16th European Headache Congress

Chair

Prof.

Inhibition of peripheral FAAH alleviates hyperalgesia induced by acute dural inflammation in experimental migraine

Abstract text (incl. figure legends and references) Peripheral and central sensitization is an important pathophysiological process in migraine and its chronification, but the underlying mechanisms are largely unclear. Trigeminal hyperalgesia and/or facial allodynia have been tested in animal models to simulate clinical symptomatology and to investigate the mechanisms underlying migraine pain. Inhibition of the fatty-acid amide hydrolase (FAAH) activity, the enzyme that deactivates the endocannabinoid anandamide, may exert anti-nociceptive effects via the activation of peripheral cannabinoid (CB1) receptors. Aim - Here, we evaluated the effects of FAAH inhibition in a migraine model of peripheral sensitization obtained by evaluating trigeminal hypersensitivity at the orofacial formalin test in animals with dural inflammation. Methods – Dural inflammation was induced in male Sprague-Dawley rats with the infusion of an inflammatory soup (IS) onto the dura. Ten min after IS infusion, rats were treated, with the peripheral FAAH inhibitor URB937 (1mg/kg, i.p.) or vehicle. A first set of rats underwent the orofacial formalin test 2h after IS application, while a second experimental set was used to evaluate the expression of CGRP and pro-inflammatory cytokines in specific brain areas in toto. Results - IS infusion induced trigeminal hyperalgesia at the orofacial formalin test, associated with an increase in gene expression of CGRP and pro-inflammatory molecules in medulla-pons and trigeminal ganglia. URB937 administration inhibited IS-induced trigeminal hyperalgesia and the prevented the increase in gene expression of CGRP and pro-inflammatory molecules in the nervous tissues. Conclusions - The findings suggest that potentiation of the endocannabinoid tone, obtained via peripheral FAAH inhibition, may modulate trigeminal hyperalgesia induced by dural IS through the reduction of pro-inflammatory cytokines and CGRP synthesis at central and peripheral sites of the nervous system.

Basic science, animal models in headache research

Migraine

Abstract lecture (online)

Aryl hydrocarbon receptors are involved in the pathogenesis of migraine

Abstract text (incl. figure legends and references) Objective: Migraine is a prevalent disorder, with episodic attacks. Some patients experience an increase in attack frequency and develop chronic migraine. However, the underlying mechanisms of this progression are uncertain. The noradrenergic locus coeruleus (LC) has been involved in migraine chronicity. Recent studies found that selective activation of LC alleviates pain by reducing neuroinflammation. As the aryl hydrocarbon receptors (AhRs) are key regulators of the brain inflammatory responses, we, therefore, investigate the contribution of LC-expressed AhRs in migraine progression. Methods: Using immunohistochemical approaches, we assessed the intracellular distribution and the expression levels of AhRs in the LC in a mice model of migraine induced by systemic administration of isosorbide dinitrate (ISDN; a nitric oxide donor). We also explored the effect of systemic AhR activation (by the indole agonist ITE) or inhibition (by the pure antagonist TMF) on cutaneous mechanical hypersensitivity (CMH) induced by ISDN. Results: In naïve mice, AhRs were detected in 47.1 ± 3.4 % of LC noradrenergic neurons. Repeated ISDN administration resulted in a significant increase in this percentage (56.7 ± 3.4%, P&lt;0.05). Interestingly, morphological alterations of LC neurons together with an increase of the soma and nucleus sizes were also observed. Behavioral testing showed that single or repeated TMF or ITE administrations did not affect cephalic mechanical sensitivity in naïve mice. In addition, ITE did not worsen the ISDN-induced CMH. However, a single administration of TMF effectively blocked ISDN-induced acute CMH, while daily administration was unable to prevent long-lasting CMH. Conclusions: These data highlight, for the first time, the involvement of AhRs in the initiation but not in the maintenance of migraine.

Abstract lecture

Miss

Orexin-A recovers sleep-deprivation induced periorbital allodynia in a preclinical migraine model

Abstract text (incl. figure legends and references) Questions: There is a bidirectional link between sleep and migraine with poor sleep reported as both a migraine trigger and symptom. Orofacial allodynia, or hypersensitivity to a non-painful stimulus, is a commonly reported migraine symptom. This study aimed to determine whether sleep deprivation results in orofacial allodynia and whether the arousal promoting neuropeptide orexin-A can recover this. Methods: Mice were sleep deprived for 6 hours using the gentle handling method or allowed to sleep as usual (n = 12 per group). Periorbital mechanical withdrawal thresholds were tested pre and post deprivation using the von Frey assay. In a series of experiments, mice were administered either 100µg/kg orexin-A/vehicle or 20mg/kg caffeine/vehicle intraperitoneal and thresholds were tested 30 minutes post injection. To measure arousal, locomotor activity after administration of each drug was tested using infrared sensors. To establish if this effect is specific to sleep deprivation, orexin-A was administered after delivery of the clinical migraine trigger nitroglycerin. Results: Caffeine significantly increased gross movements, and both caffeine and orexin-A significantly increased fine movements compared to vehicle. Sleep deprived mice had significantly lower sensory thresholds than non-sleep deprived mice. Injection of orexin-A, but not caffeine, significantly increased thresholds in sleep deprived mice, despite similar effects on arousal. Nitroglycerin significantly decreased thresholds, however orexin-A did not recover this. Conclusions: These findings demonstrate that sleep deprivation leads to orofacial allodynia in mice. This can be reversed with administration of orexin-A, but not caffeine, indicating that this is unlikely to be driven by arousal. However, orexin-A was not analgesic when delivered after nitroglycerin-induced allodynia. Further research should explore the role of orexin-A in migraine.

Neuropeptides, channels in headache

Involvement of the hypothalamus in migraine-related pathways﻿Authors: Isabella Mai Christiansen, Anja Holm Nordvang, Lars Edvinsson, Kristian Agmund Haanes

Abstract text (incl. figure legends and references) Objective According to the current hypothesis of migraine pathophysiology, attacks are initiated in the hypothalamus, whereafter an activation cascade commences, which results in the activation of the trigeminal nucleus caudalis (TNC) and then trigeminal ganglion (TG). Calcitonin gene-related peptide (CGRP) release from the trigeminal system is thought to play a central role in migraine pathology, supported by the fact that currently available antimigraine drugs act by blocking CGRP signalling. The aim was to perform a preliminary examination of the hypothalamus in migraine-related pathways. Methods CGRP release from hypothalamus-containing acute brain slices was examined using an enzyme-linked immunosorbent assay. In addition, we investigated expression levels of Calca (encoding CGRP) and other genes related to dopaminergic pathways in the hypothalamus and the TNC. Results An extracellular concentration of 60 mM potassium significantly induced CGRP release, as compared to baseline (p = 0.01, n = 6), whereas application of 100 nM capsaicin did not (p = 0.16, n = 6). Using quantitative real-time polymerase chain reaction (qRT-PCR), we found that Calca was expressed in the hypothalamus, but no difference was observed between male/female mice. Drd2 (encoding the D2 receptor) was the most highly expressed dopamine receptor in both the male and female hypothalamus and TNC, indicating a dominantly inhibitory effect of dopamine in these regions. In addition, results from qRT-PCR demonstrated a tendency towards lower expression levels of Drd2 in the TNC of female mice, as compared to males. Conclusion Central CGRP release can be detected from brain slices of mice, but the expression of Calca did not show any male/female difference. However, we found some sex differences in the dopaminergic pathways, suggesting that lower expression levels of Drd2 in females as compared to males could contribute to increased excitability of the TNC.

Headache, thalamus and other brain nuclei

P2Y14 receptor in trigeminal ganglion contributes to neuropathic pain in mice

Abstract text (incl. figure legends and references) Question: Trigeminal nerve injury usually induces trigeminal neuropathic pain but lacks effective treatments. Recent reports implied that P2Y14 receptor (P2Y14R) activation promoted orofacial inflammatory pain and migraine. However, the role and mechanism of P2Y14R in trigeminal neuropathic pain remain unknown. Methods: Trigeminal neuropathic pain was induced by chronic constriction injury of the infraorbital nerve (CCI-ION). Orofacial mechanical threshold was measured by Von-Frey tests. The ATF3 (a mark of nerve injury) and P2Y14R were detected by immunofluorescence in the trigeminal ganglion (TG). The P2Y14R agonist (UDP-glucose) or antagonist (PPTN) was delivered by trigeminal ganglion injection in a stereotaxic apparatus. Furthermore, the expression of P2Y14R and its potential downstream cellular signalings were measured by RT-qPCR and/or western blot (WB) in the TG. Results: Firstly, CCI-ION induced orofacial mechanical hypersensitivity. The increased ATF3 expression in the TG confirmed trigeminal nerve injury. P2Y14R was expressed in trigeminal ganglion neurons and satellite glial cells. RT-qPCR and WB showed that CCI-ION increased the expression of P2Y14R, interleukin-1β, interleukin-6, C-C chemokine CCL2, and tumor necrosis factor-α in TG. Secondly, PPTN alleviated CCI-ION-induced mechanical hypersensitivity and proinflammatory cytokines production. UDP-glucose evoked orofacial mechanical hypersensitivity and upregulated proinflammatory cytokines above. Thirdly, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 were increased in the TG after CCI-ION, which also were reduced by PPTN. Furthermore, the inhibitors of ERK1/2 (U0126) and p38 (SB203580) decreased these CCI-ION-induced proinflammatory cytokines. Conclusions: The present study demonstrated that P2Y14R in the TG contributed to trigeminal neuropathic pain via ERK- and p38-dependent neuroinflammation. P2Y14R may be a potential drug target against trigeminal neuropathic pain.

Oro-facial and temporomandibular disorders

Kir4.1 in satellite glial cells contributes to trigeminal neuropathic pain

Abstract text (incl. figure legends and references) Question: Astrocyte Kir4.1 emerged as a novel therapeutic target for nervous system diseases, such as depression and pain. However, the role and mechanism of Kir4.1 in satellite glial cells (SGCs) in trigeminal neuropathic pain remain unknown. Methods: In vivo, chronic constriction injury of the infraorbital nerve (CCI-ION) and Von-Frey tests were used. The ATF3, GFAP, and Kir4.1 were detected by immunofluorescence in the trigeminal ganglion (TG). The AAV2/8 aimed at Kir4.1 was delivered to TG in WT and Kir4.1f/f mice to knockdown or overexpress Kir4.1. In vitro, cultured SGCs were transfected with siRNA to knock down Kir4.1. The expression of Kir4.1 and its potential downstream cellular signalings were measured by RT-qPCR, western blot (WB), and/or ELISA. Results: Firstly, CCI-ION induced orofacial mechanical hypersensitivity. The increased ATF3 confirmed trigeminal nerve injury and increased GFAP indicated SGCs activation. Kir4.1 was expressed on SGCs of TG. RT-qPCR and WB showed that CCI-ION decreased Kir4.1 expression but increased the expression of GFAP, brain-derived neurotrophic factor (BDNF), and glial cell-derived neurotrophic factor (GDNF) in TG. Secondly, knockdown of Kir4.1 in mice evoked orofacial mechanical hypersensitivity and upregulated GFAP, BDNF, and GDNF expression in TG. Overexpression of Kir4.1 alleviated CCI-ION-induced mechanical hypersensitivity and GFAP, BDNF, and GDNF production. In vitro, Kir4.1-siRNA treated SGCs increased GFAP, BDNF, and GDNF expression. Thirdly, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) was increased in TG after CCI-ION, knockdown of Kir4.1 mice, and Kir4.1-siRNA treated SGCs, which also were reduced by Kir4.1 rescue. Furthermore, the inhibitors of ERK1/2 (U0126) decreased these above upregulated BDNF and GDNF. Conclusions: This study indicated that Kir4.1 in SGCs contributed to trigeminal neuropathic pain by regulating BDNF and GDNF production via ERK signaling pathway and SGCs activation.

Prof. Dr.

CGRP-induced relaxation remains unaffected in a porcine acute ischaemic stroke model

Abstract text (incl. figure legends and references) Objective Calcitonin gene-related peptide (CGRP) plays an important role in migraine attacks and is thought to be protective in ischaemic events. We investigated functional responses to exogenous CGRP of the middle cerebral artery (MCA) harvested from a gyrencephalic model of focal cerebral ischaemia. Methods Female pigs (n=25; weight 51±3kg) underwent a craniotomy to occlude right-sided MCAs with aneurysm clips for 1 (n=5), 2 (n=5), or 4 (n=5) hours followed by 4-h recanalization, or for 8 h without recanalization (n=5). Two animals underwent a sham procedure. After euthanasia, the right-sided MCA was dissected for tension measurements. KCl-induced contraction (30–100 mM) was measured and endothelial function was assessed by bradykinin (BK)- or substance P (SP)-induced relaxation (10–100 nM) after precontraction with thromboxane A2 analogue U46619 (10–100 nM). Concentration-response curves were constructed to CGRP (0.1–100 nM) after precontraction with 30 mM KCl. Results Relaxation to SP and BK was significantly reduced after 8 h occlusion (P&lt;0.05), but not after occlusion followed by recanalization. Contraction to KCl was only reduced after 8 h occlusion (P&lt;0.001). No differences were found in response to CGRP between groups (Figure). Conclusions CGRP-induced relaxation remained unaffected after 8 h MCA occlusion, whereas SP- and BK-induced relaxation did not. These results suggest that CGRP acts via an endothelium-independent mechanism and that its potential protective effects after stroke depend on its local release rather than changes at the CGRP receptor level. Figure: (A) No altered CGRP-induced relaxation after (recanalized) MCA occlusion. Reduced endothelial (B) and contractile (C) function after 8 h MCA occlusion. Results depicted as mean ± SEM.

Pathophysiology others than migraine and cluster

A powerful dual MAGL/FAAH inhibitor AKU-005 against migraine pain of peripheral meningeal origin

Abstract text (incl. figure legends and references) Question. Migraine is a neurological multifactorial disease whose worst symptom is pain. To deal with disturbing migraine pain, we propose the engaging of endocannabinoid system (ECS) via inhibition of enzymes monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), which are degrading 2-arachidonoylglicerol (2-AG) and anandamide (AEA), respectively. To this end, we explored the analgesic effect of enhanced endocannabinoids (endoCBs) in peripheral meningeal tissues where pain signaling is of often originating in migraine, leading later to central sensitization. Methods. To develop the functional platform for such purpose, we measured by activity-based protein profiling (ABPP) the activity of the main endoCBs-hydrolases, MAGL and FAAH and by LC-MS/MS the levels of 2-AG and AEA in rat meninges. We explored the analgesic effect of enhanced endoCBs with electrophysiological recordings from rat peripheral meningeal afferents. Results. We found that in the meninges, 2-AG is the main endoCB, much exceeding the level of AEA. However, local depolarization increased the AEA ~2 folds without affecting 2-AG levels. The dual MAGL/FAAH inhibitor AKU-005 slightly increased basal nociceptive activity of trigeminal nerves. Instead, it significantly decreased, through CB1 receptors, meningeal nociceptive activity induced by depolarizing action of KCl, indicating analgesic effect. Conclusions. These results suggest that 2-AG and AEA can differently contribute to counteract migraine pain by tonic or transient activity dependent release from meninges. These findings support the therapeutic perspective for engagement of both endoCBs analgesic molecules at early stages of attack. Thus, our novel dual ultrapotent MAGL/FAAH inhibitor AKU-005 appeared to be a promising tool in reducing migraine nociception originating in the meninges.

SciSe 13
Abstracts basic

Appointment

Chair

Programme

SciSe 13Abstracts basic

Appointment

Chair

Programme

SciSe 13
Abstracts basic