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.