Rat Model Of Migraine Research Articles

Shaoyao Gancao decoction alleviates the central hyperalgesia of recurrent NTG-induced migraine in rats by regulating the NGF/TRPV1/COX-2 signal pathway

Ethnopharmacological relevanceShaoyao Gancao Decoction (SGD) is well known as an effective prescription for analgesia composed of two herbs, and is noted as traditional Chinese medicine morphine. It is widely used in various conditions causing pain, including migraine. However, there is currently no research exploring the mechanism of action in the treatment of migraines. Aim of the studyThe current research was devised to determine the underlying regulatory mechanism of SGD, by verifying its role in the NGF/TRPV1/COX-2 signal pathway. Materials and methodsThe active components in SGD were identified by UHPLC-MS. A migraine model was prepared by subcutaneous (s.c.) injection of nitroglycerin (NTG) into the neck to detect migraine-like behavior, orbital hyperalgesia threshold changes, and the therapeutic effect of SGD. The mechanism of SGD in remedying migraine was studied through transcriptome sequencing (RNA-seq), which was further validated utilizing Elisa, Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting (WB) experiments. ResultsIn the SGD chemical composition analysis, 45 components were identified including gallic acid, paeoniflorin and albiforin. In the behavioral experiments, SGD treatment significantly decreased the score of migraine-like head scratching in the NTG-induced migraine model (Mod) rats, while the hyperalgesia threshold increased outstandingly on days 10, 12, and 14 (P < 0.01, P < 0.001 or P < 0.0001). In migraine biomarkers experiment, compared with the Mod group, the 5-hydroxytryptamine (5-HT) contents were outstandingly enhanced by SGD treatment, while nitric oxide (NO) contents were markedly declined (P < 0.01). In the RNA-seq test, the down-regulated genes of SGD inhibiting hyperalgesia migraine included the neurotrophic factor (NGF) and transient receptor potential vanillic acid subfamily protein 1 receptor (TRPV1). The down-regulation pathway is the inflammatory mediator regulation of TRP channels. In gene set enrichment analysis (GSEA), SGD decreased the over-expression of protooncogene tyrosine-protein kinase Src (SRC) and TRPV1 in this pathway, and the two genes clustered at its lower end, with similar functions. PPI network results show that NGF interacts with TRPV1. Further verification shows that when compared with Mod group, the plasma cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) protein expression levels and the dura mater calcitonin gene-related peptide (CGRP), extracellular signal-regulated kinase (ERK), p-ERK, SRC and NGF protein expression levels in the SGD group were remarkably decreased (P < 0.01, P < 0.001 or P < 0.0001), and the expression level of TRPV1 protein showed a downward trend (P = 0.06). The expression levels of COX-2, NO, CGRP, TRPV1, SRC and NGF mRNA in the dura mater was overtly down-regulated (P < 0.05, P < 0.01 or P < 0.001). ConclusionsSGD has a significant inhibitory effect on the NGF/TRPV1/COX-2 signaling pathway that mediates central hyperalgesia migraine, thus suggesting the molecular mechanism of SGD in improving the symptoms of migraine may be related to the central hyperalgesia neurotransmitter that regulates the pathogenesis of migraine.

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

AimsChronic 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.MethodsSprague–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.ResultsThe 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.ConclusionsOur 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.

Dysfunction of inhibitory interneurons contributes to synaptic plasticity via GABABR-pNR2B signaling in a chronic migraine rat model.

According to our previous study, the loss of inhibitory interneuron function contributes to central sensitization in chronic migraine (CM). Synaptic plasticity is a vital basis for the occurrence of central sensitization. However, whether the decline in interneuron-mediated inhibition promotes central sensitization by regulating synaptic plasticity in CM remains unclear. Therefore, this study aims to explore the role of interneuron-mediated inhibition in the development of synaptic plasticity in CM. A CM model was established in rats by repeated dural infusion of inflammatory soup (IS) for 7 days, and the function of inhibitory interneurons was then evaluated. After intraventricular injection of baclofen [a gamma-aminobutyric acid type B receptor (GABABR) agonist] or H89 [a protein kinase A (PKA) inhibitor), behavioral tests were performed. The changes in synaptic plasticity were investigated by determining the levels of the synapse-associated proteins postsynaptic density protein 95 (PSD95), synaptophysin (Syp) and synaptophysin-1(Syt-1)]; evaluating the synaptic ultrastructure by transmission electron microscopy (TEM); and determining the density of synaptic spines via Golgi-Cox staining. Central sensitization was evaluated by measuring calcitonin gene-related peptide (CGRP), brain-derived neurotrophic factor (BDNF), c-Fos and substance P (SP) levels. Finally, the PKA/Fyn kinase (Fyn)/tyrosine-phosphorylated NR2B (pNR2B) pathway and downstream calcium-calmodulin-dependent kinase II (CaMKII)/c-AMP-responsive element binding protein (pCREB) signaling were assessed. We observed dysfunction of inhibitory interneurons, and found that activation of GABABR ameliorated CM-induced hyperalgesia, repressed the CM-evoked elevation of synapse-associated protein levels and enhancement of synaptic transmission, alleviated the CM-triggered increases in the levels of central sensitization-related proteins, and inhibited CaMKII/pCREB signaling via the PKA/Fyn/pNR2B pathway. The inhibition of PKA suppressed the CM-induced activation of Fyn/pNR2B signaling. These data reveal that the dysfunction of inhibitory interneurons contributes to central sensitization by regulating synaptic plasticity through the GABABR/PKA/Fyn/pNR2B pathway in the periaqueductal gray (PAG) of CM rats. Blockade of GABABR-pNR2B signaling might have a positive influence on the effects of CM therapy by modulating synaptic plasticity in central sensitization.

ABHD6 and MAGL control 2-AG levels in the PAG and allodynia in a CSD-induced periorbital model of headache.

The high prevalence and severe symptoms of migraines in humans emphasizes the need to identify underlying mechanisms that can be targeted for therapeutic benefit. Clinical Endocannabinoid Deficiency (CED) posits that reduced endocannabinoid tone may contribute to migraine development and other neuropathic pain conditions. While strategies that increase levels of the endocannabinoid n-arachidonoylethanolamide have been tested, few studies have investigated targeting the levels of the more abundant endocannabinoid, 2-arachidonoylgycerol, as an effective migraine intervention. Cortical spreading depression was induced in female Sprague Dawley rats via KCl (potassium chloride) administration, followed by measures of endocannabinoid levels, enzyme activity, and neuroinflammatory markers. Efficacy of inhibiting 2-arachidonoylglycerol hydrolysis to mitigate periorbital allodynia was then tested using reversal and prevention paradigms. We discovered reduced 2-arachidonoylglycerol levels in the periaqueductal grey associated with increased hydrolysis following headache induction. Pharmacological inhibition of the 2-arachidonoylglycerol hydrolyzing enzymes, α/β-hydrolase domain-containing 6 and monoacylglycerol lipase reversed and prevented induced periorbital allodynia in a cannabinoid receptor-dependent manner. Our study unravels a mechanistic link between 2-arachidonoylglycerol hydrolysis activity in the periaqueductal grey in a preclinical, rat model of migraine. Thus, 2-arachidonoylglycerol hydrolysis inhibitors represent a potential new therapeutic avenue for the treatment of headache.

Activation of CB1R alleviates central sensitization by regulating HCN2-pNR2B signaling in a chronic migraine rat model

BackgroundCentral sensitization has been widely accepted as an underlying pathophysiological mechanism of chronic migraine (CM), activation of cannabinoid type-1 receptor (CB1R) exerts antinociceptive effects by relieving central sensitization in many pain models. However, the role of CB1R in the central sensitization of CM is still unclear.MethodsA CM model was established by infusing inflammatory soup (IS) into the dura of male Wistar rats for 7 days, and hyperalgesia was assessed by the mechanical and thermal thresholds. In the periaqueductal gray (PAG), the mRNA and protein levels of CB1R and hyperpolarization-activated cyclic nucleotide-gated cation channel 2 (HCN2) were measured by qRT–PCR and western blotting. After intraventricular injection of Noladin ether (NE) (a CB1R agonist), ZD 7288 (an HCN2 blocker), and AM 251 (a CB1R antagonist), the expression of tyrosine phosphorylation of N-methyl-D-aspartate receptor subtype 2B (pNR2B), calcium-calmodulin-dependent kinase II (CaMKII), and phosphorylated cAMP-responsive element binding protein (pCREB) was detected, and central sensitization was evaluated by the expression of calcitonin gene-related peptide (CGRP), c-Fos, and substance P (SP). Synaptic-associated protein (postsynaptic density protein 95 (PSD95) and synaptophysin (Syp)) and synaptic ultrastructure were detected to explore synaptic plasticity in central sensitization.ResultsWe observed that the mRNA and protein levels of CB1R and HCN2 were both significantly increased in the PAG of CM rats. The application of NE or ZD 7288 ameliorated IS-induced hyperalgesia; repressed the pNR2B/CaMKII/pCREB pathway; reduced CGRP, c-Fos, SP, PSD95, and Syp expression; and inhibited synaptic transmission. Strikingly, the application of ZD 7288 relieved AM 251-evoked elevation of pNR2B, CGRP, and c-Fos expression.ConclusionsThese data reveal that activation of CB1R alleviates central sensitization by regulating HCN2-pNR2B signaling in CM rats. The activation of CB1R might have a positive influence on the prevention of CM by mitigating central sensitization.

2-Deoxyglucose alleviates migraine-related behaviors by modulating microglial inflammatory factors in experimental model of migraine.

Targeting metabolic pathways has emerged as a new migraine treatment strategy as researchers realize the critical role metabolism plays in migraine. Activated inflammatory cells undergo metabolic reprogramming and rely on glycolysis to function. The objective of this study was to investigate the glycolysis changes in the experimental model of migraine and the effect of glycolysis inhibitor 2-Deoxy-D-glucose (2-DG) in the pathophysiology of migraine. We used a rat model of migraine that triggered migraine attacks by applying inflammatory soup (IS) to the dura and examined changes in glycolysis. 2-DG was used to inhibit glycolysis, and the effects of 2-DG on mechanical ectopic pain, microglial cell activation, calcitonin gene-related peptides (CGRP), c-Fos, and inflammatory factors induced by inflammatory soup were observed. LPS stimulated BV2 cells to establish a model in vitro to observe the effects of 2-DG on brain-derived neurotrophic factor (BDNF) after microglia activation. In the experimental model of migraine, key enzymes involved in glycolysis such as phosphofructokinase platelet (PFKP), hexokinase (HK2), hypoxia inducible factor-1α (HIF-1α), lactate dehydrogenase (LDH) and pyruvate kinase (PKM2) were expressed in the medullary dorsal horn. While the expression of electronic respiratory transport chain complex IV (COXIV) decreased. There were no significant changes in glucose 6-phosphate dehydrogenase (G6PD), a key enzyme in the pentose phosphate pathway. The glycolysis inhibitor 2-DG alleviated migraine-like symptoms in an experimental model of migraine, reduced the release of proinflammatory cytokines caused by microglia activation, and decreased the expression of CGRP and c-Fos. Further experiments in vitro demonstrated that glycolysis inhibition can reduce the release of Iba-1/proBDNF/BDNF and inhibit the activation of microglia. The migraine rat model showed enhanced glycolysis. This study suggests that glycolytic inhibitor 2-DG is an effective strategy for alleviating migraine-like symptoms. Glycolysis inhibition may be a new target for migraine treatment.

Electroacupuncture at Fengchi(GB20) and Yanglingquan(GB34) Ameliorates Paralgesia through Microglia-Mediated Neuroinflammation in a Rat Model of Migraine

Multiple studies have suggested that paralgesia (hyperalgesia and cutaneous allodynia) in migraine reflects the activation and sensitisation of the trigeminovascular system (TGVS). In particular, it reflects the second-order and higher nerve centre sensitisation, which is caused and maintained by neuroinflammation. Microglia activation leads to the release of proinflammatory cytokines involved in inflammatory responses. Accumulating evidence indicates that electroacupuncture (EA) is effective in ameliorating paralgesia, but the underlying mechanisms of EA in migraine attacks caused by microglia and microglia-mediated inflammatory responses are still unclear. The purpose of this study was to explore whether EA could ameliorate the dysregulation of pain sensation by suppressing microglial activation and the resulting neuroinflammatory response, and to evaluate whether this response was regulated by Toll-like receptor 4 (TLR4)/nuclear factor-kappa B(NF-κB) in the trigeminal nucleus caudalis (TNC) in a rat model of migraine. Repeated Inflammatory Soup (IS) was infused into the dura for seven sessions to establish a recurrent migraine-like rat model, and EA treatment was administered at Fengchi (GB20) and Yanglingquan (GB34) after daily IS infusion. Facial mechanical withdrawal thresholds were measured to evaluate the change in pain perception, and plasma samples and the TNC tissues of rats were collected to examine the changes in calcitonin gene-related peptide (CGRP), the Ibal-1-labelled microglial activation, and the resulting inflammatory response, including interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and their regulatory molecules TLR4/NF-κB, via enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) and Western blot analysis. Repeated IS injections into the dura induced facial mechanical paralgesia, which is the manifestation of migraine attacks, and increased the expression of CGRP, Ibal-1, microglial mediated inflammatory cytokines (IL-1β, TNF-α, IL-6), and regulatory molecules TLR4/NF-κB. EA at GB20/34 significantly attenuated repetitive IS-induced pain hypersensitivity. This effect was consistent with decreased levels of CGRP and inflammatory cytokines in the plasma and the TNC via the inhibition of microglia activation, and this response may be regulated by TLR4/NF-κB. EA ameliorated paralgesia in repetitive IS-induced migraine-like rats, which was mainly mediated by a reduction in microglial activation and microglial-mediated inflammatory responses that could be regulated by TLR4/NF-κB.

The plasma fibrinogen levels in the nitroglycerin-induced chronic migraine rat model and its association between migraine-associated vestibular dysfunction.

The purpose of this study was to measure the vestibular function and plasma fibrinogen level in the nitroglycerin (NTG)-induced chronic migraine rats, and explore the effect of defibrinogenation on migraine and associated vestibular dysfunction. The chronic migraine rat model was built by recurrent NTG injection. Batroxobin was administrated as a defibrinogenating drug. We measured the mechanical withdrawal threshold, vestibular function, and fibrinogen level of the rats 30 min before and 2 h following the model establishment, as well as 1 h after batroxobin administration. The results showed that vestibular function was impaired in NTG-induced chronic migraine rats. The fibrinogen levels were increased following repeated NTG injections. However, defibrinogenation did not affect either aggravating or alleviating mechanical hyperalgesia or vestibular dysfunction in the migraine model rats. These findings suggest that the NTG-induced chronic migraine rat model can be used for research on migraine-associated vestibular symptoms. Albeit the association between elevated fibrinogen levels and migraine attacks can be observed, the role of excessive fibrinogen in the pathogenesis of chronic migraine is yet to be determined.

Inhibiting PAC1 receptor internalization and endosomal ERK pathway activation may ameliorate hyperalgesia in a chronic migraine rat model.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multipotent neuropeptide widely distributed in the trigeminovascular system (TVS) and higher brain regions. At present, the underlying mechanism of PACAP/PACAP type1 (PAC1) receptor in migraine generation remains unclear. The rat model of chronic migraine (CM) was established by repeated intraperitoneal injection of nitroglycerin (NTG). Von Frey filaments and hot plate tests were used to measure the mechanical and thermal thresholds. The expression levels of c-Fos, calcitonin gene-related peptide (CGRP), PACAP, PAC1, protein kinase A (PKA) and phosphorylated extracellular signal-regulated kinase (ERK) were assessed by western blotting or immunofluorescence staining. The internalization of PAC1 receptor was visualized by fluorescence microscope and laser scanning confocal microscope. The results showed that c-Fos and CGRP expression significantly increased after repeated administrations of NTG or PACAP. Pitstop2 notably improved hyperalgesia in CM rats, while PACAP6-38 offered no benefit. In addition, PACAP-induced PAC1 receptor internalization, PKA and ERK pathways activation were blocked by Pitstop2 instead of PACAP6-38. Our results demonstrate that inhibition of PAC1 receptor internalization could effectively improve allodynia in CM rats by restraining ERK signaling pathway activation in a chronic migraine rat model. Modulation of receptor internalization may be a novel perspective to explore specific mechanisms of PACAP signaling activation in the trigeminal vascular system.

Hangover headache and its behavioral changes in rats.

The present study aims to establish and evaluate a rat model for hangover headaches caused by alcoholic drinks. Chronic migraine (CM) model rats were divided into 3 groups, and intragastrically administered alcoholic drinks (sample A, B, or C) to simulate hangover headache attacks. The withdrawal threshold for the hind paw/face and the thermal latency of hind paw withdrawal were detected after 24 hr. Serum was collected from the periorbital venous plexus of rats in each group, and enzymatic immunoassays were used to determine the serum levels of calcitonin gene-related peptide (CGRP), substance P (SP), and nitric oxide (NO). Compared with the control group, the mechanical hind paw pain threshold was significantly lower in rats administered Samples A and B after 24 hr; however, no significant difference was observed across groups for the thermal pain threshold. The mechanical threshold for periorbital pain was only significantly reduced in rats administered Sample A. Immunoassays further indicated that serum levels of SP in the group administered Sample A were significantly higher than those in the control group; the serum levels of NO and CGRP were significantly higher in the group of rats receiving Sample B. We successfully developed an effective and safe rat model for investigating alcohol drink induced hangover headaches. This model could be used to investigate the mechanisms associated with hangover headaches for the development of novel and promising candidates for the future treatment or prophylaxis of hangover headaches.

The Microbiota-Dependent Treatment of Wuzhuyu Decoction for Chronic Migraine Model Rat Associated with Anxiety-Depression Like Behavior.

We previously found that Wuzhuyu Decoction (WZYD) could affect central and peripheral 5-HT to relieve hyperalgesia in chronic migraine (CM) model rats, possibly related to gut microbiota. However, the exact role of gut microbiota has not been elucidated. Accumulating evidence points to the possibility of treating central nervous system disease via the gut-brain axis. In our study, the inflammatory soup-induced CM model rats presented depression- and anxiety-like behaviors which both related to insufficient 5-HT. It was found that antibiotic administration caused community dysbiosis, and proteobacteria became the main dominant bacteria. The bacteria related to short-chain fatty acids and 5-HT generation were reduced, resulting in reduced levels of 5-HT, tryptophan hydroxylase, and secondary bile acids. Functional prediction-revealed sphingolipid signaling pathway in CM rats was significantly decreased and elevated after WZYD treatment. The effect of WZYD could be weakened by antibiotics. The CM rats exhibited anxiety- and depression-like behavior with 5-HT and number of neurons decreased in the CA1 and CA2 regions of hippocampal. The treatment of WZYD could recover to varying degrees. Antibiotics combined with WZYD attenuate the effect of WZYD on increasing the 5-HT content and related protein expression in the brain stem, plasma and colon, reducing CGRP, c-Fos, and inflammatory factors. And antibiotics also led to colon length increasing and stool retention, so that the antimigraine effect was weakened compared with WZYD. This experiment revealed that gut microbiota mediated WZYD treatment of CM rats with anxiety-depression like behavior.

Effects of electroacupuncture on ethology, microglia activation and P2X7 receptor expression in periaqueductal gray in rats with migraine

To observe the effects of electroacupuncture(EA) at "Fengchi"(GB20) on the ethology, microglia activation and P2X7 receptor(P2X7R) expression in the periaqueductal gray(PAG) in recurrent migraine rat model, so as to explore the underlying mechanism of EA reducing central sensitization of migraine. Thirty-six male SD rats were randomly divided into control, model and EA groups, with 12 rats in each group. Recurrent migraine model was induced using repea-ted dural electrical stimulation once another day(the 1st, 3rd, 5th, 7th and 9th days), for a total of 5 times; rats in the EA group received EA treatment(2 Hz/15 Hz, 0.8-1 mA) at GB20 after dural electrical stimulation, for 10 min every time, once a day for 9 days; rats in the control group only received electrode placement. The facial and hindpaw mechanical withdrawal threshold was detected by using an electronic von-Frey on the 0th(baseline), 2nd, 4th, 6th, and 8th days. Microglia activation in the PAG was evaluated by using immunofluorescence staining to detect the number of ionized calcium binding adaptor molecule-1(Iba-1)-labeled microglia. Expression levels of microglia marker Iba-1, inflammatory factor interleukin(IL)-1β and P2X7R were detected by Western blot. Compared with the control group, the facial and hindpaw mechanical withdrawal threshold of rats were significantly reduced on the 2nd, 4th, 6th, and 8th days(P<0.01，P<0.001); the microglia in the PAG area were significantly activated, with the number of Iba-1-positive microglia, and the expression levels of Iba-1, IL-1β and P2X7R proteins significant increased(P<0.001, P<0.05) in the model group. Compared with the model group, the facial and hindpaw mechanical withdrawal threshold of rats were significantly increased on the 4th, 6th, and 8th days(P<0.05，P<0.001，P<0.01), and the above indicators were significantly reversed (P<0.05) in the EA group. EA at GB20 can significantly improve facial and hindpaw mechanical withdrawal threshold of migraine rats, and its possible mechanism may be related to inhibiting microglia activation mediated by P2X7R in the PAG.

Estradiol Treatment Enhances Behavioral and Molecular Changes Induced by Repetitive Trigeminal Activation in a Rat Model of Migraine.

A migraine is a neurological condition that can cause multiple symptoms. It is up to three times more common in women than men, thus, estrogen may play an important role in the appearance attacks. Its exact pathomechanism is still unknown; however, the activation and sensitization of the trigeminal system play an essential role. We aimed to use an animal model, which would better illustrate the process of repeated episodic migraine attacks to reveal possible new mechanisms of trigeminal pain chronification. Twenty male (M) and forty ovariectomized (OVX) female adult rats were used for our experiment. Male rats were divided into two groups (M + SIF, M + IS), while female rats were divided into four groups (OVX + SIF, OVX + IS, OVX + E2 + SIF, OVX + E2 + IS); half of the female rats received capsules filled with cholesterol (OVX + SIF, OVX + IS), while the other half received a 1:1 mixture of cholesterol and 17β-estradiol (OVX + E2 + SIF, OVX + E2 + IS). The animals received synthetic interstitial fluid (SIF) (M + SIF, OVX + SIF, OVX + E2 + SIF) or inflammatory soup (IS) (M + IS, OVX + IS, OVX + E2 + IS) treatment on the dural surface through a cannula for three consecutive days each week (12 times in total). Behavior tests and immunostainings were performed. After IS application, a significant decrease was observed in the pain threshold in the M + IS (0.001 &lt; p &lt; 0.5), OVX + IS (0.01 &lt; p &lt; 0.05), and OVX + E2 + IS (0.001 &lt; p &lt; 0.05) groups compared to the control groups (M + SIF; OVX + SIF, OVX + E2 + SIF). The locomotor activity of the rats was lower in the IS treated groups (M + IS, 0.01 &lt; p &lt; 0.05; OVX + IS, p &lt; 0.05; OVX + E2 + IS, 0.001 &lt; p &lt; 0.05), and these animals spent more time in the dark room (M + IS, p &lt; 0.05; OVX + IS, 0.01 &lt; p &lt; 0.05; OVX + E2 + IS, 0.001 &lt; p &lt; 0.01). We found a significant difference between M + IS and OVX + E2 + IS groups (p &lt; 0.05) in the behavior tests. Furthermore, IS increased the area covered by calcitonin gene-related peptide (CGRP) immunoreactive (IR) fibers (M + IS, p &lt; 0.01; OVX + IS, p &lt; 0.01; OVX + E2 + IS, p &lt; 0.001) and the number of neuronal nitric oxide synthase (nNOS) IR cells (M + IS, 0.001&lt; p &lt; 0.05; OVX + IS, 0.01 &lt; p &lt; 0.05; OVX + E2 + IS, 0.001 &lt; p &lt; 0.05) in the caudal trigeminal nucleus (TNC). There was no difference between M + IS and OVX + IS groups; however, the area was covered by CGRP IR fibers (0.01 &lt; p &lt; 0.05) and the number of nNOS IR cells was significantly higher in the OVX + E2 + IS (p &lt; 0.05) group than the other two IS- (M + IS, OVX + IS) treated animals. Overall, repeated administration of IS triggers activation and sensitization processes and develops nociceptive behavior changes. CGRP and nNOS levels increased significantly in the TNC after IS treatments, and moreover, pain thresholds and locomotor activity decreased with the development of photophobia. In our model, stable high estradiol levels proved to be pronociceptive. Thus, repeated trigeminal activation causes marked behavioral changes, which is more prominent in rats treated with estradiol, also reflected by the expression of the sensitization markers of the trigeminal system.

The study of the Mongolian traditional medicine Khurtsiin deed-6 analgesic and anti-inflammatory effect by inhibiting glial cell activation in nitroglycerin-induced migraine in rats

BackgroundMigraine is a highly prevalent neurovascular disorder with complicated treatment due to multifactorial pathophysiology. During the recent years, the prevalence of migraine in Mongolia is increasing and has reached 26.5% by 2019 which indicates an increase of 5.2% over the past 10 years. The industry of traditional medicine in Mongolia has a long history of development, hence, it is evident that various herbal medicines such as but not limited to Khurtsiin deed-6 (KhD-6) can be considered as generally safe and effective against several diseases including migraine. KhD-6’s single component has shown excellent analgesic, anti-inflammatory and neuroprotective effect, while the whole compound effectiveness and molecular mechanism had not been assessed for the treatment of migraine. ObjectivesTo study the effects of Khurtsiin deed-6 compound on inflammation and neuronal cell damage in migraine model in rats. MethodsA total of 36 rats were randomly divided into the following six groups including control group, Nitroglycerin-induced (NTG-induced) migraine model group, KhD-6 groups of 50 mg/kg, 100 mg/kg and 150 mg/kg doses, and Ibuprofen (IBU) 85 mg/kg treated group. Corresponding treatments were administered intragastrically for 10 days to animals of experimental groups. Then NTG-induced migraine model group, three KhD-6 groups and IBU group were subcutaneously injected with Nitroglycerin (10mg/kg) 1 h after the last treatment. Experimental animals were observed for two hours after Nitroglycerin injection to confirm the model establishment. After four hours, all rats were anesthetized and blood was collected from three rats from each group in order to detect levels of Nitric oxide (NO), calcitonin gene-related peptide (CGRP) and endothelin (ET) by ELISA method. The remaining three animals of each experimental group were perfused under anesthesia for the removal of their brains and isolation of the trigeminal nucleus caudalis (TNC). A polymerase chain reaction (PCR) method was used to analyze OX42, COX2, RAGE, GFAP, BDNF, BAX, and Bcl expressions. ResultsNTG-induced rat migraine model presents increased NO and CGRP levels in serum as well as c-Fos expression in the brain trigeminal nucleus area, which indicates that the migraine model was successfully generated. NO and CGRP levels in serum were suppressed in KhD-6 and IBU treated groups. The c-Fos expression decreased significantly in the TNC not only in Khurtsiin-6 compound, but also in IBU treated groups. Furthermore, KhD-6 compound inhibits neuro-inflammatory pathways by reducing COX2 expression, OX42 and GFAP activation. It inhibits neuronal cell death through suppressing RAGE, BAX, Bcl expression and increasing BDNF expression. ConclusionKhD-6 offers analgesic and neuroprotective effects in the NTG-induced migraine rat model by inhibiting the activation of glial cells and the secretion of neuroinflammatory factors in the TNC region.

Quercetin Attenuates Nitroglycerin-Induced Migraine Headaches by Inhibiting Oxidative Stress and Inflammatory Mediators.

This study aimed to investigate the antimigraine potential of quercetin in migraine pain induced by nitroglycerin (NTG), 10 mg/kg, intraperitoneal injection in rats. Quercetin was administered orally for 1 week, and behavioral parameters associated with pain were assessed 30 min after NTG injection. At the end of the study, the rats were killed so that immunohistochemical examination of their brains could be performed. The time and frequency of rearing and sniffing in the category of exploratory behavior, walking in the category of locomotor behavior, and total time spent in the light chamber were reduced in the disease control group compared with the normal group during the assessment of behavioral parameters. Pathologic migraine criteria, such as increased levels of calcitonin gene-related peptide and increased release of c-fos cells, were more prominent in the caudal nucleus triceminalis of the NTG control group. In the treatment groups, behavioral and pathological measures were less severe after pretreatment with quercetin at doses of 250 and 500 mg/kg. Therefore, it was concluded that quercetin improved the pain behavior of migraine patients in the NTG-induced migraine rat model. Quercetin is thought to have antimigraine effects due to its antioxidant and anti-inflammatory potential. Quercetin may therefore be a novel agent that can treat or prevent migraine pain and associated avoidance behaviors.

An Analysis of Khurtsiin Deed-6 Compound’s Effect on IL-1β Expression in Nitroglycerin Induced Migraine and Alcohol Exposed Neurodegeneration Model in Rat

Objectives: To investigate the anti-inflammatory activity of Khurtsiin deed-6 in migraine and neurodegeneration rat models. Methods: This study uses nitroglycerin induced migraine was model and alcohol exposed neurodegeneration model in Wistar rats to evaluate anti-inflammatory effect of Khurtsiin deed-6 at the 3 different doses of 50, 100and 150 mg/kg, orally. After anesthesia, the brains were removed, then trigeminal nucleus caudal is and hippocampus region isolated from fresh brain. And followed by protein and RNA extraction. Interleukin-1β expression was analyzed by real time polymerase chain reaction. Results: Nitroglycerin induced rat migraine model had increased Interleukin-1β expression in brain TNC area (p&lt;0.001). It has been decreased dramatically after treatment of Khurtsiin deed-6 by doses of 50mg/kg, 100mg/kg and 150mg/kg treatment in brain trigeminal nucleus caudal is region (p&lt;0.01) comparing ibuprofen treated group. Moreover alcohol exposed neurodegeneration rat model had observed increased Interleukin-1β expression in brain Hp area (p&lt;0.001). But Khurtsiin deed-6 doses of 50mg/kg, 100mg/kg and 150mg/kg treatment reduced neuroinflammatory cytokines IL1β expression in hippocampus region (p&lt;0.01, p&lt;0.001) of alcoholic rat models comparing pyridoxamine administrated animals. Conclusion: The present finding indicates that Khurtsiin deed-6shows anti-inflammatory activity decreasing the level of Interleukin -1β cytokine in nitroglycerin induced migraine and alcohol exposed neurodegeneration rat models

Effect of Electroacupuncture at Fengchi on Facial Allodynia, Microglial Activation, and Microglia-Neuron Interaction in a Rat Model of Migraine.

The purpose of the work was to investigate whether electroacupuncture (EA) could ameliorate migraine central sensitization by modulating microglial activation and the subsequent release of inflammatory cytokines in the trigeminal nucleus caudalis (TNC) in a rat model. Establishment of a rat model of recurrent migraine was achieved through repeated dural electrical stimulation (DES). After nine sessions of acupuncture treatment at Fengchi (GB20), facial mechanical thresholds were measured by electronic von Frey measurements. Microglial activation and cytokine receptors of TNC were evaluated by immunofluorescence staining. The expression of microglial biological marker Ibal-1, proinflammatory cytokines, and cytokine receptors in the TNC were evaluated by Western blot and/or real-time polymerase chain reaction. In addition, the effects of inhibition of microglial activation on facial thresholds and neuronal activation (i.e., expression of c-Fos in the TNC) induced by DES were observed. After consecutive EA-GB20 treatments, the facial withdrawal threshold was significantly higher than in the model group at different time points (p < 0.05). The hyperreactivity of microglia induced by DES was significantly inhibited, and the expressions of Ibal-1, interleukin-1β, tumor necrosis factor-α, and their receptors in the TNC were also significantly decreased (p < 0.05). Inhibition of microglia by minocycline demonstrated an acupuncture-like role, which was manifested by ameliorated mechanical hyperalgesia and decreased neuronal expression of c-Fos, Iba-1, and inflammatory factors. EA at GB20 could ameliorate migraine facial allodynia by inhibiting microglial activation and the subsequent release of inflammatory cytokines and their receptors in the TNC.

Dopamine receptor D2 regulates GLUA1-containing AMPA receptor trafficking and central sensitization through the PI3K signaling pathway in a male rat model of chronic migraine

BackgroundThe pathogenesis of chronic migraine remains unresolved. Recent studies have affirmed the contribution of GLUA1-containing AMPA receptors to chronic migraine. The dopamine D2 receptor, a member of G protein-coupled receptor superfamily, has been proven to have an analgesic effect on pathological headaches. The present work investigated the exact role of the dopamine D2 receptor in chronic migraine and its effect on GLUA1-containing AMPA receptor trafficking.MethodsA chronic migraine model was established by repeated inflammatory soup stimulation. Mechanical, periorbital, and thermal pain thresholds were assessed by the application of von Frey filaments and radiant heat. The mRNA and protein expression levels of the dopamine D2 receptor were analyzed by qRT‒PCR and western blotting. Colocalization of the dopamine D2 receptor and the GLUA1-containing AMPAR was observed by immunofluorescence. A dopamine D2 receptor agonist (quinpirole) and antagonist (sulpiride), a PI3K inhibitor (LY294002), a PI3K pathway agonist (740YP), and a GLUA1-containing AMPAR antagonist (NASPM) were administered to confirm the effects of the dopamine D2 receptor, the PI3K pathway and GULA1 on central sensitization and the GLUA1-containing AMPAR trafficking. Transmission electron microscopy and Golgi-Cox staining were applied to assess the impact of the dopamine D2 receptor and PI3K pathway on synaptic morphology. Fluo-4-AM was used to clarify the role of the dopamine D2 receptor and PI3K signaling on neuronal calcium influx. The Src family kinase (SFK) inhibitor PP2 was used to explore the effect of Src kinase on GLUA1-containing AMPAR trafficking and the PI3K signaling pathway.ResultsInflammatory soup stimulation significantly reduced pain thresholds in rats, accompanied by an increase in PI3K-P110β subunit expression, loss of dopamine receptor D2 expression, and enhanced GLUA1-containing AMPA receptor trafficking in the trigeminal nucleus caudalis (TNC). The dopamine D2 receptor colocalized with the GLUA1-containing AMPA receptor in the TNC; quinpirole, LY294002, and NASPM alleviated pain hypersensitivity and reduced GLUA1-containing AMPA receptor trafficking in chronic migraine rats. Sulpiride aggravated pain hypersensitivity and enhanced GLUA1 trafficking in CM rats. Importantly, the anti-injury and central sensitization-mitigating effects of quinpirole were reversed by 740YP. Both quinpirole and LY294002 inhibited calcium influx to neurons and modulated the synaptic morphology in the TNC. Additional results suggested that DRD2 may regulate PI3K signaling through Src family kinases.ConclusionModulation of GLUA1-containing AMPA receptor trafficking and central sensitization by the dopamine D2 receptor via the PI3K signaling pathway may contribute to the pathogenesis of chronic migraine in rats, and the dopamine D2 receptor could be a valuable candidate for chronic migraine treatment.

Network Pharmacology Analysis of the Effects of Achyranthis Bidentatae Radix Plus Semen Vaccariae on Migraine-induced Erectile Dysfunction.

Achyranthis Bidentatae Radix plus Semen Vaccariae are traditional Chinese medicines, which have been widely applied in the treatment of migraine and Erectile Dysfunction (ED) for many years. This study verified the effect of Achyranthis Bidentatae Radix plus Semen Vaccariae in improving migraine-induced ED and explored its potential mechanism. Key targets and signaling pathways of Achyranthis Bidentatae Radix plus Semen Vaccariae in migraine-induced erectile dysfunction treatment were predicted by network pharmacology. A rat model of migraine was established by nitroglycerin injection. Apomorphine was injected into rats to screen the migraine-induced erectile dysfunction model, Achyranthis Bidentatae Radix-Semen Vaccariae granule suspension administered, and erectile function evaluated. Hematoxylin and eosin staining was used to compare the histological structure of the penile tissue, while RT-qPCR and Western blotting were used to determine mRNA and protein levels, respectively. Screening allowed us to identify common targets for migraine and ED; the signaling pathway exhibiting the greatest change was the Myosin light chain kinase- Calcium (MLCK-CaM) signal pathway. From Western blotting and RT-qPCR, we found that the levels of MLCK mRNA and protein in rats from Group B rats were significantly higher (P <0.05) than those in Groups A and C. Furthermore, the mRNA and protein levels of CaM were significantly higher in Group B (P <0.05) than in Groups A and C. Data indicate that the regulatory effects of Achyranthis Bidentatae Radix plus Semen Vaccariae on migraine-induced ED in a rat model are mediated by the MLCK-CaM signaling pathway.

Xiongshao Zhitong Recipe Attenuates Nitroglycerin-Induced Migraine-Like Behaviors via the Inhibition of Inflammation Mediated by Nitric Oxide Synthase.

Migraine is a major cause of disability worldwide, particularly in young adults and middle-aged women. Xiongshao Zhitong Recipe (XZR) is a traditional Chinese medicine prescription used for treating migraine, but its bioactive components and therapeutic mechanisms remain unclear. We aimed to confirm the therapeutic effect of XZR on migraine and to determine the possible mechanism and bioactive components of XZR. Here, a sensitive UHPLC-LTQ-Orbitrap MS assay was carried out to analyze the ingredients of XZR, and a total of 62 components were identified, including coumarins, phenolic acids, phthalides, flavonoids, and terpenoids; among them, 15 components were identified in the serum samples after XZR treatment. We established a rat model of migraine via nitroglycerin (NTG) injection. The in vivo experiments demonstrated that XZR attenuated allodynia and photophobia in rats with NTG-induced migraine, and XZR also demonstrated analgesic effects. XZR reversed the abnormal levels of nitric oxide, 5-hydroxytryptamine (5-HT), calcitonin gene-related peptide (CGRP), and substance P (SP) to normal levels. XZR also downregulated inflammatory reactions, including mast cell degranulation and serum IL-1β, IL-6, and TNF-α levels. In terms of mechanism, we revealed that XZR treated NTG-induced migraine through the inhibition of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) expression in both the trigeminal nucleus caudalis (TNC) and periaqueductal gray matter (PAG), as well as the total NOS enzyme activity, which regulated the NF-κB signaling pathway. Additionally, imperatorin and xanthotoxin, two major ingredients of XZR, showed a high binding affinity to nNOS (Gly468-Leu616). In vitro, XZR, imperatorin, and xanthotoxin inhibited the nNOS expression and the NF-κB signaling pathway in lipopolysaccharide (LPS)-stimulated PC12 cells. In conclusion, we demonstrated the therapeutic effects of XZR and provided evidence that XZR played a critical anti-inflammatory role by suppressing NOS and NF-κB signaling pathway activation. Imperatorin and xanthotoxin were potential bioactive components of XZR. The findings from this study supported that XZR was a candidate herbal drug for migraine therapy.