DISCUSSION
Migraine has a higher prevalence in female and negatively affects the quality of life of patients [21]. Stress is a common trigger reported in migraine patients, but the underlying mechanisms are poorly understood [30,53]. Indeed, different types of stress, such as repetitive restrain stress paradigm, social defeat stress, chronic variable stress, and early life stress have been used as models of migraine-like behavior in animals [8,20]. Besides, previous studies described that unpredictable sound stress model causes plantar allodynia and chemical hyperalgesia in male rats [17,32]. In this sense, the unpredictable sound stress was used as a model to induce painful hypersensitivity in rats [17,32]. Sound stress also represents a type of stress that normally occurs in humans [26,55]. However, nociceptive behaviors had not been evaluated in mice in this model of stress, and it had not yet been characterized as a model of migraine. Thus, here we characterize the migraine-like behaviors in a model of stress mediated by unpredictable sound in male and female mice.
In this sense, previous studies have shown that a significant proportion of individuals with migraine had experienced cutaneous allodynia and spontaneous pain during episodes of headache [28]. Thus, different models of migraine evaluate the development of periorbital and hind paw allodynia, and grimacing pain behavior [8,29]. Here, we detected these nociceptive parameters after unpredictable sound stress induction, with a nociceptive peak at 7 days after stress induction. Recently, using a model of repeated restrain stress in mice, Avona and colleagues [8] showed periorbital mechanical allodynia and grimacing pain behavior in mice. Similar, to this previous study, we also observed that nociception was not detected after 14 days of stress induction, either for mechanical allodynia or grimacing pain behavior. Thus, we characterize a new model of migraine-like pain induced by unpredictable sound stress induction. This is an interesting model, because migraine patients are more sensible to sound, and studies described that sound could be a stress factor to induce migraine pain attacks [26,55].
Moreover, migraine is also associated to psychiatric disorders, such as anxiety [27]. Some previous studies described that nociception in migraine-like models could be accompanied by exploratory behavior alteration and anxiety-like symptoms [5,50]. In this view, we also detected that stress induction decreased the time of different exploratory behaviors, such as rearing and sniffing, and the number of crossings. Besides, stress was correlated with an increased time of grooming and the permanence at the peripheral zone. In previous studies using models of migraine, the reduction in rearing behavior, was accepted as an indicative of spontaneous pain. Decrease in feeding and exploratory behavior [19], an increase in facial grooming [22] were also correlated to migraine-like pain. Besides, after exposure to repeated stress mice stayed more time in the peripheral zone than controls [25,29], and it is a behavior parameter widely used for assessing anxiety [46]. Additionally, in migraine-like model induced by nitroglycerin authors verified increased anxiety-like symptoms in the open field test in rats [50]. Thus, our study also presented other aspects related to migraine-like pain, such as anxiety-like symptoms and decrease of exploratory behavior, these are relevant aspects of a model of migraine [8,25,29].
Migraine affects three times more women than men, but the mechanisms underlying this dimorphism are not known [8,52]. Some studies showed a sexual dimorphism in migraine-like models, with female rodents showing higher nociceptive and anxiety-like responses [5]. Recently, using the migraine model induced by nitroglycerin i.p. injection, a sexual dimorphic effect was detected in which females had higher nociception than males [2]. However, most of the studies using models of migraine did not evaluate the sexual differences, and male mice or rats are normally tested [32,50]. Previous studies, utilizing sound stress demonstrated hind paw nociception, but have been performed only in male rats [15]. Only one study evaluated sexual differences in mice after stress induction (repeated restrain stress) but did not find any difference for periorbital mechanical allodynia and grimacing pain behavior [8]. Nevertheless, here we demonstrated that unpredictable sound stress induces higher nociceptive and anxiety-like parameters in females than ST males. Probably, the differences detected could be induced by the stress model used.
In migraine patients, it was already demonstrated increased TNF-α and IL-6 plasmatic levels compared to healthy controls [34,40]. Similarly, in rodents, pro-inflammatory cytokines were described as inductors of primary afferent nociceptors sensitization [33,34]. Also, high plasmatic levels of CGRP have already been demonstrated in external jugular venous blood, saliva, and cerebrospinal fluid of migraine patients during an attack [3]. Besides, migraine animal model induce an increase in circulating CGRP levels in plasma [13,49], cerebrospinal fluid of the nestin/hRAMP1 transgenic mice [45]. Additionally, CGRP may trigger the release of pro-inflammatory cytokines [44], particularly IL-6 and TNF-α, which are also increased in the plasm of migraine patients [14,56].
In accordance with these findings, we had demonstrated that unpredictable sound-stress induced elevated the levels of circulating of IL-6, TNF-α, and CGRP in male and female mice. Acute stress models, including restrain and social isolation, caused high plasmatic levels of IL-6 [43,47]. However, no model of stress inducing migraine-like behavior had done this measure before [8,20,29]. The injection of IL-6 (dura mater and cisterna magna) and CGRP (dura mater and intraganglionar injection) induces nociception [5,7]. Dural meningeal nociceptors were also sensitized by TNF-α application [57]. Besides, unpredictable sound stress model induced muscle nociception could be reduced by antisense injection to IL-6 or TNF-α receptors [17]. Seeking to elucidate the sexual dimorphism presented in nociceptive parameters, the ST females presented higher levels of both TNF-α, IL-6, and CGRP levels than compared to male ST mice. Our results corroborate the study of Avona and colleagues, in which the sexually dimorphic effect of CGRP female-specific hypersensitivity responses was seen in mice, where increased grimace responses were also observed [6]. In fact, estrogen has been shown that was able to regulate the release of CGRP [42,48]. Therefore, data are innovative and to our knowledge already have not been assessed in migraine models. Thus, it can be explored in future pre-clinical studies and in research with migraine patients.
Recent reports indicate that mechanisms of pain may differ between the sexes, and a potential role for spinal prolactin has been implicated in the production of IL-6 induced hind paw allodynia [1,38]. Similarly, co-injection of prolactin with IL-6 increases hind paw hypersensitivity in female mice [41]. Furthermore, several studies indicate that fluctuations of ovarian steroid hormone (mainly estrogen) levels modulate CGRP in the trigeminovascular system during different reproductive milestones migraine and suggest that female-specific mechanisms downstream of CGRP receptor activation contribute to the higher prevalence of migraine in women [35].
Recent clinical studies have further confirmed a protagonist of CGRP in migraine due to the use of inhibitors of CGRP signaling, such as antagonists and monoclonal antibodies to CGRP receptor [24]. Here, we demonstrated that a CGRP receptor antagonist had antinociceptive and anxiolytic-like effect and revert the reduced exploratory behavior alterations, showing similar efficacy in male and female ST mice. Olcegepant (BIBN4096BS) was also used to reduce nociception observed in other models of migraine [16]. Besides, using a model of repetitive retrain stress authors showed that CGRP signaling may be involved in migraine-like behaviors detected in this model of stress. In this study calcitonin gene-related peptide monoclonal antibody ALD405 reduced the nociception mainly in female mice, but authors used a priming effect of a nitric oxide donor [8]. In migraine patients, olcegepant can reduce migraine pain in both male and female patients [54]. Intracerebroventricular CGRP infusions is reported to be involved in various behaviors suggestive of anxiety [12]. In this sense, CGRP antagonist showed antidepressant-like effects in stressed mice, and in rats, CGRP antagonism has been shown to suppress anxiety-like behaviors [22].
Our data showed that unpredictable sound stress model in mice causes periorbital/hind paw mechanical allodynia, and grimacing pain behavior. Besides, we detected in this model anxiety-like symptoms and decrease exploratory activity. The nociception, anxiety-like behavior, and exploratory activity alterations detected in this model showed sexual dimorphism. Thus, female mice after stress induction presented higher levels of hind paw mechanical allodynia, PMA, grimacing pain behavior, and anxiety-like symptoms. In addition, we demonstrated that ST female presented lower levels of exploratory behavior than compared with ST male group. Besides, the plasmatic levels of inflammatory cytokines (IL-6 and TNF-α) and CGRP were increased in ST mice, particularly in female ST mice. The CGRP antagonist also caused an antinociceptive effect in male and female ST mice and reduced the anxiety-like behavior and associated exploratory alterations related to this model. Therefore, these data support the use of this stress priming model to the study of the mechanisms by which stress contributes to migraine-related pain and anxiety-like symptoms.