Responses of spinal trigeminal neurons to noxious stimulation of paranasal cavities - a rat model of rhinosinusitis headache.

BACKGROUND: The pathophysiology of headaches associated with rhinosinusitis is poorly known. Since the generation of headaches is thought to be linked to the activation of intracranial afferents, we used an animal model to characterise spinal trigeminal neurons with nociceptive input from the dura mater and paranasal sinuses. METHODS: In isoflurane anaesthetised rats, extracellular recordings were made from neurons in the spinal trigeminal nucleus with afferent input from the exposed frontal dura mater. Dural and facial receptive fields were mapped and the paranasal cavities below the thinned nasal bone were stimulated by sequential application of synthetic interstitial fluid, 40 mM potassium chloride, 100 µM bradykinin, 1% ethanol (vehicle) and 100 µm capsaicin. RESULTS: Twenty-five neurons with input from the frontal dura mater and responses to chemical stimulation of the paranasal cavities were identified. Some of these neurons had additional receptive fields in the parietal dura, most of them in the face. The administration of synthetic interstitial fluid, potassium chloride and ethanol was not followed by significant changes in activity, but bradykinin provoked a cluster of action potentials in 20 and capsaicin in 23 neurons. CONCLUSION: Specific spinal trigeminal neurons with afferent input from the cranial dura mater respond to stimulation of paranasal cavities with noxious agents like bradykinin and capsaicin. This pattern of activation may be due to convergent input of trigeminal afferents that innervate dura mater and nasal cavities and project to spinal trigeminal neurons, which could explain the genesis of headaches due to disorders of paranasal sinuses.

Activation orexin 1 receptors in the ventrolateral periaqueductal gray matter attenuate nitroglycerin-induced migraine attacks and calcitonin gene related peptide up-regulation in trigeminal nucleus caudalis of rats.

This study aims to explore whether orexin 1 receptors (Orx1R) in the ventrolateral periaqueductal gray matter (vlPAG) play a role in the modulation of migraine headaches in adult male Wistar rats. To model chronic migraine-associated pain, nitroglycerin (NTG) (5 mg/kg/IP) was administered to test subjects every second day for 9 days. After the last NTG injection, rats were randomly separated into the following groups (n = 6): orexin-A (OrxA) groups that received intra-vlPAG OrxA (25, 50, and 100 pM), an Orx1R antagonist group, a SB-334867 (20 µM) group; and a SB-334867 (20 µM) + OrxA (100 pM) group. After 10 min, migraine-associated behavioral symptoms were recorded in all animals for up to 90 min. Light-dark chamber and hot plate tests were used for assessing light aversion and thermal hyperalgesia, respectively. Calcitonin gene-related peptide (CGRP)-positive cells were detected in the trigeminal nucleus caudalis (Vc) by immunofluorescence microscopy. NTG caused significant freezing behavior, which was prevented by all OrxA doses. Moreover, OrxA (100 pM) could obstruct NTG-induced increases in facial rubbing and decreases in climbing and body grooming. Furthermore, NTG-induced light aversion and thermal hyperalgesia were attenuated by OrxA at doses of 50 and 100 pM. The effects of OrxA were significantly blocked by SB-334867 (20 µM). Besides, OrxA (100 pM) decreased NTG-induced CGRP upregulation. The data revealed that the activation of Orx1Rs in the vlPAG is effective in relieving NTG-induced migraine symptoms mainly by the downregulation of CGRP in the Vc of rats.

Involvement of nNOS in the antinociceptive activity of melatonin in inflammatory pain at the level of sensory neurons.

OBJECTIVE: The efficacy of melatonin as an analgesic agent has been well documented in animals and humans. However, the underlying mechanisms by which melatonin exerts antinociceptive effects on inflammatory pain are poorly understood. Here, we investigated the potential of melatonin to ameliorate inflammatory pain. MATERIALS AND METHODS: In vitro, ND7/23 neurons were treated with capsaicin. We used PCR and Western blot analyses to detect the expression of neuronal nitric oxide synthase (nNOS) in response to melatonin. Orofacial inflammatory pain was induced by 4% formalin administration on the right whisker pad of Sprague Dawley (SD) rats. The analgesic effect of melatonin was evaluated using mechanical threshold analyses. The expression level of nNOS in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (Vc) neurons was assessed by RNAscope and immunohistochemistry. RESULTS: In vitro, capsaicin upregulated the expression of nNOS, which was dose-dependently reversed by melatonin pretreatment (p < 0.001). In a rat model of orofacial inflammatory pain, melatonin pretreatment significantly attenuated mechanical allodynia in both the acute and chronic phases (p < 0.05). Furthermore, melatonin decreased the formalin-evoked elevated nNOS mRNA and protein levels in the TG and Vc neurons in the acute and chronic phases (p < 0.05). CONCLUSIONS: Taken together, these results suggest that nNOS may play an active role in both peripheral and central processing of nociceptive information following orofacial inflammatory pain induction. The regulatory effect of melatonin on nNOS in inflammatory pain may have potential implications for the development of novel analgesic strategies.

Afferents of the mouse linear nucleus.

The linear nucleus (Li) was identified in 1978 from its projections to the cerebellum. However, there is no systematic study of its connections with other areas of the central nervous system possibly due to the challenge of injecting retrograde tracers into this nucleus. The present study examines its afferents from some nuclei involved in motor and cardiovascular control with anterograde tracer injections. BDA injections into the central amygdaloid nucleus result in labeled fibers to the ipsilateral Li. Bilateral projections with an ipsilateral dominance were observed after injections in a) jointly the paralemniscal nucleus, the noradrenergic group 7/ Köllike -Fuse nucleus/subcoeruleus nucleus, b) the gigantocellular reticular nucleus, c) and the solitary nucleus/the parvicellular/intermediate reticular nucleus. Retrogradely labeled neurons were observed in Li after BDA injections into all these nuclei except the central amygdaloid and the paralemniscal nuclei. Our results suggest that Li is involved in a variety of physiological functions apart from motor and balance control it may exert via its cerebellar projections.

Analgesic and Neuroprotective Effects of Electroacupuncture in a Dental Pulp Injury Model-A Basic Research.

Irreversible pulpitis is an extremely painful condition and its consequence in the central nervous system (CNS) remains unclear. A mouse model of dental pulp injury (DPI) resembles the irreversible pulpitis profile in humans. This study sought to determine whether pain induced by DPI activates microglia and astrocytes in the trigeminal subnucleus caudalis (Vc), as well as increases levels of proinflammatory cytokines, and whether electroacupuncture (EA) can be a potential analgesic and neuroprotective therapy following DPI. Pain behavior was measured via head-withdrawal threshold (HWT) and burrowing behavior at days 1, 3, 7, 14 and 21 after DPI. A marked decrease in HWT and burrowing activity was observed from day 1 to 14 after DPI and no changes were seen on day 21. Microglial and astrocytes activation; along with high cytokine (TNFα, IL-1ß, and IL-6) levels, were observed in the Vc at 21 days after DPI. These effects were attenuated by verum (local and distal) EA, as well as oral ibuprofen administration. The results suggest that DPI-induced pain and glial activations in the Vc and EA exert analgesic efficacy at both local and distal acupoints. Furthermore, verum (local and distal) EA might be associated with the modulations of microglial and astrocytes activation.

NOP receptor agonist attenuates nitroglycerin-induced migraine-like symptoms in mice.

Migraine is an extraordinarily prevalent and disabling headache disorder that affects one billion people worldwide. Throbbing pain is one of several migraine symptoms including sensitivity to light (photophobia), sometimes to sounds, smell and touch. The basic mechanisms underlying migraine remain inadequately understood, and current treatments (with triptans being the primary standard of care) are not well tolerated by some patients. NOP (Nociceptin OPioid) receptors, the fourth member of the opioid receptor family, are expressed in the brain and periphery with particularly high expression known to be in trigeminal ganglia (TG). The aim of our study was to further explore the involvement of the NOP receptor system in migraine. To this end, we used immunohistochemistry to examine NOP receptor distribution in TG and trigeminal nucleus caudalus (TNC) in mice, including colocalization with specific cellular markers, and used nitroglycerin (NTG) models of migraine to assess the influence of the selective NOP receptor agonist, Ro 64-6198, on NTG-induced pain (sensitivity of paw and head using von Frey filaments) and photophobia in mice. Our immunohistochemical studies with NOP-eGFP knock-in mice indicate that NOP receptors are on the majority of neurons in the TG and are also very highly expressed in the TNC. In addition, Ro 64-6198 can dose dependently block NTG-induced paw and head allodynia, an effect that is blocked by the NOP antagonist, SB-612111. Moreover, Ro 64-6198, can decrease NTG-induced light sensitivity in mice. These results suggest that NOP receptor agonists should be futher explored as treatment for migraine symptoms. This article is part of the special issue on Neuropeptides.

Potentiation of the Glycine Response by Bisphenol A, an Endocrine Disrupter, on the Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis in Mice.

Lamina II, also called the substantia gelatinosa (SG) of the medullary dorsal horn (the trigeminal subnucleus caudalis, Vc), is thought to play an essential role in the control of orofacial nociception because it receives the nociceptive signals from primary afferents, including thin myelinated Aδ- and unmyelinated C-fibers. Glycine, the main inhibitory neurotransmitter in the central nervous system, plays an essential role in the transference of nociceptive messages from the periphery to higher brain regions. Bisphenol A (BPA) is reported to alter the morphological and functional characteristics of neuronal cells and to be an effector of a great number of ion channels in the central nervous system. However, the electrophysiological effects of BPA on the glycine receptors of SG neurons in the Vc have not been well studied. Therefore, in this study, we used the whole-cell patch-clamp technique to determine the effect of BPA on the glycine response in SG neurons of the Vc in male mice. We demonstrated that in early neonatal mice (0-3 postnatal day mice), BPA did not affect the glycine-induced inward current. However, in the juvenile and adult groups, BPA enhanced the glycine-mediated responses. Heteromeric glycine receptors were involved in the modulation by BPA. The interaction between BPA and glycine appears to have a significant role in regulating transmission in the nociceptive pathway.

P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine.

BACKGROUND: Microglial activation contributes to the development of chronic migraine (CM). The P2Y12 receptor (P2Y12R), a metabolic purinoceptor that is expressed on microglia in the central nervous system (CNS), has been indicated to play a critical role in the pathogenesis of chronic pain. However, whether it contributes to the mechanism of CM remains unknown. Thus, the present study investigated the precise details of microglial P2Y12R involvement in CM. METHODS: Mice subjected to recurrent nitroglycerin (NTG) treatment were used as the CM model. Hyperalgesia were assessed by mechanical withdrawal threshold to electronic von Frey and thermal withdrawal latency to radiant heat. Western blot and immunohistochemical analyses were employed to detect the expression of P2Y12R, Iba-1, RhoA, and ROCK2 in the trigeminal nucleus caudalis (TNC). To confirm the role of P2Y12R and RhoA/ROCK in CM, we systemically administered P2Y12R antagonists (MRS2395 and clopidogrel) and a ROCK2 inhibitor (fasudil) and investigated their effects on microglial activation, c-fos, and calcitonin gene-related peptide (CGRP) expression in the TNC. To further confirm the effect of P2Y12R on microglial activation, we preincubated lipopolysaccharide (LPS)-treated BV-2 microglia with MRS2395 and clopidogrel. ELISA was used to evaluate the levels of inflammatory cytokines. RESULTS: The protein levels of P2Y12R, GTP-RhoA, ROCK2, CGRP, c-fos, and inducible nitric oxide synthase (iNOS) in the TNC were increased after recurrent NTG injection. A double labeling study showed that P2Y12R was restricted to microglia in the TNC. MRS2395 and clopidogrel attenuated the development of tactile allodynia and suppressed the expression of CGRP, c-fos, and GTP-RhoA/ROCK2 in the TNC. Furthermore, fasudil also prevented hyperalgesia and suppressed the expression of CGRP in the TNC. In addition, inhibiting P2Y12R and ROCK2 activities suppressed NTG-induced microglial morphological changes (process retraction) and iNOS production in the TNC. In vitro, a double labeling study showed that P2Y12R was colocalized with BV-2 cells, and the levels of iNOS, IL-1ß, and TNF-α in LPS-stimulated BV-2 microglia were reduced by P2Y12R inhibitors. CONCLUSIONS: These data demonstrate that microglial P2Y12R in the TNC plays a critical role in the pathogenesis of CM by regulating microglial activation in the TNC via RhoA/ROCK pathway.

Altered activity in the nucleus raphe magnus underlies cortical hyperexcitability and facilitates trigeminal nociception in a rat model of medication overuse headache.

BACKGROUND: The pathogenesis of medication overuse headache (MOH) involves hyperexcitability of cortical and trigeminal neurons. Derangement of the brainstem modulating system, especially raphe nuclei may contribute to this hyperexcitability. The present study aimed to investigate the involvement of the nucleus raphe magnus (NRM) in the development of cortical and trigeminal hyperexcitability in a rat model of MOH. RESULTS: Chronic treatment with acetaminophen increased the frequency of cortical spreading depression (CSD) and the number of c-Fos-immunoreactive (Fos-IR) neurons in the trigeminal nucleus caudalis (TNC). In the control group, muscimol microinjected into the NRM increased significantly the frequency of CSD-evoked direct current shift and Fos-IR neurons in the TNC. This facilitating effect was not found in rats with chronic acetaminophen exposure. In a model of migraine induced by intravenous systemic infusion of nitroglycerin (NTG), rats with chronic exposure to acetaminophen exhibited significantly more frequent neuronal firing in the TNC and greater Fos-IR than those without the acetaminophen treatment. Muscimol microinjection increased neuronal firing in the TNC in control rats, but not in acetaminophen-treated rats. The number of Fos-IR cells in TNC was not changed significantly. CONCLUSION: Chronic exposure to acetaminophen alters the function of the NRM contributing to cortical hyperexcitability and facilitating trigeminal nociception.

Effect of Paeonia lactiflora, a traditional Chinese herb, on migraines based on clinical application and animal behavior analyses.

BACKGROUND: Paeonia lactiflora (PL) was widely used for pain relief, but its effects on migraine headaches remain unclear. PURPOSE: The aim of the present study was to investigate the effects of PL on migraine headaches. METHODS: First, we found that PL was frequently used in Taiwan for headache treatment based on data from Taiwan's National Health Insurance Research Database. Migraine was induced through the intraperitoneal injection (i.p.) of nitroglycerin (NTG, 10 mg/kg) in rats. Pretreatment with PL was administered orally 30 min prior to the NTG i.p. Migraine headache behavior was observed by video-recordings. Finally, the rats were sacrificed and brain was removed for immunohistochemistry staining analysis. RESULTS: The frequency and total time spent rearing up and sniffing in exploratory behavior, and walking in locomotor behavior, were reduced in the NTG group compared with the control group (all p <  0.001). This reduction could be ameliorated by pretreatment with PL 1.0 g/kg (all p <  0.05). Total time spent in the light chamber was lower in the NTG group compared with the control group (p <  0.05); this could be ameliorated by pretreatment with 1.0 g/kg PL (p <  0.05). The rats in the NTG group spent longer time on the smooth surface than those in the control group (p <  0.001); this could be shortened by pretreatment with 0.5 and 1.0 g/kg PL (both p <  0.01). The traveling distance of rats in the NTG group was shorter than in the control group (p <  0.001); rats given 1.0 g/kg PL had a longer traveling distance than those in the NTG group (p <  0.01). Both c-fos and CGRP immunoreactive cells increased in the TNC in the NTG group compared with that of the control group (both p <  0.001); this increased could be reduced by pretreatment with PL 0.5 and 1.0 g/kg (both p <  0.05). CONCLUSION: Pretreatment with PL ameliorated migraine headache behaviors in the NTG-induced migraine rat model, suggesting pretreatment with PL is beneficial for migraine headache treatment. This effect of PL is related to the decrease of c-fos and CGRP in the TNC. However, still there are too many methodological limitations which need to be overcome in further experiments to support the data.

Rhynchophylline attenuates migraine in trigeminal nucleus caudalis in nitroglycerin-induced rat model by inhibiting MAPK/NF-кB signaling.

Migraine causes severe health and social issues worldwide. Rhynchophylline (Rhy) is one of the major active components of Uncaria rhynchophylla that is used for the treatment of headache in Traditional Chinese Medicine. In the current study, the effect of Rhy on nitroglycerin (NTG)-induced migraine was assessed and the associated mechanism was also explored to explain its function. Rats were pre-treated with Rhy of two doses (10 mg/kg and 30 mg/kg) and then subjected to NTG to induce migraine symptoms. Thereafter, the electroencephalogram (EEG) signaling, spontaneous behaviors, levels of indicators related to oxidative stress, and expression of calcitonin gene-related peptide (CGRP) were measured to assess the anti-migraine function of Rhy. Moreover, the activities of MAPK/NF-κB pathway under the administrations of Rhy were also detected. The results showed that NTG induced EEG and behavior disorders in rats, which was associated with the initiation of oxidative stress and increased expression of CGRP. Nevertheless, the pre-treatments with Rhy attenuated the damages induced by NTG by reversing the levels of all the above indicators. The results of western blotting demonstrated that the anti-migraine effect of Rhy was accompanied by the inhibition of MAPK/NF-кB pathway. The findings outlined in the current study revealed an alternative mechanism of Rhy in protecting brain tissues against migraine: the agent exerted its effect by suppressing MAPK/NF-кB pathway, which would ameliorate impairments associated with migraine.

Pathophysiological Mechanisms in Migraine and the Identification of New Therapeutic Targets.

Migraine is a strongly disabling disease characterized by a unilateral throbbing headache lasting for up to 72 h for each individual attack. There have been many theories on the pathophysiology of migraine throughout the years. Currently, the neurovascular theory dominates, suggesting clear involvement of the trigeminovascular system. The most recent data show that a migraine attack most likely originates in the hypothalamus and activates the trigeminal nucleus caudalis (TNC). Although the mechanisms are unknown, activation of the TNC leads to peripheral release of calcitonin gene-related protein (CGRP), most likely from C-fibers. During the past year monoclonal antibodies against CGRP or the CGRP receptor have emerged as the most promising targets for migraine therapy, and at the same time established the strong involvement of CGRP in the pathophysiology of migraine. The viewpoint presented here focuses further on the activation of the CGRP receptor on the sensory Aδ-fiber, leading to the sensation of pain. The CGRP receptor activates adenylate cyclase, which leads to an increase in cyclic adenosine monophosphate (cAMP). We hypothesize that cAMP activates the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, triggering an action potential sensed as pain. The mechanisms behind migraine pain on a molecular level, particularly their importance to cAMP, provide clues to potential new anti-migraine targets. In this article we focus on the development of targets related to the CGRP system, and further include novel targets such as the pituitary adenylate cyclase-activating peptide (PACAP) system, the serotonin 5-HT1F receptor, purinergic receptors, HCN channels, adenosine triphosphate-sensitive potassium channels (KATP), and the glutaminergic system.

N-Methyl-d-aspartate receptor open-channel blockers memantine and magnesium modulate nociceptive trigeminovascular neurotransmission in rats.

Experimental and clinical studies suggest that the low-affinity N-methyl-d-aspartate (NMDA) receptor open-channel blockers Mg2+ and memantine are effective in reducing trigeminal nociceptive activation. The aim of this study was to investigate the apparent effectiveness of these channel blockers using a model of trigeminal activation in vivo. Rats were anaesthetized before electrically stimulating the dura mater adjacent the middle meningeal artery. Neurons responding to stimulation were recorded extracellularly using electrophysiological methods. l-Glutamate or NMDA, and Mg2+ , memantine, or sodium controls were applied locally using microiontophoresis. Microiontophoretic application of Mg2+ or memantine into the trigeminocervical complex inhibited mechanically and electrically stimulated craniovascular afferents,  and l-glutamate or NMDA-evoked neuronal activity at the second-order trigeminal synapse of craniovascular afferents. By contrast, intravenous administration of MgSO4 (100 mg/kg) or memantine (10 mg/kg) did not significantly affect electrically stimulated afferent-evoked activity within the trigeminocervical complex. The Mg2+ and memantine concentrations achieved after systemic administration may not effectively inhibit activation of the trigeminocervical complex, perhaps providing an explanation for the relatively poor efficacy of these NMDA receptor open-channel blockers for headache treatment in clinical studies. Nevertheless, the present results suggest blocking of NMDA-receptor open channels inhibits nociceptive activation of the trigeminocervical complex. Further exploration of such channel blockers as a therapeutic strategy for primary head pain is warranted.

Activation of orexin-1 receptors in the ventrolateral periaqueductal grey matter (vlPAG) modulates pulpal nociception and the induction of substance P in vlPAG and trigeminal nucleus caudalis.

AIM: To characterize the role of orexin-1 receptors (OX1Rs) in ventrolateral periaqueductal grey matter (vlPAG) on modulation of capsaicin-induced pulpal nociception in rats. METHODOLOGY: Sixty-six adult male Wistar rats (2 months old) weighing between 230 and 260 g were used. The animals were cannulated for microinjection of drugs into the vlPAG matter. Pulpalgia was induced by intradental application of capsaicin solution (100 µg) into the incisor teeth of the rats. Ten min prior to capsaicin application, orexin-A (50, 100 and 150 pmol L-1 per rat) was administered. Orexin-A (150 pmol L-1 ) was also co-administrated with SB-334867 (40 nmol L-1 per rat), an OX1Rs antagonist; or bicuculline (1 µg per rat), a GABAA receptors antagonist. Moreover, treatment effects on the release of pro-nociceptive modulator substance P (SP) in vlPAG and trigeminal nucleus caudalis (Vc) of rats were explored using an immunofluorescence technique. One-way analysis of variance was used for the statistical analysis. RESULTS: Orexin-A dose-dependently decreased capsaicin-induced nociceptive behaviour. However, SB-334867 (40 nmol L-1 per rat) pretreatment (P < 0.05), but not bicuculline (1 µg per rat), attenuated the analgesic effect of orexin-A (150 pmol L-1 ). The level of SP was significantly increased in Vc and decreased in vlPAG of capsaicin-treated rats (P < 0.05). Capsaicin-induced changes in SP levels, however, were prohibited by orexin-A treatment (150 pmol L-1 ) (P < 0.05). CONCLUSIONS: Orexin-A administration into the vlPAG was associated with an inhibitory effect on capsaicin-induced pulpal nociception and bidirectional effects on the induction of SP in vlPAG and Vc of rats. Central activation of OX1Rs is a potential therapeutic tool for pulpalgia.

Induction of chronic migraine phenotypes in a rat model after environmental irritant exposure.

Air pollution is linked to increased emergency department visits for headache and migraine patients frequently cite chemicals or odors as headache triggers, but the association between air pollutants and headache is not well understood. We previously reported that chronic environmental irritant exposure sensitizes the trigeminovascular system response to nasal administration of environmental irritants. Here, we examine whether chronic environmental irritant exposure induces migraine behavioral phenotypes. Male rats were exposed to acrolein, a transient receptor potential channel ankyrin-1 (TRPA1) agonist, or room air by inhalation for 4 days before meningeal blood flow measurements, periorbital cutaneous sensory testing, or other behavioral testing. Touch-induced c-Fos expression in trigeminal nucleus caudalis was compared in animals exposed to room air or acrolein. Spontaneous behavior and olfactory discrimination was examined in open-field testing. Acrolein inhalation exposure produced long-lasting potentiation of blood flow responses to a subsequent TRPA1 agonist and sensitized cutaneous responses to mechanical stimulation. C-Fos expression in response to touch was increased in trigeminal nucleus caudalis in animals exposed to acrolein compared with room air. Spontaneous activity in an open-field and scent preference behavior was different in acrolein-exposed compared with room air-exposed animals. Sumatriptan, an acute migraine treatment blocked acute blood flow changes in response to TRPA1 or transient receptor potential vanilloid receptor-1 agonists. Pretreatment with valproic acid, a prophylactic migraine treatment, attenuated the enhanced blood flow responses observed after acrolein inhalation exposures. Environmental irritant exposure yields an animal model of chronic migraine in which to study mechanisms for enhanced headache susceptibility after chemical exposure.

Functional rhythmogenic domains defined by astrocytic networks in the trigeminal main sensory nucleus.

Stimuli that induce rhythmic firing in trigeminal neurons also increase astrocytic coupling and reveal networks that define the boundaries of this particular population. Rhythmic firing depends on astrocytic coupling which in turn depends on S100ß. In many nervous functions that rely on the ability of neuronal networks to generate a rhythmic pattern of activity, coordination of firing is an essential feature. Astrocytes play an important role in some of these networks, but the contribution of astrocytic coupling remains poorly defined. Here we investigate the modulation and organization of astrocytic networks in the dorsal part of the trigeminal main sensory nucleus (NVsnpr), which forms part of the network generating chewing movements. Using whole-cell recordings and the dye coupling approach by filling a single astrocyte with biocytin to reveal astrocytic networks, we showed that coupling is limited under resting conditions, but increases importantly under conditions that induce rhythmic firing in NVsnpr neurons. These are: repetitive electrical stimulation of the sensory inputs to the nucleus, local application of NMDA and decrease of extracellular Ca2+ . We have previously shown that rhythmic firing induced in NVsnpr neurons by these stimuli depends on astrocytes and their Ca2+ -binding protein S100ß. Here we show that extracellular blockade of S100ß also prevents the increase in astrocytic coupling induced by local application of NMDA. Most of the networks were small and remained confined to the functionally distinct area of dorsal NVsnpr. Disrupting coupling by perfusion with the nonspecific gap junction blocker, carbenoxolone or with GAP26, a selective inhibitor of connexin 43, mostly expressed in astrocytes, abolished NMDA-induced rhythmic firing in NVsnpr neurons. These results suggest that astrocytic coupling is regulated by sensory inputs, necessary for neuronal bursting, and organized in a region specific manner.

PPARγ Agonists Attenuate Trigeminal Neuropathic Pain.

OBJECTIVES: The aim of this study is to investigate the role of peroxisome proliferator-activated receptor-gamma isoform (PPARγ), in trigeminal neuropathic pain utilizing a novel mouse trigeminal inflammatory compression (TIC) injury model. RESULTS: The study determined that the PPARγ nuclear receptor plays a significant role in trigeminal nociception transmission, evidenced by: 1) Intense PPARγ immunoreactivity is expressed 3 weeks after TIC nerve injury in the spinal trigeminal caudalis, the termination site of trigeminal nociceptive nerve fibers. 2) Systemic administration of a PPARγ agonist, pioglitazone (PIO), attenuates whisker pad mechanical allodynia at doses of 300 mg/kg i.p. and 600 mg/kg p.o. 3) Administration of a PPARγ antagonist, GW9662 (30 mg/kg i.p.), prior to providing the optimal dose of PIO (300 mg/kg i.p.) blocked the analgesic effect of PIO. DISCUSSION: This is the first study localizing PPARγ immunoreactivity throughout the brainstem trigeminal sensory spinal nucleus (spV) and its increase three weeks after TIC nerve injury. This is also the first study to demonstrate that activation of PPARγ attenuates trigeminal hypersensitivity in the mouse TIC nerve injury model. The findings presented here suggest the possibility of utilizing the FDA approved diabetic treatment drug, PIO, as a new therapeutic that targets PPARγ for treatment of patients suffering from orofacial neuropathic pain.

Analgesia effect of baicalein against NTG-induced migraine in rats.

BACKGROUND: Migraine is a complex nervous system disease characterized by typical throbbing and unilateral headache, which causes severe healthy and social issues worldwide. The purpose of this study was to investigate the effect of baicalein (BAI) on the treatment of migraine. MATERIAL AND METHODS: Twenty-four rats were randomly divided equally into four groups, including a blank group, model group, positive group (ibuprofen tablets 82mg/kg), and BAI group (60mg/kg). All rats were intragastrically treated with the corresponding treatment for 10 consecutive days, and they were subcutaneously injected with NTG (10mg/kg) 1h after the last treatment, except in the blank group. After model establishment, the behaviors of all rats, including scratching head and shaking body were observed continuously for 100min. Four hours after NTG treatment, all rats were anaesthetized and the blood was collected. Thereafter, nitric oxide (NO) in plasma was determined by colorimetric method, the level of calcitonin gene-related peptide (CGRP) and endothelin (ET) were detected by radioimmunoassay method. In addition, immunohistochemistry was applied to detect c-Fos neuronal activity in trigeminal nucleus caudalis (TNC). RESULTS: Behavioral research showed that BAI administration alleviated the hyperalgesia in migraine rats. Compared with the model group, the levels of NO and CGRP in BAI administration groups were markedly decreased (p<0.01), and the levels of ET was significantly increased (p<0.01). Meanwhile, immunohistochemistry results showed that NTG treatment significantly activated c-Fos neurons while BAI treatment inhibited the expression of c-Fos. CONCLUSIONS: BAI could alleviate the migraine-like headache induced by NTG, which is related to the regulation of vasoactive substances. These findings may contribute to the further study of BAI as a potential drug for migraine pharmacotherapy.

Neural hyperactivity in the amygdala induced by chronic treatment of rats with analgesics may elucidate the mechanisms underlying psychiatric comorbidities associated with medication-overuse headache.

BACKGROUND: Patients with medication-overuse headache suffer not only from chronic headache, but often from psychiatric comorbidities, such as anxiety and depression. The mechanisms underlying these comorbidities are unclear, but the amygdala is likely to be involved in their pathogenesis. To investigate the mechanisms underlying the comorbidities we used elevated plus maze and open field tests to assess anxiety-like behavior in rats chronically treated with analgesics. We measured the electrical properties of neurons in the amygdala, and examined the cortical spreading depression (CSD)-evoked expression of Fos in the trigeminal nucleus caudalis (TNC) and amygdala of rats chronically treated with analgesics. CSD, an analog of aura, evokes Fos expression in the TNC of rodents suggesting trigeminal nociception, considered to be a model of migraine. RESULTS: Increased anxiety-like behavior was seen both in elevated plus maze and open field tests in a model of medication overuse produced in male rats by chronic treatment with aspirin or acetaminophen. The time spent in the open arms of the maze by aspirin- or acetaminophen-treated rats (53 ± 36.1 and 37 ± 29.5 s, respectively) was significantly shorter than that spent by saline-treated vehicle control rats (138 ± 22.6 s, P < 0.001). Chronic treatment with the analgesics increased the excitability of neurons in the central nucleus of the amygdala as indicated by their more negative threshold for action potential generation (-54.6 ± 5.01 mV for aspirin-treated, -55.2 ± 0.97 mV for acetaminophen-treated, and -31.50 ± 5.34 mV for saline-treated rats, P < 0.001). Chronic treatment with analgesics increased the CSD-evoked expression of Fos in the TNC and amygdala [18 ± 10.2 Fos-immunoreactive (IR) neurons per slide in the amygdala of rats treated with aspirin, 11 ± 5.4 IR neurons per slide in rats treated with acetaminophen, and 4 ± 3.7 IR neurons per slide in saline-treated control rats, P < 0.001]. CONCLUSIONS: Chronic treatment with analgesics can increase the excitability of neurons in the amygdala, which could underlie the anxiety seen in patients with medication-overuse headache.

Effect of PGD2 on middle meningeal artery and mRNA expression profile of L-PGD2 synthase and DP receptors in trigeminovascular system and other pain processing structures in rat brain.

BACKGROUND: Prostaglandins (PGs), particularly prostaglandin D2 (PGD2), E2 (PGE2), and I2 (PGI2), are considered to play a role in migraine pain. In humans, infusion of PGD2 causes lesser headache as compared to infusion of PGE2 and PGI2. Follow-up studies in rats have shown that infusion of PGE2 and PGI2 dilate the middle meningeal artery (MMA), and mRNA for PGE2 and PGI2 receptors is present in rat trigeminovascular system (TVS) and in the brain structures associated with pain. In the present study, we have characterized the dilatory effect of PGD2 on rat MMA and studied the relative mRNA expression of PGD2 receptors and lipocalin-type of PGD2 synthase (L-PGDS). METHOD: Rat closed-cranial window (CCW) model was used to study the effect of the DP1 receptor antagonist, MK-0524, on PGD2-induced vasodilation of middle meningeal artery. The qPCR technique was used for mRNA expression analysis. RESULTS: PGD2 infusion evoked a dose-dependent dilation of the rat MMA. The calculated mean pED50 value was 5.23±0.10 and Emax was 103±18% (n=5). MK-0524 significantly (∼61%, p<0.05) blocked the PGD2-induced dilation of MMA. mRNA for the DP1, DP2 and L-PGDS were expressed differentially in all tested tissues. DP1 receptor mRNA was expressed maximally in trigeminal ganglion (TG) and in cervical dorsal root ganglion (DRG). CONCLUSIONS: High expression of DP1 mRNA in the TG and DRG suggest that PGD2 might play a role in migraine pathophysiology. Activation of the DP1 receptor in MMA was mainly responsible for vasodilation induced by PGD2 infusion.