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.

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.

Hypothalamic functional MRI activity in the initiation phase of spontaneous and glyceryl trinitrate-induced migraine attacks.

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.

P2X7R-mediated autophagic impairment contributes to central sensitization in a chronic migraine model with recurrent nitroglycerin stimulation in mice.

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.

Therapeutic targeting of nitroglycerin-mediated trigeminovascular neuronal hypersensitivity predicts clinical outcomes of migraine abortives.

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.

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

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

Anti-migraine effect of wine-processed Radix scutellariae: Pharmacodynamic verification in nitroglycerin-induced rats and correlation study between compounds dissolution and the fractal dimension.

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.

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

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

No central action of CGRP antagonising drugs in the GTN mouse model of migraine.

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.

Dimethyl fumarate alleviates the nitroglycerin (NTG)-induced migraine in mice.

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.