was reversed after aerobic exercise training. In addition, we confirm the involvement of cannabinoid receptors since both antagonists decreased the nociceptive threshold of PD animals. These data suggest that aerobic exercise effectively improved the orofacial pain associated with the PD model, and may be mediated by pain-related neuropeptides and cannabinoid receptors in the trigeminal system.

Suppression of the excitability of rat nociceptive secondary sensory neurons following local administration of the Phytochemical, (-)-Epigallocatechin-3-gallate

The phytochemical, polyphenolic compound, (-)-epigallocatechin-3-gallate (EGCG), is the main catechin found in green tea. Although a modulatory effect of EGCG on voltage-gated sodium and potassium channels has been reported in excitable tissues, the in vivo effect of EGCG on the excitability of nociceptive sensory neurons remains to be determined. Our aim was to investigate whether local administration of EGCG to rats attenuates the excitability of nociceptive spinal trigeminal nucleus caudalis (SpVc) neurons in response to mechanical stimulation in vivo. Extracellular single unit recordings were made from SpVc neurons in response to orofacial mechanical stimulation of anesthetized rats. The mean firing frequency of SpVc wide-dynamic range neurons following both non-noxious and noxious mechanical stimuli was significantly inhibited by EGCG in a dose-dependent and reversible manner. The mean magnitude of inhibition by EGCG on SpVc neuronal discharge frequency was similar to that of the local anesthetic, 1% lidocaine. Local injection of half-dose of lidocaine replaced the half-dose of EGCG. These results suggest that local injection of EGCG suppresses the excitability of nociceptive SpVc neurons, possibly via the inhibition of voltage-gated sodium channels and opening of voltage-gated potassium channels in the trigeminal ganglion. Therefore, administration of EGCG as a local anesthetic may provide relief from trigeminal nociceptive pain without side effects.

Noradrenergic Input from Nucleus of the Solitary Tract Regulates Parabrachial Activity in Mice.

The parabrachial complex (PB) is critically involved in aversive processes, and chronic pain is associated with amplified activity of PB neurons in rodent models of neuropathic pain. Here, we demonstrate that catecholaminergic input from the caudal nucleus of the solitary tract (cNTScat), a stress responsive region that integrates interoceptive and exteroceptive signals, causes amplification of PB activity and their sensory afferents. We used a virally mediated expression of a norepinephrine (NE) sensor, NE2h, fiber photometry, and extracellular recordings in anesthetized mice to show that noxious mechanical and thermal stimuli activate cNTS neurons. These stimuli also produce prolonged NE transients in PB that far outlast the noxious stimuli. Similar NE transients can be evoked by focal electrical stimulation of cNTS, a region that contains the noradrenergic A2 cell group that projects densely on PB. In vitro, optical stimulation of cNTScat terminals depolarized PB neurons and caused a prolonged increase the frequency of excitatory synaptic activity. A dual opsin approach showed that sensory afferents from the caudal spinal trigeminal nucleus are potentiated by cNTScat terminal activation. This potentiation was coupled with a decrease in the paired pulse ratio (PPR), consistent with an cNTScat-mediated increase in the probability of release at SpVc synapses. Together, these data suggest that A2 neurons of the cNTS generate long lasting NE transients in PB which increase excitability and potentiate responses of PB neurons to sensory inputs. These reveal a mechanism through which stressors from multiple modalities may potentiate the aversiveness of nociceptive stimuli.

Effects of Peripheral Nerve Injury on the Induction of c-Fos and Phosphorylated ERK in the Brainstem Trigeminal Sensory Nuclear Complex.

Sequential changes in brainstem and spinal cord neurons after traumatic injury to peripheral nerves are related to neuropathic pain symptoms. This study was conducted to elucidate the influence of nerve insult on stimulus-induced c-Fos expression and ERK phosphorylation by brainstem neurons. The brainstem trigeminal sensory nuclear complex (BTSNC) was examined for neuronal profiles immunolabeled with c-Fos and phosphorylated ERK (p-ERK) antibodies elicited by stimulation of the tongue with capsaicin after lingual or inferior alveolar nerve (IAN) injury. Abundant neuronal profiles immunolabeled for c-Fos and p-ERK elicited by capsaicin were distributed in the spinal trigeminal nucleus caudalis (Vc) without nerve injury. The spinal trigeminal nucleus oralis (Vo) contained limited numbers of these neuronal profiles after stimulation of the tongue. A significant reduction of these neuronal profiles in the ipsilateral Vc was detected after lingual nerve injury. After IAN injury, an increased number of neuronal profiles immunolabeled for c-Fos elicited by capsaicin was noted, while that of p-ERK was left unchanged in the ipsilateral Vc. On the both sides of the Vo, an increased number of capsaicin-induced neuronal profiles immunolabeled for c-Fos and p-ERK was detected after lingual or IAN injury. Differential effects of lingual or IAN injury on stimulus-induced c-Fos expression and ERK phosphorylation by Vo and Vc neurons may be involved in the complex nature of symptoms of trigeminal neuralgia.

Electroacupuncture Reduces Ocular Surface Neuralgia in Dry-Eyed Guinea Pigs by Inhibiting the Trigeminal Ganglion and Spinal Trigeminal Nucleus Caudalis P2X3R-PKC Signaling Pathway

Purpose To observe the effects of electroacupuncture on ocular surface neuralgia and the P2X3R-PKC signaling pathway in guinea pigs with dry eye. Methods A dry eye guinea pig model was established by subcutaneous injection of scopolamine hydrobromide. Guinea pigs were monitored for body weight, palpebral fissure height, number of blinks, corneal fluorescein staining score, phenol red thread test, and corneal mechanical perception threshold. Histopathological changes and mRNA expression of P2X3R and protein kinase C in the trigeminal ganglion and spinal trigeminal nucleus caudalis were observed. We performed a second part of the experiment, which involved the P2X3R-specific antagonist A317491 and the P2X3R agonist ATP in dry-eyed guinea pigs to further validate the involvement of the P2X3R-protein kinase C signaling pathway in the regulation of ocular surface neuralgia in dry eye. The number of blinks and corneal mechanical perception threshold were monitored before and 5 min after subconjunctival injection and the protein expression of P2X3R and protein kinase C was detected in the trigeminal ganglion and spinal trigeminal nucleus caudalis of guinea pigs. Results Dry-eyed guinea pigs showed pain-related manifestations and the expression of P2X3R and protein kinase C in the trigeminal ganglion and spinal trigeminal nucleus caudalis was upregulated. Electroacupuncture reduced pain-related manifestations and inhibited the expression of P2X3R and protein kinase C in the trigeminal ganglion and spinal trigeminal nucleus caudalis. Subconjunctival injection of A317491 attenuated corneal mechanoreceptive nociceptive sensitization in dry-eyed guinea pigs, while ATP blocked the analgesic effect of electroacupuncture. Conclusions Electroacupuncture reduced ocular surface sensory neuralgia in dry-eyed guinea pigs, and the mechanism of action may be associated with the inhibition of the P2X3R-protein kinase C signaling pathway in the trigeminal ganglion and spinal trigeminal nucleus caudalis by electroacupuncture.

Effects of high-frequency near infrared laser irradiation on experimental tooth movement-induced pain in rats.

Discomfort and dull pain are known side effects of orthodontic treatment. Pain is expected to be reduced by near-infrared (NIR) lasers; however, the mechanism underlying effects of short-pulse NIR lasers in the oral and maxillofacial area remains unclear. This study aimed to examine the effects of high-frequency NIR diode laser irradiation on pain during experimental tooth movement (ETM) on 120J. NIR laser with 910nm wavelength, 45W maximum output power, 300 mW average output power, and 200ns pulse width (Lumix 2; (Lumix 2; Fisioline, Verduno CN, Italy) was used for the experiment. A nickel-titanium-closed coil was used to apply a 50-gf force between the maxillary left-side first molar and incisor in 7-week-old Sprague-Dawley rats (280-300g) to induce ETM. We measured facial-grooming frequency and vacuous chewing movement (VCM) period between laser-irradiation and ETM groups. We performed immunofluorescent histochemistry analysis to quantify levels of Iba-1, astrocytes, and c-fos protein-like immunoreactivity (Fos-IR) in the trigeminal spinal nucleus caudalis (Vc). Compared with the ETM group, the laser irradiation group had significantly decreased facial-grooming frequency (P = 0.0036), VCM period (P = 0.043), Fos-IR (P = 0.0028), Iba-1 levels (P = 0.0069), and glial fibrillary acidic protein (GFAP) levels (P = 0.0071). High-frequency NIR diode laser irradiation appears to have significant analgesic effects on ETM-induced pain, which involve inhibiting neuronal activity, microglia, and astrocytes, and it inhibits c-fos, Iba-1, and GFAP expression, reducing ETM-induced pain in rats. High-frequency NIR diode laser application could be applied to reduce pain during orthodontic tooth movement.

Characterization of the expression of gastrin-releasing peptide and its receptor in the trigeminal and spinal somatosensory systems of Japanese macaque monkeys: Insight into humans.

Gastrin-releasing peptide (GRP) and its receptor (GRPR) have been identified as itch mediators in the spinal and trigeminal somatosensory systems in rodents. In primates, there are few reports of GRP/GRPR expression or function in the spinal sensory system and virtually nothing is known in the trigeminal system. The aim of the present study was to characterize GRP and GRPR in the trigeminal and spinal somatosensory system of Japanese macaque monkeys (Macaca fuscata). cDNA encoding GRP was isolated from the macaque dorsal root ganglion (DRG) and exhibited an amino acid sequence that was highly conserved among mammals and especially in primates. Immunohistochemical analysis demonstrated that GRP was expressed mainly in the small-sized trigeminal ganglion and DRG in adult macaque monkeys. Densely stained GRP-immunoreactive (ir) fibers were observed in superficial layers of the spinal trigeminal nucleus caudalis (Sp5C) and the spinal cord. In contrast, GRP-ir fibers were rarely observed in the principal sensory trigeminal nucleus and oral and interpolar divisions of the spinal trigeminal nucleus. cDNA cloning, in situ hybridization, and Western blot revealed substantial expression of GRPR mRNA and GRPR protein in the macaque spinal dorsal horn and Sp5C. Our Western ligand blot and ligand derivative stain for GRPR revealed that GRP directly bound in the macaque Sp5C and spinal dorsal horn as reported in rodents. Finally, GRP-ir fibers were also detected in the human spinal dorsal horn. The spinal and trigeminal itch neural circuits labeled with GRP and GRPR appear to function also in primates.