Rat Dura Mater Research Articles

Topiramate Inhibits Capsaicin-Induced Mast Cell Degranulation and CGRP Release in Rat Dura Mater

Background/Objectives: Migraine is a disease that stands out for its high prevalence and socioeconomic costs. It involves the entire trigeminovascular system, the signaling substances, and their targets. However, the role of meningeal mast cells in migraine is still unclear. To better understand one of the components of neurogenic inflammation underlying migraine pathophysiology, we developed an in vivo rat model in which the dura mater was exposed bilaterally to investigate the influence of topiramate on capsaicin-induced mast cell degranulation and CGRP release from dura mater. Methods: On the day of the experiment, rats were anesthetized, and a craniectomy was performed on each parietal bone. Test substances were applied in situ over the dura mater using the right and left sides of the dura mater for the test and control, respectively. After exposure, the dura mater was processed for mast cell staining and counting. Using this setup, the effect of capsaicin (10−3 M) was evaluated in rats of both sexes, and subsequently the effect of in situ (10−3 M, 20 µL) and (20 mg/kg/day for 10 days) topiramate treatment on mast cell degranulation and CGRP release were evaluated. Results: In both female and male rats, there was a greater amount of degranulated mast cells in the side stimulated by capsaicin compared to the control side in both females (18 ± 3% vs. 74 ± 3%; p = 0.016) and males (28 ± 2% vs. 74 ± 3%, p = 0.016). In the group treated with topiramate for 10 days prior to the experiments, capsaicin did not induce mast cell degranulation (control 20 ± 1% vs. capsaicin 22 ± 1%, p = 0.375) in contrast to animals treated for 10 days with gavage control (control 25 ± 1% vs. capsaicin 76 ± 1%, p = 0.016). Topiramate applied in situ concomitant with capsaicin did not protect the mast cells from degranulation in response to capsaicin (38 ± 2% vs. 44 ± 1%, p = 0.016). There was a significant reduction in CGRP release from the dura mater in the group treated with topiramate for 10 days compared to the control. Conclusions: This study demonstrates a novel experimental model wherein systemic administration of topiramate is observed to modulate the impact of capsaicin on meningeal mast cell degranulation.

Anti-CGRP antibody galcanezumab modifies the function of the trigeminovascular nocisensor complex in the rat

BackgroundMonoclonal antibodies directed against the neuropeptide calcitonin gene-related peptide (CGRP) are effective in the prevention of chronic and frequent episodic migraine. Since the antibodies do not cross the blood brain barrier, their antinociceptive effect is attributed to effects in meningeal tissues. We aimed to probe if such an antibody can be visualized within the dura mater and the trigeminal ganglia following its administration to rats and to examine if the activity of the trigeminovascular nocisensor complex is influenced by this treatment.MethodsEffects of the anti-CGRP antibody galcanezumab on the trigeminovascular nocisensor complex was examined by measuring release of sensory neuropeptides and histamine from the rat dura mater. Deposits of galcanezumab were visualized by fluorescence microscopy in the trigeminal ganglion and the dura mater.ResultsFluorophore-labelled galcanezumab was detected in the dura mater and the trigeminal ganglion up to 30 days after treatment affirming the long-lasting modulatory effect of this antibody. In female rats, seven days after systemic treatment with galcanezumab the capsaicin-induced release of CGRP was decreased, while that of substance P (SP) was increased in the dura mater. In control rats, release of the inhibitory neuropeptide somatostatin (SOM) was higher in females than in males. Stimulation with high concentration of KCl did not significantly change the release of SOM in control animals, while in rats treated with galcanezumab SOM release was slightly reduced. Galcanezumab treatment also reduced the amount of histamine released from dural mast cells upon stimulation with CGRP, while the effect of compound 48/80 on histamine release was not changed.ConclusionsGalcanezumab treatment is followed by multiple changes in the release of neuropeptides and histamine in the trigeminal nocisensor complex, which may contribute to the migraine preventing effect of anti-CGRP antibodies. These changes affecting the communication between the components of the trigeminal nocisensor complex may reduce pain susceptibility in migraine patients treated with CGRP targeting monoclonal antibodies.

Progesterone distribution in the trigeminal system and its role to modulate sensory neurotransmission: influence of sex

BackgroundWomen are disproportionately affected by migraine, representing up to 75% of all migraine cases. This discrepancy has been proposed to be influenced by differences in hormone levels between the sexes. One such hormone is progesterone. Calcitonin gene-related peptide (CGRP) system is an important factor in migraine pathophysiology and could be influenced by circulating hormones. The purpose of this study was to investigate the distribution of progesterone and its receptor (PR) in the trigeminovascular system, and to examine the role of progesterone to modulate sensory neurotransmission.MethodsTrigeminal ganglion (TG), hypothalamus, dura mater, and the basilar artery from male and female rats were carefully dissected. Expression of progesterone and PR proteins, and mRNA levels from TG and hypothalamus were analyzed by immunohistochemistry and real-time quantitative PCR. CGRP release from TG and dura mater were measured using an enzyme-linked immunosorbent assay. In addition, the vasomotor effect of progesterone on male and female basilar artery segments was investigated with myography.ResultsProgesterone and progesterone receptor -A (PR-A) immunoreactivity were found in TG. Progesterone was located predominantly in cell membranes and in Aδ-fibers, and PR-A was found in neuronal cytoplasm and nucleus, and in satellite glial cells. The number of positive progesterone immunoreactive cells in the TG was higher in female compared to male rats. The PR mRNA was expressed in both hypothalamus and TG; however, the PR expression level was significantly higher in the hypothalamus. Progesterone did not induce a significant change neither in basal level nor upon stimulated release of CGRP from dura mater or TG in male or female rats when compared to the vehicle control. However, pre-treated with 10 µM progesterone weakly enhanced capsaicin induced CGRP release observed in the dura mater of male rats. Similarly, in male basilar arteries, progesterone significantly amplified the dilation in response to capsaicin.ConclusionsIn conclusion, these results highlight the potential for progesterone to modulate sensory neurotransmission and vascular responses in a complex manner, with effects varying by sex, tissue type, and the nature of the stimulus. Further investigations are needed to elucidate the underlying mechanisms and physiological implications of these findings.Graphical

Rodent behavior following a dural inflammation model with anti-CGRP migraine medication treatment.

Migraine is a widespread and prevalent disease with a complex pathophysiology, of which neuroinflammation and increased pain sensitivity have been suggested to be involved. Various studies have investigated the presence of different inflammatory markers in migraineurs and investigated the role of inflammation in inflammatory models with complete Freund's adjuvant (CFA) or inflammatory soup added to the dura mater. The aim of the current study was to examine whether application of CFA to the dura mater would cause behavioral alterations that are migraine relevant. In addition, we investigated the potential mitigating effects of fremanezumab, a CGRP (calcitonin gene-related peptide) specific antibody, following CFA application. Male Sprague-Dawley rats were randomly divided into six groups: fresh (n = 7), fresh + carprofen (n = 6), fresh + anti-CGRP (n = 6), sham (n = 7), CFA (n = 16), CFA + anti-CGRP (n = 8). CFA was applied for 15 min on a 3 × 3 mm clearing of the skull exposing the dura mater of male Sprague-Dawley rats. We applied the Light/Dark box and Open Field test, combined with the electronic von Frey test to evaluate outcomes. Finally, we observed CGRP immunoreactivity in the trigeminal ganglion. No differences were observed in the Light/Dark box test. The Open Field test detected behavior differences, notably that sham rats spend less time in the central zone, reared less and groomed more than fresh + carprofen rats. The other groups were not significantly different compared to sham rats, indicating that activation of the TGVS is present in sham surgery and cannot be exacerbated by CFA. However, for the allodynia, we observed specific periorbital sensitization, not observed in the sham animals. This could not be mitigated by fremanezumab, although it clearly reduced the amount of CGRP positive fibers. CFA surgically administered to the dura causes periorbital allodynia and increases CGRP positive fibers in the trigeminal ganglion. Fremanezumab does not reduce periorbital allodynia even though it reduces CGRP positive fibers in the TG. Further work is needed to investigate whether CFA administered to the dura could be used as a non-CGRP inflammatory migraine model.

Release of CGRP in vivo from rat dura mater: Influence of capsaicin and topiramate

Introduction Migraine is a disease that stands out for its high prevalence with changes in the nervous system, abnormal levels of neurotransmitters, neuromodulators, and neuropeptides. The main mechanisms of action of capsaicin are chemical induction through the activation of TRPV1 channels, allowing calcium influx into neurons in the trigeminal ganglion of the dura mater, activating mast cell degranulation, releasing pro-inflammatory (e.g., histamine, oxide nitric) and vasoactive (e.g., CGRP and substance P) substances. For treatment, classes of drugs are used to act on blood vessels and to prevent vasodilation, as well as depolarization of sensory fibers of the dura mater. Objectives To better understand sterile inflammation after exposing the dura mater bilaterally, we created an experimental model to study mechanisms of action of topiramate and capsaicin in mast cell degranulation and release of CGRP in a rat skull preparation in vivo using anesthetized animals. Methods Thirty-five Wistar rats were used, divided into two groups of chronic topiramate (GTC) treated with 20mg/kg/day, gavage/10 days, and acute topiramate (GTA) in situ in the dura mater (10-3M). The animals were anesthetized and cranial windows between the coronal and lambda sutures in the hemicraniums were performed with a drill to expose the dura mater bilaterally. 10-3M capsaicin was placed on the right side and synthetic interstitial fluid on the left side and exposed to contact for 10 minutes to a small cotton ball soaked with the respective solutions so that there is no leakage of the treatment, and posteriorly kept in the freezer (-20°C) for later quantification of CGRP. The percentage of degranulated mast cells was quantified after removal of the dura mater by staining it with toluidine blue. A commercial enzyme immunoassay quantified the release of CGRP from the cranial dura mater... (Too see the complet abstract, please, check out the PDF.)

The Anti-CGRP Antibody Fremanezumab Lowers CGRP Release from Rat Dura Mater and Meningeal Blood Flow.

Monoclonal antibodies directed against the neuropeptide calcitonin gene-related peptide (CGRP) belong to a new generation of therapeutics that are effective in the prevention of migraine. CGRP, a potent vasodilator, is strongly implicated in the pathophysiology of migraine, but its role remains to be fully elucidated. The hemisected rat head preparation and laser Doppler flowmetry were used to examine the effects on CGRP release from the dura mater and meningeal blood flow of the subcutaneously injected anti-CGRP monoclonal antibody fremanezumab at 30 mg/kg, when compared to an isotype control antibody. Some rats were administered glycerol trinitrate (GTN) intraperitoneally to produce a migraine-like sensitized state. When compared to the control antibody, the fremanezumab injection was followed by reduced basal and capsaicin-evoked CGRP release from day 3 up to 30 days. The difference was enhanced after 4 h of GTN application. The samples from the female rats showed a higher CGRP release compared to that of the males. The increases in meningeal blood flow induced by acrolein (100 µM) and capsaicin (100 nM) were reduced 13–20 days after the fremanezumab injection, and the direct vasoconstrictor effect of high capsaicin (10 µM) was intensified. In conclusion, fremanezumab lowers the CGRP release and lasts up to four weeks, thereby lowering the CGRP-dependent meningeal blood flow. The antibody may not only prevent the released CGRP from binding but may also influence the CGRP release stimulated by noxious agents relevant for the generation of migraine pain.

<em>Ex Vivo</em> Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in migraine pathophysiology has been well established, first by its potent vasodilator properties and subsequently by its presence and function as a neurotransmitter in the sensory trigeminovascular system. The migraine-provoking ability of CGRP gave support to the pharma industry to develop monoclonal antibodies and antagonists inhibiting the effect of CGRP. A new treatment paradigm has proven effective in the prophylactic treatment of migraine. One of the useful tools to further understand migraine mechanisms is the ex vivo model of CGRP release from the trigeminovascular system. It is a relatively simple method that can be used with various pharmacological tools to achieve know-how to further develop new effective migraine treatments. The present protocol describes a CGRP release model and the technique to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents. A procedure describing the experimental approach from euthanasia to the measurement of protein levels is provided. The essential isolation of the trigeminal ganglion and the trigeminal nucleus caudalis from both mice and rats and the preparation of rat dura mater are described in detail. Furthermore, representative results from both species (rats and mice) are presented. The technique is a key tool to investigate the molecular mechanisms involved in migraine pathophysiology by using various pharmacological compounds and genetically modified animals.

Ex vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents.

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in migraine pathophysiology has been well established, first by its potent vasodilator properties and subsequently by its presence and function as a neurotransmitter in the sensory trigeminovascular system. The migraine-provoking ability of CGRP gave support to the pharma industry to develop monoclonal antibodies and antagonists inhibiting the effect of CGRP. A new treatment paradigm has proven effective in the prophylactic treatment of migraine. One of the useful tools to further understand migraine mechanisms is the ex vivo model of CGRP release from the trigeminovascular system. It is a relatively simple method that can be used with various pharmacological tools to achieve know-how to further develop new effective migraine treatments. The present protocol describes a CGRP release model and the technique to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents. A procedure describing the experimental approach from euthanasia to the measurement of protein levels is provided. The essential isolation of the trigeminal ganglion and the trigeminal nucleus caudalis from both mice and rats and the preparation of rat dura mater are described in detail. Furthermore, representative results from both species (rats and mice) are presented. The technique is a key tool to investigate the molecular mechanisms involved in migraine pathophysiology by using various pharmacological compounds and genetically modified animals.

Distribution and possible function of galanin about headache and immune system in the rat dura mater

Galanin (GAL) is a nociceptive transmitter or modulator in the trigeminal sensory system. In this study, GAL expression was investigated in the rat dura mater to demonstrate its possible function in headache using immunohistochemical techniques. The cerebral falx and cerebellar dura mater received abundant blood and nerve supply, and were significantly thicker compared to other portions in the cerebral dura mater. GAL-immunoreactivity was expressed by cell and nerve fiber profiles. Presumed macrophages and dendritic cells contained GAL-immunoreactivity, and co-expressed with CD11b-immunoreactivity. Many isolated and perivascular nerve fibers also showed GAL-immunoreactivity. In addition, GAL-immunoreactive nerve fibers were present in the vicinity of macrophages and dendritic cells with either GAL- or ED1-immunoreactivity. GAL-immunoreactive cells and nerve fibers were common in the cerebral falx and cerebellar dura mater and infrequent in other portions. And, GAL-immunoreactive nerve fibers usually co-expressed calcitonin gene-related peptide (CGRP)-immunoreactivity. In the trigeminal ganglion, a substantial proportion of sensory neurons innervating the dura mater contained GAL-immunoreactivity (mean ± SD, 3.4 ± 2.2%), and co-expressed CGRP-immunoreactivity (2.7 ± 2.1%). The present study may suggest that GAL is associated with nociceptive transduction or modulation in the dura mater. GAL also possibly plays a role in the immune mechanism of the dura mater.

A new anatomical-functional model: effect of topiramate on capsaicin-induced mast cell degranulation in rat dura mater (Abstract)

Dissertation (Master’s Degree in Biotechnology). Postgraduation in Biological Sciences. Federal University of Pernambuco, Recife, Brazil. 2020. 64 f. Supervisor: Prof. Dr. Marcelo Moraes Valença.

Результаты применения противоспаечного геля после моделирования контузионного повреждения спинного мозга у крыс

Objective was to investigate an effect of anti-adhesion hyaluronic-containing resorbable gel on the course of traumatic spinal cord disease caused by a simulated contusion spinal cord injury (SCI) in rats. Material and methods A moderate spinal cord injury was simulated at the Th9 level of 40 female Wistar rats that were randomly divided into 2 groups. Group I received hyaluronic-containing absorbable antiadhesion gel Antiadgezin® that was intraoperatively applied to the dura mater of experimental rats; Group II of control rats received no gel. The BBB locomotor rating scale was used to evaluate the extent of recovered pelvic limb function. The animals were sacrificed on days 5, 15, 30, 60 and 90. Paraffin and epoxy semi-thin 1 μm sections were used for morphological examination of the spinal cord using light microscopy. Results The gel used for contusion SCI in rats caused neither significant changes during postoperative period nor local infectious and inflammatory complications, and showed no negative impact on functional results. Morphological examination revealed pathomorphological changes that were identical for both groups and characteristic of contusion SCI showing softening, necrosis of the nervous tissue, tissue and cell detritus of gray and white matter, small cysts in the early stages of the experiment; progression of syringomyelia and formation of large cysts primarily seen in the gray matter and the onset of regenerative processes in the white matter at 60 and 90 days of the experiment. Conclusion The absence of a negative effect of the anti-adhesion hyaluronic-containing resorbable gel on the functional results, and morphological characteristics of the spinal cord offer the opportunity of the further experimental use as a carrier of substance composition initiating beneficial metabolic and neuroprotective action for transdural delivery in simulated SCI.

CGRP outflow into jugular blood and cerebrospinal fluid and permeance for CGRP of rat dura mater

BackgroundCalcitonin gene-related peptide (CGRP) is released from activated meningeal afferent fibres in the cranial dura mater, which likely accompanies severe headache attacks. Increased CGRP levels have been observed in different extracellular fluid compartments during primary headaches such as migraine but it is not entirely clear how CGRP is drained from the meninges.MethodsWe have used an in vivo preparation of the rat to examine after which time and at which concentration CGRP applied onto the exposed parietal dura mater appears in the jugular venous blood and the cerebrospinal fluid (CSF) collected from the cisterna magna. Recordings of meningeal (dural) and cortical (pial) blood flow were used to monitor the vasodilatory effect of CGRP. In a new ex vivo preparation we examined how much of a defined CGRP concentration applied to the arachnoidal side penetrates the dura. CGRP concentrations were determined with an approved enzyme immunoassay.ResultsCGRP levels in the jugular plasma in vivo were slightly elevated compared to baseline values 5-20 min after dural application of CGRP (10 μM), in the CSF a significant three-fold increase was seen after 35 min. Meningeal but not cortical blood flow showed significant increases. The spontaneous CGRP release from the dura mater ex vivo was above the applied low concentration of 1 pM. CGRP at 1 nM did only partly penetrate the dura.ConclusionsWe conclude that only a small fraction of CGRP applied onto the dura mater reaches the jugular blood and, in a delayed manner, also the CSF. The dura mater may constitute a barrier for CGRP and limits diffusion into the CSF of the subarachnoidal space, where the CGRP concentration is too low to cause vasodilatation.

Effect of dural inflammatory soup application on activation and sensitization markers in the caudal trigeminal nucleus of the rat and the modulatory effects of sumatriptan and kynurenic acid

BackgroundThe topical inflammatory soup can model the inflammation of the dura mater causing hypersensitivity and activation of the trigeminal system, a phenomenon present in migraineurs. Calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase are important in the sensitization process there.5-HT1B/1D receptor agonists, triptans are used as a treatment of migraine. Kynurenic acid an NMDA antagonist can act on structures involved in trigeminal activation.AimWe investigated the effect of inflammatory soup induced dural inflammation on the calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase levels in the caudal trigeminal nucleus. We also tested whether pretreatment with a well-known antimigraine drug, such as sumatriptan and kynurenic acid, a compound with a different mechanism of action, can affect these changes and if their modulatory effects are comparable.Material and methodsAfter subcutaneous sumatriptan or intraperitoneal kynurenic acid the dura mater of adult male Sprague-Dawley rats (n = 72) was treated with inflammatory soup or its vehicle (synthetic interstitial fluid). Two and a half or four hours later perfusion was performed and the caudal trigeminal nucleus was removed for immunohistochemistry.Results and conclusionInflammatory soup increased calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase in the caudal trigeminal nucleus compared to placebo, which was attenuated by sumatriptan and kynurenic acid. This suggests the involvement of 5-HT1B/1D and NMDA receptors in neurogenic inflammation development of the dura and thus in migraine attacks.

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.

Identification of Lyve-1 positive macrophages as resident cells in meninges of rats.

Meningeal immunity along with its associated lymphatic vasculatures is widely discussed recently. Lymphatic vessels in meninges drain interstitial fluid into the deep-cervical lymph nodes. The vessels are composed of cells that express the cardinal marker for lymphatic endothelium-the lymphatic vessel hyaluronan receptor-1 (Lyve-1). However, studies also show the presence of nonendothelial Lyve-1 expressing cells in certain tissues. Therefore, we were curious if nonendothelial Lyve-1+ cells are also present in dura mater of meninges. We show that Lyve-1+ endothelial cells are distributed adjacent to the blood vessels in the brain dura mater of rats. We did not observe any lymphatic vessels in spinal dura mater. Interestingly, we also observed isolated population of nonlymphatic Lyve-1+ cells in both brain and spinal dura mater. Morphologically, the Lyve-1+ cells were extensively pleomorphic, sometimes elongated or round. Surprisingly, the thoracolumbal meningeal Lyve-1+ cells were predominantly round in morphology. Using endothelial specific marker VEGFR3 and macrophage markers CD68 and CD169, we observed that the isolated Lyve-1+ cells lacked endothelial cell signature, but were either CD68+ or CD169+ macrophages. Moreover, we observed that the Lyve-1+ cells colocalized with collagen fibers in the meninges, and some of Lyve-1+ cells had intracellular collagen. The study for the first time demonstrates the presence of Lyve-1 positive macrophages in the lymphatic and nonlymphatic regions in the meninges of rats.

High-dose phenylephrine increases meningeal blood flow through TRPV1 receptor activation and release of calcitonin gene-related peptide.

The α1 -adrenoceptor agonist, phenylephrine, is used at high concentrations as a mydriatic agent and for the treatment of nasal congestion. Among its adverse side-effects transient burning sensations are reported indicating activation of the trigeminal nociceptive system. Neuropeptide release, calcium imaging and meningeal blood flow recordings were applied in rodent models of meningeal nociception to clarify possible receptor mechanisms underlying these pain phenomena. Phenylephrine above 10mM dose-dependently released calcitonin gene-related peptide (CGRP) from the dura mater and isolated trigeminal ganglia, whereas hyperosmotic mannitol at 90mM was ineffective. The phenylephrine-evoked release was blocked by the transient receptor potential vanilloid 1 (TRPV1) antagonist BCTC and did not occur in trigeminal ganglia of TRPV1-deficient mice. Phenylephrine at 30mM caused calcium transients in cultured trigeminal ganglion neurons responding to the TRPV1 agonist capsaicin and in HEK293T cells expressing human TRPV1. Local application of phenylephrine at micromolar concentrations to the exposed rat dura mater reduced meningeal blood flow, whereas concentrations above 10mM caused increased meningeal blood flow. The flow increase was abolished by pre-application of the CGRP receptor antagonist CGRP8-37 or the TRPV1 antagonist BCTC. Phenylephrine at high millimolar concentrations activates TRPV1 receptor channels of perivascular afferents and, upon calcium inflow, releases CGRP, which increases meningeal blood flow. Activation of TRPV1 receptors may underlie trigeminal nociception leading to cranial pain such as local burning sensations or headaches caused by administration of high doses of phenylephrine. Phenylephrine is used at high concentrations as a mydriaticum and for treating nasal congestion. As adverse side-effects burning sensations and headaches have been described. Phenylephrine at high concentrations causes calcium transients in trigeminal afferents, CGRP release and increased meningeal blood flow upon activation of TRPV1 receptor channels, which is likely underlying the reported pain phenomena.

Dural Calcitonin Gene-Related Peptide Produces Female-Specific Responses in Rodent Migraine Models.

Migraine is the second leading cause for disability worldwide and the most common neurological disorder. It is also three times more common in women; reasons for this sex difference are not known. Using preclinical behavioral models of migraine, we show that application of calcitonin gene-related peptide (CGRP) to the rat dura mater produces cutaneous periorbital hypersensitivity. Surprisingly, this response was observed only in females; dural CGRP at doses from 1 pg to 3.8 μg produce no responses in males. In females, dural CGRP causes priming to a pH 7.0 solution after animals recover from the initial CGRP-induced allodynia. Dural application of interleukin-6 causes acute responses in males and females but only causes priming to subthreshold dural CGRP (0.1 pg) in females. Intracisternal application of BDNF also causes similar acute hypersensitivity responses in males and females but only priming to subthreshold dural CGRP (0.1 pg) in females. Females were additionally primed to a subthreshold dose of the NO-donor sodium nitroprusside (0.1 mg/kg) following dural CGRP. Finally, the sexually dimorphic responses to dural CGRP were not specific to rats as similar female-specific hypersensitivity responses were seen in mice, where increased grimace responses were also observed. These data are the first to demonstrate that CGRP-induced headache-like behavioral responses at doses up to 3.8 μg are female-specific both acutely and following central and peripheral priming. These data further implicate dural CGRP signaling in the pathophysiology of migraine and propose a model where dural CGRP-based mechanisms contribute to the sexual disparity of this female-biased disorder.SIGNIFICANCE STATEMENT Calcitonin gene-related peptide (CGRP) has long been implicated in the pathophysiology of migraine, and CGRP-based therapeutics are efficacious for the treatment of migraine in humans. However, the location of action for CGRP in migraine remains unclear. We show here that application of CGRP to the cranial meninges causes behavioral responses consistent with headache in preclinical rodent models. Surprisingly, however, these responses are only observed in females. Acute responses to meningeal CGRP are female-specific and sensitization to CGRP after two distinct stimuli are also female-specific. These data implicate the dura mater as a primary location of action for CGRP in migraine and suggest that female-specific mechanisms downstream of CGRP receptor activation contribute to the higher prevalence of migraine in women.

Dynamic changes in CGRP, PACAP, and PACAP receptors in the trigeminovascular system of a novel repetitive electrical stimulation rat model: Relevant to migraine.

Migraine is the seventh most disabling disorder globally, with prevalence of 11.7% worldwide. One of the prevailing mechanisms is the activation of the trigeminovascular system, and calcitonin gene-related peptide (CGRP) is an important therapeutic target for migraine in this system. Recent studies suggested an emerging role of pituitary adenylate cyclase-activating peptide (PACAP) in migraine. However, the relation between CGRP and PACAP and the role of PACAP in migraine remain undefined. In this study, we established a novel repetitive (one, three, and seven days) electrical stimulation model by stimulating dura mater in conscious rats. Then, we determined expression patterns in the trigeminal ganglion and the trigeminal nucleus caudalis of the trigeminovascular system. Electrical stimulation decreased facial mechanical thresholds, and the order of sensitivity was as follows: vibrissal pad >inner canthus >outer canthus (P < 0.001). The electrical stimulation group exhibited head-turning and head-flicks (P < 0.05) nociceptive behaviors. Importantly, electrical stimulation increased the expressions of CGRP, PACAP, and the PACAP-preferring type 1 (PAC1) receptor in both trigeminal ganglion and trigeminal nucleus caudalis (P < 0.05). The expressions of two vasoactive intestinal peptide (VIP)-shared type 2 (VPAC1 and VPAC2) receptors were increased in the trigeminal ganglion, whereas in the trigeminal nucleus caudalis, their increases were peaked on Day 3 and then decreased by Day 7. PACAP was colocalized with NEUronal Nuclei (NeuN), PAC1, and CGRP in both trigeminal ganglion and the trigeminal nucleus caudalis. Our results demonstrate that the repetitive electrical stimulation model can simulate the allodynia during the migraine chronification, and PACAP plays a role in the pathogenesis of migraine potentially via PAC1 receptor.

Intracerebroventricular infusion of D-serine decreases nociceptive behaviors induced by electrical stimulation of the dura mater of rat

ABSTRACTObjective and method: D-serine acts as an obligatory ligand for the glycine sites of N-methyl-D-aspartate receptors. To investigate its effect on head-facial pain, nociceptive behaviors of rats induced by electrical stimulation of the dura mater were observed after cerebroventricular infusion of D-serine.Results: Rats in the D-serine infusion group showed significantly fewer nociceptive behaviors, including grooming and head flicking, than rats in the saline infusion group and control rats.Conclusions: This is the first study to introduce D-serine to a trigeminovascular headache model that demonstrates an antinociceptive-like effect in rats.

Medication overuse headache following repeated morphine, but not [INCREMENT]9-tetrahydrocannabinol administration in the female rat.

The potential of [INCREMENT]-tetrahydrocannabinol (THC) as a treatment for migraine depends on antinociceptive efficacy with repeated administration. Although morphine has good antinociceptive efficacy, repeated administration causes medication overuse headache (MOH) - a condition in which the intensity/frequency of migraine increases. The present study compared the effect of repeated morphine or THC administration on the magnitude and duration of migraine-like pain induced by a microinjection of allyl isothiocyanate (AITC) onto the dura mater of female rats. Acute administration of THC or morphine prevented AITC-induced depression of wheel running. This antinociception was maintained in rats treated repeatedly with THC, but not following repeated administration of morphine. Moreover, repeated morphine, but not THC administration, extended the duration of AITC-induced depression of wheel running. These data indicate that tolerance and MOH develop rapidly to morphine administration. The lack of tolerance and MOH to THC indicates that THC may be an especially effective long-term treatment against migraine.