Oxidative stress plays an important role in the central regulatory mechanism of orofacial hyperalgesia under low estrogen conditions.

Hyperalgesia occurs in the orofacial region of rats when estrogen levels are low, although the specific mechanism needs to be investigated further. Furthermore, oxidative stress plays an important role in the transmission of pain signals. This study aimed to explore the role of oxidative stress in orofacial hyperalgesia under low estrogen conditions. We firstly found an imbalance between oxidative and antioxidant capacity within the spinal trigeminal subnucleus caudalis (SP5C) of rats after ovariectomy (OVX), resulting in oxidative stress and then a decrease in the orofacial pain threshold. To investigate the mechanism by which oxidative stress occurs, we used virus as a tool to silence or overexpress the excitatory amino acid transporter 3 (EAAT3) gene. Further investigation revealed that the regulation of glutathione (GSH) and reactive oxygen species (ROS) can be achieved by regulating EAAT3, which in turn impacts the occurrence of oxidative stress. In summary, our findings suggest that reduced expression of EAAT3 within the SP5C of rats in the low estrogen state may decrease GSH content and increase ROS levels, resulting in oxidative stress and ultimately lead to orofacial hyperalgesia. This suggests that antioxidants could be a potential therapeutic direction for orofacial hyperalgesia under low estrogen conditions, though more research is needed to understand its mechanism.

Modulation of excitatory synaptic transmissions by TRPV1 in the spinal trigeminal subnucleus caudalis neurons of neuropathic pain rats.

The present study examined contribution of the transient receptor potential vanilloid 1 channel (TRPV1) to the chronic orofacial pain. Bilateral partial nerve ligation (PNL) of the mental nerve, a branch of trigeminal nerve, was performed to induce neuropathic pain. The withdrawal threshold in response to mechanical stimulation of the lower lip skin was substantially reduced after the surgery in the PNL rats while it remained unchanged in the sham rats. This reduction in the PNL rats was alleviated by pregabalin injected intraperitoneally (10 mg/kg) and intracisternally (10, 30, 100 µg). Furthermore, an intracisternal injection of AMG9810, an antagonist of TRPV1, (1.5, 5.0 µg) attenuated the reduction of withdrawal threshold. Spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) were recorded from the spinal trigeminal subnucleus caudalis (Vc) neurons in the brainstem slice, which receive the orofacial nociceptive signals. In the PNL rats, superfusion of capsaicin (0.03, 0.1 µM) enhanced their frequency without effect on the amplitude and the highest concentration (0.3 µM) increased both the frequency and amplitude. In the sham rats, only 0.3 µM capsaicin increased their frequency. Thus, capsaicin-induced facilitation of sEPSCs and mEPSCs in the PNL rats was significantly stronger than that in the sham rats. AMG9810 (0.1 µM) attenuated the capsaicin's effect. Capsaicin was ineffective on the trigeminal tract-evoked EPSCs in the PNL and sham rats. These results suggest that the chronic orofacial pain in the PNL model results from facilitation of the spontaneous excitatory synaptic transmission in the Vc region through TRPV1 at least partly.

Trigeminal activation patterns evoked by chemical stimulation of the dura mater in rats.

BACKGROUND: Although migraine is one of the most common primary headaches, its therapy is still limited in many cases. The use of animal models is crucial in the development of novel therapeutic strategies, but unfortunately, none of them show all aspects of the disease, therefore, there is a constant need for further improvement in this field. The application of inflammatory agents on the dura mater is a widely accepted method to mimic neurogenic inflammation in rodents, which plays a key role in the pathomechanism of migraine. Complete Freund's Adjuvant (CFA), and a mixture of inflammatory mediators, called inflammatory soup (IS) are often used for this purpose. METHODS: To examine the activation pattern that is caused by chemical stimulation of dura mater, we applied CFA or IS over the right parietal lobe. After 2 h and 4 h (CFA groups), or 2.5 h and 4 h (IS groups), animals were perfused, and c-Fos immunoreactive cells were counted in the caudal trigeminal nucleus. To explore every pitfall, we examined whether our surgical procedure (anesthetic drug, stereotaxic apparatus, local lidocaine) can alter the results under the same experimental settings. c-Fos labeled cells were counted in the second-order neuron area based on the somatotopic organization of the trigeminal nerve branches. RESULTS: We could not find any difference between the CFA and physiological saline group neither 2 h, nor 4 h after dural stimulation. IS caused significant difference after both time points between IS treated and control group, and between treated (right) and control (left) side. Stereotaxic frame usage had a substantial effect on the obtained results. CONCLUSIONS: Counting c-Fos immunoreactive cells based on somatotopic organization of the trigeminal nerve helped to examine the effect of chemical stimulation of dura in a more specific way. As a result, the use of IS over the parietal lobe caused activation in the area of the ophthalmic nerve. To see this effect, the use of lidocaine anesthesia is indispensable. In conclusion, application of IS on the dura mater induces short-term, more robust c-Fos activation than CFA, therefore it might offer a better approach to model acute migraine headache in rodents.

The effect of orofacial complete Freund's adjuvant treatment on the expression of migraine-related molecules.

BACKGROUND: Migraine is a neurovascular primary headache disorder, which causes significant socioeconomic problems worldwide. The pathomechanism of disease is enigmatic, but activation of the trigeminovascular system (TS) appears to be essential during the attack. Migraine research of recent years has focused on neuropeptides, such as calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide 1-38 (PACAP1-38) as potential pathogenic factors and possible therapeutic offensives. The goal of present study was to investigate the simultaneous expression of CGRP and precursor of PACAP1-38 (preproPACAP) in the central region of the TS in a time-dependent manner following TS activation in rats. METHODS: The right whisker pad of rats was injected with 50 µl Complete Freund's Adjuvant (CFA) or saline. A mechanical allodynia test was performed with von Frey filaments before and after treatment. Transcardial perfusion of the animals was initiated 24, 48, 72 and 120 h after injection, followed by the dissection of the nucleus trigeminus caudalis (TNC). After preparation, the samples were stored at - 80 °C until further use. The relative optical density of CGRP and preproPACAP was analyzed by Western blot. One-way ANOVA and Kruskal-Wallis followed by Tukey post hoc test were used to evaluate the data. Regression analysis was applied to explore the correlation between neuropeptides expression and hyperalgesia. RESULTS: Orofacial CFA injection resulted in significant CGRP and preproPACAP release in the TNC 24, 48, 72 and 120 h after the treatment. The level of neuropeptides reached its maximum at 72 h after CFA injection, corresponding to the peak of facial allodynia. Negative, linear correlation was detected between the expression level of neuropeptides and value of mechanonociceptive threshold. CONCLUSION: This is the first study which suggests that the expression of CGRP and preproPACAP simultaneously increases in the central region of activated TS and it influences the formation of mechanical hyperalgesia. Our results contribute to a better understanding of migraine pathogenesis and thereby to the development of more effective therapeutic approaches.

Cilostazol induces C-fos expression in the trigeminal nucleus caudalis and behavioural changes suggestive of headache with the migraine-like feature photophobia in female rats.

Introduction Research in development of new migraine therapeutics is hindered by the lack of suitable, predictive animal models. Cilostazol provokes headache in healthy humans and migraineurs by increasing intracellular cAMP levels. We aimed to investigate whether cilostazol could provoke headache-like behaviours and c-fos expression in rats. In order to evaluate the predictive validity of the model, we examined the response to the migraine specific drug sumatriptan. Methods The effect of cilostazol (125 mg/kg p.o.) in female Sprague Dawley rats was evaluated on a range of spontaneous behavioural parameters, light sensitivity and mechanical sensitivity thresholds. We also measured c-fos expression in the trigeminal nucleus caudalis. Results Cilostazol increased light sensitivity and grooming behaviour. These manifestations were not inhibited by sumatriptan. Cilostazol also induced c-fos expression in the trigeminal nucleus caudalis. Furthermore, trigeminal - but not hind paw hyperalgesia was observed. Conclusion The altered behaviours are suggestive of cilostazol induced headache with migraine-like features, but not specific. The presence of head specific hyperalgesia and the c-fos response in the trigeminal nucleus caudalis imply that the model involves trigeminal nociception. The model will be useful for studying mechanisms related to the cAMP pathway in headache, but its predictive properties appear to be more limited due to the lack of response to sumatriptan.

Orofacial neuropathic pain induced by oxaliplatin: downregulation of KCNQ2 channels in V2 trigeminal ganglion neurons and treatment by the KCNQ2 channel potentiator retigabine.

Abstract: Neuropathic pain induced by chemotherapy drugs such as oxaliplatin is a dose-limiting side effect in cancer treatment. The mechanisms underlying chemotherapy-induced neuropathic pain are not fully understood. KCNQ2 channels are low-threshold voltage-gated K+ channels that play a role in controlling neuronal excitability. Downregulation of KCNQ2 channels has been proposed to be an underlying mechanism of sensory hypersensitivity that leads to neuropathic pain. However, it is currently unknown whether KCNQ channels may be downregulated by chemotherapy drugs in trigeminal ganglion neurons to contribute to the pathogenesis of chemotherapy-induced orofacial neuropathic pain. In the present study, mechanical sensitivity in orofacial regions is measured using the operant behavioral test in rats treated with oxaliplatin. Operant behaviors in these animals show the gradual development of orofacial neuropathic pain that manifests with orofacial mechanical allodynia. Immunostaining shows strong KCNQ2 immunoreactivity in small-sized V2 trigeminal ganglion neurons in controls, and the numbers of KCNQ2 immunoreactivity positive V2 trigeminal ganglion neurons are significantly reduced in oxaliplatin-treated animals. Immunostaining is also performed in brainstem and shows strong KCNQ2 immunoreactivity at the trigeminal afferent central terminals innervating the caudal spinal trigeminal nucleus (Vc) in controls, but the KCNQ2 immunoreactivity intensity is significantly reduced in oxaliplatin-treated animals. We further show with the operant behavioral test that oxaliplatin-induced orofacial mechanical allodynia can be alleviated by the KCNQ2 potentiator retigabine. Taken together, these findings suggest that KCNQ2 downregulation may be a cause of oxaliplatin-induced orofacial neuropathic pain and KCNQ2 potentiators may be useful for alleviating the neuropathic pain.

Baicalin Alleviates Nitroglycerin-induced Migraine in Rats via the Trigeminovascular System.

Migraine is a common neurological disorder with a serious impact on quality of life. The aim of this study was to explore the effect of baicalin on nitroglycerin-induced migraine rats. We carried out a behavioral research within 2 h post-nitroglycerin injection, and blood samples were drawn for measurements of nitric oxide (NO), calcitonin gene-related peptide, and endothelin (ET) levels. Immunohistochemistry was adopted to detect the activation of C-fos immunoreactive neurons in periaqueductal gray. The number, area size, and integrated optical density of C-fos positive cells were measured using Image-Pro Plus. As a result, baicalin administration (0.22 mm/kg) alleviated pain responses of migraine rats. It profoundly decreased NO and calcitonin gene-related peptide levels, increased ET levels, and rebuilt the NO/ET balance in migraine rats. Besides, baicalin pretreatment significantly reduced the number, the stained area size, and integrated optical density value of C-fos positive cells. In brief, this paper supports the possibility of baicalin as a potential migraine pharmacotherapy. Copyright © 2017 John Wiley & Sons, Ltd.

Supradural inflammatory soup in awake and freely moving rats induces facial allodynia that is blocked by putative immune modulators.

Facial allodynia is a migraine symptom that is generally considered to represent a pivotal point in migraine progression. Treatment before development of facial allodynia tends to be more successful than treatment afterwards. As such, understanding the underlying mechanisms of facial allodynia may lead to a better understanding of the mechanisms underlying migraine. Migraine facial allodynia is modeled by applying inflammatory soup (histamine, bradykinin, serotonin, prostaglandin E2) over the dura. Whether glial and/or immune activation contributes to such pain is unknown. Here we tested if trigeminal nucleus caudalis (Sp5C) glial and/or immune cells are activated following supradural inflammatory soup, and if putative glial/immune inhibitors suppress the consequent facial allodynia. Inflammatory soup was administered via bilateral indwelling supradural catheters in freely moving rats, inducing robust and reliable facial allodynia. Gene expression for microglial/macrophage activation markers, interleukin-1ß, and tumor necrosis factor-α increased following inflammatory soup along with robust expression of facial allodynia. This provided the basis for pursuing studies of the behavioral effects of 3 diverse immunomodulatory drugs on facial allodynia. Pretreatment with either of two compounds broadly used as putative glial/immune inhibitors (minocycline, ibudilast) prevented the development of facial allodynia, as did treatment after supradural inflammatory soup but prior to the expression of facial allodynia. Lastly, the toll-like receptor 4 (TLR4) antagonist (+)-naltrexone likewise blocked development of facial allodynia after supradural inflammatory soup. Taken together, these exploratory data support that activated glia and/or immune cells may drive the development of facial allodynia in response to supradural inflammatory soup in unanesthetized male rats.

Metoclopramide inhibits trigeminovascular activation:evidence for effective acute attack treatment in migraine.

BACKGROUND/AIM: Metoclopramide is an effective and commonly used medication in acute migraine treatment but an experimental evidence base is lacking. We aimed to investigate the antimigraine effect of metoclopramide in a migraine model and whether the analgesic effect of metoclopramide was likely to be D2 receptor-mediated. MATERIALS AND METHODS: Cortical spreading depression (CSD) was used to model migraine in adult male Wistar rats. Five CSDs were induced by pinprick. Metoclopramide (two different doses), raclopride, or 0.9% saline were administered 30 min before CSD induction. Two hours after the experiments, brain tissues were examined for c-fos activation. RESULTS: In metoclopramide groups brain stem c-fos expression was significantly lower than in the CSD side of the saline group (P = 0.002). In the raclopride group, ipsilateral brain stem c-fos expression was also lower than in the saline group (P = 0.002). No difference in c-fos expression in the ipsilateral trigeminal nucleus caudalis between the raclopride and metoclopramide groups was observed (P > 0.05). CONCLUSION: Metoclopramide is shown to suppress trigeminovascular activation for the first time, providing an experimental basis for its role in migraine. The analgesic effect of metoclopramide is likely to be mediated by D2 receptors since raclopride, a selective D2 receptor antagonist, suppresses trigeminovascular activation similarly.

The effect of capsaicin on expression patterns of CGRP in trigeminal ganglion and trigeminal nucleus caudalis following experimental tooth movement in rats

ABSTRACT Objectives The aim of this study was to explore the effect of capsaicin on expression patterns of calcitonin gene-related peptide (CGRP) in the trigeminal ganglion (TG) and trigeminal subnucleus caudalis (Vc) following experimental tooth movement. Material and Methods Male Sprague-Dawley rats were used in this study and divided into small-dose capsaicin+force group, large-dose capsaicin+force group, saline+force group, and no force group. Closed coil springs were used to mimic orthodontic forces in all groups except for the no force group, in which springs were inactivated. Capsaicin and saline were injected into periodontal tissues. Rats were euthanized at 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d following experimental tooth movement. Then, TG and Vc were obtained for immunohistochemical staining and western blotting against CGRP. Results Immunohistochemical results indicated that CGRP positive neurons were located in the TG, and CGRP immunoreactive fibers were distributed in the Vc. Immunohistochemical semiquantitative analysis and western blotting analysis demonstrated that CGRP expression levels both in TG and Vc were elevated at 12 h, 1 d, 3 d, 5 d, and 7 d in the saline + force group. However, both small-dose and large-dose capsaicin could decrease CGRP expression in TG and Vc at 1 d and 3 d following experimental tooth movement, as compared with the saline + force group. Conclusions These results suggest that capsaicin could regulate CGRP expression in TG and Vc following experimental tooth movement in rats.

Activation of the nuclear factor E2-related factor 2/anitioxidant response element alleviates the nitroglycerin-induced hyperalgesia in rats.

BACKGROUND: Antioxidants have been proven to weaken hyperalgesia in neuropathic pain. Endogenous antioxidant defense system may have a role in the prevention of hyperalgesia in migraine. In this study, we aimed to evaluate the role of nuclear factor E2-related factor 2/antioxidant response element (Nrf2/ARE) pathway in regulating the activation of the trigeminovascular system (TGVS) and hypersensitivity in nitroglycerin (NTG)-induced hyperalgesia rats. METHODS: The expression levels of Nrf2, HO, HO1, and NQO1 in the trigeminal nucleus caudalis (TNC) were detected by western blot. Immunofluorescence was used to demonstrate the cell-specific localization of Nrf2 in TNC. Sulforaphane, a Nrf2 activator, was administered to NTG-induced rats. Then, the number of c-Fos- and nNOS-immunoreactive neurons in TNC was evaluated using immunofluorescence, and c-Fos and nNOS protein levels were quantified using western blot. Von Frey hair testing was used to evaluate the tactile thresholds of rats at different time points in different groups. RESULTS: Total cellular and nuclear levels of the proteins Nrf2, HO1, and NQO1 were elevated in TNC after NTG injection, and Nrf2 was found to be located in the nucleus and cytoplasm of the neurons. Sulforaphane pretreatment significantly increased the nuclear Nrf2, HO1, and NQO1 levels in TNC. In addition, sulforaphane exposure effectively inhibited the expression of nNOS and c-Fos, reduced the number of nNOS and c-Fos immunoreactive neurons in TNC, and attenuated the tactile thresholds induced by NTG injection. CONCLUSION: Oxidative stress was involved in nitroglycerin-induced hyperalgesia. Activation of the Nrf2/ARE pathway inhibited the activation of TGVS and prevented the induction of hyperalgesia. Sulforaphane might therefore be an effective agent for hyperalgesia. Further studies are needed to discover the underlying mechanisms of the process.

Valproate ameliorates nitroglycerin-induced migraine in trigeminal nucleus caudalis in rats through inhibition of NF-кB.

BACKGROUND: As a complex nervous system disease, migraine causes severe healthy and social issues worldwide. Valproate (VPA) is a widely used treatment agent against seizures and bipolar disorder, and its function to alleviate damage due to migraine has also been verified in clinical investigations. However, the mechanism underlying the protective effect of VPA against migraine remains poorly revealed. In the current study, the major purpose was to uncover the mechanism which drove VPA to antagonize migraine. METHODS: Nitroglycerin (NTG) was employed to induce a migraine model in rats and the migraine animals were exposed to treatment of VPA of different doses. Thereafter, the levels of indicators related to oxidative stress were measured and used to evaluate the anti-oxidant potential of VPA. The expression of calcitonin gene-related peptide (CGRP) and c-Fos was also quantified with ELISA and immunohistochemistry, respectively. Western blotting and electrophoretic mobility shift assays (EMSA) were conducted to explore the effect of VPA treatment on NF-кB pathway. RESULTS: NTG induced the activation of oxidative stress and led to migraine in model animals, but pre-treatment with VPA attenuated the damage due to migraine attack in brain tissues. The level of lipid peroxidation was significantly reduced while the prodcution of anti-oxidant factors was restored. Furthermore, expressions of CGRP and c-Fos, which represented the neuronal activation, were also down-regulated by VPA. The results of western blotting and EMSA demonstrated that the above mentioned effect of VPA acted through the inhibition of NF-кB pathway. CONCLUSIONS: Although controversies on the effect of VPA on NF-кB pathway existed, our study revealed an alternative mechanism of VPA in protecting against migraine, which would promote the development of therapeutic strategies of migraine.

Coactivation of µ- and κ-Opioid Receptors May Mediate the Protective Effect of Testosterone on the Development of Temporomandibular Joint Nociception in Male Rats.

AIMS: To investigate whether the protective effect of testosterone on the development of temporomandibular joint (TMJ) nociception in male rats is mediated by the activation of central opioid mechanisms. METHODS: Experiments were performed on 156 male Wistar rats. A pharmacologic approach was used to assess the ability of opioid receptor antagonists infused into the dorsal portion of the brainstem and adjacent to the caudal component (subnucleus caudalis) of the spinal trigeminal nucleus to block the protective effect of testosterone in male rats. The TMJ injection of 0.5% formalin was used as a nociceptive stimulus. One-way or two-way ANOVA was used for data analyses. RESULTS: The injection of 0.5% formalin into the TMJ induced a significant nociceptive behavior in gonadectomized male rats (P < .05), but not in naïve, sham, and testosterone-replaced gonadectomized rats, confirming that testosterone prevents the development of TMJ nociception. The injection of either the nonselective opioid receptor antagonist naloxone (15 µg) or the simultaneous injection of the µ-opioid receptor antagonist Cys2, Tyr3, Orn5, Pen7amide (CTOP, 30 µg) and the κ-opioid receptor antagonist Nor-Binaltorphimine (Nor-BNI, 90 µg) significantly increased the 0.5% formalin-induced behavioral response in sham and testosterone-replaced gonadectomized rats (P < .05) but had no effect in gonadectomized rats. However, the injection of each selective opioid receptor antagonist alone or the simultaneous injection of µ- or κ- and δ-opioid receptor antagonists had no effect. CONCLUSION: These findings indicate that the protective effect of endogenous testosterone on the development of TMJ nociception in male rats is mediated by the activation of central opioid mechanisms. Furthermore, the coactivation of central µ- and κ-opioid receptors is necessary for testosterone to protect male rats from developing TMJ nociception.

Systemic administration of resveratrol suppress the nociceptive neuronal activity of spinal trigeminal nucleus caudalis in rats.

Although a modulatory role has been reported for the red wine polyphenol resveratrol on several types of ion channels and excitatory synaptic transmission in the nervous system, the acute effects of resveratrol in vivo, particularly on nociceptive transmission of the trigeminal system, remain to be determined. The aim of the present study was to investigate whether acute intravenous resveratrol administration to rats attenuates the excitability of wide dynamic range (WDR) spinal trigeminal nucleus caudalis (SpVc) neurons in response to nociceptive and non-nociceptive mechanical stimulation in vivo. Extracellular single unit recordings were made from 18 SpVc neurons in response to orofacial mechanical stimulation of pentobarbital-anesthetized rats. Responses to both non-noxious and noxious mechanical stimuli were analyzed in the present study. The mean firing frequency of SpVc WDR neurons in response to both non-noxious and noxious mechanical stimuli was inhibited by resveratrol (0.5-2 mg/kg, i.v.) and maximum inhibition of the discharge frequency of both non-noxious and noxious mechanical stimuli was seen within 10 min. These inhibitory effects were reversed after approximately 20 min. The relative magnitude of inhibition by resveratrol of SpVc WDR neuronal discharge frequency was significantly greater for noxious than non-noxious stimulation. These results suggest that, in the absence of inflammatory or neuropathic pain, acute intravenous resveratrol administration suppresses trigeminal sensory transmission, including nociception, and so resveratrol may be used as a complementary and alternative medicine therapeutic agent for the treatment of trigeminal nociceptive pain, including hyperalgesia.

Botulinum toxin in migraine: Role of transport in trigemino-somatic and trigemino-vascular afferents.

Migraine secondary to meningeal input is referred to extracranial regions innervated by somatic afferents that project to homologous regions in the trigeminal nucleus caudalis (TNC). Reported efficacy of extracranial botulinum toxin (BoNT) in treating migraine is surprising since a local extracranial effect of BoNT cannot account for its effect upon meningeal input. We hypothesize that intradermal BoNT acts through central transport in somatic afferents. Anesthetized C57Bl/6 mice (male) received unilateral supraorbital (SO) injections of BoNT-B (1.5 U/40 µl) or saline. 3 days later, mice received ipsilateral (ipsi)-SO capsaicin (20 µl of 0.5mM solution) or meningeal capsaicin (4 µl of 0.35 µM). Pre-treatment with ipsi-SO BoNT-B i) decreased nocicsponsive ipsilateral wiping behavior following ipsi-SO capsaicin; ii) produced cleavage of VAMP in the V1 region of ipsi-TG and in TG neurons showing WGA after SO injection; iii) reduced expression of c-fos in ipsi-TNC following ipsi-SO capsaicin; iv) reduced c-fos activation and NK-1 internalization in ipsi-TNC secondary to ipsi-meningeal capsaicin; and vi) SO WGA did not label dural afferents. We conclude that BoNT-B is taken up by peripheral afferents and transported to central terminals where it inhibits transmitter release resulting in decreased activation of second order neurons. Further, this study supports the hypothesis that SO BoNT exerts a trans-synaptic action on either the second order neuron (which receives convergent input from the meningeal afferent) or the terminal/TG of the converging meningeal afferent.

Local group I mGluR antagonists reduce TMJ-evoked activity of trigeminal subnucleus caudalis neurons in female rats.

Group I metabotropic glutamate receptors (mGluR1 and mGluR5) are functionally linked to estrogen receptors and play a key role in the plasticity of central neurons. Estrogen status strongly influences sensory input from the temporomandibular joint (TMJ) to neurons at the spinomedullary (Vc/C1-2) region. This study tested the hypothesis that TMJ input to trigeminal subnucleus caudalis/upper cervical cord (Vc/C1-2) neurons involved group I mGluR activation and depended on estrogen status. TMJ-responsive neurons were recorded in superficial laminae at the Vc/C1-2 region in ovariectomized (OvX) female rats treated with low-dose estradiol (2 µg/day, LE) or high-dose estradiol (20 µg/day, HE) for 2 days. TMJ-responsive units were activated by adenosine triphosphate (ATP, 1mM) injected into the joint space. Receptor antagonists selective for mGluR1 (CPCCOEt) or mGluR5 (MPEP) were applied topically to the Vc/C1-2 surface at the site of recording 10 min prior to the intra-TMJ ATP stimulus. In HE rats, CPCCOEt (50 and 500 µM) markedly reduced ATP-evoked unit activity. By contrast, in LE rats, a small but significant increase in neural activity was seen after 50 µM CPCCOEt, while 500 µM caused a large reduction in activity that was similar in magnitude as that seen in HE rats. Local application of MPEP produced a significant inhibition of TMJ-evoked unit activity independent of estrogen status. Neither mGluR1 nor mGluR5 antagonism altered the spontaneous activity of TMJ units in HE or LE rats. High-dose MPEP caused a small reduction in the size of the convergent cutaneous receptive field in HE rats, while CPCCOEt had no effect. These data suggest that group I mGluRs play a key role in sensory integration of TMJ nociceptive input to the Vc/C1-2 region and are largely independent of estrogen status.

CGRP infusion in unanesthetized rats increases expression of c-Fos in the nucleus tractus solitarius and caudal ventrolateral medulla, but not in the trigeminal nucleus caudalis.

BACKGROUND AND AIMS: Calcitonin gene-related peptide (CGRP) and glyceryl trinitrate (GTN) infusion in migraineurs provokes headache resembling spontaneous migraine, and CGRP receptor antagonists are effective in the treatment of acute migraine. We hypothesized that CGRP infusion would increase molecular markers of neuronal activation in migraine-relevant tissues of the rat. METHODS: CGRP was infused intravenously (i.v.) in freely moving rats to circumvent factors like anesthesia, acute surgery and severe hypotension, the three confounding factors for c-Fos expression. The trigeminal nucleus caudalis (TNC) was isolated at different time points after CGRP infusion. The level of c-Fos mRNA and protein expression in TNC were analyzed by qPCR and immunohistochemistry. c-Fos-stained nuclei were also counted in the nucleus tractus solitarius (NTS) and caudal ventrolateral medulla (CVLM), integrative sites in the brain stem for processing cardiovascular signals. We also investigated Zif268 protein expression (another immediate early gene) in TNC. The protein expression of p-ERK, p-CREB and c-Fos was analyzed in dura mater, trigeminal ganglion (TG) and TNC samples using Western blot. RESULTS: CGRP infusion caused a significant dose-dependent fall in mean arterial blood pressure. No significant activation of c-Fos in the TNC at mRNA and protein levels was observed after CGRP infusion. A significant increase in c-Fos protein was observed in the NTS and CVLM in the brain stem. Zif268 expression in the TNC was also not changed after CGRP infusion. p-ERK was increased in the dura mater 30 minutes after CGRP infusion. CONCLUSION: CGRP infusion increased the early expression of p-ERK in the dura mater but did not increase c-Fos and Zif268 expression in the TNC. The rats may, thus, differ from migraine patients, in whom infusion of CGRP caused headache and a delayed migraine attack. The rat CGRP infusion model with c-Fos or Zif268 as neuronal pain markers in TNC is unsuitable for antimigraine drug testing.

The thalamic reticular nucleus is activated by cortical spreading depression in freely moving rats: prevention by acute valproate administration.

This study investigated the effect of repetitive cortical spreading depression (CSD) on behaviour and the anatomical and physiological patterns of cellular activation of cortical and subcortical areas in awake, moving rats. Rat behaviours in response to repetitive CSD events evoked by the application of KCl were quantified with electrophysiological recording. Immunohistochemistry was used to quantify anatomical regions of cellular activation. The effects of acute valproic acid administration on the behavioural parameters and cellular activation were evaluated. CSD significantly decreased locomotor activity and induced freezing in awake, moving rats, and stimulated c-Fos expression in the cortex, trigeminal nucleus caudalis (TNC), and amygdala. CSD also resulted in a prominent increase in c-Fos expression in the ipsilateral thalamic reticular nucleus (TRN) visual sector. Electrophysiological recordings revealed propagation of CSD into the TRN. Valproic acid pretreatment decreased the duration of CSD-induced freezing episodes and reversed the CSD-induced reduction in locomotor activity. Acute valproic acid administration also significantly blocked CSD-induced c-Fos expression in the TNC and TRN. These findings show that CSD events cause consistent behavioural responses and activate specific brain regions in awake, freely moving rats. Selective activation of TRN by CSD and the suppression of this activation by valproic acid suggest that this brain region may play an important role in migraine pathogenesis and may represent a novel target for migraine therapy.

Differential trigeminovascular nociceptive responses in the thalamus in the familial hemiplegic migraine 1 knock-in mouse: a Fos protein study.

Familial hemiplegic migraine type 1 (FHM-1) is a monogenic subtype of migraine with aura caused by missense mutations in the CACNA1A gene, which encodes the pore-forming α1 subunit of voltage-gated neuronal CaV2.1 (P/Q-type) calcium channels. Transgenic knock-in mice expressing the CACNA1A R192Q mutation that causes FHM-1 in patients show a greater susceptibility to cortical spreading depression, the likely underlying mechanism of typical human migraine aura. The aim of this study was to compare neuronal activation within the trigeminal pain pathways in response to nociceptive trigeminovascular stimulation in wild-type and R192Q knock-in mice. After sham surgery or electrical stimulation of the superior sagittal sinus for 2h, or stimulation preceded by treatment with naratriptan, mice underwent intracardiac perfusion, and the brain, including the brainstem, was removed. Fos expression was measured in the trigeminocervical complex (TCC) and the lateral (ventroposteromedial, ventrolateral), medial (parafascicular, centromedian) and posterior thalamic nuclei. In the TCC of wild-type animals, the number of Fos-positive cells increased significantly following dural stimulation compared to the sham control group (P<0.001) and decreased after naratriptan treatment (P<0.05). In R192Q knock-in mice, there was no significant difference between the stimulated and sham (P=0.10) or naratriptan pre-treated groups (P=0.15). The number of Fos-positive cells in the R192Q stimulated group was significantly lower compared to the wild-type stimulated mice (P<0.05). In the thalamus, R192Q mice tended to be more sensitive to stimulation compared to the sham control in the medial and posterior nuclei, and between the two strains of stimulated animals there was a significant difference in the centromedian (P<0.005), and posterior nuclei (P<0.05). The present study suggests that the FHM-1 mutation affects more rostral brain structures in this experimental paradigm, which offers a novel perspective on possible differential effects of mutations causing migraine in terms of phenotype-genotype correlations.

Pain fiber anesthetic reduces brainstem Fos after tooth extraction.

We recently demonstrated that pain-sensing neurons in the trigeminal system can be selectively anesthetized by co-application of QX-314 with the TRPV1 receptor agonist, capsaicin (QX cocktail). Here we examined whether this new anesthetic strategy can block the neuronal changes in the brainstem following molar tooth extraction in the rat. Adult male Sprague-Dawley rats received infiltration injection of anesthetic 10 min prior to lower molar tooth extraction. Neuronal activation was determined by immunohistochemistry for the proto-oncogene protein c-Fos in transverse sections of the trigeminal subnucleus caudalis (Sp5C). After tooth extraction, c-Fos-like immunoreactivity (Fos-LI) detected in the dorsomedial region of bilateral Sp5C was highest at 2 hrs (p < .01 vs. naïve ipsilateral) and declined to pre-injury levels by 8 hrs. Pre-administration of the QX cocktail significantly reduced to sham levels Fos-LI examined 2 hrs after tooth extraction; reduced Fos-LI was also observed with the conventional local anesthetic lidocaine. Pulpal anesthesia by infiltration injection was confirmed by inhibition of the jaw-opening reflex in response to electrical tooth pulp stimulation. Our results suggest that the QX cocktail anesthetic is effective in reducing neuronal activation following tooth extraction. Thus, a selective pain fiber 'nociceptive anesthetic' strategy may provide an effective local anesthetic option for dental patients in the clinic.