Mechanisms of GTN-induced migraine: Role of NOS isoforms, sGC and peroxynitrite in a migraine relevant mouse model.

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 (Ntotal = 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.

1H-MRS reveals abnormal energy metabolism and excitatory-inhibitory imbalance in a chronic migraine-like state induced by nitroglycerin in mice.

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 (1H-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.

Targeting IGF1/IGF1r signaling relieve pain and autophagic dysfunction in NTG-induced chronic migraine model of mice.

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.

Brain-wide mapping of c-Fos expression in nitroglycerin-induced models of migraine.

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.

Paeonol and glycyrrhizic acid in combination ameliorate the recurrent nitroglycerin-induced migraine-like phenotype in rats by regulating the GABBR2/TRPM8/PRKACA/TRPV1 pathway.

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-1ß 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.

Environmental enrichment alleviates hyperalgesia by modulating central sensitization in a nitroglycerin-induced chronic migraine model of mice.

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.

Extrasynaptic δGABAA receptors mediate resistance to migraine-like phenotype in rats.

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.

Fenofibrate ameliorates nitroglycerin-induced migraine in rats: Role of CGRP/p-CREB/P2X3 and NGF/PKC/ASIC3 signaling pathways.

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.

Genistein mitigates nitroglycerine-induced migraine: modulation of nitric oxide-mediated vasodilation and oxidative stress.

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.

Effects of Transcranial Ultrasound Stimulation on Blood Oxygen Metabolism and Brain Rhythms in Nitroglycerin-Induced Migraine Mice.

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.

Proteomics profiling reveals mitochondrial damage in the thalamus in a mouse model of chronic migraine.

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.

PACAP6-38 improves nitroglycerin-induced central sensitization by modulating synaptic plasticity at the trigeminal nucleus caudalis in a male rat model of chronic migraine.

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.

Modelling migraine-related features in the nitroglycerin animal model: Trigeminal hyperalgesia is associated with affective status and motor behavior.

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.

Endocrine disruption: Reproductive toxicity of glyceryl trinitrate and isosorbide mononitrate in male Wistar rats.

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.

Hyperhomocysteinemia Increases Cortical Excitability and Aggravates Mechanical Hyperalgesia and Anxiety in a Nitroglycerine-Induced Migraine Model in Rats.

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.

Transient receptor potential melastatin 8 is required for nitroglycerin- and calcitonin gene-related peptide-induced migraine-like pain behaviors in mice.

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.

Classical prescription Dachuanxiong Formula delays nitroglycerin-induced pain response in migraine mice through reducing endothelin-1 level and regulating fatty acid biosynthesis.

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.

IL-17 crosses the blood-brain barrier to trigger neuroinflammation: a novel mechanism in nitroglycerin-induced chronic migraine.

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.

MicroRNA-155-5p promotes neuroinflammation and central sensitization via inhibiting SIRT1 in a nitroglycerin-induced chronic migraine mouse model.

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.

Involvement of TRPM2 in the Neurobiology of Experimental Migraine: Focus on Oxidative Stress and Apoptosis.

Excessive Ca2+ 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 Ca2+ levels, and cytokine generations (TNF-α, IL-1ß, 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.