16th European Headache Congress

Chair

Prof.

The TRPA1 pain pathway is a target for multiple environmental pollutants: Possible relevance for migraine

Basic science, animal models in headache research

Migraine

Abstract text (incl. figure legends and references) The prevalence of the disabling pain disorder migraine is rising. Here, we test the hypothesis that ubiquitous environmental pollutants evoke the release of migraine-inducing neuropeptide calcitonin gene-related peptide (CGRP), via the activation of transient receptor potential (TRP) channels, thereby increasing pain. To understand if environmental pollutants target migraine-associated TRP channels ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), we used a calcium imaging-based screen of pollutants known to be abundant in industrialized regions. Based on this screen, patch clamping and in silico docking, we selected a pesticide (pentachlorophenol; PCP) to perform proof-of-concept experiments. We tested ex vivo release of CGRP and ex vivo vasodilatory responses of isolated cerebral arteries to PCP. Finally, we tested in vivo induction of cutaneous hypersensitivity in wild type and Trpa1 deficient mice. 16 of 53 screened environmental pollutants activated TRPA1, while none of the investigated compounds activated TRPV1. Focusing on PCP, in silico molecular modelling suggested that PCP is stabilized in a known lipid binding pocket of TRPA1. In vitro, ex vivo and in vivo experiments showed that PCP induced calcium influx in neurons, TRPA1-dependent CGRP release from the brainstem, dilation of cerebral arteries, and TRPA1-dependent increased pain response in mice. These findings establish that abundant pollutants from the environment interact with the TRPA1-CGRP migraine pain pathway. Therefore, exposure to ubiquitous pollutants might be a contributing factor to the increased migraine prevalence.

ePoster

Prof. Dr.

Functional responses in a patient-specific iPSC-derived vascular model: a novel approach to study migraine

Abstract text (incl. figure legends and references) Objective Blood vessels from migraine patients are useful for studying migraine pathophysiology and drug development, but are difficult to obtain. Here, we develop a 3D vessel-on-chip model incorporating induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (VSMCs) and endothelial cells, allowing to study patient-specific blood vessels. The vascular responses of the cultured blood vessel model are compared to native human blood vessels to validate the model. Methods In iPSC-derived VSMCs, grown in 2D or in a 3D conformation in a vessel-on-chip model, cAMP responses are measured using the cADDis live cell cAMP assay after stimulation with CGRP and in the presence of phosphodiesterase 3 (PDE3) inhibitors milrinone or cilostazol or the CGRP receptor antagonists rimegepant or olcegepant. Responses to CGRP in the presence of these gepants in iPSC-derived VSMCs were compared to responses in human coronary arteries from heart valve donors (8 F and 7 M, age 48±3 years), as measured in a Mulvany myograph system. Intracellular calcium responses in VSMCs were measured using the calcium dye Cal-520 after stimulation with 10 nM endothelin-1 (ET-1). Results PDE3 inhibitors milrinone and cilostazol significantly augment the cAMP response to CGRP in iPSC-derived VSMCs (p=0.034 and p=0.002, resp.), while rimegepant inhibits the cAMP response to CGRP, similarly as observed in human isolated arteries. In the 3D cultured blood vessels, olcegepant potently blocked the response to CGRP, while ET-1 potently increased the intracellular calcium concentrations. Comparable results were obtained in human isolated arteries (Labruijere et al. 2013). Conclusion Functional measurements can be performed in human IPSC-derived VSMCs, in both 2D and 3D conformation, with comparable results in cultured blood vessels and isolated human arteries. These patient-specific vessel-on-chip models could be used in the future to study migraine pathophysiology and improve drug development.

Professor

Relevance of mouse vasculature in investigating side-effects of anti-migraine medications.

Abstract text (incl. figure legends and references) Objective The gold standard treatment in migraine, the triptans, are usually not prescribed to patients with cardiovascular risk factors. Further it has been debated whether, the Gepants and anti-CGRP/CGRP receptor antibodies (collectible named anti-CGRP treatments) poses an additional risk in patients in relation to cardiovascular disease. There are some studies aiming to address this experimentally, particular using mice models. We therefore set out to investigate if the mouse is a good model for cardiovascular effects of anti-migraine treatments. Methods ∼2mm long isolated mouse coronary (40µm wire) and basilar segments (25 µm wire) were mounted on a Mulvany–Halpern myograph. The smooth muscle cell contractile function was confirmed by challenging the segments two times with K+. We performed a concentration response curve to 5-HT with a maximal concentration of 10 µM. Following an addition of U46619 (~ 0.1 µM), the segments where challenged with CGRP (1 µM) followed by PACAP (0.1 µM). Results Mouse coronary arteries contracted to 5-HT (110 ± 13% of the K+), and receptor characterization showed that the 5-HT2A receptor was responsible for the contraction. To ensure visible dilation, we used minimal precontraction for the coronary arteries (62 ± 4.9 % of K+) and basilar arteries (19 ± 6.1 % of K+). The coronary arteries dilated to CGRP (50 ± 7.8 %) but not to PACAP (-6 ± 2.7%). These results contrast to the basilar artery, which did not significantly contract to 5-HT (5.4 ± 1.8%, p=0.06). More important, the basilar artery did not dilate in response to CGRP (9±12%. P=0.46), whereas PACAP had a significant dilatory response (49 ± 4.4%. P=0.002). Conclusion These data show that using the mouse as a model organism for side effects of anti-CGRP treatments should be analyzed with care, as the arteries do not have similar expression profile as human arteries. In relation to migraine, it is interesting that cerebral arteries appear to lack CGRP receptors in mice.

Calcitonin gene-related peptide receptor antagonist BIBN4096BS regulates synaptic transmission in the vestibular nucleus and improves vestibular function via PKC/ERK/CREB pathway in an experimental chronic migraine rat model

Abstract text (incl. figure legends and references) Questions: Vestibular symptoms are frequently reported in patients with chronic migraine (CM). The neuropeptide calcitonin gene-related peptide (CGRP) and CGRP1 receptor are essential to facilitate central sensitization in CM. However, the role of CGRP/CGRP1 receptor signaling in vestibular dysfunction after CM remains unclear. Methods: A CM rat model was established by recurrent intermittent administration of nitroglycerin. Migraine- and vestibular-related behaviors were assessed. BIBN4096BS and protein kinase C (PKC) inhibitor-chelerythrine chloride were administered intracerebroventricularly. The expressions of CGRP and CGRP1 receptor components in the vestibular nucleus (VN) were evaluated. Synaptic associated proteins and synaptic morphological characteristics were explored. The expressions of down-stream molecules including PKC, phosphorylated extracellular signal regulated kinase (p-ERK), and phosphorylated cAMP response element-binding protein at serine 133 site (p-CREB-S133) were detected. Results: The expressions of CGRP and CGRP1 receptor components were significantly upregulated in CM rats. CGRP1 receptor components were expressed mainly in neurons. BIBN4096BS treatment and PKC inhibition alleviated mechanical allodynia, thermal hyperalgesia and vestibular dysfunction in CM rats. Additionally, BIBN4096BS treatment and PKC inhibition markedly inhibited the overexpression of synaptic associated proteins and restored the abnormal synaptic structure in VN after CM. Furthermore, BIBN4096BS treatment dysregulated the expression levels of PKC, p-ERK and p-CREB-S133, and attenuated neuronal activation in VN after CM. Conclusions: The present study demonstrated that CGRP1 receptor inhibition improved vestibular function after CM by reversing the aberrant synaptic transmission via downregulating PKC/ERK/CREB signaling pathway. Therapeutic interventions by inhibiting CGRP/CGRP1 signaling may be a new target for the treatment of vestibular symptoms in CM.

An open-source electrophysiology system to explore visual evoked potentials in migraine

Abstract text (incl. figure legends and references) Question: Many studies have highlighted altered sensory processing underlying headache and non-headache symptoms in migraine, however a consensus is yet to be reached due to conflicting clinical data. Further electrophysiological investigation is needed to shed a light on pathophysiology and potential therapeutic targets, but existing tools are limited and expensive. We chose to study visual evoked potentials in a murine model of migraine using an open-source electrophysiology suite (Open-Ephys). Methods: Visual evoked potentials (VEP) were obtained by LED flash stimulation (100 trials @ 1Hz) in anaesthetised mice (n=8) and acquired into Open-Ephys. Mice were injected with an acute dose of the migraine trigger nitroglycerin (10mg/kg i.p.) and VEPs were measured at 30min and 1hr. Data analysis was performed using custom MATLAB scripts to assess VEP amplitude, latency and spontaneous activity. Results: We were able to record visual evoked potentials from V1 in response to flash stimuli using Open-Ephys. Preliminary data show NTG resulted in an increased VEP amplitude and a decreased latency of the P1 peak, associated with the extrastriate cortex. Additionally, spontaneous activity is shown to decrease after NTG injection, potentially reflecting vasodilatory effects of the compound. Conclusion: Open-source electrophysiology systems offer a low-cost alternative to traditional systems and offer customiseable experimental tools tailored to research questions. Its open-source nature enables researchers to share and access neurophysiology tools used in other labs, which is particularly relevant to the headache field, as its pathophysiology most likely involves the interaction of diverse neural circuits which require equally complex tools to unravel. This study demonstrates its feasibility in investigating migraine-relevant cortical areas and further work will be conducted to explore multi-modal thalamocortical circuits in migraine.

Effects of caffeine on intracranial pressure and pain perception in freely moving rats

Abstract text (incl. figure legends and references) Question: Caffeine, a nonselective adenosine receptor antagonist, is the most commonly consumed psycho-stimulant in the world. Caffeine has been suggested to regulate cerebrospinal fluid (CSF) secretion and is known both to alleviate and to trigger headache. However, its effect on the regulation of intracranial pressure (ICP) is not known. Therefore, we aimed to investigate the effects of caffeine on ICP and pain perception. Methods: Female Sprague Dawley rats (n=21) were implanted with a novel telemetric device for continuous ICP recordings which allowed for continuously recordings in freely moving rats. Single dose of caffeine (30 or 120 mg/kg i.p.) was given. In a second group (non-implanted), the acute effects of 30mg/kg caffeine on periorbital threshold using Von Frey and spontaneous behavior were utilized using an automated behavioral registration platform (LABORAS) in a randomized cross-over study. Immunofluorescence was performed to localize adenosine receptor (ARs) at choroid plexus (CP). Results: Single dose of 30 mg/kg caffeine lowered ICP 5h after administration (saline: 0.16±0.9 vs caffeine: -1.18±0.9ΔmmHg, p=0.0098) and lasted up to 11h. Administration of 120 mg/kg caffeine showed a faster onset of decrease in ICP already within 15min (p=0.0018) and lasted up 12h. The periorbital pain thresholds were higher after 1h (saline: 224.6±15.1 vs caffeine: 289.5±8.7 g, p=0.005) and lasted up to 5h. After 5h of administration, the hind paw threshold was higher relative to vehicle (saline: 200.1±7.7 vs caffeine: 245.7±9.1g, p=0.03). Caffeine treated rats had increased locomotor activity, speed and rearing behavior. Expression of A1 was found at CP. Conclusions: This study demonstrates that caffeine has a lowering effect on ICP as acute treatment and may act on the A1 receptor expressed at CP. Interestingly caffeine caused increased response in cephalic thresholds which was developed in an earlier stage of ICP reduction.

Capsaicin induced cell death in primary cultured neurons

Neuromodulation and nerve blocks

Abstract text (incl. figure legends and references) Purpose To determine the effect of capsaicin to central nervous system, we prepared morphologic changes and biochemical assay were investigated in mouse primary cultured CNS neuron. Methods The susceptability of capsaicin differs for different brain area. Cerebral cortex and hippocampus were more sensitive, and striatum, thalamus and midbrain area were less sensitive to capsaicinsusceptability. After capsaicin treatment, cortical and hippocampal neurons were died in dose-and time-dependent manner. By observation of nuclear fragmentation of capsaicin treated neuron, it is thought that the type of cell death is apoptosis rather than necrosis. The capsaicin receptor immunoreactive cells were observed in the cortex and hippocampus.It is consistent with area of damaged neuron. In case of capsaicin treated neurons, NOS activity stain was positive , the product of nitrite and anti-nitrotyrosineimmunoreactivity were increased, and agmatine, which is a competitive nitric oxide synthases (NOSs) inhibitor significantly protect cortical and hippocampal neurons from capsaicin-induced apoptosis. Results These results indicated that capsaicin induced influx of cation ions. These results showed that capsaicin induced influx of Ca2+, followed by neuronal NOS is activated by Ca2+ and induced cell death. Also, the activity of caspase 3 was increased after capsacin treatment in the cortical and hippocampal neurons. Conclusions These results demonstrate that capsaicin induced the apoptosis through acting with capsaicin receptors. Calcium influx due to capsaicin recptor activation may induce apoptosis, which is triggered the formation of peroxynitrite by activating NOS activity or is mediated by activating caspase 3 pathway.

Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in contributing to cortical spreading depression propagation

Neuropeptides, channels in headache

Abstract text (incl. figure legends and references) Purinergic P2X7 receptor plays an important role in migraine pathophysiology. Yet precise molecular mechanism underlying P2X7R signaling in migraine remains unclear. This study aims to test the hypothesis that P2X7 receptor transmits signaling to Src family kinases (SFKs) during cortical spreading depression (CSD). Methods: CSD was recorded using electrophysiology in rats or intrinsic optical imaging in mouse brain slices. Results: The data showed that deactivation of SFKs by systemic injection of PP2 reduced cortical susceptibility to CSD in rats. Consistently, in mouse brain slices, inhibition of SFKs activity by saracatinib and P2X7 receptor by A740003 similarly reduced cortical susceptibility to CSD. When the interaction of P2X7 receptor and SFKs was disrupted by TAT-P2X7, a marked reduction of cortical susceptibility to CSD were observed in mouse brain slices. The reduced cortical susceptibility to CSD by TAT-P2X7 was restored by NMDA, and disrupting the Fyn-NMDA interaction using TAT-Fyn (39-57) but not disrupting Src-NMDA receptor interaction using TAT-Src (40-49) reduced cortical susceptibility to CSD. Furthermore, activation of P2X7 receptor by BzATP restored the TAT-Fyn (39-57)-reduced cortical susceptibility to CSD. Conclusion: This study reveals that SFKs activity transmits P2X7 receptor signaling to facilitate CSD propagation via glutamatergic pathway, which is of particular relevance to migraine.

Evaluation of a human prolonged-release buprenorphine formulation in rats

Multidisciplinary clinical assessments

Abstract text (incl. figure legends and references) Objective: The development of novel targets within brain research often requires surgical procedures in rodents, and opioid analgesia is frequently needed postoperatively. Buprenorphine is a partial agonist of the μ-opioid receptors and has a strong analgesic effect when acting on the central nervous system. A long-acting buprenorphine formulation would be highly beneficial to avoid frequent dosing of postoperative animals. However, buprenorphine depot formulations developed for animal use are not available in Europe. The purpose of the present study was therefore to evaluate the effect on rats of a long-acting subcutaneous (s.c) depot injection with buprenorphine available for humans (Buvidal). Methods: Spraque Dawley rats were used. The depot formulation (Buvidal, 1.5mg/kg s.c, n=24) was evaluated and compared to a short-acting buprenorphine formulation commonly used in laboratory rodents (Temgesic, 0.1 mg/kg s.c, n=18) and negative control (s.c saline, n=24). The analgesic effect was assessed using the von Frey pressure test on the plantar surface of the right hind paw, allowing analgesic efficacy to be evaluated without exposing the animals to any other pain. Post-dose results at 3h, 6h and 24h following injection were compared to pre-dose levels. Results: At 3h and 6h post-dose both buprenorphine formulations showed a significant analgesic effect compared to baseline. At 6h the effect was more pronounced in rats that had received the depot injection. At 24h, only the depot formulation still showed a significant effect. Saline did not alter the sensitivity to the pressure test at any time-point. Conclusion: A human prolonged-release buprenorphine formulation (Buvidal) available in Europe has long-term analgesic effect in rats (up to 24h), as evaluated using the von Frey pressure test. This formulation should be considered as an alternative to multiple injections of short-acting buprenorphine formulations in rat studies where opioid analgesia is desired.

Pharmacological characterization of gepants in human and porcine vasculature

Abstract text (incl. figure legends and references) Objectives We aim to perform an in-depth pharmacological characterization of the potency of several gepants in porcine vasculature, in comparison with the potency in human blood vessels. Methods Distal coronary artery segments from 6 swine, obtained from the local slaughterhouse, were isolated and mounted in Mulvany myographs for isometric contraction measurements. Concentration response curves to human α-CGRP (10-10 – 3*10-6 M) were constructed in the absence or presence of increasing concentrations of olcegepant, rimegepant, zavegepant and telcagepant. The potency of the antagonists was determined by calculating pKb values. Results were compared to these obtained earlier in human isolated distal coronary arteries. Results Our results on olcegepant confirm our earlier observations (Gupta, et al. Eur J Pharmacol. 2006) of a lower potency in porcine coronary artery compared to human coronary artery (pKb 7.59±0.27 vs 9.13±0.17 respectively at 100 nM olcegepant). Preliminary results on rimegepant point to a similar difference in potency, as exemplified by the pKb values (100 nM: 6.35±0.04 vs 8.71±0.16, 1 μM: 6.35±0.04 vs 8.43±0.25 respectively) (Mulder, et al. Ann Neurol. 2020). Similarly, preliminary results on zavegepant again show a lower potency in porcine coronary arteries compared to human coronary artery (unpublished data) (pKb 100nM: 7.00±0.54 vs 9.91±0.15 respectively). For telcagepant an insufficient number of experiments was performed at the time of submission of the abstract. Conclusions Our initial analyses suggest that the difference in potency between porcine and human vasculature is similar for different gepants. Knowledge on this difference in potency between species, combined with molecular information about the structure of the CGRP receptor and the antagonists, provides information on the implications of the potency of gepants for translational research in experimental animals.

Hypothalamic paraventricular stimulation inhibits trigeminal nociceptive transmission via oxytocin receptors

Abstract text (incl. figure legends and references) Question. Recent data suggest that exogenous oxytocin exerts antinociception at the trigeminal level. Although this peptide is released from the hypothalamic paraventricular nucleus (PVN), little is known about the role of endogenous oxytocinergic neurotransmission modulating trigeminal nociception. This study tested the effect of PVN stimulation on the trigeminal nociceptive responses elicited by activation of the trigeminal nerve. Methods. In vivo electrophysiological recordings of trigeminal WDR cells and immunohistological studies were performed in rats. The animals were anesthetized with sevoflurane, assisted with mechanical ventilation, and mounted in a stereotaxic frame. A surgery to access the medullary dorsal horn was performed, and a small craniotomy was made to place a concentric stainless-steel stimulation electrode (1 MW) in the PVN. Under this condition, extracellular unitary recordings of trigeminal WDR cells with input from the first branch of the trigeminal nerve (V1) were made with quartz-Pt-W microelectrodes (4-10 MW). In addition, retrograde neuronal tracing with fluoro-gold® from the spinal trigeminal region to the PVN was assessed in search of oxytocinergic fibers. Results. PVN electrical stimulation (6 sec, 60 Hz, 1 msec pulse duration, 300 mA) inhibited the peripheral evoked trigeminal nociceptive responses. This inhibition was reversed by a peptide OTR antagonist given spinally (dOVT, d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]OVT). Furthermore, the retrograde labeling showed that direct oxytocinergic projections from trigeminal nucleus caudalis to PVN exist. Conclusion. Coupled with previous reports showing that exogenous oxytocin administration at the trigeminal level inhibited the periorbital nociceptive responses via OTR, our data strongly support the notion that PVN via oxytocinergic transmission inhibits trigeminal nociception, suggesting that enhancement of oxytocinergic transmission could be used as a potential therapy to treat headaches.

Poster session 1

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