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Ethnopharmacological Relevance: Paeonol (PAE) and glycyrrhizic acid (GLY) are predominate components of 14 blood-entering ones of Piantongtang No. 1, which is a traditional Chinese medicine prescription for chronic migraine with minimal side effects. Both paeonol and glycyrrhizic acid exhibit analgesic, neuroprotective and anti-inflammatory properties individually. Our previous research has highlighted their combined effect (PAE + GLY) in ameliorating migraine symptoms. However, there are not yet any studies exploring the mechanism of action of PAE + GLY in the treatment of migraine., Aim of the Study: This research aimed to determine the mechanism of PAE + GLY in ameliorating the recurrent nitroglycerin-induced migraine-like phenotype in rats., Materials and Methods: Using a nitroglycerin-induced migraine model via subcutaneous injection in the neck, we evaluated the effect of PAE + GLY on migraine-like symptoms. Behavioural tests and biomarkers analysis were employed, alongside transcriptome sequencing (RNA-seq). Mechanistic insights were further verified utilising reverse transcription quantitative PCR (RT-qPCR), Western blot (WB), ELISA and immunofluorescence (IF) techniques., Results: Following treatment with PAE + GLY, hyperalgesia threshold and 5-hydroxytryptamine (5-HT) levels increased, and migraine-like head scratching, histamine and calcitonin gene-related peptide (CGRP) levels were reduced. RNA-Seq experiments revealed that PAE + GLY upregulated the expression of Glutamate decarboxylase 2 (GAD2) and γ-aminobutyric acid type B receptor subunit 2 (GABBR2) genes. This upregulation activated the GABAergic synapse pathway, effectively inhibiting migraine attacks. Further validation demonstrated an increase in γ-aminobutyric acid (GABA) content in cerebrospinal fluid post PAE + GLY treatment, coupled with increased expression of dural GAD2, GABBR2 and transient receptor potential channel M8 (TRPM8). Consequently, this inhibited the expression of dural cAMP-dependent protein kinase catalytic subunit alpha (PRKACA) and transient receptor potential channel type 1 (TRPV1), subsequently downregulating p-ERK1/2, p-AKT1, IL-1β and TNF-α., Conclusions: Our findings underscore that PAE + GLY ameliorates inflammatory hyperalgesia migraine by upregulating inhibitory neurotransmitters and modulating the GABBR2/TRPM8/PRKACA/TRPV1 pathway., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare, and all the authors have approved this manuscript for publication., (Copyright © 2024. Published by Elsevier B.V.)

Background: Chronic migraine is closely related to the dysregulation of neurochemical substances in the brain, with metabolic imbalance being one of the proposed causes of chronic migraine. This study aims to evaluate the metabolic changes between energy metabolism and excitatory and inhibitory neurotransmitters in key brain regions of mice with chronic migraine-like state and to uncover the dysfunctional pathways of migraine., Methods: A chronic migraine-like state mouse model was established by repeated administration of nitroglycerin (NTG). We used von Frey filaments to assess the mechanical thresholds of the hind paw and periorbital in wild-type and familial hemiplegic migraine type 2 mice. After the experiments, tissue was collected from five brain regions: the somatosensory cortex (SSP), hippocampus, thalamus (TH), hypothalamus, and the spinal trigeminal nucleus caudalis (TNC). Proton magnetic resonance spectroscopy ( 1 H-MRS) was employed to study the changes in brain metabolites associated with migraine, aiming to explore the mechanisms underlying metabolic imbalance in chronic migraine-like state., Results: In NTG-induced chronic migraine-like state model, we observed a significant reduction in energy metabolism during central sensitization, an increase in excitatory neurotransmitters such as glutamate, and a tendency for inhibitory neurotransmitters like GABA to decrease. The TNC and thalamus were the most affected regions. Furthermore, the consistency of N-acetylaspartate levels highlighted the importance of the TNC-TH-SSP pathway in the ascending nociceptive transmission of migraine., Conclusion: Abnormal energy metabolism and neurotransmitter imbalance in the brain region of NTG-induced chronic migraine-like state model are crucial mechanisms contributing to the chronicity of migraine., (© 2024. The Author(s).)

Background: Chronic migraine is a severe and common neurological disorder, yet its precise physiological mechanisms remain unclear. The IGF1/IGF1r signaling pathway plays a crucial role in pain modulation. Studies have shown that IGF1, by binding to its receptor IGF1r, activates a series of downstream signaling cascades involved in neuronal survival, proliferation, autophagy and functional regulation. The activation of these pathways can influence nociceptive transmission. Furthermore, alterations in IGF1/IGF1r signaling are closely associated with the development of various chronic pain conditions. Therefore, understanding the specific mechanisms by which this pathway contributes to pain is of significant importance for the development of novel pain treatment strategies. In this study, we investigated the role of IGF1/IGF1r and its potential mechanisms in a mouse model of chronic migraine., Methods: Chronic migraine was induced in mice by repeated intraperitoneal injections of nitroglycerin. Mechanical and thermal hypersensitivity responses were assessed using Von Frey filaments and radiant heat, respectively. To determine the role of IGF1/IGF1r in chronic migraine (CM), we examined the effects of the IGF1 receptor antagonist ppp (Picropodophyllin) on pain behaviors and the expression of calcitonin gene-related peptide (CGRP) and c-Fos., Result: In the nitroglycerin-induced chronic migraine model in mice, neuronal secretion of IGF1 is elevated within the trigeminal nucleus caudalis (TNC). Increased phosphorylation of the IGF1 receptor occurs, predominantly co-localizing with neurons. Treatment with ppp alleviated basal mechanical hypersensitivity and acute mechanical allodynia. Furthermore, ppp ameliorated autophagic dysfunction and reduced the expression of CGRP and c-Fos., Conclusion: Our findings demonstrate that in the chronic migraine (CM) model in mice, there is a significant increase in IGF1 expression in the TNC region. This upregulation of IGF1 leads to enhanced phosphorylation of IGF1 receptors on neurons. Targeting and inhibiting this signaling pathway may offer potential preventive strategies for mitigating the progression of chronic migraine., (© 2024. The Author(s).)

Background: Migraine research has highlighted the pivotal role of nitric oxide (NO) in migraine pathophysiology. Nitric oxide donors such as glyceryl trinitrate (GTN) induce migraine attacks in humans, whereas spontaneous migraine attacks can be aborted by inhibiting NO production. The present study aimed to investigate how GTN triggers migraine through its three nitric oxide synthase (NOS) isoforms (neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)) via a suspected feed-forward phenomenon., Methods: Migraine-relevant hypersensitivity was induced by repeated injection of GTN in an in vivo mouse model. Cutaneous tactile sensitivity was assessed using von Frey filaments. Signaling pathways involved in this model were dissected using non-selective and selective NOS inhibitors, knockout mice lacking eNOS or nNOS and their wild-type control mice. Also, we tested a soluble guanylate cyclase inhibitor and a peroxynitrite decomposition catalyst (N total  = 312)., Results: Non-selective NOS inhibition blocked GTN-induced hypersensitivity. This response was partially associated with iNOS, and potentially nNOS and eNOS conjointly. Furthermore, we found that the GTN response was largely dependent on the generation of peroxynitrite and partly soluble guanylate cyclase., Conclusions: Migraine-relevant hypersensitivity induced by GTN is mediated by a possible feed-forward phenomenon of NO driven mainly by iNOS but with contributions from other isoforms. The involvement of peroxynitrite adds to the notion that oxidative stress reactions are also involved., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Background: Migraine is a neurological disorder characterized by complex, widespread, and sudden attacks with an unclear pathogenesis, particularly in chronic migraine (CM). Specific brain regions, including the insula, amygdala, thalamus, and cingulate, medial prefrontal, and anterior cingulate cortex, are commonly activated by pain stimuli in patients with CM and animal models. This study employs fluorescence microscopy optical sectioning tomography (fMOST) technology and AAV-PHP.eB whole-brain expression to map activation patterns of brain regions in CM mice, thus enhancing the understanding of CM pathogenesis and suggesting potential treatment targets., Methods: By repeatedly administering nitroglycerin (NTG) to induce migraine-like pain in mice, a chronic migraine model (CMM) was established. Olcegepant (OLC) was then used as treatment and its effects on mechanical pain hypersensitivity and brain region activation were observed. All mice underwent mechanical withdrawal threshold, light-aversive, and elevated plus maze tests. Viral injections were administered to the mice one month prior to modelling, and brain samples were collected 2 h after the final NTG/vehicle control injection for whole-brain imaging using fMOST., Results: In the NTG-induced CMM, mechanical pain threshold decreased, photophobia, and anxiety-like behavior were observed, and OLC was found to improve these manifestations. fMOST whole-brain imaging results suggest that the isocortex-cerebral cortex plate region, including somatomotor areas (MO), somatosensory areas (SS), and main olfactory bulb (MOB), appears to be the most sensitive area of activation in CM (P < 0.05). Other brain regions such as the inferior colliculus (IC) and intermediate reticular nucleus (IRN) were also exhibited significant activation (P < 0.05). The improvement in migraine-like symptoms observed with OLC treatment may be related to its effects on these brain regions, particularly SS, MO, ansiform lobule (AN), IC, spinal nucleus of the trigeminal, caudal part (Sp5c), IRN, and parvicellular reticular nucleus (PARN) (P < 0.05)., Conclusions: fMOST whole-brain imaging reveals c-Fos + cells in numerous brain regions. OLC improves migraine-like symptoms by modulating brain activity in some brain regions. This study demonstrates the activation of the specific brain areas in NTG-induced CMM and suggests some regions as a potential treatment mechanism according to OLC., (© 2024. The Author(s).)

Migraine, a debilitating neurological condition, significantly affects patients' quality of life. Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPAR-α) agonist approved for managing dyslipidemia, has shown promise in treating neurological disorders. Therefore, this study aims to investigate the protective effects of fenofibrate against nitroglycerin (NTG)-induced chronic migraine in rats. Migraine was induced in rats by administering five intermittent doses of NTG (10 mg/kg, i. p.) on days 1, 3, 5, 7, and 9. Rats were treated with either topiramate (80 mg/kg/day, p. o.), a standard drug, or fenofibrate (100 mg/kg/day, p. o.) from day 1-10. Fenofibrate significantly improved mechanical and thermal hypersensitivity, photophobia, and head grooming compared to topiramate. These effects were associated with reduced serum levels of nitric oxide (NO), calcitonin gene-related peptide (CGRP), and pituitary adenylate cyclase-activating polypeptide (PACAP). Furthermore, fenofibrate down-regulated c-Fos expression in the medulla and medullary pro-inflammatory cytokine contents. Additionally, fenofibrate attenuated NTG-induced histopathological changes in the trigeminal ganglia and trigeminal nucleus caudalis. These effects were associated with the inhibition of CGRP/p-CREB/purinergic 2X receptor 3 (P2X3) and nerve growth factor (NGF)/protein kinase C (PKC)/acid-sensing ion channel 3 (ASIC3) signaling pathways. This study demonstrates that fenofibrate attenuated NTG-induced migraine-like signs in rats. These effects were partially mediated through the inhibition of CGRP/p-CREB/P2X3 and NGF/PKC/ASIC3 signaling pathways. The present study supports the idea that fenofibrate could be an effective candidate for treating migraine headache without significant adverse effects. Future studies should explore its clinical applicability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: In this study, we aimed to investigate the regulatory mechanism of transcranial ultrasound stimulation (TUS) on nitroglycerin-induced migraine in mice., Materials and Methods: The experiment was divided into four groups, namely, the normal saline control group (n = 9), ultrasound stimulation control group (n = 6), nitroglycerin-induced migraine group (n = 9), and ultrasound stimulation group (n = 9). The behavior, blood oxygen metabolism, and brain rhythm distribution of the four groups were analyzed., Results: We found that after TUS, the movement time and speed of mice with migraine are modulated to those of the control groups, and the number of head scratching and grooming events is significantly reduced. TUS increased the deoxygenated hemoglobin, and the power of the 4-to-40 Hz frequency band of local field potentials in the cortex of migraine mice. TUS also decreased the expression of plasma calcitonin gene-related peptide and cortical c-Fos protein., Conclusions: Ultrasound stimulation can regulate brain rhythm and blood oxygen metabolism and reduce migraine symptoms in mice. The regulatory mechanism may be related to reducing calcitonin gene-related peptide in blood vessels., Competing Interests: Conflict of Interest The authors reported no conflict of interest., (Copyright © 2024 International Neuromodulation Society. Published by Elsevier Inc. All rights reserved.)

Central sensitization is an important pathophysiological mechanism underlying chronic migraine (CM). Previous studies have shown that microglial activation and subsequent inflammation in the trigeminal nucleus caudalis (TNC) contribute to central sensitization. Toll-like receptor 2 (TLR2) is a receptor expressed on the membrane of microglia and participates in central sensitization in inflammatory and chronic pain; however, its role in CM is unclear. Therefore, this study investigated TLR2 involvement in CM in detail. Mice treated with recurrent nitroglycerin (NTG) were used as a CM model. Hyperalgesia was assessed using a 50% paw mechanical threshold and a 50% periorbital threshold on a Von Frey filament pain meter. Western blotting and immunofluorescence analyses were used to detect the expression of TLR2, microglia, c-fos and CGRP in TNC. The expression of inflammatory factors (IL-6, IL-1β、 IL-10、TNF-α and IFN-β1) was detected using quantitative real-time polymerase chain reaction (qRT-PCR). A selective TLR2 antagonist (C29) was systematically administered to observe its effect on hyperalgesia, microglia activation and the expression of c-fos, CGRP and inflammatory factors. Recurrent administration of NTG resulted in acute and chronic hypersensitivity, accompanied by upregulation of TLR2 expression and microglial activation in TNC. C29 partially inhibited pain hypersensitivity. C29 suppressed microglial activation induced by NTG administration. Inhibition of TLR2 reduced the expression of c-fos and CGRP in TNC after NTG treatment. C29 inhibited the expression of inflammatory mediators in TNC. These data showed that microglial TLR2 plays a critical role in the pathogenesis of CM by regulating microglial activation in TNC., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Migraine is a widespread brain condition described by frequent, recurrent episodes of incapacitating, moderate-to-severe headaches with throbbing pain that are usually one-sided. It is the 2nd most debilitating state lived with disability in terms of years, with a prevalence rate of 15-20%. Significant drops in estrogen levels have been associated with triggering acute migraine attacks in certain cases. Phytoestrogens are plant-derived compounds that resemble estrogen in structure, enabling them to imitate estrogen's functions in the body by attaching to estrogen receptors. Thus, the study was aimed to explore the protective effect of genistein against migraine. Moreover, the role of nitric oxide was also studied in the observed effect of genistein. Nitric oxide (NO) is implicated in migraine pathophysiology due to its role in promoting cerebral vasodilation and modulation of pain perception. Exploring L-NAME, a nitric oxide synthase inhibitor in migraine research helps scientists better understand the role of NO in migraine. Nitroglycerine treatment significantly increased the facial-unilateral head pain and spontaneous pain, as evidenced by the increased number of head scratching and groomings. Nitroglycerine treatment also induced anxiogenic behavior in mice. A significant reduction in the number of entries in the light phase and open arm, respectively. Biochemical analysis indicated a significant increase in inflammatory and oxidative stress in the nitroglycerin group. A significant increase and decrease in brain TBARS and GSH were observed with nitroglycerine treatment, respectively. Moreover, nitroglycerine treatment has uplifted the serum TNF-α level. Genistein (20 mg/kg) significantly mitigated the facial-unilateral head pain, spontaneous pain, photophobia, and anxiety-like behavior induced by nitroglycerine. Biochemical analysis showed that genistein (20 mg/kg) significantly abrogated the nitroglycerine-induced lipid peroxidation and increased serum TNF-α level. Genistein treatment also upregulated the brain GSH level and downregulated the serum TNF-α level. The L-NAME-mediated alleviation of the protective effect of genistein might be attributed to the vasodilatory effect of L-NAME. Conclusively, it can be suggested that genistein might provide relief from migraine pain by inhibiting nitric oxide-mediated vasodilation and oxidative stress., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Chronic migraine (CM) is a debilitating neurofunctional disorder primarily affecting females, characterized by central sensitization. Central sensitization refers to the enhanced response to sensory stimulation, which involves changes in neuronal excitability, synaptic plasticity, and neurotransmitter release. Environmental enrichment (EE) can increase the movement, exploration, socialization and other behaviors of mice. EE has shown promising effects in various neurological disorders, but its impact on CM and the underlying mechanism remains poorly understood. Therefore, the purpose of this study was to determine whether EE has the potential to serve as a cost-effective intervention strategy for CM., Methods: A mouse CM model was successfully established by repeated administration of nitroglycerin (NTG). We selected adult female mice around 8 weeks old, exposed them to EE for 2 months, and then induced the CM model. Nociceptive threshold tests were measured using Von Frey filaments and a hot plate. The expression of c-Fos, calcitonin gene-related peptide (CGRP) and inflammatory response were measured using WB and immunofluorescence to evaluate central sensitization. RNA sequencing was used to find differentially expressed genes and signaling pathways. Finally, the expression of the target differential gene was investigated., Results: Repeated administration of NTG can induce hyperalgesia in female mice and increase the expression of c-Fos and CGRP in the trigeminal nucleus caudalis (TNC). Early exposure of mice to EE reduced NTG-induced hyperalgesia in CM mice. WB and immunofluorescence revealed that EE inhibited the overexpression of c-Fos and CGRP in the TNC of CM mice and alleviated the inflammatory response of microglia activation. RNA sequencing analysis identified that several central sensitization-related signaling pathways were altered by EE. VGluT1, a key gene involved in behavior, internal stimulus response, and ion channel activity, was found to be downregulated in mice exposed to EE., Conclusion: EE can significantly ameliorate hyperalgesia in the NTG-induced CM model. The mechanisms may be to modulate central sensitization by reducing the expression of CGRP, attenuating the inflammatory response, and downregulating the expression of VGluT1, etc., suggesting that EE can serve as an effective preventive strategy for CM., (© 2024. The Author(s).)

Background: GABA, a key inhibitory neurotransmitter, has synaptic and extrasynaptic receptors on the postsynaptic neuron. Background GABA, which spills over from the synaptic cleft, acts on extrasynaptic delta subunit containing GABAA receptors. The role of extrasynaptic GABAergic input in migraine is unknown. We investigated the susceptibility to valid migraine-provoking substances with clinically relevant behavioral readouts in Genetic Absence Epilepsy of Rats Strasbourg (GAERS), in which the GABAergic tonus was altered. Subsequently, we screened relevant GABAergic mechanisms in Wistar rats by pharmacological means to identify the mechanisms., Methods: Wistar and GAERS rats were administered nitroglycerin (10 mg/kg) or levcromakalim (1 mg/kg). Mechanical allodynia and photophobia were assessed using von Frey monofilaments and a dark-light box. Effects of GAT-1 blocker tiagabine (5 mg/kg), GABAB receptor agonist baclofen (2 mg/kg), synaptic GABAA receptor agonist diazepam (1 mg/kg), extrasynaptic GABAA receptor agonists gaboxadol (4 mg/kg), and muscimol (0.75 mg/kg), T-type calcium channel blocker ethosuximide (100 mg/kg) or synaptic GABAA receptor antagonist flumazenil (15 mg/kg) on levcromakalim-induced migraine phenotype were screened., Results: Unlike Wistar rats, GAERS exhibited no reduction in mechanical pain thresholds or light aversion following nitroglycerin or levcromakalim injection. Ethosuximide did not reverse the resistant phenotype in GAERS, excluding the role of T-type calcium channel dysfunction in this phenomenon. Tiagabine prevented levcromakalim-induced mechanical allodynia in Wistar rats, suggesting a key role in enhanced GABA spillover. Baclofen did not alleviate mechanical allodynia. Diazepam failed to mitigate levcromakalim-induced migraine phenotype. Additionally, the resistant phenotype in GAERS was not affected by flumazenil. Extrasynaptic GABAA receptor agonists gaboxadol and muscimol inhibited periorbital allodynia in Wistar rats., Conclusion: Our study introduced a rat strain resistant to migraine-provoking agents and signified a critical involvement of extrasynaptic δGABAergic receptors. Extrasynaptic δ GABAA receptors, by mediating constant background inhibition on the excitability of neurons, stand as a novel drug target with a therapeutic potential in migraine., (© 2024. The Author(s).)

Background: Migraine, a complex brain disorder, is regarded as a possible clinical manifestation of brain energy dysfunction. The trigeminovascular system is considered the basis for the pathogenesis of migraine, hence we depicted the proteomics profiling of key regions in this system, then focusing on protein alterations related to mitochondrial function. The aim of this study is to illustrate the role of mitochondria in migraine., Methods: A mouse model of chronic migraine (CM) was established by repeated nitroglycerin (NTG) stimulation and evaluated by von-Frey filaments, a hot plate and a light-dark box. Differentially expressed proteins (DEPs) in some subcortical brain regions of the trigeminovascular system were screened through liquid chromatography-tandem mass spectrometry (LC‒MS/MS) to analyse the specificity of key signaling pathways in different brain regions. And then mitochondrial function, structure and dynamics were determined by qPCR, ELISA, and transmission electron microscope (TEM). Finally, the effect of mitochondrial intervention-Urolithin A (UA) on CM was investigated., Results: Repeated NTG injection triggered photophobia, periorbital and hind paw allodynia in mice. The proteomics profiling of CM model showed that 529, 109, 163, 152 and 419 DEPs were identified in the thalamus, hypothalamus, periaqueductal grey (PAG), trigeminal ganglion (TG) and trigeminocervical complex (TCC), respectively. The most significant changes in the brain region-specific pathways pointed to thalamic mitochondrial impairment. NTG induced mitochondrial structural disruption, dysfunction and homeostatic dysregulation, which could be partially attenuated by UA intervention., Conclusion: Our findings highlight the involvement of mitochondrial damage in the thalamus in central sensitization of CM, which provides evidence of possible metabolic mechanisms in migraine pathophysiology., (© 2023. Springer-Verlag Italia S.r.l., part of Springer Nature.)

Aims: Chronic migraine (CM) is a common neurological disorder with complex pathogenesis. Evidence suggests that pituitary adenylate cyclase-activating peptide (PACAP) induces migraine-like attacks and may be potential a new target for migraine treatment, but the therapeutic results of targeting PACAP and its receptors are not uniform. Therefore, the aim of this study was to investigate the regulatory effect of PACAP type I receptor (PAC1R) antagonist, PACAP6-38, on nitroglycerin (NTG)-induced central sensitization in a CM model., Methods: Sprague-Dawley (SD) rats received repeated injections of NTG to construct a CM model. Mechanical and thermal thresholds were measured using Von Frey filaments and hot plate tests. C-Fos expression was measured by western blotting and immunofluorescence staining to assess the central sensitization. PACAP6-38 was intracerebrally injected into the trigeminal nucleus caudalis (TNC), and then the changes in c-Fos, the synaptic-associated proteins, phospho-ERK1/2 (p-ERK1/2), phosphorylation of cyclic adenosine monophosphate response element-binding protein (p-CREB) and brain-derived neurotrophic factor (BDNF) were detected. Transmission electron microscopy (TEM) and Golgi-Cox staining were used to observe the ultrastructure of synapses and dendritic structures of TNC neurons., Results: The results showed that PACAP and PAC1R expression were significantly raised in the TNC after repeated NTG injections. Additionally, PACAP6-38 treatment alleviated nociceptive sensitization, inhibited NTG-induced overexpression of c-Fos and synaptic-associated proteins in the TNC of CM rat, restored aberrant synaptic structures. Furthermore, the expression of ERK/CREB/BDNF pathway was depressed by PACAP6-38., Conclusions: Our results demonstrated that abnormal synaptic structure in the TNC of CM, which could be reversed by inhibition of PAC1R via down-regulating the ERK/CREB/BDNF signaling pathway. PACAP6-38 improves NTG-induced central sensitization by regulating synaptic plasticity in the TNC of CM rat, which may provide new insights into the treatments targeting PACAP/PAC1R in migraine., (© 2023. The Author(s).)

Abstract: Migraine is a complex neurovascular disorder that is one of the leading causes of disability and a reduced quality of life. Even with such a high societal impact, our understanding of the cellular and molecular mechanisms that contribute to migraine headaches is limited. To address this complex disorder, several groups have performed genome-wide association studies to elucidate migraine susceptibility genes, with many identifying transient receptor potential melastatin 8 (TRPM8), a cold-sensitive cation channel expressed in peripheral afferents innervating the trigeminovascular system, and the principal mediator of cold and cold pain associated with injury and disease. Interestingly, these migraine-associated single-nucleotide polymorphisms reside in noncoding regions of TRPM8, with those correlated with reduced migraine risk exhibiting lower TRPM8 expression and decreased cold sensitivity. Nonetheless, as a role for TRPM8 in migraine has yet to be defined, we sought to address this gap in our knowledge using mouse genetics and TRPM8 antagonism to determine whether TRPM8 channels or neurons are required for migraine-like pain (mechanical allodynia and facial grimace) in inducible migraine models. Our results show that both evoked and spontaneous pain behaviors are dependent on both TRPM8 channels and neurons, as well as required in both acute and chronic migraine models. Moreover, inhibition of TRPM8 channels prevented acute but not established chronic migraine-like pain. These results are consistent with its association with migraine in genetic analyses and establish that TRPM8 channels are a component of the underlying mechanisms of migraine., (Copyright © 2022 International Association for the Study of Pain.)

Migraine is a complex neurovascular disorder characterized by recurrent attacks of pain and other associated symptoms. Emotional-affective aspects are important components of pain, but so far they have been little explored in animal models of migraine. In this study, we aimed to explore the correlation between trigeminal hyperalgesia and affective status or behavioral components in a migraine-specific animal model. Male Sprague-Dawley rats were treated with nitroglycerin (10 mg/kg, i.p.) or its vehicle. Four hours later, anxiety, motor/exploratory behavior and grooming (a nociception index) were evaluated with the open field test. Rats were then exposed to formalin in the orofacial region to evaluate trigeminal hyperalgesia. The data analysis shows an inverse correlation between trigeminal hyperalgesia and motor or exploratory behavior, and a positive association with anxiety-like behavior or self-grooming. These findings further expand on the translational value of the migraine-specific model based on nitroglycerin administration and prompt additional parameters that can be investigated to explore migraine disease in its complexity., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022. Published by Elsevier Inc.)

Glyceryl trinitrate (GTN) and isosorbide mononitrate (IM) are organic nitrates which release nitric oxide upon metabolism with potential to adversely affect male reproductive function. Therefore, this study was designed to evaluate the sub-chronic effect of these organic nitrates on reproductive system in male rats. Wistar rats were separately treated with GTN and IM at 2.5, 5 and 7.5 mg/kg/day by oral gavage for 45 days. At the end of treatment, serum blood samples were taken from anaesthetized rats for assessment of hormonal profile. Epididymis was removed to analyse sperm parameters. Rat testes were dissected to perform histopathological evaluation and oxidative stress biomarkers. The GTN and IM treated groups showed a significant decrease in sperm parameters (count, motility and viability) and serum testosterone in comparison to normal control group. The GTN and IM treatment also altered sperm morphology such as bent tail and head deformities as compared to control. A significant decrease in catalase activity and, increase in nitric oxide and malondialdehyde were observed in high dose drug treated groups. Moreover, a significant increase in follicle stimulating hormone and decrease in testosterone levels were evident in all drug treated groups. The level of luteinizing hormone was raised in rats treated with medium doses of drugs while it decreased at the highest dose of both drugs. Histological study showed vacuolization and degeneration of seminiferous tubules. It is concluded that GTN and IM treatment adversely affected the male reproductive function by altering sperm parameters and disrupting the reproductive hormone profile which may be attributed to the increased level of nitric oxide and oxidative stress., (© 2022 Wiley-VCH GmbH.)

Homocysteine is a sulfur-containing endogenous amino acid leading to neurotoxic effects at high concentrations. Population studies suggest an association between plasma homocysteine levels and the risk of migraine headaches. The aim of this study was to analyze the sensitivity of rats with prenatal hyperhomocysteinemia (hHCY) in respect of the development of behavioral correlates of headache and spreading cortical depolarization (CSD) in a migraine model induced by the administration of the nitric oxide (NO) donor nitroglycerin. Animals with hHCY were characterized by migraine-related symptoms such as mechanical hyperalgesia, high-level anxiety, photophobia, as well as an enhanced level of neuronal activity in the somatosensory cortex along with a lower threshold of CSD generation. Likewise, acute or chronic intermittent administration of nitroglycerin also induced the development of mechanical allodynia, photophobia and anxiety in control groups. However, these symptoms were more pronounced in rats with hHCY. Unlike hHCY, nitroglycerin administration did not affect the threshold of CSD generation, but like hHCY, increased the background neuronal activity in layers 2/3 and 4 of the cerebral cortex. The latter was more pronounced in animals with hHCY. Thus, the migraine profile associated with hHCY can be further exaggerated in conditions with enhanced levels of migraine triggering the gaseous transmitter NO. Our data are consistent with the view that high levels of plasma homocysteine can act as a risk factor for the development of migraine.

Ethnopharmacological Relevance: Dachuanxiong Formula (DCXF) is a classical Chinese medicine prescription and is composed of dried rhizomes from Ligusticum striatum DC. (Chuanxiong Rhizoma) and Gastrodia elata Bl. (Gastrodiae Rhizoma) at the ratio of 4:1 (w/w). It has been used as Chinese medicine prescription for thousands of years. DCXF is used traditionally to treat many diseases, including migraine, atherosclerosis and ischemic stroke., Aim of the Study: This study aimed to investigate the effects of DCXF on pain response in migraine mice, and the underlying mechanisms using proteomics and bioinformatics analyses., Materials and Methods: DCXF extract was prepared by mixing Chuanxiong Rhizoma and Gastrodiae Rhizoma at a mass ratio of 4:1 (w/w). After extraction, the extract was filtered prior to high performance liquid chromatography (HPLC) analysis. Nitroglycerin (NTG) was used to establish a mouse migraine model, and a behaviour study was conducted by hot plate test. In addition, proteomics and bioinformatics studies were conducted to investigate the mechanisms of DCXF-mediating anti-migraine treatment., Results: Our results showed that there were significant differences in the latencies between NTG-treated and DCXF low dose- and high doses-treated groups at 30 min after NTG injection, this suggested that DCXF could ameliorate pain response in migraine mice. Besides, the plasma levels of endothelin-1 were also measured. NTG group significantly enhanced the endothelin-1 level compared to the control group. In contrast, DCXF low dose and high dose groups significantly reduced this level compared to NTG group. In addition, the underlying mechanisms were also investigated. Our results demonstrated that the anti-migraine treatment of DCXF was highly associated with fatty acid synthesis, suggesting that DCXF ameliorated pain response through reducing endothelin-1 level and regulating fatty acid synthesis., Conclusions: The present study revealed the anti-migraine effect of DCXF in migraine mice and provided insights into the mechanisms of DCXF-mediating anti-migraine treatment., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Chronic migraine places a disabling burden on patients, which is extensively modeled by the nitroglycerin (NTG)-treated animal model. Although the NF-κB pathway is involved in an increase in CGRP levels and activation of the trigeminal system in the NTG model, the relationship between NTG and neuroinflammation remains unclear. This study aimed to optimize a chronic NTG rat model with hyperalgesia and the ethological capacity for estimating migraine therapies and to further explore the underlying mechanism of NTG-induced migraine., Methods: Rats were administered different doses of NTG s.c. daily or every 2 d; 30 min and 2 h later, the mechanical threshold was tested. After 9 d, the rats were injected with EB or Cy5.5 for the permeability assay. The other animals were sacrificed, and then, brainstem and caudal trigeminal ganglion were removed to test CGRP, c-Fos and NOS activity; Cytokines levels in the tissue and serum were measured by ELISA; and NF-κB pathway and blood-brain barrier (BBB)-related indicators were analyzed using western blotting. Immunohistochemistry was performed to observe microglial polarization and IL-17A + T cell migration in the medulla oblongata., Results: NTG (10 mg/kg, s.c., every 2 d for a total of 5 injections) was the optimal condition, resulting in progressive hyperalgesia and migraine behavior. TNC neuroinflammation with increases in cytokines, CGRP and c-Fos and activation of the NF-κB pathway was observed, and these changes were alleviated by ibuprofen. Furthermore, NTG administration increased BBB permeability by altering the levels functional proteins (RAGE, LRP1, AQP4 and MFSD2A) and structural proteins (ZO-1, Occludin and VE-cadherin-2) to increase peripheral IL-17A permeation into the medulla oblongata, activating microglia and neuroinflammation, and eventually causing hyperalgesia and migraine attack., Conclusions: This study confirmed that NTG (10 mg/kg, s.c., every 2 d for a total of 5 injections) was the optimal condition to provoke migraine, resulting in mechanical hyperalgesia and observable migraine-like behavior. Furthermore, IL-17A crossed the blood-brain barrier into the medulla oblongata, triggering TNC activation through microglia-mediated neuroinflammation. This process was a novel mechanism in NTG-induced chronic migraine, suggesting that IL-17A might be a novel target in the treatment of migraine., (© 2021. The Author(s).)

Background: Previous studies have confirmed that the microglial activation and subsequent inflammatory responses in the trigeminal nucleus caudalis (TNC) are involved in the central sensitization of chronic migraine (CM). MicroRNA-155-5p has been shown to modulate the polarization of microglia and participate in inflammatory processes in a variety of neurological diseases. However, its role in CM remains unclear. The purpose of this study was to determine the precise role of miR-155-5p in CM., Methods: A model of CM in C57BL/6 mice was established by recurrent intraperitoneal injection of nitroglycerin (NTG). Mechanical and thermal hyperalgesia were evaluated by Von Frey filaments and radiant heat. The expression of miR-155-5p was examined by qRT-PCR, and the mRNA and protein levels of silent information regulator 1(SIRT1) were measured by qRT-PCR, Western blotting (WB) and immunofluorescence (IF) analysis. The miR-155-5p antagomir, miR-155-5p agomir, SRT1720 (a SIRT1 activator) and EX527 (a SIRT1 inhibitor) were administered to confirm the effects of miR-155-5p and SIRT1 on neuroinflammation and the central sensitization of CM. ELISA, WB and IF assays were applied to evaluate the expression of TNF-α, myeloperoxidase (MPO), IL-10, p-ERK, p-CREB, calcitonin gene-related peptide (CGRP), c-Fos and microglial activation. The cellular localization of SIRT1 was illustrated by IF., Results: After the NTG-induced mouse model of CM was established, the expression of miR-155-5p was increased. The level of SIRT1 was decreased, and partly colocalized with Iba1 in the TNC. The miR-155-5p antagomir and SRT1720 downregulated the expression of p-ERK, p-CREB, CGRP, and c-Fos, alleviating microglial activation and decreasing inflammatory substances (TNF-α, MPO). The administration of miR-155-5p agomir or EX527 exacerbated neuroinflammation and central sensitization. Importantly, the miR-155-5p agomir elevated CGRP and c-Fos expression and microglial activation, which could subsequently be alleviated by SRT1720., Conclusions: These data demonstrate that upregulated miR-155-5p in the TNC participates in the central sensitization of CM. Inhibiting miR-155-5p alleviates neuroinflammation by activating SIRT1 in the TNC of CM mice., (© 2021. The Author(s).)

Excessive Ca 2+ influx and mitochondrial oxidative stress (OS) of trigeminal ganglia (TG) have essential roles in the etiology of migraine headache and aura. The stimulation of TRPM2 channel via the generation of OS and ADP-ribose (ADPR) induces pain, inflammatory, and oxidative neurotoxicity, although its inhibition reduces the intensity of pain and neurotoxicity in several neurons. However, the cellular and molecular effects of TRPM2 in the TG of migraine model (glyceryl trinitrate, GTN) on the induction of pain, OS, apoptosis, and inflammation remain elusive. GTN-mediated increases of pain intensity, apoptosis, death, cytosolic reactive oxygen species (ROS), mitochondrial ROS, caspase -3, caspase -9, cytosolic Ca 2+ levels, and cytokine generations (TNF-α, IL-1β, and IL-6) in the TG of TRPM2 wild-type mouse were further increased by the TRPM2 activation, although they were modulated by the treatments of GSH, PARP-1 inhibitors (PJ34 and DPQ), and TRPM2 blockers (ACA and 2APB). However, the effects of GTN were not observed in the TG of TRPM2 knockout mice. The current data indicate that the maintaining activation of TRPM2 is not only important for the quenching OS, inflammation, and neurotoxicity in the TG neurons of mice with experimental migraine but also equally critical to the modulation of GTN-induced pain., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The hypothalamus has been suggested to be important in the initiation cascade of migraine attacks based on clinical and biochemical observations. Previous imaging studies could not disentangle the changes due to the attack and those due to the trigger compound. With a novel approach, we assessed hypothalamic neuronal activity in early premonitory phases of glyceryl-trinitrate (GTN)-induced and spontaneous migraine attacks. We measured the hypothalamic blood oxygen level-dependent (BOLD) response to oral glucose ingestion with 3T-functional magnetic resonance imaging (MRI) in 27 women, 16 with migraine without aura and 11 controls group matched for age and body mass index (BMI), on 1 day without prior GTN administration and on a second day after GTN administration (to coincide with the premonitory phase of an induced attack). Interestingly, subgroups of patients with and without GTN-triggered attacks could be compared. Additionally, five migraineurs were investigated in a spontaneous premonitory phase. Linear mixed models were used to study between- and within-group effects. Without prior GTN infusion, the BOLD response to glucose was similar in migraine participants and controls (P = .41). After prior GTN infusion, recovery occurred steeper and faster in migraineurs (versus Day 1; P < .0001) and in those who developed an attack versus those who did not (P < .0001). Prior GTN infusion did not alter the glucose-induced response in controls (versus baseline; P = .71). Just before spontaneous attacks, the BOLD-response recovery was also faster (P < .0001). In this study, we found new and direct evidence of altered hypothalamic neuronal function in the immediate preclinical phase of both GTN-provoked and spontaneous migraine attacks., (© 2021 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Abstract: Cranial hypersensitivity is a prominent symptom of migraine, exhibited as migraine headache exacerbated with physical activity, and cutaneous facial allodynia and hyperalgesia. The underlying mechanism is believed to be, in part, activation and sensitization of dural-responsive trigeminocervical neurons. Validated preclinical models that exhibit this phenotype have great utility for understanding putative mechanisms and as a tool to screen therapeutics. We have previously shown that nitroglycerin triggers cranial allodynia in association with migraine-like headache, and this translates to neuronal cranial hypersensitivity in rats. Furthermore, responses in both humans and rats are aborted by triptan administration, similar to responses in spontaneous migraine. Here, our objective was to study the nitroglycerin model examining the effects on therapeutic targets with newly approved treatments, specifically gepants and ditans, for the acute treatment of migraine. Using electrophysiological methods, we determined changes to ongoing firing and somatosensory-evoked cranial sensitivity, in response to nitroglycerin, followed by treatment with a calcitonin gene-related peptide receptor antagonist, gepant (olcegepant), a 5-HT1F receptor agonist, ditan (LY344864), and an NK1 receptor antagonist (GR205171). Nitroglycerin induced activation of migraine-like central trigeminocervical neurons, and intracranial and extracranial neuronal hypersensitivity. These responses were aborted by olcegepant and LY344864. However, GR205171, which failed in clinical trial for both abortive and preventive treatment of migraine, had no effect. These data support the nitroglycerin model as a valid approach to study cranial hypersensitivity and putative mechanisms involved in migraine and as a screen to dissect potentially efficacious migraine therapeutic targets., (Copyright © 2020 International Association for the Study of Pain.)

Background: Central sensitization is an important pathophysiological mechanism of chronic migraine (CM). According to our previous studies, microglial activation and subsequent inflammation in the trigeminal nucleus caudalis (TNC) contribute to the central sensitization. The P2X7 receptor (P2X7R) is a purinergic receptor expressed in microglia and participates in central sensitization in chronic pain, but its role in CM is unclear. Numerous studies have shown that P2X7R regulates the level of autophagy and that autophagy affects the microglial activation and inflammation. Recently, autophagy has been shown to be involved in neuropathic pain, but there is no information about autophagy in CM. Therefore, the current study investigated the role of P2X7R in CM and its underlying mechanism, focusing on autophagy regulation., Methods: The CM model was established by repeated intraperitoneal injection of nitroglycerin (NTG) in mice. A Von Frey filament and radiant heat were used to assess the mechanical and thermal hypersensitivity. Western blotting and immunofluorescence assays were performed to detect the expression of P2X7R, autophagy-related proteins, and the cellular localization of P2X7R. To determine the role of P2X7R and autophagy in CM, we detected the effects of the autophagy inducer, rapamycin (RAPA) and P2X7R antagonist, Brilliant Blue G (BBG), on pain behavior and the expression of calcitonin gene-related peptide (CGRP) and c-fos. In addition, the effect of RAPA and BBG on microglial activation and subsequent inflammation were investigated., Results: The expression of P2X7R was increased and was mainly colocalized with microglia in the TNC following recurrent NTG administration. The autophagic flux was blocked in CM, which was characterized by upregulated LC3-II, and accumulated autophagy substrate protein, p62. RAPA significantly improved the basal rather than acute hyperalgesia. BBG alleviated both basal and acute hyperalgesia. BBG activated the level of autophagic flux. RAPA and BBG inhibited the activation of microglia, limited the inflammatory response, and reduced the expression of CGRP and c-fos., Conclusions: Our results demonstrate the dysfunction of the autophagic process in CM. Activated autophagy may have a preventive effect on migraine chronification. P2X7R contributes to central sensitization through mediating autophagy regulation and might become a potential target for CM.

Ethnopharmacological Relevance: Wine-processed Radix scutellariae (RS) is the processed product of RS, which is the dried root of Scutellaria baicalensis Georgi. It is recorded in Chinese traditional formula that wine-processed RS has the effect of anti-migraine, while the effect has not been confirmed and the possible mechanism remains unclear., Aim of the Study: To verify the anti-migraine effect of wine-processed RS in nitroglycerin (NTG)-induced rats and explore the correlation between compounds dissolution and the pore structure based on fractal theory., Materials and Methods: In the validation of pharmacodynamics, the effects of wine-processed RS on migraines were firstly evaluated by observing the number of head-scratching of rats, then investigated by determining the levels of nitric oxide (NO), calcitonin gene-related peptide (CGRP) and the expression of c-Fos in the brain of NTG-induced rat models using ELISA and immunohistochemical assessments. In the correlation study, the stir-frying time of RS was set to 5 min, 10 min and 15 min. The scanning electron microscope (SEM) and mercury intrusion method were used to explore the pore structure and main parameters of the pore structure including pore size distribution, pore volume, porosity, surface area and fractal dimension. The compounds dissolution of total flavonoids and five major components containing baicalein, baicalin, scutellarin, wogonin and wogonoside was determined by UV-Vis spectrophotometry and HPLC separately., Results: The animal experiments had shown that wine-processed RS could significantly reduce the head-scratching times of NTG-induced rat models (p < 0.01) and markedly decrease the levels of NO (p < 0.01), CGRP (p < 0.05) and the expression of c-Fos (p < 0.01) compared with model group. The data indicated that wine-processing would affect the dissolution of compounds by changing the pore structure of RS. The order of positive correlation between pore structure parameters and compounds' dissolution was total surface area > fractal dimension (r > 0) and the order of negative correlation was average pore size > total porosity > total volume (r < 0). Compared with the other sample groups (p < 0.05), the wine-processed RS stir-fried for 10 min had a pore structure which was more favorable for compounds dissolution., Conclusions: Wine-processing could strengthen the anti-migraine effect of RS by changing the pore structure of RS, which is linked to the dissolution of compounds. The RS stir-fried for 10 min may be more effective in treating migraine., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: Clinically, calcitonin gene-related peptide antagonising drugs are recognized as effective in migraine treatment, but their site of action is debated. Only a small fraction of these compounds pass the blood-brain barrier and accesses the central nervous system. Regardless, it has been argued that the central nervous system is the site of action. Here, we test this hypothesis by bypassing the blood-brain barrier through intracerebroventricular injection of calcitonin gene-related peptide antagonising drugs., Methods: We used the glyceryl trinitrate (GTN) mouse model, which is well validated by its response to specific migraine drugs. The calcitonin gene-related peptide receptor antagonist olcegepant and the calcitonin gene-related peptide monoclonal antibody ALD405 were administered either intraperitoneally or intracerebroventricularly. The outcome measure was cutaneous mechanical allodynia., Results: Mice given olcegepant intraperitoneally + GTN on day 1 had a mean 50% withdrawal threshold of 1.2 g in contrast to mice receiving placebo + GTN, which had a threshold of 0.3 g ( p  < 0.001). Similarly, in the ALD405 + GTN group, mice had thresholds of 1.2 g versus 0.2 g in the placebo + GTN group ( p  < 0.001). However, both drugs were ineffective when delivered intracerebroventricularly, as control and active groups had identical mechanical sensitivity thresholds, 0.2 g versus 0.1 g and 0.1 g versus 0.1 g for olcegepant and ALD405, respectively ( p  > 0.99 in both cases)., Discussion: The site of action of olcegepant and of the monoclonal antibody ALD405 is outside the blood-brain barrier in this mouse model of migraine. It is likely that these results can be generalised to all gepants and all antibodies and that the results are relevant for human migraine.

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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Neurogenic inflammation, mediated by the activation of primary neurons, is thought to be an important factor in migraine pathophysiology. Programmed cell death ligand-1 (PD-L1) can suppress the immune response through the Programmed cell death-1 receptor. However, the role of PD-L1/PD-1 in migraine remains unclear. In this study we evaluated the expression and role of PD-L1/PD-1 in the trigeminal ganglia in an animal model of acute migraine., Methods: Acute nitroglycerin induces acute mechanical hyperalgesia that can be used as a readout of migraine-like pain. We investigated the expression of PD-L1 and PD-1 in the trigeminal ganglia in a mouse model by means of immunofluorescence labeling, quantitative reverse transcription-polymerase chain reaction and western blotting. We explored the effects of PD-1 in a migraine model by the von Frey test and by analyzing the expression of calcitonin gene-related peptide, interleukin-1β (IL-1β), interleukin-18 (IL-18), Tumor Necrosis Factor-α (TNF-α), interleukin-6 (IL-6) and transient receptor potential vanilloid (TRPV4) after the intravenous injection of a PD-1 inhibitor., Results: PD-L1 and PD-1 immunoreactivity were present in healthy trigeminal ganglia neurons. The mRNA levels of PD-L1 and PD-1 were significantly elevated 2 h, 4 h and 6 h after acute nitroglycerin treatment ( p  < 0.05). The protein levels of PD-L1 were significantly increased 2 h, 4 h and 6 h after treatment, and PD-1 was significantly increased at 2 h and 6 h. The blockade of PD-1 increased acute nitroglycerin-induced hyperalgesia, and this effect was accompanied by a more significant increase in calcitonin gene-related peptide, IL-1β, TNF-α, IL-6 and IL-18 in the trigeminal ganglia., Conclusion: These findings suggest that PD-L1 and PD-1 might inhibit migraine-like pain by downregulating CGRP and inflammatory factors in the trigeminal ganglia. The use of PD-L1 and PD-1 as analgesics should be further studied.

Ethnopharmacological Relevance: Migraine is a disabling neurovascular disorder, which increases risk of cardiovascular events and is a social burden worldwide. The present first-line anti-migraine medications can cause overwhelming side-effects, of which one includes the onset of cardiovascular disease. As one of the marketed Tibetan drugs, Ru-yi-Zhen-bao Pills (RYZBP) have been clinically used to treat cardiovascular disorders and as anti-migraine medication. However, there is currently no research exploring the anti-migraine actions of RYZBP., Aim of the Study: The current research was designed to assess the anti-migraine roles of RYZBP and explore the underlying mechanisms in a nitroglycerin (NTG)-induced migraine rat model trial., Materials and Methods: 120 rats were randomly divided into the following six groups of 20 rats each: normal control group, model control group, positive control group, and RYZBP high/medium/low-dose groups (Ru-yi-Zhen-bao Pills; TH 1.00 g/kg, TM 0.50 g/kg and TL 0.25 g/kg). All rats were administered intragastrically for 7 consecutive days, which were subcutaneously injected with the NTG (10 mg/kg) after the last gavage (except in the normal control group). 3min after NTG treatment, 30 rats (5 rats from each group) were anesthetized and devoted to electroencephalogram(EEG) testing, which was used to evaluate the analgesic effect of RYZBP. One hour after NTG treatment, the rest of the 90 rats (15 rats from each group) were anesthetized and midbrain tissue sample was dissected. The dissection was then washed with physiological saline and collected. The histopathological changes in the periaqueductal gray(PAG) of 5 tissue samples were determined by aematoxylin-eosin (H&E) staining, as well as an estimation of substance P (SP) and neurokinin 1 receptor (NK1R) expression through immunohistochemically staining(IHC). Another 5 midbrain preparations were carried out to evaluate calcitonin gene-related peptide (CGRP), proenkephalin (PENK), SP, and cholecystokinin (CCK) expressions by real-time quantitative polymerase chain reaction (RT-qPCR). The rest of the 5 brainstem tissues were then used to measure CCK, CGRP, and opioid peptide receptor (DORR) levels by western blotting(WB)., Results: In the EEG test, RYZBP (TM 0.50 g / kg) treatment transformed the EEG pain-wave of the NTG-induced migraine model rats in different time period. In the mechanism assay, compared with the model control group, RYZBP pretreatment reduced inflammatory cell infiltration, fibrosis and vacuolation of neuronal cells of PAG tissue seen by HE staining. IHC experiments further showed that RYZBPTM up-regulated SP expression levels and enhanced NK1R levels in the NTG-induced migraine rats (P < 0.05). Therapeutic administration of RYZBP also increased PENK mRNA expression and DORR protein level. Both RT-qPCR and western blotting trials indicated that RYZBP treatment significantly decreased CCK and CGRP expression levels (P < 0.01 or P < 0.05) in the NTG-induced migraine rats., Conclusions: RYZBP has the potential to be an effective anti-migraine treatment through suppressing the EEG pain-wave, increasing the levels of SP, PENK, DORR and reducing expression of CCK and CGRP. Mediating the PAG anti-nociceptive channel and inhibiting central sensitization were the two potential mechanisms, which offers further evidence for clinical therapy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Oxidative stress and inflammatory pathways are involved in migraine and endogenous antioxidant defense system has a role in the prevention of hyperalgesia in migraine. In this study, we aimed to evaluate the role of the most pharmacologically effective molecules among the fumaric acid esters (FAEs), dimethyl fumarate, nuclear factor E2-related factor 2/antioxidant response element (Nrf-2/ARE) pathway-mediated, in regulating the hypersensitivity in a mouse model of nitroglycerine (NTG)-induced migraine., Methods: Mice were orally administered with DMF at the doses of 10, 30, and 100 mg/kg, 5 min after NTG intraperitoneal injections. We performed histological and molecular analysis on the whole brain and behavioral tests after 4 h by NTG-migraine induction. The expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) subunit p65, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), inducible nitrite oxide synthase (iNOS), cyclooxygenase 2 (COX-2), Nrf-2, manganese superoxide dismutase (Mn-SOD), and heme-oxygenase-1 (HO-1) were detected by Western blot. Tail flick, hot plate, orofacial formalin, and photophobia tests were used to evaluate migraine-like pain and migraine-related light sensitivity. Moreover, we evaluate Nrf-2-dependent mechanism by the in vitro stimulation of cells extracted by trigeminal ganglia with diethylenetriamine/nitric oxide (DETA/NO), a nitric oxide (NO) donor. The cells were pre-treated with DMF and an antagonist of Nrf-2, trigonelline (TR) 2 h before DETA/NO stimulation., Results: DMF treatment notably reduced histological damage as showed by cresyl violet staining; also, regulating both NF-κB and Nrf-2 pathway, DMF treatment decreased the severity of inflammation and increased the protective antioxidant action. Moreover, the headache was significantly reduced. The protective effect of DMF treatment, via Nrf-2, was confirmed in in vitro studies, through inhibition of Nrf-2 by trigonelline. Cytotoxicity, iNOS, and MnSOD expression were evaluated., Conclusion: These results provided the evidence that DMF, by Nrf-2 modulation, has a protective effect on central sensitization induced by NTG, suggesting a new insight into the potential application of DMF as novel candidates in drug development for migraine.

Background: Fatty-acid amide hydrolase (FAAH) is an intracellular serine hydrolase that catalyzes the cleavage of endogenous fatty-acid amides, including the endocannabinoid anandamide (AEA). We previously reported that the peripherally restricted FAAH inhibitor URB937, which selectively increases AEA levels outside the central nervous system, reduces hyperalgesia and c-Fos expression in the trigeminal nucleus caudalis (TNC) and the locus coeruleus in an animal model of migraine based on nitroglycerin (NTG) administration., Aim: To further investigate the relevance of FAAH inhibition in the NTG animal model of migraine by testing the effects of the globally active FAAH inhibitor URB597., Methods: Our experimental approach involved mapping neuronal c-Fos protein expression, measurement of AEA levels in brain areas and in trigeminal ganglia, evaluation of pain-related behavior and quantification of molecular mediators in rats that received URB597 (2 mg/kg i.p.) either before or after NTG administration (10 mg/kg, i.p.)., Results: Pre-treatment with URB597 significantly reduced c-Fos immunoreactivity in the TNC and inhibited NTG-induced hyperalgesia in the orofacial formalin test. This behavioral response was associated with a decrease in neuronal nitric oxide synthase, calcitonin gene-related peptide and cytokine gene expression levels in central and peripheral structures. Administration of URB597 after NTG had no such effect., Conclusions: The findings suggest that global FAAH inhibition may offer a therapeutic approach to the prevention, but not the abortive treatment, of migraine attacks. Further studies are needed to elucidate the exact cellular and molecular mechanisms underlying the protective effects of FAAH inhibition., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Two main contributors of sterile neurogenic inflammation underlying migraine pain, calcitonin gene-related peptide (CGRP), and meningeal mast cells (MMCs) play a key role in the activation of the inflammatory cascade resulting in the sensitization of trigeminal nociceptors. It is well established that phytochemical agent thymoquinone exhibits multiple anti-inflammatory effects in different in vitro and in vivo models of neuroinflammation. But its effects on the CGRP release and meningeal mast cells are unknown. In the present study, we investigated the effects of thymoquinone on the CGRP release in migraine-related strategic structures which are crucial targets for anti-migraine drugs, and on the MMCs in glyceryl trinitrate (GTN)-induced in vivo migraine model as well as in the ex vivo meningeal preparations in rats. Anti-inflammatory thymoquinone ameliorated GTN-stimulated CGRP levels in plasma, and migraine-related structures including trigeminal ganglion and brainstem; moreover, thymoquinone inhibited degranulation of MMCs and prevented the increase in the number of MMCs in GTN-induced in vivo migraine model. However, in the ex vivo meningeal preparations, thymoquinone did not inhibit the GTN-induced CGRP release from trigeminal meningeal afferents. Our findings suggest that thymoquinone mediates modulation of CGRP release in trigeminal ganglion neurons and brainstem, and stabilization of MMCs. Thus, thymoquinone may be a promising candidate to prevent the meningeal neurogenic inflammation and consequently migraine.

Aim: Migraine is a neurological debilitating disorder. Previous studies have shown that cannabinoid receptor agonists have analgesic effects in various models of pain. In this study, therefore, we investigated anti-nociceptive effects of WIN 55,212-2, and the role of either CB 1 or CB 2 receptors in nitroglycerine (NTG)-induced animal model of migraine., Methods: The present study was conducted on both male and female rats receiving NTG (10 mg/kg, i.p.) to induce acute (single dose of NTG) and chronic (repetitive doses of NTG) models of migraine. Additionally, three groups received WIN 55,212-2 (0.33, 1, 3 mg/kg, i.p.) 45 min before behavioral tests. Additionally, AM251 and AM630 (CB 1 and CB 2 receptor antagonist, respectively, 1 mg/kg, i.p.) were used to evaluate the possible involvement of CB1 and CB2 receptors during the protective effects of WIN 55,212-2., Key Findings: We found that NTG (10 mg/kg, i.p.) in both acute and chronic models increased sensitivity to pain. In acute model, we found that WIN 55,212-2 (almost high doses) decreases the level of pain mainly through CB 1 receptor due to CB 1 antagonist abrogates its protective effects, however, in formalin test CB 2 receptors also had crucial roles in both phases at 3 mg/kg of WIN 55,212-2. In chronic model, WIN 55,212-2 (0.33, 1 and 3 mg/kg) significantly attenuated NTG-induced hyperalgesia through both CB 1 and CB 2 receptors., Significance: Our data supported the argument that activation of CB 1 and CB 2 receptors by WIN 55,212-2 may be considered a new medication for migraine, however in lack of each receptor leads to different responses from deletion to the reduction of analgesic effects., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Migraine is a disease for which there is still no defined pathophysiological etiology and few translational models. The organic nitrate nitroglycerin has been in use as an experimental model of migraine in both human and animal studies for several years. The drug produces a number of effects within the head, that includes blood vessels, nerves and brain areas that may produce a response similar to a migraine attack in predisposed subjects. A better understanding of the nature of these changes and how well they parallel a true migraine attack would allow for a translational model to better understand some of the mechanisms involved in the generation of a migraine attack. The present review summarizes the known body of knowledge of nitroglycerin effects evaluated in humans and animals as it relates to potential mechanisms associated with migraine headaches., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Central sensitization is an important mechanism of chronic migraine (CM) and is related to the inflammatory response of microglia. The NOD-like receptor protein 3 (NLRP3) inflammasome may regulate the inflammatory process of microglia in several neurological diseases, but its role in CM is largely unknown. Therefore, the aim of this study was to identify the precise role of microglial NLRP3 in CM., Methods: An experimental CM mouse model was established by repeated intraperitoneal (i.p) injection with nitroglycerin (NTG). We evaluated the expression levels of NLRP3 and its downstream interleukin (IL)-1β protein in the trigeminal nucleus caudalis (TNC; which is a central area relevant to migraine pain) at different time points. To further examine the effects of the NLRP3 inflammasome pathway on central sensitization of CM, we examined MCC950, an NLRP3 inflammasome-specific inhibitor, and IL-1ra, an IL-1β antagonist, whether altered NTG-induced mechanical hyperalgesia of the periorbital area and hind paw. The effect of MCC950 and IL-1ra on c-Fos, phosphorylated extracellular signal-regulated kinase (p-ERK) and calcitonin gene-related peptide (CGRP) expression in the TNC were also analyzed. The cell localization of NLRP3 and IL-1β in the TNC was evaluated by immunofluorescence staining., Results: Repeated NTG administration induced acute and chronic mechanical hyperalgesia and increased expression of NLRP3 and IL-1β. Blockade of NLRP3 or IL-1β reduced NTG-induced hyperalgesia, and this effect was accompanied by a significant inhibition of the NTG-induced increase in p-ERK, c-Fos and CGRP levels in the TNC. Immunofluorescence staining revealed that NLRP3 and IL-1β were mainly expressed in microglia in the TNC, and the IL-1β receptor, IL-1R, was mainly expressed in neurons in the TNC., Conclusions: These results indicate that NLRP3 activation in the TNC participates in the microglial-neuronal signal by mediating the inflammatory response. This process contributes to the central sensitization observed in CM.

Background According to the neurovascular theory of migraine, activation of the trigeminovascular system contributes to the development of migraine. This study examined the effects of chronic intraperitoneal ghrelin (150 µg/kg) treatment on the development of chronic migraine induced by intermittent injection of nitroglycerin 10 mg/kg. Methods Baseline and post-drug (2 h following nitroglycerin injection) mechanical and thermal sensitivity were assessed by von Frey hair and tail immersion tests, respectively on days 1, 3, 5, 7, 9 and 11. Moreover, we investigated the effect of ghrelin treatment on nitroglycerin-induced aversive behavior by using a two-chamber conditioned place aversion paradigm. At the end of behavioral testing, on day 11, animals were sacrificed and plasma concentration of calcitonin gene-related peptide was measured using a rat-specific enzyme-linked immunosorbent assay kit. Also, real time polymerase chain reaction was used to quantify mRNA expression of calcitonin gene-related peptide and transient receptor potential vanilloid 1 in the trigeminal ganglion. Results Our results indicated that nitroglycerin activated the trigeminovascular system, which was reflected by mechanical and thermal hypersensitivity and elevation of mRNA expression of calcitonin gene-related peptide and transient receptor potential vanilloid-1, as migraine markers, and plasma calcitonin gene-related peptide levels. Moreover, chronic nitroglycerin injection induced conditioned place aversion and body weight loss. Nevertheless, ghrelin modulated nitroglycerin-triggered changes in transient receptor potential vanilloid-1 and calcitonin gene-related peptide expression, and mitigated nitroglycerin-induced hyperalgesia. Conclusion These results provide the first convincing evidence that ghrelin has a modulating effect on central sensitization induced by chronic intermittent nitroglycerin, and its antinociceptive effect may be related to a reduction of these factors in the trigeminal ganglion.

Individual effects of nitrogen-based energetic materials (EMs) 2,4-dinitrotoluene (2,4-DNT), 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), nitroglycerin (NG), and 2,4,6,8,10,12-hexanitrohexaazaisowurtzitane (CL-20) on litter decomposition, an essential biologically-mediated soil process, were assessed using Orchard grass (Dactylis glomerata) straw in Sassafras sandy loam (SSL) soil, which has physicochemical characteristics that support "very high" qualitative relative bioavailability for organic chemicals. Batches of SSL soil were separately amended with individual EMs or acetone carrier control. To quantify the decomposition rates, one straw cluster was harvested from a set of randomly selected replicate containers from within each treatment, after 1, 2, 3, 4, 6, and 8 months of exposure. Results showed that soil amended with 2,4-DNT or NG inhibited litter decomposition rates based on the median effective concentration (EC50) values of 1122 mg/kg and 860 mg/kg, respectively. Exposure to 2-ADNT, 4-ADNT or CL-20 amended soil did not significantly affect litter decomposition in SSL soil at ≥ 10,000 mg/kg. These ecotoxicological data will be helpful in identifying concentrations of EMs in soil that present an acceptable ecological risk for biologically-mediated soil processes., (Published by Elsevier Inc.)

Background: Curcumin has antioxidative properties that could be useful in various diseases due to its ability to act on multiple targets of various cellular pathways. We aimed to assess the efficacy of liposomal curcumin compared with curcumin solution, when in addition to sumatriptan (ST) treatment, in an experimental migraine model induced with nitroglycerin (NTG) in rats., Methods: Seven groups of 9 rats each were investigated: control group without migraine (1 mL saline solution intraperitoneal injection [ip]), control group with induced migraine, NTG+ST group (ST), NTG+ST+curcumin1 (CC1) group - 1 mg/100 g body weight (bw), NTG+ST+CC2 - 2 mg/100 g bw, NTG+ST+liposomal curcumin1 (lCC1) group - 1 mg/100 g bw, and NTG+ST+lCC2 (lCC2) group - 2 mg/100 g bw. NTG and ST were administered as 1 mL ip NTG | 1 mg/100 g bw and 1 mL ip ST | 1 mg/100 g bw, respectively. Plasma total oxidative stress (TOS), malondialdehyde (MDA), nitric oxide (NOx), thiol levels, as well as total antioxidative capacity (TAC) were assessed. The nociception process was assessed by counting the number of flinches and shakes after the formalin test., Results: The plasma TOS, MDA, and NOx levels, as oxidative stress parameters, were significantly decreased in the curcumin-treated groups, especially where curcumin was in liposomal form. The thiol and TAC were also improved by the curcumin treatment, with the best results obtained for the liposomal curcumin. The closest number of flinches and shakes to the control group was obtained for the group treated with liposomal curcumin at a dose of 2 mg/100 g bw., Conclusion: Liposomal curcumin in a dose of 2 mg/100 g bw when in addition to ST treatment could be an optimum therapeutic strategy for migraine attacks and could represent a base for future clinical research and application., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Background A common characteristic of migraine-inducing substances is that they cause headache and no pain in other areas of the body. Few studies have compared pain mechanisms in the trigeminal and spinal systems and, so far, no major differences have been noted. We compared signalling molecules in the trigeminal and spinothalamic system after infusion of the migraine-provoking substance glyceryltrinitrate. Method A catheter was placed in the femoral vein of rats and one week later glyceryltrinitrate 4 µg/kg/min was infused for 20 min. Protein expression in the dura mater, trigeminal ganglion, nucleus caudalis, dorsal root ganglion and the dorsal horn of the thoracic spinal cord was analysed at different time points using western blotting and immunohistochemistry. Results Glyceryltrinitrate caused a threefold increase in expression of phosphorylated extracellular signal-regulated kinases at 30 min in the dura mater and nucleus caudalis ( P < 0.05) and at 2 h in the trigeminal ganglion with very few expressions in the dorsal root ganglion. In the nucleus caudalis, expression of phosphorylated extracellular signal-regulated kinases and Cam KII increased 2.6-fold and 3.2-fold, respectively, at 2 h after glycerytrinitrate infusion ( P < 0.01). p-CREB/ATF-1 upregulation was observed only at 30 min ( P < 0.05) in the nucleus caudalis. None of these markers showed increased expression in the regions of thoracic spinal cord dorsal horn. Conclusion The dura, trigeminal ganglion and nucleus caudalis are activated shortly after glycerytrinitrate infusion with long-lasting expression of phosphorylated extracellular signal-regulated kinases observed in the nucleus caudalis. These activations were not observed at the spinal level.

Migraine is the third most common disease worldwide; however, the mechanisms underlying migraine headache are still not fully understood. Previous studies have demonstrated that α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor phosphorylation plays an important role in central sensitization of pain transmission. In the present study, we observed that AMPA receptor GluA1 Ser831 phosphorylation was enhanced in the spinal trigeminal nucleus caudalis (Sp5C) after intraperitoneal injection of nitroglycerin (NTG). The NTG injection induced acute migraine-like pain including photophobia and mechanical hypersensitivity as reported previously. Interestingly, targeted mutation of GluA1 Ser831 site to prevent phosphorylation significantly inhibited NTG-induced migraine-like pain. Moreover, NTG incubation caused a robust Ca 2+ influx in cultured brainstem neurons, which was dramatically inhibited by GluA1 S831A (serine at the 831 site of GluA1 is mutated to alanine) phospho-deficient mutation, and treatment with 1-naphthyl acetyl spermine (NASPM), a selective Ca 2+ -permeable AMPA receptor channel blocker, dose-dependently blocked the NTG-evoked increase of Ca 2+ influx in the cultured neurons. We further found that intra-Sp5C injection of NASPM significantly inhibited NTG-produced mechanical hypersensitivity. These results suggest that AMPA receptor phosphorylation at the Ser831 site in the Sp5C is critical for NTG-induced migraine-like pain., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Migraine is a common disease with a high morbidity. Valproate (VP) is used as an anti-epilepsy drug in clinic. This study aimed to investigate the role of VP in nitroglycerin (NTG)-induced migraine using a mouse model. NTG was employed by intraperitoneal injection to induce a migraine model in mice. The NTG administration caused mouse head discomforts, decreased tolerance to cold or hot stimulation and increased content of nitric oxide, calcitonin gene-related peptide and neuropeptide Y in serum, which were ameliorated by intraperitoneal injection of VP. The levels of two inflammatory factors, interleukin (IL)-1β and inducible nitric oxide synthase, in dura mater were increased by NTG treatment, while the increase was attenuated by application of VP. In addition, the phosphorylation levels of protein kinase C (PKC) α, γ, δ and ε were increased by NTG and decreased by VP. However, their total expression at the transcriptional and translational levels did not change significantly. Two substrates of PKC, cAMP-response element binding protein 1 and signal transducer and activator of transcription 1 were also phosphorylated by NTG application, and the phosphorylation level was attenuated by VP, consistent with the change of PKC informs. Together, we demonstrated that VP prevented damage due to migraine by inhibiting PKC signalling in NTG-injected mice, which may provide a basis for investigating the clinical treatment of migraine., (© 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Despite the relatively high prevalence of migraine or headache, the pathophysiological mechanisms triggering headache-associated peripheral hypersensitivities, are unknown. Since nitric oxide (NO) is well known as a causative factor in the pathogenesis of migraine or migraine-associated hypersensitivities, a mouse model has been established using systemic administration of the NO donor, nitroglycerin (NTG). Here we tried to investigate the time course development of facial or hindpaw hypersensitivity after repetitive NTG injection. NTG (10 mg/kg) was administrated to mice every other day for nine days. Two hours post-injection, NTG produced acute mechanical and heat hypersensitivity in the hind paws. By contrast, cold allodynia, but not mechanical hypersensitivity, occurred in the facial region. Moreover, this hindpaws mechanical hypersensitivity and the facial cold allodynia was progressive and long-lasting. We subsequently examined whether the depletion of capsaicin-sensitive primary afferents (CSPAs) with resiniferatoxin (RTX, 0.02 mg/kg) altered these peripheral hypersensitivities in NTG-treated mice. RTX pretreatment did not affect the NTG-induced mechanical allodynia in the hind paws nor the cold allodynia in the facial region, but it did inhibit the development of hind paw heat hyperalgesia. Similarly, NTG injection produced significant hindpaw mechanical allodynia or facial cold allodynia, but not heat hyperalgesia in transient receptor potential type V1 (TRPV1) knockout mice. These findings demonstrate that different peripheral hypersensitivities develop in the face versus hindpaw regions in a mouse model of repetitive NTG-induced migraine, and that these hindpaw mechanical hypersensitivity and facial cold allodynia are not mediated by the activation of CSPAs.

Background Trigeminal sensitization represents a major mechanism underlying migraine attacks and their recurrence. Nitroglycerin (NTG) administration provokes spontaneous migraine-like headaches and in rat, an increased sensitivity to the formalin test. Kynurenic acid (KYNA), an endogenous regulator of glutamate activity and its analogues attenuate NTG-induced neuronal activation in the nucleus trigeminalis caudalis (NTC). The anti-hyperalgesic effect of KYNA analogue 1 (KYNA-A1) was investigated on animal models specific for migraine pain. Aim Rats made hyperalgesic by NTG administration underwent the plantar or orofacial formalin tests. The effect of KYNA-A1 was evaluated in terms of nocifensive behavior and of neuronal nitric oxide synthase (nNOS), calcitonin gene-related peptide (CGRP) and cytokines expression in areas involved in trigeminal nociception. Results KYNA-A1 abolished NTG-induced hyperalgesia in both pain models; NTG alone or associated to formalin injection induced an increased mRNA expression of CGRP, nNOS and cytokines in the trigeminal ganglia and central areas, which was reduced by KYNA-A1. Additionally, NTG caused a significant increase in nNOS immunoreactivity in the NTC, which was prevented by KYNA-A1. Conclusion Glutamate activity is likely involved in mediating hyperalgesia in an animal model specific for migraine. Its inhibition by means of a KYNA analogue modulates nNOS, CGRP and cytokines expression at peripheral and central levels.

Migraine is a debilitating condition that affects hundreds of millions of people worldwide. A subset of these patients experience chronic migraine, resulting in long-term disability and a severely lowered quality of life. The development of novel migraine therapies has been slow, partially due to the small number of predictive animal models. We have recently developed a novel model of chronic migraine-associated pain, using the known human migraine trigger, nitroglycerin. Injection of nitroglycerin evokes an acute mechanical hyperalgesia, which is sensitive to the acute migraine therapy sumatriptan. In addition, chronic administration of nitroglycerin produces a progressive and sustained decrease in basal mechanical responses, and this hypersensitivity is blocked by migraine preventatives such as topiramate. This mouse model of chronic migraine can be used to study the mechanisms underlying progression of migraine from an episodic to a chronic disorder, and for identifying and screening novel acute and preventive migraine therapies. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

Chronic migraine is a debilitating disorder that has a significant impact on patients and society. Nearly all migraineurs frequently reported light sensitivity during a headache attack. Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the activation of trigeminal system and migraine pain. To identify the effect of chronic ghrelin treatment on endogenous PACAP and associated symptoms of migraine, an experimental chronic migraine model was induced by intermittent intraperitoneal (i.p) injection of nitroglycerin (NTG). Photophobia and anxiety-like behaviors were determined in the modified elevated plus maze on days 2, 4, 6, 8, and 10 and in the light/dark box on days 3, 5, 7, 9, and 11. Blood levels of PACAP and cortisol were assessed by enzyme-linked immunosorbent (ELISA) kits. Chronic injection of NTG evoked photophobia and anxiety-like behaviors and treatment with ghrelin (150 μg/kg) for 11 days effectively attenuated photophobia and anxiety-like behaviors in the both paradigms. We further found that NTG increased the blood levels of PACAP and cortisol, which was significantly reduced by ghrelin treatment. Additionally, staining with Hematoxylin and Eosin (H&E) revealed that ghrelin reduced NTG-induced increase in the number of satellite glial cells in the trigeminal ganglion. Furthermore, for the first time we showed that repeated administrations of NTG increased white blood cell (WBC) counts and mean platelet volume (MPV), and decreased platelet counts. These results indicated that ghrelin decreased migraine associated symptoms possibly through attenuating endogenous PACAP and cortisol levels. Therefore, ghrelin may hold therapeutic potentialities in managing the chronic migraine., (© 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Objective: Our study aimed to investigate the analgesic and antioxidative stress effects of Curcumin (CC) in experimental migraine induced by Nitroglycerin (NTG) on rats, compared with Indomethacin (ID) and Propranolol (PP) treatments., Material and Methods: Five groups of 10 rats treated i.p. were investigated: control group (healthy rats) injected with saline solution (0.9%), NTG-control group injected with NTG (1 mg/100 gbw, bw = body weight), and three groups with pretreatment applied 30 min previous to the formalin test (NTG + CC group: Curcumin (10 mg/100 gbw), NTG + PP group: Propranolol (100  μ g/100 gbw), and NTG + ID group: Indomethacin (0.5 mg/100 gbw)). Formalin test was performed and number of flinches and shakes were counted. Several oxidative stress parameters were also assessed., Results: The smallest values of malondialdehyde (MDA), nitric oxide (NOx), and total oxidative status (TOS) were observed on NTG + CC with significant differences as compared with the control group ( p < 0.0001). The group pretreated with Curcumin proved significantly smaller number of flinches and shakes compared with both NTG + PP and NTG + ID., Conclusion: Our study demonstrates a superior activity of Curcumin not only versus control, but also versus Propranolol and Indomethacin.

Background: One of the human and animal models of migraine is the systemic administration of the nitric oxide donor (NO) nitroglycerin (NTG). NO can provoke migraine-like attacks in migraineurs and initiates a self-amplifying process in the trigeminal system, probably leading to central sensitization. Recent studies suggest that the endocannabinoid system is involved in nociceptive signal processing and cannabinoid receptor (CB) agonists are able to attenuate nociception in animal models of pain., Aim: The purpose of the present study was to investigate the modulatory effects of a CB agonist anandamide (AEA) on the NTG-induced expression of transient receptor potential vanilloid type 1 (TRPV1), neuronal nitric oxide synthase (nNOS), nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2) and kynurenine aminotransferase-II (KAT-II) in the upper cervical spinal cord (C1-C2) of the rat, where most of the trigeminal nociceptive afferents convey., Methods: A half hour before and one hour after NTG (10 mg/kg) or placebo injection, adult male Sprague-Dawley rats (n = 44) were treated with AEA (2 × 5 mg/kg). Four hours after placebo/NTG injection, the animals were perfused and the cervical spinal cords were removed for immunohistochemistry and Western blotting., Results and Conclusion: Our results show that NTG is able to increase TRPV1, nNOS, NF-κB and COX-2 and decrease KAT-II expression in the C1-C2 segments. On the other hand, we have found that AEA modulates the NTG-induced changes, thus it influences the activation and central sensitization process in the trigeminal system, probably via CBs., (© International Headache Society 2015.)

Nitroglycerin (GTN) and other organic nitrates are widely used vasodilators. Their side effects are development of nitrate tolerance and endothelial dysfunction. Given the potential of GTN to induce nitro-oxidative stress, we investigated the interaction between nitro-oxidative DNA damage and vascular dysfunction in experimental nitrate tolerance. Cultured endothelial hybridoma cells (EA.hy 926) and Wistar rats were treated with GTN (ex vivo: 10-1000 µM; in vivo: 10, 20 and 50 mg/kg/day for 3 days, s.c.). The level of DNA strand breaks, 8-oxoguanine and O (6)-methylguanine DNA adducts was determined by Comet assay, dot blot and immunohistochemistry. Vascular function was determined by isometric tension recording. DNA adducts and strand breaks were induced by GTN in cells in vitro in a concentration-dependent manner. GTN in vivo administration leads to endothelial dysfunction, nitrate tolerance, aortic and cardiac oxidative stress, formation of DNA adducts, stabilization of p53 and apoptotic death of vascular cells in a dose-dependent fashion. Mice lacking O (6)-methylguanine-DNA methyltransferase displayed more vascular O (6)-methylguanine adducts and oxidative stress under GTN therapy than wild-type mice. Although we were not able to prove a causal role of DNA damage in the etiology of nitrate tolerance, the finding of GTN-induced DNA damage such as the mutagenic and toxic adduct O (6)-methylguanine, and cell death supports the notion that GTN based therapy may provoke adverse side effects, including endothelial function. Further studies are warranted to clarify whether GTN pro-apoptotic effects are related to an impaired recovery of patients upon myocardial infarction.

Nitroglycerin (glycerol trinitrate, GTN) induces headache in migraineurs, an effect that has been used both diagnostically and in the study of the pathophysiology of this neurovascular pain syndrome. An important feature of this headache is a delay from the administration of GTN to headache onset that, because of GTN's very rapid metabolism, cannot be due to its pharmacokinetic profile. It has recently been suggested that activation of perivascular mast cells, which has been implicated in the pathophysiology of migraine, may contribute to this delay. We reported that hyperalgesia induced by intradermal GTN has a delay to onset of ∼ 30 min in male and ∼ 45 min in female rats. This hyperalgesia was greater in females, was prevented by pretreatment with the anti-migraine drug, sumatriptan, as well as by chronic pretreatment with the mast cell degranulator, compound 48/80. The acute administration of GTN and compound 48/80 both induced hyperalgesia that was prevented by pretreatment with octoxynol-9, which attenuates endothelial function, suggesting that GTN and mast cell-mediated hyperalgesia are endothelial cell-dependent. Furthermore, A-317491, a P2X3 antagonist, which inhibits endothelial cell-dependent hyperalgesia, also prevents GTN and mast cell-mediated hyperalgesia. We conclude that delayed-onset mechanical hyperalgesia induced by GTN is mediated by activation of mast cells, which in turn release mediators that stimulate endothelial cells to release ATP, to act on P2X3, a ligand-gated ion channel, in perivascular nociceptors. A role of the mast and endothelial cell in GTN-induced hyperalgesia suggests potential novel risk factors and targets for the treatment of migraine., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)