Migraine is a complex condition that causes more disability than all other neurological disorders combined. Characterized by an extreme throbbing headache, increased sensitivity to light, touch, and sound, nausea and vomiting, this disorder is two to three times more likely to occur in women than in men, but why these sex differences exist is unknown.
Now, new research led by Gregory Dussor, University of Texas at Dallas, US, reveals that calcitonin gene-related peptide (CGRP) injected directly onto the dura mater produced mechanical hypersensitivity in the facial region of female but not male rats. Similar sex differences were also observed in priming responses to subthreshold doses of CGRP, as well as in facial grimace scores. The investigators further showed that these were not rat-specific phenomena, as female mice also exhibited mechanical hypersensitivity and increased facial grimace scores in response to CGRP.
“This is a really important study because it demonstrates that CGRP has a dimorphic mechanism between males and females that had not yet been studied before in CGRP research," said Jes Olesen, University of Copenhagen, Denmark, who was not involved with the research.
“The gender issue is very important in migraine,” Olesen continued, “because migraine is grossly underrated and understudied as compared to its enormous societal impact. Therefore, these findings are extremely important because they could help explain why this disabling neurological disorder is so much more prevalent in females than it is in males, as it appears that a lower dose of CGRP is sufficient to trigger migraine-like pain behavior in females.”
The study also has implications for migraine treatment. “Our work demonstrates the need for sex-specific migraine therapeutics, as the mechanisms leading to migraine pain might be very different between males and females,” said Dussor.
The work was published online on April 8, 2019, in the Journal of Neuroscience.
Studying CGRP in female animals—finally!
CGRP has been implicated as a player in migraine for 30 years, and antibodies to the peptide or its receptor have been approved for clinical use in patients with episodic and chronic migraine. However, where CGRP acts was unknown, nor did researchers have a clear grasp as to why migraine is more common in women than in men and whether sexually dimorphic responses to CGRP could explain this difference in the prevalence of the condition.
Based on the CGRP antibody literature, it appeared that the peptide most likely acted in the periphery. In addition, the dura mater, which covers the spinal cord and brain, is highly innervated by nociceptive nerve endings, which can release CGRP, and contains many blood vessels where CGRP acts as a strong vasodilator. So Dussor and colleagues wanted to test if CGRP might be acting on the dura mater during a migraine attack.
Previous work from Dan Levy and colleagues found that CGRP failed to sensitize or excite meningeal nociceptors in anesthetized rats (Levy et al., 2005). However, when looking at the experimental detail of that study, the authors of the new paper quickly realized that, like many other pain studies, the prior investigation had only used male animals.
“Because migraines are more prevalent in females, we wanted to determine whether CGRP could trigger migraine-like pain in female rats using the same concentration of CGRP that Levy at al. used,” according to Amanda Avona, first author of the new study, also at UT Dallas.
To test that idea, the group injected CGRP directly onto the dura and tested the animals’ response at multiple time points. Dural CGRP produced facial hypersensitivity at three, five, and 24 hours following injection in females rats, but had no effect on male rats. The authors also found that even a 1,000-fold lower dose of CGRP was sufficient to elicit decreased facial withdrawal thresholds in females but not in males.
Prime time
Subthreshold doses (that is, doses that do not elicit pain in normal controls) of migraine triggers are typically sufficient to elicit a migraine in migraineurs. This suggests that these individuals are in a primed state even in between migraine attacks.
To model priming in rats, the investigators used a paradigm where the inflammatory cytokine interleukin-6 (IL-6) is injected onto the dura, or where brain-derived neurotrophic factor (BDNF) is injected intracisternally. In each case, males and females show mechanical hypersensitivity for 48 hours, after which they return to baseline. When the IL-6- or BDNF-treated animals then receive a dural injection of a pH7 subthreshold solution, they become hypersensitive again, for 24 hours, whereas there is no such effect in non-primed animals (Burgos-Vega et al., 2016).
In the current study, to see if there were sex differences in response to CGRP in primed animals, the authors first injected IL-6 onto the dura and observed similar mechanical hypersensitivity in both males and females. After the animals returned to baseline, the group challenged the animals with a subthreshold dose of CGRP and found that this elicited mechanical hypersensitivity only in female rats and not in male rats.
To make sure that this was not an IL-6-specific priming phenomenon, the researchers used intracisternal BDNF as a second priming stimulus in rats. Similar to IL-6, BDNF elicited facial hypersensitivity that lasted 48 hours in both males and females. However, consistent with the IL-6 experiment, only females exhibited mechanical allodynia following subthreshold CGRP injection.
Thus, once again, a female-specific response to CGRP had emerged, this time in primed animals.
As in rats, as in mice
The authors next wanted to see if what they found in rats also held true for other species. So they repeated the experiment where they injected CGRP directly onto the dura in male and female mice. Similar to rats, low doses of CGRP caused facial mechanical hypersensitivity in female but not male mice.
Up to this point, the investigators had obtained all of their behavioral data using evoked hypersensitivity assays. So they also asked whether mice that received dural CGRP showed changes in spontaneous pain behavior and whether sex differences arose in this case too.
To test this, Dussor and his colleagues looked at facial grimace responses using the mouse grimace scale. They found that, similar to the evoked behavioral assays, only female mice showed increases in facial grimace scores. This, too, revealed that facial hypersensitivity elicited by dural CGRP in females was not a rat-specific phenomenon.
Just say NO
Nitric oxide (NO) donors can cause migraines in humans and migraine-like behavior in rodents (Olesen et al., 1993Olesen and Jansen-Olesen, 2000). Because NO donors can also elicit CGRP release from the dura (Strecker et al., 2002), the authors next asked whether CGRP could prime animals to a subthreshold dose of NO donor.
Dural CGRP provoked mechanical hypersensitivity in female rats, which returned to baseline 24 hours after injection. At this time the animals received the NO donor sodium nitroprusside. One hour after intraperitoneal injection, female rats once again exhibited facial allodynia, which returned to baseline three hours later, compared to control females.
All in all, the results showed that dural CGRP is sufficient to elicit a primed state in female rats, and that a known migraine trigger can produce migraine-like behaviors in this primed state in females.
Finally, to test if the effects of CGRP were specific to dural administration, the authors injected a low dose of CGRP subcutaneously into the hindpaw of naïve male and female rats. In response, females but not males developed mechanical hypersensitivity. Neither sex developed facial hypersensitivity in response to the intraplantar injection of CGRP.
“This came as a surprise to me, as I was expecting that the sensitization due to CGRP might be a dura-specific phenomenon,” said Avona.
“Overall I think this is a very convincing paper that utilized a variety of techniques, drugs, and two rodent species to show that CGRP has a sexually dimorphic response and thus should encourage further research,” Olesen said.
Open questions
In terms of future research, it’s uncertain whether other compounds that provoke migraine will also produce sexually dimorphic effects.
“Are females in general more sensitive to other migraine triggers? There are many more compounds that could be investigated, such as pituitary adenylate cyclase-activating polypeptide [PACAP], which has been shown to induce migraines in humans,” according to Olesen.
Another important question is the physiological mechanism by which the sex differences arise. The immune system may play a role, since CGRP could have sexually dimorphic effects on mast cell degranulation in the meninges, which has been linked to excitation of nociceptors there. Perhaps females also express different amounts of CGRP receptor and receptor components, allowing them to respond to lower concentrations of CGRP.
Where exactly the peptide is acting is also uncertain, according to Dussor. “Is CGRP truly directly acting on neurons within the dura, or is it acting on blood vessels or immune cells which then signal back to the nervous system leading to the sexually dimorphic response?”
Francie Moehring, PhD, is a postdoctoral research fellow at the Medical College of Wisconsin, Milwaukee, US.
Featured image credit: Ilya Zaytsev/123RF Stock Photo.