Protease activated receptor 2 (PAR2) activation causes migraine-like pain behaviors in mice

Background Pain is the most debilitating symptom of migraine. The cause of migraine pain likely requires activation of meningeal nociceptors. Mast cell degranulation, with subsequent meningeal nociceptor activation, has been implicated in migraine pathophysiology. Degranulating mast cells release serine proteases that can cleave and activate protease activated receptors. The purpose of these studies was to investigate whether protease activated receptor 2 is a potential generator of nociceptive input from the meninges by using selective pharmacological agents and knockout mice. Methods Ratiometric Ca++ imaging was performed on primary trigeminal and dural cell cultures after application of 2at-LIGRL-NH2, a specific protease activated receptor 2 agonist. Cutaneous hypersensitivity and facial grimace was measured in wild-type and protease activated receptor 2−/− mice after dural application of 2at-LIGRL-NH2 or compound 48-80, a mast cell degranulator. Behavioral experiments were also conducted in mice after dural application of 2at-LIGRL-NH2 (2AT) in the presence of either C391, a selective protease activated receptor 2 antagonist, or sumatriptan. Results 2at-LIGRL-NH2 evoked Ca2+ signaling in mouse trigeminal neurons, dural fibroblasts and in meningeal afferents. Dural application of 2at-LIGRL-NH2 or 48-80 caused dose-dependent grimace behavior and mechanical allodynia that were attenuated by either local or systemic application of C391 as well as in protease activated receptor 2−/− mice. Nociceptive behavior after dural injection of 2at-LIGRL-NH2 was also attenuated by sumatriptan. Conclusions Functional protease activated receptor 2 receptors are expressed on both dural afferents and fibroblasts and activation of dural protease activated receptor 2 produces migraine-like behavioral responses. Protease activated receptor 2 may link resident immune cells to meningeal nociceptor activation, driving migraine-like pain and implicating protease activated receptor 2 as a therapeutic target for migraine in humans.

Periaqueductal gray calcitonin gene-related peptide modulates trigeminovascular neurons

Background Calcitonin gene-related peptide (CGRP) receptor antagonism is an approach to migraine therapy. The locus of action of antimigraine treatment is not resolved. The objective was to investigate CGRP receptors in the ventrolateral periaqueductal gray (vlPAG) involved in the modulation of trigeminovascular nociception by descending influences on neurotransmission. Methods The presence of calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1), which form functional CGRP receptors, was investigated. CGRP and its receptor antagonists, olcegepant and CGRP ( 8 – 37 ), were microinjected into the vlPAG while changes of neural responses in the trigeminocervical complex (TCC) were monitored. Results Immunoreactivity indicated the presence of functional CGRP receptor components in the vlPAG and adjacent mesencephalic trigeminal nucleus. Inhibition of TCC responses to stimulation of dural afferents and ophthalmic cutaneous receptive fields after microinjection of bicuculline into vlPAG indicated a connection between the vlPAG and TCC neurons. CGRP facilitated these TCC responses, whereas olcegepant and CGRP ( 8 – 37 ) decreased them. Conclusions CGRP and its receptor antagonists act on neurons in the region of vlPAG to influence nociceptive transmission in the TCC. This suggests CGRP receptor antagonists may act at loci outside of the TCC and reinforces the concept of migraine as a disorder of the brain.

Meningeal norepinephrine produces headache behaviors in rats via actions both on dural afferents and fibroblasts

Background Stress is commonly reported to contribute to migraine although mechanisms by which this may occur are not fully known. The purpose of these studies was to examine whether norepinephrine (NE), the primary sympathetic efferent transmitter, acts on processes in the meninges that may contribute to the pain of migraine. Methods NE was applied to rat dura using a behavioral model of headache. Primary cultures of rat trigeminal ganglia retrogradely labeled from the dura mater and of rat dural fibroblasts were prepared. Patch-clamp electrophysiology, Western blot, and ELISA were performed to examine the effects of NE. Conditioned media from NE-treated fibroblast cultures was applied to the dura using the behavioral headache model. Results Dural injection both of NE and media from NE-stimulated fibroblasts caused cutaneous facial and hindpaw allodynia in awake rats. NE application to cultured dural afferents increased action potential firing in response to current injections. Application of NE to dural fibroblasts increased phosphorylation of ERK and caused the release of interleukin-6 (IL-6). Conclusions These data demonstrate that NE can contribute to pro-nociceptive signaling from the meninges via actions on dural afferents and dural fibroblasts. Together, these actions of NE may contribute to the headache phase of migraine.

Activation of TRPV4 on dural afferents produces headache-related behavior in a preclinical rat model

Background: The mechanisms contributing to the pain of migraine are poorly understood although activation of afferent nociceptors in the trigeminovascular system has been proposed as a key event. Prior studies have shown that dural-afferent nociceptors are sensitive to both osmotic and mechanical stimuli. Based on the sensitivity to these stimuli we hypothesized that dural afferents express the osmo/mechano-sensitive channel transient receptor-potential vanilloid 4 (TRPV4). Methods: These studies used in vitro patch-clamp electrophysiology of trigeminal neurons retrogradely labeled from the dura to examine the functional expression of TRPV4. Additionally, we used a rat headache model in which facial/hind paw allodynia following dural stimulation is measured to determine whether activation of meningeal TRPV4 produces responses consistent with migraine. Results: These studies found that 56% and 49% of identified dural afferents generate currents in response to hypotonic solutions and 4α-PDD, respectively. The response to these stimuli indicates that dural afferents express TRPV4. Activation of meningeal TPRV4 using hypotonic solution or 4α-PDD in vivo resulted in both facial and hind paw allodynia that was blocked by the TRPV4 antagonist RN1734. Conclusion: These data indicate that activation of TRPV4 within the meninges produces afferent nociceptive signaling from the head that may contribute to migraine headache.

Sex differences in the inflammatory mediator-induced sensitization of dural afferents

Approximately 20% of the adult population suffers from migraine. This debilitating pain disorder is three times more prevalent in women than in men. To begin to evaluate the underlying mechanisms that may contribute to this sex difference, we tested the hypothesis that there is a sex difference in the inflammatory mediator (IM)-induced sensitization of dural afferents. Acutely dissociated retrogradely labeled dural afferents from adult Sprague-Dawley rats were examined with whole cell patch-clamp recordings. Baseline passive and active electrophysiological properties of dural afferents from both sexes were comparable. However, while IM-induced increases in the excitability of dural afferents from male and female rats were also comparable, the proportion of dural afferents from female rats sensitized by IM (∼100%) was significantly greater than that of dural afferents from male rats (∼50%). This appeared to be due to differences downstream of IM receptors, as tetrodotoxin-resistant sodium current was increased by IM in a majority of male dural afferents (13/14). These data indicate that there are both quantitative and qualitative differences in the IM-induced sensitization of dural afferents that may contribute to the sex difference in the manifestation of migraine.