Migraine Model Research Articles

CaV3.2 T-type calcium channels contribute to CGRP- induced allodynia in a rodent model of experimental migraine

BackgroundMigraine is a painful neurological syndrome characterized by attacks of throbbing headache, of moderate to severe intensity, which is associated with photo- and phono- sensitivity as well as nausea and vomiting. It affects about 15% of the world’s population being 2–3 times more prevalent in females. The calcitonin gene-related peptide (CGRP) is a key mediator in the pathophysiology of migraine, and a significant advance in the field has been the development of anti-CGRP therapies. The trigeminal ganglion (TG) is thought to be an important site of action for these drugs. Moreover, experimental migraine can be induced by CGRP injection in the TG. The signaling pathway induced by CGRP in the TG is not fully understood, but studies suggest that voltage-gated calcium channels contribute to CGRP effects relevant to migraine.ObjectiveWe hypothesised that CGRP injection in the TG enhances CaV3.2 T-type calcium channel currents to contribute to the development of periorbital mechanical allodynia.ResultsA Co-Immunoprecipitation assay in tsA-201 cells revealed that CaV3.2 channels form a complex with RAMP-1, a component of the CGRP receptor. Constitutive CGRPR activity was able to inhibit CaV3.2 channels and induce a depolarizing shift in both activation and inactivation curves. Incubation of TG neurons with CGRP increased T-type current density by ~ 3.6 fold, an effect that was not observed in TG neurons from CaV3.2 knockout mice. Incubation of TG neurons with Z944, a pan T-type channel blocker, resulted in an approximately 80% inhibition of T-type currents. In vivo, this treatment abolished the development of periorbital mechanical allodynia induced by CGRP in male and female mice. Likewise, CaV3.2 knockout mice did not develop periorbital mechanical allodynia after intraganglionic CGRP injection. Finally, we demonstrated that the CGRP effect depends on the activation of its canonical GPCR, followed by protein kinase A activation.ConclusionThe present study suggests that CGRP modulates CaV3.2 in the TG, an effect possibly mediated by the canonical CGRP receptor and PKA activation. The increase in T-type currents in the TG may represent a contributing factor for the initiation and maintenance of the headache pain during migraine.Graphical abstract

Metformin attenuates central sensitization by regulating neuroinflammation through the TREM2-SYK signaling pathway in a mouse model of chronic migraine

BackgroundChronic migraine (CM) is a serious neurological disorder. Central sensitization is one of the important pathophysiological mechanisms underlying CM, and microglia-induced neuroinflammation conduces to central sensitization. Triggering receptor expressed on myeloid cells 2 (TREM2) is presented solely in microglia residing within the central nervous system and plays a key role in neuroinflammation. Metformin has been shown to regulate inflammatory responses and exert analgesic effects, but its relationship with CM remains unclear. In the study, we investigated whether metformin modulates TREM2 to improve central sensitization of CM and clarified the potential molecular mechanisms.MethodsA CM mouse model was induced by administration of nitroglycerin (NTG). Behavioral evaluations were conducted using von Frey filaments and hot plate experiments. Western blot and immunofluorescence techniques were employed to investigate the molecular mechanisms. Metformin and the SYK inhibitor R406 were administered to mice to assess their regulatory effects on neuroinflammation and central sensitization. To explore the role of TREM2-SYK in regulating neuroinflammation with metformin, a lentivirus encoding TREM2 was injected into the trigeminal nucleus caudalis (TNC). In vitro experiments were conducted to evaluate the regulation of TREM2-SYK by metformin, involving interventions with LPS, metformin, R406, siTREM2, and TREM2 plasmids.ResultsMetformin and R406 pretreatment can effectively improve hyperalgesia in CM mice. Both metformin and R406 significantly inhibit c-fos and CGRP expression in CM mice, effectively suppressing the activation of microglia and NLRP3 inflammasome induced by NTG. With the administration of NTG, TREM2 expression gradually increased in TNC microglia. Additionally, we observed that metformin significantly inhibits TREM2 and SYK expression in CM mice. Lv-TREM2 attenuated metformin-mediated anti-inflammatory responses. In vitro experiments, knockdown of TREM2 inhibited LPS-induced SYK pathway activation and alleviated inflammatory responses. After the sole overexpression of TREM2, the SYK signaling pathway is activated, resulting in the activation of the NLRP3 inflammasome and an increased expression of pro-inflammatory cytokines; nevertheless, this consequence can be reversed by R406. The overexpression of TREM2 attenuates the inhibition of SYK activity mediated by metformin, and this effect can be reversed by R406.ConclusionsOur findings suggest that metformin attenuates central sensitization in CM by regulating the activation of microglia and NLRP3 inflammasome through the TREM2-SYK pathway.Graphical

The neurotrophic factor artemin and its receptor GFRα3 mediate migraine-like pain via the ion channel TRPM8.

Migraine has a strong genetic foundation, including both monogenic and polygenic types. The former are rare, with most migraine considered polygenic, supported by genome-wide association studies (GWAS) identifying numerous genetic variants linked with migraine risk. Surprisingly, some of the most common mutations are associated with transient receptor potential melastatin 8 (TRPM8), a non-selective cation channel that is the primary sensor of cold temperatures in cutaneous primary afferents of the somatosensory system. However, it is unlikely that the temperature sensitivity of TRPM8 is relevant in migraine-related tissues, such as the meninges, suggesting other activation mechanisms underly its role in migraine pathogenesis. Thus, to define the basis of the channel's involvement, we reasoned that cellular processes that increase cold sensitivity in the skin, such as the neurotrophic factor artemin, via its receptor glial cell-line derived neurotrophic factor family receptor alpha-3 (GFRα3), also mediate TRPM8-associated migraine-like pain in the meninges. To investigate the role of artemin and GFRα3 in preclinical rodent migraine models, we infused nitroglycerin acutely and chronically, and measured changes in periorbital and hind paw mechanical sensitivity in male and female mice lacking GFRα3, after neutralization of free artemin with specific monoclonal antibodies, or by systemic treatment with a TRPM8-specific antagonist. Further, in mice lacking GFRα3 we tested the effects of supradural infusions of a mix of inflammatory mediators, as well as tested if dura stimulation with artemin or a TRPM8-specific agonist induce migraine-related pain in mice. We find that mechanical allodynia induced by systemic nitroglycerin, or supradural infusion of inflammatory mediators, involves GFRα3. In addition, neutralization of circulating artemin reduces the nitroglycerin phenotype, demonstrating the importance of this neurotrophic pathway in headaches. Further, we show TRPM8 expression in the meninges, and that direct supradural infusion of either a TRPM8-specific agonist or artemin itself produces mechanical allodynia, with the latter dependent on TRPM8 and ameliorated by concurrent treatment with sumatriptan. These results indicate that neuroinflammatory events in the meninges can produce migraine-like pain in mice via artemin and GFRα3, likely acting upstream of TRPM8, providing a novel pathway that may contribute to headaches or migraine pathogenesis.

Estrous cycle regulates cephalic mechanical sensitivity and sensitization of the trigemino-cervical complex in a female rat model of chronic migraine.

The higher incidence of migraines in women compared with men has led to the inclusion of female animals in pain research models. However, the critical role of the hormonal cycle is frequently overlooked, despite its clear correlation with migraine occurrences. In this study, we show in a rat model of migraine induced by repeated dural infusions of an inflammatory soup (IS) that a second IS (IS2) injection performed in proestrus/estrus (PE, high estrogen) female rats evokes higher cephalic mechanical hypersensitivities than when performed in metestrus/diestrus (MD, low estrogen) or ovariectomized (OV) rats. This hypersensitivity induced by IS2 correlates with increased c-Fos expression in outer lamina II (IIo) neurons located in the periorbital projection area of the trigemino-cervical complex (TCC), in PE only. Four IS (IS4) repetition induced an enlargement of c-Fos expression in adjacent territories areas in PE, but not MD or OV animals. Unexpectedly, c-Fos expression in locus coeruleus neurons does not potentiate after IS2 or IS4 injections. To examine the impacts of the hormonal cycle on the physiology of lamina IIo TCC neurons, we performed whole-cell patch-clamp recordings. Second inflammatory soup depolarizes neurons in PE and MD but not in OV rats and enhances excitatory synaptic inputs in PE animals to a greater extent compared with MD and OV rats. These findings show that central TCC sensitization triggered by meningeal nociceptor activation and the resulting cephalic hypersensitivity are modulated by the estrous cycle. This highlights the crucial need to account for not just sex, but also the female estrous cycle in pain research.

Chronic Hyperhomocysteinemia Impairs CSD Propagation and Induces Cortical Damage in a Rat Model of Migraine with Aura.

Hyperhomocysteinemia (hHCY) is a metabolic disorder characterized by elevated levels of homocysteine in plasma. hHCY correlates with a high risk of migraine headaches, especially migraine with aura. Cortical spreading depression (CSD) is a wave of depolarization passing through neurons and glial cells of the cortex and is considered an electrophysiological correlate of migraine aura. The aim of the present study was to analyze neuronal activity and CSD in the somatosensory cortex of rats in vivo with prenatal hHCY and to assess cortex viability after 2 h of CSD generation. Female rats were fed a diet high in methionine, and their offspring with high homocysteine levels in plasma were further used in experiments. Recurrent CSD was evoked by local KCl application on the dura surface. Neuronal viability was assessed by measuring the activity of lactate dehydrogenase (LDH) in the brain and 2,3,5-triphenyltetrazolium chloride staining of the somatosensory cortex after two hours of CSD generation. Animals with hHCY exhibited higher neuronal activity, and more CSDs were generated in response to KCl, indicating higher cortical excitability. Propagation of recurrent CSD was impaired in supragranular cortical layers, and the recovery of multiple unit activity and evoked sensory potentials after CSD was delayed in the hHCY group. Finally, in animals with prenatal hHCY, an ischemic focus was identified as a consequence of multiple CSDs, along with elevated levels of LDH activity in brain tissues, suggestive of diminished neuronal viability. These findings imply that prolonged elevated levels of homocysteine may not only predispose to migraine with aura but also potentially elevate the risk of migrainous infarction.

TRESK channel activation ameliorates migraine-like pain via modulation of CGRP release from the trigeminovascular system and meningeal mast cells in experimental migraine models

AimsAccumulating evidence indicates the involvement of TRESK potassium channels in migraine, however, effects of TRESK activation on migraine-related mechanisms remain unclear. We explored effects of TRESK channel modulation on migraine-related behavioral and molecular markers in in-vivo and ex-vivo rat models of migraine. Main methodsThe selective TRESK activator cloxyquin at different doses, the TRESK inhibitor A2764, and the migraine drug sumatriptan were tested alone or in different combinations in nitroglycerin (NTG)-induced in-vivo model, and in ex-vivo meningeal, trigeminal ganglion and brainstem preparations in which CGRP release was induced by capsaicin. Mechanical allodynia, CGRP and c-fos levels in trigeminovascular structures and meningeal mast cells were evaluated. Key findingsCloxyquin attenuated NTG-induced mechanical allodynia, brainstem c-fos and CGRP levels, trigeminal ganglion CGRP levels and meningeal mast cell degranulation and number, in-vivo. It also diminished capsaicin-induced CGRP release from ex-vivo meningeal, trigeminal ganglion and brainstem preparations. Specific TRESK inhibitor A2764 abolished all effects of cloxyquin in in-vivo and ex-vivo. Combining cloxyquin and sumatriptan exerted a synergistic effect ex-vivo, but not in-vivo. SignificanceOur findings provide the experimental evidence for the anti-migraine effect of TRESK activation in migraine-like conditions. The modulation of TRESK channels may therefore be an attractive alternative strategy to relieve migraine pain.

The neurotrophic factor artemin and its receptor GFRα3 mediate migraine-like pain via the ion channel TRPM8.

Migraine has a strong genetic foundation, including both monogenic and polygenic types. The former are rare, with most migraine considered polygenic, supported by genome-wide association studies (GWAS) identifying numerous genetic variants associated with migraine risk. Surprisingly, some of the most common mutations are associated with TRPM8, a non-selective cation channel that is the primary sensor of cold temperatures in primary afferent neurons of the somatosensory system. However, it is unlikely that the temperature sensitivity of TRPM8 underlies its role in migraine pathogenesis. To define the basis of the channel's involvement, we reasoned that cellular processes that increase cold sensitivity in the skin, such as the neurotrophic factor artemin, via its receptor GFRα3, also mediate TRPM8-associated migraine-like pain in the meninges. To investigate the role of artemin and GFRα3 in preclinical rodent migraine models, we infused nitroglycerin acutely and chronically, and measured changes in periorbital and hind paw mechanical sensitivity in male and female mice lacking GFRα3, after neutralization of free artemin with specific monoclonal antibodies, or by systemic treatment with a TRPM8-specific antagonist. Further, in wildtypes and mice lacking either GFRα3 or TRPM8, we tested the effects of supradural infusions of a mix of inflammatory mediators, artemin, and a TRPM8-specific agonist on migraine-related pain in mice. We find that mechanical allodynia induced by systemic nitroglycerin, or supradural infusion of inflammatory mediators, involves GFRα3. In addition, neutralization of circulating artemin reduces the nitroglycerin phenotype, demonstrating the importance of this neurotrophic pathway. Further, we show TRPM8 expression in the meninges and that direct supradural infusion of either a TRPM8-specific agonist or artemin itself produces mechanical allodynia, the latter dependent on TRPM8 and ameliorated by concurrent treatment with sumatriptan. These results indicate that neuroinflammatory events in the meninges can produce migraine-like pain in mice via artemin and GFRα3, likely acting upstream of TRPM8, providing a novel pathway that may contribute to migraine pathogenesis.

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

BackgroundChronic 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.MethodsChronic 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.ResultIn 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.ConclusionOur 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.

SIRT1 activation by Ligustrazine ameliorates migraine via the paracrine interaction of microglia and neurons

BackgroundNeuroinflammation with associated oxidative stress aggravates the pathogenesis and progression of migraine. Ligustrazine (LGZ) is a key component from traditional edible-medicinal herb Ligusticum chuanxiong Hort., and has the effects of anti-platelet aggregation, expanding small arteries, improving microcirculation and promoting blood circulation and removing blood stasis in clinic. Hypothesis/PurposeThis study aims to investigate the pharmacological effect and mechanism of LGZ in migraine. Study Design/MethodsA mouse model of migraine was induced by nitroglycerin (NTG), and LPS/IFN-γ stimulated microglial cell model was conducted to investigate neuroinflammation, the paracrine interactions between microglia and neurons were determined by the co-culture system, and the effect of LGZ on stability of SIRT1 protein was measured by cellular thermal shift assay (CETSA). Whilst, the SIRT1 inhibitor EX527 was used alone or co-treatment with LGZ in vitro or in vivo. ResultsLGZ significantly attenuated migraine-like behaviors in NTG-induced mice, and ameliorated neuroinflammation and related oxidative damage in brain tissue, but co-treatment with SIRT1 inhibitor EX527 abolished the protective effects of LGZ. Mechanistically, LGZ mitigated neuroinflammation by upregulating SIRT1 expression and subsequently inhibiting the activation of NF-κB pathway in microglia. CETSA indicated that LGZ significantly maintained the stability of SIRT1 protein in microglia. While, in the co-culture system, culture medium from LPS/IFN-γ-treated microglia exacerbated neuronal damage and oxidative stress, which was suppressed by treating LPS/IFN-γ-induced microglia with LGZ, this effect might be related to the activation of Nrf2 signals in neurons. Notably, SIRT1 inhibitor EX527 abrogated the effects of LGZ both in vitro and in vivo. ConclusionConsequently, SIRT1 might be an important pharmacological target of LGZ, which attenuates migraine associated neuroinflammation and oxidative stress by interfering the crosstalk between microglia and neurons, thereby relieving migraine.

Higher sodium in older individuals or after stroke/reperfusion, but not in migraine or Alzheimer’s disease – a study in different preclinical models

Sodium serves as one of the primary cations in the central nervous system, playing a crucial role in maintaining normal brain function. In this study, we investigated alterations in sodium concentrations in the brain and/or cerebrospinal fluid across multiple models, including an aging model, a stroke model, a nitroglycerin (NTG)-induced rat migraine model, a familial hemiplegic migraine type 2 (FHM2) mouse model, and a transgenic mouse model of Alzheimer’s disease (AD). Our results reveal that older rats exhibited higher sodium concentrations in cerebrospinal fluid (CSF), plasma, and various brain regions compared to their younger counterparts. Additionally, findings from the stroke model demonstrated a significant increase in sodium in the ischemic/reperfused region, accompanied by a decrease in potassium and an elevated sodium/potassium ratio. However, we did not detect significant changes in sodium in the NTG-induced rat migraine model or the FHM2 mouse model. Furthermore, AD transgenic mice showed no significant differences in sodium levels compared to wild-type mice in CSF, plasma, or the hippocampus. These results underscore the nuanced regulation of sodium homeostasis in various neurological conditions and aging, providing valuable insights into potential mechanisms underlying these alterations.

Clinical decision support system using hierarchical fuzzy diagnosis model for migraine and tension-type headache based on International Classification of Headache Disorders, 3rd edition.

To determine whether the diagnostic ability of the newly designed hierarchical fuzzy diagnosis method is consistent with that of headache experts for probable migraine (PM) and probable tension-type headache (PTTH). Clinical decision support systems (CDSS) are computer systems designed to help doctors to make clinician decisions by information technology, and have proven to be effective in improving headache diagnosis by making medical knowledge readily available to users in some studies. However, one serious drawback is that the CDSS lacks the ability to deal with some fuzzy boundaries of the headache features utilized in diagnostic criteria, which might be caused by patients' recall bias and subjective bias. A hybrid mechanism of rule-based reasoning and hierarchical fuzzy diagnosis method based on International Classification of Headache Disorders, 3rd edition (ICHD-3) was designed and then validated by a retrospective study with 325 consecutive patients and a prospective study with 380 patients who were clinically diagnosed with migraine and TTH at the headache clinic of Chinese PLA General Hospital. The results of the diagnostic test in the retrospective study indicated that the fuzzy-based CDSS can be used in the diagnosis of migraine without aura (MO) (sensitivity 97.71%, specificity 100%), TTH (sensitivity 98.57%, specificity 100%), PM (sensitivity 91.25%, specificity 98.75%) and PTTH (sensitivity 90.91%, specificity 99.63%). While in the prospective study, the diagnostic performances were MO (sensitivity 91.62%, specificity 96.52%), TTH (sensitivity 92.17%, specificity 95.47%), PM (sensitivity 85.48%, specificity 98.11%) and PTTH (sensitivity 87.50%, specificity 98.60%). Cohen's kappa values for the consistency test were 0.984 ± 0.018 (MO), 0.991 ± 0.018 (TTH), 0.916 ± 0.051 (PM), 0.932 ± 0.059 (PTTH) in the retrospective study and 0.884 ± 0.047 (MO), 0.870 ± 0.055 (TTH), 0.853 ± 0.073 (PM), 0.827 ± 0.118 (PTTH) in the prospective study, which indicated good consistency with the fuzzy-based CDSS and the gold standard (p < 0.001). We developed a fuzzy-based CDSS performs much more similarly to expert diagnosis and performs better than the routine CDSS method in the diagnosis of migraine and TTH, and it could promote the application of artificial intelligence in the area of headache diagnosis.

Transcriptomic changes in the hypothalamus of mice with chronic migraine: Activation of pathways associated with neuropathic inflammation and central sensitization

Chronic migraine is a common central nervous system disorder characterized by recurrent, pulsating headaches. However, the extent and mechanisms of hypothalamic involvement in disease progression have not been thoroughly investigated. Herein, we created a chronic migraine mouse model using repeated intraperitoneal injections of nitroglycerin. We performed transcriptomic sequencing on the hypothalamus of mice with chronic migraine and control mice under normal physiological conditions, followed by differential gene set enrichment and functional analysis of the data. Additionally, we examined the intrinsic connection between chronic migraine and sleep disorders using transcriptomic sequencing data from sleep-deprived mice available in public databases. We identified 39 differentially expressed genes (DEGs) in the hypothalamus of a mouse model of chronic migraine. Functional analysis of DEGs revealed enrichment primarily in signaling transduction, immune-inflammatory responses, and the cellular microenvironment. A comparison of the transcriptomic data of sleep-deprived mice revealed two commonly expressed DEGs. Our findings indicate that the hypothalamic DEGs are primarily enriched in the PI3K/AKT/mTOR pathway and associated with the NF-κB/NLRP3/IL-1 β pathway activation to maintain the central sensitization of the chronic migraine. Chronic migraine-induced gene expression changes in the hypothalamus may help better understand the underlying mechanisms and identify therapeutic targets.

Glial activation in pain and emotional processing regions in the nitroglycerin mouse model of chronic migraine.

Our aim was to survey astrocyte and microglial activation across four brain regions in a mouse model of chronic migraine. Chronic migraine is a leading cause of disability, with higher rates in females. The role of central nervous system neurons and glia in migraine pathophysiology is not fully elucidated. Preclinical studies have shown abnormal glial activation in the trigeminal nucleus caudalis of male rodents. No current reports have investigated glial activation in both sexes in other important brain regions involved with the nociceptive and emotional processing of pain. The mouse nitroglycerin model of migraine was used, and nitroglycerin (10 mg/kg) or vehicle was administered every other day for 9 days. Prior to injections on days 1, 5, and 9, cephalic allodynia was determined by periorbital von Frey hair testing. Immunofluorescent staining of astrocyte marker, glial fibrillary protein (GFAP), and microglial marker, ionized calcium binding adaptor molecule 1 (Iba1), in male and female trigeminal nucleus caudalis, periaqueductal gray, somatosensory cortex, and nucleus accumbens was completed. Behavioral testing demonstrated increased cephalic allodynia in nitroglycerin- versus vehicle-treated mice. An increase in the percent area covered by GFAP+ cells in the trigeminal nucleus caudalis and nucleus accumbens, but not the periaqueductal gray or somatosensory cortex, was observed in response to nitroglycerin. No significant differences were observed for Iba1 staining across brain regions. We did not detect significant sex differences in GFAP or Iba1 quantification. Immunohistochemical analysis suggests that, at the time point tested, immunoreactivity of GFAP+ astrocytes, but not Iba1+ microglia, changes in response to chronic migraine-associated pain. Additionally, there do not appear to be significant differences between males and females in GFAP+ or Iba1+ cells across the four brain regions analyzed.

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

BackgroundMigraine 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.MethodsBy 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.ResultsIn 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).ConclusionsfMOST 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.Graphical

Exploring the synergistic effects of chuanxiong rhizoma and Cyperi rhizoma in eliciting a rapid anti-migraine action based on pharmacodynamics and pharmacokinetics

Ethnopharmacological relevanceHerb-herb combination has been used to maximize the therapeutic efficacy in the theory of traditional Chinese medicine. Chuanxiong rhizoma (called Chuanxiong in Chinese, CX) and Cyperi rhizoma (called Xiangfu in Chinese, XF) have been used alone or in combination (CRCR) to treat migraine dating back to Eastern Jin Dynasty (AD317) of China. But no data demonstrate the possible necessities or advantages of combining CX and XF for migraine. Aim of the studyThis study explores the combination mechanism based on pharmacodynamics and pharmacokinetics. Materials and methodsA nitroglycerin-induced acute migraine model in rats was used to evaluate the anti-migraine effects of CRCR and the individual herbs using behavior, real time polymerase chain reaction and Western blot experiments. The absorption characteristics of active components involved in the anti-migraine action were analyzed by UPLC-MS/MS. ResultsCX and CRCR significantly reversed the abnormal levels of vasoactive substances (5-HT, CGRP, MMP-2 and MMP-9) to normal levels, but XF did not. XF and CRCR significantly decreased the pro-inflammatory cytokines (IL-1β, IL-6, and TNF-a), and increased the anti-inflammatory cytokines (IL-4 and IL-10). CRCR significantly decreased the mRNA expression levels of c-fos, iNos and nNos, and the corresponding protein expression levels of c-Fos, iNOS, and nNOS. CRCR inhibited NOS/NO pathway by downregulating the expression levels of NOS and NO. Furthermore, CRCR significantly increased the intestinal absorption rate and amount, and changed the pharmacokinetic parameters of active components in comparison with the individual herbs. ConclusionsCX had an advantage in regulating vasoactive substances, and XF focused on regulating inflammatory cytokines. CRCR is more effective in treating migraine than the individual herbs by depending on the synergistic action of CX and XF. This research provided some critical evidences on synergistic action between herb-herb interactions, and revealed the potential advantages of herb-herb combination in traditional Chinese medicine.

Pharmacological investigation of genistein for its therapeutic potential against nitroglycerin-induced migraine headache.

Migraine, typically occurs on one side of the head, lasts for hours to days. Trigemino-vascular system (TVS) plays a vital role in pain generation, with neurogenic inflammation and oxidative stress playing key roles in its pathophysiology. This study aimed to investigate genistein's potential as anti-inflammatory and anti-oxidant agent in mitigating migraine pain. Genistein (20 and 50mg/kg) was administered intraperitoneally (IP) to nitroglycerin (NTG; 10mg/kg)-induced migraine model in rats. Behavioral analysis, antioxidant assay, immunohistochemistry (IHC), histopathological examination, ELISA, and RT-PCR were conducted to evaluate the antimigraine potential of genistein. In-silico analysis showed genestien's ACE values of -4.8 to -9.2 Kcal/mol against selected protein targets. Genistein significantly reversed mechanical and thermal nociception, light phobicity, and head scratching; increased the intensities of GST, GSH, catalase; and down regulated lipid peroxidase (LPO) in cortex and trigeminal nucleus caudalis (TNC). It also reduced Nrf2, NF-kB, and IL6 expression, analyzed through IHC, improved histopathological features, and increased COX-2 and decreased PPAR-γ expressions, while RT-PCR analysis revealed increased PPAR-γ expressions in genistein-treated rats. Genistein exhibited potent antioxidant and anti-inflammatory properties in migraine treatment, acting through multifactorial mechanisms by modulating the expression of numerous proteins in the region cortex and TNC.

Trigeminal ganglion neurons are directly activated by influx of CSF solutes in a migraine model.

Classical migraine patients experience aura, which is transient neurological deficits associated with cortical spreading depression (CSD), preceding headache attacks. It is not currently understood how a pathological event in cortex can affect peripheral sensory neurons. In this study, we show that cerebrospinal fluid (CSF) flows into the trigeminal ganglion, establishing nonsynaptic signaling between brain and trigeminal cells. After CSD, ~11% of the CSF proteome is altered, with up-regulation of proteins that directly activate receptors in the trigeminal ganglion. CSF collected from animals exposed to CSD activates trigeminal neurons in naïve mice in part by CSF-borne calcitonin gene-related peptide (CGRP). We identify a communication pathway between the central and peripheral nervous system that might explain the relationship between migrainous aura and headache.

Animal Models of Chronic Migraine: From the Bench to Therapy.

Chronic migraine is a disabling progressive disorder without effective management approaches. Animal models have been developed and used in chronic migraine research. However, there are several problems with existing models. Therefore, we aimed to summarize and analyze existing animal models to facilitate translation from basic to clinical. The most commonly used models are the inflammatory soup induction model and the nitric oxide donor induction model. In addition, KATP openers have also been used in model induction. Based on the above models, some molecular targets have been identified, such as glutamate receptors. However, each model has its shortcomings and characteristics, and there are still some common problems that need to be solved, such as spontaneous headache, evaluation criteria after model establishment, and identification methods. In this review, we summarized and highlighted the advantages and limitations of the currently commonly used animal models of chronic migraine with a special focus on drug discovery and current therapeutic strategies, and discussed the directions that can be worked on in the future.

4D-DIA proteomics reveals mechanism of Sanpian Decoction in treating chronic migraine in rats

To explore the mechanism of the classic formula Sanpian Decoction in treating chronic migraine, this study employed the four-dimensional data-dependent acquisition(4D-DIA) proteomics to analyze the effect of the decoction on chronic migraine in rats and experimentally verified the key differentially expressed proteins. Firstly, SD male rats were randomly divided into groups and repeatedly injected with nitroglycerin to prepare a chronic migraine model. After 7 consecutive days of gavage, rat grimace scale(RGS) was employed to evaluate the treatment efficacy. The trigeminal ganglion was collected for 4D-DIA proteomics, on the basis of which the diffe-rentially expressed proteins between groups were screened. Multiple databases were used for the Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment of the differentially expressed proteins. STRING and Cytoscape were employed to establish the protein-protein interaction(PPI) network. Western blot was employed to determine the expression level of the key diffe-rentially expressed protein TRPV1. The results showed that there were 517 differentially expressed proteins between blank group and model group and 221 differentially expressed proteins between model group and medium-dose Sanpian Decoction group. The GO and KEGG enrichment results showed that these differentially expressed proteins were mainly related to inflammatory response, injurious sensory stimulation, triglyceride metabolism, immune regulation, etc., which mainly involved the inflammation-related TRP, AMPK, PI3K-Akt, and TGF-β signaling pathways. The PPI network showed that the target proteins such as IGF, TOP2A, APOA1, CDK1, TTN, RYR1, and CSRP3 had high degrees. Compared with that in model group, the expression level of TRPV1 altered in medium-and high-dose Sanpian Decoction group(P&lt;0.05). In conclusion, Sanpian Decoction may treat chronic migraine by regulating the inflammation-related pathways such as TRP, AMPK, and PI3K-Akt. It plays an important role in the regulation of TRPV1 protein and potentially modulates the perception of injurious stimuli, lipid metabolism, and immune responses.

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 relevancePaeonol (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 studyThis research aimed to determine the mechanism of PAE + GLY in ameliorating the recurrent nitroglycerin-induced migraine-like phenotype in rats. Materials and methodsUsing 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. ResultsFollowing 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. ConclusionsOur findings underscore that PAE + GLY ameliorates inflammatory hyperalgesia migraine by upregulating inhibitory neurotransmitters and modulating the GABBR2/TRPM8/PRKACA/TRPV1 pathway.