Migraine is one of the most disabling disorders worldwide but the underlying mechanisms are poorly understood. Stress is consistently reported as a common trigger of migraine attacks. Here, we show that repeated stress in mice causes migraine-like behaviors that are responsive to a migraine therapeutic. Adult female and male mice were exposed to 2 hours of restraint stress for 3 consecutive days, after which they demonstrated facial mechanical hypersensitivity and facial grimace responses that were resolved by 14 days after stress. Hypersensitivity or grimace was not observed in either control animals or those stressed for only 1 day. After return to baseline, the nitric oxide donor sodium nitroprusside (SNP; 0.1 mg/kg) elicited mechanical hypersensitivity in stressed but not in control animals, demonstrating the presence of hyperalgesic priming. This suggests the presence of a migraine-like state, because nitric oxide donors are reliable triggers of attacks in migraine patients but not controls. The stress paradigm also caused priming responses to dural pH 7.0 treatment. The presence of this primed state after stress is not permanent because it was no longer present at 35 days after stress. Finally, mice received either the calcitonin gene-related peptide monoclonal antibody ALD405 (10 mg/kg) 24 hours before SNP or a coinjection of sumatriptan (0.6 mg/kg). ALD405, but not sumatriptan, blocked the facial hypersensitivity due to SNP. This stress paradigm in mice and the subsequent primed state caused by stress allow further preclinical investigation of mechanisms contributing to migraine, particularly those caused by common triggers of attacks.

Migraine is thought to involve sensitization of the trigeminal nociceptive system. In preclinical pain models, activation of MNK-eIF4E signaling contributes to nociceptor sensitization and the development of persistent pain. Despite these observations, the role of MNK signaling in migraine remains unclear. Here, we investigate whether activation of MNK contributes to hypersensitivity in two rodent models of migraine. Female and male wild-type (WT) and MNK1 knock-out (KO) mice were subjected to repeated restraint stress or a dural injection of interleukin-6 (IL-6) and tested for periorbital hypersensitivity and grimacing. Upon returning to baseline thresholds, stressed mice were administered a low dose of the nitric oxide donor sodium nitroprusside (SNP) and mice previously injected with IL-6 were given a second dural injection of pH 7.0 to test for hyperalgesic priming. MNK1 KO mice were significantly less hypersensitive than WTs following dural IL-6 and did not prime to pH 7.0 or SNP. Furthermore, treatment with the selective MNK inhibitor, eFT508, in WT mice prevented hypersensitivity caused by dural IL-6 or pH 7.0. Together, these results implicate MNK-eIF4E signaling in the development of pain originating from the dura and strongly suggest that targeting MNK inhibition may have significant therapeutic potential as a treatment for migraine.

Background Migraine attacks are often triggered by normally innocuous stimuli, suggesting that sensitization within the nervous system is present. One mechanism that may contribute to neuronal sensitization in this context is translation regulation of new protein synthesis. The goal of this study was to determine whether protein synthesis contributes to behavioral responses and priming in preclinical models of migraine. Methods Mice received a dural injection of interleukin-6 in the absence or presence of the protein synthesis inhibitor anisomycin or the translation initiation inhibitor 4EGI-1 and were tested for facial hypersensitivity. Upon returning to baseline, mice were given a second, non-noxious dural injection of pH 7.0 to test for priming. Additionally, eIF4ES209Amice lacking phosphorylation of mRNA cap-binding protein eIF4E received dural interleukin-6 or were subjected to repeated restraint stress and then tested for facial hypersensitivity. After returning to baseline, mice were given either dural pH 7.0 or a systemic sub-threshold dose of the nitric oxide donor sodium nitroprusside and tested for priming. Results Dural injection of interleukin-6 in the presence of anisomycin or 4EGI-1 or in eIF4ES209Amice resulted in the partial attenuation of acute facial hypersensitivity and complete block of hyperalgesic priming. Additionally, hyperalgesic priming following repeated restraint stress was blocked in eIF4ES209Amice. Conclusions These studies show that de novo protein synthesis regulated by activity-dependent translation is critical to the development of priming in two preclinical models of migraine. This suggests that targeting the regulation of protein synthesis may be a novel approach for new migraine treatment strategies.

Administration of a nitric oxide (NO) donor triggers migraine attacks but mechanisms by which this occurs are unknown. Reactive nitroxidative species, including NO and peroxynitrite (PN), have been implicated in nociceptive sensitization and neutralizing PN is anti-nociceptive. We determined whether PN contributes to nociceptive responses in two distinct models of migraine headache. Female and male mice were subjected to three consecutive days of restraint stress or to dural stimulation with the pro-inflammatory cytokine interleukin-6. Following resolution of the initial post-stimulus behavioral responses, animals were tested for hyperalgesic priming using a normally non-noxious dose of the NO donor sodium nitroprusside (SNP) or dural pH 7.0, respectively. We measured periorbital von Frey and grimace responses in both models and measured stress-induced changes in 3-nitrotyrosine (3-NT) expression (a marker for PN activity) and trigeminal ganglia (TG) mitochondrial function. Additionally, we recorded TG neuronal activity in response to the PN generator SIN-1. We then tested the effects of the PN decomposition catalysts, FeTMPyP and FeTPPS, or the PN scavenger MnTBAP against these behavioral, molecular, and neuronal changes. Neutralizing PN attenuated stress-induced periorbital hypersensitivity and priming to SNP, with no effect on priming to dural pH 7.0. These compounds also prevented stress-induced increases in 3-NT expression in both the TG and dura mater and attenuated TG neuronal hyperexcitability caused by SIN-1. Surprisingly, FeTMPyP attenuated changes in TG mitochondrial function caused by SNP in stressed males only. Together, these data strongly implicate PN in migraine mechanisms and highlight the therapeutic potential of targeting PN.SIGNIFICANCE STATEMENT:Among the most reliable experimental triggers of migraine are nitric oxide donors. Mechanisms by which nitric oxide triggers attacks are unclear but may be due to reactive nitroxidative species such as peroxynitrite. Utilizing mouse models of migraine headache, we show that peroxynitrite-modulating compounds attenuate behavioral, neuronal, and molecular changes caused by repeated stress and nitric oxide donors (two of the most common triggers of migraine in humans). Additionally, our results show a sex-specific regulation of mitochondrial function by peroxynitrite following stress, providing novel insight into the ways in which peroxynitrite may contribute to migraine-related mechanisms. Critically, our data underscore the potential in targeting peroxynitrite formation as a novel therapeutic for the treatment of migraine headache.

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