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. Author manuscript; available in PMC: 2025 Jun 27.
Published in final edited form as: Cephalalgia. 2024 Sep;44(9):3331024241281493. doi: 10.1177/03331024241281493

A male-specific mechanism of meningeal nociceptor sensitization promoting migraine headache

Caroline M Kopruszinski 1, Grace Lee 1, Laurent K Martin 1,2, Kara R Barber 1, Aubin Moutal 3, David W Dodick 4,5, Edita Navratilova 1,4, Frank Porreca 1,4
PMCID: PMC12204761  NIHMSID: NIHMS2087145  PMID: 39233656

Abstract

Background:

We wished to explore possible sexual dimorphism in mechanisms sensitizing or activating meningeal nociceptors that can promote the headache phase of migraine.

Methods:

Male and female C57BL6J mice received either supradural orexin B and an inflammatory mediator cocktail (IM) with migraine-like pain behaviors and photophobia recorded. Expression of orexin 2 receptor (OX2R) in trigeminal ganglion (TG) and phosphorylated extracellular signal-regulated kinases (ERK) levels in trigeminal nucleus caudalis (TNC) were evaluated. Orexin B-induced excitability of TG cells was assessed with patch-clamp electrophysiology. Intranasal delivery of CRISPR/Cas9 plasmids was used to edit the expression of OX2R in the TG.

Results:

Supradural orexin B induced migraine-like pain behaviors, photophobia and increased TNC ERK phosphorylation exclusively in males. Blockade of orexin signaling with supradural suvorexant, a dual orexin receptor antagonist, prevented, but did not reverse, migraine-like pain in males induced by supradural IM cocktail. OX2R expression was higher in male TG and orexin B increased TG neuron excitability in males. Intranasal OX2R CRISPR/Cas9 reduced TG receptor expression and orexin B-induced TNC ERK phosphorylation and prevented migraine-like pain induced by supradural orexin B in males.

Conclusions:

Our studies reveal a male-specific mechanism of TG nociceptor sensitization and migraine-like pain behavior mediated by orexin B/OX2R signaling. Sexually dimorphic mechanisms of trigeminal nociceptor sensitization and activation offer opportunities to improve patient outcomes by considering patient sex and may influence clinical trial design and interpretation.

Keywords: Sexual dimorphism, male nociceptors, orexin B, orexin receptor 2, migraine, trigeminal system

Introduction

Migraine is one of the world’s most prevalent and disabling neurological disorders impacting over one billion individuals (1,2). While migraine predominantly affects women, about one third of individuals with migraine are men (3,4). Human and preclinical evidence have revealed that mechanisms promoting pain are sexually dimorphic, suggesting the possible existence of qualitative sex differences in migraine pathophysiology and raising the question of whether personalized therapies considering patient sex could improve outcomes (5–14). Consistent with this possibility, existing migraine medications targeting the calcitonin-gene related peptide (CGRP) receptor have been shown to be effective in acute treatment of migraine in women, but evidence suggesting efficacy for acute therapy in men is lacking (15,16). However, sex differences in migraine prevention were not observed with anti-CGRP peptide or receptor monoclonal antibodies likely reflecting differences both in patient populations treated with these medications as well as outcome measures for termination of ongoing pain or inhibition of initiation of migraine (15,16). Identification of satisfactory migraine therapies for men is an important unmet medical need that may require engagement of novel, non-CGRP based mechanisms.

Disruption of sleep has been linked to migraine (17). Orexin is produced by neurons in the lateral hypothalamus that promote wakefulness (18–23). Narcolepsy is associated with deficits in orexin signaling (19,21,22). Hypothalamic orexinergic neurons project to multiple brain areas through ascending pathways, but critically, these cells also project to the spinal and medullary dorsal horns where orexin can influence nociceptive afferent fibers that express orexin receptors (24–29). There are two main forms of orexin, termed orexin A and orexin B that interact with orexin 1 (OX1R) and orexin 2 (OX2R) receptors (19–23). While orexin A can act at both OX1R and OX2R, orexin B demonstrates higher affinity and selectivity for OX2R (19–23,25). Orexin receptors are expressed in trigeminal ganglion (TG) and dorsal root ganglion (DRG) neurons of both male and female animals (24,30–34). Activation of OX2R can signal through a Gs pathway leading to activation of adenylate cyclase and increased cAMP production, a mechanism that has been linked to increased pain and migraine (35). Suvorexant is a dual orexin receptor antagonist (DORA) approved in the United States for the treatment of insomnia disorder (36,37).

A recent preclinical study investigating the relationship between chronic pain induced by partial sciatic nerve ligation (PSNL) and sleep reported that suvorexant mitigated pain-induced sleep disruption but unexpectedly also alleviated nerve injury induced mechanical allodynia (38). This finding led us to investigate whether the observed effects of suvorexant on allodynia might be the consequence of a direct effect of orexin in promoting nociceptor activation or sensitization. We found that orexin B can activate OX2R receptors to promote migraine-like pain behaviors only in males, revealing a qualitative sex difference that may be exploited to improve the treatment of migraine in men.

Materials and methods

Animals

All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Arizona and were performed in accordance with the ARRIVE guidelines, the ethical guidelines of the International Association for the Study of Pain regulations on animal welfare and the National Institutes of Health guidelines for the care and use of laboratory animals. A total of 227 male and female 8– 10-week-old C57BL/6J mice (Jackson Laboratories) were used in these studies. The estrous cycle of females was not monitored. Animals were housed four per cage under standard animal husbandry conditions of temperature-, humidity- and light cycle-controlled environment, with free access to food and water in the University of Arizona animal facility. The experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Arizona. Mice were randomly divided into control and experimental groups. Experimenters were blinded to treatments.

Drugs

Orexin B (Bachem, #4028264) was freshly diluted in 1x PBS at 1, 10 and 30 µg/5 µL for supradural injections and 180 nM for electrophysiology studies. Synthetic interstitial fluid consisted of 10 mM HEPES, 5 mM KCl, 135 mM NaCl, 1 mM MgCl2, 2 mM CaCl2, and 10 mM glucose at pH 7.4 (all components were from Sigma). Inflammatory mediator (IM) cocktail was given supradurally or subcutaneously into the hindpaw at 5 µL and consisted of 1 mM bradykinin, 1 mM histamine, 1 mM serotonin (Sigma), and 100 mM prostaglandin E2 (PGE2; Cayman Chemicals) diluted in synthetic interstitial fluid at a pH 4.0. Suvorexant (Adooq Bioscience, MK-4305 #A11500) was prepared at 100 pg/5 µL with 0.2% of DMSO in PBS for supradural injections. Controls received the respective vehicles at 5 µL.

Cutaneous allodynia evaluation

Mice were placed in elevated individual Plexiglass chambers with mesh flooring and allowed to habituate for three days for 2 h each day. Periorbital (cephalic) and hindpaw (extracephalic) tactile frequency of response were measured in the same mice following a 2 h habituation period after the third day. The 0.4 g (3.61N) and 1 g (4.08N) von Frey filaments (Stoelting, Wood Dale, IL, USA) were applied 10 times, with just enough pressure to cause the filament to display a slight arch to the periorbital (0.4 g) and hindpaw (1 g) region. Swiping of the face (facial grooming) was considered a positive periorbital response. Sharp withdrawal, shaking and/or licking the paw were considered positive hindpaw responses. Increased frequency of response was taken as a measure of cutaneous allodynia (CA). Frequency of response was calculated as (number of positive responses/10 × 100%) (8,14,39,40).

Spontaneous migraine-like headache behavior assays

Counts of rearing/vertical episodes behavior and conditioned place aversion (CPA) were used as an outcome measurement for spontaneous migraine-like headache (41,42). Details can be found in the Online Supplementary methods.

Photophobia assay

Light and dark equipment was used to access photophobia-like headache (43). Assay details can be found in the Online Supplementary methods.

Supradural injection

Injectors were modified from commercially available cannulas (Plastic One/Invivo1, part #C313I/SPC) by inserting a stopper to establish a length of 0.65–0.7 mm to maintain dura mater integrity. The injectors were connected to Tygon tubing (Cole Parmer Co. Vernon Hills, Illinois) which was then attached to a 25 μL Hamilton syringe (Hamilton, Reno, Nevada). The injector was inserted through the sagittal and lambdoid suture junction for supradural delivery under brief isoflurane anesthesia (40,44).

Viral vector, gRNA design, cloning and in vivo transfection for CRISPR/Cas9 targeting OX2R

Our strategy to delete the OX2R aimed to target the first exon (ENSMUST00000063140.15, gRNA: gATGACGACGAGGAATTCCTG, on-score 79.7, off-score 44.2) common to all mRNA arising from the mouse Hcrtr2 gene. Complete description of CRISPR/Cas9 plasmids targeting OX2R is in the Online Supplementary methods.

Intranasal CRISPR plasmid administration

The intranasal delivery of CRISPR plasmid solutions targeting the TG was performed as previously described (7,14). Briefly, animals were anesthetized with isoflurane, placed on a heating pad in a supine position and 30 μL of OX2R CRISPR/Cas9 or control plasmid was administered into each nasal cavity with a 2 min interval between administrations (3 μL each) over 20 min (15 μL in each nasal cavity). The administrations occurred in alternating fashion in the left and right nasal passages throughout the 20 min. No unusual behaviors were observed after the intranasal administration of plasmids. Animals received two intranasal administrations of OX2R CRISPR/Cas9 or control with three days apart from each administration.

Mouse trigeminal ganglion culture

Male and female mice were euthanized with an overdose of isoflurane before decapitation. The TG were dissected out and placed in cell culture media before being transferred to media containing neutral protease at 3.125 mg/mL (Worthington, #LS02104) and collagenase type 1 at 5 mg/mL (Worthington, #LS004194). The ganglia were incubated with the enzyme for approximately 45 min before mechanical dissociation with a fire polished Pasteur pipette. Neurons were maintained in neurobasal-A cell culture media (Thermo Fisher, #10888022) supplemented with 2% B-27 (Thermo Fisher, #17504044), 1% Penicillin/Streptomycin (Hyclone, #16777–164), 1% GlutaMax (Thermo Fisher, #35050061), 10% fetal bovine serum (Sigma, #F0926–50ML), 25 ng/mL GDNF (VWR, #10788–106), and 100 ng/mL mouse NGF (VRW, #76046–694). Dissociated TG neurons were plated onto 12 mm poly-D-lysine and laminin-coated glass coverslips (Neuvitro Corporation, #GG-12-LAMININ) and cultured for up to 24 h. After 12 h in culture, cells were incubated with orexin B or control and electrophysiological recordings were subsequently performed 30 min post incubation.

Patch-clamp electrophysiology

Current-clamp recordings were performed in TGV1 neurons of both female and male naïve mice. Details of current-clamp recordings can be found in the Online Supplementary methods.

Fluorescence microscopy

Naïve male and female mice were anesthetized with isoflurane and perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. TG were collected, post-fixed in paraformaldehyde for 4 h and transferred to a sucrose (15% then 30%) PBS solution at 4°C. TG sections (10 µm) were obtained using a microtome. Sections with V1 area cell bodies were selected for staining, rehydrated in PBS, then underwent antigen retrieval (citrate buffer, Sigma-Aldrich, #C9999) at 90°C for 10 min. Sections were then incubated with TrueBlack Autofluorescence Quencher (Biotium, #23014) for 1 min, washed in PBS and blocked to suppress non-specific antibody binding using TrueBlack IF Background Suppressor System (Biotium, #23012). Sections were incubated with primary antibodies rabbit anti-Orexin 2 Receptor (OX2R) (1:200; Millipore, #AB3094) and goat anti-Calcitonin gene-related peptide (CGRP) (1:200; Abcam, #ab36001), overnight at 4 °C. After washing in PBS, secondary antibodies donkey anti-goat AF488 (1:600; Jackson ImmunoResearch, #705-546-147) and donkey anti-rabbit AF555 (1:600; Invitrogen, #A31572) were incubated for 1 h. Slides were subsequently counterstained in DAPI and mounted with Prolong Glass Antifade media (Invitrogen, #P36980).

The TGV1 area was identified from a longitudinal section and imaged at 4×/0.13 (Echo Revolve R4 microscope) and stitched together using Pairwise Stitching in FIJI (45). Confocal z-stacks from TGV1 were acquired using an Olympus Fluoview FV1200 microscope, with images captured at magnifications of 20×/0.8. Consistent equipment and software settings were maintained throughout the duration of the experiments. Maximum intensity projections were subjected to background subtraction (meaning intensity of all pixels in the images).

Western blot analysis

Total lysate (30–40 μg of proteins) of TNC and TGV1 samples were subjected to western blot analysis as described previously (14,46,47). The following primary antibodies were used (anti-extracellular signal-regulated kinase (ERK) antibody, 1:1000, Cell signaling technology, #4696; anti-phosphorylated ERK, 1:1000, Cell signaling technology, #4370; and anti-β-Actin antibody: 1:4000, #A1978, Sigma Aldrich). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch (1:10000, #115-035-003 and #111-035-003). Protein bands were detected with Azure Sapphire Biomolecular Imager (Azure Biosystems) after applying a chemiluminescent reagent for 2 min (Thermo Fisher Scientific, #34577). Bands were quantified densitometrically with Image J software (National Institutes of Health). Original full-length blots images used in this study can be found in the Online Supplementary material.

General experimental design overview

See the detailed general experimental design overview in the Online Supplementary methods.

Statistical analysis

Sample size was determined using the GPower 3.1 software, with an established significance level of p < 0.05. Data are expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software). Mean differences of two group comparison of time course experiments for sensory thresholds and quantification of the number of action potentials fired for each depolarizing current pulse were analyzed using two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons post hoc test. An unpaired student t-test was used to analyze spontaneous migraine-like headache and photophobia-like behaviors. Data for electrophysiology rheobase and resting membrane potential, and protein expression levels were analyzed using the Mann-Whitney U-test or ANOVA following confirmation of normality distribution and homoscedasticity (Shapiro-Wilk and Brown Forsythe tests). Statistically significant differences were considered when p < 0.05. The statistical analysis, numbers of animals used (n), p values and interaction F ratios are reported in Online Supplementary Table S1.

Results

Sexually dimorphic effect of supradural orexin B in inducing migraine-like phenotypes and increased pERK expression in the trigeminal nucleus caudalis

Administration of orexin B onto the dura mater produced dose- and time-dependent development of allodynia in the periorbital (Figure 1(a) and (b)) and hindpaw (Figure 1(c) and (d)) regions of male (Figure 1(a) and (c)) but not female (Figure 1(b) and (d)) mice, revealed by increased frequency of response to tactile stimulation after orexin B when compared to control group. Supradural orexin B at 10 µg decreased rearing (Figure 1(e) and (f)) and produced conditioned place aversion (Figure 1(g) and (h)) demonstrated by decreased time spent in orexin B-paired chamber on the test day only in male mice. Administration of orexin B onto the dura mater also produced male-selective photophobia-like behavior revealed by the decrease in time spent in the light chamber in comparison to control (Figure 1(i) and (j)).

Figure 1.

Figure 1.

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Injection of orexin B onto the dura mater induced migraine-like behaviors and increased activity of the TNC exclusively in male mice. (a and b) Periorbital and (c and d) hindpaw tactile frequency of response was collected at baseline (BL) in (a and c) male and female (b and d) mice, followed by supradural administration of orexin B at 1, 10 and 30 µg/5 µL or vehicle control (5 µL). (a–d) Cutaneous allodynia was assessed 30 min, 1–5 h post supradural injection. (e and f) Count of rearing/vertical episodes as an outcome measurement of spontaneous migraine-like headache behavior for 1 h immediately after orexin B at 10 µg/5 µL or control in (e) male and (f) female mice. (g and h) CPA was performed in male and female mice with orexin B at 10 µg/5 µL or control used for conditioning. The difference in time spent in orexin B or control-paired chambers between test and baseline evaluation confirms the development of CPA in male mice treated with orexin B when compared to control group. (i and j) Adapted light and dark assay was assessed to evaluate photophobia-like behavior in male and female mice. Reduction of the percentage was considered an outcome measurement of photophobia-like behavior. TNC was collected 30 min after supradural orexin B at 10 µg/5 µL or control in male and female mice. (k) Representative western blot illustrating the levels of pERK and ERK in TNC of male and female mice. (l) Western blot quantification of ERK phosphorylation status (pERK/ERK ratio) in TNC of male and female mice treated with orexin B or control. Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by (a – d; n = 8–24) Tukey’s multiple comparison test, (e – j; n = 6–14) Student t-test and (k – l; n = 4) Mann-Whitney U-test with * and ** representing p < 0.05 and p < 0.01, respectively. Details of statistical analysis are found in Online Supplementary Table 1.

Western Blot analysis demonstrated that 10 µg of supradural orexin B increased ERK phosphorylation on Thr202/Tyr204 (phospho-ERK/total-ERK ratio) in the trigeminal nucleus caudalis (TNC) of male but not female mice (Figure 1(k) and (l)) while total ERK was unchanged (Figure 1(l)). Supradural administration of vehicle control had no effect on ERK expression or phosphorylation in the TNC of male and female mice (Figure 1(l)). Altogether, these data suggest that orexin B produces a male-specific mechanism of nociceptor sensitization in the trigeminal system.

Orexin B elicits male-specific increased excitability of TG neurons

Neuronal excitability of cultured TGV1 neurons from male, but not female, mice was significantly increased after a 30 min incubation with orexin B (180 nM) (Figure 2(a) and (b)). The number of action potential firing was evaluated by injecting current from 0 to 800 pA in 50 pA steps. Treatment with orexin B increased the number of action potentials starting at the 600 pA step only in TG neurons prepared from male mice (Figure 2(c)). In contrast, orexin B did not alter the excitability of female TG neurons (Figure 2(d)). Rheobase, which is the current required to fire a single action potential, was not changed in TG neurons of male (Figure 2(e)) and female (Figure 2(f)) mice pretreated with orexin B. However, orexin B significantly decreased the resting membrane potential (RMP) of isolated TG neurons from males (Figure 2(g)) but not females (Figure 2(h)). These results confirm that orexin B produces a male-specific sensitization of TG neurons.

Figure 2.

Figure 2.

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Pretreatment with orexin B increases neuronal excitability in trigeminal ganglia (TG) primary afferent nociceptors from male but not female mice. (a and b) Representative traces showing action potentials elicited from TG neurons following injection of 800 pA of depolarizing current into small TG neurons of (a) male and (b) female mice. Neurons were pretreated 30 min prior to the recording with either 180 nM orexin B or control. (c and d) Quantification of action potentials evoked by sequentially increasing current steps injected into the soma of (c) male and (d) female mice TG neurons pretreated with 180 nM orexin B or control. (e and f) Rheobase or the current required to fire a single action potential and (g and h) resting membrane potential (RMP) were recorded in TG neurons of (g) male and (h) female mice. Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by (c and d) Sidak’s multiple comparison test, (e – h) Mann-Whitney U-test with *, ** and *** representing p < 0.05, p < 0.01 and p < 0.001, respectively (n = 17–19). Details of statistical analysis are found in Online Supplementary Table 1.

Higher expression of orexin 2 receptor in the V1 region from male trigeminal ganglia

Images of longitudinal sections of mouse TGV1 tissues immunostained for anti-OX2R in magenta, anti-CGRP in green, and DAPI in blue illustrate an increase of neuronal immunoreactivity of OX2R in the TGV1 of male (Figure 3(a)) in comparison to female (Figure 3(b)) mice, as well as neurons with positive immunoreactivity for both OX2R and CGRP (Figure 3(a)–(d)). Western blot confirmed a higher expression of OX2R in male compared to female TG (Figure 3(e) and (f)).

Figure 3.

Figure 3.

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Enhanced orexin receptor 2 (OX2R) expression in male mice trigeminal ganglion (TG). (a and b) Representative photomicrograph of a longitudinal section of naïve (a) male and (b) female mouse trigeminal ganglion (TG) immunostained for anti-OX2R (magenta), anti-CGRP (green), and DAPI (blue). Scale bar: 500 µm. (c and d) Insets are representative confocal photomicrograph panels of (c) male and (d) female from TGV1 region (n = 4 per group). White arrow heads show neurons with positive immunoreactivity for both OX2R and CGRP. Scale bar: 50 µm. (e) Representative Western Blot of OX2R expression in TGV1 of male and female mice. (f) Western blot quantification of OX2R expression in TGV1 of male and female mice. Data are presented as mean ± SEM and analyzed using (f) Mann-Whitney U-test with * representing p < 0.05. Details of statistical analysis are found in Online Supplementary Table 1.

Suvorexant administration onto the dura mater prevents, but does not reverse, pain-like behavior induced by supradural administration of inflammatory mediator (IM) cocktail, specifically in male mice

Supradural administration of IM in rodents is well established model of migraine (42,48). Supradural administration of suvorexant immediately before IM (Figures 4) prevented the development of periorbital (Figure 4(a)) and hindpaw (Figure 4(b)) allodynia selectively in male mice, demonstrated by the blockade of increased frequency of response to tactile stimulation at the periorbital and hindpaw region (Figure 4). Local suvorexant failed to prevent the development of periorbital (Figure 4(c)) and hindpaw (Figure 4(c)) allodynia induced by supradural IM in female mice. Administration of suvorexant per se onto the dura mater did not modify periorbital (Online Supplementary Figure 1a) or hindpaw (Online Supplementary Figure 1b) tactile frequency of response of male mice. Supradural suvorexant did not modify hindpaw allodynia of male mice injected with hindpaw IM (Online Supplementary Figure 2), confirming the local effect of the orexin receptor antagonist.

Figure 4.

Figure 4.

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Supradural administration of suvorexant prevented migraine-like pain behavior induced by IM selectively in male mice. Periorbital tactile frequency of response was collected at baseline (BL) in (a and b) male and female (c and d) mice, followed by administration of suvorexant at 100 pg/5 µL or vehicle control (5 µL). Immediately after suvorexant or control, animals received supradural IM at 5 µL. Allodynia was assessed 30 min, 1–5 h post IM injection. Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by Sidak’s multiple comparison test with * representing p < 0.05 (n = 8). Details of statistical analysis are found in Online Supplementary Table 1.

Suvorexant injection onto the dura mater 30 min after supradural IM (Figure 5), when the migraine-like pain behavior was already established, failed to modify periorbital (Figure 5(a)) or hindpaw (Figure 5(b)) allodynia of male mice when compared to control-treated groups.

Figure 5.

Figure 5.

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Supradural administration of the orexin receptor antagonist suvorexant did not reverse migraine-like pain behavior induced by IM in male mice. (a) Periorbital and (b) hindpaw tactile frequency of response was collected at baseline (BL) in male mice, followed by supradural IM at 5 µL. Thirty minutes after IM, when allodynia was confirmed, animals received supradural administration of suvorexant at 100 pg/5 µL or vehicle control (5 µL). Cutaneous allodynia (CA) was reassessed 1–5 h post migraine-like pain induction. Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by Sidak’s multiple comparison test with * representing p < 0.05 (n = 8). Details of statistical analysis are found in Online Supplementary Table 1.

OX2R-gene editing in TGV1 blocked migraine-like pain in male mice

Intranasal administration of OX2R CRISPR/Cas9 plasmid produced significant loss of OX2R in the TGV1 region of male mice (Figure 6(a) and (b)). Deletion of TGV1 OX2R with CRISPR/Cas9-gene editing prevented periorbital (Figure 6(c)), and hindpaw (Figure 6(d)) allodynia produced by supradural orexin B in male mice confirming the specificity of the OX2R CRISPR/Cas9. Transfection of the CRISPR/Cas9 control construct did not alter the increased phosphorylation of ERK compared to naïve animals, while downregulation of TGV1 OX2R prevented this increased ERK phosphorylation in the TNC following supradural orexin B administration (Figure 6(e) and (f)). Editing of OX2R prevented periorbital (Figure 7(a)) and hindpaw (Figure 7(b)) allodynia induced by IM. Intranasal administration of control plasmid did not impact IM-induced allodynia (Figure 7) in male mice. These data confirm the pivotal role of TG OX2R signaling in migraine-like pain in males.

Figure 6.

Figure 6.

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Editing of OX2R in TGs prevent migraine-like pain behavior and activation of TCN induced by supradural orexin B in males. Periorbital tactile frequency of response was collected at baseline (BL) and animals received two intranasal administrations of OX2R CRISPR/Cas9 or control plasmid that resulted in down-regulation of OX2R in TGV1 region. (a) Representative Western Blot of OX2R and actin expression in TGV1 of male mice injected with OX2R CRISPR/Cas9 or control plasmid. (b) Western blot quantification of OX2R expression in TGV1 after intranasal OX2R CRISPR/Cas9 or control plasmid. (c and d) Fourteen days after the first CRISPR exposure, tactile frequency of response was collected (0) followed by administration of supradural orexin B at 10 µg/5 µL. Allodynia was evaluated 15- and 30-min post orexin B. TNC was collected immediately after the 30 min behavioral evaluation. (e) Representative western blot images of pERK, ERK and actin expression in TNC of male and female mice. (f) Quantitative verification of pERK (pERK/ERK ratio) expression in TNC of male mice treated with intranasal administrations of OX2R CRISPR/Cas9 or control plasmid that received supradural orexin B. Data are presented as mean ± SEM and analyzed using (b and f) Mann-Whitney U-test (c and d) two-way ANOVA followed by Sidak’s multiple comparison test or with * representing p < 0.05 (n = 8). Details of statistical analysis are found in Online Supplementary Table 1.

Figure 7.

Figure 7.

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Editing of OX2R in TGs reduced periorbital allodynia induced by IM in male mice. (a) Periorbital and (b) hindpaw tactile frequency of response was collected at baseline (BL) and animals received two intranasal administrations of OX2R CRISPR/Cas9 or control plasmid that resulted in down-regulation of OX2R in TGV1 region. Fourteen days after the first CRISPR exposure, tactile frequency of response was collected (0) followed by administration of supradural IM at 5 µL. Allodynia was evaluated 30 min and 1–5 h post IM. Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by Sidak’s multiple comparison test with * representing p < 0.05 (n = 8). Details of statistical analysis are found in Online Supplementary Table 1.

Discussion

Sexually dimorphic mechanisms relevant to nociception have recently been suggested by post-mortem analysis of transcript, protein and function of human, Rhesus macaque and rodent nociceptors (5–14). Female-specific pain mechanisms relevant to migraine have been previously described in the preclinical literature (7,8,14,49,50). However, mechanisms underlying migraine-like pain in males remain unexplored. Data from the present study revealed an unexpected male selective mechanism resulting from orexin B/OX2R signaling relevant to migraine pathophysiology. Our studies demonstrated that: (a) orexin B injection on the dura induced migraine-like pain behaviors, photophobia, increased TNC signaling and TG neuron excitability in male mice only; (b) TG OX2R expression was higher in males; (c) supradural suvorexant pretreatment prevented, but did not reverse, migraine-like pain in male mice; (d) intranasal OX2R CRISPR/Cas9 plasmid reduced TG OX2R expression and prevented migraine-like pain and TNC ERK phosphorylation induced by orexin B in males. These findings are the first to unveil a male-specific meningeal signaling mechanism that may contribute to migraine headache.

While preclinical models cannot capture the complex nature of migraine, they provide crucial mechanistic insights into migraine symptoms (42,48). Migraine headache likely arises from trigeminal nociceptors in the cranial meninges (51–53) assessed in animals through direct afferent activation and behavioral evaluation (42,48,54). Orexin A and B exhibit distinct and often opposing roles in nociception (55–57) including preclinical evaluation in migraine (55,56,58). Microinjection of orexin A or B into the posterior hypothalamus of rats respectively produced antinociceptive and pronociceptive modulation of meningeal and facial trigeminal input (55). Orexin B also increased intracellular calcium in cultured DRG neurons, and increased excitability of spinal dorsal horn neurons of rats (59,60). Interestingly, all of these studies were performed in male animals or in tissues taken from male animals (55,56,58). Our data are consistent with the central pronociceptive effects of orexin B demonstrated in male animals or in neurons from male animals. Additionally, however, we now demonstrate that these effects occur in peripheral neurons and are male specific. We found that orexin B sensitized cultured TG neurons and that supradural orexin B induced migraine-like pain behaviors in male, but not female mice. Thus, orexin B modulated the activity of neurons in the TNC of males only, as evidenced by the modulation of ERK phosphorylation. These data reveal a previously unknown sexually dimorphic pronociceptive mechanism in the peripheral trigeminal system.

Orexins signal through OX1R and OX2R, which are widely distributed in both central and peripheral tissues (57,61–63) including in the TG (30). Sex differences in the expression of prepro-orexin, orexin, and OXR mRNA have been reported in preclinical investigations (61,64–66). Female rodents express higher levels of prepro-orexin and OX1R in the CNS than males (61,65,66). Prepro-orexin mRNA is present in rat testes but not ovaries (61) and male rats show higher OX2R expression in the adrenal gland compared to females (61). We observed higher expression of OX2R in the TG of male mice. The reasons for differential receptor expression require further investigation but are likely influenced by sex hormones. Expression of OX2R in the adrenal gland was increased in ovariectomized female rats and attenuated with estrogen treatment (67). In males, orchiectomy decreased expression OX2R in the adrenal gland and this was reversed by treatment with testosterone (67). OX2R expression in the CNS remains unchanged by gonadectomy or hormone treatments (67). These data suggest that peripheral expression of the OX2R is negatively modulated by estrogen in females and positively modulated by testosterone in males (67). Interestingly, our data also revealed a very low level of co-localization of OX2R expression with CGRP positive neurons in the TG of both male and female mice suggesting that OX2R activation may promote nociception independently of CGRP which has been suggested to be female selective (5). Further studies are needed to comprehensively characterize TG cells expressing OX2R, especially in humans as these become available.

Pharmacological administration of orexin B demonstrated male specific effects but whether orexin B/OX2R signaling played a role in either preventing or reversing migraine-like pain was unknown. We therefore administered suvorexant onto the dura mater before or after inducing migraine-like pain via supradural IM cocktail. Pretreatment with supradural suvorexant fully prevented migraine-like pain in male, but not female, mice. Local actions of supradural administration were confirmed with a failure to affect hindpaw allodynia elicited by subcutaneous IM in the hindpaw of male mice. Supradural IM is a well characterized migraine model with presumed different mechanisms (42,48). IM components include bradykinin, serotonin, prostaglandins, and histamine in a low pH that are thought to activate respective meningeal receptors (42,48). The prevention of migraine-like pain behavior by suvorexant suggests that multiple activating mechanisms ultimately converge onto orexin/OXR pathway selectively in males. Remarkably, these observations suggest that in male mice, OX2R may be a common regulator of the activation of several meningeal receptors, including serotonin, bradykinin, histamine, PGE2, and ASIC receptors as well as receptors activated by substances released from mast cells. Alternatively, it is plausible to speculate that low pH might be the major driver of allodynia in the IM cocktail as evidenced by the heightened periorbital and hindpaw allodynia observed in both male and female mice following supradural administration of low pH compared to supradural IM (44). Therefore, blockade of OX2R may be producing its preventive effects primarily by preventing the effects of low pH. These possibilities require future investigation.

The preventive effect of blockade of orexin B/OX2R signaling was also confirmed by genetic editing of OX2R expression using CRISPR/Cas9 plasmids. We delivered the OX2R targeting CRISPR/Cas9 plasmid by the nasal route, an approach that we previously reported allows to edit the expression of prolactin receptors in the mouse TG. Importantly, we have previously confirmed that intranasal administration of CRISPR/Cas9 plasmids does not decrease receptor expression in tissues beyond the TG. In the present studies, we found that decreased TG expression of OX2R prevented migraine-like behavior as well as the increase of TNC pERK after supradural orexin B in male mice.

It should be noted that while pretreatment with suvorexant prevented migraine-like pain behaviors in male mice, reversal studies with suvorexant administration following establishment of pain behaviors from IM-migraine model was ineffective in mice of either sex. Although these data imply that OX2R blockade may be effective for migraine prevention but not for acute treatment, clinical experience will ultimately determine the potential utility of orexin receptor antagonists for either preventive or acute migraine therapy. The source of orexin B in activation of OX2R in migraine remains unclear. While previous studies indicate that orexin-containing neurons are mainly in the lateral hypothalamus and adjacent regions, with projections throughout the CNS and PNS (68,69), notable levels of prepro-orexin mRNA have been observed in peripheral regions that do not receive hypothalamic projections (30,61). Widespread expression of OXRs outside the CNS suggests that orexin production may occur in locations beyond the hypothalamus and importantly blood levels of orexin have been detected in humans (61,65,67).

The data reported have numerous implications. Migraine and sleep disorders are common co-morbidities. The strong link between orexinergic system and sleep/wakefulness states led to the investigation of blocking orexin signaling as a potential strategy for migraine prevention in a clinical trial (70). Filorexant, a dual orexin receptor antagonist (DORA) or placebo were administered orally for three months before bedtime in patients with episodic migraine to assess potential benefits in prevention. Filorexant failed to improve the number of headaches per month of patients recruited in the study (70). The authors suggested reasons that could have contributed to the failure of the clinical trial, including the fact that the majority of the migraine patients recruited for the study did not experience sleep problems (70). Notably, however, a compelling aspect of this clinical trial is the fact that the study predominantly involved a female population, comprising approximately 85% women (70). This raises the possibility that any potential signals or therapeutic benefits in males may have been overlooked, potentially altering the outcomes and conclusions had the population been predominantly or selectively male.

In conclusion, targeting peripheral orexin B/OX2R signaling could ultimately represent a significant male-specific therapy for migraine prevention. These findings highlight the importance of sex-specific considerations in migraine research and therapy. The sexually dimorphic nature of nociceptor sensitization and activation offer avenues for personalized therapeutic interventions and improving clinical outcomes through sex-based considerations. Furthermore, our study underscores the need to reassess past clinical trials and to consider patient sex in the design of future migraine clinical trials. Recognizing and leveraging sexual dimorphism in pain mechanisms may lead to more effective, personalized migraine therapies, alleviating the burden of this neurological disorder in both men and women.

Supplementary Material

Supplementary Material

Supplemental material for this article is available online.

Article highlights.

  • Identification of sexual dimorphism in sensitization and activation of meningeal nociceptors promoting migraine-like pain.

  • Supradural orexin B induced migraine-like pain and increased trigeminal nucleus caudalis ERK phosphorylation exclusively in males.

  • Higher trigeminal OX2R expression and increased orexin B-induced trigeminal neuron excitability were observed in male than in female mice.

  • Blocking orexin signaling with a dual orexin receptor antagonist prevented, but did not reverse, migraine-like pain induced by dural activators only in males.

  • Intranasal CRISPR/Cas9 editing of OX2R in the trigeminal ganglion prevented migraine-like pain in males.

  • These findings reveal a qualitative sex difference in mechanisms promoting migraine headache and suggest consideration of patient sex in choice of therapy.

  • These findings also suggest consideration of patient sex in the design of clinical trials of new mechanisms and possible re-evaluation of past failed trials.

Acknowledgments

We kindly thank Dr. Harrison Stratton, Dr. Xu Yue, Mahdi Dolatyari, Shizhen Luo, Gege Guzman, Kris Narisawa, and Aruna Sreenivasan for the technical support.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the National Institutes of Health (NIH) to Frank Porreca and Edita Navratilova (R01 NS120395 and R01 NS129552) and in part by grant from the U.S. Department of Defense (DOD) to Caroline M. Kopruszinski (PR230220).

Footnotes

Declaration of Conflicting Interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: C.M.K., G.L., L.K.M., K.B., A.M. and E.N. declare that they have no personal, financial, or relational conflicts of interest with this work. F.P. has served as a consultant or received research funding from Voyager, SiteOne Therapeutics, Nektar, Amgen, Acadia, Blackthorn, Teva, Eli Lilly, Hoba, Allergan, Ipsen, and Proximagen and has served as a founder of Catalina Pharma, Axon Therapeutics, Scientific Advisory Board Regulonix, and Condor Pharma. D.W.D. reports the following conflicts within the past 12 months: Consulting: AEON, Amgen, Clexio, Cerecin, Ctrl M, Allergan, Alder, Biohaven, Linpharma, Lundbeck, Promius, Eli Lilly, eNeura, Novartis, Impel, Satsuma, Theranica, Vedanta, WL Gore, Nocira, XoC, Zosano, Upjohn (Division of Pfizer), Pieris, Revance, Equinox. Honoraria: CME Outfitters, Curry Rockefeller Group, DeepBench, Global Access Meetings, KLJ Associates, Academy for Continued Healthcare Learning, Majallin LLC, Medlogix Communications, MJH Lifesciences, Miller Medical Communications, Southern Headache Society (MAHEC), WebMD Health/Medscape, Wolters Kluwer, Oxford University Press, Cambridge University Press. Research Support: Department of Defense, National Institutes of Health, Henry Jackson Foundation, Sperling Foundation, American Migraine Foundation, Patient Centered Outcomes Research Institute (PCORI). Stock Options/Shareholder/Patents/Board of Directors: Ctrl M (options), Aural analytics (options), ExSano (options), Palion (options), Healint (Options), Theranica (Options), Second Opinion/Mobile Health (Options), Epien (Options/Board), Nocira (options), Matterhorn (Shares/Board), Ontologics (Shares/Board), King-Devick Technologies (Options/Board), Precon Health (Options/Board). Patent 17189376.1–1466: vTitle: Botulinum Toxin Dosage Regimen for Chronic Migraine Prophylaxis.

Data availability

All data are available upon reasonable request.

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Supplementary Materials

Supplementary Material
NIHMS2087145-supplement-Supplementary_Material.docx (435.8KB, docx)

Data Availability Statement

All data are available upon reasonable request.

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