Inflammatory Soup Research Articles

Microglia-Astrocyte Cross Talk through IL-18/IL-18R Signaling Modulates Migraine-like Behavior in Experimental Models of Migraine

Interleukin-18 (IL-18) is an important regulator of innate and immune responses, and is involved in the pain process, including neuropathic and cancer pain. The current study demonstrated that inflammatory soup (IS) dural infusions elicited the activation of microglia and astrocytes. In comparison, IS dural infusions induced the upregulation of IL-18 and IL-18R in microglia and astrocytes, respectively. Blocking the IL-18 signaling pathway attenuated nociceptive behavior. In comparison, blocking IL-18 signaling also suppressed the activation of astrocytes and nuclear factor-kappa B (NF-κB). IL-18 dural infusions induced nociceptive behavior and glia activation. IL-18 is a product of the activation of microglial toll-like receptor 4 (TLR4), and it acted on IL-18R expressed in astrocytes. Subsequently, it stimulated the activation of nuclear factor-kappa B (NF-κB), leading to the activation of astrocytes. In conclusion, IL-18-mediated microglia/astrocyte interactions in the medullary dorsal horn likely contribute to the development of hyperpathia or allodynia induced by migraines.

Deficiency in the function of inhibitory interneurons contributes to glutamate-associated central sensitization through GABABR2-SynCAM1 signaling in chronic migraine rats.

The occurrence of pain has always been closely related to a break in the balance between excitatory and inhibitory systems, and the internal relationship between these two systems has not been studied in the pathogenesis of chronic migraine (CM). In this study, we explored how inhibitory interneurons specifically modulate the glutamate-induced hyperexcitability in the periaqueductal gray (PAG) of CM rats. The CM model was established by repeated dural infusion of inflammatory soup (IS) in rats. Then, Baclofen, a gamma-aminobutyric acid type B receptor (GABABR) agonist; CGP35348, a GABABR antagonist; H89, a protein kinase A (PKA) inhibitor; and 8-Bromo-cAMP, a PKA agonist, were applied by intraventricular injection to investigate the detailed CM mechanism. Our results showed that GABABR2 mRNA and protein levels were significantly downregulated (P<.01) in the PAG of CM rats. Similarly, gamma-aminobutyric acid (GABA) and its synthetase glutamate decarboxylase 65/67 (GAD65/67) seriously decreased (P<.01), implying a deficit in the function of inhibitory interneurons in the PAG of CM rats. Afterward, the application of Baclofen and H89 alleviated the IS-evoked hyperalgesia and extenuated vesicular glutamate transporter 2 (VGLUT2), glutamate, calcitonin gene-related peptide (CGRP), and c-Fos expression by regulating the GABABR2/PKA/SynCAM1 pathway in the PAG of CM rats, while the application of CGP35348 and 8-Bromo-cAMP exactly exerted the opposite effect. Importantly, CGP35348 induced an elevation of CGRP, and VGLUT2 expression was relieved by H89. These data suggest that the loss in the function of inhibitory interneurons contributes to glutamate-associated central sensitization through the GABABR2/PKA/SynCAM1 pathway in the PAG of CM rats.

Trigeminal activation patterns evoked by chemical stimulation of the dura mater in rats

BackgroundAlthough migraine is one of the most common primary headaches, its therapy is still limited in many cases. The use of animal models is crucial in the development of novel therapeutic strategies, but unfortunately, none of them show all aspects of the disease, therefore, there is a constant need for further improvement in this field. The application of inflammatory agents on the dura mater is a widely accepted method to mimic neurogenic inflammation in rodents, which plays a key role in the pathomechanism of migraine. Complete Freund’s Adjuvant (CFA), and a mixture of inflammatory mediators, called inflammatory soup (IS) are often used for this purpose.MethodsTo examine the activation pattern that is caused by chemical stimulation of dura mater, we applied CFA or IS over the right parietal lobe. After 2 h and 4 h (CFA groups), or 2.5 h and 4 h (IS groups), animals were perfused, and c-Fos immunoreactive cells were counted in the caudal trigeminal nucleus. To explore every pitfall, we examined whether our surgical procedure (anesthetic drug, stereotaxic apparatus, local lidocaine) can alter the results under the same experimental settings. c-Fos labeled cells were counted in the second-order neuron area based on the somatotopic organization of the trigeminal nerve branches.ResultsWe could not find any difference between the CFA and physiological saline group neither 2 h, nor 4 h after dural stimulation. IS caused significant difference after both time points between IS treated and control group, and between treated (right) and control (left) side. Stereotaxic frame usage had a substantial effect on the obtained results.ConclusionsCounting c-Fos immunoreactive cells based on somatotopic organization of the trigeminal nerve helped to examine the effect of chemical stimulation of dura in a more specific way. As a result, the use of IS over the parietal lobe caused activation in the area of the ophthalmic nerve. To see this effect, the use of lidocaine anesthesia is indispensable.In conclusion, application of IS on the dura mater induces short-term, more robust c-Fos activation than CFA, therefore it might offer a better approach to model acute migraine headache in rodents.

When the Efferent Becomes Afferent ‐ the Cellular Response of Sympathetic Neurons to Sensory Stimuli

Responding to the so called “inflammatory soup” has often been regarded as the sole domain of sensory afferent neurons. We tested the hypothesis that efferent sympathetic neurons can also directly respond to traditionally afferent stimuli. We used single cell RNA‐sequencing (scRNAseq) to explore transcript expression of membrane G protein and ion channel coupled receptors in the sympathetic neuronal population within the stellate ganglia of 5–6‐week‐old Wistar rats. Agonist responses were then evaluated using Fura‐2 AM calcium imaging and perforated patch clamp recordings in isolated cultured neurons.ScRNAseq identified transcript expression of transient receptor potential (TRP) channels (TRPV2&gt;TRM7&gt;TRPC1&gt;&gt;TRM4). Certain TRP channel activators (10 mM camphor n=18, 10 mM cannabidiol n=16, and 100 mM 2‐APB n=15) caused large baseline calcium influxes, which were blocked by the TRP channel inhibitor ruthenium red (RuR, 10 mM; Camphor n=7; Cannabidiol n=24; 2‐APB n=11). An accompanying depolarization of the resting membrane potential and spontaneous firing was also observed, which was reduced by RuR. Other TRP channel activators (100 mM menthol n=15, 1 mM capsaicin n=16, 500 mM AITC n=9, 1 mM vanillin n=15, 300 nM JT010 n=15, 100 mM cavracol n=13, 10 mM icillin n=10, 50 mM naltriben n=15, pH=6.5 n=16 and 100 ng/ml IGF‐1 n=12) failed to produce significant responses.ScRNAseq also identified transcript expression of receptors for inflammatory mediators, for example histamine (HRH3) and prostaglandin E2 (PTGER2 and PTGER3). Stimulation of these receptors (100 mM histamine n=11 and 1 mM prostaglandin E2 n=8) had no effect on baseline [Ca2+]I, but were able to reduce Ca2+ influx in response to KCl depolarization (50 mM) or nicotine (100 mM).Overall, these data are important when interpreting stellate ganglia sympathetic neurons as purely efferent, with evidence that neuronal excitability can be directly influenced by sensory signaling pathways.Support or Funding InformationThis project was supported by the Wellcome trust and the British Heart Foundation

Altered functional connectivity of the insula in a rat model of recurrent headache.

Migraine is a pain disorder accompanied by various symptoms. The insula, a “cortical hub,” is involved in many functions. Few studies have focused on the insula in migraine. We explored the resting-state functional connectivity between the insula and other brain areas in rats subjected to repeated meningeal nociception which was commonly used as animal model of migraine. Inflammatory soup was infused through supradural catheters in conscious rats. The rats were subdivided based on the frequency of the inflammatory soup infusions. Magnetic resonance imaging data were acquired on rats 21 days after inflammatory soup infusion and functional connectivity seeded on the insula was analyzed. In the low-frequency inflammatory soup group, magnetic resonance imaging was performed again 1 h after the glyceryl trinitrate injection following baseline scanning. The cerebellum showed increased functional connectivity with the insula in the inflammatory soup groups. The insula showed increased functional connectivity with the medulla and thalamus in the ictal period in the low-frequency inflammatory soup rats. In the high-frequency inflammatory soup group, several areas showed increased functional connectivity with the insula, including the pons, midbrain, thalamus, temporal association cortex, and retrosplenial, visual, and sensory cortices. Our findings support the hypothesis that the headache phase of migraine depends on the activation and sensitization of the trigeminovascular system, and that the chronification of migraine may be related to higher brain centers and limbic cortices. The insula may be a new target for treatment of migraine.

Cognitive impairment in a classical rat model of chronic migraine may be due to alterations in hippocampal synaptic plasticity and N-methyl-D-aspartate receptor subunits

Although migraine is a major global public health problem, its impact on cognitive abilities remains controversial. Thus, the present study investigated the effects of repeated administration of inflammatory soup to the dura of rats, over three weeks, on spatial cognition, hippocampal synaptic plasticity, and the expression of N-methyl-D-aspartate receptor subunits. Additionally, low doses of amitriptyline (5 mg/kg) were applied to assess its therapeutic effects. The inflammatory soup group exhibited significant reductions in the cutaneous stimulation threshold, presence of mild cognitive impairment, and decreased long-term potentiation in right hippocampus. However, amitriptyline improved pain behaviors, enhanced cognitive function, and increased synaptic plasticity in the inflammatory soup rats. On the other hand, the administration of amitriptyline to normal rats negatively influenced synaptic plasticity and reduced the expression of N-methyl-D-aspartate receptor subunits. The present results indicate that inflammatory soup-induced dural nociception led to impairments in spatial cognition that could be attributed to reductions in hippocampal long-term potentiation and the decreased expression of N-methyl-D-aspartate receptor subunits.

Prophylactic Electroacupuncture on the Upper Cervical Segments Decreases Neuronal Discharges of the Trigeminocervical Complex in Migraine-Affected Rats: An in vivo Extracellular Electrophysiological Experiment.

PurposeThis rat experiment aims to demonstrate the efficacy of electrical acupuncture in preventing migraine attacks by stimulating the acupoint GB20.IntroductionMigraine, which takes 2ed at level four causes of GBD’s disease hierarchy, becomes a public health issue. It is important for physicians to supplement their knowledge of its treatment and consider alternative methods of therapy, such as acupuncture. However, the neurobiological and pathophysiological mechanisms of this prophylactic effect were unclear. The trigeminocervical complex is thought to be an important relay station in migraine pathophysiology as the key nuclei of the trigeminovascular system and the brainstem descending pain modulation system.MethodsThere were six groups involved in this study: control, model, electroacupuncture, non-acupoint electroacupuncture, saline+electroacupuncture and saline+non-acupoint electroacupuncture. We injected saline or inflammatory soup into dura mater to induce control or migraine in the rats. The mechanical pain threshold and the single-cell extraneural neurophysiology of the C1 spinal dorsal horn neurons in the trigeminocervical complex were detected.ResultsPre-electroacupuncture could significantly increase the mechanical pain threshold of the periorbital region receptive field of the trigeminal nerve and decrease the discharges of neurons in the trigeminocervical complex. Acupuncture also reversed the abnormal skin pain response of the periorbital region receptive field of the trigeminal nerve caused by low-intensity stimulation.DiscussionWe believe that our study makes a significant contribution to the literature because it is the first of its kind to use GB20 to provide relief from migraine attacks and mechanical cephalic cutaneous hypersensitivity by regulating the neuronal discharge from trigeminocervical complex.

EphrinB/EphB Signaling Contributes to the Synaptic Plasticity of Chronic Migraine Through NR2B Phosphorylation

The specific mechanism of migraine chronification remains unclear. We previously demonstrated that synaptic plasticity was associated with migraine chronification. EphB receptors and their ligands, ephrinBs, are considered to be key molecules regulating the synaptic plasticity of the central nervous system. However, whether they can promote the chronification of migraine by regulating synaptic plasticity is unknown. Therefore, we investigated the role of ephrinB/EphB signaling in chronic migraine (CM). Male Sprague-Dawley rats were used to construct a chronic migraine model by dural infusion of an inflammatory soup for 7 days. We used qPCR, western blot, and immunofluorescence to detect the mRNA and protein levels of EphB2 and ephrinB2. The paw withdrawal latency and paw withdrawal threshold were measured after lateral ventricle treatment with EphB1-Fc (an inhibitor of EphB receptor). Changes in synaptic plasticity were explored by examining synaptic-associated proteins by western blot, dendritic spines of neurons by Golgi-Cox staining, and synaptic ultrastructure by transmission electron microscopy. We found that the expression of EphB2 and ephrinB2 increased in CM. The administration of EphB1-Fc relieved hyperalgesia and changes in synaptic plasticity induced by CM. In addition, EphB1-Fc inhibited the upregulation of NR2B phosphorylation. These results indicate that ephrinB/EphB signaling may regulate synaptic plasticity in CM via NR2B phosphorylation, which suggests the novel idea that ephrinB/EphB signaling may be a target for the treatment of migraine chronification.

Cutaneous inflammation differentially regulates the expression and function of Angiotensin-II types 1 and 2 receptors in rat primary sensory neurons.

Neuropathic and inflammatory pain results from cellular and molecular changes in dorsal root ganglion (DRG) neurons. The type-2 receptor for Angiotensin-II (AT2R) has been involved in this type of pain. However, the underlying mechanisms are poorly understood, including the role of the type-1 receptor for Angiotensin-II (AT1R). Here, we used a combination of immunohistochemistry and immunocytochemistry, RT-PCR and in vitro and in vivo pharmacological manipulation to examine how cutaneous inflammation affected the expression of AT1R and AT2R in subpopulations of rat DRG neurons and studied their impact on inflammation-induced neuritogenesis. We demonstrated that AT2R-neurons express C- or A-neuron markers, primarily IB4, trkA, and substance-P. AT1R expression was highest in small neurons and co-localized significantly with AT2R. In vitro, an inflammatory soup caused significant elevation of AT2R mRNA, whereas AT1R mRNA levels remained unchanged. In vivo, we found a unique pattern of change in the expression of AT1R and AT2R after cutaneous inflammation. AT2R increased in small neurons at 1day and in medium size neurons at 4days. Interestingly, cutaneous inflammation increased AT1R levels only in large neurons at 4days. We found that in vitro and in vivo AT1R and AT2R acted co-operatively to regulate DRG neurite outgrowth. In vivo, AT2R inhibition impacted more on non-peptidergic C-neurons neuritogenesis, whereas AT1R blockade affected primarily peptidergic nerve terminals. Thus, cutaneous-induced inflammation regulated AT1R and AT2R expression and function in different DRG neuronal subpopulations at different times. These findings must be considered when targeting AT1R and AT2R to treat chronic inflammatory pain. Cover Image for this issue: doi: 10.1111/jnc.14737.

Migraine: Experimental Models and Novel Therapeutic Approaches.

Migraine is a disorder affecting an increasing number of subjects. Currently, this disorder is not entirely understood, and limited therapeutic solutions are available. Migraine manifests as a debilitating headache associated with an altered sensory perception that may compromise the quality of life. Animal models have been developed using chemical, physical or genetic modifications, to evoke migraine-like hallmarks for the identification of novel molecules for the treatment of migraine. In this context, experimental models based on the use of chemicals as nitroglycerin or inflammatory soup were extensively used to mimic the acute state and the chronicity of the disorder. This manuscript is aimed to provide an overview of murine models used to investigate migraine pathophysiology. Pharmacological targets as 5-HT and calcitonin gene-related peptide (CGRP) receptors were evaluated for their relevance in the development of migraine therapeutics. Drug delivery systems using nanoparticles may be helpful for the enhancement of the brain targeting and bioavailability of anti-migraine drugs as triptans. In conclusion, the progresses in migraine management have been reached with the development of emerging agonists of 5-HT receptors and novel antagonists of CGRP receptors. The nanoformulations may represent a future perspective in which already known anti-migraine drugs showed to better exert their therapeutic effects.

Modulation of brain networks by sumatriptan-naproxen in the inflammatory soup migraine model.

Migraine is a debilitating condition; however, the pharmacological effects on central nervous system networks after successful therapy are poorly understood. Defining this neurocircuitry is critical to our understanding of the disorder and for the development of antimigraine drugs. Using an established inflammatory soup model of migraine-like pathophysiology (N = 12) compared with sham synthetic interstitial fluid migraine induction (N = 12), our aim was to evaluate changes in network-level functional connectivity after sumatriptan-naproxen infusion in awake, conscious rodents (Sprague-Dawley rats). Sumatriptan-naproxen infusion functional magnetic resonance imaging data were analyzed using an independent component analysis approach. Whole-brain analysis yielded significant between-group (inflammatory soup vs synthetic interstitial fluid) alterations in functional connectivity across the cerebellar, default mode, basal ganglia, autonomic, and salience networks. These results demonstrate the large-scale antimigraine effects of sumatriptan-naproxen co-administration after dural sensitization.

Atypical functional connectivity between the anterior cingulate cortex and other brain regions in a rat model of recurrent headache.

We explored the atypical functional connectivity between the anterior cingulate cortex and other brain areas in rats subjected to repeated meningeal nociception. The rat model was established by infusing an inflammatory soup through supradural catheters in conscious rats. Rats were subdivided according to the frequency of the inflammatory soup infusions. Functional connectivity analysis seeded on the anterior cingulate cortex was performed on rats 21 days after inflammatory soup infusion. Glyceryl trinitrate was injected following baseline scanning in the low-frequency inflammatory soup group and magnetic resonance imaging data were acquired 1 h after the injection. The rats exhibited nociceptive behavior after high-frequency inflammatory soup infusion. The anterior cingulate cortex showed increased functional connectivity with the cerebellum in the inflammatory soup groups. The medulla showed increased functional connectivity with the anterior cingulate cortex in the ictal period in the low-frequency inflammatory soup rats. Several areas showed increased functional connectivity with the anterior cingulate cortex in the high-frequency inflammatory soup group, including the pontine tegmentum, midbrain, thalamus, corpus callosum, hippocampus, and retrosplenial, visual, sensory, and motor cortices. This study indicated that the medulla participates in the early stage of a migraine attack and may be associated with the initiation of migraine. Sensitization of the trigeminal nociceptive pathway might contribute to the cutaneous allodynia seen in chronic migraine. Brain areas important for memory function may be related to the chronification of migraine. Electrophysiological studies should examine those migraine-related areas and provide new targets for migraine treatment and prevention.

A novel large animal model of recurrent migraine established by repeated administration of inflammatory soup into the dura mater of the rhesus monkey.

Several animal models of migraine have been established, and those based on trigeminovascular system activation are widely accepted. However, most of these models have been established on lower animals, such as rodents, and involve only a single administration of a noxious stimulus. In this study, an inflammatory soup (10 μL), consisting of prostaglandin E2 (0.2 mM), serotonin (2 mM), bradykinin (2 mM) and histamine (2 mM), was injected into the dura mater of conscious rhesus monkeys through an indwelling catheter. The infusion started on day 8 and was repeated every 3 days, for a total of six administrations, to induce neurogenic inflammation. We performed behavioral assessments and measured the expression of the oncogene c-fos, neuronal nitric oxide synthase (nNOS) and calcitonin gene related peptide (CGRP) in the trigeminal system and in multiple brain regions involved in pain processing by immunohistochemical staining. Compared with monkeys in the control group, three of the four animals in the inflammatory soup group displayed decreased motor behaviors, and two showed increased ipsilateral nose and mouth secretions during the stimulus period. Higher expression levels of c-fos, nNOS and CGRP were found in various brain areas of experimental animals compared with controls, including the trigeminal nucleus caudalis, thalamus, hypothalamus, midbrain, pons and other areas involved in pain perception. These results suggest that repeated inflammatory soup stimulation of the dura activates the trigeminovascular system and produces migraine-like pathological changes and abnormal behaviors in conscious rhesus monkeys.

Inhibition of Nerve Growth Factor Signaling Alleviates Repeated Dural Stimulation-induced Hyperalgesia in Rats

Our previous study showed that acid-sensing ion channel 3 (ASIC3) in the trigeminal nucleus caudalis (TNC) is involved in the pathogenesis of recurrent migraine. ASIC3 is regulated by nerve growth factor (NGF), which induces hyperalgesia in various pain disorders. Neutralization of NGF is considered an effective treatment method. However, the contribution of NGF to repeated migraine-like attacks in chronic migraine (CM) remains unclear. Therefore, this study investigated the effect of NGF on ASIC3 expression in the TNC and the role of NGF signaling in chemical dural stimulation-induced hyperalgesia. A rat model was established by repeated dural infusions of inflammatory soup (IS) for seven days to simulate CM attacks. After repeated IS infusions, cutaneous hyperalgesia appeared in the rats’ periorbital region and hind paws, which showed significantly lower pain thresholds. IS infusions upregulated the mRNA and protein of NGF in the TNC, and NGF was mainly expressed in the cytoplasm of TNC neurons. An intracerebroventricular injection of an anti-NGF-neutralizing antibody relieved the cutaneous hyperalgesia of CM rats and decreased protein kinase C (PKC), ASIC3, calcitonin gene-related peptide (CGRP) and c-Fos expression in the TNC. Moreover, intracerebroventricular injection with the PKC blocker chelerythrine chloride alleviated IS infusion-induced hyperalgesia and reduced ASIC3, CGRP and c-Fos levels in the TNC. These results indicate that NGF might regulate ASIC3 expression via PKC activity in the TNC following repeated IS dural stimulation, and this signaling pathway might participate in IS-induced hyperalgesia.

Recurrent Headache Increases Blood-Brain Barrier Permeability and VEGF Expression in Rats

Background: The blood-brain barrier (BBB) is an important anatomical structure of the central nervous system (CNS) that limits the penetration of a variety of substances from the blood into the parenchyma. Dysfunction of the BBB is involved in various CNS disorders, including stroke, inflammation, and pain. However, the evidence concerning its role in migraine is insufficient. Objective: This study will investigate whether recurrent headache increases BBB permeability and vascular endothelial growth factor (VEGF) expression in a rat model. Study Design: This study used an experimental design. Setting: The research took place in the Laboratory Research Center at The First Affiliated Hospital of Chongqing Medical University. Methods: Eighty male Sprague-Dawley rats were randomly divided into 3 groups: inflammatory soup (IS), control (PBS), and treatment (IS+Sumatriptan) groups. Recurrent headache was induced by episodic IS stimulation: 20 µL of IS were pumped into the dura 3 times per week in rats. The control group was administered 20 µL of PBS. The rats in the treatment group were simultaneously treated with sumatriptan (300 ug/kg, intraperitoneal) at the same time that IS was applied to the dura. Mechanical nociceptive thresholds were examined by electronic von Frey filaments with rigid tips. BBB permeability changes were measured with Evans blue (EB). The expression of VEGF was measured by double labeling and Western blotting. Results: After 4 IS applications, the mechanical nociceptive thresholds significantly decreased. In addition, the mechanical hypersensitivity persisted for 4 hours after 9 applications. Only after 9 applications did the BBB permeability increase, as demonstrated by the EB tracer. The BBB disruption was accompanied by an elevation in VEGF expression. Sumatriptan treatment significantly reduced the mechanical hypersensitivity induced by IS stimulations and decreased the BBB disruption and VEGF expression. Limitations: Potential mechanisms that underlie the relationship between BBB and VEGF were not examined in this study. Conclusions: The present study showed that repeated IS stimulations induced long-lasting allodynia, increased BBB permeability, and upregulated VEGF expression, all of which could be attenuated by early sumatriptan treatment. Key words: Migraine, inflammatory soup, blood-brain barrier, vascular endothelial growth factor, sumatriptan

NR2B-Tyr phosphorylation regulates synaptic plasticity in central sensitization in a chronic migraine rat model

BackgroundAlthough the mechanism of chronic migraine (CM) is unclear, it might be related to central sensitization and neuronal persistent hyperexcitability. The tyrosine phosphorylation of NR2B (NR2B-pTyr) reportedly contributes to the development of central sensitization and persistent pain in the spinal cord. Central sensitization is thought to be associated with an increase in synaptic efficiency, but the mechanism through which NR2B-pTyr regulates synaptic participation in CM-related central sensitization is unknown. In this study, we aim to investigate the role of NR2B-pTyr in regulating synaptic plasticity in CM-related central sensitization.MethodsMale Sprague-Dawley rats were subjected to seven inflammatory soup (IS) injections to model recurrent trigeminovascular or dural nociceptor activation, which is assumed to occur in patients with CM. We used the von Frey test to detect changes in mechanical withdrawal thresholds, and western blotting and immunofluorescence staining assays were performed to detect the expression of NR2B-pTyr in the trigeminal nucleus caudalis (TNC). NR2B-pTyr was blocked with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)-pyrazolo [3,4-d] pyrimidine (PP2) and the protein tyrosine kinase inhibitor genistein to detected the changes in calcitonin gene-related peptide (CGRP), substance P (SP), and the synaptic proteins postsynaptic density 95 (PSD95), synaptophysin (Syp), synaptotagmin1 (Syt-1). The synaptic ultrastructures were observed by transmission electron microscopy (TEM), and the dendritic architecture of TNC neurons was observed by Golgi-Cox staining.ResultsStatistical analyses revealed that repeated infusions of IS induced mechanical allodynia and significantly increased the expression of NR2B Tyr-1472 phosphorylation (pNR2B-Y1472) and NR2B Tyr-1252 phosphorylation (pNR2B-Y1252) in the TNC. Furthermore, the inhibition of NR2B-pTyr by PP2 and genistein relieved allodynia and reduced the expression of CGRP, SP, PSD95, Syp and Syt-1 and synaptic transmission.ConclusionsThese data indicate that NR2B-pTyr might regulate synaptic plasticity in central sensitization in a CM rat model. The inhibition of NR2B tyrosine phosphorylation has a protective effect on threshold dysfunction and migraine attacks through the regulation of synaptic plasticity in central sensitization.

Expression of ASIC3 in the Trigeminal Nucleus Caudalis Plays a Role in a Rat Model of Recurrent Migraine.

Acid-sensing ion channel 3 (ASIC3) is abundant in the trigeminal nervous system and is most sensitive to a slight pH decrease. Recent studies have indicated that ASIC3 in the peripheral trigeminal ganglia is likely involved in the pathogenesis of migraine pain. However, it is unclear whether this receptor plays a role in recurrent migraine, namely, migraine chronicity. Here, we aimed to investigate the role of ASIC3 in an animal model of recurrent migraine (RM). In this study, we established a rat model of RM through repeated administration of inflammatory soup (IS) onto the dura. Then, we tested the mechanical pain thresholds of the face and hindpaws by von Frey filaments. qRT-PCR, Western blot and immunofluorescence labelling were used to detect the expression and localization of ASIC3 in the trigeminal nucleus caudalis (TNC). The protein levels of calcitonin gene-related peptide (CGRP), its receptor component receptor activity modifying protein 1 (RAMP1) and c-Fos were analysed following treatment with the ASIC3 inhibitor APETx2 and activator 2-guanidine-4-methylquinazoline (GMQ). We found decreased pain thresholds after repeated dural inflammatory stimulation, which suggested the establishment of an RM model. Based on this model, we observed elevated expression of ASIC3 in the TNC group compared to that in the Sham group. ASIC3 was primarily expressed in neurons but not in astrocytes of the TNC. Moreover, APETx2 attenuated tactile allodynia and significantly decreased the expression of c-Fos, CGRP and RAMP1, while GMQ aggravated these effects compared to those observed in the IS + vehicle group. These findings indicate a critical role of ASIC3 channels in the pathophysiology of RM, and ASIC3 might represent a potential therapeutic target to prevent the progression of migraine.

α7 Nicotinic acetylcholine receptor-mediated anti-inflammatory effect in a chronic migraine rat model via the attenuation of glial cell activation.

BackgroundEvidence suggests that the activation of α7 nicotinic acetylcholine receptor (α7nAChR) can greatly decrease the neuroinflammation response. Neuroinflammation plays a pivotal role in the pathogenesis of chronic migraine (CM). Clinical observations also show that nicotine gum induces analgesic effects in migraine patients. However, whether α7nAChR is involved in CM is unclear.ObjectiveTo investigate the role of α7nAChR in CM and provide a new therapeutic target for CM.Materials and methodsThirty-six male Sprague–Dawley rats were distributed randomly into control, CM, PNU-282987, and α-bungarotoxin groups (n=9 rats in each group). The CM model was established by the recurrent daily administration of inflammatory soup on the dura over the course of 1 week. The hind paw threshold and facial allodynia were assessed by the von Frey test. The expression levels of α7nAChR, tumor necrosis factor-alpha, and interleukin-1 beta were analyzed by Western blot and real-time fluorescence quantitative polymerase chain reaction. The location of α7nAChR in the hippocampus was quantified by immunofluorescence, as well as the microglial and astrocyte alterations. Changes in the calcitonin gene-related peptide and the phosphorylated JNK protein among different groups were measured by Western blot.ResultsWe found that the expression of α7nAChR was reduced after repeated inflammatory soup administration. The increased expression of tumor necrosis factor-alpha, interleukin-1 beta, and calcitonin gene-related peptide in CM group were significantly decreased by PNU-282987 and aggravated by α-bungarotoxin. Moreover, PNU-282987 decreased the numbers of astrocytes and microglia compared with the numbers in the CM group in both hippocampal CA1 and CA3 regions. In contrast, α-bungarotoxin activated the astrocytes and microglia, but the differences with respect to the CM group were not significant. Activated c-Jun N-terminal kinase signaling was observed in CM rats and was also blocked by PNU-282987.ConclusionThe activation of α7nAChR increased the mechanical threshold and alleviated pain in the CM rat model. α7nAChR activation also decreased the upregulation of astrocytes and microglia through the p-c-Jun N-terminal kinase–mitogen-activated protein kinase signaling pathway.

Non-invasive dural stimulation in mice: A novel preclinical model of migraine.

Migraine is characterized by a collection of neurological symptoms in the absence of injury or damage. However, several common preclinical migraine models require significant damage to the skull to stimulate the dura mater, the likely source of afferent signaling leading to head pain. The goal of this study was to determine whether dural stimulation can be performed in mice using an injection that does not cause injury or damage. Using mice, injections of stimuli were administered to the dura mater through the soft tissue at the intersection between the lambdoidal and sagittal sutures. This technique did not require a permanent cannula nor did it cause damage to the skull or dura. Following injection of noxious stimuli, migraine-like behaviors were measured including cutaneous allodynia and facial grimace. The retrograde tracer fluorogold was applied onto the dura using the same injection technique to label trigeminal ganglion cell bodies, which were then testing invitro using patch-clamp electrophysiology. Dural injection of allyl-isothiocyanate, low pH, interleukin-6, or inflammatory soup but not vehicles, led to cephalic/extracephalic allodynia. Facial grimace responses were also observed with allyl-isothiocyanate, pH 6.0, and interleukin-6. Stimulation with interleukin-6 causes priming to normally subthreshold pH 7.0 stimulation of the dura following resolution of the initial interleukin-6 behavior. Systemic injection of sumatriptan at the time of dural stimulation with inflammatory soup decreased the resulting cutaneous hypersensitivity. Trigeminal ganglion cell bodies retrogradely labeled from the dura had low pH-evoked currents similar to those generated by acid-sensing ion channels. Non-invasive dural stimulation in mice can be used as a model of migraine in the absence of injury.

Animal Models of Chronic Migraine.

Chronic migraine (CM) is a recalcitrant subtype of migraine which causes high degrees of disability, poor treatment responses, and frequent recurrences in sufferers. However, the pathophysiological mechanisms underlying the development and chronification of migraine attacks remain incompletely understood. A validated animal model could help to decipher the pathogenic mechanism of the disease, facilitating the development of possible therapeutic strategies for CM. In this review, we aimed to summarize current animal models of CM and discuss the validity of these models. Several methods have been available to induce recurrent headache-like behaviors or biochemical changes in rodents, including repeated dural application of inflammatory soup, chronic systemic infusion of nitroglycerin, repeated administration of acute migraine abortive treatment to simulate medication overuse headache, or genetic modification. These models exhibit some features that are believed to be associated with migraine; however, none of the model can recapitulate all the clinical phenotypes found in humans and each has its own weakness. The complex features of CM increase the difficulty of constructing a proper animal model. Nonetheless, currently available models are valid to certain degrees. Future directions might consider simulating the spontaneity and chronicity of migraine by combining known genetic substrates and allostatic loads into the same model.