Rat Trigeminal Ganglion Research Articles

Attenuation of Sensory Transmission Through the Rat Trigeminal Ganglion by GABA Receptor Activation

While the trigeminal ganglion is often considered a passive conduit of sensory transmission, neurons and satellite glial cells (SGCs) within it can release neurotransmitters and express neuroreceptors. Some trigeminal ganglion neurons contain the neurotransmitter γ-aminobutyric acid (GABA) and express GABA receptors. There is behavioral evidence that increased GABA levels in the trigeminal ganglion decreases nociception, while a loss of GABA receptors results in hyperalgesia, although the neural mechanisms for this remain to be investigated. In this study, the expression of GABA receptors by trigeminal ganglion neurons that innervate rat labial skin and masseter muscle was compared using immunohistochemistry. The effect of intraganglionic administration of GABA receptor agonists was investigated by single unit recording of trigeminal brainstem and ganglion neuron responses to stimulation of the labial skin and/or masseter muscle in anesthetized rats. The mean frequency of expression of GABAA and GABAB receptors by masseter and labial skin ganglion neurons was 62.5% and 92.7%, and 55.4% and 20.3%, respectively. The expression of both GABA receptors was significantly greater in skin ganglion neurons. Masticatory muscle evoked brainstem trigeminal neuron responses were significantly attenuated by intraganglionic injection of muscimol (GABAA) but not baclofen (GABAB). The mechanical sensitivity of slow and fast conducting masticatory muscle afferent fibers was decreased and increased, respectively, by intraganglionic injection of both muscimol and baclofen. Activation of GABAA receptors may exert a gating effect on sensory transmission through the trigeminal ganglion by decreasing putative nociceptive input and enhancing innocuous sensory input.

Inhibitory Effects of Palmatine on P2X7 Receptor Expression in Trigeminal Ganglion and Facial Pain in Trigeminal Neuralgia Rats

Trigeminal Neuralgia (TN) refers to recurrent severe paroxysmal pain in the distribution area of the trigeminal nerve, which seriously affects the quality of life of patients. This research applied the chronic constriction injury of the infraorbital nerve (CCI—ION) approach to induce an animal model of TN in rats. The mechanical pain threshold of each group of rats was determined postoperatively; the expression of P2X7 receptor in trigeminal ganglion (TG) was assessed by qRT-PCR, immunofluorescence and Western blot; and the changes of the proinflammatory cytokines IL-1β and TNF-α in serum of rats were detected by ELISA. The results showed that the administration of palmatine in the TN rats could reduce the mechanical pain threshold, significantly decrease the expression of P2X7 receptor in TG, and lower the serum concentrations of IL-1β and TNF-α, compared to the sham group. In addition, the phosphorylation level of p38 in TG of TN rats was significantly decreased after treatment with palmatine. Likewise, inhibition of P2X7 expression by shRNA treatment could effectively counteract the adversary changes of pain sensitivity, IL-1β and TNF-α production, and p38 phosphorylation in TN rats. Our data suggest that palmatine may alleviate mechanical facial pain in TN rats possibly by reducing the expression of P2X7 receptor in TG of TN rats, which may be attributable to inhibiting p38 phosphorylation and reducing the release of IL-1β and TNF-α.

Intracellular potassium depletion enhances apoptosis induced by staurosporine in cultured trigeminal satellite glial cells

Purpose Satellite glial cells (SGC) surrounding neurons in sensory ganglia can buffer extracellular potassium, regulating the excitability of injured neurons and possibly influencing a shift from acute to neuropathic pain. SGC apoptosis may be a key component in this process. This work evaluated induction or enhancement of apoptosis in cultured trigeminal SGC following changes in intracellular potassium [K]ic. Materials and methods We developed SGC primary cultures from rat trigeminal ganglia (TG). Purity of our cultures was confirmed using immunofluorescence and western blot analysis for the presence of the specific marker of SGC, glutamine synthetase (GS). SGC [K]ic was depleted using hypo-osmotic shock and 4 mM bumetanide plus 10 mM ouabain. [K]ic was measured using the K+ fluorescent indicator potassium benzofuran isophthalate (PBFI-AM). Results SGC tested positive for GS and hypo-osmotic shock induced a significant decrease in [K]ic at every evaluated time. Cells were then incubated for 5 h with either 2 mM staurosporine (STS) or 20 ng/ml of TNF-α and evaluated for early apoptosis and late apoptosis/necrosis by flow cytometry using annexin V and propidium iodide. A significant increase in early apoptosis, from 16 to 38%, was detected in SGC with depleted [K]ic after incubation with STS. In contrast, TNF-α did not increase early apoptosis in normal or [K]ic depleted SGC. Conclusion Hypo-osmotic shock induced a decrease in intracellular potassium in cultured trigeminal SGC and this enhanced apoptosis induced by STS that is associated with the mitochondrial pathway. These results suggest that K+ dysregulation may underlie apoptosis in trigeminal SGC.

Nerve Growth Factor Enhances Tooth Mechanical Hyperalgesia Through C-C Chemokine Ligand 19 in Rats.

The nerve growth factor (NGF) plays an important role in the regulation of neuropathic pain. It has been demonstrated that calcitonin gene-related peptide (CGRP), a well-known contributor to neurogenic inflammation, increases neuroinflammatory pain induced by NGF. The inflammatory mediator that NGF most strongly induces is C-C chemokine ligand 19 (CCL19), which can recruit inflammatory cells by binding to the receptor CCR7 followed by promoting the response of neuroinflammation. However, the regulatory mechanism of NGF and CCL19 in tooth movement orofacial pain and the interaction between both are still unclear. In this study, male Sprague–Dawley rats were used to study the modulation of NGF on orofacial pain through CCL19 and the role of each in tooth movement pain in rats. The expression levels of CCL19 mRNA and protein were determined by real-time PCR and immunofluorescence, respectively. Pain levels were assessed by measuring the rats' bite force, which drops as pain rises. Meanwhile, by verifying the relationship between CGRP and CCL19, it was laterally confirmed that NGF could modulate tooth movement-induced mechanical hyperalgesia through CCL19. The results showed that the expression level of CCL19 rose with the increased NGF, and neurons expressing CGRP can express stronger CCL19. Compared with the baseline level, the bite force for all rats dropped sharply on day 1, reached its lowest level on day 3, and recovered gradually on day 5. All results indicated that NGF played an important role in tooth movement orofacial pain via positively regulating CCL19 expression in the trigeminal ganglia of rats. Additionally, CCL19 increased the sensitivity to experimental tooth movement orofacial pain. NGF can regulate CCL19 expression, although it may regulate other inflammatory pathways as well. This is the first report on the interactions and modulations of tooth movement orofacial pain by NGF through CCL19 in rats.

Functional Coupling between the P2X7 Receptor and Pannexin-1 Channel in Rat Trigeminal Ganglion Neurons.

The ionotropic P2X receptor, P2X7, is believed to regulate and/or generate nociceptive pain, and pain in several neuropathological diseases. Although there is a known relationship between P2X7 receptor activity and pain sensing, its detailed functional properties in trigeminal ganglion (TG) neurons remains unclear. We examined the electrophysiological and pharmacological characteristics of the P2X7 receptor and its functional coupling with other P2X receptors and pannexin-1 (PANX1) channels in primary cultured rat TG neurons, using whole-cell patch-clamp recordings. Application of ATP and Bz-ATP induced long-lasting biphasic inward currents that were more sensitive to extracellular Bz-ATP than ATP, indicating that the current was carried by P2X7 receptors. While the biphasic current densities of the first and second components were increased by Bz-ATP in a concentration dependent manner; current duration was only affected in the second component. These currents were significantly inhibited by P2X7 receptor antagonists, while only the second component was inhibited by P2X1, 3, and 4 receptor antagonists, PANX1 channel inhibitors, and extracellular ATPase. Taken together, our data suggests that autocrine or paracrine signaling via the P2X7-PANX1-P2X receptor/channel complex may play important roles in several pain sensing pathways via long-lasting neuronal activity driven by extracellular high-concentration ATP following tissue damage in the orofacial area.

A Tale of Two Peptides: Identifying Targets for Migraine

Calcitonin gene related-peptide (CGRP) and amylin are closely-related peptides that are both reported to be elevated in the plasma of migraine sufferers. CGRP is highly expressed in neural tissues and migraine relevant structures, including the trigeminal ganglia (TG). Amylin is secreted from pancreatic islets in response to food intake, with conflicting reports of expression in the TG. CGRP potently activates two receptors, the CGRP receptor and Amylin 1 (AMY1) receptor, whereas amylin potently activates the AMY1 receptor. CGRP has been proposed to be auto regulated in an autocrine manner in the TG, however CGRP does not colocalise with the CGRP receptor in the TG. The AMY1 receptor is also expressed in the TG, but where it is expressed and whether it is found together with CGRP or amylin is poorly understood. Therefore, our aim was to determine whether amylin is expressed in the TG and the relative distribution of AMY1 receptor components with CGRP or amylin using thoroughly validated antibodies. To study the distribution of CGRP, amylin and the AMY1 receptor component, calcitonin receptor (CTR), in the TG a range of antibodies were validated. Anti-CGRP and amylin antibodies were validated using immunoblotting and rat pancreas histology. Anti-CTR antibodies were validated using transfected HEK293S cells and knock out tissue. Lead antibodies were then applied to mouse, rat and human TG sections with neural markers to investigate their distribution. Anti-amylin antibodies frequently cross-reacted with CGRP, whereas anti-CGRP antibodies displayed no cross-reactivity with amylin. Lead anti-amylin antibodies displayed little or no detection of amylin in rodent and human β-tubulin III-positive neurons, respectively, whereas abundant CGRP-like immunoreactivity was observed across all species. When co-incubated the little amylin-like immunoreactivity observed strongly colocalised with anti-CGRP staining. This was particularly apparently with T4145, the more cross-reactive amylin antibody. The lead anti-CTR antibody, 188/10, displayed robust detection of rodent CTR in transiently transfected HEK293S cells and a marked loss of staining in knock out mouse nucleus accumbens compared to wild type. CTR-like and CGRP-like immunoreactivity frequently colocalised in rat TG sections, primarily in C-fibre neurons, and rarely in NF200-positive A-fibre neurons. These results demonstrate the importance of thoroughly validating antibodies. The data suggest that in the TG, CGRP, rather than amylin, is the endogenous agonist for the AMY1 receptor. Additionally, it suggests that CGRP autocrine signalling occurs via the AMY1 receptor.

Petasin and isopetasin reduce CGRP release from trigeminal afferents indicating an inhibitory effect on TRPA1 and TRPV1 receptor channels

BackgroundButterbur root extract with its active ingredients petasin and isopetasin has been used in the prophylactic treatment of migraine for years, while its sites of action are not completely clear. Calcitonin gene-related peptide (CGRP) is known as a biomarker and promoting factor of migraine. We set out to investigate the impact of petasins on the CGRP release from trigeminal afferents induced by activation of the calcium conducting transient receptor potential channels (TRPs) of the subtypes TRPA1 and TRPV1.MethodsWe used well-established in vitro preparations, the hemisected rodent skull and dissected trigeminal ganglia, to examine the CGRP release from rat and mouse cranial dura mater and trigeminal ganglion neurons, respectively, after pre-incubation with petasin and isopetasin. Mustard oil and capsaicin were used to stimulate TRPA1 and TRPV1 receptor channels. CGRP concentrations were measured with a CGRP enzyme immunoassay.ResultsPre-incubation with either petasin or isopetasin reduced mustard oil- and capsaicin-evoked CGRP release compared to vehicle in an approximately dose-dependent manner. These results were validated by additional experiments with mice expressing functionally deleted TRPA1 or TRPV1 receptor channels.ConclusionsEarlier findings of TRPA1 receptor channels being involved in the site of action of petasin and isopetasin are confirmed. Furthermore, we suggest an important inhibitory effect on TRPV1 receptor channels and assume a cooperative action between the two TRP receptors. These mechanisms may contribute to the migraine prophylactic effect of petasins.

Involvement of the BNP/NPR-A/BKCa pathway in rat trigeminal ganglia following chronic constriction injury

Accumulating evidence indicates that the brain natriuretic peptide (BNP) and its receptor (natriuretic peptide receptor, NPR) are widely distributed in a variety of tissues including trigeminal ganglion (TG). Furthermore, recent studies support the involvement of the BNP-NPR-A pathway in acute and chronic pain. To investigate the role of this pathway in chronic pain, an infraorbital nerve-chronic constriction injury (ION-CCI) model of trigeminal neuralgia (TN) was produced in the rat. The time course of changes in mechanical pain threshold was examined. We observed an upregulation of BNP and NPR-A and a downregulation of large-conductance Ca2+-activated K+ (BKCa) mRNA and protein in rats subjected to ION-CCI. Patch clamping experiments in vitro found that BKCa currents were significantly reduced in rats subjected to ION-CCI. BNP increased BKCa currents in ION-CCI rats. These results suggest that BNP and NPR-A might serve as endogenous pain relievers in ION-CCI rats. Modulation of the BNP/NPR-A/BKCa channel pathway in TG may be a viable strategy for the treatment of TN.NEW & NOTEWORTHY BNP has been known to activate its receptor, NPR-A, to modulate inflammatory pain. However, the potential modulatory roles of BNP in TN have not been investigated in detail. We established an ION-CCI model of TN in the rat and observed an upregulation of BNP and NPR-A and a downregulation of BKCa in rats subjected to ION-CCI. Moreover, BNP can increase BKCa currents in ION-CCI rats. Thus, BNP and NPR-A might have inhibitory effects on trigeminal neuralgia through activating the BNP/NPR-A/BKCa channel pathway.

Neurokinins and their receptors in the rat trigeminal system: Differential localization and release with implications for migraine pain.

Substance P (SP) and calcitonin gene-related peptide (CGRP) have both been considered potential drug candidates in migraine therapy. In recent years, CGRP receptor inhibition has been established as an effective treatment, in particular as a prophylactic for chronic migraine. Curiously, inhibition of neurokinin receptor 1 (NK1R) failed to alleviate acute migraine attacks in clinical trials, and the neurokinins were consequently abandoned as potential antimigraine candidates. The reason behind this has remained enigmatic.Utilizing immunohistochemistry and semi-quantitative cell counts the expression of neurokinins and their associated receptors was examined in the rat trigeminal ganglion.Immunohistochemistry results revealed SP co-localization in CGRP positive neurons and C-fibres, where it mainly concentrated at boutons. Neurokinin A (NKA) was observed in a population of C-fibres and small neurons where it could co-localize with SP. In contrast, neurokinin B (NKB) did not co-localize with SP and was observed in large/medium sized neurons and Aδ-fibres. All neurokinin receptors (NK1-3R) were found to be expressed in a majority of trigeminal ganglion neurons and A-fibres.The functional release of SP and CGRP in the trigeminovascular system was stimulated with either 60 mM K+ or 100 nM capsaicin and measured with an enzyme-linked immunosorbent assay (ELISA). ELISA results established that SP can be released locally from trigeminovascular system. The released SP was comparatively minor compared to the CGRP release from stimulated dura mater, trigeminal ganglion neurons and fibres. We hypothesize that SP and CGRP signalling pathways may work in tandem to exacerbate painful stimuli in the TGV system.

Activation of oxytocin receptor in the trigeminal ganglion attenuates orofacial ectopic pain attributed to inferior alveolar nerve injury.

This study explores the effects of oxytocin receptor (OXTR) in the trigeminal ganglion (TG) on orofacial neuropathic pain. We demonstrate that OXTR activation in the TG relieves the orofacial ectopic pain as well as inhibits the upregulated expression of calcitonin gene-related peptide (CGRP), IL-1β, and TNFα in the TG and spinal trigeminal nucleus caudalis (SpVc) of rats with inferior alveolar nerve transection. OXTR, a G protein-coupled receptor, has been demonstrated to play a significant role in analgesia after activation by its canonical agonist oxytocin (OXT) in the dorsal root ganglion. However, the role of OXTR in the trigeminal nervous system on the orofacial neuropathic pain is still little known. In the present study, we aimed to investigate the regulation effect and mechanism of OXTR in the TG) and SpVc) on orofacial ectopic pain induced by trigeminal nerve injury. The inferior alveolar nerve (IAN) was transected to establish a ectopic pain model. A behavioral test with electronic von Frey filament demonstrated IAN transection (IANX) evoked mechanical hypersensitivity in the whisker pad from day 1 to at least day 14 after surgery. In addition, administration of OXT (50 and 100 μM) into the TG attenuated the mechanical hypersensitivity induced by IANX, which was reversed by pretreatment with L-368,899 (a selective antagonist of OXTR) into the TG. In addition, immunofluorescence showed the expression of OXTR in neurons in the TG and SpVc. Furthermore, Western blot analysis indicated that the upregulated expression of OXTR, CGRP, IL-1β, and TNFα in the TG and SpVc after IANX was inhibited by the administration of OXT into the TG. And the inhibition effect of OXT on the expression of CGRP, IL-1β, and TNFα was abolished by preapplication of OXTR antagonist L-368,899 into the TG.NEW & NOTEWORTHY This study explores the effects of oxytocin receptor (OXTR) in the trigeminal ganglion (TG) on orofacial neuropathic pain. We demonstrate that OXTR activation in the TG relieves the orofacial ectopic pain as well as inhibits the upregulated expression of calcitonin gene-related peptide, IL-1β, and TNF-α in the TG and spinal trigeminal nucleus caudalis of rats with inferior alveolar nerve transection.

MiR-34a-5p up-regulates the IL-1β/COX2/PGE2 inflammation pathway and induces the release of CGRP via inhibition of SIRT1 in rat trigeminal ganglion neurons.

Migraine is a debilitating neurological condition, with a global prevalence rate of 10.68% in men and 18.79% in women. Elucidation of the molecular mechanisms underlying migraines is of great importance for improving the quality of life of patients. The release of the neuropeptide calcitonin gene-related peptide (CGRP) from trigeminal nerve terminals is involved in the pathogenesis of migraine. Recent studies have shown that up-regulation of miR-34a-5p expression is associated with acute migraine attacks. Here, we investigated whether alteration of the expression of miR-34a-5p induces the release of the vasoactive peptide CGRP. We isolated primary rat trigeminal ganglion neurons and performed gain- and loss-of-function assays to alter the expression level of miR-34a-5p. Down-regulation of miR-34a-5p inhibited the expression of interleukin-1β (IL-1β)/cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2), decreased IL-1β, PGE2 and CGRP release, and up-regulated the expression of silencing information regulator 1 (SIRT1) in trigeminal ganglion, whereas overexpression of miR-34a-5p enhanced the expression of IL-1β/COX2/PGE2, increased the release of IL-1β, PGE2 and CGRP, and decreased the expression of SIRT1 in trigeminal ganglion. In addition, overexpression of miR-34a-5p induced apoptosis in primary rat trigeminal neurons. In summary, these findings suggest that miR-34a-5p up-regulates the IL-1β/COX2/PGE2 inflammation pathway, induces apoptosis and enhances release of CGRP via inhibition of SIRT1 expression in trigeminal ganglion neurons; thus, miR-34a-5p may have potential as a therapeutic target for the treatment of migraine.

Effects of electroacupuncture on miR-34a-5p/SIRT1 signaling in the trigeminal ganglion of rats with migraine

To investigate the effect of electroacupuncture(EA) on miR-34a-5p, silent mating type information regulation 2 homolog-1 (SIRT1) and nuclear factor-κB subunit p65 (NF-κB p65) in the trigeminal ganglion of rats with migraine, so as to explore the mechanisms of EA underlying prevention of migraine. Male SD rats were randomly divi-ded into normal, sham operation, model, EA, and EA plus EX527(a SIRT1 inhibitor) groups, with 10 rats in each group. The rat model of migraine was established by electrical stimulation of the trigeminal ganglion. Before modeling, EA was applied at "Waiguan"(TE5) and "Fengchi"(GB20) for 20 min each time, once a day for 5 consecutive days, and intraperitoneal injection of EX527 (5 mg/kg) every day simultaneously. Serum prostaglandin E2 (PGE2) and calcitonin gene-related peptide (CGRP) concentrations were measured by enzyme-linked immunosorbent assay. The levels of miR-34a-5p, SIRT1 and interleukin-1β (IL-1β) mRNA，and protein expression of SIRT1, IL-1β, NF-κB p65, NF-κB Ac-p65 and cyclooxygenase-2 （COX2） in trigeminal ganglia were detected by real-time quantitative PCR and Western blot, separately. The serum concentrations of PGE2 and CGRP， the expression of miR-34a-5p, IL-1β mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expression in the trigeminal ganglion were remarkably increased (P<0.05), while the SIRT1 mRNA and protein decreased (P<0.05) in the model group in contrast to the normal group. Following EA intervention， the serum PGE2 and CGRP concentrations, miR-34a-5p expression, IL-1β mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expression were significantly down-regulated (P<0.05), and SIRT1 mRNA and protein significantly up-regulated (P<0.05). Compared with the EA group, the serum concentrations of PGE2 and CGRP, IL-1β mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expressions increased (P<0.05), and SIRT1 protein decreased (P<0.05) in the EA plus EX527 group. In migraine rats, EA can inhibit miR-34a-5p expression in the trigeminal ganglion, increase SIRT1 expression, down-regulate IL-1β/COX2 inflammation signals, reduce PGE2 synthesis, and thus redue CGRP released from the peripheral terminals, which may be one of the mechanisms of EA in preventing migraine.

Vesicular glutamate transporter-immunopositive axons that coexpress neuropeptides in the rat and human dental pulp.

To examine the type of vesicular glutamate transporter (VGLUT)-immunopositive (+) axons that coexpress neuropeptides in the rat and human dental pulp, which may help understand peripheral mechanism of pulpal inflammatory pain in rats and humans. The trigeminal ganglia (TG) and the dental pulp of the maxillary molar teeth from three male Sprague-Dawley rats weighing 300-330g and dental pulps of three healthy human (male) maxillary premolar teeth from three 16 to 28-year-old patients extracted for orthodontic treatment were used. The type of VGLUT+axons that coexpress substance P (SP)- and/or calcitonin gene-related peptide (CGRP) and parvalbumin in the rat TG and in the axons of the rat and the human dental pulp was examined by double fluorescence immunohistochemistry and quantitative analysis. Results were analyzed using one-way anova and the Kruskal-Wallis test. SP and CGRP were expressed in many human VGLUT1+pulpal axons but not in the rat VGLUT1+TG neurons and pulpal axons (P<0.05). SP and CGRP were expressed in a considerable number of human VGLUT2+pulpal axons and also in many rat TG neurons and pulpal axons. The fraction of VGLUT1+axons expressing parvalbumin was about three times higher in the rat than in the human dental pulp (P<0.05). These findings suggest that the types of VGLUT+axons, which release neuropeptides, may be different between the rat and the human dental pulp, raising a possibility that peripheral mechanism of pulpal inflammatory pain may be different between rats and humans.

Effects of trigeminal nerve injury on the expression of galanin and its receptors in the rat trigeminal ganglion

In the spinal nervous system, the expression of galanin (GAL) and galanin receptors (GALRs) that play important roles in the transmission and modulation of nociceptive information can be affected by nerve injury. However, in the trigeminal nervous system, the effects of trigeminal nerve injury on the expression of GAL are controversy in the previous studies. Besides, little is known about the effects of trigeminal nerve injury on the expression of GALRs. In the present study, the effects of trigeminal nerve injury on the expression of GAL and GALRs in the rat trigeminal ganglion (TG) were investigated by using quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemistry. To identify the nerve-injured and nerve-uninjured TG neurons, activating transcription factor 3 (ATF3, the nerve-injured neuron marker) was stained by immunofluorescence. The levels of GAL mRNA in the rostral half and caudal half of the TG dramatically increased after transection of infraorbital nerve (ION) and inferior alveolar nerve (IAN), respectively. Immunohistochemical labeling of GAL and ATF3 revealed that GAL level was elevated in both injured and adjacent uninjured small and medium-sized TG neurons after ION/IAN transection. In addition, the levels of GAL2R-like immunoreactivity were reduced in both injured and adjacent uninjured TG neurons after ION/IAN transection, while levels of GAL1R and GAL3R-like immunoreactivity remained unchanged. Furthermore, the number of small to medium-sized TG neurons co-expressing GAL- and GAL1R/GAL2R/GAL3R-like immunoreactivity was significantly increased after ION/IAN transection. In line with previous studies in other spinal neuron systems, these results suggest that GAL and GALRs play functional roles in orofacial neuropathic pain and trigeminal nerve regeneration after trigeminal nerve injury.

Hejie Zhitong prescription promotes sleep and inhibits nociceptive transmission-associated neurotransmitter activity in a rodent migraine model

BackgroundMigraine is painful disease in which neurotransmitters related to pain transmission play an important role. Hejie Zhitong prescription (HJZT) has been used in the clinic as an effective prescription for the treatment of migraine for many years. Our team aimed to further explore its antimigraine mechanism based on previous research results and to explore the inhibitory effect of HJZT on the transmission of pain related to nitroglycerine (NTG)-induced migraine as well as the synergistic effect of HJZT with pentobarbital sodium on promoting sleep.MethodsSixty mice were randomly assigned to groups and received the corresponding interventions. Sleep latency and sleep time were recorded to calculate the incidence of sleep. Forty-eight Wistar rats were randomly assigned and administered an intervention corresponding to their group. Calcitonin gene-related peptide (CGRP), serotonin (5-HT), substance P (SP), and cholecystokinin (CCK) levels were measured using ELISAs. Levels of the cannabinoid receptor type 1 (CB1R) and cyclooxygenase-2 (COX-2) protein were assessed using immunohistochemistry. The expression of the CGRP and CCK mRNAs in the midbrain and trigeminal ganglion (TG) were measured using real-time quantitative PCR.ResultsHJZT promoted the occurrence of sleep in mice. HJZT downregulated COX-2 expression in the midbrain and TG of rats but upregulated the expression of the CB1R, and decreased the plasma level of the CGRP protein and expression of its mRNA in the midbrain and TG. It also downregulated the expression of the CCK mRNA in the midbrain and TG. The high-dose HJZT treatment increased plasma 5-HT levels, but did not induce changes in the plasma levels of the SP or CCK protein.ConclusionsHJZT exerts a synergistic effect with pentobarbital sodium on promoting sleep. As for anti-migraine, HJZT can inhibits the expression of nociceptive transmission-associated neurotransmitters, including 5-HT, CGRP and CCK, which may be related to its upregulation of CB1R and downregulation of COX-2.

Orofacial skin inflammation increases the number of macrophages in the maxillary subregion of the rat trigeminal ganglion in a corticosteroid-reversible manner

Inflammation of the cutaneous orofacial tissue can lead to a prolonged alteration of neuronal and nonneuronal cellular functions in trigeminal nociceptive pathways. In this study, we investigated the effects of experimentally induced skin inflammation by dithranol (anthralin) on macrophage activation in the rat trigeminal ganglion. Tissue localization and protein expression levels of ionized calcium-binding adaptor molecule 1 (Iba1), a macrophage/microglia-specific marker, and proliferation/mitotic marker antigen identified by the monoclonal antibody Ki67 (Ki67), were quantitatively analyzed using immunohistochemistry and western blots in control, dithranol-treated, dithranol- and corticosteroid-treated, and corticosteroid-treated trigeminal ganglia. Chronic orofacial dithranol treatment elicited a strong pro-inflammatory effect in the ipsilateral trigeminal ganglion. Indeed, daily dithranol treatment of the orofacial skin for 3–5 days increased the number of macrophages and Iba1 protein expression in the maxillary subregion of the ipsilateral ganglion. In the affected ganglia, none of the Iba1-positive cells expressed Ki67. This absence of mitotically active cells suggested that the accumulation of macrophages in the ganglion was not the result of resident microglia proliferation but rather the extravasation of hematogenous monocytes from the periphery. Subsequently, when a 5-day-long anti-inflammatory corticosteroid therapy was employed on the previously dithranol-treated orofacial skin, Iba1 immunoreactivity was substantially reduced in the ipsilateral ganglion. Collectively, our findings indicate that both peripheral inflammation and subsequent anti-inflammatory therapy affect macrophage activity and thus interfere with the functioning of the affected sensory ganglion neurons.

Oxytocin as a regulatory neuropeptide in the trigeminovascular system: Localization, expression and function of oxytocin and oxytocin receptors.

Recent clinical findings suggest that oxytocin could be a novel treatment for migraine. However, little is known about the role of this neuropeptide/hormone and its receptor in the trigeminovascular pathway. Here we determine expression, localization, and function of oxytocin and oxytocin receptors in rat trigeminal ganglia and targets of peripheral (dura mater and cranial arteries) and central (trigeminal nucleus caudalis) afferents. The methods include immunohistochemistry, messenger RNA measurements, quantitative PCR, release of calcitonin gene-related peptide and myography of arterial segments. Oxytocin receptor mRNA was expressed in rat trigeminal ganglia and the receptor protein was localized in numerous small to medium-sized neurons and thick axons characteristic of A∂ sensory fibers. Double immunohistochemistry revealed only a small number of neurons expressing both oxytocin receptors and calcitonin gene-related peptide. In contrast, double immunostaining showed expression of the calcitonin gene-related peptide receptor component receptor activity-modifying protein 1 and oxytocin receptors in 23% of the small cells and in 47% of the medium-sized cells. Oxytocin immunofluorescence was observed only in trigeminal ganglia satellite glial cells. Oxytocin mRNA was below detection limit in the trigeminal ganglia. The trigeminal nucleus caudalis expressed mRNA for both oxytocin and its receptor. K+-evoked calcitonin gene-related peptide release from either isolated trigeminal ganglia or dura mater and it was not significantly affected by oxytocin (10 µM). Oxytocin directly constricted cranial arteries ex vivo (pEC50 ∼ 7); however, these effects were inhibited by the vasopressin V1A antagonist SR49059. Oxytocin receptors are extensively expressed throughout the rat trigeminovascular system and in particular in trigeminal ganglia A∂ neurons and fibers, but no functional oxytocin receptors were demonstrated in the dura and cranial arteries. Thus, circulating oxytocin may act on oxytocin receptors in the trigeminal ganglia to affect nociception transmission. These effects may help explain hormonal influences in migraine and offer a novel way for treatment.

GCaMP as an indirect measure of electrical activity in rat trigeminal ganglion neurons

While debate continues over whether somatosensory information is transmitted via labeled line, population coding, frequency coding, or some combination therein, researchers have begun to address this question at the level of the primary afferent by using optical approaches that enable the assessment of neural activity in hundreds to even thousands of neurons simultaneously. However, with limited availability of tools to optically assess electrical activity in large populations of neurons, researchers have turned to genetically encoded Ca2+ indicators (GECIs) including GCaMP to enable the detection of increases in cytosolic Ca2+ concentrations as a correlate for neuronal activity. One of the most widely used GECIs is GCaMP6, which is available in three different versions tuned for sensitivity (GCaMP6s), speed (GCaMP6f), or a balance of the two (GCaMP6m). In order to determine if these issues were unique to GCaMP6 itself, or if they were inherent to more than one generation of GCaMP, we also characterized jGCaMP7. In the present study, we sought to determine the utility of the three GCaMP6 isoforms to detect changes in activity in primary afferents at frequencies ranging from 0.1–30 Hz. Given the heterogeneity of sensory neurons, we also compared the performance of each GCaMP6 isoform in subpopulations of neurons defined by properties used to identify putative nociceptive afferents: cell body size, isolectin B4 (IB4) binding, and capsaicin sensitivity. Finally, we compared results generated with GCaMP6 with that generated from neurons expressing the next generation of GCaMP, jGCaMP7s and jGCaMP7f. A viral approach, with AAV9-CAG-GCaMP6s/m/f, was used to drive GECI expression in acutely dissociated rat trigeminal ganglion (TG) neurons, and neural activity was driven by electrical field stimulation. Infection efficiency with the AAV serotype was high >95 %, and the impact of GCaMP6 expression in TG neurons over the period of study (<10 days) on the regulation of intracellular Ca2+, as assessed with fura-2, was minimal. Having confirmed that the field stimulation evoked Ca2+ transients were dependent on Ca2+ influx secondary to the activation of action potentials and voltage-gated Ca2+ channels, we also confirmed that the signal-to-noise ratio for each of the isoforms was excellent, enabling detection of a single spike in>90% of neurons. However, the utility of the GCaMP6 isoforms to enable an assessment of the firing frequency let alone changes in firing frequency of each neuron was relatively limited and isoform specific: GCaMP6s and 6m had the lowest resolution, enabling detection of spikes at 3 Hz in 15% and 32% of neurons respectively, but it was possible to resolve discrete single spikes up to 10 Hz in 36% of GCaMP6f neurons. Unfortunately, using other parameters of the Ca2+ transient, such as magnitude of the transient or the rate of rise, did not improve the range over which these indicators could be used to assess changes in spike number or firing frequency. Furthermore, in the presence of ongoing neural activity, it was even more difficult to detect a change in firing frequency. The frequency response relationship for the increase in Ca2+ was highly heterogeneous among sensory neurons and was influenced by both the GCaMP6 isoform used to assess it, the timing between the delivery of stimulation trains (inter-burst interval), and afferent subpopulation. Notably, the same deficiencies were observed with jGCaMP7s and 7f in resolving the degree of activity as were present for the GCaMP6 isoforms. Together, these data suggest that while both GCaMP6 and jGCaMP7 are potentially useful tools in sensory neurons to determine the presence or absence of neural activity, the ability to discriminate changes in firing frequency ≥ 3 Hz is extremely limited. As a result, GECIs should probably not be used in sensory neurons to assess changes in activity within or between subpopulations of neurons.

Palmatine alleviates hyperalgesia by inhibiting the expression of calcitonin gene-related peptide in the trigeminal ganglion of rats with chronic constriction injury of the infraorbital nerve.

Trigeminal neuralgia is one of the most common of the neuropathic pains, and it can seriously influence patients' quality of life. Calcitonin gene-related peptide (CGRP) is a type of nociceptive neurotransmitter that is expressed in neurons of the trigeminal ganglion and plays a major part in transmitting pain. The rat model of trigeminal neuralgia was established by causing a chronic constriction injury of the infraorbital nerve (CCI-ION). Male Sprague-Dawley rats (n=24) were randomly divided into a sham control group (sham, n=6), sham-treated with palmatine group (sham+palmatine, n=6), trigeminal nerve model group (TN, n=6), and trigeminal nerve treated with palmatine group (TN+palmatine, n=6). Fifteen days after the operation the mechanical response threshold was decreased in the TN group compared with the sham group. From postoperative day 7 to day 15, the mechanical response threshold in the TN+palmatine group significantly increased compared with the TN group. On postoperative day 15 the results of quantitative polymerase chain reaction (qPCR), immunohistochemical staining, and western blotting showed an obvious increase in expression of CGRP and its receptors, serum concentrations of interleukin-1β (IL-1β), and tumour necrosis factor-α (TNF-α), and phosphorylation of protein kinase C (PKC) in the trigeminal ganglia of the TN group compared with the sham group, but these increases could be down-regulated by treatment with palmatine. Palmatine might therefore have therapeutic potential for the treatment of trigeminal neuralgia by inhibiting the expression of CGRP and its receptors in trigeminal ganglia, suppressing the serum concentrations of IL-1β and TNF-α, and decreasing the phosphorylation of PKC in the trigeminal ganglia of affected rats.

Expression of the CGRP Family of Neuropeptides and their Receptors in the Trigeminal Ganglion

The calcitonin gene-related peptide (CGRP) family of neuropeptides, consists of CGRP, adrenomedullin, amylin, and calcitonin. The receptors consist of either calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) which for function needs an accessory protein, receptor activity-modifying proteins (RAMPs). CGRP has a pivotal role in primary headaches but the role of the other members of the CGRP family of peptides in headaches is not known. Here, we describe the expression of these molecules in the trigeminal ganglion (TG) to understand more on their possible role(s). Single or double immunohistochemistry were applied on frozen sections of rat TG using primary antibodies against CGRP, procalcitonin, calcitonin, adrenomedullin, amylin, RAMP1/2/3, CLR, and CTR. In addition, mRNA expression was measured by quantitative qPCR on TGs. CGRP and calcitonin showed rich expression in the cytoplasm of small to medium-sized neurons, and co-localized sometimes. Procalcitonin was observed in the glial cells. Immunoreactive fibers storing both CGRP and calcitonin were also observed. Adrenomedullin immunoreactivity was found in the satellite glial cells and in fibers, probably the myelinating Schwann cells. Amylin was found in the cytoplasm in many TG neurons. Levels of mRNA expression for adrenomedullin, amylin, CLR, RAMP1, RAMP2, RAMP3, and CTR were measured using qPCR. The experiments verified the expression of mRNA in the TG with the exception of CTR, which was above the limit of detection indicating little or no mRNA expression. In addition to the well-known CGRP receptor (CLR/RAMP1) and the receptor for calcitonin—CTR, we propose that other receptors exist in the rat TG: adrenomedullin receptor AM2 (CLR/RAMP3) in mainly the satellite glial cells, amylin receptors AMY1 (CTR/RAMP1) in mainly neurons, and AMY3 (CTR/RAMP3) in the satellite glial cells. It is important to compare peptides and receptors side-by-side in studies to help address questions of actions resulting from cross-reactivity between receptors. Several of the diverse biological actions of the CGRP family of peptides are clinically relevant. Our findings demonstrate the specific ligand and receptor sites in the rat trigeminal ganglion, highlighting recognition mechanisms to facilitate drug development.